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Article #8 Summary


Department
Cell and Systems Biology
Course Code
CSB428H1
Professor
U.Tepass

Page:
of 2
CSB428H1F Article #8: Siegrist et al.Drosophila MT induced Pins/G๎€i
Introduction
Astral MT, kinesin, Dlg induce cortical polarization of Pins/G๎€i 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), G๎€i 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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