Article #4 Summary

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24 Dec 2010
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CSB428H1F Article #4: Martin-Belmonte et al. PTEN control PIP and Cdc42
PTEN localizes to apical PM to increase PI45P2 by decreasing levels of PI345P3. Ectopic PI45P2 at
basolateral surface cause apical proteins to relocate to basolateral side.
Annexin 2 (Anx2) binds PI45P2 and is recruited to apical surface to bind Cdc42, which recruits
aPKC. Loss of PTEN, Anx2, Cdc42 or aPKC prevents apical surface and lumen formation.
Madin-Darby-canine kidney (MDCK) cells embedded in gels for cysts with lumen.
PI45P2 and PTEN localize to AP PM
PH domain of PLC fused to GFP (PHD-GFP) detects PI45P2. PHD-GFP localized mainly to AP PM
and little bit in BL PM, similar to that of actin.
PH-Akt-GFP detects both PI34P2 and PI345P2 which are enriched at BL PM.
Before lumen formation, PI345P3 and PI45P2 localized to cell-cell and cell-ECM contacts. During
lumen formation, PI45P2 became confined to AP PM, but PI345P3 became exclusive to BL.
In early stages, GFP-PTEN is localized to the cytoplasm but later is localized to AP PM and
overlapped with PI45P2 and cell-cell junctions.
Loss of PTEN disrupts PI45P2
SiRNA treated cells formed multiple small lumen with decreased actin levels but BL marker -cat
was normal.
PTEN inhibitor bpV(pic) also inhibited lumen formation and both PI345P3 and PI45P2 became
homogenous in PM resulting in cyst with no lumen.
Therefore PTEN is required for apical enrichment of PI45P2 and AP PM lumen formation.
Exogenous PI45P2 localize AP protein to BL
Exogenous PI45P2 to BL PM induced shrinkage of lumen and patchy redistribution of PHD-GFP to
BL surface. AP marker gp135, ezrin, ZO-1 also re-localized to BL PM.
Lateral markers p58 and PI345P3 ended up in puncta at periphery and disappeared from basal
region. This shows ectopic PI45P2 to BL PM is sufficient to relocalize AP and TJ to periphery.
Anx2 bind PI45P2 at AP PM
GFP-Anx2 binds PI45P2 and is localized to AP PM overlapping with actin with small amount in BL
PM. Localization during cyst formation is identical to PI45P2.
Loss of Anx2 inhibits lumen formation
SiRNA of Anx2 caused cells to form smaller cyst and multiple small lumens with reduced actin.
GFP-Anx2-DN inhibited formation of central lumen. Zerin and actin was absent in GFP-Anx2-DN.
Exogenous BL PM PI45P2 relocalized Anx2 from AP BL to BL PM.
PTEN inhibition caused Anx2-GFP to be homogenously distributed in PM. This shows Anx2 in
combination with PI45P2 is required for cortical actin, AP PM and lumen formation.
Anx2 binds Cdc42 at AP PM
Rac1-GFP was localized to BL PM and cell-cell junctions but Cdc42-GFP was enriched at AP PM
and colocalized with actin.
CBD-GFP detects activated Ccdc42. CBD-GFP and Cdc42-GFP was initially localized at cell-cell
and cell-ECM contacts. As lumen forms, CBD-GFP relocalized to AP PM with Cdc42.
Endogenous Cdc42 co-precipitated with Anx2-GFP and interaction was increased by non-
hydrolyzable GTPS. Anx2 interacted with itself and p11 to form heterotrimers
Anx2-GFP bind p11 stronger with GDP, while co-precipitation with Cdc42 decreased, suggesting
Cdc42 competes with p11 for Anx2 binding.
Chimera of N’NH2-N’p11 lead to cell aggregates with Anx2, p11, Cdc42-GFP and chimera and
disrupted AP cytoskeleton. Rac1-GFP and BL marker -cat was normal.
Cdc42 depletion inhibits lumen formation
SiRNA of Cdc42 reduced lumen formation and caused multiple small lumens to form with
decreased cortical F-actin and associated proteins, similar to PTEN or Anx2 knockdown. BL marker
-cat was normal.
AP marker gp135 was initially at external PM and later internalized in vesicles. Cdc42 SiRNA did
not affect relocalization but now vesicle cannot fuse with PM. TJ marker ZO-1 was normal.
Exogenous PI45P2 target activated Cdc42 to BL PM
PTEN and Cdc42 reduction reduced Cdc42 activity as measured by GST pull-down. Cdc42-DN
reduced activated Cdc42, but level of actin, gp135, ezrin and ZO-1 was normal. PTEN and Anx2
knockdown caused relocalization of Cdc42 from PM to cytoplasm.
Exogenous PI45P2 to BL PM induced relocalization of Cdc42-GFP and CBD-GFP from AP to
cytoplasm with enrichment at basal portion, similar to the redistribution pattern of PHD-GFP, Anx2,
gp135, ezrin and ZO-1.
Cdc42 targets Par6/aPKC to AP PM for lumen formation
Par6 links aPKC to activated Cdc42. cells depleted of Cdc42, PTEN or Anx2 show intracellular
aPKC and show co-localization with Anx2-GFP PI45P2 localization was normal.
DN-aPKC inhibited aPKC phosphorylation and formation of AP PM and lumen formation. ZO-1
distribution was normal.
Exogenous PI45P2 relocalized aPKC from AP to basal PM and followed similar redistribution
pattern to PHD-GFP, Anx2, active Cdc42, gp135, ezrin and ZO-1.
PTEN exclude PI345P3 at AP PM by forming PI45P2. PI45P2 binds Anx2 which binds Cdc42 which
binds Par6/aPKC at AP PM.