THESIS
2017
xiv, 103 pages : illustrations (some color) ; 30 cm
Abstract
The spatial and temporal organization of the microtubule cytoskeleton requires γ-tubulin ring
complexes (γTuRCs), which initiate microtubule growth and mediate microtubule attachment
at microtubule-organizing centers, such as centrosomes and the Golgi complex. In a search for
γTuRC-interacting proteins, I identified the catalytic subunit of DNA polymerase δ (PolD1).
Polδ is a major DNA polymerase which is responsible for DNA replication. It is also involved
in DNA repair and recombination. PolD1 binds directly to γTuRCs and potently inhibits
γTuRC-mediated microtubule nucleation. Whereas PolD1 depletion through RNA interference
augments microtubule nucleation at the Golgi, PolD1 overexpression interferes with Golgi-based
nucleation. By contrast, the depletion of PolD1 does not i...[
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The spatial and temporal organization of the microtubule cytoskeleton requires γ-tubulin ring
complexes (γTuRCs), which initiate microtubule growth and mediate microtubule attachment
at microtubule-organizing centers, such as centrosomes and the Golgi complex. In a search for
γTuRC-interacting proteins, I identified the catalytic subunit of DNA polymerase δ (PolD1).
Polδ is a major DNA polymerase which is responsible for DNA replication. It is also involved
in DNA repair and recombination. PolD1 binds directly to γTuRCs and potently inhibits
γTuRC-mediated microtubule nucleation. Whereas PolD1 depletion through RNA interference
augments microtubule nucleation at the Golgi, PolD1 overexpression interferes with Golgi-based
nucleation. By contrast, the depletion of PolD1 does not influence centrosome-based
microtubule growth. Suppression of PolD1 expression impairs Golgi reassembly after
nocodazole-induced disassembly and causes defects of Golgi reorientation during cell
polarization. These results reveal a mechanism for the control of non-centrosomal γTuRC
activity and a mechanism that regulates Golgi-mediated microtubule organization and
functions.
A body of evidence has shown that CDK5RAP2 and Nedd1 play key roles in γTuRC
attachment to centrosomes, microtubule nucleation, and mitotic spindle organization. I
investigated the γTuRC association with CDK5RAP2 and Nedd1, and found that most of the
CDK5RAP2- and Nedd1-associated γTuRCs contain either CDK5RAP2 or Nedd1 and that
their binding with γTuRCs is controlled by unknown protein factors. I found that CDK5RAP2 and Nedd1-bound γTuRCs undergo dynamic changes of the complex composition during the
cell cycle. Moreover, the interphase and mitotic γTuRCs showed different microtubule-nucleating
activities in an in vitro assay. Therefore, γTuRCs are regulated in a cell cycle-dependent
manner. My results also suggest that CDK5RAP2 and Nedd1 may be under distinct
controls for interacting with γTuRCs.
In summary, I identified PolD1 to be a γTuRC inhibitor and revealed a new control mechanism
of γTuRC-mediated microtubule nucleation in the Golgi complex. In addition, I showed that
γTuRCs undergo cell cycle-dependent changes of subunit stoichiometry and associated
proteins. My findings expand our knowledge on γTuRC regulation studies and provide insights
into the organization mechanism on microtubule networks during the cell cycle.
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