cell migration

chemotaxis. definitions. amoeboid chemotaxis. cell migration. morphology. PI3K. challenges. transcellular/paracellular routes. actin cytoskeleton. examples. metabolic pathways. inositol phosphatases. bioprobes. protein constructs. Dictyostelium (WT/PTEN -/-). neutrophils. PTEN -/-. SHIP -/-. PI3K -/-. HL60 cells. Jurkat cells. zebrafish model. DOCK2 -/-. cell compass. KLF2. mTORC 1/2. FOXO. inhibitors (Akt/PKC). cAMP. FRET. adenylyl cylase. WNK1. aquaporins.

cell migration

orchestrated wound healing, regeneration, development, tumour metastases, and angiogenesis.

innate and adaptive immune system.

highly motile cells (lymphocytes and neutrophils).

wounded cell releases inflammatory mediators which attract cells in direction of concentration gradient into inflammation site.

termed chemotaxis.

diverse roles of chemotaxis

sourcing of nutrients by prokaryotes (amoebae).

formation of multi-cellular structures in protozoa.

embryonic development.

metastatic cancer migration to growth factors.

immune cell development.

immunosurveillance and activation.

chemokinesis definition

migration driven by soluble chemokines, without any cue gradient to provide a directional bias.

haptokinesis definition

migration along a surface, utilising immobilised ligands such as chemokines or integrins, without any cue gradient to provide a directional bias.

chemotaxis definition

migration driven by a gradient of soluble chemokines, which occurs when an asymmetry in chemoattractant (such as rate and density) exists and local cue gradients can be followed.

haptotaxis definition

migration along a surface that is guided by a gradient of immobilised chemoattractants or adhesion receptor ligands, which provides a directional bias.

diseases that involve migration

vascular disease.

chronic inflammatory disease (RA/MS).

cancer - metastatic spreading.

steps to amoeboid chemotaxis

pseudopod formation.

polarisation.

directional sensing.

pseudopod formation

well defined size, shape, and duration.

extended at 60s intervals at sites regulated by chemoattractant.

basic premise of amoeboid chemotaxis relies on firstly the formation of pseudopod.

extensions of cell membrane at cell’s leading edge in response to navigational cue, to swim towards source.

polarisation

elongated cell shape (starts round them polarised).

cells turn towards new gradient because anterior is more sensitive than posterior as it has more receptors to detect chemokine.

as cell develops pseudopods searching for chemokine stimulus, cell is polarised (develops front and back end leading to elongated shape).

directional sensing

can occur in immobilised cells (can immobilise cells by blocking actin polymerisation).

accumulation PH domain containing proteins at membrane facing high end of gradient.

sense direction don’t necessarily move (if manipulated with drugs).

occurs even in mobilised cells (switching off actin machinery that drives polarisation).

directional sensing and physical migration are distinct.

steps in cell migration

cell polarisation.

formation of protrusion.

rear retraction.

cell polarisation

Cdc42, PAR proteins and aPKC are involved in generation of polarity (intrinsic to migration).

directed vesicle trafficking towards leading edge.

organisation of microtubules MTOC and Golgi apparatus in front of nucleus.

PTEN (phosphatase) and myosin II implicated in restricting protrusions to cell front.

side/rear regulators of polarity

PTEN - phosphatase and tensin homolog.

myosin II.

front regulators of polarity

activated Cdc42 (cell division control protein 42 homolog) and Rac (Ras-related C3 botulinum toxin substrate 1).

Cdc42/PARs/aPKC.

PIP3.

activated integrin.

MTOC (microtubule organising complex)/Golgi.

microtubules.

formation of protrusion

profilin controls availability of activated actin monomers.

cofilin controls debranching and depolymerising proteins.

capping and severing proteins.

WASP/WAVE proteins are targets of Rac and Cdc42 and regulate formation of actin branches via action on Arp2/3 complex at the front of cell.

protrusions are stabilised by formation of adhesions.

at front of cell there is actin meshwork, whilst back and sides of cell have long thin actin strands.

front of cell - protrusion and ruffling effect.

actin polymerisation - nucleation

Arp2/3 complex - actin-related protein 2/3.

WAVE (WASP-family verprolin-homologous protein)/WASP (Wiskott-Aldrich syndrome protein).

Rac/Cdc42.

actin polymerisation - polymerisation/organisation

profilin.

ENA/VASP.

ADP/cofilin.

capping proteins.

cross linkers.

adhesion formation - integrin activation

Talin.

PKC.

Rap1.

PI3K.

adhesion formation - integrin aggregation

Rac/Cdc42.

rear retraction

at cell rear, adhesions disassemble as rear retracts.

as cell moves forward, rear of cell becomes detached otherwise gets stuck.

proteins responsible for detachment.

calcium dependent.

back of cell - uropod.

rear retraction - adhesion disassembly and retraction

FAK (focal adhesion kinase)/Src/ERK (extracellular signal-regulated kinases).

myosin II.

microtubules.

Rho - Ras homolog family member A.

Ca2+.

calpain.

calcineurin.

uropod - morphology, surface receptors, and signalling

adhesive and contractile rear.

contains microtubules and mitochondria.

mid zone - morphology, surface receptors, and signalling

actomyosin filaments.

controlled by Rho and ROCK.

leading edge - morphology, surface receptors, and signalling

PI3K, Ras, and p38, DOCK2 - feed into actin cytoskeleton.

Rac is central effector of pathways.

mediates formation of actin filaments that move the plasma membrane forward.

PI3K signals

antigen engagement.	cell proliferation. cell survival. differentiation. acquisition of “address” code.
homing to sites of infection/inflammation.	cell movement/migration. chemokines. inflammatory mediators.

role of PI3K in T lymphocyte biology

T cells circulate blood and constantly seek presented antigens (by DCs) in lymph nodes.

when engaged with antigens, T cells become activated/armed.

T cells are primed against antigens.

migrate back to tissue - T cell destination determined by receptor barcode expressed on surface.

PI3K is driven/activated by T cell antigen receptor.

involved in proliferation, cell survival, differentiation, and acquisition of address codes.

T cell migration challenges

crossing endothelium barrier follows tethering and rolling on luminal surface of endothelial cells.

squeeze from blood vessels to tissues.

navigate across extracellular matrices within tissue.

chemokines in combination with adhesion molecules triggers adhesion to endothelial surface via emission of filopodia-like protrustions.

T cells use either transcellular or paracellular route.

transcellular route

emit invadopodium-like protrusions that span the entire endothelial cell body.

immune cells pass through body of endothelial cells.

paracellular route

squeezing through junction between two adjacent endothelial cells.

immune cells pass through the gaps between endothelial cells.

micron sized pores in PECAM labelled regions between adjacent endothelial cells that represent sites of paracellular migration.

involves coordinated disassembly of VE-cadherin assemblies and other homophilic molecular interactions by serial leukocyte occupancy event.

T cell migration steps

sentinel cells release chemokines and other chemoattractants.

starts slow rolling, arrest, adhesion, strengthening, speading processes.

constantly rolling across endothelial cells - crawling.

scanning for the right signals and then protrusion.

paracellular/transcellular migration.

tight junctions.

PECAMs, JAMs, junction adhesion molecules, intracellular adhesion molecules 1/2.

form homophilic interactions.

bind partner and form tight zip like structure that prevents cells from crossing blood vessel to tissue.

migrating cell responding to chemoattractants, squeezes through these tight junctions and unzips tight homophilic interactions.

how cell motility relies on remodelling cell shape.

highly controlled to allow cell polarisation and coordinated movement in response extracellular cues.

integration of motility signals into coordinated cell shape remodelling ensured by actin cytoskeleton.

actin cytoskeleton

acts as coordinator/facilitator to ensure everything integrated and occurs right order.

building block = globular actin (polymerises into complex filament with branching and crosslinking).

actin networks undergo rapid/dynamic remodelling.

basis for emission of diverse protrusion types at leading edge (e.g. filopodia, lamellipodia, pseudopodia).

allows cell to sense physical constraints of environment and assemble force generating protrusions that lead to cell body translation (migration/movement).

actin stain

Dendra2-LifeAct.

migrating T cell example (Dendra2-LifeAct)

primary CD8+ hT cell expressing Dendra2-LifeAct moving along CXCL12 (SDF1) gradient created in a collagen IV-coated slide.

cell extends dynamic protrusions and moves towards source of CXCL12.

spike like protrusions at leading edge.

dynamic changes to actin cytoskeleton as cell migrates.

lamellipodium (lamellipodia) and filopodia.

actin projection on leading edge.

supported by actin meshwork.

contains 2D actin mesh.

whole structure propels cell across substrate.

within are ribs/spikes of actin (microspikes).

microspikes spread beyond lamellipodium frontier (filopodia - sharper form of microspike).

pseudopodium

temporary cytoplasm-filled projections of cell membrane.

used for motility or ingesting nutrients/other particle matter (depending on cell).

cell guided system into three networks (+ PI3K)

gradient sensing (chemoattractant cue) - cell adapts/amplifies cue into biochemical signals. chemical signals underpin gradient signalling.

these signals regulate actin.

leads to cell polarisation and movement.

PI3K is involved in all three (shared).

F-actin and cell migration (and others)

need continuous F-actin formation at leading edge for migration.

chemoattractants stabilise polarisation by stimulating actin polymerisation at leading edge.

polarised cell, F-actin and lipids show polarised distribution.

PIP3 appears prior to polarisation and, through PH domain containing proteins, facilitates translocation of protein to membranes, some amplify signal directionally.

PI3K class 1 subunits

PI3K is lipid kinase.

class 1 - consist of regulatory p85 subunit and catalytic subunit.

class 1a catalytic subunit isoforms - p110a, b, d.

share overall structure (regulatory subunit, binding site, RAS GTPase binding sites, PIK domain, catalytic domain).

class 1b - p110g - regulated by GPCRs.

lipid interactions

interact with proteins via PH and FYVE domains.

leads to recruitment of proteins to membrane surface.

PI3K

phosphorylates phosphatidylinositol lipids.

inositol head group sitting on diacyl glycerol backbone with several fatty acid side chains.

phosphorylates 3’ hydroxyl prosition on inositol head group.

singularly phosphorylated - PI3 monophosphate.

or combination - bisphosphate (3,4) or triphosphate (3,4,5).

only PI3K phosphorylates 3’ position.

3’ phosphorylaed lipid (PIP, PIP2, PIP3) interact with structural regions such as PH and FYVE domains.

PI3K lipid - metabolic pathways

receptor activated PI3K (GPCR or protein tyrosine kinase) generates PIP3.

3,4 bisphosphate occasionaly activated by receptors (but by-product of PIP3 metabolism).

both lipids interact with PH domains containing proteins, (PKB/Akt).

downregulate downstream effectors (PLC gamma).

GEF - guanine nucleotide exchange factor.

PI3K dependent signalling cascade by inositol phosphatases

regulated by phosphatases to suppress PIP3 levels (cal lead/generate cancer).

SHIP (SH2 domain containing inositol phosphate) leaves phosphate from 5’ hydroxide position to generate 3,4 bisphosphate (interacts PH domains).

PTEN cleaves 3’ hydoxyl and converts PLC isoforms giving IP3 and DAG.

PTEN mutated in many cancers.

bioprobes

used to visualise actin.

GFP - green fluorescent proteins.

take DNA that encodes fluorescent protein and insert into mammalian gene (can be expresses in multiple cell types).

act as probe to visualise intracellular proteins or act as surrogate markers for lipid localisation etc.

GFP-PH domains expressed in cells

fuse GFP DNA to DNA that codes PH domain (protein construct).

put in cell.

if express protein in cell, exists in cytoplasm (green under microscope).

stimulate with T cell antigen receptor antibody, see polarisation of fluorescence to cell membrane (away from cytoplasm).

relocates close to receptor which stimulated by PI3K and produces PIP3.

attracts and interacts with PH domain, taking GFP along to visualise intracellular localisation of PIP3 through accumulation.

used to study mechanism of cell migration in amoebae.

Dicytostelium discoideum amoebae

excellent model for chemotaxis.

contains 3 PI3Ks related to class 1 PI3Ks - PI3K1 and PI3K2 (similar to P110a) and PI3K (similar to PI3Ky).

chemotax/migrate via g protein coupled cAMP or folic acid receptors.

cAMP - attractant to bring cells/community together.

released from discoideum when want to build population (starvation instances).

binds specific cell surface receptors (same as folic acid).

expression of GPCRs and Gb subunits are mostly uniform.

PH-CRAC-GFP in WT Dictyostelium

probes.

GFP-PH protein construct.

PH domain from cytosolic regulator of adenylyl cyclase (CRAC).

exposed Dictyostelium to source of chemoattractant at end of pipette.

see migration to source (swimming towards source).

PIP3 localisation lights up due to GFP-PH domain probe - well defined leading edge.

GFP-tagged PH domain of CRAC translocated to leading edge within 20-60 secs.

PH-CRAC-GFP PTEN -/- Dictyostelium

negative for PTEN.

multiple leading edges.

broad irregular front.

three or more pseudopodia.

lots of PIP3 generated.

move but not straight to source/misguided.

GFP-PI3K accumulates at leading edge.

similar studies on neutrophils (GFP-Akt PH domains).

Ca2+ release activated Ca2+ channel.

positive feedback loop evidence

neutrophil-like cell line model.

exogenous PIP3 elicits accumulation of endogenous PIP3 at leading edge.

dependent on functional PI3K and Rho family GTPase (inhibited by LY294002 and Toxin B).

inhibition prevents PIP3 induced accumulation of endogenous PIP3.

amplification of internal PIP3 gradient disrupted by agents which inhibit reorganisation (latrunculin, jasplakinolide or toxins).

actin reorganisation facilitates PIP3 induce accumulation of endogenous PIP3.

block actin polymerisation with latrunculin, jasplakinolide or toxins.

reciprocal interplay between PIP3 and polymerised actin.

maintains asymmetry of intracellular signals responsible for cell polarity and directed motility.

subcellular re-localisation of PTEN is Dictyostelium

exposed to gradient from cAMP filled micropipette.

PTEN-GFP accumulates at lateral regions and back of cells.

neutrophils and PIP3 production

PI3K gamma required for PIP3 production.

PI3K senses and interprets spatial cue provided by gradient and facilitates orientation of polarised cell.

PI3K deficient cells lose directionality but still polarise.

PTEN -/- neutrophils

didn’t have elevated PIP3 (lipid product of PI3K).

migrate normally - detect gradient and migrate towards it.

different cell types use SHIP or PTEN to control PIPs and regulate actin cytoskeleton.

SHIP -/- neutrophils

flat un-polarised profile.

increased membrane extensions labelled with Akt-PH GFP (fluorescent marker).

impaired speed of migration.

different cell types use SHIP or PTEN to control PIPs and regulate actin cytoskeleton.

neutrophils from PI3K -/- mice

still respond/migrate to chemoattractant gradient (e.g. fMLP - bacterial product).

migrate at half speed of WT.

strong adhesion defects to substrate - explains slow migration.

reduced number of cells that are polarised after a long period of time.

PI3Ky sustains level of migration but reduced rate over long period time.

cytosolic compartment marker

cell tracker organge.

cell membrane marker

DilC16.

HL60 cells responding to fMLP

neutrophilic cell line.

fMLP-induced chemotaxis of DMSO differentiated HL60 cells expressing PTEN-GFP and polarisation marker RFP-GPI (raft associated protein).

PTEN-GFP evenly distributed in cytosol.

RFP-GPI accumulated at leading edge and uropod in moving cells.

RFP-GPI

glycerol phosphatidylinositol lipid stain.

stains membrane rafts which denotes leading edge.

Jurkat

leukaemia T cell line widely used in T cell migration assays.

don’t express PTEN or SHIP.

migrate towards chemotactic gradient in absence PTEN and SHIP.

Jurkat responding to SDF-1

reconstitution with PTEN-GFP chimera dynamics during Jurkat cell chemotaxis is evenly distributed in cytosol.

RFG-GPI accumulated at leading edge and uropod.

lots of PIP3 accumulation.

lots of growth (no suppression).

SHIP -/- mice (inflammation phenotype in vivo)

dies because of unregulated accumulation of leukocytes to lung.

SHIP - germline mutation.

zebrafish model

depletion of SHIP increased neutrophil 3D motility and neutrophil infiltration into wounds.

rescued by PI3Ky inhibition.

overexpression SHIP impaired neutrophil 3D migration.

use morpholino knockdown.

WT - induce laser wound to tail fin, saw accumulation of inflammatory responses.

morpholino (knockdown SHIP) - increase accumulation of neutrophils (pre-treatment of PI3Ky inhibitor can rescue).

DOCK2 -/- mice

DOCK2 - downstream of Crk180 homolog 2).

DOCK2 - Rac-specific guanine nucleotide exchange factor.

DOCK2 is predominant signal.

normal activation of PI3K.

major defect in T cell migration in vitro and homing T cells to lymph nodes in vivo.

cell compass

receptor/g-protein signalling orient’s cell compass.

activates PIP3 pathway and unknown parallel pathways.

signals from compass integrated with inputs before regulating PI3K, PTEN, and SHIP activity.

PI3Ky -/- cells - signalling through parallel pathways hence moderate effects to migration.

navigation systems

cells navigate through complex chemoattractant fields.

PI3K - dispensible signal for directional cell migration.

trafficking of naive versus effector T cells

balance and differentiation between them is crucial for adaptive immune responses.

naive:

• express CCR7 and CD62L.

• attracted by chemokines (CCL21 - released by lymph nodes).

• scan/identify antigens presented by DCs in lymph nodes,

• activated, egress/released and migrate to anatomical sites due to address code.

T cell trafficking and TCR

H-Y antigen - male tissue specific antigen.

PI3K well documented and robust signal elicited upon TCR ligation.

PI3K p110-delta required for tissue infiltration by antigen specific T cells.

female and male mice treated with IFNy

treated with i.p. IFNy.

PKH26 labelled WT or p110-delta D910A H-Y-Ab restricted CD4+ T cells injected i.p.

presence of labelled T cells in peritoneal membrane and lavage analysed after 24h by wide-field fluorescent microscopy.

two mechanisms for KLF2 regulation

metalloprotenase (TACE-ADAM17):

• caused CD62L shedding.

• removing receptor important for recruiting to lymph nodes.

mTOR activation:

• decreased homing of activated T cells.

• mTOR suppresses transcription of KLF2 (transcription factor).

• remove expressed receptors on activated T cells.

  • homing to peripheral tissue.

serine/threonine kinase

integrates signals from growth factors, nutrients, and stress.

regulates mRNA translation, cell cycle progression, autophagy, and cell survival.

mTOR (a serine/threonine kinase) is target of rapamycin.

mTORC1

sensitive to rapamycin.

contains mTOR and associated proteins (raptor, deptor, PRA40, and mLST8).

controls protein synthesis and cell growth.

translation regulator of eIF4E binding protein 1 (4E-BP1).

s6 kinase 1 (S6K1).

mTORC2

insensitive to rapamycin.

composed of mTOR, rictor, mLST8, mSin1, and protor.

modulates cell survival in response to growth factors.

regulate actin cytoskeleton organisation and cell migration.

mTORC2 in Dictyostelium

cells lacking LST8, Rip3 (mSin1 homolog), or Pianissimo (rictor homolog) all exhibit cell polarity and chemotaxis defects.

Akt via FOXO

FOXO - (p)-forkhead box O.

FOXO sequestered in cytoplasm.

Akt binds 14-3-3 proteins - prevents transcription of target genes (KLF2).

FOXO accesses nucleus - drives KLF2 expression.

expression of adhesion and chemokine receptors (e.g. CD62L, CCR7, S1P1).

strong Akt leads to FOXO phosphorylation.

once p-FOXO binds 14-3-3 it is sequestered - prevents relocation to nucleus (no KLF2).

Rheb

Akt phosphorylates and inactivates Rheb GTPase activating protein (TSC2).

leads to accumulation

cAMP and FRET

cAMP FRET sensor.

reporter construct - eYFP (acceptor) and eCFP (donor).

PKA regulatory subunit cAMP binding domain flanked by eYFP and eCFP.

visualise cellular amounts of cAMP.

low cAMP levels, probe remains closed state - FRET response maximal (optimal FRET).

presence cAMP, conformational change and FRET response lost.

cAMP in cells

levels lowest at front of cells (extensive actin remodelling takes place).

FRET signal highest at front of cells and lowest in cell body.

dynamic change in FRET levels (fluorescence) at back of chemotaxing cells - signal intermittently went up and down suring chemotaxis.

high cAMP levels = low fluorescence levels.

TORC2-mediated and AC9-dependent cAMP production in RhoA-ROCK-myosine pathway

AC9 knockdown cells, distribution of P-MLC dramatically extends to front of cells (multiple back sites in single cell).

rictor shRNA cells, devoid of P-MLC mediated increase in cAMP levels and exhibit strong polarity defects.

AC9 knockdown leads to increased RhoA activity - decreased cAMP production.

causes increased ROCK-mediated phosphorylation of MLC - enhanced back activity.

P-MLC - phosphorylated mysosin light chain (always stains at uropod).

no PKA stimulation with reduced cAMP levels.

cells don’t know which way is back.

TORC2 controls neutrophil polarity and chemotaxis

TORC2 at front of cells.

controls polarity of F-actin.

local activation of PKC.

PKC —> AC9 activation and cAMP production.

TORC2/PKC dependent cAMP production oscillates (constantly changes).

cAMP enriched at uropod.

PKA acts on Rhoa and/or MLCK.

cAMP and PKA

cAMP through PKA actiavtion, provides antagonistic signals to RhoA/MyoII signalling pathway by phosphorylating RhoA.

inhibitory effects of PKA - mediated through phosphorylation of MLCK (inhibits activity).

PKA negatively regulates at least 2 RhoAGEFs (RhoAGEF Lfc and AKAP-Lbc).

MyoII activity stimulated by phosphorylation of MLC at Ser19.

stability of MyoII filaments more significantly regulated by MLC dephosphorylation at Thr18 and Ser19.

dephosphorylation step required for rapid filament assemble through rapid disassembly of preexisting filaments.

as cells chemotax up chemoattractant gradients, experience increased receptor activation that triggers activating/inhibiting signals leading to cycling of MyoII phosphorylation/dephosphorylation and optimal migration.

WNK1 regulation of actin reorganisation

WNK1 - member of WNK family of serine/threonine proteins.

regulate ion homeostasis in kidney epithelial cells - promote uptake of Na+, K+, and Cl- ions from urine.

activated, WNK1 phosphorylates/activates OXSR1 and STK39.

these phosphorylate SLC12A family members (electroneutral ion co-transporters) - alters activity.

phosphorylation of SLC12A2 (NKCC1) - influx Na+, K+, and Cl- ions.

phosphorylation of SLC12A6 (KCC3) - blocks wffulx of K+ and Cl- ions through transporter.

signalling from WNK1 results in net influx of ions.

WNK1 and immune functions

WNK1 - strongly expressed in thymus and spleen.

RNAi screens identified WNK1 as regulator of integrin-mediated adhesion and T cell migration.

NKCC1 inhibition (bumetanide) decreased chemotaxis effect of CCL21 (chemokine) in T cells.

WNK1 pathway proteins required for CCL21 induced migration of CD4+ T cells.

CCL21 increases K+ ions.

WNKi (inhibition) - reduced amount of K+ in T cells and blocked CCL21 induced increase.

WNK1 pathway proteins regulate water entry and cell volume.

WNK1 and water entry in migration of T cells

CCL21L binding CCR7 at leading edge activated WNK1.

phosphorylates/activates OXSR1 and STK39 (kinases).

phosphorylates SLC12A family members, resulting in entry of Na+, K+, Cl- ions into cell.

water entry through AQP3 (and other aquaporins) by osmosis.

water entry swells plasma membrane.

creates space for actin filament extension.

protrudes the cell forward.

water entry via aquaporin channels

RNA-seq analysis shows naive CD4+ T cells predominantly express AQP3 and loswer levels of AQP9 and 11.

DFP00173 (AQP3i) - selective AQP3 inhibitor (causes significant descrease in speed of CCL21 induced T cell migration).

AQP3i - reduced CCL21 induced polarisation.

SLC12A2i - AQP3i didn’t change basal volume T cells but eliminated CCL21 increase in volume.