Quiz 2 Flashcards

Question

What causes the different Cc rates on the two ends?

Answer 1

During hydrolysis of ATP and the subsequent release of Pi from subunits in a filament, actin undergoes a conformational change that is responsible for the different dissociation and association rates at the two ends. When ATP-G-actin binds to the (+) end, ATP is hydrolyzed to ADP and Pi. The Pi is slowly released from the subunits so that the filament is asymmetric: the front has ATP, the middle is ADP + Pi, and the end (minus end) is ADP. Practically speaking, the (-) end is always in ADP form. F-actin (ADP) depolymerizes more readily than F-actin (ATP)

Answer 2

Treadmilling occurs when the G-actin concentration is between the critical concentrations (Cc) of the plus and minus ends:

Answer 3

Plus end: Continues to grow as monomers add. Minus end: Continues to shrink as monomers dissociate. The filament maintains a constant length because the rate of addition at the plus end equals the rate of loss at the minus end.

Answer 4

Yes, ATP is consumed. Actin monomers bind ATP before polymerizing. After incorporation, ATP-actin hydrolyzes to ADP-actin, releasing inorganic phosphate (Pi). ADP-actin is less stable, leading to depolymerization at the minus end. This ATP hydrolysis cycle fuels treadmilling and maintains actin filament dynamics.

Answer 5

If no additional actin monomers are supplied and ATP hydrolysis continues: Eventually, all filaments depolymerize, leaving only G-actin monomers in solution. Most G-actin will be in the ADP-bound form, since ATP hydrolysis occurs during polymerization.

Answer 6

- Binds at the interface between subunits in F-actin, locking adjacent subunits together and preventing actin filaments from depolymerizing (stabilizes F-actin) - Popular reagent for staining of F-actin *Because many processes rely on actin filament turnover, the introduction of phalloidin paralyzes the cells and makes them die

Answer 7

- Bind to the (+) end and blocks polymerization, leading to depolymerization

Answer 8

- Binds to and sequesters G-actin, inhibiting it from adding to the filament end. *When cytochalasin or latrunculin are added to live cells, the actin cytoskeleton disassembles and certain cell movements like locomotion and cytokinesis are inhibited.

Answer 9

- Binds G-actin on the side opposite the nucleotide-binding cleft. When profilin binds ADP-actin, it opens the cleft and greatly enhances the loss of ADP, which is replaced by the more abundant cellular ATP, yielding a profilin–ATP-actin complex. This complex cannot bind to the (−) end because profilin blocks the sites on G-actin for (−) end assembly. However, the profilin–ATP-actin complex can bind efficiently to the (+) end, and profilin dissociates after the new actin subunit is bound. Profilin provides a supply of ATP-actin from released ADP-actin; as a consequence, essentially all the free G-actin in a cell has bound ATP

Answer 10

binds specifically to F-actin in which the subunits contain ADP. Cofilin binding destabilizes the filament, breaking it into short pieces. By breaking the filament in this way, cofilin generates many more free (−) ends and therefore greatly enhances the net disassembly of the filament

Answer 11

a small protein that binds to ATP–G-actin in such a way that it inhibits addition of the actin subunit to either end of the filament. Free actin and free thymosin-β4 are in a dynamic equilibrium with actin–thymosin-β4. If some of the free actin is used up for polymerization, more actin–thymosin-β4 will dissociate, providing more free actin for polymerization. Thus thymosin-β4 behaves as a buffer of unpolymerized actin, making it available when it is needed.

Answer 12

Faster in vivo because of profilin and cofilin (cofilin increases turnover)

Answer 13

binds with a very high affinity (~0.1 nM) to the (+) end of an actin filament, thereby inhibiting subunit addition or loss. Certain regulatory proteins are able to bind the (+) end and protect it from CapZ while still allowing assembly there *redo if you forget the regulatory protein part

Answer 14

binds to the (−) end of an actin filament, also inhibiting its assembly and disassembly. This protein is found predominantly in cells in which actin filaments need to be highly stabilized. Two examples are the short actin filaments in the cortex of the red blood cell and the actin filaments in muscle (later lectures). In both cases, tropomodulin works with another protein, tropomyosin, which lies along the filament, to stabilize it. Tropomodulin binds to both tropomyosin and actin at the (−) end to greatly stabilize the filament.

Answer 15

Gelsolin can sever actin filaments and cap the (+) ends. Its activity is regulated by Ca2+ ion concentrations. On binding Ca2+, gelsolin undergoes a conformational change that allows it to bind to the side of an actin filament and then insert itself between subunits of the helix, thereby breaking the filament. It then remains bound to and caps the (+) end, generating a new (−) end that can disassemble.

Answer 16

Cross-linking of actin filaments turns a solution of F-actin into a gel. If gelsolin is added to such a gel and the level of Ca2+ is elevated, gelsolin will sever the actin filaments and turn the gel back into a liquid solution. This ability to turn a gel into a sol is why the protein was named gelsolin. *therefore, its important for regulating consistency

Answer 17

Formin proteins nucleate the assembly of the long unbranched actin filaments. Two FH2 domains from 2 individual Formin monomers associate to form a doughnut shaped complex This complex has the ability to nucleate actin assembly by binding two actin subunits, holding them so that the (+) end of the nascent filament is toward the FH2 domains. The filament can grow at the (+) end while the FH2 domain remains attached to it.

Answer 18

- To nucleate the assembly of branched actin, Arp2/3 needs to be activated by a nucleation promoting factor (NPF), in addition to associating with the side of a preexisting actin filament. -Two NPFs each bind an actin subunit at their WH2 domains, and together, they activate the Arp2/3 complex through its interaction with their connector and acidic domains. - The actin subunits brought in by the WH2 domains of the NPFs binds to the Arp2/3 template to nucleate filament assembly at the (+) end. The NPFs are released, and the new (+) end then grows as long as ATP–G-actin is available or until it is capped by a (+) end capping protein such as CapZ.

Answer 19

70 degrees

Answer 20

- To move from one host cell to another, Listeria moves around the cell by polymerizing actin in to a comet tail, and when it runs into the plasma membrane, it pushes its way into the adjacent cell to infect it

Answer 21

The bacterium avoids being exposed to the extracellular medium and minimizes the possibility of being recognized and attacked by the host immune system

Answer 22

Listeria has on its surface a protein called ActA, which mimics NPF by having an actin-binding site and an acidic region that efficiently activates the Arp2/3 complex. When Listeria activates Arp2/3 through its ActA protein, actin filaments begin polymerizing at its rear. These filaments are physically connected to the surrounding cytoskeleton, which means they are essentially locked in place within the dense actin network of the host cell. Since the growing ends of actin filaments (the barbed ends) push against the bacterium, but their other ends remain embedded in the host cytoskeleton, the only direction Listeria can move is forward—it can't slide backward because the actin network behind it is rigid and resists movement

Answer 23

Just four: - ATP-G-Actin for growing the actin filament - Arp 2/3 complex to act as the start site for the actin filament - CapZ to ensure the stability of the actin filaments - Cofilin to increase turnover from only the end so that there's free G-actin to keep adding

Answer 24

- The process that cells use to take up particles, molecules, or fluid from the external medium by enclosing them in plasma membrane and then internalizing them --> The uptake of molecules or liquid is called receptor mediated endocytosis or fluid phase endocytosis, and the uptake of large particles is phagocytosis

Answer 25

- Endocytosis assembly factors recruit NPFs so that as the endocytic vesicles invaginate and pinch off from the membrane, they are driven into the cytoplasm, powered by a rapid and very short-lived burst of actin polymerization driven by the Arp 2/3 complex

Answer 26

- Upon the signals that occur when a cell is opsonizing an antibody w pathogen, the signal also tells the cell to assemble microfilaments at the site of interaction and the assembled microfilaments, together with myosin motor proteins, provide the force necessary to draw the bacterium into the cell, ultimately fully enclosing the pathogen in the plasma membrane *It then fuses with the lysosome and is killed via lysosomal enzymes.

Answer 27

Cytosolic proteins can organize the actin filaments in many ways: - (Bundling): parallel cross-linking of the filaments - (Gel): Crosslinking to make a gel structure - Proteins can be used to anchor the actin filaments to the membrane

Answer 28

To be able to organize actin filaments, an actin cross-linking protein must have 2 F-actin-binding sites - This can exist if a single polypeptide has two F- actin binding sites (ex. Fimbrin) - Can also exist if a polypeptide has one F-actin binding site but is a dimer so it will make two F-actin binding sites upon dimerization (alpha-actinin)

Answer 29

A tetramer with two actin-binding sites; spectrin spans an even greater distance between actin filaments and makes networks under the plasma membrane

Answer 30

Spectrin forms a tough meshwork of actin underneath the plasma membrane. It is linked to short actin filaments and to two different integral membrane proteins (Band 4.1 and Ankyrin).This extra stability and strength is important for cells like RBCs, that undergo a lot of stress - They are associated with short and highly crosslinked F actin filaments that are bound to the membrane

Answer 31

Ezrin links actin filaments laterally to the plasma membrane in surface structures such as microvilli; attachment of Ezrin can be direct or indirect

Answer 32

Ezrin, activated by phosphorylation can be bound... - Directly to the cytoplasmic region of transmembrane bound proteins - Indirectly to the EBP50 protein scaffold

Answer 33

Complex with 6 polypeptide subunits - Two of the subunits are identical heavy chains - Each heavy chain consist of a globular head domain and a long tail domain, connected by a flexible neck domain - The tails of the two heavy chains intertwine so that the head regions are in close proximity - The remaining 4 subunits of the myosin complex are the light chains - There are two types of light chains: the essential light chain and regulatory light chain. One chain of each associates with the neck region of each heavy chain

Answer 34

- Use Chymotrypsin to generate HMM and LMM. HMM consist of the globular heads, light chains, and a tiny portion of the tail while the LMM is just the tail - Use Papain to further digest the HMM into subfragment 1 (S1) and subfragment (2)

Answer 35

The intrinsic ATPase activity of the myosin resides in this fragment and its F-actin binding site (on the globular heads). Contains the head and the neck domains, associated with the light chains

Answer 36

ATPase activity is greatly enhanced by the presence of filamentous actin (meaning: myosin heads don't efficiently break down ATP until they are bound to actin)

Answer 37

- All myosin head domains convert ATP hydrolysis into mechanical work using the same general mechanisms - All are motor proteins that walk - All have related S1 domains with considerable similarity in their primary amino acid sequence

Answer 38

- Most myosins walk towards the plus end of the actin filament, powered by ATP to move something - Most myosin are directly or indirectly affected by Ca2+

Answer 39

Head domains with specific properties have co-evolved with specific classes of tail domains, suggesting that each class of myosin has evolved to carry out a specific function

Answer 40

Myosin II molecules assemble into a bipolar filament with opposite orientations in each half of the filament, so that there is a cluster of head domains at each end of the filament. This organization is important for its involvement in contraction. Myosin II is the only class involved in contractive function

Answer 41

The myosin I class has a variable number of light chains associated with the neck region, and the only one in which the two heavy chains are not associated through their tail domains and so are single-headed. Some members of this family connect actin filaments to membranes, and others are implicated in endocytosis.

Answer 42

Members of the myosin V class have two heavy chains, resulting in a motor with two heads, long neck regions with six light chains each, and tail regions that dimerize and terminate in domains that bind to specific organelles to be transported.

Answer 43

*Myosin head binds actin in filament. *ATP binding to myosin head causes its release from the filament (1). *ATP hydrolysis causes a conformational change, “cocking” the head domain (2). *Myosin head binds to the filament displaced from the original binding site (3). *Release of the phosphate causes straightening = “power stroke” (4). This step is triggered by binding of F-actin. *ADP release allows new ATP to bind (5). *The “step size” of the different types of myosin varies. It depends on the length of the neck domain. *All myosins convert the energy released by ATP hydrolysis into mechanical work, yet different myosins can perform very different types of functions.

Answer 44

In the sliding-filament assay, myosin molecules are tethered to a coverslip to which is added stabilized, fluorescently labeled actin filaments. Because the myosin molecules are tethered, they cannot slide; thus any force generated by the interaction of myosin heads with actin filaments forces the filaments to move relative to the myosin. If ATP is present, the added actin filaments can be seen to glide along the surface of the coverslip; if ATP is absent, no filament movement is observed. The rate at which myosin moves an actin filament can be determined from video recordings of sliding-filament assays.

Answer 45

Remember to visualize the diagram so you know that the actin is always pushing the filament towards the minus end while the myosin itself tries to go towards the plus end. * just take your time to understand this and visualize the diagram

Answer 46

In the optical trap approach, myosin is immobilized on beads at a low density. An actin filament, held between two optical traps, is lowered toward the bead until it contacts a myosin molecule on the bead. When ATP is added, the myosin pulls on the actin filament. Using a mechanical feedback mechanism controlled by a computer, one can measure the distance pulled and the forces and duration of the movement

Answer 47

The results of optical trap studies show that myosin II takes discrete steps, which average out to about 8 nm, and generates 3–5 piconewtons (pN) of force, approximately the same force as that exerted by gravity on a single bacterium. Myosin II does not interact with the actin filament continuously, but rather binds, moves, and releases it.

Answer 48

Myosin II spends on average only about 10 percent of each ATPase cycle in contact with F-actin—it is said to have a duty ratio of 10 percent. In contracting muscle, hundreds of myosin heads pull on actin filaments, so that at any one time, 10 percent of the heads are engaged to provide a smooth contraction

Answer 49

When measured by optical traps, the overall myosin V motor moves at a step size of 36 nm without releasing from the actin—it is said to move processively. To determine if the motor moves according to the hand-over-hand model or the inchworm model, scientists have managed to attach a fluorescent probe to just one of the two neck regions of a myosin V molecule and watch the fluorescent image as the molecule moves along an actin filament. The labeled head takes many 72-nm steps, showing that the hand-over-hand model is correct. This step size is twice the 36-nm length between helical repeats in the actin filament. So each 36-nm helical repeat site on the filament presents binding sites as each head alternately takes 72-nm steps.

Answer 50

Myosin V has presumably evolved to have a long neck domain—the lever arm—to take large steps to match the size of the helical repeat of the filament. Moreover, its ATPase cycle has been modified to have a much higher duty ratio (>70 percent) by slowing the rate of ADP release; thus the head remains in contact with the actin filament for a much larger percentage of the cycle. Since a single myosin V molecule has two heads, a duty ratio greater than 50 percent ensures that it maintains contact at all times as it moves down an actin filament, so that it does not fall off. These are exactly the properties one would expect for a motor designed to transport cargo along an actin filament.

Answer 51

a processive motor, meaning it "walks" along actin filaments without detaching completely.

Answer 52

The duty ratio is the fraction of the time that a motor protein spends attached to actin during its ATPase cycle.

Answer 53

A high duty ratio (above 50%) means that at least one of myosin V’s heads is always bound to actin—this is important because myosin V functions as a processive motor, meaning it "walks" along actin filaments without detaching completely.

Answer 54

A sarcomere, which is about 2 μm long in resting muscle, shortens by about 70 percent of its length during contraction. Each sarcomere contains two major types of filaments: thick filaments, composed of myosin II, and thin filaments, containing actin and associated proteins.

Answer 55

The thick filaments are myosin II bipolar filaments, in which the heads on each half of the filament have opposite orientations. The thin actin filaments are assembled with their (+) ends embedded in a densely staining structure known as the Z disk, so that the two sets of actin filaments in a sarcomere have opposite orientations.

Answer 56

During the cyclical interactions, also called the cross-bridge cycle, the hydrolysis of ATP is coupled to the movement of a myosin head toward the Z disk, which corresponds to the (+) end of the actin thin filament. Because the thick filament is bipolar, the action of the myosin heads at opposite ends of the thick filament draws the thin filaments toward the center of the thick filament and therefore toward the center of the sarcomere. This movement shortens the sarcomere until the ends of the thick filaments abut the Z disk

Answer 57

- ATP and Ca2+

Answer 58

Skeletal muscle contraction is initiated by an increase in the cytosolic Ca2+ concentration. In skeletal muscle cells, a low cytosolic Ca2+ level is maintained primarily by a unique Ca2+ ATPase that continually pumps Ca2+ ions from the cytosol containing the myofibrils into the sarcoplasmic reticulum (SR), a specialized endoplasmic reticulum of muscle cells

Answer 59

The arrival of a nerve impulse at a neuromuscular junction triggers an action potential in the muscle-cell plasma membrane and stimulates the opening of voltage-gated Ca2+ channels in the SR membrane - ensuring the release of Ca2+ from the SR raises the cytosolic Ca2+ concentration in the myofibrils

Answer 60

In this context, myofibrils refer to the long, thread-like structures within muscle cells (also called muscle fibers). These myofibrils are composed of repeating units called sarcomeres, which are the basic contractile units of muscle tissue. The myofibrils are responsible for muscle contraction and are made up primarily of two proteins, actin and myosin, which interact to produce force and movement.

Answer 61

The elevated Ca2+ concentration induces changes in two accessory proteins, tropomyosin and troponin, which are bound to the actin thin filaments and normally block myosin binding. Changes in the positions of these proteins on the actin thin filaments in turn permit the myosin-actin interactions and hence contraction. This type of regulation is very rapid and is known as thin-filament regulation

Answer 62

(TM) a ropelike molecule that binds to seven actin subunits in an actin filament. Associated with each tropomyosin molecule is a calcium-binding protein complex troponin (TN)

Answer 63

Troponin controls the position of TM on the surface of an actin filament.

Answer 64

Smooth muscle surrounds blood vessels to regulate blood pressure, surrounds the intestine to move food through the gut, and restricts airway passages in the lung. Smooth muscle cells contain large, loosely aligned contractile bundles. Smooth muscle contraction is regulated by the cycling of myosin II between on and off states. This myosin II cycling is regulated in response to many extracellular signaling molecules

Answer 65

Myosin II is regulated primarily by a pathway in which the myosin regulatory light chain (LC) undergoes phosphorylation and dephosphorylation. When the regulatory LC is not phosphorylated, the smooth muscle myosin II adopts a folded conformation, and its ATPase cycle is inactive. When the regulatory LC is phosphorylated by the enzyme myosin light- chain kinase (MLC kinase), whose activity is regulated by the level of cytosolic free Ca2+, the myosin II unfolds, assembles into active bipolar filaments, and becomes active to induce contraction. The Ca2+-dependent regulation of MLC kinase activity is mediated through the Ca2+-binding protein calmodulin. Calcium first binds to calmodulin, which induces a conformational change in the protein, and the Ca2+/calmodulin complex then binds to MLC kinase and activates it. When the Ca2+ returns to its resting level, MLC kinase becomes inactive, and myosin light-chain (MLC) phosphatase removes the phosphates to allow the system to return to its relaxed state.

Answer 66

The reason Ca²⁺ has to travel a longer distance in smooth muscle is due to the differences in structure between smooth and skeletal muscle cells. In skeletal muscle, the sarcomeres are highly organized, and the release of Ca²⁺ from the sarcoplasmic reticulum (SR) is well-localized within the muscle fibers, allowing for rapid and direct activation of the contractile machinery. The Ca²⁺ released from the SR quickly interacts with the thin filaments (actin), triggering muscle contraction. In smooth muscle, however, the arrangement is less organized. The Ca²⁺ is released more diffusely into the cytoplasm, and smooth muscle cells do not have the highly structured, repeating sarcomeres that skeletal muscle does. Because of this less organized structure, the Ca²⁺ needs to diffuse over longer distances within the cell to reach the areas where it can activate the contractile proteins. This slower, less efficient diffusion process contributes to the slower contraction speed of smooth muscle compared to skeletal muscle. Additionally, the regulation of smooth muscle contraction involves protein kinases, which add another layer of complexity and slower regulation, compared to the more direct interaction between Ca²⁺ and the thin filaments in skeletal muscle.

Answer 67

Contractile ring is a contractile bundle in animal cells that assembles at the equator of a dividing cell, encircling the cell midway between the poles of the mitotic spindle (later lecture). As the ring contracts, pulling the plasma membrane in, the cytoplasm is divided and eventually pinched into two parts in a process known as cytokinesis, giving rise to two daughter cells. Dividing cells stained with antibodies against myosin I and myosin II show that myosin II is localized to the contractile ring, whereas myosin I is at the distal regions, where it links the cell cortex to the plasma membrane

Answer 68

The Ras superfamily is composed of protein families with related functions, and all play key roles in signaling. Examples: Ran (nuclear transport, later lecture); the Rab family (vesicle trafficking, later lectures), and Rac, Rho, and Cdc42 (cytoskeleton remodeling) *Ras itself is the product of an oncogene (later lectures) and the founding member of a large group of ~100 proteins that act as bimolecular switches.

Answer 69

Principle: an ‘on’ GTP state and an ‘off’ GDP state, with GTP hydrolysis and GDP/GTP exchange mediated by several other proteins.

Answer 70

Certain mutations in these small GTPase proteins can turn them “on” constitutively (“dominant active”). Such mutations helped unravel the function of the proteins (e.g. by microinjection). Other mutations turn the switch “off” or “down” (“dominant negative”) GTPase refers to the Ras family protein, not GEF or GAPs *ex. for dominant active, induce a mutation where Rho can bind to the GTP molecule but cannot unbind (irreversibly) * ex. for dominant negative, induce a mutation where Rho binsd to the GDP molecule but cannot unbind (irreversibly)

Answer 71

- GEF= guanine nucleotide exchange factor. => turns switch “on”. In the cell [GTP] >> [GDP] - GAP= GTPase activating protein. => turns switch “off”. Ras GTPase can hydrolyze GTP, but this enzyme activity is regulated (stimulated by GAP) - GDI = guanine nucleotide dissociation inhibitor, preventing exchange of GDP for GTP and therefore keeping Ras GTPases off

Answer 72

Cdc42, Rac, and Rho were implicated in the regulation of microfilament organization because introduction of dominant-active mutant proteins had dramatic effects on the actin cytoskeleton, even in the absence of growth factors. Dominant-active Cdc42 results in the appearance of filopodia, dominant-active Rac results in the appearance of membrane ruffles, and dominant-active Rho results in the formation of stress fibers, which then contract.

Answer 73

- Like all small GTPases of the Ras superfamily, Cdc42, Rac, and Rho act as molecular switches, inactive in the GDP-bound state and active in the GTP-bound state. In their GDP-bound state, they exist free in the cytoplasm in an inactive form bound to a protein known as guanine nucleotide dissociation inhibitor (GDI) (this is because we don't want the Rho to acquire an ATP on its own, we want to keep it sequestered until we get a signal from the body that it's needed). - Growth factors can bind and activate their receptors to turn on specific membrane-bound regulatory proteins, called guanine nucleotide exchange factors (GEFs), which activate Rho proteins at the membrane by releasing them from GDI and catalyzing the exchange of GDP for GTP. - The GTP-bound active Rho protein associates with the plasma membrane, where it binds effector proteins to transmit the biological response. The small GTPase remains active until the GTP is hydrolyzed to GDP, a process that is stimulated by specific GTPase-activating proteins (GAPs).

Answer 74

- Many formins exist in a folded, inactive conformation as a result of an interaction between the first half of the protein and its C-terminal tail. These formins are activated by membrane-bound Rho-GTP. - When Rho is switched from its inactive Rho-GDP form to its activated membrane-bound Rho-GTP state, it can bind and activate these formins (RBD domain). Interaction with Rho at plasma membrane “opens” formin, allowing it to nucleate actin filaments. Such “closed” (INTRA-molecular interaction) and “open” (INTER-molecular interaction) conformational switches are very common in cell biology (Ezrin is another example). - The formin FH1 domain adjacent to the FH2 domain behaves as a landing site for profilin–ATP–G-actin to increase the local concentration of these complexes. The actin from these profilin-actin complexes is then fed into the FH2 domain to add actin to the (+) end of the filament, and the profilin is released. - Since the formin allows rapid addition of actin subunits to the (+) end, long filaments with a formin at their (+) end are generated. To ensure the continued growth of the filament, formins bind to the (+) end in such a way that stops binding of a (+) end capping protein such as CapZ, which would normally terminate assembly.

Answer 75

- WASp is a NPF. It exists in a folded inactive conformation that makes the WCA domain unavailable. Its activation requires two signals. - One signal is the presence of the regulatory phospholipid PI(4,5)P2, which is characteristically enriched in the plasma membrane. WASp binds PI(4,5)P2 through its basic domain. The second signal is binding of the activated form of the small GTP-binding protein Cdc42. This type of two-signal input, called coincidence detection, ensures that the protein is activated only at the right place—at the plasma membrane—and by the right signaling pathway. Once bound to the two input signals, WASp is opened, and the WCA domain becomes accessible

Answer 76

- Classes of microfilaments involved in cell migration - Focal adhesions - Arp2/3 complexes nucleate the dynamic actin network in the leading edge.

Answer 77

* The network of actin filaments in the leading edge advances the cell forward. * Contractile fibers in the cell cortex squeeze the cell body forward. * Stress fibers terminating in focal adhesions pull the bulk of the cell body up as the rear adhesions are released

Answer 78

* Structure – stress fiber actin filament ends attach through integrins attached to the underlying extracellular matrix * Signaling – contain many signaling molecules important for regulating cell locomotion

Answer 79

Arp2/3 complexes nucleate the dynamic actin network in the leading edge. Arp2/3 complexes nucleate the dynamic actin network in the leading edge.

Answer 80

- Growth factors, such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), stimulate cells to move and then to divide by activating Rho GTPases. - Activation of Cdc42 stimulates actin assembly by Arp2/3 through activation of WASp, a nucleation promoting factor (NPF), resulting in the formation of filopodia. - Activation of Rac also induces Arp2/3, mediated by the WAVE complex, leading to the assembly of branched actin filaments in the leading edge. - Activation of Rho has at least two effects. First, it can activate a formin for unbranched actin filament assembly. Second, through activation of Rho kinase, it can phosphorylate the myosin light chain to activate nonmuscle myosin II and can also inhibit light-chain dephosphorylation by phosphorylating myosin light-chain phosphatase to inhibit its activity. Both actions of Rho kinase lead to a higher level of myosin light-chain phosphorylation and therefore higher myosin activity and contraction. The three Rho proteins, Cdc42, Rac, and Rho, are also linked by activation and inhibition pathways

Answer 81

- Lamellipodia - Filopodia - Stress fibers

Answer 82

- Signals from the environment are transmitted to Cdc42, which orients the cell (To orient the cell means to establish front-rear polarity, determining which direction the cell will migrate. This involves organizing the cytoskeleton so that the cell has a defined leading edge (front) and a trailing edge (rear) in response to external signals (such as chemical gradients or substrate cues)) -. Cdc42 activation promotes Rac activity in the leading edge (front), which activates Arp2/3 dependent actin polymerization. - Rac activates Rho at the back of the cell (rear), which induces the assembly of formin-dependent contractile structures and activates the myosin-II-based contractile machinery. Active Rho inhibits Rac activation, ensuring no leading-edge structures form at the rear of the cell

Answer 83

* Step 1: An Arp2/3-dependent mechanism extends one or more lamellipodia at the cell leading edge. * Step 2: Lamellipodia adhere to the substratum by formation of focal adhesions in which integrin mediates a connection between the actin cytoskeleton and extracellular matrix proteins such as fibronectin and collagen. * Step 3: Actin-myosin II-dependent contraction at the rear of the cell propels th bulk of the cytoplasm forward. * Step 4: Deadhesion and endocytic recycling at the back of the cell: * The trailing edge of the cell remains attached to the substratum until the tail eventually detaches and contractile force retracts into the cell body. * The endocytic cycle internalizes membrane and integrins at the rear of the cell and transports them to the front of the cell (arrows) for reuse in making new adhesions. * Cells fail to move if they are either too strongly or too weakly attached to a surface

Answer 84

Under certain conditions, extracellular chemical cues guide the locomotion of a cell in a particular direction. For example, cells can sense soluble molecules and follows them, along a concentration gradient, to their source—a process known as chemotaxis. - Chemotactic molecules all work through a common mechanism: binding to cell- surface receptors, activation of intracellular signaling pathways, and remodeling of the cytoskeleton through the activation or inhibition of various actin-binding proteins. - Dictyostelium cells migrate toward the source of extracellular cAMP (left), which stimulates aggregation and migratory slug formation in nature. Human neutrophils migrate toward a pipette releasing fMLP (formylated Met-Leu-Phe), a chemotactic peptide produced by bacteria (right). The internal signal transduction pathways used in chemotaxis have been conserved between Dictyostelium amoebae and human leukocytes despite almost a billion years of evolution.

Answer 85

1. Building Blocks 2. Filament structure and organization (polarity) 3. Assembly and disassembly 4. Binding proteins (side and end binding) 5. Drugs 6. Motor proteins 7. Cellular structures, cellular processes

Answer 86

- Tubulin consists of two closely related subunits called α- and β-tubulin. The α- and β-subunits of the tubulin dimer can each bind one molecule of GTP. The GTP in the α-tubulin subunit is never hydrolyzed and is trapped by the interface between the α- and β-subunits. By contrast, the GTP-binding site on the β-subunit is at the surface of the dimer. GTP bound by the β-subunit can be hydrolyzed, and the resulting GDP can be exchanged for free GTP. The tubulin heterodimer is very stable (essentially all depolymerized tubulin is dimeric), but tubulin monomers are very unstable.

Answer 87

A microtubule is composed of 13 laterally associated protofilaments, which form a tubule whose external diameter is about 25 nm. Each of the 13 protofilaments is a string of αβ-tubulin dimers, longitudinally arranged so that the subunits alternate down a protofilament, with each subunit type repeating every 8 nm. Because the αβ- tubulin dimers in a protofilament are all oriented in the same way, the protofilaments have an intrinsic polarity. In a microtubule, all the laterally associated protofilaments have the same polarity, so the microtubule also has an overall polarity. The end with exposed β-subunits is the (+) end, while the end with exposed α-subunits is the (−) end

Answer 88

Add a fragment of a microtubule bundle, as a nucleus for in vitro αβ-tubulin assembly, to a pool of unpolymerized tubulin. Tubulin assembles preferentially on one end ([+] end) compared to assembly at the other end ([–] end).

Answer 89

People have learned some lessons learned from early in vitro studies. First, for assembly to occur, the αβ-tubulin concentration must be above the critical concentration (Cc), just as we saw for actin polymerization. Second, at αβ-tubulin concentrations higher than Cc, dimers are added faster to one end of the microtubule than to the other. As with F-actin assembly, the preferred end for assembly, which is the end with β-tubulin exposed, is designated the (+) end. The (−) end has α-tubulin exposed. Although a tremendous amount of research effort was devoted to characterizing the bulk polymerization properties of microtubular proteins in solution, its general relevance was superseded by subsequent studies examining the properties of individual microtubules (*look at the diagram for this please, it's the same idea which is that the two ends have different critical concentrations that allows for the GTP ab tubulin dimers to preferentially add to the Beta end.

Answer 90

Early experiments revealed that most microtubules in animal cells disassemble when the cells are cooled to 4 °C and reassemble when the cells are rewarmed to 37 °C. Researchers took advantage of this intrinsic property of microtubules to purify their components (use cool/worm cycles).

Answer 91

Individual microtubules can grow and then suddenly experience a catastrophe: an abrupt transition to a shrinking phase during which the microtubule would undergo rapid depolymerization. Moreover, sometimes a depolymerizing microtubule end could go through a rescue and begin growing again. This alternation between growing and shrinking states is known as dynamic instability. Thus the dynamic life of a microtubule end is determined by the rate of growth, the frequency of catastrophes, the rate of depolymerization, and the frequency of rescues. These features of microtubule dynamics are controlled in vivo. Since the (−) ends of microtubules in animal cells are generally anchored to an MTOC, this dynamism is most relevant to the (+) end of the microtubule. Assembly and disassembly each proceed at uniform rates, but disassembly is much more rapid (7 μm/min) than assembly (1 μm/min).

Answer 92

- the rate of growth - the frequency of catastrophes - the rate of depolymerization - the frequency of rescues

Answer 93

Assembly and disassembly each proceed at uniform rates, but disassembly is much more rapid (7 μm/min) than assembly (1 μm/min)

Answer 94

ince the (−) ends of microtubules in animal cells are generally anchored to an MTOC, this dynamism is most relevant to the (+) end of the microtubule.

Answer 95

Fluorescently labeled tubulin was microinjected into cultured human fibroblasts. The cells were chilled to depolymerize preexisting microtubules into tubulin dimers and were then incubated at 37 °C to allow repolymerization, which incorporated the fluorescent tubulin into all the cells’ microtubules

Answer 96

- GTP-Beta-tubulin - Protofilament is less curved - Sheet like *Using a GDP analog, researchers have found that artificially made single protofilaments—which are not exposed to lateral interactions—made up of repeating αβ-tubulin dimers containing GDP-β-tubulin are curved, like a ram’s horn. However, artificially made single protofilaments made up of αβ-tubulin dimers in which β-tubulin has a bound GTP analog are only slightly curved.

Answer 97

- GDP-Beta-tubulin - Protofilament is more curved - Ram's horn like

Answer 98

Therefore, if the GTP molecules in the terminal β-tubulins become hydrolyzed on a microtubule that has stopped growing, a formerly blunt-ended microtubule will curl and a catastrophe will ensue.

Answer 99

- The addition of a dimer to the (+) end of a protofilament on a growing microtubule enhances the hydrolysis of the GTP in the formerly terminal β-subunit to GDP and Pi. - The inorganic phosphate is then released to yield a microtubule with predominantly GDP-β-tubulin down its length. - However, the β-tubulin in the newly added dimer contains GTP. Thus each protofilament in a growing microtubule has mostly GDP-β- tubulin down its length and is capped by a few terminal dimers containing GTP-β-tubulin and GDP-Pi-β-tubulin. - The lateral protofilament-protofilament interactions in the GTP-β-tubulin cap are sufficiently strong that they do not allow the microtubule to unpeel at its end—and so the protofilaments behind the GTP-β-tubulin cap are constrained from unpeeling. - The energy released by GTP hydrolysis in the subunits behind the cap is stored within the lattice as structural strain waiting to be released when the GTP-β-tubulin cap is lost. If the GTP-β-tubulin cap is lost, the stored energy can do work. (The energy from GTP hydrolysis is "trapped" in the microtubule lattice as structural strain. As long as the GTP cap is there, the microtubule remains stable. If the cap disappears, the built-up energy is released, leading to rapid depolymerization.)

Answer 100

Using an antibody that recognizes only GTP-β-tubulin and not GDP-β-tubulin, researchers have found that “islands” of GTP-β-tubulin can persist along the length of an assembled microtubule. It seems likely that when a disassembling microtubule encounters one of these GTP-β-tubulin islands, disassembly pauses, and a rescue may be provoked

Answer 101

Colchicine is present in extracts of the meadow saffron and binds tubulin dimers so that they cannot polymerize into microtubules. Since most microtubules are in a dynamic state between dimers and polymers, the addition of colchicine sequesters all free dimers in the cytoplasm, resulting in loss of microtubules due to their natural turnover.

Answer 102

a synthetic drug, also binds the tubulin dimer and restrains it from forming polymers.

Answer 103

binds microtubules and stabilizes them against depolymerization. Because taxol stops cells from dividing by inhibiting mitosis, it has been used to treat some cancers, such as those of the breast and ovary, where the cells are especially sensitive to the drug

Answer 104

In the cell, microtubules are assembled from specific sites to generate many different configurations. - The nucleation phase of microtubule assembly is such an energetically unfavorable reaction that spontaneous nucleation does not play a significant role in microtubule assembly in vivo. - Rather, all microtubules are nucleated from structures known as microtubule-organizing centers, or MTOCs. In most cases, the (−) end of the microtubule stays anchored in the MTOC while the (+) end extends away from it.

Answer 105

- The centrosome is the main MTOC in animal cells. During interphase, the centrosome is generally located near the nucleus, producing an array of microtubules with their (+) ends radiating toward the cell periphery. This radial array provides tracks for microtubule-based motor proteins to organize and transport membrane-bounded compartments, such as those constituting the secretory and endocytic pathways. - During mitosis, cells completely reorganize their microtubules to form a bipolar spindle extending from two centrosomes, also known as spindle poles, that can accurately segregate copies of the duplicated chromosomes. Neurons have long processes called axons, in which organelles are transported in both directions along microtubules (Figure 18-5e). - In cilia and flagella (Figure 18-5f), microtubules are assembled from an MTOC called a basal body.

Answer 106

- Centrosome - Spindle poles (which are centrosomes) - Basal body

Answer 107

In addition to α- and β-tubulin, all eukaryotes also have genes specifying a third tubulin, γ-tubulin, which is involved in microtubule assembly. - γ-tubulin is a relative of α and β. γ-tubulin ring complex (γ-TuRC) consists of many copies of γ-tubulin associated with several other proteins. It is believed that γ-TuRC acts like a split- washer template to bind αβ-tubulin dimers for the formation of a new microtubule, whose (−) end is associated with γ-TuRC and whose (+) end is free for further assembly. *γ-tubulin ring complex (γ-TuRC) serves as a template for microtubule nucleation—essentially acting as a starting point for new microtubule growth.

Answer 108

Most microtubules in a cell consist of a simple tube, a singlet microtubule (like cytoplasmic), built from 13 protofilaments. In addition to this 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 doublet or triplet contains one complete 13-protofilament microtubule (called the A tubule) and one or two additional tubules (B and C) consisting of 10 protofilaments each.

Answer 109

- Motor protein for microfilament is actin and the different types - The motor protein for microtubules are kinesin and dyneins

Answer 110

Each centrosome in an animal cell consists of a pair of orthogonally arranged cylindrical centrioles surrounded by apparently amorphous material called pericentriolar material (Figure 18-6a). The centrioles, which are about 0.5 μm long and 0.2 μm in diameter, are highly organized and stable structures that consist of nine sets of triplet microtubules (Figure 18-6b). They are closely related in structure to the basal bodies found at the bases of cilia and flagella. It is not the centrioles themselves that nucleate the cytoplasmic microtubule array, but rather other factors (including γ- TuRC) in the pericentriolar material. The centrosome consists of two centrioles surrounded by pericentriolar material (PCM). The PCM—not the centrioles—contains the necessary factors (like γ-TuRC) to nucleate microtubules. The (-) ends of microtubules are anchored in the PCM, while the (+) ends grow outward into the cytoplasm. *Not all eukaryotic cells have centrioles. The function of centrioles is not clear. *MTs emanate from the granular diffuse region around the centriole, ie. the minus end is embedded in this region

Answer 111

No, making it hard for us to understand the function of centrioles

Answer 112

- The stored energy comes from GTP hydrolysis in the microtubule lattice. - When the microtubule depolymerizes, this stored energy is released as mechanical force that helps pull chromosomes toward spindle poles in anaphase. *The energy stored in the microtubule lattice is converted into mechanical force through protofilament peeling during microtubule depolymerization

Answer 113

Is a dimer of two heavy chains, each associated with a light chain. The molecule comprises of a head domain: binds microtubules and ATP and is responsible for the motor activity of kinesin. It also has the linker which is critical for forward movement. Has the stalk which is involved in the dimerization of the coiled coil interaction of the two heavy chains; and the tail domain is responsible for binding to receptors on the membranes of cargoes

Answer 114

X-ray crystallography revealed that the catalytic core of kinesin has the same overall structure as myosin. This is the case despite the fact that there is no amino acid sequence conservation between the two proteins arguing strongly that convergent evolution has twice generated a fold that can use hydrolysis of ATP to generate work

Answer 115

Directionality: Most kinesins move toward the plus end of microtubules, helping transport cargo (e.g., organelles, vesicles) outward toward the cell periphery. Some (like kinesin-14) move toward the minus end, which is crucial in spindle organization during mitosis, pulling microtubules inward toward spindle poles. Processivity (How long they stay attached before detaching): High-processivity kinesins (like kinesin-1) are needed for long-distance transport of cargo along microtubules. Lower-processivity kinesins (like kinesin-13) specialize in microtubule depolymerization rather than transport. Specialized Mitotic Functions: Kinesin-5 crosslinks and pushes apart overlapping microtubules to help separate spindle poles. Kinesin-13 promotes microtubule disassembly at the spindle poles and kinetochores, helping chromosome movement during anaphase. Kinesin-14 pulls microtubules inward, counteracting outward forces in the spindle.

Answer 116

- Initial state: The leading head is bound to the microtubule with no nucleotide (after releasing ADP), while the trailing head has ADP and is detached. ATP binding: ATP binds to the leading head, triggering a conformational change in the linker region, which zippers against the motor domain. Power stroke: The linker movement swings the trailing head forward toward the next binding site on the microtubule. New head attachment: The now-forward head (previously trailing) docks onto the microtubule and releases ADP, securing its attachment. ATP hydrolysis in the original leading head: The trailing head (now the new leading head) hydrolyzes ATP to ADP + Pi, weakening its grip and preparing for detachment. Pi release and detachment: The trailing head (now with ADP) detaches, resetting for the next step.

Answer 117

Transports organelles in a retrograde direction toward the (-) ends of microtubules. The motor consist of two large, two intermediate, and two small subunits. - The first is the stem which binds to the other subunits and which associates with cargo through another protein complex, dynactin. - The next part of the heavy chain is a linker that plays a critical role during ATP-dependent motor activity

Answer 118

Dynactin is a protein complex that helps dynein move cargo along microtubules. It has a structure where one part is made up of actin and a related protein called Arp1, which forms a short filament. The (+) end of this filament is capped by a protein called CapZ, and other proteins are attached to the (-) end. Essentially, dynactin helps dynein connect to its cargo and controls its movement. The second domain of dynactin consists of a long protein called p150Glued. - Dynamitin: holding the two dynactin domains together is a protein called dynamitin (because when it is overexpressed it blows apart the domains)

Answer 119

- Transportation of organelles within a cell - Forming microtubule-based surface structures (like cilia and flagella) - Mitosis

Answer 120

The Golgi complex collegcts in the vicinity of the centrosome, where the (-) ends of microtubules lie, and is driven there by dynein-dnactin.

Answer 121

Secretory cargo emerging from the ER is transported to the Golgi through dynein-dynactin

Answer 122

Endoplasmic reticulum is spread throughout the cytoplasm and is transported there by kinesin 1, which moves towards the peripheral ends of microtubules

Answer 123

Dynein-dynactin

Answer 124

Dynein- dynactin

Answer 125

Toward the cell periphery

Answer 126

Towards the centrally-located MTOC or the cell center

Answer 127

Melanophores are cells of the vertebrate skin that contain hundreds of dark melanin-filled pigment granules called melanosomes. Melanophores either have their melanosomes dispersed, in which case they make the skin darker, or aggregated at the cell center, which makes the skin paler. These changes in skin color, mediated by neurotransmitters in the fish and regulated by hormones in the frog, serve to camouflage the fish and enhance social interactions in the frog. The movement of the melanosomes is mediated by changes in intracellular cAMP and is dependent on microtubules. Melanosome dispersion requires kinesin-2, whereas melanosome aggregation requires cytoplasmic dynein-dynactin.

Answer 128

Neurons need to transport proteins and membranes from the cell body to the axon terminal to replace those used in neurotransmitter release. This is done using microtubules, with kinesin-1 moving materials toward the axon terminal (anterograde), and dynein carrying them back (retrograde). Both motors can attach to the same organelle, and there are mechanisms that control when each motor is active, though they are not fully understood.

Answer 129

In the classical pulse-chase experiments, radioactive amino acids were microinjected into the dorsal-root ganglia near the spinal cord to allow for their incorporation into proteins in spinal neurons, and the radioactivity was then tracked along the axons of those cells showed that transport can also occur down the axon. Other experiments showed that the transport can occur in the reverse directions.

Quiz 2 Flashcards

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