Cell Migration Flashcards
Discuss the tole of PI3K-dependent & independent pathways that are involved in leukocyte migration & homing response (1500-WL)
What is PI3K (structure & classes; class1a (activation), class 1b (activation); PIP3-(in)dep signalling) (1) (1/13)
- group of PM-ass lipid kinases
- consist of 3 SU
- p85 reg SU
- p55 reg SU
- p110 cata SU
- categorised into 3 classes by diff struct & specific SBTs
- class 1a & 1b
CLASS 1a
- α, β, δ
- activated by memb-bound RTKs
- Bind to adaptor SU p85, wh is recruited by its pY binding domain (x2 SH2) to phosphorylated receptors or adaptor proteins (i.e., insulin-receptor substrate)
CLASS 1b
- γ
- activated by GPCR (generally Gi subtype)
- binds to adaptor/regulators (p101, p84/87) and to free G-proteins that = liberated by active GPCRs
ALL CLASS1 PI3Ks:
- phosphorylate PIP2 → PIP3 (2° messenger membrane-lipid)
- PIP3 = docking site for many prot that have PH domains → prolif, survival, growth, migration etc
- occurs via PDK1/AKT/mTOR pathway
OR
- prolif can be PIP3 independent
- RAS = recruited to activated RTK and induces prolif via Raf/Mek/Erk/Msk/Akt/mTOR pathway
Discuss the tole of PI3K-dependent & independent pathways that are involved in leukocyte migration & homing response (1500-WL)
What is migration, and trans-endothelial migration (1) (2/13)
- a central process in the development & maint of multicellular organisms
- T formation during embryonic development, wound healing & IR all require the orchestrated movement of cells in particular directions to specific locations
- leukocyte trans-endothelial migration (TEM) = crucial for app innate immunity & inflam responses
- when T = damaged/infected, leukocyte exit BV via
1) adhering to & probing vascular endothelial cells (VECs)
2) breaching endothelial C-C junctions
3) transmigrating across endothelium - TEM = critical rate limiting step
Discuss the tole of PI3K-dependent & independent pathways that are involved in leukocyte migration & homing response (1500-WL)
Steps in migration - Cell polarisation (molecules @ front (Cdc42, PAR, PKC, MTOC, Golgi); molecules @ back (PTEN, myosin II) (1) (3/13)
- some molecules accumulate @ back of the cell, & some @ front
- Actv Cdc42, PAR proteins & atypical PKC = involved in generation of polarity
- Directed vesicle trafficking towards leading edge of cell
- MTOC → relocalizes behind the nucleus and MT align along the axis of migration w/ individual filaments orienting toward the uropod, F-actin rich lamellipodium becomes populated with only few filaments, extending to the leading edge cell membrane, the MT deliver mRNA, secretory vesicles, integrins & sig molecules to the leading edge of migrating cells
- Golgi apparatus towards mid/front region
- PTEN & myosin II → involved in ≠ making protrusions @ back of cell
Discuss the tole of PI3K-dependent & independent pathways that are involved in leukocyte migration & homing response (1500-WL)
Steps in migration - Formation of a protrusion (nucleation & actin polymerisation; integrin activation & aggregation) (1) (4/13)
- protrusion & adhesion to SBT i.e., surf of endo cell
- req nucleation & growth of polymerised actin processes
- Nucleation = followed by polymerisation of actin, driven by profillin, ENA/VASP etc
- Adhesion via integrin activation and integrin aggregation (Talin/PKC/Rap1/PI3K & Rac/Cdc42 respectively)
- protrusions = stabilised by formation of adhesions
Discuss the tole of PI3K-dependent & independent pathways that are involved in leukocyte migration & homing response (1500-WL)
Steps in migration - Rear retraction (1) (5/13)
- cell moves through interstitial matrix/BV
- front of the cell becomes the back of the cell
- back has to be detached
- req disassembly of adhesion molecules
- req retraction of the back of the cell as it becomes the front
- Ca-activated prot involved (calpain & calcineurin)
- other molec inv. = FAK, Src, ERK, myosin II, MT, Rho & Ca
- FAK → reg cell adhesion & migration signals via integrin engagement & ass of focal adhesions by cata downstream sig
- auto-phos of FAK → PI3K engagement (via p85), → Akt → regulates FAK indirectly
- FAK/Akt/Rho-GTPas = triple complex inv in signal integration & cross talk.
- Rho → reg formation of stress fibres
Discuss the tole of PI3K-dependent & independent pathways that are involved in leukocyte migration & homing response (1500-WL)
PI3Kδ characterisation (dominant isoform; models (i.e., APDS)) (1) (6/13)
- dominant PI3K isoform in T-cells
- characterised using:
- PI3Kδ mutant mouse models & PI3K-Inhib
- people w/ ‘Activated PI3K Delta Syndrome’ (APDS)
- mouse models w/ hyperactive PI3Kδ
- studies showed how increased PI3K activity FXs T-cell functions
Discuss the tole of PI3K-dependent & independent pathways that are involved in leukocyte migration & homing response (1500-WL)
PI3Kδ general functions in migration (entry, exit; recruitment, retention; LFA-1; CD62L + CCR7, S1PR1) (1) (7/13)
- PI3Kδ cont:
- entry & exit of T-cells from lymph nodes
- recruit. to & retention of T-cells within inflam T
- PI3Kδ does this via:
- acting DS of TCR stim to actv major T-cell integrin (LFA-1) wh controls TEM of T-cells and their interac. w/ APCs
- suppresses cell-surf XPS’ion of CD62L and CCR7 = control migration of T-cells across high endo venules in lymph nodes, and S1PR1 controls lymph node egress
Discuss the tole of PI3K-dependent & independent pathways that are involved in leukocyte migration & homing response (1500-WL)
PI3Kδ signalling in T-cells (via PIP2) (1) (8/13)
- C1 PI3Kδ phos the D3-pos of the inositol ring of PIP2 to PIP3
- PIP3 bound by subset of SH2- and PH-DC prot
- so these prot will be recruited to memb
- initiates DS pathways
Discuss the tole of PI3K-dependent & independent pathways that are involved in leukocyte migration & homing response (1500-WL)
PI3K-dep specific example #1: PI3Kδ in Crk-dep integrin signalling general pathway (LFA-1 engagement, SFK, Csk/CasL complexes & c-Cbl, PI3K, PIP3, Rho-GTPase, actin polymerisation) (1) (9/13)
- LFA-1 engagement by ligands actv Src family kinases (SFK)
→ Csk/CasL complexes bind to c-Cbl
→ promotes SFK-dep phos of c-Cbl
→ creates binding site for p85 SU of PI3K
→ activates PI3K cata func i.e., localised production of PIP3
→ recruitment of GEF to PIP3-rich memb
→ Rho-GTPase actv.
→ Actin polymerisation & leading edge formation
Discuss the tole of PI3K-dependent & independent pathways that are involved in leukocyte migration & homing response (1500-WL)
PI3K-dep specific example #1: PI3Kδ in Crk-dep integrin signalling (PI3K, LFA-1 & Crk; outside-in signalling) (1) (10/13)
- PI3Kδ = actv DS of TCR & by co-stimulatory & cytokine receptors that stim phos of Y within YXXM motifs that bind to the SH2 domains of the p85 SU
- LFA-1 also induces PI3Kδ actv via outside-in signalling
- outside-in: mediates cellular resp induced by ligand binding to integrins
- engaged integrins direct CS-reorgan
- T-cells from Crk-/- mice had defects in actin polymerisation, leading edge formation & 2D cell migration
- Crk = essential mediator of LFA-1 signalling-induced phos of E3-UL, c-Cbl.
- Crk = essential to phos c-Cbl to recruit PI3K (CS-remodelling)
- Crk promotes integrin med-PI3K sig
Discuss the tole of PI3K-dependent & independent pathways that are involved in leukocyte migration & homing response (1500-WL)
PI3K-dep specific example #2: PI3Kδ in Klf2 & L-selectin signalling (L-selectin downregulation) (1) (11/13)
- TCR trig actv of PI3Kδ → ectodomain shedding & concomitant inhibition of Klf2-dependent XPS’ion of L-selectin in mouse T-cells
- Terminal diff. of naive T-cells → effector mem-T-cells involves L-selectin shedding & TC’ion shutdown of L-selectin gene
- drives trafficking of T effector memory cells from 2° lymphoid organs → peripheral T
- PIP3 generation (PI3K) actv Akt (phos) wh inhibits FOXO1-mediated TC of Klf2
- L-selectin = adhesion molecules, needs to be downreg so cell can be mobile
- L-selectin role is complex
- XPS’ion of L-selectin in human CD8 naïve central memory cells = essential for trafficking toward, & the immune surveillance of, peripheral lymph nodes
Discuss the tole of PI3K-dependent & independent pathways that are involved in leukocyte migration & homing response (1500-WL)
PI3K-indep specific example #1: PI3K accelerates, but is not needed for neutrophil chemotaxis to fMLP (accelerated intial, p38 MAPK, ATF-2, MMP-3) (1) (12/13)
- generation of PIP3 at the leading edge of chemotaxing cells ≈ dispensable
- may be needed for certain stimuli that attract migrating cells, b other sig cascade ≈ used for other ligands
- study looked at role, data showed that although PI3K enhances early responses to fMLP, it ≠ req for migration towards fMLP
- in vivo/vitro models of neutro chemotaxis to show PI3K = minor roles
- PI3K accelerated initial polarisation & chemotaxis
- had ≠ role in maintaining directionality & speed
- showed that the p38 MAPK pathway is key for chemotaxis to fMLP (initial polarisation & motility towards fMLP)
- can be actv by osmotic shock, inflam cytokines, LPS, UV light, and growth factors
- i.e., TGFβ receptor actv can actv p38 DS, actv TCF (i.e., cyclic AMP-dependent transcription factor-2; ATF-2) wh induces TC of genes needed for motility (MMP-3)
Discuss the tole of PI3K-dependent & independent pathways that are involved in leukocyte migration & homing response (1500-WL)
Combined role - proposed model for PI3K-(in)depen polarisation in mouse neutrophils (1) (13/13)
PROPOSED MODEL
- chemoattractant → neutrophils partially polarised without PI3K/PIP3
- @ this point, neutro = directional movement @ IMD speed
- PI3Kγ & SHIP1 confine PIP3 to prospective leading edge
- facilitates assembly of complete leading edge & full polarisation
- req for full motility
- PIP3 stim local actin reorg = supports membrane protrusion
- PI3K-/- (or inhib) cells migrate slowly
- disruption of SHIP1 = expansion of PM around entire cell circumference (≈ caused by ↑ PIP3)
- despite morphological irregularity → leading edge is established in both PI3K-/SHIP1-def neutro → orientation of chemotaxis = intact
- ∴ PIP3 facilitates formation of leading lamella protrusions needed for full polarisation/motility
- b/ ≠ needed for direction
- XS PIP3 outside leading edge = obstructs motility
- PI3K-I
- ↓ migration speed of WT-cells
- ↑ migration speed of SHIP1-/- cells
- ∴ SHIP1 ≈ role in restricting where PIP3 and by association F-actin can accumulate
- ∴ ≈ essential for formation of a single, stable pseudopod
