The Rho-associated protein kinase p160ROCK is required for centrosome positioning

The p160–Rho-associated coiled-coil–containing protein kinase (ROCK) is identified as a new centrosomal component. Using immunofluorescence with a variety of p160ROCK antibodies, immuno EM, and depletion with RNA interference, p160ROCK is principally bound to the mother centriole (MC) and an intercentriolar linker. Inhibition of p160ROCK provoked centrosome splitting in G1 with the MC, which is normally positioned at the cell center and shows little motion during G1, displaying wide excursions around the cell periphery, similar to its migration toward the midbody during cytokinesis. p160ROCK inhibition late after anaphase in mitosis triggered MC migration to the midbody followed by completion of cell division. Thus, p160ROCK is required for centrosome positioning and centrosome-dependent exit from mitosis

The Centrosomal Protein C-Nap1 Is Required for Cell Cycle–Regulated Centrosome Cohesion

Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle–regulated manner. To test this model, we have performed a detailed structure–function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle–regulated structure that mediates centriole–centriole cohesion

Centrosomes : methods for preparation

The centrosome of higher eukaryotic cells is the main microtubule-organising centre. To understand the molecular mechanisms underlying this organelle's biogenesis and important functions in several cellular processes, such as microtubule nucleation, cell division and stress response, it was critical to develop methods for isolating biochemically meaningful quantities of centrosomes. Centrosomes have been isolated from a variety of organisms and based on these preparations, numerous aspects of this intriguing organelle's morphological, functional and biochemical properties have been uncovered. Better isolation procedures along with the development of new technologies, like RNAi (ribonucleic acid interference) and the increasing accuracy of mass spectrometry and electron microscopy techniques, have profoundly improved our knowledge of the centrosome, leading to a better understanding of its implications in various cellular processes and in diseases

The Mother Centriole Plays an Instructive Role in Defining Cell Geometry

Centriole positioning is a key step in establishment and propagation of cell geometry, but the mechanism of this positioning is unknown. The ability of pre-existing centrioles to induce formation of new centrioles at a defined angle relative to themselves suggests they may have the capacity to transmit spatial information to their daughters. Using three-dimensional computer-aided analysis of cell morphology in Chlamydomonas, we identify six genes required for centriole positioning relative to overall cell polarity, four of which have known sequences. We show that the distal portion of the centriole is critical for positioning, and that the centriole positions the nucleus rather than vice versa. We obtain evidence that the daughter centriole is unable to respond to normal positioning cues and relies on the mother for positional information. Our results represent a clear example of “cytotaxis” as defined by Sonneborn, and suggest that centrioles can play a key function in propagation of cellular geometry from one generation to the next. The genes documented here that are required for proper centriole positioning may represent a new class of ciliary disease genes, defects in which would be expected to cause disorganized ciliary position and impaired function

Control of daughter centriole formation by the pericentriolar material

Author Posting. © The Author(s), 2008. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature Cell Biology 10 (2008): 322-328, doi:10.1038/ncb1694.Controlling the number of its centrioles is vital for the cell as supernumerary centrioles result in multipolar mitosis and genomic instability. Normally, just one daughter centriole forms on each mature (mother) centriole; however, a mother centriole can produce multiple daughters within a single cell cycle. The mechanisms that prevent centriole ‘overduplication’ are poorly understood. Here we use laser microsurgery to test the hypothesis that attachment of the daughter centriole to the wall of the mother inhibits formation of additional daughters. We show that physical removal of the daughter induces reduplication of the mother in Sarrested cells. Under conditions when multiple daughters simultaneously form on a single mother, all of these daughters must be removed to induce reduplication. Intriguingly, the number of daughter centrioles that form during reduplication does not always match the number of ablated daughter centrioles. We also find that exaggeration of the pericentriolar material (PCM) via overexpression of the PCM protein pericentrin in S-arrested CHO cells induces formation of numerous daughter centrioles. We propose that that the size of the PCM cloud associated with the mother centriole restricts the number of daughters that can form simultaneously.This work was supported by grants from the National Institutes of Health (GM GM59363) and the Human Frontiers Science Program (RGP0064). Construction of our laser microsurgery workstation was supported in part by a fellowship from Nikon/Marine Biological Laboratory (A.K.)

C-Nap1, a Novel Centrosomal Coiled-Coil Protein and Candidate Substrate of the Cell Cycle–regulated Protein Kinase Nek2

Nek2 (for NIMA-related kinase 2) is a mammalian cell cycle–regulated kinase structurally related to the mitotic regulator NIMA of Aspergillus nidulans. In human cells, Nek2 associates with centrosomes, and overexpression of active Nek2 has drastic consequences for centrosome structure. Here, we describe the molecular characterization of a novel human centrosomal protein, C-Nap1 (for centrosomal Nek2-associated protein 1), first identified as a Nek2-interacting protein in a yeast two-hybrid screen. Antibodies raised against recombinant C-Nap1 produced strong labeling of centrosomes by immunofluorescence, and immunoelectron microscopy revealed that C-Nap1 is associated specifically with the proximal ends of both mother and daughter centrioles. On Western blots, anti–C-Nap1 antibodies recognized a large protein (>250 kD) that was highly enriched in centrosome preparations. Sequencing of overlapping cDNAs showed that C-Nap1 has a calculated molecular mass of 281 kD and comprises extended domains of predicted coiled-coil structure. Whereas C-Nap1 was concentrated at centrosomes in all interphase cells, immunoreactivity at mitotic spindle poles was strongly diminished. Finally, the COOH-terminal domain of C-Nap1 could readily be phosphorylated by Nek2 in vitro, as well as after coexpression of the two proteins in vivo. Based on these findings, we propose a model implicating both Nek2 and C-Nap1 in the regulation of centriole–centriole cohesion during the cell cycle