Article #8 Summary

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24 Dec 2010
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CSB428H1F Article #8: Siegrist et al.Drosophila MT induced Pins/Gi
Introduction
Astral MT, kinesin, Dlg induce cortical polarization of Pins/Gi in neuroblasts. The cortical domain
generates spindle asymmetry, daughter cell size asymmetry and sibling fates.
Kinesin localizes to (+) ends, and Dlg/kinesin, Dlg/Pins co-immunoprecipitate, suggesting polarity
is induced by Dlg/kinesin interactions
MT/Kinesin/Dlg pathway acts in parallel to Inscuteable/Par pathway, but Inscuteable pathway is at
prophase coordinating with CNS and MT pathway is at metaphase coordinating mitotic spindle axis.
Neuroblast cortical polarity involves Baz/aPKC/Par-6 and Insc, Partner of Insc (Pins), Gi to the
apical cortex during interphase.
At metaphase NMY-2, Dlg, Scrib and Lgl localize to apical as well.
Astral MT induce pins/Gi/Dlg cortical polarity at metaphase
insc mutant lacks apical Insc/Baz/aPKC/Par-6, but Pins/Gi/Dlg still formed crescents. The crescents
were found all around cortex abnormally and delayed from prophase to metaphase.
This suggests formation of Pins/Gi/Dlg crescents at metaphase is independent of Insc/Par pathway.
MT disrupting agent Colcemid treatment of insc mutant caused loss of Pins/Gi/Dlg crescents. Pins
were cytoplasmic, and Gi/Dlg was uniformly cortical.
Colcemid treatment did not affect WT embryo because Pins/Gi/Dlg binds Insc/Par complex apically.
aPKC mutant lacking aPKC/Par-6 localization but retained Baz/Insc localization still formed
Pins/Gi/Dlg crescents in absence of MT showing Pins/Gi/Dlg pathway only require Insc and Baz.
WT cells treated with nocodazole lacked astral MT and cannot align their spindle, but Pins/Gi/Dlg
with Insc/Par complex at cortex was normal.
insc mutant treated with nocodazole lost Pins/Gi/Dlg crescent. insc/fizzy double mutant showed
same loss of crescents, Pins was delocalised from the cortex, and Gi/Dlg was uniformly cortical
This suggests astral MT is required for Pins/Gi/Dlg crescent formation in absence of Insc/Par.
Dlg is necessary for MT induced Pins/Gi polarity
dlg/insc double mutants had defects in Pins/Gi cortical polarity: Pins was cytoplasmic and Gi was
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uniformly cortex but astral MT was present.
With Insc/Par pathway present, Pins/Gi crescent form even without Dlg or MT, therefore Dlg is
required specifically for MT-induced Pins/Gi polarity.
insc mutant failed to localize Mira to basal bur showed Insc/Par independent rescue of basal Mira at
telophase.
dlg/insc double mutant lacked telophase rescue of Mira showing MT/Dlg pathway both directly and
indirectly induce basal polarity.
Mutant for both SH3 and GK domain of Dlg in dlg/insc showed uniformly cortical Dlg/Gi,
cytoplasmic Pins, so both domains are required for crescent formation and Pins/Gi polarity.
Dlg GK domain is necessary for Pins/Gi/Dlg spindle alignment
Mitotic spindle was aligned with Pins/Gi/Dlg in both WT and insc mutants but showed poor
alignment in dlg/insc double mutants. Those with SH3 domain mutated showed normal alignment
but those with GK domain truncated did not align.
Kinesin binds Dlg and is necessary for MT-induced Pins/Gi/Dlg crescent formation
Both full length Kinesin and only its MBS domain co-immunoprecipitated with endogenous Dlg but
not Pins.
Kinesin knockdown in showed strong Pins crescent localization with apical Insc/Par. But in insc
mutants, Pins delocalised into cytoplasmic and Dlg was uniform around cortex but astral MT was
present.
Overexpression of MBS in insc mutant caused cytoplasmic Pins and uniform Dlg but MBS alone
did not cause defect. Therefore Kinesin/Dlg interaction is important for cortical polarity.
Kinesin localized to (+) tip of astral MT and binds Dlg at the same time.
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