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Chapter 16 (Part 1)
The Cytoskeleton
CONTENT
Function and origin of the cytoskeleton
Actin and actin-binding proteins
Myosin and actin
Lecturer: Yu-Ling Shih, IBC, AS
Nov. 28. 2016
0"
FUNCTION AND ORIGIN OF THE CYTOSKELETON
•  Types of cytoskeleton (best known functions)
Microfilament (cell shape, locomotion, cytokinesis)
Microtubule (organelle position, intracellular transport,
chromosome segregation)
Intermediate filament (mechanical strength)
•  Organization of cytoskeleton
Polarity, dynamic 3D networks, constantly remodel
(assemble/disassemble)
•  For each type of cytoskeleton, you need to knowSubunit, nucleotide usage, dynamic and mechanical
properties, cellular localization, motor and associating
proteins, functions
Introduction
Cultured cell
Green- microtubule
Red- microfilament
Blue- DNA
Dividing cell
Green- microtubule
Red- Intermediate filament
Blue- DNA
Three Major Types of Protein Filaments that Form the Cytoskeleton
Three Major Types of Protein Filaments that Form the Cytoskeleton
Three Major Types of Protein Filaments that Form the Cytoskeleton
FUNCTION AND ORIGIN OF THE CYTOSKELETON
•  Summary of microfilament-based structures:
microvilli
cell cortex
stress fiber
focal adhesion
lamellipodium (leading edge)
adhesion belt
contractile ring
Diagram of changes in cytoskeletal organization associated with cell division
•  The polarization of the actin cytoskeleton is
assisted by the microtubule-organizing center
(MTOC) located in front of the nucleus.
•  When the cell divides, the polarized microtubule
array rearranges to form a bipolar mitotic
spindle, which is responsible for aligning and
Lamellipodia
Filopodia
then segregating the duplicated chromosomes.
•  The actin microfilaments form a contractile ring
at the center of the cell that pinches the cell in
two after the chromosome segregation.
Green- microtubule
Red- microfilament
Brown- DNA
Dynamic and constantly remodeled
Contractile ring
Organization of the cytoskeleton in polarized epithelial cells
Microfilament:
microvilli, cell
adherens junction
IF: link to adhesive
structure, ECM
Microtubule:
Vesicle/organelle
transport
The bacterial cytoskeleton
• 
• 
• 
• 
Tubulin homolog FtsZ- Cytokinesis
Actin homolog MreB- Cell morphology
Actin homolog ParM- Plasmid segregation
Intermediate filament Crescentin- Cell morphology
FtsZ
CreS
MreB
ParM
ACTIN STRUCTURE AND POLYMERIZATION DYNAMICS"
•  Actin monomer, globular actin- G-actin
•  Filamentous polymer – F-actin
•  Filament and functional polarity
(-) end: pointed end
(+) end: barbed end
•  Actin subunits assemble head-to-tail to create flexible, polar
filaments
•  Polymerization dynamics
nucleation-> elongation -> steady state -> treadmilling
•  Nucleation is the rate-limiting step in the formation of actin
filaments.
•  Actin filaments have two distinct ends that grow at different rates.
•  ATP hydrolysis within actin filaments leads to treadmilling at steady
state.
The structures of an actin monomer and actin filament
Molecular asymmetry
Structural polarity of the actin filament
The Polymerization of Actin and Tubulin- ON Rates and Off Rates
Nucleation Is the Rate-Limiting Step in the Formation of Actin Filaments
The Polymerization of Actin and Tubulin- The Critical Concentration
The Polymerization of Actin and Tubulin- The Time Course of Polymerization
Nucleation Is the Rate-Limiting Step in the Formation of Actin Filaments
Protein conformational changes induced by • 
Nucleotide binding
• 
Hydrolysis
• 
Phosphate release
The Polymerization of Actin and Tubulin- ATP Caps and GTP Caps
Dynamic Instability and Treadmilling
The Polymerization of Actin and Tubulin- Treadmilling
The time course of actin polymerization in a test tube
ATP Hydrolysis Within Actin Filaments Leads to Treadmilling at Steady State
The Polymerization of Actin and Tubulin- Dynamic Instability
The Functions of Actin Filaments Are Inhibited by Both Polymer-stabilizing
and Polymer-destabilizing Chemicals
•  The functions of actin filaments are inhibited by both polymer-stabilizing and
polymer-destabilizing chemicals
ACTIN AND ACTIN-BINDING PROTEINS
•  Actin-binding proteins influence filament dynamics and organization
•  Monomer availability controls actin filament assembly
•  Actin-nucleating factors accelerate polymerization and generate branched or
straight filaments
•  Function
Monomer-binding: Profillin, Cofillin, Thymosin β4
Nucleation and elongation: Formin
Branching nucleation: Arp2/3
Reaction of filamentCapping: CapZ
Severing: Cofillin, Gelsolin
Crosslinking: Fimbrin, α-actinin, Spectrin, Filamin
Stabilization: Tropomyosin
Actin-Binding Proteins Influence Filament Dynamics and Organization
Monomer Availability Controls Actin Filament Assembly
Actin-Nucleating Factors Accelerate Polymerization and Generate Branched or
Straight Filaments
Actin-Nucleating Factors Accelerate Polymerization and Generate Branched or
Straight Filaments
Actin-Nucleating Factors Accelerate Polymerization and Generate Branched or
Straight Filaments
Higher-Order Actin Filament Arrays Influence Cellular Mechanical Properties and
Signaling
Higher-Order Actin Filament Arrays Influence Cellular Mechanical Properties
and Signaling
Bacteria Can Hijack the Host Actin Cytoskeleton"
The actin-based movement of Listeria monocytogenes
Bacteria Can Hijack the Host Actin Cytoskeleton
The actin-based movement of Listeria monocytogenes
ACTIN AND ACTIN-BINDING PROTEINS"
SUMMARY
•  Actin-binding proteins influence filament dynamics and organization.
•  Monomer availability controls actin filament assembly.
•  Actin-nucleating factors accelerate polymerization and generate
branched or straight filaments.
•  Actin filament-binding proteins alter filament dynamics.
•  Severing proteins regulate actin filament depolymerization.
•  Higher-order actin filament arrays influence cellular mechanical
properties and signaling.
•  Bacteria can hijack the host actin cytoskeleton.
MYOSIN AND ACTIN
•  Actin-based motor proteins are members of the myosin superfamily.
•  Myosin generates mechanical force by coupling ATP hydrolysis to
conformational changes.
•  Myosin II is the first motor protein discovered in muscle. Sliding of
myosin II along actin filaments causes muscles to contract.
•  Myosin V binds to vesicular cargo to transport it along actin filaments.
Table 12.2 Some Types of Myosins and Their Functions
Molecular Weight of
Heavy Chain (kDa)
Main Function
I
110-150
Membrane binding
II
220
Filament sliding in muscle
V
170-220
Vesicle transport
VI
140
Transport of endocytic vesicles,
moves towards the “-” end of the
actin fiber
Type
Textbook of Structural Biology
55"
Myosin superfamily members
MyosinFigure
and17.31
Muscle
FunctionMuscle
Architecture
Structure
of the skeletal
muscle sarcomere.
Myofibrils: bundles of contractile protein fibers
Sarcomere:
Z-disc
Thin filament: actin fiber
Thick filament: 300 myosin molecules
M-band (M-line)
A-band
I-band
Associating protein- Titin: a large and long
protein (2.7MDa); extend from M-band to Z-disc
to maintain the length of the sarcomere.
MOLECULAR CELL BIOLOGY
SIXTH EDITION
57"
Sliding of Myosin II Along Actin Filaments Causes Muscles to Contract
Actin-Based Motor Proteins Are Members of the Myosin Superfamily- Myosin II
Actin-Based Motor Proteins Are Members of the Myosin Superfamily
300 myosin molecules
Myosin Generates Force by Coupling ATP Hydrolysis to Conformational
Changes
MYOSIN AND ACTIN
1.  Muscle contraction
Striated muscle cells
Smooth muscle cells
Cardiac muscle cells
2.  Contractile bundles in nonmuscle cells are less organized. It
is regulated by myosin phosphorylation.
Examples: adherens belt in epithelial cell
stress fibers in migrating cells
contractile ring during cytokinesis
72"
Myosin V carries cargo along actin filaments