CSB331H1 Lecture Notes - Lecture 8: Proline, Fibronectin, Paxillin
8 Jun 2018
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Lecture'8:'Cell'Migration'
Lipid4Protein'Docking'Sites!
" there!is!a!polarized!distribution!of!
phosphoinositides!in!epithelial!plasma!
membrane!
" kinase!phosphorylates!the!third!position!of!
phosphotidylinositol!
" tight!junctions!activate!PTEN!=!high!
concentration!of!PIP(3,4)P2!apically!
" integrin!signaling!activates!PI3"K!=!high!
PIP(3,4,5)P3!basally!
!
Model'Connecting'PI34K'Activation'and'Actin'
Nucleation'at'the'Leading'Edge'of'Migrating'Cells!
" growth!factors!have!to!recruit!and!activate!
kinases!that!turn!them!off!or!on!
" kinases!creates!PIP3!
" Activation!of!actin!filament!assembly!by!
PI(3,4,5)P3!through!Rho!GTPases!
A. extracellular!cues!can!activate!signal!
transduction!pathways!that!result!in!local!
activation!of!PI3K!at!the!plasma!
membrane!
B. increased!PI(3,4,5)P3!concentration!
recruits!PI(3,4,5)P3"binding!proteins!
such!as!Vav,!a!Rac1'GEF!protein,!to!the!
plasma!membrane!
C. when!you!phosphorylate!Rac1!GEF!=!
autoinhibitory!domain!is!released!
" exchange!of!Rac1!from!GDP"!to!
GTP"bound!state!to!activate!this!
protein!=!Rac!is!now!activated!
D. activated!Rac1!signaling!leads!to!induction!of!the!Arp2/3"mediated!actin!filament!assembly!
" growth!factor!stimulant!!!tyrosine!kinase!activity!!!creates!a!docking!site!for!phosphorylating!of!targets!!!
exchange!of!GDP!to!GTP!!!activated!Arp!complex!
!
Actin'filament'nucleation'and'web'formation'by'
the'ARP'complex'of'lamellipodia'
" Keratocyte!with!actin!filaments!labeled!in!
red!by!fluorescent!phalloidin!and!the!ARP!
complex!labeled!in!green!with!antibody!
raised!against!one!of!its!component!protein!
" The!regions!where!the!two!overlap!appear!
in!yellow!
" The!ARP!complex!is!highly!concentrated!
near!the!front!of!the!lamellipodium,!where!
actin!nucleation!is!most!active!
" Although!the!dense!actin!network!reaches!
all!the!way!through!the!lamellipodium,!
cofilin!is!not!found!at!the!very!leading!edge!
o Cofilin!is!found!abundantly!
throughout!the!cell!
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RhoA'activity'is'required'for'cell'traction'4'“pull”'phase''
" focal!contract/focal!adhesion!formation!
" Myosin!II!bipolar!filaments!bind!to!actin!filaments!
in!the!dendritic!lamellipodia!meshwork!and!cause!
network!contraction!
" The!myosin"driven!reorientation!of!the!actin!
filaments!in!the!dendritic!meshwork!forms!an!
actin!bundle!that!recruits!more!myosin!II!and!
contributes!to!generating!the!contractile!forces!
required!for!retraction!of!the!trailing!edge!of!the!
moving!cell!
" Many!of!the!fragments!continue!to!move!rapidly!
with!the!same!overall!cytoskeletal!organization!as!
the!intact!keratocyte!
o Actin!(blue)!forms!a!protrusive!
meshwork!at!the!front!of!the!fragment!
o Myosin!II!(pink)!is!gathered!into!a!band!at!
the!rear!
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Adhesive'Cells'Exert'Force'on'Substratum'
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Focal'Complexes'and'Focal'Adhesions'are'Signaling'Centres'
" Actin!cytoskeletal!dynamics!are!required!for!integrin!clustering!as!well!as!
for!the!maturation!and!remodelling!of!these!complexes!during!migration!
" Actin!filaments!terminate!at!focal!adhesions!(FAs),!where!integrins!link!
to!ECM!
" Concentration!of!proteins!containing!P4tyr!at!these!sites!
o Lots!of!phospho"tyrosine!residues!at!focal!adhesion!points!
o Reflects!local!activation!of!protein!tyrosine!kinases!
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