D103 Cell Migration (ALS 4, Video 7 and 8)

when do cells migrate

• during embryonic development

• immune response

• wound healing

• cancer: metastasis

• involves actin

components of cell migration

stress fibers: parallel bundles of f-actin

leading edge: branched actin 

trailing edge

contractile actin fibers (stress fibers)

• actin bundles are anchored to substratum 

• cell body is translocated

• integrins connect to the ECM 

leading edge 

branched actin meshwork 

• front membrane is pushed out 

steps of cell migration

1. membrane extension: enhanced actin mesh formation at the leading edge of the cell (protrusion)

2. formation of new attachment: dynamic attachment of cells to the substratum via focal adhesions (attachment)

3. cell body translocation: myosin-dependent constration at the trailing edge (traction)

4. breaking cell attachment; recycling of the membrane and attachment machinery

how can we study cell migration in tissue culture cells

wound healing assay

• indicates cell migration, not directional migration

how is cell migration regulated 

via small GTPases (Rho, Rac, and Cdc42)  

• found inside cytosol as a part of the signal transduction pathways for cytoskeleton structure

small GTPases function

function as tightly controlled molecular timers

• gtp binding portein with 2 different conformational states (GTP or GDP bound)

GAPs and GEFs

regulators of activation states

• localization of GAPs and GEFs provides spatial regulation of GTPase activity

• GAP: turns off GTPase

• GEF: turns on GTPase

lipid anchored

transient association with the cytosolic leaflet of plasma membrane 

GDI

prevents GTPase from associating with membrane

dominant active

mutant whihc mimics the GTP-bound state

dominant negative 

mutant, which mimics hte GDP-bound state 

• can be locked in inactive state 

result of dominant active Rho

gain stress fiber formation and contraction

• involves formin 

result of dominant active Rac 

gain of lamellopodia formation 

• requires WAVE → Arp2/3

• branched network

result of dominant active Cdc42

filopodia formation

• requires WASP → Arp2/3

• linear f-actin (filopodia) to establish polarity 

what does Rho activation lead to

formation of linear stress F-actin fibers

• binding induces conformational change to bind protein

• formin and myosin (via ROCK) are activated; stress fibers and contraction for movement

what does rac and Cdc42 activation promote

polymerization of f-actin structures

• both use Arp2/3 for movement

• activated via conformational change

wiskott-aldrich syndrome 

WASp is mutated 

• x-linked, causing eczema and autoimmiune def

what leads to directional cell migration

coordinated interplay between small GTPases

• front: Cdc42 and RAC activation

• rear: RHO activation

location of GEFs

dictates where F-actin polymerizes and how f-actin organized

listeriosis

caused by listeria monocytogenes

• food poisoning, diarrhea, meningitis, pregnant women

• bacteria in rapid movement

how does listeria move inside a “host” cell 

actin-based motor/tail 

• without ActA, the bacteria cannot move inside a cell 

• ActA is necessary for bacterial motility; sufficient for motility 

actin dynamics at leading edge in listeria

Rac1 (GTP) activates WAVE to activate Arp2/3

• treadmilling actin network for migration in eukaryote; reuse building blocks and pushing plasma membrane forward

• ActA of listeria activates the Arp2/3 complex (WASp independent)

intrinsic property of actin 

polymerization/depolymerization that depends on the cytosolic conc of g-actin atp 

• thymosin, profilin, cofilin

regulation of actin binding proteins

actin binding protein control organization and dynamics

• formin, arp complex, capping protein, toropomyosin 

nucleating factors

formin and ARP complex

formin 

nucleates assembly and remains associated with the growing + end 

ARP complex

nucleates assembly to form a web and remains assorted with the - end

thymosin

binds subunits; prevents assembly

profilin 

binds subunits; sppeds elongation by promoting ADP/ATP exchange 

cofilin 

binds adp-actin filaments; accelerates disassembly 

• destabilizes - end 

gelsolin

severs filaments and binds to + end

capping protein

prevents assembly and disassembly at + end

• critical regulators of f-actin in skeletal muscle cells (CapZ and tropomodulin)

sarcomeres

capped f-actin that cannot depolymerize (f-actin binding proteins)

• bipolar filaments of myosin 2

motor domain of myosin

in all myosins

• binds atp

• bind f-actin

tail of myosin

unique to each myosin

tropomyosin 

occupies the same site on f-actin as myosin → myosin cannot bind, muscle cannot contract

troponin

ca²+ sensing protein complex

muscle contraction in presence of Ca2+ and ATP

• troponin undergoes conformational change

• displaces tropomyosin

• myosin can bind to f-actin because tropomyosin no longer blocks the myosin binding site

• myosin “walks” towards the f-actin + end

where does the ca2+ come form during skeletal muscle contraction

action potential opens the voltage-gated ca²+ channel to release ca2+ to cytosol → binds to troponin → moves tryptomyosin → contracts 

smooth muscle and Ca2+

• sheets fo elongated spindle-shaped cells

• cells have a single nucleus

• contractile filaments without sarcomere organization

• contain specialized forms of actin and myosin

• ca2+ dependent contraction involves calmodulin, not troponin

focal adhesions

connecting a cell's actin cytoskeleton to the extracellular matrix (ECM), allowing cells to adhere to their environment and sense mechanical forces

how were actin and focal adhesions visualized in this cell in the video 

express G-actin with GFP tag; express vinculin with RFP tag

• bc it was in a video, both structures are dynamic and require tagging for imaging

regulators of contractile actin bundle in trailing edge

• formin

• f-actin bundling proteins, myosin

• rho

• focal adhesion components

regulators of this branched actin org

• WASP/ Arp2/3

• profilin and cofilin

• Rac and Cdc42

GTPS is commonly used as GTP analog that cannot be hydrolyzed. which of the following conditions does the addition of gtps mimic

gtp hydrolysis = gtp → GDP and Pi

• thus activation of GEF to create the activated GTP 

which of the following proteins is most upstream in the pathway that is activated by the bac chemoattractant

upstream = earliest

• rac gef bc it must be activated first to do anything

why are f-actin capping protein critical for muscle contraction

prevent f-actin dynamics at both ends

titin 

positions bipolar myosin fiber 

nebulin

f-actin stabilizer