Elucidating the mechanisms of Orb2 mediated neural stem cell asymmetry and division

Project Summary:
The centrosome is a membraneless organelle comprising a pair of centrioles surrounded by pericentriolar
material, which nucleates microtubules to direct cellular trafficking and mitosis. Cancer cells frequently possess
extra or aberrant centrosomes, which are associated with poor prognosis. Multiple mechanisms for centrosome
overexpansion in cancer cells are proposed, including failures in cytokinesis and unregulated centrosome
duplication. Centrosome amplification produces erroneous mitotic spindles, which lead to chromosomal
segregation defects, contributing to tumorigenesis and cancer progression. Drosophila neural stem cells
(NSCs) represent a genetically tractable model to study mechanisms by which centrosomes assure proper
mitotic potency. High grade brain cancers frequently exhibit centrosome amplifications, illustrating how the
Drosophila NSC model system informs foundational cancer cell biology. NSCs undergo repeated rounds of
asymmetric cell division along an invariant apical-basal polarity axis to regenerate a self-renewing stem cell
and a daughter cell fated for differentiation. Our lab recently discovered that loss of the RNA-binding protein
Orb2 results in centrosome amplification, dysregulation of centrosome asymmetry, and spindle alignment
errors in NSCs, but the mechanisms behind these defects remain elusive. Orb2 is a conserved cytoplasmic
polyadenylation element binding protein (CPEB) ortholog involved in the translational regulation of mRNAs. I
hypothesize that Orb2 represses the translation of specific centrosome and spindle-associated RNAs
to control NSC asymmetric cell division. Importantly, my preliminary data show that the basal centrosome is
hyperactivated in orb2 null NSCs. To determine the mechanism by which Orb2 influences asymmetric
centrosome maturation (Aim1), I will 1) test whether Orb2 represses the translation of the centrosome
activation targets aurA, polo, cnb, or wdr62, and 2) determine if Orb2 requires RNA-binding activity to promote
NSC centrosome asymmetry. To determine the mechanism by which Orb2 influences spindle alignment and
centrosome segregation (Aim2), I will 1) test whether Orb2 represses the translation of spindle stability targets
msps, tacc, or eb1; and 2) live image control and orb2 null NSCs to characterize the formation of dysmorphic
spindles and supernumerary centrosomes. This proposed study will reveal how centrosome and mitotic
asymmetry is guided by translational control of centrosome and spindle proteins, providing insight into how
post transcriptional regulation of the centrosome cycle can lead to cancer.

Grant Number: 5F31NS134380-03
NIH Institute/Center: NIH
Principal Investigator: Joseph Buehler