Microtubule Nucleation and Organization During the Cell Cycle

How a cell organizes its interior and divides are central questions in cell and developmental biology. Research in my lab focuses on understanding how the cell nucleates and organizes microtubules to achieve intracellular organization and cell division.

The major microtubule nucleation site inside an animal cell is the centrosome. One research avenue in my lab is to understand the structure and function of the centrosome using Xenopus, Drosophila, and mammalian tissue culture cells. The centrosome consists of a pair of centrioles and an electron-dense pericentriolar material (PCM) which harbors the activity for microtubule nucleation and organization. We discovered a γ-Tubulin containing Ring Complex (γTuRC) and showed that it can nucleate microtubules in vitro. Furthermore, we found, that γTuRC is essential for centrosomes to nucleate microtubules. Our current hypothesis is that the γTuRC is the major microtubule nucleator at the PCM. γTuRC consists of approximately 6 presently uncharacterized proteins in addition to γ-tubulin. We are using a combination of molecular genetic, biochemical and genetic approaches to understand this ring complex. We are particularly interested in addressing how the γTuRC is involved in regulating microtubule nucleating activity of the centrosome, how it is recruited to and assembled at the PCM and whether and how it is involved in centrosome duplication.

In animal cells, the transition from interphase to mitosis is accompanied by dramatic changes in cellular architecture such as nuclear envelope break down, chromosome condensation and spindle formation. Another direction in my lab is to understand the signals that regulate spindle assembly during mitosis. The reorganization of the interphase microtubule array into a highly dynamic mitotic spindle requires more than the presence of centrosomes and the conversion of cytosol into a mitotic state. Several studies have shown that nuclear signals released into the cytoplasm upon nuclear envelope breakdown exert many different effects on microtubule arrays. Recently, we discovered that the nuclear GTPase, Ran, can stimulate microtubule aster and spindle formation in the absence of both centrosomes and chromosomes. Our findings suggest that Ran is the nuclear signal that regulates microtubule assembly in mitosis.

Recently we found that a carboxyl-terminal region of the nuclear mitotic apparatus protein (NuMA), a nuclear protein required for organizing mitotic spindle poles, mimics Ran’s ability to induce microtubule asters. This NuMA fragment also specifically interacted with the nuclear transport factor, importin-β, a receptor for protein import into the nucleus. Importin-β is an inhibitor of microbutule assembly in Xenopus egg extracts, and Ran regulates the interaction between importin-β and NuMA. Importin-β therefore links NuMa to regulation by Ran. This suggests that similar mechanisms regulate nuclear import during interphase and spindle assembly during mitosis.

Considering the complexity of spindle assembly, other mitotic microtubule regulators are likely to be regulated by Ran through importin-β. Indeed, several characterized microtubule regulators were found to be nuclear in interphase, suggesting an interaction with the nuclear import pathway. Based on our findings, we hypothesize that Ran may regulate the activity of these proteins in mitosis via importin-β. The next challenge is to understand the mechanism of importin-β-mediated regulation of spindle assembly.

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