BIO Test IV

When/where is a tubulin dimer bound to GTP, and when/where does it hydrolyze this GTP to become bound to GDP? Understand why a microtubule depolymerizes so rapidly once it loses its "GTP cap."

GTP when growing rapidly (GTP while added, hydrolyzes to GDP shortly after added) end of growing MT is a GTP cap. GDP when shrinking. depolymerizes bc GDP less tightly bound, randomness of chem. processes will have end hydrolyze b4 next added, ends now GDP bound. now favors disassembly 》 to pool of free unpolym. tubulin

function of centrosome

In animal cells, microtubules radiate outward to the cell periphery from a microtubule organizing center called the centrosome This spokelike arrangement of microtubules helps cells withstand compression and thereby maintain their shape. Many organelles are tethered to microtubules, and thus microtubules guide the arrangement of organelles in the cell.

role of microtubules in establishing cell polarity (e.g., in a neuron)

MT systems in interior help position organelles in req. locations in cell and guide vesic. macromolec. traffic in cell nerve cell- MT in axon point in same direcfion, plus ends towaeds terminals w oriented tracks, guides organelles/vesicles/maxeomolec from cell body to axon term, vice versa

Explain how the dynamic nature of microtubules and microfilaments is important for their functions.

Microtubules and microfilaments are dynamic in structure because they can become longer or shorter with the addition or subtraction of their subunits. This addition or deletion is influenced by many factors, including the concentration of free subunits and the activity of regulatory proteins. The dynamic nature of microtubules in spindles allows them to explore the space of a cell and find chromosomes during cell division. The dynamic nature of microfilaments is important for some forms of cell movement.

Name three types of cytoskeletal element, the subunits they are composed of, their relative sizes, and the major functions of each type.

Microtubules are made up of α- and β-tubulin dimers; microfilaments are made up of actin monomers; and intermediate filaments are made up of intermediate filament protein subunits. Microtubules have the largest diameter (25 nm); microfilaments have the smallest diameter (7 nm); and intermediate filaments have an intermediate diameter (10 nm). Microtubules help to maintain cell shape, provide tracks for vesicles and other cargo inside of cells, and make up the spindles that attach to chromosomes during cell division. Microfilaments also help to maintain cell shape and associate with myosin to cause muscle contraction. Intermediate filaments provide cells with mechanical strength.

How tubulin monomers assemble into microtubules

One alpha tubulin and one beta tubulin combine to make a tubulin dimer and the tubulin heterodimers are assembled to form the microtubule, a hollow tube the ability of spindle microtubules to "explore" the space of the cell and encounter chromosomes is driven by a unique property of microtubules: plus ends undergo random cycles of rapid depolymerization followed by slower polymerization. cycles of depolymerization and polymerization are called dynamic instability- allow spindle microtubules to quickly find and attach to chromosomes during cell division.

reductionist approach

Reduce the problem being studied down to its component 'parts'. By understanding the behavior of the 'parts', you can assemble an understanding of the behavior of the 'whole'.

Describe the polarity of microtubules and microfilaments. How does the dynamic behavior of these cytoskeletal elements depend on polymer polarity? (NOTE: In lecture, we will refer to the ends of an actin filament as "barbed" and "pointed." You should be able to talk about microfilaments in these terms.)

The faster assembling end is called the plus end and the slower-assembling end is called the minus end cell movement, such as a single-celled amoeba foraging for food or a mammalian white blood cell chasing down foreign bacteria, depend on actin polymerization and depolymerization. required when a single cell divides in two during cytokinesis

Describe the functions of the three major motor proteins and state which cytoskeletal element each interacts with.

The three major motor proteins are myosin, kinesin, and dynein. Myosin binds to microfilaments in the cell and can cause these filaments to move relative to each other, as in muscle cell contraction. Myosin can also attach to various types of cellular cargo and move along a microfilament, transporting these materials from one part of the cell to another. Kinesin transports cargo toward the plus end of microtubules, while dynein transports cargo in the opposite way, toward the minus end of the microtubule. Dynein also associates with microtubules to cause movement of cilia and flagella.

main function of intermediate filaments

They are polymers of intermediate filament proteins that combine to form strong, cable-like structures in the cell. As a result, they provide cells with mechanical strength the proteins making up intermediate filaments differ from one cell type to another.

microfilaments and microtubules are dynamic

They become longer by the addition of subunits to their ends, and become shorter by the loss of subunits. The rate @ protein subunits are added depends on concentrations of tubulin and actin @ high concentrations of subunits, microtubules and microfilaments can become longer at both ends- assembled more quickly on one end faster assembling end is called the plus end and the slower-assembling end = the minus end

dendritic-nucleation model of actin assembly in the protrusive end of a crawling cell. Protein functions within the model: capping protein, Arp2/3, profilin, ADF/cofilin, and the aging process of actin filaments in terms of the nucleotide state bound to actin.

a. External cues (step 1) activate signalling pathways that lead to GTPases (2). These then activate Wiskott-Aldrich syndrome protein (WASP) and related proteins (3), which in turn activate Arp2/3 complex. Arp2/3 complex initiates a new filament as a branch on the side of an existing filament (4).Each new filament grows rapidly (5), fed by a high concentration of profilin-bound actin stored in the cytoplasm, and this pushes the plasma membrane forward (6). Capping protein binds to the growing ends, terminating elongation (7). Actin-depolymerizing factor (ADF)/cofilin then severs and depolymerizes the ADP filaments, mainly in the 'older regions of the filaments (8, 9). Profilin re-enters the cycle at this point, promoting dissociation of ADP and binding of ATP to dissociated subunits (10). ATP-actin binds to profilin, refilling the pool of subunits available for assembly

Know that cilia and flagella

are constructed around a core of stable microtubules that interact with dynein and do not show dynamic instability.

capping protein

binds to the growing ends, terminating elongation

§ Compare and contrast treadmilling in actin filaments with dynamic instability in microtubules

both rely on hydrol. of bound nucleoside triphosphate to regulate length, result is different. treadmilling- simult. gain and loss, when equal, filament same size dyn. inst.- rapid switch fro. growth to shrink, v.v. more drastic change in length

Define, describe the structure, and briefly explain the functional importance of centrosomes, centrioles, and g-tubulin ring complexes in the growth and organization of microtubules

centrosome: organizes array of microtubules that radiate outward through cytoplasm (near nucleus interphase) pair of centrioles make up centrosome: surrounded by matrix of proteins formed from y tubulin. each gamma tubulin ring complex serves as starting pt (nucleation site) for growth of microtubule. the alpha/beta dimers add to each g tubulin ring complex (minus embedded in centrosome, growing plus end extends into cytoplasm)

When presented with information about how a particular drug (e.g., colchicine, Taxol, vincristine, vinblastine) interacts with tubulin, be able to predict its effects on microtubule polymerization and the function of the mitotic spindle. Understand why these drugs can be useful forms of cancer treatment.

colchicine: binds tightly to free tubulin dimers and prevents polym. of MT( spindle disappears) taxol: binds to MT and prevents them from losing subunits. MT can grow but not shrink, same overall effect though in stopping cells in mitosis( for mitotic spindle to function,must assemble and disassemble) taxol-binds and stabilizes MT colchicine,colcemid- binds dimers and prevents polym. vinblastine, vincristine, binds dimers and prevents pklym. inactiv. or destruction of spindle eventually kills dividing cells, antimitotic drugs can kill cancer cells (divide uncontrollable, can be stopped by MT- stabilizing/destabilizng drugs)

dynamic instability and name the type of cytoskeletal element that exhibits this behavior. Briefly describe the importance of dynamic instability for spindle assembly and chromosome segregation during cell division

cycles of depolymerization and polymerization by microtubules They allow spindle microtubules to quickly find and attach to chromosomes during cell division. make up the spindles that attach to chromosomes during cell division- the ability of spindle microtubules to "explore" the space of the cell and encounter chromosomes

When presented with information about how a particular drug (e.g., cytochalasin, latrunculin, phalloidin) interacts with actin, be able to predict its effects on actin polymerization

cytochlasin (caps plus ends, prevent there) and latruncilin(binds monomers)- prevent actin polym. phalloidin- stabilize actin fil. against depolym. can freeze cell movements like cell locomotion (binds and stabilizes filaments)

Know that there are...

cytoskeletal proteins found in prokaryotes (but the prokaryotic cytoskeleton is far less extensive and dynamic than the eukaryotic cytoskeleton).

intermediate filaments function

enable cell to withstand mechanical stress when cell is stretched (great tensile strength), network surrounding nucleus and extending out network throughout cytoplasm, anchored to desmosomes **intermediate filaments contribute to desmosomes and make up the nuclear lamina. § Know that intermediate filaments are ropelike polymers without polarity.

Name the intermediate filament protein found in epithelial cells. Name the intermediate filament protein found in the nuclear lamina

epithelial cells, protein subunits are keratins;. Intermediate filaments bind to cell junctions called desmosomes, forming a strong, interconnected network. lamins, found inside the nucleus, where they provide support for the nuclear envelope

Cilia and flagella: explain how these structures differ from each other. Name one animal cell type that has cilia and one animal cell type that has flagella and briefly describe the importance of these structures for cellular function.

fiberlike organelles that propel the movement of cells or substances surrounding the cell, (microtubules assoc. w/ dynein) move cells or allow cells to propel substances. motion of cilia (which are short) or flagella (which are long) the sperm cells of algae, some plants, and many animals are propelled by one or more flagella. Epithelial cells in a number of animal tissues, such as the lining of the trachea and the upper respiratory tract, have cilia that move substances along the surface of the cell layer. Coordinated beating of the cilia that cover the paramecium moves the cell through its environment. The cilia in these human airway epithelial cells propel mucus containing debris out of the lungs. Some cells of multicellular organisms, including these sperm cells, swim by movement of a flagellum.

Centrosomes and Centrioles

form mitotic spindle; needed to form cilia and flagella each centriole perpendicular made of cylindrical array of short microtubules, no role in nucleation. organizing centers for microtubules in cilia and flagella(basal bodies)

Describe the architecture of the actin filaments associated with this leading edge and explain how ARPs (Arp2/3 complex) generate this architecture

grow out in side branches , continual assembly at leading edge and disassembly further back, moving lamellipodium forward nucleation mediated by arp

how actin polymerization pushes the leading edge of a lamellopodium forward

has dense meshwork of actin filament, most have + ends close to plasma membrane. also have thin protrusions filopodia @ leading edge form and retract w great speed by local growth of fil. assemble close to membrane and elongate by addit. of polymers (push without tearing) at plus ends web of polym. fil. pushes assisted by actin related proteins (ARP) promote web

Microtubules

hollow tubelike structures with the largest diameter of the three cytoskeletal elements, about 25 nm protein dimers (alpha and beta) maintain cell shape and the cell's internal structure. Many organelles are tethered to microtubules, and thus microtubules guide the arrangement of organelles in the cell. microtubules radiate outward to the cell periphery from a microtubule organizing center called the centrosome. This spokelike arrangement of microtubules helps cells withstand compression and thereby maintain their shape.

Rho GTPases are...

important in signaling pathways that regulate the location, organization, and behavior of actin filaments. ecm sign. moleculues regulate cytoskeleton activate receptor proteins, converge on rho protein family. molecular switches that control intracell. processes( can shape actin and drive bundling, promote lamellipodia formation, etc)

Define the term cytoskeleton

intricate network of protein filaments throughout the cytoplasm, support the large volume, for shapes of cell, coord. movements, organize components inside..mostly for complex euk. cell dynamic structure constantly reorganizing

functional importance of intermediate filaments composed of keratin and lamin

keratin for epithelial cells: anchored to desmosomes interconnecting cells, cabling of high tensile strength, distributes stress when stretched lamin for nuclear lamina: line (support) and strengthen inner surface of nucleus, disassembly and reassembly of nuclear lamina controlled by its phosph. and dephosph.

Name two motor proteins associated with microtubules. Know which one moves along a microtubule toward the plus end and which one moves along a microtubule toward the minus end.

kinesin and dynein. Kinesin transports cargo toward the plus end of microtubules, located at the periphery of the cell. By contrast, dynein carries its load away from the plasma membrane toward the minus end, located at the centrosome in the interior of the cell.

MT motor proteins

kinesins move along, towards plus end outward from cell body dyneins minus end(towards cell ody) globular atp binding heads and single tail, atp hydrolysis provides energy for confirm. change in head to make it move (binding,release, rebind) to MT. play role in positioning organelles. kineains pull ER out, dyneins pull golgi inwards toward nucleus

extracellular matrix

meshwork of proteins and polysaccharides outside the cell- provides structural and biochemical support to the surrounding cells. for strong, properly shaped tissue or organ- ability of cells to adhere to ECM

Know that...

microtubules and microfilaments are found in all eukaryotes and that animals also have intermediate filaments.

compare and contrast the 3 cytoskeletal elements as listed and described in Table One. Specifically, for each cytoskeletal elements, list protein subunit, polymerization scheme, bound nucleotide, properties of filament aging, molecular motors, and the shape and thickness from the electron micrograph.

monomer: -Actin monomer -Tubulin heterodimer --Various proteins with an a-helical coiled-coil Ageing by nucleotide hydrolysis + phosph. release: Actin: Yes, allows binding of proteins promote disassembly (actin) MT: Yes, destabilizes polymer IF: No

actin-binding proteins. Describe how different types of actin-binding proteins can influence the behavior of actin filaments.

most bind to assembled fil. than monomers and control their behavior of filaments. (can hold together in bundles for microvilli, cross-link for cortex)

nucleation of a cytoskeletal filament. How is g-tubulin related to nucleation of microtubules?

much harder to start new microtubule from nothing, easier to add to existing g tubulin ring complex than assembling ring. the ab tubulin concentration is too low in a living cell to assemble initial ring (providing organizing centers at specific sites and keeping concentration of ab dimers low, cells control where microtubules form) in lab vitro concentration higher so this natural assembly more likely to happen (ab dimers polymerize)

Name a motor protein associated with actin microfilaments.

myosin- to transport various types of cellular cargo, such as vesicles, inside of cells. microfilaments are responsible for changes in the shape of many types of cell. One of the most dramatic examples of cell shape change is the shortening (contraction) of a muscle cell. Muscle contraction depends on the interaction of myosin with microfilaments, and is powered by ATP

Describe the structure of a tubulin dimer and how these dimers polymerize into a microtubule.

noncovalent interactions bind alpha/beta tubulin dimers stack together by noncov. interact. to form wall of hollow tubule (alternating chains)

Explain why microtubules are important for trafficking cargo in large eukaryotic cells.

oriented movement is faster and more efficient than by free diffusion. (weeks as opposed to years) tracks of directional transport, help to maintain org.

Microfilaments

polymers of actin monomers, arranged to form a helix. They are the thinnest of the three cytoskeletal fibers, about 7 nm in diameter, and are present in various locations in the cytoplasm At the cortex, microfilaments reinforce the plasma membrane and organize proteins associated with it.

importance of profilin in maintaining a pool of actin monomers in a cell

since actin conc. much higher, proteins keep them from polym. totally into filaments (thymosin, profilin) stop them from being added and keep them in reserve. important in regulation of polym. filament severing proteins fragment fil. and make actin gel more fluid, also assoc. with motor proteins

motor proteins

small jerky steps (saltatory mov) can occur along MT or MF and are driven by motor proteins, use the atp hydrolysis energy to travel steadily along microtubule/filament in single direction. also attach to other components, can transfer cargo along , differ in tyle of filament, direction, and cargo carried

the structure and function of the cell cortex

specialized later of actin filament rich cytoplasm beneath membrane (crosslinked in gel meshwork actin filaments) support membrane and give it mechanical strength (shape and mov.) ex. disc shape in RBC

Cytoskeleton

structural protein networks in cytoplasm that maintain and determines shape of cells

structural polarity of a microtubule and explain how this polarity is related to the way tubulin dimers add to each end of a growing microtubule.

structure has definite direction and chemically + functionally distinct ends, important for vesicle transport alpha tubulin exposed @ one end(minus end), and beta at the other (plus end) added more rapidly to the plus end

cell junctions

structures that lead to the connection of cells to one another. (shape and structural integrity of tissues and organs depend on the ability of cells to connect to one another.)

Role of GTP in microtubule polymerization and describe how GTP hydrolysis controls dynamic instability

switching back and forth between polymerization/depolymerization for rapid remodeling crucial for function tubulin dimers hydrolyze gtp. each free dimer has one gtp bound to beta, which hydrolyzes after added to growing MT. GDP remains tightly bound to beta. in rapid growth, dimers add faster than gtp hydrolysis, microtubule composed of gtp-tubulin dimers , bind more strongly to neighbors than w gdp, pack efficiently, tend to keep growing MT if growth slow, dimers in gtp cap will 》》GDP before binding to fresh gtp, gtp cap is lost. less tightly bound, protofilaments peel away, dimers released, shrink MT

Know that microtubules form

the mitotic spindle (chromosome segregation), cilia, and flagella. (stable structures, rhythmically beating)- hairlike and extend from surface (core contains microtub.) function: to transport and position organelles and guiding intracellular transport

Arp2/3

the nucleating complex (actin-binding protein) that is used both for initial nucleation of MFs and for nucleation of side branches is In the dendritic (branched) networks of actin filaments in the leading edge of a crawling cell, nucleation of actin polymerization is mostly performed by ... (a seven-subunit nucleator of actin filaments) activated by wasp

role of integrins in cell crawling. Recall the categories of actin-linked cell-matrix adhesions discussed earlier in the quarter** and name type depicted in Figure 17-33

transmembrane integrins adhere to molecules in ecm) or neighboring cell over it is crawling. interxellular side integrins capture actin filaments in cortex, making Anchorage for crawling to drag forward (actin linked cell matrix junction)

identify each type of cytoskeletal element based on the organization of filaments within the cell, the diameter and general structure of the filaments, and the identity of the monomeric subunits.

7nm(microf.), 10nm (int. f), 25 nm (microtub.) cortex (layer under cytoplasm) for microfilaments, throughout cytoplasm for intermed., from centrosomes outwards for microtubules double helix for microfil, rope-like for intermed., and hollow tub. for microtubules

type of energetic coupling that powers eukaryotic motor proteins.

Movement along microtubules by kinesin and dynein is driven by conformational changes in the motor proteins and is powered by energy harvested from ATP atp energetic coupling-released by the hydrolysis of ATP to drive a thermodynamically unfavorable reaction.

How myosin motor proteins interact with actin filaments and explain the role of ATP hydrolysis in generating these movements.

all actin dep. motor proteins belong to myosin fam. bind and hydrolyzes atp for energy to move along actin filaments toward plus end. (head w atp activity and tail ina cycle of binding and detaching) tail drags cargo

Describe the architecture of the actin filaments associated with protrusion of a filopodium and explain how formins generate this architecture

formin is a nucleating protein that attaches to growing plus ends of actin fil. promotes growth for straight, unbranched filaments. also used for contractile ring to pinch cell in two (filopodium cell protrusion)

Explain the role of ATP in actin polymerization. Compare and contrast the role of ATP in actin microfilaments with the role of GTP in microtubules

free actin monomers added faster at plus end(barbed) than neg.(pointed) like tubules. free monomers have ATP tightly bound and hydrolyzes to ADP after added, in filament hydrolys. to adp can weaken stability of polymer, promote depolymerization assembly rate on concentration., if high both ends grow, but if intermediate conc. then faster at one end and loses subunits at minus end. (treadmilling)

g-tubulin ring complex

role in nucleation, each microtubule grows and shrinks indep. of neighbors

5 functions of microtubules

1. Intracellular transport - Movement of vesicles & organelles 2. Cell motility - Movement of cilia and flagella - Cell elongation and movement 3. Mitotic Spindle (movement of chromosomes) - Attachment of chromosomes & their movement during cell division 4. Rigid intracellular skeleton - Maintenance of cell shape and polarity 5. Assemble and disassemble as the needs of the cell changes, movement of organelles -maintain cell shape -enables movement of substances -guide movement of organelles

three essential processes associated with cell crawling

1. cell pushes out protrusions @ front/leading edge 2. protrusions stick to surface over cell is crawling 3. rest of cell drags itself forward by traction at these anchorage points

Describe three "articles of faith" - evolution, structure and function of individual components and their effects on the ensemble, and reconstitution of a complex process from purified components

1. owing to evolution from common ancestors, modern cells use a common set of molecular mechanisms to carry out their basic functions. believe that analysis of any experimentally tractable organism provides insights about general principles that will apply to most cells. (Despite diversity at the cellular level, the underlying mechanistic unity is now clear at the molecular level) 2. knowledge of the structures and functions of the individual parts of molecular machines reveals much about the workings of ensembles of molecules. 3. critical test for understanding is reconstitution of a complex process from purified components in 'wet' biochemical experiments and/or in computer simulations.

how actin monomers assemble into microfilaments

A microfilament is a double helix of actin monomers.

motor protein

Accessory protein that interacts with cytoskeletal elements (microfil. microtub) and other cell components, producing movement of the whole cell or parts of the cell.

Compare and contrast the structural properties, relative diameters, and general functions of microtubules, microfilaments (= actin filaments), and intermediate filaments

All 3 made of polymers (long chains) microtubules + microfilaments - structural support, enable the movement of substances within cells, changes in cell shape. Microtubules: Tubulin dimers for Cell shape and support Cell movement (by cilia, flagella) Cell division (chromosome segregation) Vesicle transport Organelle arrangement Microfilaments:Actin monomers for Cell shape and support Cell movement (by crawling) Cell division (cytokinesis) Vesicle transport Muscle contraction Intermediate filaments: Diverse subunits Cell shape and support