To establish a functional bipolar mitotic spindle, the centrosome expands and matures, acquiring enhanced activities for microtubule (MT) nucleation and assembly at the onset of mitosis. However, the regulatory mechanisms of centrosome maturation and MT assembly from the matured centrosome are largely unknown. In this study, we showed that heat shock protein (HSP) 70 considerably accumulates at the mitotic centrosome during prometaphase to metaphase and is required for bipolar spindle assembly. Inhibition or depletion of HSP70 impaired the function of mitotic centrosome and disrupted MT nucleation and polymerization from the spindle pole, and may thus result in formation of abnormal mitotic spindles. In addition, HSP70 may associate with NEDD1 and γ-tubulin, two pericentriolar material (PCM) components essential for centrosome maturation and MT nucleation. Loss of HSP70 function disrupted the interaction between NEDD1 and γ-tubulin, and reduced their accumulation at the mitotic centrosome. Our results thus demonstrate a role for HSP70 in regulating centrosome integrity during mitosis, and indicate that HSP70 is required for the maintenance of a functional mitotic centrosome that supports the assembly of a bipolar mitotic spindle.

Background A previous screen of a human kinase and phosphatase shRNA library to select genes that mediate arsenite induction of spindle abnormalities resulted in the identification of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), a phosphatidylinositol 4,5-bisphosphate (PIP2)-synthesizing enzyme. In this study, we explored how PIP4KIIγ regulates the assembly of mitotic spindles. Results PIP4KIIγ accumulates at the spindle pole before anaphase, and is required for the assembly of functional bipolar spindles. Depletion of PIP4KIIγ enhanced the spindle pole accumulation of mitotic centromere-associated kinesin (MCAK), a microtubule (MT)-depolymerizing kinesin, and resulted in a less stable spindle pole-associated MT. Depletion of MCAK can ameliorate PIP4KIIγ depletion-induced spindle abnormalities. In addition, PIP2 binds to polo-like kinase (PLK1) and reduces PLK1-mediated phosphorylation of MCAK. These results indicate that PIP4KIIγ and PIP2 may negatively regulate the MT depolymerization activity of MCAK by reducing PLK1-mediated phosphorylation of MCAK. Consequently, depletion of PLK1 has been shown to counteract the PIP4KIIγ depletion-induced instability of spindle pole-associated MT and cell resistance to arsenite. Conclusions Our current results imply that PIP4KIIγ may restrain MT depolymerization at the spindle pole through attenuating PLK1-mediated activation of MCAK before anaphase onset.

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