cell migration

cell polarization and migration

defines the front and the rear, allowing cells to move in one direction

guided by the nucleus, golgi apparatus, and MTs → i.e., asymmetrical emanation of MTs from the centrosome pulls the nucleus behind them

defines the direction of polarization in most mesenchymal cells via nuclear translocation and MT polarization

protrusion or microspikes

refers to filopodia, lamellipodia, invadopodia, and blebbing

filopodia

common in migrating growth cones of neurons and fibroblasts

one dimensional, long bundles of parallel actin → "thin “finger-like” projections

contains receptors, allowing response to the immediate, surrounding cellular environment

function of filopodia

acts as cellular “antennae”, probing the cell microenvironment

constructs cell-cell adhesions and guides growing dendrites to chemoattractants

lamellipodia

common in epithelial cells, fibroblasts, and neurons → wound-healing

two-dimensional, mesh-like network of branched actin that stretch out to the cell periphery

lamellipodia machinery

actin filaments for rapid movement

MTs and IFs restricted to the immediate area around the nucleus

dependent on the ARP2/3 complex

the role of ARP2/3 complex in lamellipodia cell motility

anchors minus ends to other actin filaments, creating actin mesh → plus ends of actin filaments orient towards the leading edge of the cell

facilitates the nucleation of new actin polymers at a 70 degree angle to the existing actin polymer

when absent → there is still some cell motility governed by filopodia, not lamellipodia

function of lamellipodia

cell motility → generates pushing forces, driving the cell forward

directs motility by: controlling cell adhesion and giving the cell a polarized structure, acting as an “internal compass”

the role of cofilin

disassembles actin filaments

has a higher affinity for ADP-actin

localizes to the rear of the leading edge → ensures actin polymerization is to the leading edge only

allows continuous unidirectional movement (i.e., that enough actin monomers are available locally for growth)

invadopodia

often found in metastatic cancer

three-dimensional actin, rich in protrusions that effectively penetrate tissue barriers → “invasive feet”

invadopodia machinery

starts from localized loss of the actin cortex → blebbing to form an immature filopodia-like structure

maturation results in degradation of the surrounding ECM → creates a point of entry for migration into other tissues

blebbing

protrusion of the membrane due to loss of the actin cortex/formation of an immature spine

membrane blebbing

blebs form when the PM detaches from the cytoskeleton/underlying actin cortex

contraction of myosin in the absence of an opposing actin cortex → membrane protrusion

reassembly of the actin cortex, establishing a new cellular position

actin polymerization

drives PM protrusion

the leading edge results from actin contraction at the rear, pushing the PM forward

movement of cells across a solid substratum

lamellipodia at the leading edge

localized contraction and actin polymerization at the rear pushes lamellipodia forward → gives room for the formation of new focal adhesions

growth cones

guides a developing axon to its synaptic target

involves actin and MT cytoskeletons that steer the axon → directional migration

growth cone dynamics

1. extension of the filopodia, contacting an adhesive substance via receptors on the actin cytoskeleton → some depolymerization at the minus end, no polymerization at the plus end

2. vesicle fusion at the PM, allowing space for actin filament polymerization → supports the growth of filopodia at the plus end/leading edge

3. actin polymerization pushes filopodia forward to extend and contract

4. advancement of MTs from the core

5. entry of cytoplasm/organelles to MTs/motor proteins → advancement of the axon

invadopodia in breast cancer

stage 0 → no lamellipodia

stage 1 → initial membrane relaxation and blebbing, resulting in F-actin polymerization pushing forward and stabilization of branched actin

stage 2a → migration through the ECM

stage 2b → MT and IF movement, pulling along other cellular components

invadopodia formation in a breast cancer cell

where actin is concentrated, laminin is less concentrated

eventually results in a gole, allowing blebbing

myosin contraction and cell adhesion

allows cells to pull themselves forward and promotes the formation of focal adhesions → coupled with de-adhesion at the rear of the cell

myosin links the actin cytoskeleton to the substratum via integrin-mediated adhesion

the role of myosin II

associates with actin filaments at the rear of lamellipodia to pull them to a new position → from perpendicular to parallel to the leading edge

pulls the cell towards the leading edge, maintaining contact with the substratum by leaving focal adhesion contacts intact

premature loss of focal adhesions

allows actin filaments to slip back away from the leading edge following actin polymerization

control of cell-substratum adhesion

A: assembly of actin monomers at the leading edge of actin filaments → integrins help form focal adhesions, linking the cell membrane to the substratum

B: no interaction between actin filaments and focal adhesions drives the retraction of the actin filament → drives the actin filament rearward via polymerization pressure at the leading edge and myosin contraction, causing retrograde slippage

C: interaction between actin filaments and focal adhesion drives the contraction of the actin filament → drives the leading edge forward via the transmission of myosin contraction through the focal adhesion, generating traction on the ECM, and new actin polymerization

cell polarization

controlled by members of the Rho family (i.e., CDC42, Rac, and Rho)

must distinguish between the leading and rear edges before migration via polarization of the cytoskeleton

the role of CDC42 in filopodia formation

triggers actin polymerization and bundling through activation on the inner surface of the PM → triggers filopodia formation

activates WASp protein family members (i.e., N-WASp)

stabilizes the open/active N-WASp protein conformation, binding profilin in filopodia and some ARP2/3 in lamellipodia

promotes primarily filamentous actin nucleation, and some branched actin nucleation

lamellipodia formation via Rac

activation triggers actin polymerization in the cell periphery → triggers the formation of sheet-like lamellipodia

activates WASp → activates the ARP2/3 complex in lamellipodia, and some profilin in filopodia

activates filamin and Rho, inhibits myosin II

Rac: the role of filamin

an actin crosslinking protein

stabilizes orthogonal actin networks in lamellipodia

Rac: the role of Rho

induces myosin contraction in the region opposite to the leading edge

only occurs after the lamellipodia is fully formed

Rac: the role of myosin II

inhibits myosin II via activation of PAK and inhibition of MLCK

results in no contraction and stable lamellipodia

Rac activation pathways

active Rac (Rac-GTP) activates WASp, which activates ARP2/3, giving branched actin in lamellipodia

Rac-GTP also activates PAK, which inhibits MHC/MLCK, resulting in decreased myosin activity/contractility, and less stress fiber (F-actin) formation

RAC-GTP also activates PAK, which activates filamin, resulting in the stabilization of branched actin in lamellipodia

contractile force and migration via Rho

activation promotes myosin activity → bundles of actin filaments/myosin II results in stress fiber formation and integrins cluster into focal adhesions

activation also promotes MLC phosphorylation → inhibits MLC phosphatase, promoting myosin contraction

activates formin and inhibits cofilin

Rho: the role of formin

promotes actin bundling

Rho: the role of cofilin

inhibits cofilin via activation of LIM kinase

blocks actin depolymerization

Rho activation pathways

active-Rho (Rho-GTP) activates ROCK, which inhibits MLC phosphatase, deactivating MLC(P), and increasing myosin activity

active ROCK also activates LIM kinase, inhibiting cofilin, resulting in decreased polymerization and actin filaments

Rho-GTP also activates formins, increasing actin bundle growth

→ more stress fiber formation and integrin clustering/focal adhesion formation

chemotaxis

dictates the direction of cell migration

chemotactic signals promoting cell migration towards/away from the signal

ECM adhesion

proteins in the matrix associate with the growth cone

cell-surface adhesion

proteins on the surface of the cell represent signals attracting the growth cone, providing a surface for migration

fasciculation

if there is already a path to follow, the growth cone follows it

chemoattraction

an area where a signaling factor is released, attracting the growth cone towards it

chemorepulsion

an area where a signaling factor is release, repulsing the growth cone away from it

contact inhibition

repulsion occurring when growth cones come into contact with something (i.e., a specific cell type)

the role of chemotaxis

initiates a signaling cascade

involves a chemoattractant binding its GPCR on the migrating cell, activating PI3K and activating RAC

RAC → activates ARP2/3, promoting lamellipodia formation

PI3K → rapidly degraded, cannot diffuse to provide directionality to the newly formed actin mesh

GPCR activation → activates Rho, promoting myosin contraction (i.e., Rac in the leading edge + Rho in the rear → polarity to the cascade)

neutrophil polarization

enables the cell to maintain functional polarity with protrusion at the leading edge and contraction at the back

steps to neutrophil polarization

chemoattractant → binds GPCR → activates PI3K and PIP3 → activates Rac → signaling cascade → forms branched actin of lamellipodia and F-actin of filopodia

components of neutrophil polarization

PI3K and PIP3 act locally at the front of the cell → does not diffuse to the back of the cell

GPCR simultaneously activates Rho at the back of the cell, which goes through its signaling cascade, promoting the formation of stress fibers and contraction

Rac and Rho

inhibit each other

gives polarity and direction of migration

putting it all together (1)

Rho-GTP → ROCK → pMLC → local myosin II contraction

CDC42 → WASp → mDia → filamentous actin

Rac1 → WASp → Arp2/3 → actin mesh

putting it all together (2)

Rac-GTP → activates WASp → activates Arp2/3 → promotes the nucleating of branched actin → forms the meshwork of the lamellipodia

AT THE FRONT OF THE LEADING EDGE OF THE CELL: active CDC42 → activates WASp → activates formin → promotes the nucleation and growth of F-actin

AT THE REAR EDGE OF THE CELL: Rho-GTP → activates ROCK → phosphorylated MLC → increases myosin contraction

putting it all together (3)

gradient between Rho-GTP and Rac-GTP maintains a separation

Rho inhibits Rac at the rear of the cell, pushing it to the front of the leading edge of the cell where Rac inhibits Rho, pushing it to the back of the cell → net force for direction of migration