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Cell Motility

Dr. Susan LaFlamme


Room: ME301
Phone: 262-6256
Email: laflams@mail.amc.edu
Reading
Molecular Biology of the Cell (6th Edition)
Chapter 16
Pages: 889-913; 923-925; 941-944; 951-959

Objective
To gain an understanding of the physiological
importance and molecular mechanisms governing
cell migration and motility

Outline
1. When and why do cells move?
Morphogenesis, wound healing, metastasis,
the immune response, neurite outgrowth,
reproduction
2. How do cells move?
Steps in migration
*Generation of cell polarity
*Membrane protrusion and adhesion at the cell front
*Loss of adhesion at the cell rear
*Movement of the cell body forward and retraction
of the rear
Regulators of cell movement
* Chemotactic agents and growth factors
* Rho GTPases: Rho, Rac and Cdc42
Cdc42 and cell polarity
Rac and actin polymerization
Rho and cell contraction
2. Microtubule-dependent cell motility (Cilia and flagella)
Structure and function of cilia and flagella
The axoneme
Ciliary dynein
Kartageners syndrome

Leukocyte Transmigration

When and Why do Cells Move?


1. Development
2. Wound healing
3. Immune Response
4. Tumor Metastasis
5. Angiogenesis
6. Reproduction

Wound healing

Tumor Metastasis

Angiogenesis

How Do Cells Move?


Stably adherent cells are
activated by an extracellular
stimulus that leads to
cell polarity.
The cell sends out a
membrane extension in
the direction of migration.
New adhesions are formed
between the membrane
extensions and the
underlying substratum.
The cell body is translocated
forward.
Adhesions are stronger
in the front and weaker
in the rear of a migrating
cell.
Adhesions in the back of the
cell are disrupted and the
cell rear is retracted.

How Do Cells Adhere?



Integrins are transmembrane cell surface receptors that
mediate cell adhesion by forming transmembrane links
between the extracellular matrix and the actin cytoskeleton.

Extracellular matrix (ECM)
provides an adhesive substrate

Actin-binding
proteins

Fibronectin is a
component of the
ECM

Adhesion
Site

Actin
Filament
Stress
Fiber

How Do Cells Move Forward?


Membrane protrusive activity is generated
by actin polymerization.

How Do Cells Regulate Actin


Polymerization?

By the following:

1. Generation of free barbed ends either by
actin severing proteins or by the
uncapping of barbed ends
2. Nucleation of actin filaments

3. Actin-binding proteins regulate these


processes.
4. Signaling proteins regulate the activities of
actin-binding proteins in response to
extracellular signals.

Actin Binding Proteins Involved in the


Regulation of Cell Migration

Thymosin 4 sequesters G actin and inhibits actin
polymerization.
Profilin binds G actin to promote actin polymerization.
Cofilin binds F actin, severs filaments generating free
barbed ends for actin polymerization. Cofilin also
promotes actin depolymerization at the minus ends of
actin filaments.
CapZ binds to the (+) end of actin filaments.
Arp2/3 Complex is a protein complex that nucleates
actin polymerization generating branching structures.
Formins can nucleate actin polymerization and
promote filament elongation generating
actin bundles.
Myosin II is a motor protein that forms bipolar
filaments that can pull two actin filaments past each
other resulting in contraction.

How Do Cells Extend Forward?



Actin polymerization at the front of the cell
is a common mechanism that promotes
membrane extension.
Membrane extensions can
form as filopodia
or lamellipodia. Their
formation is
driven by actin
polymerization

Filopodia are spike-like membrane extensions.


Lamellipodia are sheet-like membrane extensions.
Invadopodia or podosomes are actin rich
protrusions that deliver matrix degrading proteases
that aid in cell invasion.
Blebbing is a distinct form of membrane protrusion.
The plasma membrane detaches from the
underlying actin cortex allowing cytoplasmic flow to
push the membrane forward
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Regulation of Actin Polymerization


And Depolymerization
***Arp2/3 Complex is a protein complex that
nucleates actin polymerization at the leading edge
that contributes to membrane extension.

***Cofilin promotes filament severing to generate
free barbed ends and actin depolymerization behind
the leading edge to increase the levels of actin
monomers needed for new actin polymerization.

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ARP2/3 Complex Promotes


Actin Polymerization

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Arp2/3 Nucleates the Assembly of


Branched Actin Filaments

Arp2/3 nucleates actin filaments by binding
to the side of a preexisting filament. This
results in an actin filament branch that grows
of the existing filament at a 70o angle.

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Arp2/3 Localizes to the Leading Edge



Migrating keratocyte
Arp2/3 labeled in green.
Actin filaments labeled
In red.

Electron micrographs of
the leading edge
showing actin filaments
in the lamellipodia at the
characteristic 70o angles
due to actin filament
nucleation by Arp2/3
complexes

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Profilin and Thymosin 4


Profilin enhances the rate of actin polymerization by


recharging actin monomers with ATP by promoting the
release of ADP and the uptake of ATP
Profilin competes with thymosin 4 for binding
actin monomers
Thymosin 4 sequesters actin monomers and inhibits
their association with the plus ends of actin filaments

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Contraction and Retraction


Weak adhesions

Activation of Myosin II
Generates Contractile Forces

Strong adhesions

Phosphorylation of the light


chain of non-muscle
myosin II (MLC) promotes
myosin II activity

Myosin II-Dependent Contraction


wrinkles
Cells adhered to a thin
sheet of silicon rubber
can pull on the rubber
substrate due to myosin II
activity resulting the
formation of wrinkles.

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Regulation of Cell Migration


Extracellular Signals

Cell Surface Receptors

Intracellular Signaling Proteins

(Rho-Family of GTPases)
Actin-Binding Proteins
Actin
Polymerization

Adhesion

Contractility

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Regulation of Rho-Family GTPases


Rho-Family of GTPases are active when bound to
GTP and inactive when bound to GDP.
Extracellular signals regulate whether they are GTP
or GDP bound.

Extracellular Signals

OFF

Rho-GDP

Rho-GTP

ON

Extracellular Signals
*Similar for Rac and Cdc42

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Rho-Family GTPases Regulate


Cell Migration
Rho-GTP: promotes focal adhesion and stress

fiber formation and cell contractility


Rac-GTP: promotes lamellipodia formation by


enhancing actin polymerization.

Cdc42-GTP: promotes filapodia formation by


enhancing actin polymerization and cell polarity
by stabilizing the association of microtubules at
the leading edge.

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How Do Cells Adhere?



Integrins are cell surface receptors that mediate
cell adhesion by forming transmembrane links between
the extracellular matrix and the actin cytoskeleton.

Actin-binding
proteins

vinculin
focal adhesion

Adhesion
Site

Actin
Filament
Stress
Fiber

Adhesions are stronger in the front and


weaker in the rear of a migrating cell.
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Rho-GTP

Rho kinase
(Rock)
LIM kinase

Formins
MLC
phosphatase

Cofilin
MLC (P)

Increased Myosin
Activity

Actin bundle
growth

Increased Stress Fibers Formation


And Increased Contractility

MLC: Myosin Light Chain
MLC(P): Phosphorylated Myosin Light Chain
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Rho-Family GTPases Regulate


Cell Migration
Rho-GTP: promotes focal adhesion and stress

fiber formation and cell contractility


Rac-GTP: promotes lamellipodia formation by


enhancing actin polymerization.

Cdc42-GTP: promotes filapodia formation by


enhancing actin polymerization and cell polarity
and by stabilizing microtubules at the leading edge.

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Rac-GTP

PI(4)P2
kinase

WASP
family

PI(4,5)P2

Arp2/3
complex

Capping
proteins

Nucleation of actin
polymerization

(Increased barbed
ends and actin
polymerization)

Lamellipodia Formation

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Rho-Family GTPases Regulate


Cell Migration
Rho-GTP: promotes focal adhesion and stress

fiber formation and cell contractility


Rac-GTP: promotes lamellipodia formation by


enhancing actin polymerization.

Cdc42-GTP: promotes filapodia formation by


enhancing actin polymerization and cell polarity
by polarizing and stabilizing microtubules at
the leading edge.

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Cdc42-GTP

Polarized microtubule growth


towards the leading edge

Polarized membrane
towards the leading edge

Delivery of membrane
and proteins such as
integrins to leading edge

Formin

Nucleation/elongation
of actin bundles

Filapodia
Formation

Directional
Migration

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Microtubule Cytoskeleton
and Cell Migration

microtubule

Membrane vesicles
from the golgi or
recycling endosomes

golgi

Rear

Front

Microtubules preferentially grow towards the leading


edge.
Microtubules support membrane trafficking to the
leading edge.
Vesicular trafficking delivers membrane, adhesion
receptors, growth factor receptors, etc, to the
leading edge to promote membrane extension, the
formation of new adhesions, and to receive signals
that promote cell polarity and directed migration

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Summary
Actin Cytoskeleton and Cell Migration
Rear

Adhesion
Disassembly

retracting rear

Front

Rho
contractility

Rac
Actin
Polymerization
Adhesion
Assembly

protruding front
Direction of Migration

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Microtubule-Dependent Motility
Motile Cilia and Flagella
Microtubule-Dependent
Cell Motility

Motile Cilia

Beating Motion
of Cilia

Where are they found and


what do they do?
On epithelial cells of the respiratory
tract --where they produce a continuous flow
of mucus that is essential for
pulmonary clearance
On ependymal cells of the brain ventricles
and spinal canal --where they facilitate the circulation of
cerebrospinal fluid
On the ventral node of the developing embryo
--- to produce a leftward fluid flow that is thought
to provide an initial cue for the asymmetric
development of visceral organs

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Microtubule-Dependent Motility
Cilia and Flagella
Flagellum/Flagella
Wave-like motion of flagella

Where are they found?


Sperm and protozoa
What is their function?
Cell Movement

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Axoneme
Cross-section of an Axoneme

Major components

**Nine doublet microtubules arranged
around a pair of single microtubules

These microtubules extend the length
of the axoneme making up the cilium or
flagellum

**Accessory proteins cross-link the


microtubules together

**Ciliary dynein is a motor protein that


forms bridges between neighboring
microtubule doublets

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Generation of Movement
**Ciliary dynein is a large multisubunit motor protein
that moves towards the minus ends of microtubules.
**The tail of ciliary dynein binds to the A tubule
independent of ATP.
**The heads of ciliary dynein bind to the B tubule in
an ATP-dependent manner.
**When the heads hydrolyze ATP, the heads move
towards the minus ends of the B tubule
**Because the A and B tubules are link together the
movement of ciliary dynein along the B tubule
causes a bending motion
ciliary
dynein

isolated
microtubule
doublet

Dynein activation
causes microtubule
sliding

Dynein activation
causes microtubule
bending
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Kartageners Syndrome
Cause
Hereditary Defects in Ciliary Dynein

Characteristics
Male sterility due to immotile sperm
High susceptibility to lung infections due
to the failure of cilia in the respiratory
tract to clear bacteria
Defects in determination of the left-right
axis of the body during early embryonic
development

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