USTH BS3 ADVANCED BIOCHEMISTRY

Pathophysiology

Course C2: cytoskeleton, cell motility and

cell movements (Part 2)

Pr Germain TRUGNAN
with the help of Dr Lan Trang VU
4. Microfilaments (1)
4.1. Structure and assembly
Microfilament polymerization consists in the addition of monomers at both ends. Addition is faster at the
plus end as compared to the minus end.
plus end
Cytochalasins, which bind to the
cytochalasins
microfilaments plus end, block Fast
microfilament polymerization polymerization

Pi
ATP/ADP exchange

phalloidins
ATP hydrolysis
ATP DEPOLYMERIZATION

ADP
Phalloidins (a group of toxin from Amanita
phalloides), which bind to microfilaments,
block their depolymerization.
Slow
polymerization

minus end

When both ends are free of microfilament-binding proteins and that

monomer concentration is favorable, monomer assembly and
dissociation occur at the same rate at both ends. In these conditions, the
microfilament lenght remains constant but the filament moves by a
movement called «treadmilling».
4. Microfilaments (2)
4.2. Actin-binding proteins: 3 main families
4.2.1. Proteins involved in microfilament (MF) organization
Examples
* fimbrin, villin : formation of tight MF bundles * a-actinin : formation of large MF bundles

* filamin : MF cross-linking * spectrin, dystrophin : MF anchoring to the plasma membrane

4.2.2. MF-associated motor proteins

Examples
* myosin II ensures muscle contraction by moving microfilaments

* myosins I, V (etc.) ensure movent along microfilaments

* tropomyosin stabilizes microfilaments

4.2.3. proteins regulating microfilaments dynamics

Examples
* profilin, ARP 2/3 : promote MF polymerization
* thymosin, ADF/cofilin, gelsolin : promote MF depolymerization and/or severing
4. Microfilaments (3)
4.3. Functions of the microfilament network (1)
Formation, maintenance and dynamic of cell structures
lamellipodes
 4. Microfilaments (4)
4.3. Functions of the microfilament network (2)

2 Extracellular 10 Other extracellular

stimulus 5 MF polymerization pushes the plasma stimulus
membrane forwards

3 Activation of WASP and Arp2/3 6 MF capping

complexes. MF nucleation limits elongation

11 Inhibition
of ADF/Cofilin

1 Binding of 8 MF depolymerization and

profilin to ATP-actin severing by ADF/Cofilin

9 ADP-ATP exchange
on actin monomer
4. Microfilaments (5)
4.3. Functions of the microfilament network (3)
4.3.1. Assembly, maintaining and dynamics of cellular structures: example of the cellular cortex
The MF network organizes beneath the plasma membrane and ensures cell movement
Tight
Loose network bundles Cellular cortex
lamelipod
filopods
microtubules

noyau
Focal adhesion plaque=
stress fiber (large and
contractile bundle)
Radial
network Free actin are recruted to the
cell front Actin polymerization pushes
lamelipods the membrane forwards
+
Newly formed focal adhesion plaques
+ Tension forces applied on the cellular cortex disassemble focal
adhesion plaques at the cell rear…

The polymerization/depolymerization of the MF … while creating new ones at the cell front…
network allows changes in cell shape

The cell moves forwards and the cycle starts

again…
4. Microfilaments (6)
4.3. Functions of the microfilament network (4)
Functions of the microfilament network are tightly linked to MF associated proteins.
4.3.2. Assembly and maintaining of cellular structures (examples).

a actinin
Cross-bridges
(villin,
fimbrin)

MF
Membrane

MF

Lateral arms
(myosin I)
integrins

fibronectin
Focal adhesion plaque

microvillus
4. Microfilaments (7)
4.3. Functions of the microfilament network (5)
4.3.3. Motile functions (1): some bacteria recruit cellular actin to promote movement from cell to cell .

bacteria

phagosome

“actin comet”

Cell to cell
movement

immunofluorescence
4. Microfilaments (8)
4. Microfilaments (7)
4.3. Functions of the microfilament network (5)
4.3.3. Motile functions (2): muscle contraction

4.3.3.1. Correlation between anatomical and histological structures and biochimical composition
4.3.3.2. Understanding the mechanism that transforms chimical energy into mechanical energy
4.3.3.3. How protein activity is controlled by their environment
4.3.3.1. Correlation between anatomical and histological structures and biochimical composition
1- Sarcomer structure (by electron microscopy) shows the presence of thin and thick filaments

Resting myofibril
Band I Band A Band I
Ligne Z Zone H Ligne Z

1mm

thin thin + thick thick thin + thick thin

Contracted myofibril
4.3.3.1. Correlation between anatomical and histological structures and biochimical composition

2. Thin filaments are mainly made with actin. Thick filaments are mainly made with myosin II

Myosin I molecules are organized as

bundles with emerging bridges
4.3.3.1. Correlation between anatomical and histological structures and biochimical composition
3. A simple model allows to correlates anatomical and biochemical data

Thin actin microfilaments slide on thick myosin

II filaments

Z-lines are mainly composed by a particular

protein: meromyosin

In a muscle, several sarcomer units are

juxtaposed
4.3.3.2. Understanding the mechanism that transforms chemical energy into mechanical energy
1. Microfilaments are polymers of globular actin
Actin monomer, actin G (43 kDa), contains one ATP molecule and one Ca2+ ion.
Actin monomer is made of two domains.

Plus end

Each domain displays several

types of secondary structures

a helix

b sheets

turns and loops C-ter

N-ter

minus end
4.3.3.2. Understanding the mechanism that transforms chimical energy into mechanical energy
2. Muscle myosin (myosin II) is a big-sized complex protein

N-terminal
2 Supercoiled
a helixes Light chaîns

head

C-terminal
tail
4.3.3.2. Understanding the mechanism that transforms chimical energy into mechanical energy
2. Muscle myosin is a big-sized complex protein
Myosin II (520 kDa) is a polymeric protein with two heavy chains (220 kDa)
and two pairs of light chains (20 kDa, each)

Enzyme digestion of myosin reveals its structure and its functions

Isolated light meromyosin is able to

form filaments that do not associate with actin

Isolated heavy meromyosin is able to bind

actin, to hydrolyze ATP,
but unable to form filaments

Papain cleaves Heavy meromyosin (= myosin head)

into two fragments (S2 et S1)
4.3.3.2. Understanding the mechanism that transforms chimical energy into mechanical energy

3. Myosin head (S1) is responsible for its motor activity

Myosin is an ATPase that catalyze a

Actin-binding
reaction that produce energy:
site
ATP + H2O ADP + Pi + H+
ATP

Active
site
ADP
Pi
ATP hydrolysis is accompanied by a
conformational change
(lever arm) 10 nm

Light chains
4.3.3.2. Understanding the mechanism that transforms chimical energy into mechanical energy

4. Interaction of myosin (S1) with actin is reversible and depends ATP fixation and ATPase activity

The lever arm movement of myosin head will be transmitted to actin filament

Myosin head

Actin filament
4.3.3.2. Understanding the mechanism that transforms chimical energy into mechanical energy
5. Coordination between ATP hydrolysis, conformational change and actin fixation allows the movement

1 5

ATP ADP

2 4

3
ATP hydrolysis Pi
4.3.3.3. How protein activity is controlled by their environment
How muscle contraction is controlled?

A complex of regulatory proteins is required: troponin and tropomyosin

This complex interacts with actin filaments: to form a macromolecular structure
Troponine is a Ca2+ sensitive protein that change its conformation in the presence
of Ca2+ and tropomyosin is displaced.

Troponin
This displacement allows the fixation of myosin
C T I

Actin Tropomyosin
C = calcium binding T = tropomyosin binding I = inhibitory

Muscle contraction is directly controlled by the concentration of Ca2+ ions

4.3.3.3. How protein activity is controlled by their environment
At the cell level, the molecular mechanic of muscle contraction is highly controlled
Nerve
Influx Channel Ca2+ voltage-sensitive

Ca2+
influx

Membrane
depolarization Acetylcholine secretion

Plasma membrane + + + + + + + + + + Coupled Na+ channel

+
+ - - - - - - - - -
+ - Na+ Na+
+ -
+ - Z Z
+ -
+ -
- actin
Tubule T + myosin
Regulatory
voltage sensitive Proteins
Protein

SER

Channel Ca2+

Myocyte
4.3.3.3. How protein activity is controlled by their environment
At the cell level, the molecular mechanic of muscle contraction is highly controlled
Influx
nerveux Canal Ca2+ sensible au voltage

Influx
de Ca2+

Sécrétion d’acetylcholine

Membrane plasmique + + + + + + + + + + Canal Na+ couplé

+
+ - - - - - - - - -
+ - Na+ Na+
+ -
+ - Z Z
+ -
+ -
- actine
Tubule T + myosine
Protéines
protéine sensible au régulatrices
voltage

REL

Canal Ca2+

Ca2+ ATPase

Myocyte