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<p>Robust regulation of spindle orientation is essential for driving asymmetric cell
divisions (ACDs), which generate cellular diversity within a tissue. During the development
of the multilayered mammalian epidermis, mitotic spindle orientation in the proliferative
basal cells is crucial not only for dictating daughter cell fate but also for initiating
stratification of the entire tissue. A conserved protein complex, including LGN, Nuclear
mitotic apparatus (NuMA) and dynein/dynactin, plays a key role in establishing proper
spindle orientation during ACDs. Two of these proteins, NuMA and dynein, interact
directly with astral microtubules (MTs) that emanate from the mitotic spindle. While
the contribution of these MT-binding interactions to spindle orientation remains unclear,
these implicate apical NuMA and dynein as strong candidates for the machinery required
to transduce pulling forces onto the spindle to drive perpendicular spindle orientation.
</p><p> In my work, I first investigated the requirements for the cortical
recruitment of NuMA and dynein, which had never been thoroughly addressed. I revealed
that NuMA is required to recruit the dynein/dynactin complex to the cell cortex of
cultured epidermal cells. In addition, I found that interaction with LGN is necessary
but not sufficient for cortical NuMA recruitment. This led me to examine the role
of additional NuMA-interacting proteins in spindle orientation. Notably, I identified
a role for the 4.1 protein family in stabilizing NuMA's association with the cell
cortex using a FRAP (fluorescence recovery after photobleaching)-based approach. I
also showed that NuMA's spindle orientation activity is perturbed in the absence of
4.1 interactions. This effect was demonstrated in culture using both a cortical NuMA/spindle
alignment assay as well as a cell stretch assay. Interestingly, I also noted a significant
increase in cortical NuMA localization as cells enter anaphase. I found that inhibition
of Cdk1 or mutation of a single residue on NuMA mimics this effect. I also revealed
that this anaphase localization is independent of LGN and 4.1 interactions, thus revealing
two independent mechanisms responsible for NuMA cortical recruitment at different
stages of mitosis. </p><p> After gaining a deeper understanding of how NuMA
is recruited and stabilized at the cell cortex, I then sought to investigate how cortical
NuMA functions during spindle orientation. NuMA contains binding domains in its N-
and C-termini that facilitate its interactions with the molecular motor dynein and
MTs, respectively. In addition to its known role in recruiting dynein, I was interested
in determining whether NuMA's ability to interact directly with MTs was critical for
its function in spindle orientation. Surprisingly, I revealed that direct interactions
between NuMA and MTs are required for spindle orientation in cultured keratinocytes.
I also discovered that NuMA can specifically interact with MT ends and remain attached
to depolymerizing MTs. To test the role of NuMA/MT interactions in vivo, I generated
mice with an epidermal-specific in-frame deletion of the NuMA MT-binding domain. I
determined that this deletion causes randomization of spindle orientation in vivo,
resulting in defective epidermal differentiation and barrier formation, as well as
neonatal lethality. In addition, conditional deletion of the NuMA MT-binding domain
in adult mice results in severe hair growth defects. I found that NuMA is required
for proper spindle positioning in hair follicle matrix cells and that differentiation
of matrix-derived progeny is disrupted when NuMA is mutated, thus revealing an essential
role for spindle orientation in hair morphogenesis. Finally, I discovered hyperproliferative
regions in the interfollicular epidermis of these adult mutant mice, which is consistent
with a loss of ACDs and perturbed differentiation. Based on these data, I propose
a novel mechanism for force generation during spindle positioning whereby cortically-tethered
NuMA plays a critical dynein-independent role in coupling MT depolymerization energy
with cortical tethering to promote robust spindle orientation accuracy. </p><p>
Taken together, my work highlights the complexity of NuMA localization and demonstrates
the importance of NuMA cortical stability for productive force generation during spindle
orientation. In addition, my findings validate the direct role of NuMA in spindle
positioning and reveal that spindle orientation is used reiteratively in multiple
distinct cell populations during epidermal morphogenesis and homeostasis.</p>
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