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1. Three systems of cytoskeletal filaments exist in most eukaryotic cells. Compare them in terms of composition, function, and structure.
Microfilaments: composed of actin, polymers of actin organized in functional bundles and networks by actin binding proteins, give shape to surface structures and serve as tracts to motor proteins.
Microtubules: alpha beta tubulin dimers organized by microtubule-associated proteins. provide organization, also form the structure of the mitotic spindle, also serve as tracts to motor proteins and like myosins are powered by ATP hydrolysis.
Intermediate filaments: contains various different elements and are tissue specific filamenotous structures that serve as structural integritiy to cells and tissues and are NOT used as tracts by motor proteins.
2. Actin filaments have a defined polarity. What is filament polarity? How is it generated at the subunit level?
Filament polarity is the binding of the subunits of an actin filament. It is visualized by the binding of the myosin subfragment (S1) to the filament, which creates barbed and pointed ends
3. The predominant forms of actin inside a cell are ATP-G-actin and ADP-F-actin. Explain how the interconversion of the nucleotide state is coupled to the assembly and disassembly of actin subunits.
ATP attaches to the G actin in the nucleation phase of actin filament polymerization forming the base for the F- actin.
F-actin is a longer molecule which is undergoing elongation and dissassembly via treadmilling (The atp hydrolysis then allows for elongation to occur when energentically unfavorable)
4. What would be the consequence for actin filament assembly/disassembly if a mutation prevented actin's ability to bind ATP? What would be the consequence if a mutation prevented actin's ability to hydrolyze ATP?
If a mutation prevented actins ability to bind to ATP then the actin would not be able assemble or undergo treadmilling as ATP would not even be able to bind to the G -actin that elongates the actin molecule. Thus, nucleation phase would not be able to occur
If you prevented actins ability to hydrolyze ATP then the elongation could not occur as it uses the energy from hydrolyzing ATP to attach to the actin molecule
5. Actin filaments at the leading edge of a crawling cell are believed to undergo treadmilling. What is treadmilling, and what accounts for this assembly behavior?
Treadmilling is the process by which a G actin attaches to the + end of the F actin at a faster rate than the G actin dissociate from the - end, giving the appearance of it moving like a treadmill.
6. Review the roles of profilin, cofilin, and thymosin-β4 on actin filament polymerization and stability.
profilin-serves to catalyze exchange of ADP for ATP making ATP bound actin available ot assemble at the positive end
cofilin-breaks apart ADP actin filament regions which makes more negative ends and depolymerization can increase
thymosin- B4: acts as a buffer by sequestering ATP G-actin and releasing it when free actin concentration is low
7. Although purified actin can assemble reversibly in vitro, various actin-binding proteins regulate the assembly of actin filaments in the cell.
Predict the effect on a cell's actin cytoskeleton if function-blocking antibodies against profilin were microinjected into cells.
Assembly at the positive end would not be able to occur since profilin serves to make ATP bound actin available to assemble at the positive end
Predict the effect on a cell's actin cytoskeleton if function-blocking antibodies against thymosin-β4 were microinjected into cells.
The actin would elongate without control mechanisms in place leading to likely improper folding as this would occur much faster than disassembly and result in a destabilized actin molecule
8. Although purified actin can assemble reversibly in vitro, various actin-binding proteins regulate the assembly of actin filaments in the cell.
Predict the effect on a cell's actin cytoskeleton if function-blocking antibodies against CapZ were microinjected into cells.
This would result in subunit addition or loss at the end of each filament. If this was blocked then the filaments would either grow uncontrollably on their + end, or continue to shorten since capz functions to block assembly or dissassembly of filaments
Predict the effect on a cell's actin cytoskeleton if function-blocking antibodies against the Arp2/3 complex were microinjected into cells.
ARP 2/3 leads to branched actin networks which can help with intracellular motility. The effect on a cell's actin cytoskeleton if function-blocking antibodies against the ARP2/3 complex were microinjected into cells would be either a reduction in branches within the within the cytoskeleton or no motility. it would also mean that it could not form the reaction creating a 70 degree angle with another myosin structure
9. Compare and contrast the ways in which formin and WASp are activated and explain how each stimulates actin filament formation.
Formin and Wasp are activated in similar formations. Both undergo nucleation by formins and ARp2/3 complexesBoth fold on itself to inhibit actiity and have a Rho GTP binding domain to activate the pathway to form the element.
Formin is used to regulate FH2 activity that results in the formation of the long actin filaments. Membrane receptors are activated by Rho binds with GTP then binds to RBD -->then profilin ATP actin is bound to the FH1 by ARP2/3 complexes-->this recruits G actin to the which then added to the FH2 domain. and long actin filaments are synthesized
Wasp is activated by two things: regulated phospholipid and membrane bound G proteins. (Unlike formin WASp RBD domain is not activated first. Instead, the W domain binds and transfers G actin to activate the ARP2/3 complex-->ARP2/3 then recruits GTP to ARP2/3 and transfers its G complex -->A then activates ARP 2/3 to form new new binding domain on the actin filament.
10. There are at least 20 different types of myosin. What properties do all types share, and what makes them different? Why is myosin II the only myosin capable of producing contractile force?
Each type of myosin head, neck, and tail domain, all act as motor proteins, but each type of myosin has its own distinct function.
Each type of myosin is also composed of one or two heavy chains and several light chains. (myosin II is involved in powering muscle contraction and cytokinesis, but myosin I and V are involved in cytoskeleton-membrane interactions like the transport of membrane vesicles).
Myosin II is the only myosin capable of producing contractile forces because it moves the actin filament at a faster rate than the other myosin. Faster movement is associated with muscle contraction. And the variations in the rate of movement shed light on the specific functions of the different myosin.
11. Contractile bundles occur in nonmuscle cells; these structures are less organized than the sarcomeres of muscle cells. What is the purpose of nonmuscle contractile bundles? Which type of myosin is found in contractile bundles?
The purpose of nonmuscle contractile bundles function in cell adhesion rather than cell movement and help maintain shape. Meanwhile myosin II is scattered throughout the contractile bundles and with actin make up "core of membrane projections" such as microvilli. So, Myosin II is found in contractile bundles.
12. Describe how myosin converts the chemical energy released by ATP hydrolysis into mechanical work.
Myosin converts the chemical energy released by ATP hydrolysis into mechanical work by binding with actin before. Thus, when the myosin head is activated by ATP a conformational change occurs allowing it to walk to the +end it is already attached to actin
Myosin is the motor, actin filaments are the tracks along which myosin moves, and ATP is the fuel that powers movement." (Lodish)
13. Myosin V can take many large steps along actin filaments without disassociating. Why might this feature be useful for transporting its cargo (vesicles, organelles, mRNA, etc.)? Describe how myosin V functions.
Because Myosin V can take large steps along actin filaments without dissociating, it can move faster. This wold be very useful when transporting cargo as it makes cell function quicker and "deposits" cruicial cargo the cell needs to function.
14. Contraction of both skeletal and smooth muscle is triggered by an increase in cytosolic Ca2+. Compare the mechanisms by which each type of muscle converts a rise in Ca2+ concentration into contraction. In other words, what binds to Ca2+ in order to produce each type of muscle contraction?
In skeletal muscle, troponin, binds to calcium. he Ca2+ binds to troponin, an actin filament-bound protein that block interaction with myosin in the relaxed state. Troponin binding by Ca2+ induces a conformational change in troponin that allows myosin to bind to the actin filaments and cause contraction. (thin-filament regulation)
In smooth muscle, higher calcium levels are bound to calmodulin (CAM) to regulate contraction of smooth muscle.
First Ca2+ binds to calmodulin in the cytosol, which can then allow the formation of a complex with the myosin light-chain kinase, which is then able to phosphorylate the myosin II light-chain domain. When the light chain is not phosphorylated it keeps myosin II in a folded and inactive conformation, but upon phosphorylation the myosin II protein can unfold and assemble into a filament with other myosin II proteins, which can then induce contraction.
15. Phosphorylation of myosin light-chain kinase (MLC kinase) by protein kinase A (PKA) inhibits MLC kinase activation by Ca2+/calmodulin. Drugs such as albuterol bind to the β-adrenergic receptor, which causes a rise in cAMP in cells and activation of PKA.
Explain why albuterol is useful for treating the severe contraction of the smooth muscle cells surrounding airway passages involved in an asthma attack.
Albuterol is useful in treating severe contraction of smooth muscles because if it elevates PKA and CAMP, that will leave less calcium available to the airways and bronchioles, thus mlc phosphotace would become activated and myosin would fold so it can no longer interact with actin rendering it inactive. This inactivation would prevent contraction and stimulate relaxation.
16. In cells, actin filaments form bundles or networks. How do cells form such structures, and what specifically determines whether actin filaments will form a bundle or a network?
Actin filaments are able to form bundles through the function-specific cross-linking proteins binding one actin filament to another and as a result need two actin-binding sites.
They will form network if the Arp2/3 facilitates the branching of actin molecules at a characteristic 70-degree angle to the main filament. This Arp2/3 facilitated branching allows for network like structures that span the cytoplasm.
17. How do Listeria bacteria use actin polymerization to power intracellular movement?
Listeria use actin polymerization to power intracellular movement by "catching a ride" on these filaments that push host cell plasma membrane to form filipodium that sticks into another cell to infect bacteria
The Listeria bacterium directs the polymerization of the host cell's actin within the host cell's cytoplasm and localizes this polymerization to the rear region of the Listeria cell. This process allows the Listeria to infect the host cell by forcefully moving forward into it. During the polymerization of the actin filaments, the actin-ATP complex is converted to actin-ADP through hydrolysis so that actin grows at the (+) end and breaks apart at the (-) end of the filaments.
The Listeria bacterium contains membrane proteins called ActA which bind actin and activate the Arp2/3 complex so that nucleation of new filaments can form from preexisting filaments. ActA binds VASP
18. Briefly describe the key steps in cell locomotion.
1. extension:extended at leading edge by arp 2/3 mechanism
2. adhesion: adhesion to the host by integration mediated focal adhesions
3. transloaction: where actin/myosin II contract at the rear of the cell to propel the cell forward
4. de-adhesion and endocytic recycling: rear releases and is recycled to the front of the cell for release
19. Several types of cells use the actin cytoskeleton to power their locomotion across surfaces. How are different assemblies of actin filaments involved in locomotion?
different assemblies lead to
20. To move in a specific direction, a migrating cell must use extracellular cues to establish which portion of the cell will act as the front and which will act as the back. Describe how small GTPase proteins appear to be involved in the signaling pathways used by migrating cells to determine direction of movement.
GTPase's are typically active when GTP bound, being able to turn a pathway off by GTP hydrolysis into GDP and vice versa. In the case of cell movement, Rho proteins (Rho, Rac, and Cdc42) are normally inactively bound to GDP and GDI (guanine nucleotide dissociation inhibitor) within the cytoplasm. Extracellular growth factors start the cascade of cell movement by stimulating guanine nucleotide exchange factors (GEF) at the cell surface. GEF is able to take the GDP/GDI bound Rho proteins, facilitate the exchange of GDP for GTP, releasing the GDI segment, activating Rho. Once activated, Rho is able to associate with different effector proteins, signaling various actin structures to form. Based on the type of Rho protein (Rho, Rac, Cdc42, etc.), different effector proteins are associated, creating a wide range of different pathways to follow, resulting in different cytoskeletal structures that form the basis of the "front and back" sections of a cell during cellular movement (
