cell bio: cytoskeleton part 2

a) describe what severing proteins are and

b) the types of severing proteins for microtubules and actin

a) they are proteins that cut filaments into shorter pieces, creating new ends for polymerization/depolymerization

b) microtubules = katanin, actin = gelsolin

describe the graph of severing proteins in the following ways:

a) x-axis

b) y-axis

c) critical concentration

d) which line is unsevered population, what line is severed population

a) monomer concentration

b) 0 in middle of graph; below 0 = shrinking rate, above 0 - growth rate

c) concentration where unsevered protein rate = severed protein rate (where lines cross on graph, usually at 0)

d) unsevered population has a slower, gradual slope.

severed population has a more rapid, sharper slope, for they grow / shrink more rapidly

briefly explain the experiment that showed how fragile fibers (specifically fra2) mutants in Arabidopsis that defines a plant katanin

- Observation: Arabidopsis fra2 mutants displayed fragile, brittle fibers and abnormal cell wall mechanics, suggesting defects in cytoskeletal regulation.

- Analysis: Microscopy revealed that microtubules were misorganized in fra2 mutants, failing to form proper parallel arrays in cells.

- Conclusion: in fra2 mutants, the microtubules were not properly severed because the plant katanin (FRA2) was nonfunctional, leading to misorganized microtubule arrays and fragile fibers

-----------> shows that severing is essential for proper microtubule organization and plant fiber strength.

explain how it is known that gelsolin is an actin-severing protein

in vitro assays showed that adding gelsolin to actin filaments caused rapid filament shortening and generation of new filament ends, demonstrating its severing activity.

what are motor proteins?

they are specialized proteins that convert chemical energy from ATP / GTP hydrolysis into mechanical work, moving along cytoskeletal filaments to transport cargo, generate force, or drive filament sliding

list the complementary motor proteins for the following types of filaments:

a) actin

b) microtubules

c) head domain

d) tail domain

e) transport cargoes

a) myosins

b) kinesins, dyneins

c) filament binding motor domain

d) cargo-binding domain

e) vesicles, organelles, Golgi stacks

describe the structure of myosin II and what it does

- it has head domains at the N-terminus that bind actin and hydrolyze ATP, generating contractile force for movement along actin filaments.

- C-terminus contains a coiled-coil of two α-helices, forming the tail that allows dimerization and filament assembly.

list the structires seen between the myosin types below:

a) myosin I

b) myosin II

why is it that myosin II is used more than myosin I?

a) single head, short tail, monomeric

b) two heads, long coiled-coil tail, dimer

- as a dimer, myosin II can generate contractile force and bulk cytoskeletal rearrangements, making it more effective for cell movement

- monomeric myosin I mainly links membranes to filaments without producing large-scale contraction, due to the lack of two heads / long coiled tail

describe how myosin motor activity was studied in vitro

- Purified myosin heads were immobilized on a glass slide, creating a fixed surface of motor domains.

- Fluorescently labeled actin filaments were then added, allowing their orientation / movement to be easily visualized under the microscope.

- Addition of ATP triggered myosin-driven sliding of the actin filaments, directly demonstrating myosin motor activity and directional movement in vitro.

explain how actin crawls in the in vitro experiment mentioned on the previous notecard

- they "crawl" because the immobilized myosin heads repeatedly bind, power-stroke, and release along the actin, causing the filament to slide across the surface

- ATP hydrolysis fuels this cyclical movement, so each ATP-driven stroke pushes the actin filament a bit further, creating continuous gliding motion in one direction.

explain how the following types of microtubule motors work:

a) kinesins

b) dyneins

a) they move toward the microtubule + end by using ATP-driven conformational changes in their motor heads to "walk" hand-over-hand along the protofilaments.

----------> structurally similar to myosins

b) Dyneins move toward the microtubule − end, pulling their cargo toward the centrosome

explain how myosin motor activity works

- myosin binds to actin filaments and, upon ATP binding and hydrolysis, undergoes a conformational "power stroke" that pulls the actin filament to the myosin head

- cycle of ATP binding, hydrolysis, and product release resets the myosin head, allowing repeated attachment-pull-release steps that generate directed movement and force.

explain how kinesin motor activity works

- Kinesin uses ATP binding and hydrolysis to drive an alternating "hand-over-hand" stepping motion of its two heads along microtubules.

- Each ATP-powered step moves kinesin toward the microtubule's plus end, allowing it to transport cargo in a consistent, directional manner.

compare and contrast myosin and kinesin cycles: how do they utilize energy to transport cargo down filaments?

- Myosin binds tightly to actin in its ADP-bound state

--------> generates movement by bending the region between its head and lever arm, using ATP binding to release from actin and reset the cycle.

- Kinesin is detached from the microtubule in its ADP-bound state

--------> moves by rotating its two heads around flexible linker regions in a coordinated, hand-over-hand stepping motion powered by ATP hydrolysis.

explain the roles of the plus and minus end of directed kinesins

- N-terminal (plus-end-directed) kinesins move cargo toward the microtubule's + end, typically carrying vesicles and organelles outward toward the cell periphery.

- C-terminal (minus-end-directed) kinesins move cargo toward the microtubule's − end, generally transporting materials inward toward the cell center near the microtubule-organizing center (MTOC).

explain how organelles move on microtubules

- Organelles attach to motor proteins through adaptor complexes that link the vesicle membrane to the motor's tail, allowing the motor to physically grab the microtubule track.

- RESULT: motor "walks" along the microtubule, pulling the organelle in a specific direction (usually dynein to the minus end or kinesin to the plus end).

If you are shown a filament with labeled polarity (+ and - ends) and the direction an organelle is moving along it, how can you determine which motor protein is responsible for its transport?

- if organelle moves toward the + end of a microtubule → kinesin (because kinesins are almost always plus-end-directed microtubule motors)

- if organelle moves toward the - end of a microtubule → dynein (because dynein is the major minus-end-directed microtubule motor)

- if organelle moves toward the + end of an actin filament → most myosins, including myosin II (myosins are mostly plus-end-directed actin motors)

If you had cloned a new kinesin form an organism and did not know its directionality, how would you try and find out? can you think of an experiment to test if your prediction is correct?

- Attach your unknown kinesin to a glass slide, add polarity-marked microtubules (with the + end labeled differently), and watch which end of the microtubule moves forward

- if the + end leads, the motor walks toward the - end; if the - end leads, the motor walks toward the + end, revealing motor directionality.

- Repeat with fluorescently labeled kinesin on immobilized polarity-marked microtubules to visually confirm that the kinesin itself steps toward either the plus or minus end, validating the directional prediction.