CSB428H1 Study Guide - Immunoprecipitation, Epistasis, Cantharidin
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CSB428H1F Article #6: Krahn et al.Formation of Baz-Sdt for epithelial polarity
Baz phosphorylation site bind PDZ of Sdt to recruit Sdt to PM.
Phosphorylation of Baz causes it to dissociate from Sdt. Non-phosphorylatable Baz blocks
dissociation causing mutation similar to crb and sdt.
Crb cytoplasmic domain binds PDZ domain of Sdt and Sdt recruit Patj and Lin-7 to apical complex.
Dlg/Lgl/Scrib localize to lateral PM and antagonize Crb-Sdt complex, restricting apical expansion.
BazS980 phosphorylation is required for localization
WT Baz-GFP and BazS980E-GFP shows the same localization basal to Crb-Sdt.
Phosphorylated BazS980-P was concentrated in most apical part, sometimes with/without Crb-Sdt.
WT Baz-GFP and BazS980E-GFP but not BazS980A-GFP rescued maternal and zygotic baz lethality.
Overexpression of BazS980A-GFP is similar to crb and sdt
Overexpression of BazS980A-GFP caused formation of aggregate containing DE-Cad, Arm, -cat,
Par-6, aPKC, Crb, Sdt, Patj and Lin7. Lateral marker Dlg was normal.
BazS980A-GFP overexpression caused cells to round up and die of apoptosis.
These dominant-negative effects of BazS980A-GFP were sell-autonomous. Deletion of Baz CR1 or
PDZ domains did not affect is dominancy. However, those lacking PM targeting signal was not
dominant, showing BazS980A-GFP must be at PM to be dominant.
In neuroblasts and oocytes, BazS980A-GFP localize apically like WT and cell fate determinants,
spindle orientation, cell division, and viability was not affected.
BazS980A-GFP mimic crb/sdt
crb, sdt, baz, aPKC, and Par-6 mutants secrete only little scraps of cuticle.
Overexpressing BazS980A-GFP caused a cuticle phenotype similar to crb or sdt mutants, but
phenotype can be rescued with lgl, dlg and scrib mutation, showing Crb antagonizes Scrib complex.
Overexpressing intracellular portion of Crb (Crbintra) caused ectopic cuticle release.
Overexpressing BazS980A-GFP with Crbintra has the same phenotype as overexpressing BazS980A-GFP
along, suggesting BazS980A-GFP is epistatic over Crbintra.
Baz is required to recruit Sdt
Sdt colocalized with Baz before Crb expression. Later Sdt colocalize with Crb, partially with Baz.
In crb mutant, Sdt remain colocalized with Baz. In baz mutant, neither Crb nor Sdt were detectable.
In Sdt mutant, Crb was mislocalized but Baz was normal.
This shows Baz is upstream of Sdt, binds Sdt independent of Crb, and Sdt is upstream of Crb.
Crb bind PDZ of Sdt
PDZ of Sdt can pull down Baz in GST assays.
Immunoprecipitation did not find Crb-Baz, but found Sdt-Baz. This excludes Par-6 as an
intermediate between Baz and Sdt.
Deletion of PDZ of Baz did not affect binding, but deletion of Baz C’ or aPKC binding region
abolished Sdt binding to Baz. This shows aPKC phosphorylation site of Baz binds PDZ of Sdt.
BazS980-P weaken Baz and Sdt binding
Upon phosphatase inhibitor cantharidin treatment, co-immunoprecipitation of Sdt and Baz was
reduced, suggesting Baz phosphorylation weakens binding to Sdt.
More Sdt co-immunoprecipitated with BazS980A-GFP, and was unaffected by cantharidin treatment,
confirming S980-P reduces Baz affinity for Sdt.
BazS980E-GFP bound less Sdt than BazS980A-GFP and less than WT Baz-GFP.
baz phosphorylation site bind pdz of sdt to recruit sdt to pm. Phosphorylation of baz causes it to dissociate from sdt. Non-phosphorylatable baz blocks dissociation causing mutation similar to crb and sdt. crb cytoplasmic domain binds pdz domain of sdt and sdt recruit patj and lin-7 to apical complex. dlg/lgl/scrib localize to lateral pm and antagonize crb-sdt complex, restricting apical expansion. wt baz-gfp and bazs980e-gfp shows the same localization basal to crb-sdt. Phosphorylated bazs980-p was concentrated in most apical part, sometimes with/without crb-sdt. wt baz-gfp and bazs980e-gfp but not bazs980a-gfp rescued maternal and zygotic baz lethality. Overexpression of bazs980a-gfp is similar to crb and sdt. overexpression of bazs980a-gfp caused formation of aggregate containing de-cad, arm, -cat, bazs980a-gfp overexpression caused cells to round up and die of apoptosis. However, those lacking pm targeting signal was not dominant, showing bazs980a-gfp must be at pm to be dominant.