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<p>During development, oriented cell divisions are crucial for correctly organizing
and shaping a tissue. Mitotic spindle orientation can be coupled with cell fate decisions
to provide cellular diversity through asymmetric cell divisions (ACDs), in which the
division of a progenitor cell results in two daughters with different cell fates.
Proper tissue morphogenesis relies on the coupling of these two phenomena being highly
regulated. The development of the mouse epidermis provides a powerful system in which
to study the many levels that regulate ACDs. Within the basal layer of the epidermis,
both symmetric and asymmetric cell divisions occur. While symmetric divisions allow
for an increase in surface area and progenitor cell number, asymmetric divisions drive
the stratification of the epidermis, directly contributing additional cell layers
(Lechler and Fuchs 2005; Poulson and Lechler 2010; Williams, Beronja et al. 2011).
</p><p>Utilizing genetic lineage tracing to label individual basal cells I show that
individual basal cells can undergo both symmetric and asymmetric divisions. Therefore,
the balance of symmetric:asymmetric divisions is provided by the sum of individual
cells' choices. In addition, I define two control points for determining a cell's
mode of division. First is the expression of the mInscuteable gene, which is sufficient
to drive ACDs. However, there is robust control of division orientation as excessive
ACDs are prevented by a change in the localization of NuMA, an effector of spindle
orientation. Finally, I show that p63, a transcriptional regulator of stratification,
does not control either of these processes, rather it controls ACD indirectly by promoting
cell polarity. </p><p>Given the robust control on NuMA localization to prevent excess
ACDs, I sought to determine how targeting of NuMA to the cortex is regulated. First,
I determined which regions within the protein were necessary and sufficient for cortical
localization. NuMA is a large coiled- coil protein that binds many factors important
for ACDs, which include but are not limited to: microtubules, 4.1, and LGN. Interestingly,
while the LGN binding domain was necessary, it was not sufficient for proper NuMA
localization at the cortex. However, a fragment of NuMA containing both the 4.1 and
LGN binding domains was able to localize to the cortex. Additionally, the NuMA-binding
domain of 4.1 was able to specifically disrupt NuMA localization at the cortex. These
data suggested an important role for a NuMA-4.1 interaction at the cortex. While
the 4.1 binding domain was not necessary for the cortical localization of NuMA, it
was important for the overall stability of NuMA at the cortex. I hypothesize that
4.1 acts to anchor/stabilize NuMA at the cortex to provide resistance against pulling
forces on the mitotic spindle to ensure proper spindle orientation.</p><p>Finally,
to determine if post-translational modifications of NuMA could regulate its localization
I tested the importance of a conserved Cdk-1 phosphorylation site. Interestingly,
a non-phosphorylatable form of NuMA localized predominately to the cortex while the
phosphomimetic protein localized strongly to spindle poles. In agreement with these
data, use of a CDK-1 inhibitor was able to enhance the cortical localization of NuMA.
Unexpectedly, the non-phosphorylatable form of NuMA did not require LGN to localize
to the cortex. Additionally, restoration of cortical localization of the phosphomimetic
form of NuMA was accomplished by the overexpression of either LGN or 4.1. Thus, phosphorylation
of NuMA may alter its overall affinity for the cortex. </p><p>Overall, my studies
highlight two important regulatory mechanisms controlling asymmetric cell division
in the epidermis. Additionally, I show a novel role for the interaction between NuMA
and 4.1 in providing stability at the cortex. This will ultimately provide a framework
for analysis of how external cues control the important choice between asymmetric
and symmetric cell divisions.</p>
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