4 Cytoskeleton|Advanced Cell Biology|Tulane

Cytoskeleton

Type

Intermediate filament; microtubule; Actin filament

• Geometry of the cell
• Fix the position of organelles
• Moves the compounds (cargos)
• Facilitate movement of the whole cells

Cytoskeletal Element	Diameter	Composition
Microfilament (MF)	thin (6-7nm)	Actin+ associated protein
Microtubules (MT)	tubular structures (25nm)	tubline + associated proteins
Intermediate filaments (IF)	rope-like fibers (~10)	No associated proteins

Actin and Microfilaments

• G-actin Highly globular protein
• F-actin Polymerizes into microfilaments (exhibits polarity)
• Filaments stabilized by other proteins

Polymerization

• Requires nucleation (activation)
  • 3 actins
• Elongation primarily at “+” end (barbed end)
• ATP/ADP
  • G-actin has bound ATP
  • ADP-actin more likely to dissociate
• Actin-binding proteins
  • monomer sequestration (profilin)
  • trimeric G-proteins
• Celluar regulation
  • hro family (ras-like G-proteins)
  • trimeric G-proteins
• Drugs
  • cytochalasins (prevent assembly)
  • phalloidin (prevent disassembly)

$$
G-actin \to F-actin
$$

Nucleation factors

Initial dimer and trimer formation is energetically unfavorable.	Assemble automatically is unfavorable
Tandem monomer-binding nucleators bring together actin monomers through tandem G-actin-binding motifs to form an actin nucleus.	Tandem: initial a pointed end (-)
A formin FH2-domain dimer associates with the barbed end of an actin filament and the FH1 domains recruit and deliver profilin-bound actin to the barbed end.	Formins helping elongate by binding the barbed ends
Arp2/3 with NPF (WASp) nucleates a new filament from the side of an existing filament, causing branching at a 70°angle.	Arp2/3 for forming a branch

Examples of Actin-Binding Proteins

Tight bundles	Loose bundles	3-D Gels	Dick shape
fimbrin	alpha actinin	filamin	Spectrin (Red blood cell)

Actin Microfilament (MF) Bundles

• Tight bundles (e.g., microvilli)
  • MFs having the same polarity
  • (+) ends point toward the microvillus tip
  • Fimbrin bundling proteins
    • Two ABD of the monomer holding two MFs together

• Loose bundles (e.g., contractile ring); which could interacted with myosine
  • MFs loosely spaced for myosin binding for contraction
  • α-actinin bundling proteins
    • Two ABD of the dimer holding two MFs together

Actin Microfilament (MF) networks

Filamin is a dimer

• Forming a flexible V-shaped molecule
• Crosslinking MFs into orthogonal networks

Submembrane cortical MF in RBC

RBC spectrin, a tetramer of α and β chains

• Two chains join laterally to form a dimer and head-to-head to form a tetramer
• ABDs of the β chain are separated by ~200 nm
• Spectrins associate with short MFs (yellow ball) to form the network.
• Protein 4.1 (pink) links MFs to PM-embedded glycophorin
• Ankyrin (green) links spectrins to PM-embedded Band 3

Stress Fibers at Focal Adhesions

Focal adhesions: ECM attachment regions of the cell
Stress fibers: α-actinin-linked contractile bundles of MFs

• Attached to the plasma membrane at focal adhesions via interactions with integrin.
• Mediated by talin and vinculin
  • Talin and α-actinin bind to the cytoplasmic domains of integrins. (out cell)
  • Talin also binds to vinculin, which in turn interacts with actin. (inner cell)

MFs at Adherens Junctions

• Adherens junctions are regions of cell-cell contact.
• In epithelial cells, the AJs form a belt-like structure
• Contractile bundle of MFs is linked to the PM
  • MFs associate with cytoplasmic α/β catenins (connect to Cadherins), which form a complex with PM-embedded cadherins
  • Cadherins (membrane protein) mediate contact between cells

Myosin and Force Generation (in loos connection)

• large family of proteins (16)
• actin-activated ATPase
• converts chemical energy into mechanical energy

Cell Locomotion-1

• Step 1: Movement begins with extension of one or more lamellipodia from the leading edge of the cell.
• Step 2: Some lamellipodia adhere to the substratum via focal adhesions.
• Step 3: Then the bulk of the cytoplasm in the cell body flows forward.
• Step 4: The trailing edge of the cell remains attached to the substratum until the tail eventually detaches and retracts into the cell body.

Proline leading the actin assembly

Cell Locomotion-2

What propels the membrane forward?

• The polymerization of actin filaments at the (+) end, stimulated by profilin located at the leading-edge membrane, pushes the membrane outward.
• Vasp (MF elongator) and Arp2/3 may participate in directing assembly.
• Myosin I links actin filaments to the leading-edge plasma membrane
• Arp2/3 and actin cross-linking proteins stabilize the actin filaments into networks and bundles.
• Cofilin induces the loss of subunits fromthe (−) ends of filaments.

Microtubules (MT)

© Linda A Amos

Major Roles:

1. Mechanical/Cell Shape
2. in Cilia(lung)/Flagella(bone)
3. Mitotic Spindle (divide)

Tubulin:

• highly conserved
• heterodimer (α, β)
• in vivo MT assembly starting from MT organization center

13 β subunits on the top only.
two subunits (one unit) added at a time

Microtubule Organizing Center (MTOC)

Centrioles of animal cell

• aka basal bodies of flagella
• Barrel-shaped (triplets of MT)

Centrosome of plant cell

• Amorphous protein matrix + two centrioles
• Organizes cytoplasmic MT array
• Forms spindle poles during cell division in animal

Spindle pole bodies of fungi

• Not contain centrioles
• Located on nuclear membrane
• Forms mitotic spindle in many lower eukaryotes

γ-Tublin and MT Nucleation

γ-Tublin: nucleation center

• MT nucleation in vivo requires γ-tubulin and associated proteins (γTu ring complex)
  • Caps minus end
  • Anchored near MTOC
  • Tubulin α/β-dimers added to plus end

Flagellar Movement-1

© Linda A Amos

Axoneme cytoskeleton of cilia and flagella

• Nine outer doublets MTs
• A central pair of singlet MTs
• A and B tubules in each MT doublet
  • 13 protofilaments in A tubule
  • 10 protofilaments in B tubule
  • Inner and outer dynein arms in A tubule

Flagellar Movement-2

Sliding forces generated by dynein arms:
The dynein arms on the A tubule of 1^st doublet “walk” along the adjacent 2^nd doublet’s B tubule toward its base, the (−) end, moving the 2^nd doublet toward the (+) end.

Flagellar Movement-3

Axonemal bending:

• Each doublet slides down only one of its two neighboring doublets,
• Active sliding in a portion of the axoneme produces bending toward one side
• Active sliding in the opposite portion produces bending toward the opposite side.

Motor Proteins

MF associated

• myosin

MT associated

• dynein
  • cilia/flagella
  • cytoplasmic
• Kinesin

© Jason Duncan

Kinesins

© Chapin Korosec

• Large superfamily of proteins (45)
  • defined by ‘motor’ domain
• Tail regions are highly divergent (cargo binding function)
• Chemomechanical cycle
  • similarities to myosin
  • two motors needed (?)

Structure:

Moto Domain	Stalk Region	Tail region
μT binding ATPase	Coiled-coil	accessory proteins specific functions

Kinesins in Mitosis

• Reorganization of MT during mitosis
  • disassembly of cytoplasmic MT
  • assembly of spindle apparatus
• Duplication of centrosome to initiate spindle apparatus formation
• Several kinesins implicated
  • separation of poles
  • migration of chromosomes

Intermediate Filaments

© Douglas Fudge, et al.

• Rope-like fibers extending from nucleus periphery
• Extremely stable
  • resistant to detergent extraction
• Only in metazoa
• Subunits are part of large multigene family
  • related to nuclear lamins
  • tissue specific expression

GFAP: Glial fibrillary acidic protein

Intermediate Filament Proteins

α-helical rod domain
C-terminus domain

• IF protein types distinguished by N- and C-termini
• Central region composed of heptad repeats
  • Forms coiled-coil structure (a-helices that twist around each other)

IF Proposed Structure

• Tetramer believed to be fundamental subunit
• Mechanism of assembly and disassembly not known
• Form stable structures $\to$ mechanical support

IF Role in Mechanical Support

• abundant in cells/structures under mechanical stress (e.g., epithelial, muscle)
• links to membranes and other cytoskeletal elements

digraph F { rankdir = "LR" node [style=filled, font=Courier, color = "salmon", shape = "box"]; A [label="Lamellipodia\nFormation"] B [label="Focal Adhesion\nDormation"] C [label="Cytoplasm\nTransportation"] D [label="Old Focal\nAdhesion Detach"] A -> B -> C -> D }