cell bio: cytoskeleton part 1

list the three major types of cytoskeleton and what they do

1) microtubules: hollow tubulin polymers that provide tracks for intracellular transport / organize cell division

2) microfilaments: thin actin polymers that drive cell movement, shape changes, & muscle contraction.

3) intermediate filaments (including nuclear lamins): rope-like fibers that provide mechanical strength & structural stability to cells and the nucleus

all filaments are assembled from smaller protein subunits. list the subunits of the following types of filaments and what they do:

a) microfilaments

b) microtubules

c) intermediate filaments

a) actin:

- forms dynamic microfilaments that support cell shape and generate force for movement and contraction.

b) tubulin:

- polymerizes into microtubules that organize intracellular transport, chromosome segregation, and cell polarity.

c) intermediate filament proteins (IF proteins)

- assemble into tough, rope-like fibers that provide mechanical strength and structure to cells & the nucleus

list the structure of the following type of cytoskeletal protein and what it does:

- microtubules / tubulin

- microtubules are hollow tubes made of α-tubulin / β-tubulin heterodimers

----------> β-tubulin binds and hydrolyzes GTP to drive polymerization dynamics.

- are polarized with a fast-growing + end and a slow-growing - end, which enables directional transport and dynamic instability.

- functions as tracks for motor proteins and organize cell division and overall intracellular organization

why is it that the cytoskeletal protein must be dynamically unstable for directional transport to occur?

it allows microtubules to remodel / search-and-capture targets, creating the changing tracks & directional bias motors needed to deliver cargo to specific cellular locations

list the structure of the following type of cytoskeletal protein and what it does:

- microfilaments / actin

- G-actin (globular actin) is an ATP-binding monomer with inherent polarity, assembling so that its ATP-bound side adds preferentially to the + end and its ADP-bound side dissociates more readily from the - end

- F-actin (filamentous actin) is a helical polymer of G-actin subunits, with a fast-growing + end and a slow-growing - end that together drive treadmilling and rapid structural remodeling.

---------> polarized F-actin filaments generate force and shape the cell, enabling processes such as cytokinesis

list the structure of the following type of cytoskeletal protein and what it does:

- IFs / IF proteins

- they are rope-like polymers formed by staggered, antiparallel coiled-coil dimers that assemble into strong, flexible fibers

- provide mechanical strength and structural support, maintaining cell integrity and protecting the nucleus and other organelles from stress

a) true or false: actins and tubulins are highly conserved among organisms

b) true or false: IF proteins are also conserved to the same levels as actins and tubulins

a) true, AA sequence is 90% identical

b) false, cytoplasmic IF proteins are less conserved, more ancestral

describe the thermal stability and structure of the following types of filaments:

a) single protofilaments

b) multiple protofilaments

a) Thermally unstable

--------> because single break in the single line of subunits causes complete filament failure, and breakage in the middle is just as easy as at the ends.

b) Thermally stable

--------> the many lateral contacts between multiple protofilaments means a break in the middle would require simultaneously disrupting many bonds at once, making end-shortening more likely than internal breakage.

explain how ATP binding to actin promotes its polymerization

- ATP-bound actin (G-actin-ATP) preferentially adds to the plus end of a growing filament, promoting polymerization and filament elongation.

- After incorporation, ATP is hydrolyzed to ADP, making actin less stable at the minus end, which dissociates the actin at the minus ends and is recycled as G-actin-ATP for further filament growth.

describe the process of treadmilling in actin

- soluble actin monomers are in the ATP-bound (T) form, which preferentially adds to the filament's fast-growing plus end, while filamentous actin contains a mix of ATP-actin (T) and ADP-actin (D).

- After polymerization, ATP-actin is hydrolyzed to ADP-actin, so the slow-growing minus end catches up to hydrolysis and loses subunits, while the plus end grows faster than hydrolysis can occur

- Treadmilling occurs because Cc(T) < Cc(D), meaning the plus end can continue to add T-actin even as the minus end loses D-actin, maintaining filament length while subunits turnover.

explain the following components of treadmilling graph and what they mean:

a) x-axis

b) y-axis

c) critical concentration

d) treadmilling range

a) subunit concentration

b) elongation rate (down = shrink / depolymerization, up means grow / polymerization)

c) monomer concentration at which polymerization and depolymerization are balanced / the same: BOTH WILL BE AT 0!!!!! (Cc(T) = Cc(D))

d) range of actin concentrations where ATP-actin adds to the plus (T) end while ADP-actin dissociates from the minus (D) end, keeping the filament length roughly constant.

-------->> is between balanced points on graph

how could you tell whether or not treadmilling occurs on a treadmilling graph?

Look for a range of actin concentrations where the plus end growth rate is positive while the minus end depolymerization rate is negative, indicating simultaneous addition and loss of subunits.

What would happen to the treadmilling graph if you were to do each of the following:

a) increase the concentration of free subunits

b) decrease the concentration of free subunits

a) polymerization rate at both ends increases, shifting the graph upward and expanding the treadmilling range.

b) polymerization rate at both ends decreases, shifting the graph downward and potentially falling below the critical concentration, stopping treadmilling.

explain the role of GTP / ATP hydrolysis in microfilaments

- ATP-actin hydrolysis after incorporation destabilizes the filament at the minus end, allowing subunit dissociation and filament turnover.

- energy from ATP hydrolysis drives treadmilling, maintaining dynamic filament remodeling for cellular processes like motility / shape changes.

explain the role of GTP / ATP hydrolysis in microtubules

- GTP-tubulin hydrolysis after polymerization destabilizes the plus end, promoting dynamic instability and rapid switching between growth and shrinkage.

- energy from GTP hydrolysis allows microtubules to search for targets and reorganize the cytoskeleton during transport and mitosis.

explain why GTP-tubulin hydrolysis destabilizes the plus end (DYNAMIC INSTABILITY)

- Tubulin grows at the plus end as long as there is a GTP-tubulin "cap" at the tip of the microtubule

- hydrolysis of GTP to GDP occurs after incorporation, so the GTP cap stabilizes the plus end and allows continued polymerization.

- Once the GTP cap is lost, the plus end becomes unstable and rapid shrinkage (catastrophe) occurs.

explain how "rescue" happens after catastrophe occurs to GTP cap

- occurs after the free tubulin concentration increases, allowing new GTP-tubulin subunits to add to the shrinking plus end.

- addition of GTP-tubulin re-establishes a GTP cap, stabilizing the microtubule and allowing growth to resume, a process called "rescue."

explain how dynamic instability leads to a subunit conformation change in tubulin

- GTP-tubulin dimers have an exchangeable GTP bound to β-tubulin, which stabilizes their straight conformation and allows incorporation into the microtubule plus end.

- After polymerization, GTP is hydrolyzed to GDP, weakening bonds in the filament and causing the tubulin subunit to adopt a curved, destabilized conformation.

- Dynamic instability allows GDP-tubulin to dissociate, and free tubulin dimers can exchange GDP for GTP in the cytoplasm, making them ready for reincorporation into a growing microtubule / straightening it again

describe what nucleation is broadly

it is the initial, rate-limiting step of filament assembly in which a small number of subunits come together to form a stable seed for further polymerization

describe the following components of the nucleation graph / what happens:

a) x-axis

b) y-axis

c) nucleation / lag phase

d) elongation / growth phase

e) steady state (equilibrium)

a) time after salt addition, which triggers filament polymerization.

b) amount or length of polymerized filament.

c) period where actin subunits bind together to form oligomers, slowly forming a stable seed

d) rapid addition of subunits to the filament / oligomer ends causes a steep increase in polymerized filament

e) filament growth and subunit loss balance, so the total filament amount remains constant.

describe the process / components of microtubule nucleation

- nucleation starts at the MTOC (microtubule organizing center), where γ-tubulin ring complexes (γ-TuRC) provide a template for α / β-tubulin dimers to assemble.

- γ-TuRC caps the minus end of the microtubule, stabilizing it and allowing growth to occur at plus end

- Once a stable seed is formed, additional GTP-tubulin dimers add to the plus end, initiating elongation and dynamic instability.

describe the process / components of actin nucleation

- occurs in the plasma membrane

- actin-related proteins (ARP2 and ARP3) form "Cap" at minus end of the actin filament

- Cap can also bind to existing filaments, promoting branched networks

- ARPs (Actin-Related Protein) nucleate new filaments and bind ATP-actin at the plus end, promoting filament growth just like regular actin.

- minus end of ARP-nucleated filaments is stabilized or capped, preventing depolymerization, unlike normal actin filaments where the minus end can lose subunits.

describe the purpose of branched actin networks in vivo

they generate a dense, dynamic meshwork that provides force and structural support for membrane protrusions, cell motility, and intracellular trafficking.

describe the experiment of ARP2 and ARP3: WURM and DISTORTED 1 in Arabidopsis and what was concluded about trichome development / formation

- Experiment: Mutations in ARP2 and ARP3 homologs (WURM and DISTORTED1) in Arabidopsis disrupted actin nucleation, leading to abnormal trichome branching and distorted morphology.

- Conclusion: Proper ARP2/3-mediated actin branching is essential for normal trichome development, demonstrating that branched actin networks control cell shape and structural patterning in plants.

filament elongation is regulated by proteins that bind to free subunits. list those two proteins and describe how they work

1) Thymosin:

- Binds ATP-actin monomers, sequestering them and preventing their addition to filament ends, thus slowing polymerization.

2) Profilin:

- Binds ADP-actin monomers and promotes exchange of ADP for ATP, increasing polymerization-competent actin for filament elongation

BOTH COMPETE WITH ONE ANOTHER

explain the role of plant profilin and how it contributes to the following phenotypes in Arabidopsis:

a) WT

b) overexpressed profilin

c) underexpressed profilin

a) supports normal actin filament elongation, allowing typical cell elongation.

b) enhances actin filament elongation, potentially causing excessive or abnormal cell elongation.

c) limits actin filament elongation, resulting in reduced or stunted cell elongation (short plant)

explain how stathmin regulates microtubule polymerization

- Stathmin binds two tubulin heterodimers, sequestering them and preventing their addition to microtubule plus ends, which inhibits polymerization.

----------> controlled by phosphorylation

- increases the rate of catastrophe by promoting microtubule depolymerization at the plus end.

- GTP hydrolysis then catches up to plus end, leading to microtubule shrinking

list the major filament-binding protein and explain what it does once bound to a microfilament

MAPs (microtubule-associated proteins)

- can stabilize microtubules and connect microtubules with other ones as well as other cellular components

it is known that end-binding proteins affect filament stability. explain how the following types of end-binding proteins affect filament stability:

a) MAPs

b) Kinesins

c) capping proteins (like EB1)

a) bind preferentially to GTP end, stabilizing it, and leading to longer, less dynamic microtubules

b) kinesins like catastrophin counteract and destabilize the plus end, leading to shorter, more dynamic microtubules

c) can target microtubule ends to cellular structures, linking growing plus ends to other filaments & guiding microtubule orientation

explain wha actin cross-linking proteins are and what they do

- they are proteins that bind and connect multiple actin filaments, organizing them into networks or bundles.

- one includes actin filaments & alpha actin

--------> results in contractile bundle with loose packing; allows myosin II to enter bundle

- another includes actin filaments & fimbrin

--------> results in parallel bundle and tight packing; prevents myosin II from entering bundle