Molecular mechanisms regulating bipolar spindle formation and microtubule depolymerization

Abstract

Microtubules are involved in a wide range of cellular functions including the establishment of cell polarity, organelle trafficking and positioning, and mitotic chromosome segregation. To accomplish such diverse functions during the cell cycle, microtubules must be organized and dynamically regulated by a large number of microtubule-associated proteins (MAPs) in space and time. Despite identification of many MAPs, the functions of these MAPs remain to be further explored and defined. In this study, I investigate the roles of several key MAPs in mitotic spindle assembly and in microtubule depolymerization during interphase, using the fission yeast Schizosaccharomyces pombe as a model organism. Fission yeast has a well-defined microtubule array and is genetically tractable. Importantly, most MAPs in fission yeast are evolutionarily conserved. All these features make fission yeast an excellent organism for microtubule studies.

During mitosis, microtubules form a bipolar spindle for regulating chromosome segregation. In bipolar spindle formation, the kinesin-5 motor protein cut7p has been shown to be an essential player. However, studies in multiple model organisms also suggest that kinesin-independent mechanisms are required to ensure the efficiency of bipolar spindle assembly. One of the components involved in the kinesin-independent mechanism could be the evolutionarily conserved complex comprising alp7p (TACC in human) and alp14p (ch-TOG in human) as depletion of alp7p or alp14p causes defects in bipolar spindle formation. The function of the alp7p-alp14p complex in spindle assembly depends on their localization to the SPB (the centrosome in human cells). However, it remains unknown how the alp7p-alp14p complex is targeted to the SPB/centrosome. In the first part of this thesis (chapter 3), I demonstrate that the alp7p-alp14p complex is recruited to the SPB by the new SPB protein csi1p through the interaction between alp7p and csi1p. Domain mapping further revealed that the csi1p-interacting region in alp7p lies within the conserved TACC domain, and the carboxyl-terminal domain of csi1p is responsible for its interaction with alp7p. Compromised interaction between csi1p and alp7p impairs the SPBs localization of alp7p during mitosis, thus leading to bipolar spindle formation defects and consequently anaphase B lagging chromosomes. Hence, this study establishes that csi1p serves as a linking molecule tethering the microtubule-stabilizing factors alp7p and alp14p to the SPB to promote bipolar spindle assembly.

In the second part of this thesis (chapter 4), I address the role of a new MAP in microtubule depolymerization in interphase. Two types of MAPs work synergistically to regulate microtubule dynamics. These MAPs function either to stabilize microtubules or to destabilize microtubules. Thus far few microtubule-destabilizing proteins have been identified and characterized. Here, I presented a new microtubule-destabilizing protein mcp1p. Mcp1p is mobile in the cell and localizes along microtubules, particularly concentrating at microtubule plus ends. Depletion of mcp1p leads to stabilized long microtubules, a phenotype also observed in cells lacking the kinesin-8 motor protein klp6p, but not klp5p. In addition, the motility of mcp1p depends on klp6p. Thus, I conclude that mcp1p works in concert with klp6p to promote microtubule depolymerization. This work provides new molecular insights into microtubule dynamics regulation.