BIO3153 - Cytoskeleton Filaments, regulation, molecular motors

Three types of filaments

actin, microtubules, intermediate filaments

Filament construction

- small subunits form filaments
- disassembly, diffusion, reassembly

Nucleation

Initiates construction of filaments - lag phase, growth phase, equilibrium phase

Tubulin

- heterodimer
- each binds GTP; hydrolyzed at only 1 site
- 13 protofilaments
- plus and minus end

Actin

- monomer
- ATP
- plus and minus end

Treadmilling and dynamic instability

- growth / shrinkage of filament proteins
- actin and tubulin catalyze hydrolysis of ATP or GTP

Critical Concentration (Cc)

Where subunit addition equals subunit loss

Treadmilling of actin filaments

- filaments added to ATP-actin
- [ATP-actin] high, addition occurs at both ends
- [ATP-actin] drops, addition greater at plus end
- steady state

Treadmilling

For it to occur - Cc(T)<C<Cc(D)

Dynamic instability - MTs

- [tubulin] within critical values
- Continuous transitions between catastrophe and resue
1. Rapid growth with GTP capped end
2. Accidental loss of GTP cap
3. Rapid shrinkage
4. Regain of GTP cap

Accessory proteins

- filaments are dynamic and under control of the cell
- form higher order structures
- modify these cytoskeletal dynamics

Nucleation by y-tubulin protein complex

- occurs at the minus end
- y-TuRC (ring complex) accelerates MT formation
- y-tubulin is highly conserved
- spindle body in yeast
-MTOC (centrosome) in animal cells
- MT initiation also occurs in cytosol
- MTs nucleate daughter MTs

Centrioles and centrosome

- centrosome contains 2 cylindrical centrioles
- 9 triplets of MTs
- centrioles organize PCM
- centriole lumen and PCM contain y-tubulin
- PCM initiates MT assembly
- centrosome near nucleus
- minus ends of MTs are anchored
- plus ends of MTs emanate in astral configuration

Nucleation of AFs

• Cell periphery (cortex), where density of AF proteins is highest.
• Location related to AF function (cell shape, movement).
• Facilitated by ABPs (actin binding proteins) including ARPs (actin related proteins).

Initiation of AFs (unbranched)

- by formin, an ABP
- correct alignment for polymerization

Intiation of AF branches

• Cell periphery
• Arp2/3 produces extensive branching.
• Complex contains 7 proteins.
• Binds at (-) end.
• 70º favourable angle.

Control of subunit pools (not build or dismantle too fast)

• Both actin and tubulin are maintained in the cytosol at high concentrations.
• Concentration can exceed Cc.
• Accessory proteins may sequester unused subunits (sequestering proteins).
• Sequestered proteins are not hydrolyzed.
• Provide control or regulation of filament elongation.

Thymosin does what to actin

- Thymosin sequesters, but profilin recruits monomers.
- Thymosin makes polymerization less favourable.
- Profilin competes with thymosin and promotes assembly.

Stathmin does what to tubulin

• Stathmin sequesters
- Stathmin binding prevents polymerization.
• Decreases effective [tubulin].
• Promotes dynamic instability (catastrophe).

MT-associated proteins (MAPs)

• Several binding domains.
• Length of projecting domain determines packing of MTs.
• MAP2 (neuronal cell bodies); tau (axons).
• tau and Alzheimer's Disease (tau unable to bind to MTs).
• Poorly soluble (hyperphosphorylated) tau may induce neurodegeneration.

AF binding proteins

Cofilin destabilizes AFs
• binds to side of proteins.
• binding induces mechanical stress.
• treadmilling, turnover.
• cell locomotion.
Tropomyosin stabilizes AFs
• binds to side of proteins.
• muscle contraction

Modifications at AF ends

• "Capping"
• Recall elongation slower at (-) end.
• e.g. CapZ (+), elongation occurs only at (-) end.
• e.g. tropomodulin (-) in muscle contraction.

Modifications at MT ends

• Capping in MTs has dramatic effect on dynamic instability.
• Important during mitosis.
- MAPs provide stabilization, frequency of catastrophes suppressed and/or growth rate enhanced, longer and less dynamic microtubules
- kinesins provide destabilization, frequency increased, and shorter and more dynamic microtubules

Cross linking proteins and AFs

• Formation of higher-order structures.
• Spacing of 2 actin binding domains of cross-linking protein determines type of structure.
• 2 major groups: bundling and gel-forming.

Actin bundling proteins

- contractile bundle: loose packing allows myosin-II to enter bundle
- parallel bundle: tight packing prevents myosin-II from entering bundle

Changes in cell shape during embryonic development

• Vertebrate embryo.
• Formation of neural tube during nervous system development.
• Cell height - MTs
• Folding into tube - AFs.

Induction by extracellular signals

1. Extracellular signal
2. Activation of WASP
3. Nucleation and branching by Arp2/3
4. Promotion of assembly by profilin
5. Elongation reduced by capping proteins
6. Destabilized by cofilin and return of subunits to pool

Motor proteins

- use energy derived from hydrolysis of ATP to produce mechanical force
- binds to cytoskeleton
- produces net movement of protein or cargo
- divided into 3 families

Families of motor proteins

myosins (move along AFs), kinesins (move along MTs +), dyneins (move along MTs -)

Functions of myosins

- conventional (type II: muscle)
- unconventional (type V: organelle)

Myosin II structure

- 2 heavy chains
- 4 light chains

Myosin cycle

1. Attached
2. Release
3. Cocked
4. Force generating

Ca+2 dependence of muscle contraction

1. No Ca+2, tropomyosin blocks myosin-AF binding
2. Ca+2 released from SR and binds to troponin C
3. Conformational change in troponin I
4. Tropomyosin (bound to troponin T) moves and exposes binding sites
5. Myosin AF binding
6. Muscle contraction

Myosin II S1 fragment

- addition of protease enzyme
- myosin cleaved between neck and tail
- S1 contains catalytic site
- can be immobilized on glass surface
- slide AFs in vitro

Contractile ring

- important during cytokinesis
- myosin II and actin filaments
- redistribution of filaments

Myosin V

- long neck region
- "hand-over-hand" steps
- processivity
- carries cargo (organelles)

Kinesins

• Similar basic structure as myosins.
• Head region conserved.
• Tail regions diverse.
• C-terminal domain attaches to cargo.
• Most travel on MTs toward (+) end.
• KIFC2 travels toward (-) end.
• ATP-binding sites similar.
• MT vs. AF binding sites differ.
• Linker region interacts with catalytic core, thus swinging arm around.

Kinesin cycle

• Processive steps along MTs.
1. ATP binding of leading head induces conformational change in its linker region; trailing head advances.
2. ADP induces weak binding of leading head.
3. Hydrolysis of trailing head induces detachment; ADP dissociates from leading head. (Repeat...)

Dyneins

• (-) end directed.
• MT motor proteins.
• 2 major divisions:

Divisions of dyneins

- Cytoplasmic, e.g. retrograde vesicular transport.
- Axonemal, e.g. beating of cilia and flagella.

Cytoplasmic dynein

• Dynein cannot bind directly to cargo.
• Attachment to MT mediated by dynactin complex
• So named because of inclusion of actin.
• Other accessory proteins.