Cell Bio Test 2

What is the purpose of the Golgi complex?

Flattened, disclike, membranous cisternae arranged in orderly stack stacks are interconnected *Cis-golgi network*: sorting station that distinguishes between proteins to be shipped back to the ER and those that are allowed to proceed to the next Golgi station. (closest to the ER) *Cis, medial, trans cisternae*: Primary activities of golgi Error checking Glycosylation Occurs at the site of glucose removal. *Trans golgi network*: Tubules that serve as sorting sites. Vesicle formation and budding

Secretory granules

In regulated secretion Large, densely packed; where materials to be secreted may be stored

Targeting the lysosome

In the tGN the carbohydrate label is modified to possess a mannose-6-phosphate group Mannose-6-phosphate receptor (MPR) recognize this and acts similar to a cargo receptor Clarion is recruited by the MPR receptor when bound to a lysosomal protein

Integrins are cell-surface receptors that mediate interactions between cells and their substratum. Can you structurally describe integrins? How do they work? Are there any conformational changes associated with integrins?

Integirns are heterodiemric integral membrane proteins whose cytoplasmic domains interact with cytoskeletal components, and whose extracellular domains contain binding sites for extracellular materials. Internal changes within a cell can send inside out signals that activate the ligand binding activity of integrins This activation is associated with a dramatic change of the structure of the integrin from a bent to an upright. Binding of an extracellular ligand to an integral may send outside in signals that initiate changes in cell activities

What is the role of the lysosome? Are the involved with the immune-system? Are there any diseases of the lysosome?

Lysosomes degrade materials, such as bacteria and debris, that are brought into the cell by phagocytosis. They degrade aging cytoplasmic organelles by autophagy. Digest macromolecules that are delivered by way of endosomes by receptor mediated endocytosis. -Important in immune system functioning -*acid hydrolases* --> 4.6 *I-cell disease*: lysosomes contain undegraded material. Lysosomal enzymes are synthesized normally but are secreted into the medium and not targeted to lysosomes. Enzymes *lacked mannose phosphate* --> *deficiency of N-acetylglucosamine phosphotransferase* *Tay-Sachs*: deficiency of enzyme that degrades ganglioside GM2 (GM2 major component of membranes of brain cells) *Gauchers*: deficiency of lysosome enzyme glucocerebrosidase. Spleen enlargement and anemia. Can digest almost every type of biological molecule Nucleases: DNA, RNA Proteases: proteins Poly and Oligosaccharideases: carbs Sphingolipid hydrolyzing enzymes Lipid hydrolyzing enzymes: acid hydrolyses Liver lysosomes digest RBC -*Kupffer cells*: a phagocytic cell that forms the lining of the sinusoids of the liver and is involved in the breakdown of red blood cells.

Regulated secretion

Materials are stored as membrane-bound packages and discharged *only* in response to a stimulus. Ex: endocrine cells with hormones. NT release. Digestive enzymes. Materials that are secreted due to signals. -Hormone release -NT release -Digestive enzyme release

Constitutive secretion

Materials are transported in secretory vesicles from their sites of synthesis and discharged into the extracellular space in a continual manner. Most common. -ECM and plasma membrane *Continue release* -ECM and plasma membrane

Intermediate filaments are ropelike cytoskeletal structures approximately 10 nm in diameter. How are these different from microtubules?

May consist of variety of different proteins capable of assembling into similar types of filaments. Unlike MT, intermediate filaments are composed of symmetric building blocks which assemble into filaments that lack polarity. IFs are resistant to tensile forces and relatively insoluble They are dynamic structures that rapidly incorporate labeled subunits that are injected into a cell Assembly and disassembly are thought to be controlled primarily by phosphorylation and dephospho rylation. IFs provide mechanical stability to cells and are required for specialized, tissue-specific functions.

Cell to cell adhesion How does this work? What types of connections are involved? What types of proteins? What about the different types of junctions? Can you name and describe them?

Mediated by *integral membrane proteins* like selectins, integrins, cadherins, and members of the immunoglobin superfamily (IgSF) -Selectins bind to specific arrangements of carb groups projecting from the surfaces of other cells and mediate calcium-dependent, transient interactions between circulating leukocytes and the walls of blood vessels at sites of inflammation and clotting -Ig superfamily mediate ca-independent cell-cell adhesion. IgSF protein on one cell may interact with an integrin or with the same or a different IgSF protein projecting from another cell. -Cadherins mediate calcium dependent cell-cell adhesion by binding to the same cadherin species on the opposing cell, thereby facilitating the formation of tissues composed of similar cell types.

Most lipids of a cell's membranes are synthesized in the ER and moved to various destinations. How does this happen? How are lipids modified?

Newly synthesized phospholipids are inserted into the half of the bilayer facing the cytosol. May be flipped by *flippases*. Lipids are carried from the ER to the Golgi and plasma membrane as part of the bilayer that makes up the walls of transport vesicles. (1) Most organelles contain enzymes that modify lipids already present within a membrane, converts one type to another (2) Cells contain *lipid-transfer proteins* that can bind and transport lipids through the aqueous cytosol from one membrane compartment to another. These proteins facilitate movement of specific lipids from the ER to other organelles WITHOUT transport vesicles. Happens when ER is close to outer membrane of other organelles.

Membrane asymmetry

One side of the plasma membrane is accessible to cellular enzymes. Membranes that bud have different types of phospholipids on each leaflet of the membrane. enzymes can move particular phospholipids from one membrane to another --> creating the asymmetry

Actin filaments are 8 nm in diameter, composed of a double-helical polymer of the protein actin. What do these do? How do they grow or shrink? Why is it important that these structures grow and shrink?

Plays a key role in virtually all types of contractility and motility Actin filaments can be organized into highly ordered arrays or loose networks or tightly held bundles Often identified by their ability to bind to the S1 myosin fragment, which also reveals the *polarity of the filament* Although both ends of an actin filament can gain or lose subunits, the *plus end* is the preferred site of of subunit addition and the *minus end* is the preferred site of subunit loss. To be incorporated on the growing end of the filament, an actin subunit must be bound to an ATP, which is hydrolyzed soon after its incorporation. Cells maintain a dynamic eq between monomeric and polymeric forms of actin, which can be altered by changes in variety of local conditions As long as concentration of ATP-actin molecules remain high, subunits continue to be added at *both ends* Plus end has a higher affinity for ATP-actin

Biosynthetic pathway

Proteins are synthesized in the ER, modified in Golgi, and transported from Golgi to various destinations. Also known as *the secretory pathway* Nucleus mRNA --> ER ER product to Golgi Golgi to Plasma membrane (for expulsion out of the cell) and lysosome (sites of digestion) Golgi provides 3 major pathways: Regulated secretory pathway

Each compartment along the biosynthetic or endocytic pathway has a characteristic protein composition. Can you describe these compartments and their proteins?

Resident proteins tend to be retained in that particular compartment and retrieved if they escape to other compartments. The sorting of proteins in the biosynthetic pathway *occurs in the TGN*. The TGN is the source of vesicles that contain particular membrane proteins that target the vesicle to a particular destination. Lysosomal enzymes produced in the rough ER are sorted in TGN and targeted for lysosomes in clathrin-coated vesicles. Lysosomal enzymes are enclosed in their budding vesicles because they possess phosphorylated mannose residues on their core oligosaccharides. -modified oligos recognized by *MPRs* --> bound to a class of adaptors or *GGAs* that form a layer between the outer clathrin coat and the vesicle membrane.

Contraction of skeletal muscle fibers. Can you describe this in detail?

Results from the *sliding of actin-containing thin filaments toward the center of the individual sarcomeres of a myofibril, and it is driven by forces generated in the myosin cross-bridges that extend outward from the thick filaments* Changes that occur during the shortening of a muscle fiber are reflected in changes in the banding pattern of the sarcomeres as the *Z lines at the ends of sarcomeres are mood toward the outer edges of A bands*. Contraction is triggered as an impulse penetrates into the interior of the muscle fiber along the membranous tubules, stimulating the release of Ca++ from storage sites in the sarcoplasmic reticulum. Binding of Ca++ to *troponin* molecules of the thin filaments causes a change in conformation that moves the tropomyosin molecules to a position that exposes the myosin-binding sites on the actin subunits of the thin filament. The subsequent interaction between myosin and actin triggers filament movement.

Fluorescent speckle microscopy

Small amounts of flourescently labeled proteins are injected -> filaments can no longer assemble -> speckles -> serve as fixed markers to follow changes in length or orientation of the filament

Microfilaments

Solid, thinner structures, often organized into a branching network and composed of the protein actin Flexible, inextensible, helical filament ATP-actin monomers Incorporates at the + end (barbed) Myosin motor proteins Motility, contractility, intracellular transport All eukaryotes

Material transport by vesicles

Soluble and membrane bound proteins load into vesicles. The vesicles bud off and are transported via motor proteins. Special receptors on the vesicle and target membrane allow for directed merging Allows for asymmetrical membranes

Gap junctions and plasmodesmata

Specialized sites of communication between adjoining cells in animals and plants, respectively Plasma membranes of a gap junction contain channels formed by a hexagonal array of connexin subunits forming a connexion. These channels connect the cytoplasm of one cell with the cytoplasm of the adjoining cell. The central channel of a connexon allows the direct diffusion of substances up to about 1000 Da. Passage of ionic current through gap junctions plays a role in numerous physio processes like spread of excitation through cardiac muscle tissue of the heart.

Axonal transport

Structures and materials traveling from the cell body toward the terminals of a neuron ---> *anterograde* From the synapse toward the cell body --> *retrograde* ((diseases)) Axons filled with MF, IF, and MT.

The ER is a system of tubules, cisternae, and vesicles that divides the cytoplasm into a lumenal space with the ER and a cytosolic space outside of the membranes. What differentiates the lumen and the cytosolic spaces? How does rough ER (RER) compare to smooth ER (SER) in function and structure?

The *ER* comprises a network of membranes that penetrates much of the cytoplasm. -Enclosed within the ER is an extensive space called the *lumen*. Composition of the *luminal* space is quite different from the *cytosolic space*. -The ER is a highly dynamic structure undergoing continual turnover and reorganization. -RER: ribosomes bound to cytosolic surface. Has *flattened* sacs called *cisternae*. Continuous with the outer membrane of the nuclear envelope --> fragment into rough-surfaced vesicles. Contains proteins involved in the movement of nascent proteins. *Cells that secrete large amounts of proteins* like pancreas has more RER. The RER... -Starting point of the biosynthetic pathway (site of synthesis of proteins, carbs and phospholipids) -*Primary starting point of the secretory pathway* -Soluble -Site of protein generation (1/3) -SER: lacking of ribosomes. Membranes of SER are highly curved and *tubular* --> fragment into smooth-surfaced vesicles. Has membrane bending proteins called *reticulons*. The SER... -Synthesizes steroid hormones in endrocine cells of gonad and adrenal cortex. -Detoxifies organic compounds in liver. *cytochrome p450s* (hydrophobic compounds --> hydrophilic compounds). -Sequestering calcium ions within the cytoplasm of cells. -Cardiac, skeletal, sarcoplasmic reticulum (Ca++ signaling is important here)

Cilia and flagella contain a core structure. What is the name of this structure, what is its structure? How does it work?

The *axoneme* is the name of the structure. It is composed of an array of MT that support the organelle as it projects from the cell surface and provides the machinery for generating the forces required for locomotion. (*9+2 arrangement*) -- signature Axoneme consists of nine outer doublet MT surrounding a pair of single MT A pair of arms projects from the A microtubule of each doublet. The arms consist of ciliary *dynein*, a motor protein that uses the energy released by ATP hydrolysis to generate the forces required fro ciliary or flagellar bending. This is accomplished as the dyne arms of one doublet attach to MT B of the neighboring doublet and then undergo a conformational change that causes the A MT to slide. Sliding on one side of the axoneme alternates with sliding on the other part so that it can bend in one direction and then in opposite.

Vesicles that bud from a donor compartment have specific proteins that are recognized by the acceptor compartment. What are these proteins, how does this work?

The docking of two membranes is mediated by tethering proteins and regulated by Rabs. Fusion of donor and acceptor membranes is mediated by v-SNAREs and t-SNAREs, which interact to form a four-stranded bundle.

Proteins to be synthesized on membrane-bound ribosomes of the RER are recognized by a hydrophobic signal sequence, which is usually situated near the N-terminus of the nascent polypeptide. Can you explain how protein insertion into the ER works? What about other signaling aspects to the lysosome or the mitochondria? How do those signals differ and how are they the same? What happens if the lumen is full?

The site of synthesis of a protein was determined by the sequence of amino acids in the N-terminal portion of the polypeptide. (1) Secretory proteins contain a *signal sequence* at their N-terminus that directs the emerging polypeptide and ribosome to the ER membrane. (2) Polypeptide moves into the cisternal space of ER through a protein-lined channel in the ER membrane. Polypeptide moves through *cotranslationally*. Polypeptides synthesized on membrane bound ribosomes contain a signal sequence that targets the *nascent* (one about to be synthesized) polypeptide to the ER membrane and leads to the compartmentalization of the polypeptide within the ER lumen. As it emerges from the ribosome, the hydrophobic signal sequence is recognized by a *signal recognition particle* (SRP). SRP binds to signal sequence on nascent polypeptide and the ribosome. Bound SRP serves as a tag and binds to cytosolic surface of ER. Binding to the ER can happen between SRP and *SRP receptor* and the other between the ribosome and *translocon*. Once SRP-ribosome nascent chain complex binds to the ER membrane, SRP is released from its ER receptor, ribosomes attach to translocon and signal sequence on the nascent polypeptide is inserted into the narrow channel of the translocon. (1) Signal sequence binds to SRP (stops further translation) (2) SRP ribosome complex collides with and binds to an SRP receptor situated with the ER membrane (3) Release of SRP and association of ribosome with a translocon of the ER membrane (4) Signal peptide binds to the interior of the translocon, displacing the plug from the channel and allowing the remainder of the polypeptide to translocate through the membrane cotranslationally (5) Signal peptide is cleaved by a membrane In the ER, nascent polypeptide is acted on by several enzymes. N-terminal portion containing the signal peptide is removed from most nascent polypeptides by *signal peptidase*.

Intermediate filaments

Tough, roselike fibers composed of a variety of proteins Tough, flexible, extensible filament. 70 proteins as tetramers Incorporate internally No motor proteins Structural support, mechanical strength Found in animals (not all eukaryotes)

Microtubules are hollow, tubule structures 25 nm in diameter. What makes these and how are they used?

Tubulin -Hollow, rigid, tubular structures and they occur in nearly every eukaryotic cell. -Includes mitotic spindle and core of cilia and flagella -Outer diameter of 25 nm and thickness of 4 nm -Wall of microtubule is composed of globular proteins in a longitudinal row called *protofilaments*. Noncovalent interactions between adjacent protofilaments are thought to play an important role in maintaining microtubule structure. -Microtubules stiff enough to resist forces that might bend fiber --> mechanical support -In plant cells -> MT play an indirect role in maintaining cell shape -MT play a key role in maintaining the internal organization of cells. *Treatment of cells with microtubule-disrupting drugs can seriously affect the location of membranous organelles* Large, stiff, hollow tubes made of an GTP--tubulin heterodimer Incorporate at the + end which is -tubulin Polarity Kinesins and Dyneins Support, intracellular transport, and cell organization All eukaryotes -*MAPS*: increase the stability of microtubules and promote their assembly. Controlled by the addition and removal of phosphate groups. -*Tau*: abnormally high phosphorylation of this MAP --> Alzheimer's --> strange tangled filaments called neurofibrillary tangles -> unable to bind to microtubules --> death of nerve cells --> FTDP-17

Targeting the vesicle

Vesicle leaves Golgi 00> carried by a motor protein This track allows vesicles to pass near targets and particular membrane integral proteins (v-SNARE) will recognize the target's membranes (t-SNAREs)

Translocon

a protein-lined channel embedded in the ER membrane through which the nascent polypeptide is able to move in its passage from the ribosome to the ER lumen.

MT destruction

form by GTP hydrolysis stress of tubule causes system to collapse. An unclosed cone that then induces to hydrolyzes its GTP --> closes tubule --> causes tubule to depolymerize --> *catastrophe*

Motor proteins

kinesins tetramers of two indeticial heavy chains and two identical light chains move towards the plasma membrane PLUS END of MT speed is dependent on ATP concentration of the cell low levels of ATP --> slower drosophila: moves towards minus end

Microtubules

long, hollow unbraced tubes composed of subunits of the protein tubulin *alpha-beta tubulin* polarity Support, intracellular transport, and cell organization Hollow rigid structure Many functions including mitotic spindle Each longitudinal row is a protofilament Staggered slightly Made of units Forms a helical structure Two isoforms that interact with the microtubule subunits. 3 subunits or 4 subunits Connect the microtubule to other cellular features. E.g. Cross bridge to other microtubules Promote stability of the tubule. Binding activity is controlled by phosphorylation. High levels of phosphorylation of the MAP Tau are implicated in Alzheimer's Disease.

SNAREs

membranes of vesicle and target component come into close contact because of this protein v-Snares: incorporated into membranes of transport vesicles t-SNAREs: located n the membranes of target compartments.

Endomembrane system Remember this

*Endoplasmic reticulum* + *Golgi complex* + endosomes + *lysosomes* + vacuoles Comes from specialization; it is a *eukaryotic trait* -Specialized locations for biochemical events and *unique environments*

Transport vesicles

-Materials are shuttled back and forth from one part of the cell to another i.e. Golgi -> plasma membrane This is accomplished by *transport vesicles*. These move through the cytoplasm in a directed manner with the help of motor proteins.

The extracellular space extends outward from the outer surface of the plasma membrane and contains a variety of secreted materials that influence cell behavior. What are basement membranes? Compare and contrast basement membrane with cellular membrane. What is extracellular matrix?

Epithelial tissues rest on a basement membrane, which consists of a thin, interwoven network of extracellular materials. Various types of connective tissue, including tendons, cartilage, and the corneal storm, contain an expansive ECM that gives the tissue its characteristic properties.

Microtubule associated protein

High levels of phosphorylation of MAP tau are implicated in *Alzheimers* Promote stability of MT Binding activity is controlled by phosphorylation

MTOCs

MT formation initiates slowly due to the need for a nucleation event MTOCs act as a pre nucleation site for MT elongation The best studied is the centrosome

Endocytic pathway

Materials move from the outer surface of the cell to compartments located within the cytoplasm. Opposite of secretory pathways (constitutive Cellular uptake Balanced by secretory pathway Material needs of the cell MPR move lysosomal enzymes to early endosomes H-ATPase pumps lower the pH of the nascent lysosomes. -pH change releases cargo from the MPR -MPR recycles back to tGN -Endocytic vesicles merge with the lysosome. -pH change release cargo allowing budding of a recycling vesicle to return to the plasma membrane

Membrane synthesis

Plasma membrane proteins are synthesized at the RER The lumen of the RER and Golgi becomes the exterior surface of the cell Carb markers are always on the luminal face. -Allows for a recognition of the sides of a membrane. -Keeps protein properly oriented

Secretory pathway

Refer to biosynthetic pathway.

cisternal maturation model need to look up evidence support model

Vesicles move back to the ER as the system matures much more dynamic system

Endosomes

distribution patterns along endocytic pathway

Dynein

move towards negative end of MT negative ends lead to the centrosome --> Golgi

Proton motive force

proton gradient has two components--*voltage and pH gradient*

Rabs

small g protein cycling between active GTP bound state and inactive GDP bound state.

The cytoskeleton functions as...

(1) a dynamic scaffold providing structural support (2) an internal framework responsible for positioning the various organelles (3) A network of tracks that direct the movement of materials and organelles within the cells (4) Moves cells from one place to another (5) Essential component of the cell's division machinery.

What are basement membranes? Compare and contrast basement membrane with cellular membrane. What is extracellular matrix?

*Basement membrane*: Basal lamina. Is a continuous sheet 50 to 200 nm thick that surrounds nerve fibers, muscles, and fat cells, and (2) underlies the basal surface of epithelial tissues, such as the epidermis of the skin, or the lining of digestive, and (3) underlies the inner endothelial lining of blood vessels. These provide *mechanical support for attached cells*, generate signals that promote cell survival, serve as substratum for cell migration, separate adjacent tissues within an organ, and act as a barrier to the passage of macromolecules. Basement membrane in capillaries: prevents passage of proteins out of blood and into tissues --> important in kidney. *Kidney failure in long-term diabetes* may result from abnormal thickening of the basement membrane. *Basement membranes also serve as a barrier to invasion of tissues by cancer cells*

Most if not all of the vesicles that transport material through the endomembrane system are encased initially in a protein coat. Several distinct types of coated vesicles have been identified. What are the proteins, what are their purposes? How does this even happen?

*COPII-coated vesicles*: move materials from the ER forward to the ERGIC and Golgi complex. -Includes small g protein *Sar1* --> plays regulatory role in vesicle formation and assembly of vesicle coat. Moves materials anterograde (forward) and combines with VTCs that can merge with the CGN. *COPI-coated vesicles*: move materials in a retrograde (backward) direction from ERGIC and Golgi backward toward ER and from trans golgi cisternae backward to cis. ER resident proteins get caught up in coated vesicles, hence its movement back. *Clathrin-coated vesicles*: move materials from the TGN to endosomes, lysosomes, and plant vacuoles. Also move materials from plasma membrane to cytoplasmic compartments along endocytic pathway. Move materials from endosomes to lysosomes Protein coats can act as a mechanical device that causes the membrane to curve and form a budding vesicle and they provide a mechanism for selecting components to be carried by the vesicle. -COPII vesicles reach the Golgi carrying ER proteins -ER proteins have a special amino acid sequence (KDEL) -Recognized by KDEL receptors for COPI vesicle formation and return to the ER.

The major components of extracellular matrices. What are they? How do they connect to cells? Can you describe and discuss collagen and proteoglycans?

*Collagen*: Fibrous glycoproteins; present only in ECM. High tensile strength. Most abundant protein in the human body. Produced by fibroblasts. *All collagen molecules are trimers consisting of three polypeptide chains (alpha) and along at least part of their length, the three polypeptide chains of a collagen molecule are wound around each other to form a unique, rod-like triple helix* -Alpha chains = proline; maintains stability of triple helix by forming H bonds between component chains. Failure of this --> Scurvy

The addition of sugars (what is the term for this?) to the asp (what amino acid is this) residues of proteins begins in the RER and continues in the Golgi. What enzymes do this? Can you describe this process (addition of sugars) and how they are modified and what purpose this serves?

*Glycosylation* *Glycosyltransferases* transfer a monosaccharide from a nucleotide sugar to the growing end of a carbohydrate chain. (1 and 2) Five mannose and two N-acetylglucosamine are transferred one at a time to the *dolichol-PP on the cytosol side of the ER membrane. *Dolichol phosphate* is a lipid carrier embedded in ER. (3) Dolichol with oligosaccharide flipped across membrane and the remaining sugars are attached on the luminal side of membrane (4) Latter sugars are attached one at a time on the cytosolic side of the membrane to the end of a dolichol phosphate, which then flips across the membrane and donates its sugar to the growing end of the oligosaccharide chain. (5) Once oligosaccharide i completely (*3 glucose molecules on the mature carbohydrate chain in the RER*), transferred to an *asparagine* residue of the nascent polypeptide. (6) Dolichol flips and is ready to begin accepting sugars. Modification begins in the ER with the enzymatic removal of two of the three glucose residues. *Quality control*: whether good or not to move n to the next compartment of pathway (1) 3 glucose chain will be acted on by Glucosidase I and II so that only one glucose will remain (2) Each glycoprotein (with one single glucose) binds to an ER chaperone like calnexin. Allowing the protein time to fold. (3) Removal of remaining glucose by glucosidase II leads to release of glycoprotein from the chaperone. GSII removes the glucose, freeing the protein. (4) UGGT corrects folding (*exposed hydrophobic residues --> not correctly folded*), adds a single glucose residue back to one of the mannose residues at the exposed end (5) Once glucose is added, tagged glycoprotein is recognized by same ER chaperones, which gives the protein another chance to fold properly. (6) If still misfolded, repeats (or moved to vesicles for transport--> Golgi) or is destroyed (7)Once one or more of these mannose residues have been removed --> sentenced to degradation by proteasome.

Three families of motor proteins have been identified and characterized. What are these families and what do they do? How do they work?

*Kinesins*: move along microtubules *Dyneins*: move along microtubules *Myosins*: move along microfilaments Both kinesin and cytoplasmic dyneins are motor proteins with globular heads that interact with microtubules and act as force-generating engines and an opposite end that binds to specific types of cargo to be transported. Kinesin moves materials towards the plus end of a microtubule, cytoplasmic dynein towards the minus end. Kinesin has been implicated in the movement of ER-derived vesicles, endosomes, lysosomes, and secretory granules and is the primary motor protein mediating anterograde transport in an axon.

The nucleation of microtubules in vivo occurs in association with a variety of MTOCs. What is an MTOC? How do centrioles and the centrosome play into this?

*MTOC*: MT organizing center. This is where nucleation of MT takes place rapidly. Microtubule formation initiates slowly. Due to the need for a nucleation event. MTOCs act as "pre-nucleation" sites for microtubule elongation. The best studied is the centrosome -Centrosome: MT of cytoskeleton are nucleated here. Contains two barrel-shaped *centrioles* surrounded by *pericentriolar material*. MT do not actually penetrate into the centrosome and make contact with the centrioles, but *terminate in dense PCM near centrosome*. -Centrioles play a role in recruiting the surrounding PCM during the assembly of the centrosome. -MTOCs *control number of MT, their polarity, the number of protofilaments that make up their walls, and the time and location of their assembly*. -Consists of *gamma tubulin*: critical in MT nucleation. -PCM serves as attachment sites for ring-shaped structures and contain gamma-tubulin.

The ETC contains five different types of carriers. What are the chemical carriers, what are the complexes?

*heme-containg cytochromes*: accepting and donating e- only *flavin nucleotide-containing flavoproteins*: can accept and donate H atoms *iron-sulfur proteins*: accepting and donating e- only *copper atoms*: accepting and donating e- only *quinones*: can accept and donate H atoms -Carriers of the ETC are arranged in order of increasingly positive redox potential. Various carriers are organized into four large, multi protein complexes. Cytochrome c and ubiquinone are mobile carriers, shuttling electrons between the larger complexes. As pairs of e- are passed through complexes I, III, and IV, protons are translocated from the matrix across the membrane into the inter membrane space. The translocation of protons by these e--transporting complexes establishes the proton gradient in which energy is stored. The last f the complexes is cytochrome oxidase, which transfers e- from cytochrome c to O2, reducing it to form water, a step that also removes protons from the matrix, contributing to the proton gradient. `

The forces responsible for microfilament-dependent processes may be generated by the assembly of the actin filament or, more often, as the result of interaction with motor proteins. Which motor protein? How does this tie to muscle contraction?

*myosin* Divided into two classes: conventional (type II) myosin and unconventional (types I and III-etc) myosins. Myosin II is the molecular motor that generates *force in various types of muscle tissue and also a variety of non muscle activities like cytokinesis.* Myosin II molecule contain a long, rod-shaped tail attached at one end to two globular heads. Heads bind the actin filament, hydrolyze ATP, and undergo the conformational changes required for force generation. Neck is thought to act as a lever arm that amplifies conformational changes of the head Fibrous tail mediates the assembly of myosin into bipolar filaments

Key products for each turn of the TCA cycle

-1 GTP -CO2 (2/3) NADH (3/4) FADH2 (1)

Mitochondria are large organelles Can you fully describe the structure and features of the mitochondria?

-Comprised of an outer porous membrane and a highly impermeable inner membrane that consists of *folds (cristae) that contain much of the machinery required for aerobic respiration.* -Membranes of mitochondrion divide organelle into two aqueous compartments --> interior (*matrix*) and an *intermembrane space*. Matrix contains high [] of water soluble proteins. -Outer membrane: Permeable to most small molecules and ions Contains Porin, a protein which is essentially a large pore -Inner membrane: Essentially impermeable Any movement between intermembrane space and matrix must be mediated by a transporter protein -Porisity of outer membrane results from integral proteins called *porins* -Fluidity of bilayer facilitate interactions of components that are required during e- transport and ATP formation -Inner membrane surrounds a gel-like matrix that contains, in addition to proteins, a genetic system that includes DNA, RNA, ribosomes, and all of the machinery necessary to transcribe and translate genetic info -Properties similar to bacteria due to evolution

The mitochondria is the center for oxidative metabolism. What biomolecules can be used in oxidative metabolism? What are the concepts of oxidative metabolism? What are the waste products? What are electron carriers?

-Converts products from carbs, fat, and protein catabolism into chemical energy stored as ATP. -Pyruvate and NADH are the two products of glycolysis. Pyruvate is transported across the inner mitochondrial membrane where it is decarboxylated and combined with coenzyme A to form acetyl coA, which is condensed with OAA to form citrate --> begins TCA. As it transverses the reactions of the TCA cycle, two of the carbons from the citrate are removed and released as CO2, which represents the most highly oxidized state of the carbon atom. *Electrons removed from substrates are transferred to FAD and NAD+ to form FADH2 and NADH*. -Fatty acids are broken down into acetyl coA, which is fed into the TCA cycle, and all 20 AA are broken down into either pyruvate, acetyl coA, or TCA cycle intermediates. *NADH and FADH2 are the carriers of free energy (∆G) and the PRODUCTS of the TCA cycle*

Kinesins

-Kinesins: Kinesin-1 --> *KRPs* 14 families. -Each kinesin-1 molecule is a tetramer constructed from two identical heavy chains and two identical light chains -Pair of globular heads that bind a MT and act as ATP-hydrolyzing engines. -All have related amino acid sequences. But tails of KRPs have diverse sequences. -*Much smaller than myosin* -*Plus end MT motor* (toward synaptic terminals) -Single kinesin moves along single protofilament of MT at a velocity *proportional to ATP []* Moves in distinct steps. Two heads alternate in taking the leading and lagging positions without an accompanying rotation of the stalk and cargo at every step. -Movement is highly *processive* (motor protein tends to move along individual MT for long distances) --> *long distance transport* -Both heads behave in coordinated manner, so that they are always present at different stages in their chemical cycles at a time. -Only minus movement: *Drosophila* -Kinesin-13 incapable of movement -Kinesins tend to move vesicles and organelles in an *outward direction towards the plasma membrane*

The cytoskeleton is composed of three distinct types of fibrous structures. What are they, and how are they composed?

-Microtubules: TUBULIN -Microfilaments: ACTIN -Intermediate filaments: VARIETY

Nonmuscle motility and contractility. Compare and contrast nonmuscle with muscle motility and contraction.

-Nonmuscle motility is arranged in less ordered, more labile, and transient configurations -Nonmuscle motility depends on *actin* with myosin. The organization and behavior of actin filaments are determined by a variety of actin-binding proteins that affect the assembly of the actin filaments, their physical properties, and their interactions.

Protofilament

-One alpha tubulin and one beta-tubulin -asymmetric -Plus end: terminated by B-tubulin subunits -Minus end: Terminated by a row of alpha-tubulin subunits. -polar

Endocytosis facilitates the uptake of fluid and suspended macromolecules, the internalization of membrane receptors and their bound ligands, and functions in the recycling of membrane between cell surface and cytoplasm. Phagocytosis is the uptake of particulate matter. Can you describe receptor-mediate endocytosis? What about pinocytosis and phagocytosis? How does this system work? What are the steps?

-Receptor mediated endocytosis: specific ligands are bound to plasma membrane receptors. Receptors collect in pits of membrane that are coated on their cytoplasmic surface by a polygonal scaffold of clathrin molecules. Coated pits give rise to coated vesicles -> lose their coat -> deliver contents to an endosome --> lysosome. -Dynamin is a large GTP binding protein that is required for the release of a clathrin coated vesicle from the membrane on which it forms. It forms a ring that pinches the plasma membrane. Hydrolysis of GTP allows dynamin to contract and pinch off -Phagocytosis: Specialized receptors pick up cellular cargo (bacteria, dead cells, etc) Form vesicle which then merges with a lysosome (phagosome). Digestion occurs and residual body undergoes exocytosis. mostly a protective mechanism -Pinocytosis: In cellular biology, pinocytosis, otherwise known as cell drinking, fluid endocytosis, and bulk-phase pinocytosis, is a mode of endocytosis in which small particles are brought into the cell, forming an invagination, and then suspended within small vesicles. These pinocytotic vesicles subsequently fuse with lysosomes to hydrolyze (break down) the particles. This process requires energy in the form of adenosine triphosphate (ATP), the chemical compound mostly used as energy in the majority of animal cells. Autophagy: -Organelle turnover -Protection from infection 1) Phagophore surrounds organelle 2) Golgi transport vesicle merges with lysosome or matures to lysosome 3) Phagophore merges with lysosome 4) Digestio n takes place 5) Materials are exocytosed 6) Result is a lipofuscin pigment granule (correlated with age of individual)

Dyneins

-Responsible for movement of cilia and flagella -Composed of two identical heavy chains and a variety of intermediate and light chains. -Large globular head with an elongated stalk. Head acts as force-generating engine. -Stalk - binding site for MT -Moves towards polymers *minus end* -Absent in higher plants -Positions the centrosome and Golgi and moving organelles, vesicles, and particles through the *cytoplasm* -Positions spindle and moving chromosomes during mitosis -*Dyactin* regulates dyne activity and binds motor protein to MT --> *increases processivity*

The F1F0 ATP Synthase Describe, in detail, the structure and function of the ATP Synthase.

ATP synthase contains two distinct parts: *F1 head that projects into the matrix and includes catalytic sites, and an Fo base that is embedded in the lipid bilayer and forms a channel through which protons are conducted from the inter membrane space into the matrix.* -Controlled movement of protons through the Fo portion of the enzyme induces the rotation of the gamma subunit of the enzyme, which forms the stalk connecting the Fo and F1 portions of the enzyme. -Rotation of th gamma subunit is brought by the movement of protons through half channels in the alpha subunit. -Rotation of gamma subunit induces changes in the conformation of the F1 catalytic sites --> makes ATP -In addition to forming ATP, proton-motive force also provides the energy required for a number of transport activities like uptake of ADP into the mitochondrion in exchange for the release of ATP to the cytosol, uptake of phosphate and calcium ions, and the import of mitochondrial proteins.

Adherens junctions and desmosomes

Adherens junctions encircle a cell near its apical surface, allowing contact between the cell and all of its neighbors. Plasma membranes of adherents junctions contain clusters of cadherins that are linked by intermediary proteins to the actin filaments of the cytoskeleton. Desmosomes occur as patches between cells and are characterized by dense cytoplasmic plaques on the inner surfaces of the membranes. Sites of concentration of cadherins, which are connected by intermediary proteins with IF that loop through cytoplasmic plaques

Proteins within peroxisomes, mitochondria, or chloroplasts that are encoded by nuclear genes must be important into the organelle post-translationally. What are the signals and steps of this process?

All of these organelles contain protein-lined channels in their membranes that promote the translocation of either folded polypeptides (in peroxisomes) or unfolded polypeptides (mitochondria and chloroplasts) into the organelle. Mitochondria and chloroplasts contain several different compartments into which newly translocated proteins are directed. -Proteins destined for a peroxisome use *peroxisomal targeting signal*. PTS receptors bind to peroxisome-destined proteins in the cytosol and shuttle to peroxisome. -Proteins destined for mitochondria can be delivered to four areas OMM, IMM, inter membrane space and matrix. Protein must be in unfolded state. Chaperones like Hsp help. OMM uses TOM complex. IMM uses TIM complex. Once the protein enters the matrix, it is bound by a mitochondrial chaperone which may either pull the polypeptide into the matrix or ensure that it diffuses into the matrix. Once inside matrix, assumes its native conformation. -Proteins contain mitochondrial localization signal -Protein binds to specific receptor protein via the MLS and is moved to region where inner and outer membranes are in close proximity -Protein is translocated through membranes in the UNFOLDED state Protein is refolded with the help of chaperone proteins MLS is cleaved

The microtubules of the cytoskeleton are dynamic polymers that are subject to shortening, lengthening, disassembly, and reassembly. Can you describe and discuss assembly and disassembly and the factors involved.

Alpha beta tubulin dimers add to the negative end Disassembly of MT cytoskeleton can be induced by a number of agents like *colchicine, cold temperature, and elevated Ca++ []*. The MT of the cytoskeleton are normally disassembled prior to cell division, and the tubulin subunits are reassembled as part of the mitotic spindle. At any given moment, some MT of cytoskeleton are growing in length while others are shrinking. When individual MT are followed over time, they are seen to switch back and forth between growing and shrinking phases -> *dynamic instability* Both growth and shrinking occur predominantly at the *plus end of the polymer*--the end opposite the MTOC. Tubular dimers that are polymerized into a MT containing a GTP molecule are hydrolyzed soon after its incorporation into the polymer. During periods of rapid polymerization, the hydrolysis of GTP bound to incorporated tubulin dimers lag behind the incorporation of new dimers, so that the end of MT contains a cap of tubulin-GTP dimers that favors the addition of subunits and growth of MT. Assembly and disassembly may also be governed by +TIP binding proteins and specific MAPs.

Cell attachment to substratum. How does this happen? What are they structures? Can you describe them structurally?

By means of cell-surface specializations like *focal adhesions and hemidesmosomes* Focal adhesions -- sites of attachment where the plasma membrane contains clusters of integrins linked to actin-containing microfilaments of cytoskeleton. Hemidesmosomes --- sites of attachment where the plasma membrane contains clusters of integrins that are connected to the basement membrane on their outer surface and indirectly to keratin-containing intermediate filaments on their inner surface. Both types of adhesions are sites of potential cell signaling.

The major components of extracellular matrices. What are they? How do they connect to cells? Can you describe and discuss collagen and proteoglycans?

Collagens Proteoglycans Fibronectin and laminin Each of the proteins of the ECM contains binding sites for one another and for receptors on the cell surface. As a result, these various extracellular materials interact to form an interconnected network that is bound to the cell surface. Collagens are highly abundant and fibrous, give ECM the capability to resist pulling forces Proteoglycans consist of protein and glycosaminoglycans and serve as an amorphous packing material that fills the extracellular space.

What are some examples of nonmuscle motility and contractility.

Crawling of cells and axonal outgrowth -Crawling of cells: accomplished by *lamellipodium*. As it extends from the cell, it adheres to the underlying substratum at specific points, providing temporary sites of anchorage for the cell to crawl over. The protrusion of this is associated with nucleation and polymerization of actin filaments. This is from actin polymerization. The tip of an elongating axon consists of a growth cone. Acts both to explore the environment and to elongate the axon.

The cytoplasm of eukaryotic cells contains a system of membranous organelles, including the ER, Golgi complex, and lysosomes Can you describe the overall structure of the membrane system, all the different parts, and what they do?

ER: Huge, highly folded, membrane network. (ER may take up bulk of cell) Fills most of the eukaryotic cell. Activities occur in *luminal (cisternae) space* as well as on the cytosolic space.