Shingo Yasuda

Cdc42 and mDia3 regulate microtubule attachment to kinetochores

During mitosis, the mitotic spindle, a bipolar structure composed of microtubules (MTs) and associated motor proteins, segregates sister chromatids to daughter cells. Initially some MTs emanating from one centrosome attach to the kinetochore at the centromere of one of the duplicated chromosomes. This attachment allows rapid poleward movement of the bound chromosome. Subsequent attachment of the sister kinetochore to MTs growing from the other centrosome results in the bi-orientation of the chromosome, in which interactions between kinetochores and the plus ends of MTs are formed and stabilized. These processes ensure alignment of chromosomes during metaphase and their correct segregation during anaphase. Although many proteins constituting the kinetochore have been identified and extensively studied, the signalling responsible for MT capture and stabilization is unclear. Small GTPases of the Rho family regulate cell morphogenesis by organizing the actin cytoskeleton and regulating MT alignment and stabilization. We now show that one member of this family, Cdc42, and its effector, mDia3, regulate MT attachment to kinetochores.

mDia2 induces the actin scaffold for the contractile ring and stabilizes its position during cytokinesis in NIH 3T3 cells.

mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell.

Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis

Although Rho regulates cytokinesis, little was known about the functions in mitosis of Cdc42 and Rac. We recently suggested that Cdc42 works in metaphase by regulating bi-orient attachment of spindle microtubules to kinetochores. We now confirm the role of Cdc42 by RNA interference and identify the mechanisms for activation and down-regulation of Cdc42. Using a pull-down assay, we found that the level of GTP-Cdc42 elevates in metaphase, whereas the level of GTP-Rac does not change significantly in mitosis. Overexpression of dominant-negative mutants of Ect2 and MgcRacGAP, a Rho GTPase guanine nucleotide exchange factor and GTPase activating protein, respectively, or depletion of Ect2 by RNA interference suppresses this change of GTP-Cdc42 in mitosis. Depletion of Ect2 also impairs microtubule attachment to kinetochores and causes prometaphase delay and abnormal chromosomal segregation, as does depletion of Cdc42 or expression of the Ect2 and MgcRacGAP mutants. These results suggest that Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis.

Triggering of neuronal cell death by accumulation of activated SEK1 on nuclear polyglutamine aggregations in PML bodies

A novel class of inherited human neurodegenerations is now known to be caused by expanded CAG repeats encoding polyglutamines. Polyglutamine‐containing protein fragments have been shown to accumulate as aggregates in the nucleus and in the cytoplasm, and to induce cell death when expressed in cultured cells, leading to the proposal that polyglutamine aggregation is an important step in the pathogenesis. Supporting this, nuclear inclusions containing expanded polyglutamines have been identified in neurones from the brains of patients and in neurones from transgenic mouse models of this class of neural disorders.

An essential role of Cdc42-like GTPases in mitosis of HeLa cells

Here we used RNA interference and examined possible redundancy amongst Rho GTPases in their mitotic role. Chromosome misalignment is induced significantly in HeLa cells by Cdc42 depletion and not by depletion of either one or all of the other four Cdc42‐like GTPases (TC10, TCL, Wrch1 or Wrch2), four Rac‐like GTPases or three Rho‐like GTPases. Notably, combined depletion of Cdc42 and all of the other four Cdc42‐like GTPases significantly enhances chromosomal misalignment. These observations suggest that Cdc42 is the primary GTPase functioning during mitosis but that the other four Cdc42‐like GTPases can also assume the mitotic role in its absence.

A New Look at Rho GTPases in the Cell Cycle: Their Role in Kinetochore-Microtubule Attachment

Rho GTPases including Rho, Rac and Cdc42 are involved in cell morphogenesis by inducing specific types of actin cytoskeleton and alignment and stabilization of microtubules. Previous studies suggest that they also regulate cell cycle progression; Rho, Rac and Cdc42 regulate the G1-S progression and Rho controls cytokinesis. However, a role of Rho GTPases in nuclear division has not been definitely shown. We have recently found that Cdc42 and its downstream effector mDia3 are involved in bi-orientation and stabilization of spindle microtubules attachment to kinetochores and regulate chromosome alignment and segregation. Here, we discuss how this is coordinated with other events in mitosis, particularly, with the action of Rho in cytokinesis and how attachment of microtubules to kinetochores is achieved and stabilized. We also discuss redundancy of Cdc42 and Cdc42-related GTPase(s) and potential mechanisms of chromosome instability in cancer

Analysis of a mitotic role of Cdc42

Rho GTPases including Rho, Rac, and Cdc42 determine the cell shape by regulating the actin and microtubule dynamics. Through these actions on cytoskeleton, Rho GTPases also regulate cell cycle progression. Specifically, Rho, Rac, and Cdc42 regulate G1-S progression, and Rho regulates cytokinesis. However, involvement of these GTPases in nuclear division has not been definitely shown. This seems to be due to the lack of standard procedures examining mitosis-specific functions of these GTPases. Recently, we have found a mitosis-specific role of Cdc42 by enrichment of cells in mitosis by cell cycle synchronization and interfering with functions of Rho GTPases. This chapter describes the procedures we used in these studies.

Impaired T lymphocyte trafficking in mice deficient in an actin-nucleating protein, mDia1

Sakata et al. 2007. J. Exp. Med. doi:10.1084/jem.20062647[OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft.jtitle%253DJ.%2BExp.%2BMed.%26rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20062647%26rft_id%253Dinfo%253Apmid%252F17682067%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%