Bio 12 third exam
focal adhesions
responsible for connecting intracellular microfilaments to protein fibers of the basal lamina
what genes are mutated in cancer cells?
those controlling cell growth, division, and survival
different types of cell junctions
tight junction adherens junction desmosome gap junction hemidesmosome
three filament systems comprise the cytoskeleton
actin filaments microtubules intermediate filaments
how is actin polymerization regulated?
actin polymerization is regulated by proteins that bind G-actin
activity of cyclin/CDK complexes
different cyclin/CDK complexes become active at different times during cell cycle G1 cyclin/CDKs G1/S cyclin/CDKs S cyclin/CDK M cyclin/CDK
dynamic instability
dynamic instability of microtubules is an intrinsic property
troponin
holds tropomyosin in place moves tropomyosin aside & exposes myosin binding sites when Ca++ is released regulatory protein that binds to actin, tropomyosin, and calcium
kinetochore attachment to MTs during prometaphase
side capture- MT catches kinetochore and then slides to plus end end capture- kinetochore is caught at the plus end so no sliding necessary
Mad2
signal that chromosomes are not completely attached to microtubule spindles by blocking Cdc20 action
what affects microtubule assembly/disassembly?
temperature and tubulin concentration affect microtubule assembly and disassembly cold leads to disassembly warm leads to assembly there is a critical concentration of tubulin, much like with G-actin for the actin filaments
Z-discs
the Z-discs are made of CapZ binding protein
cell motility
Cell motility is one of the crowning achievements of evolution. Primitive cells were probably immobile, carried by currents in the primordial milieu. With the evolution of multicellular organisms, primitive organs were formed by migrations of single cells and groups of cells from distant parts of the embryo. In adult organisms, movements of single cells in search of foreign organisms are integral to the host's defenses against infection; on the other hand, uncontrolled cell migration is an ominous sign of a cancerous cell. Most cells in the body are stationary, but many of these exhibit dramatic changes in their morphology — the contraction of muscle cells, the elongation of nerve axons, the formation of cell-surface protrusions, the constriction of a dividing cell during mitosis. Perhaps the most subtle movements are those within cells — the active separation of chromosomes, the streaming of cytosol, the transport of membrane vesicles. These internal movements are essential elements in the growth and differentiation of cells, carefully controlled by the cell to take place at specified times and in particular locations. All cell movements are a manifestation of mechanical work; they require a fuel (ATP) and proteins that convert the energy stored in ATP into motion. The cytoskeleton, a cytoplasmic system of fibers, is critical to cell motility. Like steel girders supporting the shell of a building, the cytoskeleton plays a structural role by supporting the cell membrane and by forming tracks along which organelles and other elements move in the cytosol. Unlike the passive framework of a building, though, the cytoskeleton undergoes constant rearrangement, which can produce movements. In the electron microscope, the cytoskeleton appears as a dense and seemingly random array of fibers. However, we now recognize that this array consists of three types of cytosolic fibers: microfilaments, 7 to 9 nm in diameter; intermediate filaments, 10 nm in diameter; and microtubules, 24 nm in diameter. These cytoskeletal fibers are well-ordered polymers built from small protein subunits held together by noncovalent bonds. Instead of being a disordered array, the cytoskeleton is organized into discrete structures — primarily bundles, geodesic-dome-like networks, and gel-like lattices. Although the primary function of intermediate filaments is structural, to reinforce cells and to organize them into tissues, we discuss them in the next chapter because microtubules and intermediate filaments are often associated with one another. Cells have evolved two basic mechanisms for gener-ating movement. One mechanism involves a special class of enzymes called motor proteins. These proteins use energy from ATP to walk or slide along a microfilament or a microtubule. Some motor proteins carry membrane-bound organelles and vesicles along the cytoskeletal fiber tracks; other motor proteins cause the fibers to slide past each other. The other mechanism responsible for many of the changes in the shape of a cell entails assembly and disassembly of microfilaments and microtubules. A few movements involve both the action of motor proteins and cytoskeleton rearrangements. The structure and functions of microtubules are discussed in Chapter 19. In this chapter we focus on microfilaments and the actin subunits that compose them, which play a role in numerous types of movement from cell migration to cytosol transport. We begin with a description of the actin cytoskeleton and its role in determining cell shape. Then we cover the biochemistry of actin assembly and cellular mechanisms for controlling this process. We next examine the structure of myosin, the actin motor protein, and how it transduces the energy of ATP into a sliding movement along actin filaments. With a basic understanding of the key components of the cytoskeleton and the interaction of actin and myosin, we can examine the mechanisms responsible for various types of cell movement, starting with the contraction of muscle and ending with the crawling movements of amebas and skin cells. The chapter ends with a discussion about the regulation of movement by cell-signaling pathways.
GTP cap model to explain dynamic instability
Dynamic instability refers to the coexistence of assembly and disassembly at the ends of a microtubule. The microtubule can dynamically switch between growing and shrinking phases in this region. Tubulin dimers can bind two molecules of GTP, one of which can be hydrolyzed subsequent to assembly. During polymerization, the tubulin dimers are in the GTP-bound state. The GTP bound to α-tubulin is stable and it plays a structural function in this bound state. However, the GTP bound to β-tubulin may be hydrolyzed to GDP shortly after assembly. The assembly properties of GDP-tubulin are different from those of GTP-tubulin, as GDP-tubulin is more prone to depolymerization. A GDP-bound tubulin subunit at the tip of a microtubule will tend to fall off, although a GDP-bound tubulin in the middle of a microtubule cannot spontaneously pop out of the polymer. Since tubulin adds onto the end of the microtubule in the GTP-bound state, a cap of GTP-bound tubulin is proposed to exist at the tip of the microtubule, protecting it from disassembly. When hydrolysis catches up to the tip of the microtubule, it begins a rapid depolymerization and shrinkage. This switch from growth to shrinking is called a catastrophe. GTP-bound tubulin can begin adding to the tip of the microtubule again, providing a new cap and protecting the microtubule from shrinking. This is referred to as "rescue". The guanine nucleotide molecule (i.e., GTP) bound to the β-tubulin molecule plays a key role in dynamic instability. Each α- and β-tubulin molecule binds one molecule of GTP. The GTP bound to the α-tubulin is neither hydrolyzed nor exchanged, whereas that bound to the β-tubulin can be exchanged when the dimer is free in solution. These exchangeable GTP molecules are located at the head-to-tail interface between subunits along a protofilament and are hydrolyzed shortly after being incorporated into the polymer. The hydrolysis of the GTP to GDP affects the conformation of the inter-dimer interface. Structural studies indicated that, while the protofilament composed of GTP-tubulin is almost straight, the natural conformation of the protofilament of GDP-tubulin is curved outward from the wall of the microtubule. Lateral interactions between GTP-tubulin molecules provide the force that holds these molecules together, resisting the natural tendency of protofilaments composed of GDP-tubulins to curve outward. Microtubule disassembly can be triggered by a subtle change in the lateral interactions between the protofilaments. The tubulin subunits near the ends of rapidly growing microtubules are more likely to be bound to GTP, and the loss of the GTP-tubulin portion, known as the GTP-cap, renders the microtubules more prone to depolymerization. This GTP-cap model is supported by experimental evidence and is widely accepted. The mechanism underlying the rescue event is relatively poorly understood. It was postulated that the addition of new tubulin dimers to the shrinking ends of multiple protofilaments stops depolymerization. Direct observations using a GTP-tubulin-specific antibody revealed that GTP-tubulin patches exist in the middle of microtubule. These GTP-tubulin patches may contribute to the rescue events following depolymerization. Although the frequency of rescue events is important for controlling microtubule organization in vivo, the mechanism underlying GTP-tubulin patch formation is unknown. A noteworthy inconsistency of the GTP-cap model is the behavior of the minus, or slower growing end. In principle, the minus end should be more inclined to undergo depolymerization than the quicker growing plus end, because the minus end would be expected to lose its GTP-cap more readily and frequently than the plus end; however, experimental evidence indicates that the opposite is true. Furthermore, the newly exposed minus ends created by physical severing of the microtubules are quite stable, as these ends used to be located in the middle of longer microtubules. By severing a microtubule, a new plus end and minus end are created and the subunits near the newly formed ends are predominantly in the GDP form. According to the GTP-cap model, both new microtubules are highly likely to depolymerize from their newly created ends. However, this is only true for the new plus ends. Although microtubule behavior after cutting is important in living cells, the details of this behavior are unknown.
regulation of CDK
make sure to know p27
what are the causes of cancer promoting mutations?
- chemical induced mutations (ex: cigarette smoke) - radiation induced mutations (ex: UV radiation in sunlight) - mutations caused by DNA replication errors - inherited mutations - infection agents - retroviruses - DNA tumor viruses (ex: HPV)
separation of sister chromatids
-Securin binds Separase and keeps it inactive -Ubiuqination of Securin by APC targets it for degradation -Separase then able to cleave a subunit of cohesion complex -Cleavage of cohesion complex opens the ring and allows separation of sister chromatids
structure of microtubules
-microtubules are hollow, cylindrical structures -13 longitudinal rows called protofilaments -each protofilament is assembled from dimers of alpha- and beta-tubulin subunits assembled into tubules with plus and minus ends staggered structure creates seam
microtubule dynamics (and experimental approaches to study it)
1. FRAP 2. cell injected with rhodamine 3. injection of a low number of tubulin dimers that are brightly labeled with fluor
methods to view microtubule dynamics in living cells
1. FRAP 2. injection of rhodamine tubulin- tells us filaments can bend (bend once they reach membrane) 3. injection of low number of tubulin dimers that are brightly labeled with fluor- generates "speckles"
how is CDK activity regulated?
1. cyclin proteins (synthesis, localization, and degradation) 2. phosphorylation of the CDK itself 3. binding of inhibitors to the CDK
new strategies for treating cancer beyond surgery, radiation, and chemotherapy
1. immunotherapy- use antibodies or immune cells to attack tumors 2. gene therapy- introduce a gene that kills tumors or causes gain of normal cellular properties 3. inhibit the activity of cancer promoting proteins 4. prevent angiogenesis
roles of actin-binding proteins
1. monomer nucleating 2. monomer-sequestering 3. end-blocking (capping) 4. monomer polymerizing 5. depolymerizing 6. bundling 7. filament-severing 8. membrane-binding 9. cross-linking
cells undergoing apoptosis have many characteristic features
3 to remember: 1. nuclear fragmentation 2. blabbing 3. cell fragmentation
cell junctions
A cell junction (or intercellular bridge) is a type of structure that exists within the tissue of some multicellular organisms, such as animals. Cell junctions consist of multiprotein complexes that provide contact between neighboring cells or between a cell and the extracellular matrix. They also build up the paracellular barrier of epithelia and control the paracellular transport. Cell junctions are especially abundant in epithelial tissues. Cell junctions are especially important in enabling communication between neighboring cells via specialized proteins called communicating junctions. Cell junctions are also important in reducing stress placed upon cells.
MPF
A cyclin-Cdk complex that causes the cell to move from interphase into mitosis. MPF activity rises during mitosis and falls during interphase A famous example of how cyclins and Cdks work together to control cell cycle transitions is that of maturation-promoting factor (MPF). The name dates back to the 1970s, when researchers found that cells in M phase contained an unknown factor that could force frog egg cells (stuck in G2 phase) to enter M phase. This mystery molecule, called MPF, was discovered in the 1980s to be a Cdk bound to its M cyclin partner. MPF provides a good example of how cyclins and Cdks can work together to drive a cell cycle transition. Like a typical cyclin, M cyclin stays at low levels for much of the cell cycle, but builds up as the cell approaches the G2/M transition. As M cyclin accumulates, it binds to Cdks already present in the cell, forming complexes that are poised to trigger M phase. Once these complexes receive an additional signal (essentially, an all-clear confirming that the cell's DNA is intact), they become active and set the events of M phase in motion. The MPF complexes add phosphate tags to several different proteins in the nuclear envelope, resulting in its breakdown (a key event of early M phase), and also activate targets that promote chromosome condensation and other M phase events.
integrin
A large family of heterodimeric transmembrane proteins that promote adhesion of cells to the extracellular matrix or to the surface of other cells.
neuromuscular junction
A neuromuscular junction (or myoneural junction) is a chemical synapse formed by the contact between a motor neuron and a muscle fiber. It is at the neuromuscular junction that a motor neuron is able to transmit a signal to the muscle fiber, causing muscle contraction.
poleward flux
A particularly dramatic manifestation of spindle dynamics is poleward flux: the minus-end-directed flow of tubulin subunits through spindle microtubules, driven by the disassembly of microtubules at their minus ends (oriented toward the spindle poles) and assembly at their plus ends (oriented toward the spindle equator). Functionally, poleward flux has been proposed to provide a mechanism for moving chromosomes toward spindle poles and for controlling spindle length. However, the overall contribution of flux to these activities has remained a point of contention
pre-replicative complex
A pre-replication complex (pre-RC) is a protein complex that forms at the origin of replication during the initiation step of DNA replication. Formation of the pre-RC is required for DNA replication to occur. Complete and faithful replication of the genome ensures that each daughter cell will carry the same genetic information as the parent cell. Accordingly, formation of the pre-RC is a very important part of the cell cycle.
what activates ARP 2/3
ARP 2/3 is activated by Cdc42
how can branched filaments form?
ARP 2/3 is similar to G-actin in shape so they form a nucleus on the side of the filament to form a daughter filament
ATP and assembly
ATP binds in the cleft between the two lobes of each actin monomer G-actin monomers assemble into long, helical F-actin polymers ATP-bind cleft opens to minus end. minus end of one subunit interacts with plus end of another subunit. leads to structured polarity F-actin has structural and functional polarity
how does ATP hydrolysis affect stability?
ATP hydrolysis decreases the stability of actin-actin interactions
mitogens
An extracellular signal molecule that stimulates cell proliferation most mammalian cells require a positive signal to undergo another round of the cell cycle these signals are called "mitogens" examples: EGF, PDGF (growth factors that initiate signal transduction pathway) one way that mitogens stimulate cell proliferation is by inhibiting the cell cycle inhibitor, Rb protein Rb=retinoblastoma
chromosome
Any of the usually linear bodies in the cell nucleus that contain the genetic material. long, continuous thread of DNA that consists of numerous genes and regulatory information
telomerase
As a cell begins to become cancerous, it divides more often, and its telomeres become very short. If its telomeres get too short, the cell may die. Often times, these cells escape death by making more telomerase enzyme, which prevents the telomeres from getting even shorter.
Metaphase
As prometaphase ends and metaphase begins, the chromosomes align along the cell equator. Every chromosome has at least two microtubules extending from its kinetochore — with at least one microtubule connected to each pole. At this point, the tension within the cell becomes balanced, and the chromosomes no longer move back and forth. In addition, the spindle is now complete, and three groups of spindle microtubules are apparent. Kinetochore microtubules attach the chromosomes to the spindle pole; interpolar microtubules extend from the spindle pole across the equator, almost to the opposite spindle pole; and astral microtubules extend from the spindle pole to the cell membrane.
anaphase
At anaphase, the paired chromatids synchronously separate to form two daughter chromosomes, and each is pulled slowly toward the spindle pole it is attached to. The kinetochore microtubules get shorter, and the spindle poles also move apart, both contributing to chromosome separation
metaphase
At metaphase, the chromosomes are aligned at the equator of the spindle, midway between the spindle poles. The paired kinetochore microtubules on each chromosome attach to opposite poles of the spindle
prophase
At prophase, the replicated chromosomes, each consisting of two closely associated sister chromatids, condense. Outside the nucleus, the mitotic spindle assembles between the two centrosomes, which have begun to move apart.
cytoplasmic versus axonemal dynein
Axonemal dynein causes sliding of microtubules in the axonemes of cilia and flagella and is found only in cells that have those structures. Cytoplasmic dynein moves things around in cell
Interphase parts
G1: cell grows and carries out normal metabolism; organelles duplicate S: DNA replication and chromosome duplication G2: cell grows and prepares for mitosis
two different "specificity factors" determine the substrates for the APC
Cdc20→ Securin Chd-1→ mitotic cyclins
caspase
Caspases are a family of intracellular cysteine proteinases involved in inflammation and apoptosis. These enzymes appear to be involved in the initial signaling events, as well as the downstream proteolytic cleavages, that result in apoptotic cell death. A "killer enzyme" that plays a role in apoptosis, or programmed cell death
Cdc25
Cdc25 is the phosphatase that removes the inhibitory phosphate on Tyr-15 (Y15) when Cdc25 activity missing, CDK does not get activated and cells get larger and larger but don't divide
chromosome condensation
Chromosome condensation is the dramatic reorganisation of the long thin chromatin strands into compact short chromosomes that occurs in mitosis and meiosis. occurs during prophase
separase
Cleaves cohesin complex, releasing sister chromatids during metaphase-anaphase transition
cytokinesis
Division of the cytoplasm during cell division
ErbB oncoprotein
Dimerization of ErbB receptors leads to induction of the kinase activity. As a result, a number of tyrosine residues at the C terminal end of the ErbB molecules become phosphorylated. The phosphorylated tyrosine residues serve as docking sites for an array of signaling molecules that contain Src homology 2 (SH2) domains or phosphotyrosine-binding domains. The 2 major signaling pathways activated by ErbB receptors are the MAPK and PI3K-AKT pathways. In addition, ErbB receptors also trigger signaling through other effectors, such as mammalian target of rapamycin (mTOR). The general paradigm is that the specific combination of ErbB receptors in the dimer defines the downstream signaling network as well as the intensity and the duration of the stimulation. For example, heterodimers that contain ErbB3 favor activation of the PI3K pathway. The signaling events triggered by the ErbB receptors promote cell proliferation and survival, which are the driving forces of malignant transformation. In addition, activation of ErbB receptors might contribute to gross chromosomal changes, leading to aneuploidy. Chromosome instability and aneuploidy are frequently seen in human cancers overexpressing ErbB receptors. Similarly, in her2/neu-transgenic mice, chromosome instability is associated with her2/neu-positive tumors. Amplification of the centrosome, the organelle that plays a critical role in spindle assembly and proper chromosomal segregation during mitosis, is also commonly detected in her2/neu-transgenic mice. Aberrant ErbB receptor activities can facilitate accumulation of genomic abnormalities by suppressing the cell death that is normally associated with mitotic failure. Moreover, dysregulation of ErbB receptor signaling might disrupt mitotic checkpoints that are essential for maintaining chromosome integrity.
Survival factor
Extracellular signaling molecule that must be present to prevent apoptosis.
what reveals microtubules are dynamic structures?
FRAP reveals microtubules are dynamic structures three possible mechanisms by which "recovery" of fluorescence might occur: 1. recovery via microtubule dynamics 2. recovery via microtubule growth 3. recovery via translating microtubule
foci
Focus is a pathologic term describing cells that can be seen only microscopically. The cells stand out from surrounding tissue based on their appearance, special stains, or other testing. Foci is the plural of focus and implies only microscopic visualization of the tumor cells. Foci are cells located in a specific organ of the body that are notably different from the surrounding cells. These differences are caused by mutation or other types of cellular damage, and they're generally the first sign of a developing lesion, tumor or other disease
elongation
G-actin added to the ends of nucleus to form F-actin
loss-of-function
Generally, loss-of-function (null) mutations are found to be recessive. In a wild-type diploid cell, there are two wild-type alleles of a gene, both making normal gene product. In heterozygotes (the crucial genotypes for testing dominance or recessiveness), the single wild-type allele may be able to provide enough normal gene product to produce a wild-type phenotype. In such cases, loss-of-function mutations are recessive. In some cases, the cell is able to "upregulate" the level of activity of the single wild-type allele so that in the heterozygote the total amount of wild-type gene product is more than half that found in the homozygous wild type. However, some loss-of-function mutations are dominant. In such cases, the single wild-type allele in the heterozygote cannot provide the amount of gene product needed for the cells and the organism to be wild type.
if no ATP hydrolysis
High [G-actin]- both ends will grow and plus end grows faster Low [G-actin]- bonds ends shrink and plus end shrinks faster
where are IFs anchored?
IFs are anchored in cell junctions this helps make epithelial cells resistant to mechanical stress
dynamic
IFs are dynamic polymers
tensile strength
IFs have great tensile strength
Drugs commonly used in studying the actin cytoskeleton
Phalloidin Cytochalasin Latrunculin
Myc transcription factor
In cancer, c-myc is often constitutively (persistently) expressed. This leads to the increased expression of many genes, some of which are involved in cell proliferation, contributing to the formation of cancer. A common human translocation involving c-myc is critical to the development of most cases of Burkitt lymphoma. Constitutive upregulation of Myc genes have also been observed in carcinoma of the cervix, colon, breast, lung and stomach. Myc is thus viewed as a promising target for anti-cancer drugs
mitosis
In cell biology, mitosis is a part of the cell cycle when replicated chromosomes are separated into two new nuclei. In general, mitosis (division of the nucleus) is preceded by the S stage of interphase (during which the DNA is replicated) and is often accompanied or followed by cytokinesis, which divides the cytoplasm, organelles and cell membrane into two new cells containing roughly equal shares of these cellular components. Mitosis and cytokinesis together define the mitotic (M) phase of an animal cell cycle—the division of the mother cell into two daughter cells genetically identical to each other. The process of mitosis is divided into stages corresponding to the completion of one set of activities and the start of the next. These stages are prophase, prometaphase, metaphase, anaphase, and telophase. During mitosis, the chromosomes, which have already duplicated, condense and attach to spindle fibers that pull one copy of each chromosome to opposite sides of the cell.The result is two genetically identical daughter nuclei. The rest of the cell may then continue to divide by cytokinesis to produce two daughter cells. Producing three or more daughter cells instead of normal two is a mitotic error called tripolar mitosis or multipolar mitosis (direct cell triplication / multiplication). Other errors during mitosis can induce apoptosis (programmed cell death) or cause mutations. Certain types of cancer can arise from such mutations. Mitosis occurs only in eukaryotic cells. Prokaryotic cells, which lack a nucleus, divide by a different process called binary fission. Mitosis varies between organisms. For example, animal cells undergo an "open" mitosis, where the nuclear envelope breaks down before the chromosomes separate, whereas fungi undergo a "closed" mitosis, where chromosomes divide within an intact cell nucleus. Most animal cells undergo a shape change, known as mitotic cell rounding, to adopt a near spherical morphology at the start of mitosis. Most human cells are produced by mitotic cell division. Important exceptions include the gametes - sperm and egg cells - which are produced by meiosis.
Fibronectin
Large glycoprotein- promotes cell adhesion and anchorage
microtubule associated proteins (MAPs)
MAP2 Tau Katanin can connect microtubules to one another and other structures Because of their inherent dynamic instability, most microtubules are frequently disassembled within the cell. This dynamic behavior can, however, be modified by the interactions of microtubules with other proteins. Some cellular proteins act to disassemble microtubules, either by severing microtubules or by increasing the rate of tubulin depolymerization from microtubule ends. Other proteins (called microtubule-associated proteins or MAPs) bind to microtubules and increase their stability. Such interactions allow the cell to stabilize microtubules in particular locations and provide an important mechanism for determining cell shape and polarity.
actin bundles
Microfilament bundles - These extremely long microfilaments are located in networks and, in association with contractile proteins such as non-muscular myosin, they are involved in the movement of substances at an intracellular level.
motor protein
Motor proteins are a class of molecular motors that can move along the cytoplasm of animal cells. They convert chemical energy into mechanical work by the hydrolysis of ATP. Flagellar rotation, however, is powered by proton pump. The best prominent example of a motor protein is the muscle protein myosin which "motors" the contraction of muscle fibers in animals. Motor proteins are the driving force behind most active transport of proteins and vesicles in the cytoplasm. Kinesins and cytoplasmic dyneins play essential roles in intracellular transport such as axonal transport and in the formation of the spindle apparatus and the separation of the chromosomes during mitosis and meiosis. Axonemal dynein, found in cilia and flagella, is crucial to cell motility, for example in spermatozoa, and fluid transport, for example in trachea.
myosin power stroke
Multiple myosin II molecules generate force in skeletal muscle through a power stroke mechanism fueled by the energy released from ATP hydrolysis. The power stroke occurs at the release of phosphate from the myosin molecule after the ATP hydrolysis while myosin is tightly bound to actin. The effect of this release is a conformational change in the molecule that pulls against the actin. The release of the ADP molecule leads to the so-called rigor state of myosin. The binding of a new ATP molecule will release myosin from actin. ATP hydrolysis within the myosin will cause it to bind to actin again to repeat the cycle. The combined effect of the myriad power strokes causes the muscle to contract.
Myosin I
Myosin I, a ubiquitous cellular protein, functions as monomer and functions in vesicle transport. It has step size of 10 nm and has been implicated as being responsible for the adaptation response of the stereocilia in the inner ear.
Myosin V
Myosin V is an unconventional myosin motor, which is processive as a dimer and has a step size of 36 nm. It translocates (walks) along actin filaments traveling towards the barbed end (+ end) of the filaments. Myosin V is involved in the transport of cargo (e.g. RNA, vesicles, organelles, mitochondria) from the center of the cell to the periphery, but has been furthermore shown to act like a dynamic tether, retaining vesicles and organelles in the actin-rich periphery of cells. A recent single molecule in vitro reconstitution study on assembling actin filaments suggests that Myosin V travels farther on newly assembling (ADP-Pi rich) F-actin, while processive runlengths are shorter on older (ADP-rich) F-actin.
necrosis
Necrosis is a form of cell injury which results in the premature death of cells in living tissue by autolysis. Necrosis is caused by factors external to the cell or tissue, such as infection, toxins, or trauma which result in the unregulated digestion of cell components. In contrast, apoptosis is a naturally occurring programmed and targeted cause of cellular death. While apoptosis often provides beneficial effects to the organism, necrosis is almost always detrimental and can be fatal. Cellular death due to necrosis does not follow the apoptotic signal transduction pathway, but rather various receptors are activated, and result in the loss of cell membrane integrity and an uncontrolled release of products of cell death into the extracellular space
neurofilaments
Neurofilaments are intermediate filaments found in the cytoplasm of neurons. They are protein polymers measuring approximately 10 nm in diameter and many micrometers in length.
nexin
Nexin is a proteinous inter-doublet linkage that prevents microtubules in the outer layer of axonemes from movement with respect to each other. Otherwise vesicular transport proteins such as dynein would dissolve the whole structure.
G0 phase
Not all cells are continually replicated. Non-replicating cells are found in a stage of the cell cycle called G0. These cells may be quiescent (dormant) or senescent (aging or deteriorating). Such cells generally enter the G0 phase from G1. Cells may remain quiescent in G0 for an indeterminate period of time (when no more new cells are needed), only to re-enter G1 phase and begin dividing again under specific conditions. While quiescent cells may re-enter the cell cycle, senescent cells do not. One reason that cells trigger senescence is to ensure that damaged or defective DNA sequences is not passed on to daughter cells.
myosin
One of a family of motor proteins with a globular head region and coiled-coil tail region that has actin-stimulated ATPase activity; drives movement along actin filaments during muscle contraction and cytokinesis (myosin II) and mediates vesicle translocation (myosins I and V).
sarcoplasmic reticulum
Organelle of the muscle fiber that stores calcium. specialized endoplasmic reticulum of muscle cells
pericentriolar material
Pericentriolar material (PCM, sometimes also called pericentriolar matrix) is an amorphous mass of protein which makes up the part of the animal centrosome that surrounds the two centrioles. The PCM contains proteins responsible for microtubule nucleation and anchoring including γ-tubulin, pericentrin and ninein.
procaspase
Procaspases are the inactive precursors to caspases.They are activated by cleaving one or two specific aspartic acids. The procaspase is split into two subunits that form a dimer. Two of these dimers combine to form the activated tetramer that is a caspase. inactive form of caspase
prometaphase
Prometaphase starts abruptly with the breakdown of the nuclear envelope. Chromosomes can now attach to spindle microtubules via their kinetochores and undergo active movement.
Rb
Rb is a tumor suppressor protein
Rho GTPases
Rho GTPases are molecular switches that control a wide variety of signal transduction pathways in all eukaryotic cells. They are known principally for their pivotal role in regulating the actin cytoskeleton, but their ability to influence cell polarity, microtubule dynamics, membrane transport pathways and transcription factor activity is probably just as significant. Underlying this biological complexity is a simple biochemical idea, namely that by switching on a single GTPase, several distinct signalling pathways can be coordinately activated. With spatial and temporal activation of multiple switches factored in, it is not surprising to find Rho GTPases having such a prominent role in eukaryotic cell biology.
securin
Securin is a key APC substrate and the one whose degradation gave rise to the name APC: securin binds to and inhibits a protease termed separase, which, when released following securin degradation, cleaves proteins that hold sister chromatids together, thereby initiating anaphase
protofilament
Set of tubulin dimers that make up a microtubule when 13 of them surround a hollow center. one of the 13 lines of tubulin dimers stacked on top of each other from one end to the other
controlled polymerization and rearrangements of actin filaments occur during cell crawling
Step 1: Protrusion of leading edge (lamellipodium) Step 2: Adhesion of lower surface of lamellipodium to substratum (mediated by integrins residing in PM) Step 3: Movement of the bulk of the cell forward over the site of attachment (stationary). Accomplished by contractile force exerted against substratum. Step 4: Cell after the attachments with the substratum have been severed and the rear of the cell has been pulled forward
CDK regulation by phosphorylation: activation and inhibition
Step 1: association of cyclin with CDK subunit Wee1: kinase that adds inhibitory phosphate CAK: CDK activating kinase adds activating phosphate Cdc25: phosphatase that removes inhibitory phosphate
conformational changes in the myosin II head couple ATP hydrolysis to movement
Step 1: binding of ATP to a cleft in the myosin head, causing the detachment of the head from the actin filament Step 2: hydrolysis of ATP to Pi and ADP which energizes the head Step 3: and causes the head to bind weakly to the actin filament Step 4: the release of Pi causes tighter attachment of the myosin head to the thin filament and the power stroke that moves the thin filament toward the center of the sarcomere Step 5: the release of the ADP sets the stage for another cycle
stress fibers
Stress fibers are contractile actin bundles found in non-muscle cells. They are composed of actin (microfilaments) and non-muscle myosin II (NMMII), and also contain various crosslinking proteins, such as α-actinin, to form a highly regulated actomyosin structure within non-muscle cells. Stress fibers have been shown to play an important role in cellular contractility, providing force for a number of functions such as cell adhesion, migration and morphogenesis.
basal body (9 triplet microtubules)
Structure at the base of cilia and flagella from which microtubules forming the axoneme radiate; structurally similar to a centriole At its point of attachment to the cell, the axoneme connects with the basal body. Like centrioles, basal bodies are cylindrical structures, about 0.4 μm long and 0.2 μm wide, which contain nine triplet microtubules. Each triplet contains one complete 13-protofilament microtubule, the A tubule, fused to the incomplete B tubule, which in turn is fused to the incomplete C tubule. The A and B tubules of basal bodies continue into the axonemal shaft, whereas the C tubule terminates within the transition zone between the basal body and the shaft. The two central tubules in a flagellum or a cilium also end in the transition zone, above the basal body. As we will see later, the basal body plays an important role in initiating the growth of the axoneme. https://www.ncbi.nlm.nih.gov/books/NBK21698/
ubiquination
The addition of ubiquitin to a substrate protein is called ubiquitination or less frequently ubiquitylation. Ubiquitination affects proteins in many ways: it can mark them for degradation via the proteasome, alter their cellular location, affect their activity, and promote or prevent protein interactions
basil lamina
The basal lamina is a layer of extracellular matrix secreted by the epithelial cells, on which the epithelium sits. It is often incorrectly referred to as the basement membrane, though it does constitute a portion of the basement membrane.
cell cortex
The cell cortex, also known as the actin cortex or actomyosin cortex, is a specialized layer of cytoplasmic protein on the inner face of the plasma membrane of the cell periphery. It functions as a modulator of plasma membrane behavior and cell surface properties. In most eukaryotic cells lacking a cell wall, the cortex is an actin-rich network consisting of F-actin filaments, myosin motors, and actin-binding proteins. The actomyosin cortex is attached to the cell membrane via membrane-anchoring proteins called ERM proteins and it plays a central role in cell shape control. The protein constituents of the cortex undergo rapid turnover, making the cortex both mechanically rigid and highly plastic, two properties essential to its function. In most cases, the cortex is in the range of 100 to 1000 nanometers thick. Functions: In mitosis, F-actin and myosin II form a highly contractile and uniform cortex to drive mitotic cell rounding. The surface tension produced by the actomyosin cortex activity generates intracellular hydrostatic pressure capable of displacing surrounding objects to facilitate rounding. In cytokinesis the cell cortex plays a central role by producing a myosin-rich contractile ring to constrict the dividing cell into two daughter cells
cdc (cell division cycle)
The cell cycle or cell-division cycle is the series of events that take place in a cell leading to its division and duplication of its DNA (DNA replication) to produce two daughter cells.
axoneme
The core of microtubules, usually in a "9+2" array, at the center of a cilium or flagellum
actin nucleator
The first step in actin polymerization is known as 'nucleation'. This step sees the formation of an actin nucleus, which is essentially a complex of three actin monomers, from which an actin filament may elongate. Although non-muscle cells have a high concentration of G-actin-ATP (~100 μM), pure G-actin monomers fail to nucleate new actin filaments efficiently due to the instability of actin oligomers. Additional factors are therefore required and although the exact mechanisms behind filament nucleation remain to be clearly defined, two models, each involving distinct mechanisms and proteins have been proposed. Importantly, these mechanisms are not mutually exclusive and it maybe be the case that nucleation of actin filaments results from a combination of both mechanisms. In the first model, known as the 'tip nucleation model', members of the formin family of proteins cluster at the plasma membrane and initiate the nucleation of actin filaments. Formin subsequently mediates filament extension with the structural integrity of the filament bundles maintained by fascin cross-linking. The alternative model is known as the "convergent elongation model". In this model, the Arp2/3 complex, which is more commonly associated with lamellipodia formation but has been found to be critical for filopodia initiation, plays a role. Here, Arp2/3 complex nucleated branches continually develop from the actin filament network located at the leading edge of the lamellipodia. These filaments are proposed to gradually converge, forming a bundle that is secured by facsin cross-linking.
G1 phase
The g₁ phase, or Gap 1 phase, is the first of four phases of the cell cycle that takes place in eukaryotic cell division. In this part of interphase, the cell synthesizes mRNA and proteins in preparation for subsequent steps leading to mitosis
MTOC (microtubule organizing center)
The microtubule-organizing center (MTOC) is a structure found in eukaryotic cells from which microtubules emerge. MTOCs have two main functions: the organization of eukaryotic flagella and cilia and the organization of the mitotic and meiotic spindle apparatus, which separate the chromosomes during cell division. two centrioles are positioned orthogonally within the MTOC most microtubules have constant orientation relative to MTOCs plant cells don't have MTOCs
hemidesmosomes
The second type of cell-matrix adhesion junction, the hemidesmosome, is found mainly on the basal surface of epithelial cells. These junctions firmly anchor epithelial cells to the underlying basal lamina. The cytosolic side of a hemidesmosome consists of a plaque composed of adapter proteins, which are attached to the ends of keratin filaments (see Figure 22-9). Integrin α6β4 is localized to hemidesmosomes and is thought to bind to an adapter protein, plectin, within the plaques and to the extracellular-matrix protein laminin. By interconnecting the intermediate filaments of the cytoskeleton with the fibers of the basal lamina, these cell-matrix junctions increase the overall rigidity of epithelial tissues.
extracellular matrix
The substance in which animal tissue cells are embedded, consisting of protein and polysaccharides.
plus end / barbed end
The terms "pointed" and "barbed" referring to the two ends of the microfilaments derive from their appearance under transmission electron microscopy when samples are examined following a preparation technique called "decoration". This method consists of the addition of myosin S1 fragments to tissue that has been fixed with tannic acid. This myosin forms polar bonds with actin monomers, giving rise to a configuration that looks like arrows with feather fletchings along its shaft, where the shaft is the actin and the fletchings are the myosin. Following this logic, the end of the microfilament that does not have any protruding myosin is called the point of the arrow (- end) and the other end is called the barbed end (+ end).[32] A S1 fragment is composed of the head and neck domains of myosin II. Under physiological conditions, G-actin (the monomer form) is transformed to F-actin (the polymer form) by ATP, where the role of ATP is essential
lamins
The type of intermediate filament found inside the nucleus. Nuclear lamins, also known as Class V intermediate filaments, are fibrous proteins providing structural function and transcriptional regulation in the cell nucleus. Nuclear lamins interact with membrane-associated proteins to form the nuclear lamina on the interior of the nuclear envelope. Lamins are present in all members of the kingdom Animalia (Metazoa), but are not found in unicellular organisms, plants, or fungi. Lamin proteins are involved in the disassembling and reforming of the nuclear envelope during mitosis, the positioning of nuclear pores, and programmed cell death. Mutations in lamin genes can result in laminopathies, some of which are potentially lethal disorders
thick and thin filaments in sarcomere
Thick filaments consist primarily of the protein myosin. Each thick filament is approximately 15 nm in diameter, and each is made of several hundred molecules of myosin. A myosin molecule is shaped like a golf club, with a tail formed of two intertwined chains and a double globular head projecting from it at an angle. Half of the myosin heads angle to the left and half of them angle to the right, creating an area in the middle of the filament known as the bare zone. Thin filaments, 7 nm in diameter, consist primarily of the protein actin, specifically fibrous (F) actin. Each F actin strand is composed of a string of subunits called globular (G) actin. Each G actin has an active site that can bind to the head of a myosin molecule. Each thin filament also has approximately 40 to 60 molecules of tropomyosin, the protein that blocks the active sites of the thin filaments when the muscle is relaxed. Each tropomyosin molecule has a smaller calcium-binding protein called troponin bound to it. All thin filaments are attached to the Z-line.
microtubules
Thick hollow tubes that make up the cilia, flagella, and spindle fibers. may assemble as singlet, doublet, or triplet tubule strucutres functions: structure and organization dynamics associated proteins locations of assembly motors
alpha-tubulin and beta-tubulin
To form microtubules, the dimers of α- and β-tubulin bind to GTP and assemble onto the (+) ends of microtubules while in the GTP-bound state. The β-tubulin subunit is exposed on the plus end of the microtubule while the α-tubulin subunit is exposed on the minus end. After the dimer is incorporated into the microtubule, the molecule of GTP bound to the β-tubulin subunit eventually hydrolyzes into GDP through inter-dimer contacts along the microtubule protofilament. Whether the β-tubulin member of the tubulin dimer is bound to GTP or GDP influences the stability of the dimer in the microtubule. Dimers bound to GTP tend to assemble into microtubules, while dimers bound to GDP tend to fall apart; thus, this GTP cycle is essential for the dynamic instability of the microtubule.
transverse tubule
Transverse tubules (T-tubules) are extensions of the cell membrane that penetrate into the centre of skeletal and cardiac muscle cells.
What are the substrates for MPF? (still an active area of research)
What we know: MPF phosphorylates... 1. proteins required for chromosome condensation 2. microtubule binding proteins (which increases dynamic instability) (MAPs) 3. nuclear lamins, which leads to nuclear envelope breakdown (animal cells) 4. core proteins of the controller that regulate order
Prometaphase
When prophase is complete, the cell enters prometaphase — the second stage of mitosis. During prometaphase, phosphorylation of nuclear lamins by M-CDK causes the nuclear membrane to break down into numerous small vesicles. As a result, the spindle microtubules now have direct access to the genetic material of the cell. Each microtubule is highly dynamic, growing outward from the centrosome and collapsing backward as it tries to locate a chromosome. Eventually, the microtubules find their targets and connect to each chromosome at its kinetochore, a complex of proteins positioned at the centromere. The actual number of microtubules that attach to a kinetochore varies between species, but at least one microtubule from each pole attaches to the kinetochore of each chromosome. A tug-of-war then ensues as the chromosomes move back and forth toward the two poles.
Listeria
a bacterial membrane protein binds to profilin and is essential for infectivity and motility of Listeria
extracellular matrix (ECM)
a complex mesh of interconnected proteins and associated carbohydrates the ECM is the major component of connective tissues such as bone and cartilage. The basic lamina is a specialized ECM to which epithelial cells are attached. carbs are covalently linked to proteins - N-linked and O-linked glycosylation in ER for lots of cells, ECM makes up most of mass A usually insoluble network consisting of polysaccharides, fibrous proteins, and adhesive proteins that are secreted by animal cells. It provides structural support in tissues and can affect the development and biochemical functions of cells.
myofibril
a long, filamentous organelle found within muscle cells that has a banded appearance
G-actin
a monomer of actin that polymerizes to form F-actin, that makes up actin filaments a globular subunit with an active site for binding myosin head
ubiquitin
a protein degradation substance that binds to proteins and marks them for destruction by proteasome
ubiquitin ligase
a protein that recruits an E2 ubiquitin-conjugating enzyme that has been loaded with ubiquitin, recognizes a protein substrate, and assists or directly catalyzes the transfer of ubiquitin from the E2 to the protein substrate
titin
a springy protein that forms the elastic filaments protein that extends from the Z line to the thick filaments and M line of skeletal muscle sarcomere
mitotic spindle
a structure made of microtubules that controls chromosome movement during mitosis
actin and myosin II in cytokinesis
actin and myosin II have essential roles in cytokinesis when actin-myosin II interaction is disrupted, nucleus replicates but cytokinesis does not occur
what does actin assembly in vitro depend on?
actin assembly in vitro depends on the concentration of of monomeric actin
actin assembly
actin can assemble into distinct structures at different locations in a single cell
activation and inactivation of CDK/cyclin complex
activation of the "mitotic" CDK/cyclin complex drives the cell into the M phase inactivation is required for the cell to "exit" the M phase ON: chromosome condensation, spindle assembly, nuclear envelope breakdown (NEB) OFF: chromosome de-condensation, spindle dis-assembly, and nuclear envelope re-formation
negative feedback with MPF
active M-Cdk negative feedback for Wee1 watch recording
positive feedback with MPF
active M-Cdk positive feedback for active cdc25 which then activates M-Cdk watch recording
addition of ATP-actin
addition of ATP-actin to polymer is followed by ATP hydrolysis minus end addition is slow so hydrolysis catches up plus end addition is fast so hydrolysis lags behind ATP hydrolysis is not required for polymer formation
bundles of doublet microtubules
all eukaryotic cilia and flagella contain bundles of doublet microtubules Nexin links the doublet microtubules 9 doublet microtubules that surround a central pair is a "9+2" arrangment
hemidesmosome
anchors intermediate filaments in a cell to the basil lamina
kinetochore
assembles at a specialized region of the chromosome called the centromere (DNA) A specialized region on the centromere that links each sister chromatid to the mitotic spindle.
axonemal dynein
beating of the cilia/flagellum is driven by the axonemal dynein attached to the doublet microtubules the head/stalk of dynein attached to the A tubule will "walk" along the B tubule
why does incidence of cancer increase with age?
because epidemiology of human cancers indicates that development of cancer requires several mutations
benign tumors
benign tumors arise with great frequency but pose little risk because they are localized and small
actin filament depolymerizing proteins
bind actin-ADP subunits for rapid turnover of actin filaments example: cofilin
monomer-sequestering proteins
bind to actin-ATP monomers and prevent them from polymerizing example: thymosin B4 By sequestering ATP-G-actin, Thymosin B4 maintains the pool of monomeric actin
kinesin heads
bind to microtubules and are place of ATP hydrolysis
use of capping protein to determine Cc
binding of a specific "capping" protein to a specific end of the filament allows experimental determination of Cc for the other end
Phalloidin
binds along side actin filaments and stabilize them
latrunculin
binds to G actin and inhibits its addition to filament
Myosin Type II
binds to other Type II tails involved in muscle contraction through power stroke
frayed ends of microtubules
bonds between adjacent subunits weakens, which leads to fraying
alpha and beta subunits and GTP
both alpha and beta subunits can bind to GTP only beta subunit possesses GTPase activity - only beta is an enzyme
what initiates branching in plant cells?
branching from pre-existing microtubules is initiated by gamma-tubulin in plant cells plant cells do not contain centrioles or centrosomes
what happens faster: catastrophe or rescue?
catastrophe happens faster than rescue
aneuploidy
cell doesn't have right number of centrosomes
basal bodies
centrioles that assemble cilia and flagella cilia and flagella are assembled from basal bodies cross sections of the cilia/flagellum at different locations have different structures basal bodies associate with minus end, therefore dynein facilitates transport
centrosome duplication
centrosome duplication precedes M phase mis-regulated centrosome duplication is common in cancer cells
Anaphase A
chromosomes move to opposite poles depolymerization shortens kinetochore MTs, bringing chromosomes closer to spindle poles
drugs that disrupt microtubule dynamics
colchicine and taxol
collagen
collagen is major component of ECM collagen polypeptides form triple helix secreted collagen molecules can assemble into ordered polymers called fibrils single collagen polypeptide chain→ tripled-stranded collagen molecule→ collagen fibril→ collagen fibrils structural protein found in the skin and connective tissue arrangement of collagen fibrils is highly ordered in many tissue types A triple-helical protein that forms fibrils of great tensile strength; a major component of the extracellular matrix and connective tissues. The numerous collagen subtypes differ in their tissue distribution and the extracellular components and cell-surface proteins with which they associate.
sarcomere
contractile unit of a muscle fiber Repeating unit of a myofibril in striated muscle that extends from one Z disk to an adjacent one and shortens during contraction
cellular response to acetylcholine: smooth muscle
contraction Acetylcholine activates a different type of receptor present in smooth muscle: the muscarinic receptor. When this receptor binds acetylcholine, one result is the release of calcium ions from internal stores. Acetylcholine's interaction with muscarinic receptors, as with nicotinic receptors, causes channels to open resulting in ion flow that depolarizes the muscle cell. As in skeletal muscle, the depolarization leads to muscle contraction.
cellular response to acetylcholine: skeletal muscle
contraction An example of a ligand-gated ion channel is the acetylcholine receptor in skeletal muscle cells. When the acetylcholine receptor binds acetylcholine, it opens allowing Na+ to enter the cell. This causes the membrane to depolarize and opens voltage-gated sodium channels. The rapid depolarization of the cell membrane generates an action potential that ultimately triggers the opening of calcium channels inside the cell. The rise in cytosolic calcium induces muscle contraction. The benefit of ligand-gated ion channels is that the cell's response to acetylcholine is fast (milliseconds).
cellular response to acetylcholine: heart muscle
decreased frequency of contraction In cardiac muscle, acetylcholine acts through G protein coupled receptor to hyper polarize membranes, making it more difficult to trigger contraction in these cells. Thus, the internal wiring of skeletal and cardiac muscle determines each cell's response to acetylcholine. Like smooth muscle, cardiac muscle has muscarinic receptors. The effect of acetylcholine on cardiac muscle, however, is very different from its effects on skeletal or smooth muscle. In the heart, acetylcholine activation of muscarinic receptors causes channels in the muscle membrane to let potassium pass. This has the effect of slowing contraction of the heart muscle and making it beat with less force.
cell control system
essential processes of the cell cycle are triggered by a cell cycle control system Main events are: 1. DNA replication 2. mitotic spindle assembly-mitosis 3. exist from mitosis and cytokinesis Job of controller: 1. make events happen 2. allow events to happen once 3. make sure events happen in right order controller has some key "parts": 1. proteins that rise and fall (cycle) with stages of cell cycle 2. regulated degradation of proteins 3. regulated phosphorylation and dephosphorylation 4. regulation by inhibitor proteins
unbranched filaments
filament does not split into branches
Ndc80
helps microtubule bind to kinetocore
p53 in cancer
in normal cells, p53 will arrest the cell cycle in response to DNA damage (or if damage is too severe, promote apoptosis) mutations in the p53 gene occur in more than 50% of human cancers because p53 functions as a tetramer, and a single mutant protein within the tetramer can poison the tetramer activity, p53 mutations can be dominant
bending
in normal cilia/flagella, dynein walking causes bending no nexin- dynein generates force against adjacent B tubule causing sliding between the doublets with nexin- because doublets are crosslinked and also anchored at the basal bodies, sliding is translated to bending
cell-ECM adhesion
involves focal adhesions and hemidesmosomes
adherens junction
joins an actin bundle in one cell to a similar bundle in a neighboring cell cell-cell adhesion a "belt" of adherens junctions around the circumference of epithelial cells links actin filaments of multiple cells and allows sheets of cells to change shape when adherens junctions are intact, epithelial able to fold properly. when not intact, cell tears apart
desmosome
joins the intermediate filaments in one cell to those in a neighbor cell-cell adhesion
IF types I and II
keratins found in epithelia
chromatid
one of two identical "sister" parts of a duplicated chromosome
what triggers muscle contraction
rise in cytosolic Ca++ triggers muscle contraction
array of actin and myosin in skeletal muscle
skeletal muscle contains a regular array of actin and myosin
formins
they direct where unbranched filaments form in a cell RBD= Rho-binding domain
actin filament functions
transport force motility shape division
troponin tropomyosin interaction
troponin binds to Ca++ which results in conformational change of troponin which moves tropomyosin out of the way, exposing myosin-binding sites
p53
tumor suppressor gene that controls cell division and apoptosis
F-actin
twisted strand compossed of two rows individual globular molecules with myosin binding sites
normal cells vs cancer cells
unlike cancer cells, normal cells respond to a number of conditions that limit cell division
IF type III
vimentin and vimentin-related found in connective tissue, muscle cells, and neuroglial cells
with ATP hydrolysis
when Cc- > [G-actin] > Cc+, plus end grows and minus end shrinks
contractile ring
where actin is found during cell division
traits of a successful cancer cell
- self-sufficiency in growth signals - insensitivity to anti-growth signals - tissue invasion and metastasis - limitless replicative potential - sustained angiogenesis - evading apoptosis
How is CDK activity regulated?
1. cyclin proteins (synthesis, localization, and degradation) 2. phosphorylation of the CDK itself 3. binding of inhibitors to the CDK example of binding inhibitors to the CDK: activation of the S-phase cyclin/CDK complex by the G1/S cyclin/CDK complex
six broad classes of proteins are encoded by tumor-suppressor genes
1. intracellular proteins that regulate or inhibit progression through a specific stage of the cell cycle (Rb, p27) 2. receptors for secreted humors that inhibit cell proliferation 3. checkpoint-control proteins that arrest the cell cycle 4. proteins that promote apoptosis (cytochrome c) 5. enzymes that participate in DNA repair 6. cell-cell adhesion proteins usually loss-of-function mutations render the tumor suppressor protein inactive and unable to carry out its normal role for most tumor-suppressor genes, loss-of-function mutations must occur in both copies of the gene for the "brakes" to be in activated
current molecular model of kinesin "processively" walking along a microtubule
1. leading head binds ATP 2. binding of ATP induces conformational change causing the neck linker to swing forward and dock into head. this motion swings the former trailing head to become leading head 3. new leading head finds a binding site on the microtubule 16 nm ahead of its previous site 4. leading head releases ADP and coordinately the trailing head hydrolyzes ATP to ADP and Pi. Pi is released and the linker becomes undocked
important roles for cytoplasmic dynein
1. positioning mitotic spindle 2. moving chromosomes 3. positioning centrosome and Golgi 4. moving organelles and vesicles
transformed cells (cells taken from tumor)
1. reduced serum independence 2. do not need to attach to surface to grow 3. cells keep dividing even at high density and pile up in "foci"
normal cells in culture
1. serum dependence: growth factors required for proliferation 2. anchorage dependence: must attach to solid surface to grow 3. contact inhibition: cells grow as a monolayer and cell division is inhibited once they reach a certain density
challenges in curing cancer
1. tumors are quite large before detected -only 1000 times smaller at first detection than at death 2. different tumors have a different constellation of mutations
gain-of-function
A type of mutation in which the altered gene product possesses a new molecular function or a new pattern of gene expression. Gain-of-function mutations are almost always Dominant or Semidominant. Because mutation events introduce random genetic changes, most of the time they result in loss of function. The mutation events are like bullets being fired at a complex machine; most of the time they will inactivate it. However, it is conceivable that in rare cases a bullet will strike the machine in such a way that it produces some new function. So it is with mutation events; sometimes the random change by pure chance confers some new function on the gene. In a heterozygote, the new function will be expressed, and therefore the gain-of-function mutation most likely will act like a dominant allele and produce some kind of new phenotype.
APC (anaphase promoting complex)
APC is a ubiquitin ligase. It covalently attaches ubiquitin to its substrate. The APC also promotes the degradation of the cohesion regulator Securin In addition to driving the events of M phase, MPF also triggers its own destruction by activating the anaphase-promoting complex/cyclosome (APC/C), a protein complex that causes M cyclins to be destroyed starting in anaphase. The destruction of M cyclins pushes the cell out of mitosis, allowing the new daughter cells to enter G1. The APC/C also causes destruction of the proteins that hold the sister chromatids together, allowing them to separate in anaphase and move to opposite poles of the cell. How does the APC/C do its job? Like a Cdk, the APC/C is an enzyme, but it has different type of function than a Cdk. Rather than attaching a phosphate group to its targets, it adds a small protein tag called ubiquitin (Ub). When a target is tagged with ubiquitin, it is sent to the proteasome, which can be thought of as the recycle bin of the cell, and destroyed. For example, the APC/C attaches a ubiquitin tag to M cyclins, causing them to be chopped up by the proteasome and allowing the newly forming daughter cells to enter G1 phase. The APC/C also uses ubiquitin tagging to trigger the separation of sister chromatids during mitosis. If the APC/C gets the right signals at metaphase, it sets off a chain of events that destroys cohesion, the protein glue that holds sister chromatids together. The APC/C first adds a ubiquitin tag to a protein called securin, sending it for recycling. Securin normally binds to, and inactivates, a protein called separase. When securin is sent for recycling, separase becomes active and can do its job. Separase chops up the cohesin that holds sister chromatids together, allowing them to separate.
oncogene
An oncogene is a gene that has the potential to cause cancer. In tumor cells, they are often mutated and/or expressed at high levels. Most normal cells will undergo a programmed form of rapid cell death (apoptosis) when critical functions are altered and malfunctioning. Activated oncogenes can cause those cells designated for apoptosis to survive and proliferate instead. Most oncogenes began as proto-oncogenes, normal genes involved in cell growth and proliferation or inhibition of apoptosis. If normal genes promoting cellular growth, through mutation, are up-regulated, (gain of function mutation) they will predispose the cell to cancer and are thus termed oncogenes. Usually multiple oncogenes, along with mutated apoptotic and/or tumor suppressor genes will all act in concert to cause cancer.
Axoneme
Bundle of microtubules and associated proteins present in cilia and flagella and responsible for their movement. Numerous eukaryotic cells carry whip-like appendages (cilia or eukaryotic flagella) whose inner core consists of a cytoskeletal structure called the axoneme. The axoneme serves as the "skeleton" of these organelles, both giving support to the structure and, in some cases, causing it to bend. Though distinctions of function and/or length may be made between cilia and flagella, the internal structure of the axoneme is common to both. Inside cilia and flagella is a microtubule-based cytoskeleton called the axoneme. The axoneme of primary cilia typically has a ring of nine outer microtubule doublets (called a 9+0 axoneme), and the axoneme of a motile cilium has two central microtubules in addition to the nine outer doublets (called a 9+2 axoneme). The axonemal cytoskeleton acts as a scaffolding for various protein complexes and provides binding sites for molecular motor proteins such as kinesin II, that help carry proteins up and down the microtubules. Motile cilia The building-block of the axoneme is the microtubule; each axoneme is composed of several microtubules aligned in parallel. To be specific, the microtubules are arranged in a characteristic pattern known as the "9x2 + 2," as shown in the image at right. Nine sets of "doublet" microtubules (a specialized structure consisting of two linked microtubules) form a ring around a "central pair" of single microtubules. Besides the microtubules, the axoneme contains many proteins and protein complexes necessary for its function. The dynein arms, for example, are motor complexes that produce the force needed for bending. Each dynein arm is anchored to a doublet microtubule; by "walking" along an adjacent microtubule, the dynein motors can cause the microtubules to slide against each other. When this is carried out in a synchronized fashion, with the microtubules on one side of the axoneme being pulled 'down' and those on the other side pulled 'up,' the axoneme as a whole can bend back and forth. This process is responsible for ciliary/flagellar beating, as in the well-known example of the human sperm. The radial spoke is another protein complex of the axoneme. Thought to be important in regulating the motion of the axoneme, this "T"-shape complex projects from each set of outer doublets toward the central microtubules. The inter-doublet connections between adjacent microtubule pairs are termed nexin linkages. Immotile The axoneme structure in non-motile ("primary") cilia shows some variation from the canonical "9x2 + 2" anatomy. No dynein arms are found on the outer doublet microtubules, and there is no pair of central microtubule singlets. This organization of axoneme is referred as "9x2 + 0". In addition, "9x2 + 1" axonemes, with only a single central microtubule, have been found to exist. Primary cilia appear to serve sensory functions.
cdc6
Cdc6, or cell division cycle 6, is a protein in eukaryotic cells that is studied in the budding yeast Saccharomyces cerevisiae. It is an essential regulator of DNA replication and plays important roles in the activation and maintenance of the checkpoint mechanisms in the cell cycle that coordinate S phase and mitosis. It is part of the pre-replicative complex (pre-RC) and is required for loading minichromosome maintenance (MCM) proteins onto the DNA, an essential step in the initiation of DNA synthesis.
checkpoints and regulators
Cdks, cyclins, and the APC/C are direct regulators of cell cycle transitions, but they aren't always in the driver's seat. Instead, they respond to cues from inside and outside the cell. These cues influence activity of the core regulators to determine whether the cell moves forward in the cell cycle. Positive cues, like growth factors, typically increase activity of Cdks and cyclins, while negative ones, like DNA damage, typically decrease or block activity. As an example, let's examine how DNA damage halts the cell cycle in G1. DNA damage can, and will, happen in many cells of the body during a person's lifetime (for example, due to UV rays from the sun). Cells must be able to deal with this damage, fixing it if possible and preventing cell division if not. Key to the DNA damage response is a protein called p53, a famous tumor suppressor often described as "the guardian of the genome." p53 works on multiple levels to ensure that cells do not pass on their damaged DNA through cell division. First, it stops the cell cycle at the G1 checkpoint by triggering production of Cdk inhibitor (CKI) proteins. The CKI proteins bind to Cdk-cyclin complexes and block their activity , buying time for DNA repair. p53's second job is to activate DNA repair enzymes. If DNA damage is not fixable, p53 will play its third and final role: triggering programmed cell death so damaged DNA is not passed on.
checkpoint
Cell cycle progression requires a sequence of processes, with later events dependent on the completion of earlier ones. This dependency ensures that each cell division accurately replicates the genome and transmits it to daughter cells. Checkpoints control the cell's progress through the cell cycle, and ensure that key processes such as DNA replication and DNA damage repair are completed before the cell cycle is allowed to progress into the next stage. Checkpoints also ensure that both daughter cells receive the same number of chromosomes and that daughter cells are genetically identical to the parents. A checkpoint is a stage in the eukaryotic cell cycle at which the cell examines internal and external cues and "decides" whether or not to move forward with division. There are a number of checkpoints, but the three most important ones are: -The G1 checkpoint, at the G1/S transition. -The G2 checkpoint, at the G2/M transition. -The spindle checkpoint, at the transition from metaphase to anaphase.
cohesion
Cohesin is a protein complex that regulates the separation of sister chromatids during cell division, either mitosis or meiosis.
Overview of Cell Cycle, Checkpoint Control and DNA Damage
Control of eukaryotic cell growth and division involves molecular circuits known as "checkpoints" that ensure proper timing of cellular events. Passage through a checkpoint from one cell cycle phase to the next requires a coordinated set of proteins that monitor cell growth and DNA integrity. Uncontrolled cell division or propagation of damaged DNA can contribute to genomic instability and tumorigenesis. The G1/S checkpoint controls progression of cells through the restriction point (R) into the DNA synthesis S-phase. During G1, the tumor suppressor Rb binds and inhibits transcription factor E2F. Phosphorylation of Rb by cyclin-bound cyclin dependent kinases (CDK) in late G1 induces dissociation of Rb and permits E2F-mediated transcription of S-phase-promoting genes. Responding to upstream signals, INK4 and Kip/Cip family inhibitors control CDK activity and prevent entry into S-phase. DNA damage activates response pathways through ATM/ ATR and Chk1/2 kinases to block CDK activity, leading to cell cycle arrest and DNA repair or cell death. The G2/M checkpoint prevents cells containing damaged DNA from entering mitosis (M). Activated CDK1 (cdc2) bound to cyclin B promotes entry into M-phase. Wee1 and Myt1 kinases and cdc25 phosphatase competitively regulate CDK1 activity; Wee1 and Myt1 inhibit CDK1 and prevent entry into M-phase, while cdc25 removes inhibitory phosphates. DNA damage activates multiple kinases that phosphorylate kinases Chk1/2 and tumor suppressor protein p53. Chk1/2 kinases stimulate Wee1 activity and inhibit cdc25C, preventing entry into M-phase. Phosphorylation of p53 promotes dissociation between p53 and MDM2 and allows binding of the transcription factor to DNA. The spindle checkpoint ensures proper chromatid attachment prior to progression from metaphase to anaphase. The SCF and APC/C protein complexes play prominent roles, with APC-cdc20 initiating the entry into anaphase by promoting ubiquitin-mediated degradation of multiple substrates, including cyclin B and the regulatory protein securin.
cyclin
Cyclins are among the most important core cell cycle regulators. Cyclins are a group of related proteins, and there are four basic types found in humans and most other eukaryotes: G1 cyclins, G1/S cyclins, S cyclins, and M cyclins. As the names suggest, each cyclin is associated with a particular phase, transition, or set of phases in the cell cycle and helps drive the events of that phase or period. For instance, M cyclin promotes the events of M phase, such as nuclear envelope breakdown and chromosome condensation.
Cytokinesis
Cytokinesis is the physical process that finally splits the parent cell into two identical daughter cells. During cytokinesis, the cell membrane pinches in at the cell equator, forming a cleft called the cleavage furrow. The position of the furrow depends on the position of the astral and interpolar microtubules during anaphase. The cleavage furrow forms because of the action of a contractile ring of overlapping actin and myosin filaments. As the actin and myosin filaments move past each other, the contractile ring becomes smaller, akin to pulling a drawstring at the top of a purse. When the ring reaches its smallest point, the cleavage furrow completely bisects the cell at its center, resulting in two separate daughter cells of equal size
activation of the S-phase cyclin/CDK complex induces DNA replication to begin
DNA replication is tightly regulated so that it occurs only once per cell cycle Cdc6 is required for ORC assembly. Phosphorylation of Cdc6 targets it for degradation and new ORC assembly will only occur after exit from M phase phosphorylation of ORC allows replication to begin
HPV
DNA tumor virus that causes cervical cancer encodes proteins that inactivate both p53 and Rb
S phase
During S phase, which follows G1 phase, all of the chromosomes are replicated. Following replication, each chromosome now consists of two sister chromatids. Thus, the amount of DNA in the cell has effectively doubled, even though the ploidy, or chromosome count, of the cell remains at 2n. Note: Chromosomes double their number of chromatids post replication but the nuclei remains diploid as the number of centromeres and chromosomes remains unchanged. Hence, the number of chromosomes in the nucleus, which determines the ploidy, remains unchanged from the beginning to the end of the S phase.
Cytokinesis
During cytokinesis of an animal cell, the cytoplasm is divided in two by a contractile ring of actin and myosin II filaments, which pinches the cell to create two daughters, each with one nucleus.
Telophase
During telophase, the chromosomes arrive at the cell poles, the mitotic spindle disassembles, and the vesicles that contain fragments of the original nuclear membrane assemble around the two sets of chromosomes. Phosphatases then dephosphorylate the lamins at each end of the cell. This dephosphorylation results in the formation of a new nuclear membrane around each group of chromosomes.
telophase
During telophase, the two sets of daughter chromosomes arrive at the poles of the spindle. A new nuclear envelope reassembles around each set, completing the formation of two nuclei and marking the end of mitosis. The division of the cytoplasm begins with the assembly of the contractile ring.
nucleus
During the nucleation phase the formation of a stable 'actin nucleus' occurs. This is usually comprised of three actin monomers in complex. In the elongation phase monomers are rapidly added to the filament at the (+ve) or barbed end and this is often facilitated by additional elongation factors such as formin. For this process to occur, the (+) end of the filament must be exposed, and this means removal of *capping protein.
dynactin complex
Dynactin or Dynein activator complex is a multi-subunit protein found in eukaryotic cells that aids in bidirectional intracellular transport by binding to dynein and kinesin-2 and linking them to the organelle or vesicle to be transported Dynactin is often essential for dynein activity and can be thought of as a "dynein receptor" that modulates binding of dynein to cell organelles which are to be transported along microtubules. Dynactin also enhances the processivity of cytoplasmic dynein and kinesin-2 motors
dynein stalk and microtubule binding domain
Dyneins are AAA+ adenosine triphos-phatases (ATPases) that power minus end-directed movement along microtubules. The cytoplasmic form of dynein serves many cellular functions including regulation of the mitotic checkpoint, organization of the Golgi apparatus, and the transport of vesicles, viruses, and mRNAs. The motor region of dynein consists of a ring of AAA+ domains (four of which bind and hydrolyze ATP), a mechanical element (termed the "linker") that is likely involved in driving motility, and a ~15-nm "stalk" that has a microtubule-binding domain (MTBD) at its tip. The stalk, which emerges from AAA4 (the fourth nucleotide-binding AAA+ domain in the ring), extends as one α helix of an antiparallel coiled coil (termed CC1), forms the small, globular MTBD, and then returns as the partner helix of the coiled coil (CC2) and joins AAA5 (a non-nucleotide-binding AAA+ domain). The separation of the AAA+ ring from the MTBD by a long and somewhat flexible coiled coil distinguishes dynein from kinesin and myosin, where the polymer-binding site and catalytic site are integrated within a single globular motor domain.
Dynein
Dyneins are microtubule motors capable of a retrograde sliding movement. Dynein complexes are much larger and more complex than kinesin and myosin motors. Dyneins are composed of two or three heavy chains and a large and variable number of associated light chains. Dyneins drive intracellular transport toward the minus end of microtubules which lies in the microtubule organizing center near the nucleus. Dynein family has two major branches. One is axonemal dynein facilitates the beating of cilia and flagella by rapid and efficient sliding movements of microtubules. Another one is cytoplasmic dynein which facilitates transport of intracellular cargos. Compared to 15 types of axonemal dynein, only two cytoplasmic forms are known.
G2 phase
Following S phase, the cell enters G2 phase. During G2, the cell synthesizes a variety of proteins. Of particular significance to the cell cycle, most microtubules - proteins that are required during mitosis - are produced during G2.
p27
For example, p27 binds to cyclin D either alone, or when complexed to its catalytic subunit CDK4. In doing so p27 inhibits the catalytic activity of Cdk4, which means that it prevents Cdk4 from adding phosphate residues to its principal substrate, the retinoblastoma (pRb) protein. Increased levels of the p27 protein typically cause cells to arrest in the G1 phase of the cell cycle. Likewise, p27 is able to bind other Cdk proteins when complexed to cyclin subunits such as Cyclin E/Cdk2 and Cyclin A/Cdk2.
Contractile ring
Formed during cytokinesis, the last step of cell division, the contractile ring is composed of filamentous actin (F-actin) and the motor protein myosin-2, along with additional structural and regulatory proteins.
nucleation
G-actin has to form dimer and then form trimer rate-limiting step which is why it takes so much more time for mass to grow when no nuclei added than when nuclei added
Her2 receptor
Human epidermal growth factor receptors (HER/ erb B) constitute a family of four cell surface receptors involved in transmission of signals controlling normal cell growth and differentiation. A range of growth factors serve as ligands, but none is specific for the HER2 receptor. HER receptors exist as both monomers and dimers, either homo- or heterodimers. Ligand binding to HER1, HER3 or HER4 induces rapid receptor dimerization, with a marked preference for HER2 as a dimer partner. Moreover, HER2-containing heterodimers generate intracellular signals that are significantly stronger than signals emanating from other HER combinations. In normal cells, few HER2 molecules exist at the cell surface, so few heterodimers are formed and growth signals are relatively weak and controllable. When HER2 is overexpressed multiple HER2 heterodimers are formed and cell signaling is stronger, resulting in enhanced responsiveness to growth factors and malignant growth. This explains why HER2 overexpression is an indicator of poor prognosis in breast tumors and may be predictive of response to treatment. HER2 is a highly specific and promising target for new breast cancer treatments. The recombinant human anti-HER2 monoclonal antibody (rhuMAb-HER2, trastuzumab, Herceptin) induces rapid removal of HER2 from the cell surface, thereby reducing its availability to heterodimers and reducing oncogenicity.
motile and immotile cilia and flagella
Immotile: kidney epithelium, rod photoreceptor In animals, immotile cilia are found on nearly every cell. In comparison to motile cilia, non-motile (or primary) cilia usually occur one per cell; nearly all mammalian cells have a single non-motile primary cilium. In addition, examples of specialized primary cilia can be found in human sensory organs such as the eye and the nose: The outer segment of the rod photoreceptor cell in the human eye is connected to its cell body with a specialized non-motile cilium. The dendritic knob of the olfactory neuron, where the odorant receptors are located, also contains non-motile cilia (about 10 cilia per dendritic knob). Motile: chlamydomonas, paramecium, respiratory epithelium, sperm Larger eukaryotes, such as mammals, have motile cilia as well. Motile cilia are usually present on a cell's surface in large numbers and beat in coordinated waves. In humans, for example, motile cilia are found in the lining of the trachea (windpipe), where they sweep mucus and dirt out of the lungs. In female mammals, the beating of cilia in the Fallopian tubes moves the ovum from the ovary to the uterus.
singlet, doublet, and triplet microtububles
In cross section, a typical microtubule, a singlet, is a simple tube built from 13 protofilaments. In a doublet microtubule, an additional set of 10 protofilaments forms a second tubule (B) by fusing to the wall of a singlet (A) microtubule. Attachment of another 10 protofilaments to the B tubule of a doublet microtubule creates a C tubule and a triplet structure. In addition to the simple singlet structure, doublet or triplet microtubules are found in specialized structures such as cilia and flagella (doublet microtubules) and centrioles and basal bodies (triplet microtubules). Each of these contains one complete 13-protofilament microtubule (the A tubule) and one or two additional tubules (B and C) consisting of 10 protofilaments
chromosomal translocation
In genetics, a chromosome translocation is a chromosome abnormality caused by rearrangement of parts between nonhomologous chromosomes. A gene fusion may be created when the translocation joins two otherwise-separated genes, it is detected on cytogenetics or a karyotype of affected cells. Translocations can be balanced (in an even exchange of material with no genetic information extra or missing, and ideally full functionality) or unbalanced (where the exchange of chromosome material is unequal resulting in extra or missing genes)
microtubule plus-end binding proteins (+TIPs)
Its certain localization at the microtubule ends makes it a highly relevant aspect in regulation, whether it is promoting growth by catalyzing the specific addition of tubulin to the microtubule ends or by balancing microtubules at the cell cortex, its role in regulation (though still not fully understood in terms of mechanisms) makes +TIP-proteins a main attribute to the overall function of microtubules generally. Another important concept for +TIP-proteins is allocating microtubule addition and dynamical regulation at mitotic kintechores. Also, they contribute in the extension of endoplasmic reticulum tubules together at expanding microtubule ends. Furthermore, +TIP-proteins aid in advocating organization of specialized microtubule array, take for example the discretely arranged bipolar microtubule bundles in fission yeast or mitotic spindles customarily. In addition to the +TIP-proteins basic known functions, the proteins also are crucial for the linkages between microtubule ends and other cellular structure. Take for instance their ability to bind microtubule tips to the cell cortex by colliding to plasma membrane associated proteins or (by some +TIPs) straight forwardly to actin fiber. Moreover, +TIP-protein complexes in budding yeast are utilized for myosin based transport of microtubule ends. Microtubule plus-end trafficking proteins engage in microtubule actin crosstalk, such as the CLIP-170 (+TIP-protein) that controls actin polymerization which is a necessity for the process of phagocytosis done by mammalian cells. +TIP-proteins have been known for an extravagant accumulation by the centrosomes and other structural organizing centers of cells. This leads to the basic assumption that +TIP-proteins may aid in microtubule nucleation and anchoring; however, its distinct role at centrosomes still awaits evidential findings. Overall, +TIPs play a critical part in morphogenesis, cell division, and motility.
Kinesin
Kinesins are a group of related motor proteins that use a microtubule track in anterograde movement. They are vital to spindle formation in mitotic and meiotic and chromosomes separation during cell division and are also responsible for shuttling mitochondria, Golgi bodies, and vesicles within eukaryotic cells. Kinesin have two heavy chains and two light chains per active motor. The two globular head motor domains in heavy chains can convert the chemical energy of ATP hydrolysis into mechanical work to move along microtubules. The direction in which cargo is transported can be towards the plus-end or the minus-end, depending on the type of kinesin. In general, kinesins with N-terminal motor domains move their cargo towards the plus ends of microtubules located at the cell periphery, while kinesins with C-terminal motor domains move cargo towards the minus ends of microtubules located at the nucleus. Fourteen distinct kinesin families are known, with some additional kinesin-like proteins that cannot be classified into these families
inactivation of MPF
MPF is inactivated when the cyclin subunit is poly-ubiquinated and degraded APC=anaphase promoting complex APC is a ubiquitin ligase. It covalently attaches ubiquitin to its substrate. Ubiquination of the mitotic cyclin by APC targets it for degradation by the proteosome
Anaphase
Metaphase leads to anaphase, during which each chromosome's sister chromatids separate and move to opposite poles of the cell. Enzymatic breakdown of cohesin — which linked the sister chromatids together during prophase — causes this separation to occur. Upon separation, every chromatid becomes an independent chromosome. Meanwhile, changes in microtubule length provide the mechanism for chromosome movement. More specifically, in the first part of anaphase — sometimes called anaphase A — the kinetochore microtubules shorten and draw the chromosomes toward the spindle poles. Then, in the second part of anaphase — sometimes called anaphase B — the astral microtubules that are anchored to the cell membrane pull the poles further apart and the interpolar microtubules slide past each other, exerting additional pull on the chromosomes
actin networks
Microfilament networks - Animal cells commonly have a cell cortex under the cell membrane that contains a large number of microfilaments, which precludes the presence of organelles. This network is connected with numerous receptor cells that relay signals to the outside of a cell. In most cells actin filaments form larger-scale networks which are essential for many key functions in cells: Various types of actin networks (made of actin filaments) give mechanical support to cells, and provide trafficking routes through the cytoplasm to aid signal transduction Rapid assembly and disassembly of actin network enables cells to migrate (Cell migration). In metazoan muscle cells, to be the scaffold on which myosin proteins generate force to support muscle contraction In non-muscle cells, to be a track for cargo transport myosins (nonconventional myosins) such as myosin V and VI. Nonconventional myosins use ATP hydrolysis to transport cargo, such as vesicles and organelles, in a directed fashion much faster than diffusion. Myosin V walks towards the barbed end of actin filaments, while myosin VI walks toward the pointed end. Most actin filaments are arranged with the barbed end toward the cellular membrane and the pointed end toward the cellular interior. This arrangement allows myosin V to be an effective motor for the export of cargos, and myosin VI to be an effective motor for import.
Gamma-tubulin ring complex
Microtubule nucleation is unfavorable under normal conditions found in most living cells. Consequently, microtubules are nucleated from a complex of γ-tubulin and other protein components known as the γ-tubulin ring complex (γ-TuRC). γ-TuRC nucleates and caps the minus end of new filaments by providing stable binding sites for tubulin dimers. Tubulin dimers primarily use longitudinal interactions to bind to each other and to γ−TuRC during the nucleation phase. As the protofilament length increases, lateral interactions between the protofilaments create additional stability that leads to a closed microtubule. After the slow nucleation phase, microtubules elongate rapidly. The minus end of γ-tubulin is anchored near the MTOC, whilst the plus end of γ-tubulin is exposed. This allows elongation to occur from the exposed γ-tubulin through interactions with the minus end of α-tubulin/β-tubulin heterodimers. Although it remains unclear whether the formation of longitudinal contacts with α-tubulin stimulates γ-tubulin hydrolysis of GTP, the rate of GTP hydrolysis on β-tubulin, along with its concentration are determining factors of microtubule assembly. Once a tubulin dimer has been added to the lattice, the more likely it is for the GTP on β-tubulin to be hydrolyzed. MAPs can also control the rate of assembly/disassembly, the rate of GTP hydrolysis and the overall length of microtubules.
polarity of microtubule
Microtubules have a distinct polarity that is critical for their biological function. Tubulin polymerizes end to end, with the β-subunits of one tubulin dimer contacting the α-subunits of the next dimer. Therefore, in a protofilament, one end will have the α-subunits exposed while the other end will have the β-subunits exposed. These ends are designated the (−) and (+) ends, respectively. The protofilaments bundle parallel to one another with the same polarity, so, in a microtubule, there is one end, the (+) end, with only β-subunits exposed, while the other end, the (−) end, has only α-subunits exposed. While microtubule elongation can occur at both the (+) and (-) ends, it is significantly more rapid at the (+) end.
stages of M phase
Mitosis and cytokinesis Mitosis comprised of: prophase, prometaphase, metaphase, anaphase, telophasestages of M phase
Mitosis
Mitosis is the process in which a eukaryotic cell nucleus splits in two, followed by division of the parent cell into two daughter cells. The word "mitosis" means "threads," and it refers to the threadlike appearance of chromosomes as the cell prepares to divide. Early microscopists were the first to observe these structures, and they also noted the appearance of a specialized network of microtubules during mitosis. These tubules, collectively known as the spindle, extend from structures called centrosomes — with one centrosome located at each of the opposite ends, or poles, of a cell. As mitosis progresses, the microtubules attach to the chromosomes, which have already duplicated their DNA and aligned across the center of the cell. The spindle tubules then shorten and move toward the poles of the cell. As they move, they pull the one copy of each chromosome with them to opposite poles of the cell. This process ensures that each daughter cell will contain one exact copy of the parent cell DNA.
Head (ATPase), neck, and tail domains of myosin
Most myosin molecules are composed of a head, neck, and tail domain. The head domain binds the filamentous actin, and uses ATP hydrolysis to generate force and to "walk" along the filament towards the barbed (+) end (with the exception of myosin VI, which moves towards the pointed (-) end). the neck domain acts as a linker and as a lever arm for transducing force generated by the catalytic motor domain. The neck domain can also serve as a binding site for myosin light chains which are distinct proteins that form part of a macromolecular complex and generally have regulatory functions. The tail domain generally mediates interaction with cargo molecules and/or other myosin subunits. In some cases, the tail domain may play a role in regulating motor activity.
Myosin II
Myosin II (also known as conventional myosin) is the myosin type responsible for producing muscle contraction in muscle cells. Myosin II contains two heavy chains, each about 2000 amino acids in length, which constitute the head and tail domains. Each of these heavy chains contains the N-terminal head domain, while the C-terminal tails take on a coiled-coil morphology, holding the two heavy chains together (imagine two snakes wrapped around each other, as in a caduceus). Thus, myosin II has two heads. The intermediate neck domain is the region creating the angle between the head and tail. In smooth muscle, a single gene (MYH11) codes for the heavy chains myosin II, but splice variants of this gene result in four distinct isoforms. It also contains 4 myosin light chains (MLC), resulting in 2 per head, weighing 20 (MLC20) and 17 (MLC17) kDa. These bind the heavy chains in the "neck" region between the head and tail. The MLC20 is also known as the regulatory light chain and actively participates in muscle contraction. The MLC17 is also known as the essential light chain. Its exact function is unclear, but is believed to contribute to the structural stability of the myosin head along with MLC20. Two variants of MLC17 (MLC17a/b) exist as a result of alternative splicing at the MLC17 gene. In muscle cells, the long coiled-coil tails of the individual myosin molecules join together, forming the thick filaments of the sarcomere. The force-producing head domains stick out from the side of the thick filament, ready to walk along the adjacent actin-based thin filaments in response to the proper chemical signals.
Myosin
Myosins are a superfamily of actin motor proteins that convert chemical energy in the form of ATP to mechanical energy, thus generating force and movement. The first identified myosin, myosin II, is responsible for generating muscle contraction. Myosin II is an elongated protein that is formed from two heavy chains with motor heads and two light chains. Each myosin head contains actin and ATP binding site. The myosin heads bind and hydrolyze ATP, which provides the energy to walk toward the plus end of an actin filament. Myosin II are also vital in the process of cell division. For example, non-muscle myosin II bipolar thick filaments provide the force of contraction needed to divide cell into two daughter cells during cytokinesis.In addition to myosin II, many other myosin types are responsible for variety of movement of non-muscle cells. For example, myosin is involved in intracellular organization and the protrusion of actin-rich structures at the cell surface. Myosin V is involved in vesicle and organelle transport
Prophase
Prophase is the first stage in mitosis, occurring after the conclusion of the G2 portion of interphase. During prophase, the parent cell chromosomes — which were duplicated during S phase — condense and become thousands of times more compact than they were during interphase. Because each duplicated chromosome consists of two identical sister chromatids joined at a point called the centromere, these structures now appear as X-shaped bodies when viewed under a microscope. Several DNA binding proteins catalyze the condensation process, including cohesin and condensin. Cohesin forms rings that hold the sister chromatids together, whereas condensin forms rings that coil the chromosomes into highly compact forms. The mitotic spindle also begins to develop during prophase. As the cell's two centrosomes move toward opposite poles, microtubules gradually assemble between them, forming the network that will later pull the duplicated chromosomes apart.
proteasome
Proteasomes are protein complexes which degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that help such reactions are called proteases. Proteasomes are part of a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins. Proteins are tagged for degradation with a small protein called ubiquitin. The tagging reaction is catalyzed by enzymes called ubiquitin ligases. Once a protein is tagged with a single ubiquitin molecule, this is a signal to other ligases to attach additional ubiquitin molecules. The result is a polyubiquitin chain that is bound by the proteasome, allowing it to degrade the tagged protein
ryanodine receptor
Ryanodine receptors mediate the release of calcium ions from the sarcoplasmic reticulum and endoplasmic reticulum, an essential step in muscle contraction. In skeletal muscle, activation of ryanodine receptors occurs via a physical coupling to the dihydropyridine receptor (a voltage-dependent, L-type calcium channel), whereas, in cardiac muscle, the primary mechanism of activation is calcium-induced calcium release, which causes calcium outflow from the sarcoplasmic reticulum
centriole (9 triplet microtubules)
Structure A centriole is composed of short lengths of microtubules arranged in the form of an open-ended cylinder about 500nm long and 200nm in diameter. The microtubules forming the wall of the cylinder are grouped into nine sets of bundles of three microtubules each. In cilia and flagella where centrioles are at the base of the structure, and are called basal bodies, the wall and cavity architecture is slightly different. In addition to cylinder walls composed of nine sets of bundles of three microtubules, there are walls of nine sets of two bundles. In both types there is a central matrix from which spokes radiate as in a cart wheel. In animal cells centrioles usually reside in pairs with the cylindrical centrioles at right angles to each other. Centrioles organise a 'cloud' of protein material around themselves; this is the pericentriolar material (PCM). Together the two constitute the all important centrosome. Function Centrioles function as a pair in most cells in animals but as a single centriole or basal body in cilia and flagella. Centrioles in pairs Cells entering mitosis have a centrosome containing two pairs of centrioles and associated pericentriolar material (PCM). During prophase the centrosome divides into two parts and a centriole pair migrates to each end or pole on the outside of the nuclear membrane or envelope. At this point microtubules are produced at the outer edge of the pericentriolar material and grow out in a radial form. The centriole pair and PCM is called an aster. Microtubules from the aster at one pole grow towards the aster at the opposite pole. These microtubules are called spindle fibres. Some of these will become attached by centromeres to chromosomes lined up on the 'equator' of the dividing cell. Others, though not attached to chromatids/chromosomes by centromeres, will assist in pushing apart the two parts of the dividing cell. A single centriole or basal body. At the base of each cilium or flagellum there is a single centriole. This structure and associated pericentriolar material, construct microtubules in a linear direction. These microtubules form most of the inside of cilia and flagella and are largely responsible, using protein motors, for the mechanical aspects of their movement. The centriole at the base of each one also appears to exert some degree of direction and control over the movement of the cilia and flagella.
Dynamic instability
The co-existence of growing and shrinking microtubules in the same conditions is termed "dynamic instability." This phenomenon was first predicted based on observations of fixed in vitro reconstituted microtubules. Because reconstituted microtubules can be generated from purified tubulin, the dynamic behavior of microtubules is considered to be an intrinsic property, and not caused by external controlling factors. The co-existence of growing and shrinking microtubules was verified by observations of individual microtubules using unfixed preparations, and the "rescue" event, i.e., the transition from shrinkage back to growth, was described. The microtubules formed from purified tubulin dimers polymerize in a concentration- and temperature-dependent manner. However, they depolymerize in a stochastic manner that is unaffected by the concentration of available tubulin or the state of the neighboring microtubules. The execution and timing of rescue events also occur in a stochastic manner. Under assembly competent conditions, microtubules remain in the growth state for the majority of the time and all microtubules grow at a similar rate determined by the concentration of tubulin dimers present in the environment. The rate of microtubule depolymerization is several fold faster than that of polymerization and does not directly correlate with the subunit concentration. The transition from a growing to a shrinking state is called "catastrophe." Catastrophe events appear to occur stochastically, and the depolymerizing microtubules may or may not be rescued and resume growth. The following parameters are frequently used to characterize microtubules in a particular set of conditions: the growth rate and duration, the shortening rate and duration, and the frequency of catastrophe and rescue events. Recent observations of in vitro reconstituted microtubules revealed that the molecular events during microtubule polymerization are more complex than simple subunit addition at the ends. The ends of each protofilament of growing microtubules randomly alternates between periods of subunit addition (growth) and loss (shrinkage), and pausing. Microtubule growth means that subunits are added more frequently than they are lost.
minus end / pointed end
The terms "pointed" and "barbed" referring to the two ends of the microfilaments derive from their appearance under transmission electron microscopy when samples are examined following a preparation technique called "decoration". This method consists of the addition of myosin S1 fragments to tissue that has been fixed with tannic acid. This myosin forms polar bonds with actin monomers, giving rise to a configuration that looks like arrows with feather fletchings along its shaft, where the shaft is the actin and the fletchings are the myosin. Following this logic, the end of the microfilament that does not have any protruding myosin is called the point of the arrow (- end) and the other end is called the barbed end (+ end).[32] A S1 fragment is composed of the head and neck domains of myosin II. Under physiological conditions, G-actin (the monomer form) is transformed to F-actin (the polymer form) by ATP, where the role of ATP is essential
vimentin and vimentin-related filaments
Vimentin plays a significant role in supporting and anchoring the position of the organelles in the cytosol. Vimentin is attached to the nucleus, endoplasmic reticulum, and mitochondria, either laterally or terminally. The dynamic nature of vimentin is important when offering flexibility to the cell. Scientists found that vimentin provided cells with a resilience absent from the microtubule or actin filament networks, when under mechanical stress in vivo. Therefore, in general, it is accepted that vimentin is the cytoskeletal component responsible for maintaining cell integrity.
A tubule and B tubule in doublet
Virtually all eukaryotic cilia and flagella are remarkably similar in their organization, possessing a central bundle of microtubules, called the axoneme, in which nine outer doublet microtubules surround a central pair of singlet microtubules (Figure 19-28). This characteristic "9 + 2" arrangement of microtubules is seen when the axoneme is viewed in cross section with the electron microscope. As shown in Figure 19-3, each doublet microtubule consists of A and B tubules, or subfibers: the A tubule is a complete microtubule with 13 protofilaments, while the B tubule contains 10 protofilaments. The bundle of microtubules comprising the axoneme is surrounded by the plasma membrane. Regardless of the organism or cell type, the axoneme is about 0.25 μm in diameter, but it varies greatly in length, from a few microns to more than 2 mm.
Wee1
Wee1 is the kinase that phosphorylates CDK at Tyr-15 (Y15) and inhibits CDK activity. When Wee1 activity missing, CDK becomes active too soon and cells divide too early, before they are grown
major steps in formation of lamellipodium
actin polymerization pushes the plasma membrane forward look at diagram in ppt
apoptosis and caspase
activation of caspase cascade Examples of caspase cascade during apoptosis: Intrinsic apoptopic pathway: During times of cellular stress, mitochondrial cytochrome c is released into the cytosol. This molecule binds an adaptor protein (APAF-1), which recruits initiator Caspase-9 (via CARD-CARD interactions). This leads to the formation of a Caspase activating multiprotein complex called the Apoptosome. Once activated, initiator caspases such as Caspase 9 will cleave and activate other executioner caspases. This leads to degradation of cellular components for apoptosis. Extrinsic apoptopic pathway: The caspase cascade is also activated by extracellular ligands, via cell surface Death Receptors. This is done by the formation of a multiprotein Death Inducing Signalling Complex (DISC) that recruits and activates a pro-caspase. For example, the Fas Ligand binds the FasR receptor at the receptor's extracellular surface; this activates the death domains at the cytoplasmic tail of the receptor. The adaptor protein FADD will recruit (by a Death domain-Death domain interaction). The other end of the adaptor contains a DED domain for pro-caspase recruitment. This FasR, FADD and pro-Caspase 8 form the Death Inducing Signalling Complex (DISC) where Caspase-8 is activated. This could lead to either downstream activation of the intrinsic pathway by inducing mitochondrial stress, or direct activation of Executioner Caspases (Caspase 3, Caspase 6 and Caspase 7) to degrade cellular components as shown in the adjacent diagram.
plectin
aids in linking IFs to other cytoskeleton protein networks such as microtubules helps stabilize and reinforce connections between the different cytoskeleton filaments crosslinks microtubules to intermediate filaments
gap junction
allows the passage of small water-soluble ions and molecules in cytosol allo diffusion of small molecules (up to 1 kDa, which includes secondary messengers like cAMP and IP3) cardiac muscle cells beat synchronously because of ion movement through gap junctions 6 connexin subunits form a connexon which is a gap junction particle
cross-linking proteins
alter the three-dimensional organization of actin filaments examples: villin, fimbrin
laminin
an ECM protein found in basil lamina like fibronectin, has binding sites for several proteins A component of the extracellular matrix that is found in all basal laminae and has binding sites for cell-surface receptors, collagen, and heparan sulfate proteoglycans.
fibronectin
an ECM protein that can bind to an Integrin protein and help attach a cell to the extracellular matrix fibronectin has binding sites for numerous components of ECM An extracellular multiadhesive protein that binds to other matrix components, fibrin, and cell-surface receptors of the integrin family. It functions to attach cells to the extracellular matrix and is important in wound healing
metaphase checkpoint (spindle checkpoint)
are all chromosomes attached to the spindle? The M checkpoint is also known as the spindle checkpoint: here, the cell examines whether all the sister chromatids are correctly attached to the spindle microtubules. Because the separation of the sister chromatids during anaphase is an irreversible step, the cycle will not proceed until all the chromosomes are firmly attached to at least two spindle fibers from opposite poles of the cell. How does this checkpoint work? It seems that cells don't actually scan the metaphase plate to confirm that all of the chromosomes are there. Instead, they look for "straggler" chromosomes that are in the wrong place (e.g., floating around in the cytoplasm). If a chromosome is misplaced, the cell will pause mitosis, allowing time for the spindle to capture the stray chromosome.
a model of IF assembly and architecture
assembly: - basic building block is a rod-like tetramer formed by two anti-parallel dimers - both the tetramer and the IF lack polarity IFs are less sensitive to chemical agents than other types of cytoskeletal elements IFs don't bind to nucleotides
where does axonal transport occur?
axonal transport occurs along microtubules different motor proteins transport cargo along microtubules kinesins- go toward plus end dynein- go toward minus end
colchicine
binds to free tubulin and prevents its polymerization. it also binds to other proteins and is anti-inflammatory, being used to treat gout for several thousand years
Myosin Type I
binds to plasma membrane involved in filament formation
cytochalasin
binds to the plus ends of actin preventing polymerization
Myosin Type IV
binds to vesicle and plasma membrane acts as a track for vesicles
cyclin dependent kinase (CDK)
binds with cyclin the kinase subunit cannot function unless the cyclin subunit is bound to it MPF is a cyclin-CDK complex that controls entry into M-phase In order to drive the cell cycle forward, a cyclin must activate or inactivate many target proteins inside of the cell. Cyclins drive the events of the cell cycle by partnering with a family of enzymes called the cyclin-dependent kinases (Cdks). A lone Cdk is inactive, but the binding of a cyclin activates it, making it a functional enzyme and allowing it to modify target proteins. How does this work? Cdks are kinases, enzymes that phosphorylate (attach phosphate groups to) specific target proteins. The attached phosphate group acts like a switch, making the target protein more or less active. When a cyclin attaches to a Cdk, it has two important effects: it activates the Cdk as a kinase, but it also directs the Cdk to a specific set of target proteins, ones appropriate to the cell cycle period controlled by the cyclin. For instance, G1/S cyclins send Cdks to S phase targets (e.g., promoting DNA replication), while M cyclins send Cdks to M phase targets (e.g., making the nuclear membrane break down). In general, Cdk levels remain relatively constant across the cell cycle, but Cdk activity and target proteins change as levels of the various cyclins rise and fall. In addition to needing a cyclin partner, Cdks must also be phosphorylated on a particular site in order to be active (not shown in the diagrams in this article), and may also be negatively regulated by phosphorylation of other sites. Cyclins and Cdks are very evolutionarily conserved, meaning that they are found in many different types of species, from yeast to frogs to humans. The details of the system vary a little: for instance, yeast has just one Cdk, while humans and other mammals have multiple Cdks that are used at different stages of the cell cycle. (Yes, this kind of an exception to the "Cdks don't change in levels" rule!) But the basic principles are quite similar, so that Cdks and the different types of cyclins can be found in each species. All eukaryotes have multiple cyclins, each of which acts during a specific stage of the cell cycle. (In organisms with multiple CDKs, each CDK is paired with a specific cyclin.) All cyclins are named according to the stage at which they assemble with CDKs. Common classes of cyclins include G1-phase cyclins, G1/S-phase cyclins, S-phase cyclins, and M-phase cyclins. M-phase cyclins form M-CDK complexes and drive the cell's entry into mitosis; G1 cyclins form G1-CDK complexes and guide the cell's progress through the G1 phase; and so on.
breakdown and formation of lamins
breakdown of lamins caused by phosphorylation (kinase activity) formation of lamins caued by dephosphorylation (phosphatase activity)
Burkitt's Lymphoma
chromosomal translocation in Burkitt's Lymphoma results in overexpression of the transcription factor Myc when a very strong promoter is placed close to the Myc gene, high levels of Myc mRNA and protein are procuded usually Myc protein is only produced at high levels in the presence of a mitogen
ciliary and flagellar beating
ciliary and flagellar beating are produced by controlled sliding of outer doublet microtubules
cell can pause the cell cycle if it senses problems with itself or environment
components of a checkpoint: 1. sensor of problem 2. communication between sensor and cell cycle machinery 3. ability to delay cell cycle
nuclear lamina
comprised of lamins (Type V IFs) two-dimensional lattice assembly and disassembly regulated by phosphorylation A netlike array of protein filaments that maintains the shape of the nucleus.
triggering of DNA replication
degradation of the CDK inhibitor Sic1 by SCF at the end of G1 triggers DNA replication
intermediate filaments
diameter: 10 nm
microtubules
diameter: 24 nm hollow
actin filaments
diameter: 7-9 nm
tubulin heterodimer
dimer formed from alpha and beta-tubulin in microtubules
embrace model vs. handcuff model
embrace: one-ring model handcuff: two-ring model see ppt for pictures
cell motility
encompasses both changes in cell location and more limited movements of parts of the cell
Rb protein
enforces the G1 checkpoint, keeping the cell in G0 keeps cells from progressing through G1 until certain environmental signals are received phosphorylation of Rb allows expression of proteins required for DNA replication including G1/S and S phase cyclins and CDKs phosphorylation of Rb necessary for cell to pass the restriction point phosphorylation of Rb by the G1 (and G1/S cyclin/CDK) complexes inactivates Rb
spindle
fanlike microtubule structure that helps separate the chromosomes during mitosis three classes of microtubules make up spindle: 1. astral spindle microtubules 2. chromosomal (kinetochore) spindle fibers 3. polar spindle microtubules minus end of microtubule is at centriole plus end is in middle with chromosomes
branched filaments
filament splits into branches
filamin location
filopodia, pseudopodia, stress fibers
examples of actin cross-linking proteins
fimbrin filamin villin
what demonstrates microtubules are nucleated from centrosome?
fluorescently-labeled tubulin demonstrates microtubules are nucleated from the centrosome 1. depolymerize MTs with drug- colchicine, can also lower temperature 2. add fluorescently labeled tubulin 3. observe reassembly
connexon
forms gap junction particle
studying cell cycle
frog and sea urchin eggs have been powerful tools for studying the cell cycle
steady state
gain of G-actin at plus end equals loss of G-actin at minus end at a Cc that is intermediate for the plus and minus ends, addition of subunits at plus end equals loss of subunits at minus end referred to as "treadmilling"
malignant tumors
generally invade surrounding tissue and spread throughout the body alterations in cell-cell interactions and the formulation of new blood vessels are associated with malignancy
GTP cap model
has been proposed to explain dynamic instability high concentration of GTP-bound free tubulin leads to stability low concentration of GTP-bound free tubulin leads to instability due to GDP cap refers only to alpha-beta subunits gamma tubules always GTP-capped
keratin filaments
help epithelial cells to stay tightly associated -over 20 different forms of keratins -different types of epithelial cells express different combinations of keratins -hair and nails contain 10 different keratins used to diagnose epithelial cell type origin of primary tumors in carcinomas keratin composition an indicator of cancer
what composes wall of microtubule?
heterodimeric tubulin subunits compose the wall of the microtubule
rise and fall of cyclin and MPF during cell cycle
high levels of cyclin "correlate" with high MPF activity
in vitro actin gliding assay
in this experiment, microtubules are stuck to the microscope slide first. then, beads to which a single motor protein is attached are added in a buffer containing ATP. the beads are observed to travel along the microtubules
cell controller discovery
initial experiments utilized oocytes, which are arrested in the G2 phase of cell cycle inject cytoplasm from M-phase cell into oocyte. oocyte is driven into M-phase inject cytoplasm from interphase cell into oocyte. oocyte does not enter M-phase MPF controls entry into M-phase MPF=maturation (or M-phase) promoting factor
dynamic instability of microtubules increases during M phase
interphase: fewer, longer, and more stable microtubules. lifetime= 10 minutes prophase: greater number of shorter and more dynamic microtubules. lifetime=30 seconds
G2 checkpoint
is all DNA replicated? is environment favorable? ensures all of the chromosomes have been replicated and that the replicated DNA is not damaged To make sure that cell division goes smoothly (produces healthy daughter cells with complete, undamaged DNA), the cell has an additional checkpoint before M phase, called the G2 checkpoint. At this stage, the cell will check: DNA integrity. Is any of the DNA damaged? DNA replication. Was the DNA completely copied during S phase? If errors or damage are detected, the cell will pause at the G2 checkpoint to allow for repairs. If the checkpoint mechanisms detect problems with the DNA, the cell cycle is halted, and the cell attempts to either complete DNA replication or repair the damaged DNA. If the damage is irreparable, the cell may undergo apoptosis, or programmed cell death. This self-destruction mechanism ensures that damaged DNA is not passed on to daughter cells and is important in preventing cancer.
G1 checkpoint
is environment favorable? is the cell big enough? checks for cell size, nutrients, growth factors and DNA damage The G1 checkpoint is the main decision point for a cell - that is, the primary point at which it must choose whether or not to divide. Once the cell passes the G1 checkpoint and enters S phase, it becomes irreversibly committed to division. That is, barring unexpected problems, such as DNA damage or replication errors, a cell that passes the G1 checkpoint will continue the rest of the way through the cell cycle and produce two daughter cells. At the G1 checkpoint, a cell checks whether internal and external conditions are right for division. Here are some of the factors a cell might assess: Size. Is the cell large enough to divide? Nutrients. Does the cell have enough energy reserves or available nutrients to divide? Molecular signals. Is the cell receiving positive cues (such as growth factors) from neighbors? DNA integrity. Is any of the DNA damaged? These are not the only factors that can affect progression through the G1 checkpoint, and which factors are most important depend on the type of cell. For instance, some cells also need mechanical cues (such as being attached to a supportive network called the extracellular matrix) in order to divide. If a cell doesn't get the go-ahead cues it needs at the G1 and enter a resting state called G0 phase. Some cells stay permanently in G0 phase, while others resume dividing if conditions improve.
CENP-E
kinesin
chromo-kinesin
kinesin motor associated with chromosome arms also important for chromosome alignment chromosomes fail to align properly in spindlse assembled in frog egg extract depleted of chromo-kinesin
dyneins
large motor proteins that are attached along each outer microtubule doublet minus-end directed microtubule motor huge protein with a globular, force-generating head requires an adaptor (dynactin) to interact with membrane-bound cargo 2 types: axonemal cytoplasmic
SCF
like APC, SCF is a ubiquitin ligase that adds a ubiquitin chain onto its substrates and polyubiquination targets the protein for degradation
membrane-binding proteins
link contractile proteins to plasma membrane
MAP2
location- dendrites assembles and cross-links MTs to one another and to intermediate filaments overexpression of MAP2 leads to MTs further apart spacing of MTs depends on length of protein arm (MAP2 has longer arm)
tau
location- dendrites and axions assembles, stabilizes, and cross-links MTs overexpression of tau leads to MTs closer together spacing of MTs depends on length of protein arm (tau has shorter arm)
katanin
location- most cell types microtubule severing
proteoglycans
major component of ECM a large number of glycosaminoglycan chains (GAGs) are attached to a core protein. Each GAG is composed of repeating disaccharides. in cartilage, individual proteoglycans bind to a specific GAG and form an aggregate consists of a small core protein with many carbohydrate chains covalently attached can resist compression and can give ECM a gel-like quality. negative charges on GAG subunits allow H-bonding with many water molecules which creates a "gel" that can act as a cushion
role of cilia in development and disease
many cells have nonmotile primary cilia that sense chemical and mechanical properties of surrounding fluids mutations in primary cilia may lead to polycystic kidney disease motile and non-motile cilia are important in developmental processes, and mutations lead to range to abnormalities
cadherin molecules
mediate cell-cell adhesion cadherins comprise large family of Ca++ dependent adhesion molecules
MCAK
microtubule depolymerase
minus end and plus end microtubule motors contribute to spindle assembly
microtubules emanating from the two centrosomes need to get oriented in an antiparallel fashion. this requires the action of minus end-directed motor Dynein spindle poles also need to move farther apart. This is achieved by the plus end-directed motors acting on the polar microtubules, and by the minus end-directed motors acting on the astral microtubules
villin location
microvilli in intestinal and kidney brush border
fimbrin location
microvilli, sterocilia, adhesion plaques, yeast actin cables
movement of chromosomes during anaphase
movement of chromosomes during anaphase caused by depolymerization of MTs at kinetochores and poles
multiple motor proteins
multiple motor proteins can be associated with membrane vesicles motor protein can only bind to vesicle with no coat
myosin: the actin motor protein
myosins have head (ATPase), neck, and tail domains with distinct functions
IF Type IV
neurofilaments neurofilaments give neurons tensile strength found in nerve cells
tumor-supressor genes
normal function is to put the brakes on cell cycle progression
IF type V
nuclear lamins found in all nucleated cells
gamma-tubulin ring complex
nucleates polymerization of tubulin subunits
three steps of actin polymerization in vitro
nucleation elongation steady state
proto-oncogenes
operate in normal cellular pathways that stimulate cell cycle progression mutations in proto-oncogenes can result in activation of a signal transduction pathway even in the absence of signal
ORC (origin recognition complex)
origin- pieces of DNA where DNA replication starts ORC recognizes origin, allowing DNA replication to start
p21
p21 is a potent cyclin-dependent kinase inhibitor (CKI). The p21 protein binds to and inhibits the activity of cyclin-CDK2, -CDK1, and -CDK4/6 complexes, and thus functions as a regulator of cell cycle progression at G1 and S phase. cell cycle regulatory protein that inhibits the cell cycle; its levels are controlled by p53 Suppresses G1/S-Cdk and S-Cdk activity following DNA damage
interphase
period of the cell cycle between cell divisions in which the cell grows
what causes breakdown of the nuclear envelope?
phosphorylation of lamins help trigger disassembly there is also evidence that nuclear envelope is "ripped" open due to forces exerted by spindle microtubule motors as dynein motors associated with the nuclear envelope walk toward the minus ends of MTs, this stretches the nuclear envelope on the side opposite the centrosome. the nuclear envelope ruptures at the site of greatest stretching/tension.
mitosis in plant cells
plants cells don't have centromeres have to deal with cell walls The phragmoplast is a plant cell specific structure that forms during late cytokinesis. It serves as a scaffold for cell plate assembly and subsequent formation of a new cell wall separating the two daughter cells.
kinesins
plus end directed motor proteins which aid in fast axonal transport in retrograde transmission
which end of actin filament grows faster?
plus end grows much faster than minus end
+TIPs
plus-end binding protein used to monitor dynamic instability in MTs
what can be used to monitor dynamic instability?
plus-end binding proteins can be used to monitor dynamic instability
activation of MPF
positive and negative feedback loops enhance activation of MPF active M-CDK decreases Wee1 activity active M-CDK increases Cdc25 activity make sure to check this
motor processivity
processivity is an enzyme's ability to catalyze "consecutive reactions without releasing its substrate"
apoptosis
programmed cell death pathway kills cells quickly and cleanly plays normal role in development and maintenance in multicellular organisms - apoptosis in developing mouse paw sculpts the digits - apoptosis helps eliminate the tail during the metamorphosis of tadpole into frog
microvilli
projections of the plasma membrane of a cell that increase the cell's surface area Small, membrane-covered projection on the surface of an animal cell containing a core of actin filaments. Numerous microvilli are present on the absorptive surface of intestinal epithelial cells, increasing the surface area for transport of nutrients.
MT dynamics contribute to chromosome alignment
prometaphase: congression- getting the chromosomes aligned/positioned on the metaphase plate metaphase: poleward flux observe using fluorescent "speckle" microscopy
monomer-polymerizing proteins
promote the growth of actin filaments example: profilin profilin binds to ADP-G-actin and promotes nucleotide exchange (ATP replaces ADP)
nucleating proteins
provide a template for adding actin monomers examples: Arp 2/3 complex, formins
leading edge
region of the plasma membrane that extends in the direction of movement
end-blocking (capping) proteins
regulate the length of actin filaments example: CapZ
tropomyosin
regulatory protein that covers myosin-binding sites to prevent actin from binding to myosin In a relaxed muscle fiber, the active sites of actin are blocked by regulates contraction in skeletal muscle
what marks onset of anaphase?
release of cohesion between sister chromatids marks onset of anaphase
apoptosis intrinsic pathway
remember release of cytochrome c
apoptosis extrinsic pathway
remember role of Fas ligand
start/restriction point
same as a checkpoint
tight junction
seals neighboring cells together in an epithelial sheet to prevent leakage of molecules between them forms a solute and water tight seal
Z-disc
sheet of proteins that anchor thin filaments and connect myofibrils
lamellipodia
sheet-like projections at the leading edge Membrane pushed outward by rapidly polymerizing branched network of actin filaments Have adhesion of lamellipodium to the surface, anchoring the leading edge Contract the rear end of the cell and pull it forward
filament-severing proteins
shorten filaments and decrease cytoplasmic viscosity example: gelsolin
DNA damage checkpoint in G1
similar pathway operates if damage occurs during G2 prevents aneuploidy key protein is p53. normally bound to Mdm2 double strand break in DNA causes phosphorylation of p53 causing p53 to dissociate from Mdm2 p53 is a transcription factor so active p53 will enter nucleus and activate downstream responses. most notable is production of p21 which is a Cdk inhibitor protein. when p21 binds to G1/S and S-Gdk complex the complex becomes inactive once DNA is fixed, p53 degrades so normal cell activity resumes. cell can enter S phase
cohesion complex
sister chromatids are held together by the cohesion complex from the time of their synthesis until anaphase onset chromatids are together along their entire length cohesion complex forms a ring integrity of ring needs to be maintained for chromatids to stick together
centrosome
site of microtubule nucleation in animal cells centrioles and pericentriolar material (including gamma-tubulin) comprise the centrosome region where the cell's microtubules are initiated; contains a pair of centrioles
epithelial cells
skin cells that cover the outside of the body and line the internal surfaces of organs
survival factor
some cells receive insufficient amounts of survival factor to keep their suicide program suppressed, and, as a consequence, undergo apoptosis
cdh-1
specificity factor that determines mitotic cyclins are substrates for APC
cdc20
specificity factor that determines securin is substrate for APC
metaphase checkpoint example
spindle attachment checkpoint senses lack of tension on unattached chromosomes and blocks progression into anaphase lack of tension/MT attachment at any kinetochore results in phosphorylation of Mad2 which activates a pathway that keeps Cdc20-APC inactive until all the sister chromatids are attached
metastasis
spread of cancer cells beyond their original site in the body tumor cells make it into blood stream and spread throughout body
taxol
stabilizes GDP-tubulin so that there is no depolymerization from the yew tree. was discovered to be cytotoxic in 1964
intermediate filaments strengthen animal cells
stretching a sheet of cells with IFs→ cells remain intact and together stretching a sheet of cells without IFs→ cells rupture -intermediate filaments withstand stress and deformation -microtubules and actin filaments eventually rupture -IFs become more resistant with more force
how can structural polarity be revealed?
structural polarity can be revealed by "decoration" of actin filaments with myosin S1 fragments
myosin S1 fragment
structural polarity of F-actin can be revealed by "decoration" of actin filaments with myosin S1 fragments The terms "pointed" and "barbed" referring to the two ends of the microfilaments derive from their appearance under transmission electron microscopy when samples are examined following a preparation technique called "decoration". This method consists of the addition of myosin S1 fragments to tissue that has been fixed with tannic acid. This myosin forms polar bonds with actin monomers, giving rise to a configuration that looks like arrows with feather fletchings along its shaft, where the shaft is the actin and the fletchings are the myosin. Following this logic, the end of the microfilament that does not have any protruding myosin is called the point of the arrow (- end) and the other end is called the barbed end (+ end). A S1 fragment is composed of the head and neck domains of myosin II. Under physiological conditions, G-actin (the monomer form) is transformed to F-actin (the polymer form) by ATP, where the role of ATP is essential
centriole
structure in an animal cell that helps to organize cell division organelle that plays a role in cell division and is made of microtubules
cytoskeleton
supporting network of protein fibers that provide a framework for the cell within the cytoplasm governs cell structure, cell shape changes, and cell motility
How does cell know if all the chromosomes are aligned at metaphase?
tension seems to indicate that chromosomes are aligned when tension is applied with a needle, the cell proceeds into anaphase, even though chromosomes aren't actually aligned. without needle tension, the cell arrests at metaphase chromosomes not under tension have kinetochore protein that is phosphorylated (Mad2)- can see this with phospho-specific antibody
how is the actin cytoskeleton organized?
the actin cytoskeleton is organized into bundles and networks of filaments
critical concentration (Cc)
the concentration of G-actin above which polymerization can take place two ends of actin filaments have different critical concentrations plus end is preferred for assembly and therefore it has a lower Cc
checkpoints
the controller has built in "eyes" that are checking if the steps of the cell cycle are completed properly and if the cell is ready to go on to the next phase the "eyes" are called checkpoints
intraflagellar transport (IFT)
the mechanism by which the axoneme is assembled and involves the activity of kinesin and ctyoplasmic dynein
myosin head
the myosin head domain has the actin and ATP binding sites walks along actin filaments minus end is leading end
Anaphase B
the poles move away from each other polymerization of polar MTs move poles away from each other. also depolymerization at poles spindle elongates -dynein anchored in plasma membrane: pulling force -kinesins bound to polar microtubules: pushing force
angiogenesis
the process through which the tumor supports its growth by creating its own blood supply formation of new blood vessels to support tumor growth without a blood supply, tumors can only grow to a diameter of 2 mm. To survive, a tumor must induce the formation of blood vessels
organization of cells into tissues
the wall of the intestine is composed of different types of tissues (epithelial, muscle, and connective tissues). In each tissue is an organized assembly of cells held together by cell-cell adhesions, cell-extracellular interactions, or both. very structured see ppt for diagram
thick and thin filaments
thick and thin filaments slide past one another during contraction thin- actin thick- myosin
intermediate filaments (IFs)
tough, rope-like fibers heterogeneous group of proteins, divided into five major classes IFs classes I-IV are used in the construction of cytoskeletal filaments; type V (lamins) are present near the inner membrane of nucleus around 70 IFs encoded in human genome
