Cytoskeleton

Question 1

Types of filaments and differences between MTs, MFs, and IFs

Answer 1

• Microtubules (MTs): Made of tubulin subunits, organized as hollow cylinders, they provide structural support and are involved in intracellular transport and mitosis.

-Microfilaments (MFs, Actin Filaments): Composed of actin subunits, forming helical polymers. They are responsible for cell shape, motility, and cytokinesis.

Intermediate Filaments (IFs): Made of various proteins like keratin and lamin. They provide mechanical strength.

Key Difference: MTs and MFs are dynamic, polar filaments with ATP/GTP-dependent assembly/disassembly. IFs are more stable and non-polar and lack the nucleotide-binding properties seen in MTs and MFs.

Question 2

Treadmilling and critical concentration

Answer 2

Treadmilling: A dynamic behavior where subunits are added at the plus end and removed at the minus end. This occurs when the concentration of free subunits is between the critical concentrations (Cc) for the plus and minus ends.

Critical Concentration (Cc): The concentration of free subunits where assembly and disassembly are balanced. The plus end has a lower Cc than the minus end.

Graph Concept: The graph would show the rate of polymerization and depolymerization against subunit concentration, highlighting the distinct Cc values for the plus and minus ends.

If subunit concentration increases above the plus-end Cc, filament elongation dominates.
If concentration decreases below the minus-end Cc, depolymerization occurs.

Question 3

Role of GTP/ATP Hydrolysis in MFs and MTs

Answer 3

MTs (GTP Hydrolysis): Tubulin dimers bind GTP; GTP-tubulin adds to the filament. Hydrolysis to GDP weakens binding and promotes depolymerization.

MFs (ATP Hydrolysis): Actin binds ATP; ATP-actin polymerizes more readily. ATP is hydrolyzed to ADP after polymerization, weakening the filament and promoting turnover​

Question 4

Dynamic Instability

Answer 4

Dynamic instability is unique to MTs and describes the rapid transition between growth and shrinkage at the plus end. It is governed by the presence of a GTP cap. Loss of this cap triggers “catastrophe” (rapid shrinkage), while its restoration leads to “rescue” (regrowth)​

Question 5

Nucleation

Answer 5

Nucleation is the initial, rate-limiting step in filament formation, where subunits assemble into a stable nucleus. It affects:

Growth Rate: Enhanced by nucleation proteins like γ-TuRC for MTs or ARP2/3 for actin.
Unaffected Parameters: Filament elongation rate, which depends on free subunit concentration​

Question 6

Proteins Modulating MT and MF dynamics

Answer 6

Various proteins regulate MT and MF dynamics, including:

MT-binding proteins: MAPs, stathmin, severing proteins like katanin.

MF-binding proteins: Thymosin, profilin, gelsolin (severing), and cross-linking proteins​

Question 7

Motors proteins and their Relationship to cytoskeletal filaments

Answer 7

Microtubule Motors
Kinesins: Typically move toward the plus end of microtubules.
Most kinesins have two motor heads that walk along the microtubule via ATP hydrolysis.
Example functions: Transporting vesicles and organelles toward the cell periphery.

Dyneins: Move toward the minus end of microtubules.
These motors are complex and require additional factors like the dynactin complex for cargo attachment.
Example functions: Transporting vesicles toward the centrosome or nucleus and driving ciliary and flagellar motion.

Actin Motors
Myosins: Myosin motors primarily interact with actin filaments. Different classes of myosins have distinct functions:
Myosin II: Powers muscle contraction.
Myosin V/XI: Involved in intracellular cargo transport.
Most myosins move toward the plus end of actin filaments, except Myosin VI, which moves toward the minus end.

Structural Features of Motor Proteins
All motor proteins have:
A head domain that binds the filament and performs ATP hydrolysis.
A tail domain that binds specific cargo.

Question 8

Identifying Kinesin Directionality

Answer 8

You could test a kinesin’s directionality using an in vitro gliding assay. Attach the kinesin to a slide, add fluorescent MTs, and observe their movement relative to filament polarity. Directionality is confirmed by comparing it with polarity markers.

Question 9

Identifying Myosin XI Cargo in Arabidopsis.

Answer 9

Using affinity purification with tagged myosin XI, identify interacting proteins or organelles. Then, follow up with co-localization studies using fluorescent markers or mutant phenotypes to confirm cargo association.

Question 10

Determining motor protein for transport: if given a scenario where an organelle is transported along a filament, consider:

Answer 10

The polarity of the Filament: Microtubules have distinct plus (growing) and minus (anchored) ends, while actin filaments have a plus (barbed) and minus (pointed) end.

Direction of Movement:
Plus-end directed movement: Kinesins (MTs) or Myosin (MFs).
Minus-end directed movement: Dyneins (MTs) or Myosin VI (MFs).

Example:
If you observe an organelle moving toward the microtubule minus end (e.g., toward the centrosome in animal cells), the likely motor is dynein.

Question 11

Energy Source for Transport

Answer 11

The energy for motor protein activity comes from ATP hydrolysis.

Process:
ATP Binding: The motor protein head binds ATP, inducing a conformational change.
Hydrolysis: ATP is hydrolyzed to ADP and inorganic phosphate (Pi). This process provides the energy needed for movement.
Release and Reset: ADP and Pi release resets the motor for another cycle.

How ATP Drives Movement:
For kinesins and dyneins, the heads take coordinated steps along the microtubule. ATP hydrolysis ensures tight binding to the filament and power-stroke motion.
For myosins, ATP hydrolysis drives the sliding of actin filaments or processive movement along actin.

Efficiency and Regulation:
ATP hydrolysis is highly efficient but tightly regulated by accessory proteins and signaling pathways. Cellular energy levels (ATP availability) can directly influence the speed and extent of cargo transport.