Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells.

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145-amino acid residues with extensive alpha-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.     

Asymmetric spindle pole formation in CPAP-depleted mitotic cells

CPAP is an essential component for centriole formation. Here, we report that CPAP is also critical for symmetric spindle pole formation during mitosis. We observed that pericentriolar material between the mitotic spindle poles were asymmetrically distributed in CPAP-depleted cells even with intact numbers of centrioles. The length of procentrioles was slightly reduced by CPAP depletion, but the length of mother centrioles was not affected. Surprisingly, the young mother centrioles of the CPAP-depleted cells are not fully matured, as evidenced by the absence of distal and subdistal appendage proteins. We propose that the selective absence of centriolar appendages at the young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells. Our results suggest that the neural stem cells with CPAP mutations might form asymmetric spindle poles, which results in premature initiation of differentiation.     

Structured illumination of the interface between centriole and peri-centriolar material

The increase in centrosome size in mitosis was described over a century ago, and yet it is poorly understood how centrioles, which lie at the core of centrosomes, organize the pericentriolar material (PCM) in this process. Now, structured illumination microscopy reveals in Drosophila that, before clouds of PCM appear, its proteins are closely associated with interphase centrioles in two tube-like layers: an inner layer occupied by centriolar microtubules, Sas-4, Spd-2 and Polo kinase; and an outer layer comprising Pericentrin-like protein (Dplp), Asterless (Asl) and Plk4 kinase. Centrosomin (Cnn) and γ-tubulin associate with this outer tube in G2 cells and, upon mitotic entry, Polo activity is required to recruit them together with Spd-2 into PCM clouds. Cnn is required for Spd-2 to expand into the PCM during this maturation process but can itself contribute to PCM independently of Spd-2. By contrast, the centrioles of spermatocytes elongate from a pre-existing proximal unit during the G2 preceding meiosis. Sas-4 is restricted to the microtubule-associated, inner cylinder and Dplp and Cnn to the outer cylinder of this proximal part. γ-Tubulin and Asl associate with the outer cylinder and Spd-2 with the inner cylinder throughout the entire G2 centriole. Although they occupy different spatial compartments on the G2 centriole, Cnn, Spd-2 and γ-tubulin become diminished at the centriole upon entry into meiosis to become part of PCM clouds.     

Unequal first cleavage in the Tubifex egg: involvement of a monastral mitotic apparatus

The first cleavage in the freshwater oligochaete Tubifex hattai is unequal and meridional, and produces a smaller cell AB and a larger cell CD. This study traces the process of furrow formation, reorganization of cortical F-actin and the assembly of a mitotic apparatus during this unequal division. Cleavage furrow formation consists of two stages: (i) when eggs are viewed from the animal pole, meridionally running furrows emerge at two points of the egg's equator that are 90° apart from each other and approach the egg axis as they deepen; and (ii) at the midpoint between the equator and the egg center, the bottoms of these furrows link to each other on the animal and vegetal surfaces of the egg and form a continuous ring of constriction in a plane parallel to the egg axis. Egg cortices, isolated during the first step and stained with rhodamine-phalloidin, show that the bottoms of recently formed furrows are underlaid by a belt of tightly packed actin bundles (i.e. a contractile arc). The transition to the second stage of furrow formation coincides with the conversion of these actin belts into a continuous ring of F-actin. Whole-mount immunocytochemistry of microtubules reveals that the first cleavage in Tubifex involves an asymmetric mitotic spindle, which initially possesses an aster at one pole but not the other. This ‘monastral’ spindle is located at the egg's center and orients itself perpendicular to the egg axis. During anaphase, astral rays elongate to reach the cell surface, so that the array of astral microtubules in the plane of the egg's equator covers a sector of 270–300°. In contrast, it is not until the transition to telophase that microtubules emanating from the anastral spindle pole approach the cell margin. If eggs are compressed along the egg axis or forced to elongate, they form monastral spindles and divide unequally. In living compressed eggs, mitotic spindles, which are recognizable as bright streaks at the egg's center, appear not to shift their position along the spindle axis during division, suggesting that without eccentric migration of spindles Tubifex eggs are able to divide unequally. These results suggest that mechanisms that translocate the mitotic spindle eccentrically do not operate in Tubifex eggs during the first cell cycle. The mechanisms that generate asymmetry in spindle organization are discussed in the light of the present results.     

Recruitment of the γ-Tubulin Ring Complex to Drosophila Salt-stripped Centrosome Scaffolds

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and γ-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the γ-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, γ-tubulin is found in two distinct protein complexes of 240,000 and ∼3,000,000 D. The larger complex, which is analogous to the Xenopus γ-tubulin ring complex (γTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578–583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the γTuRC is required for microtubule nucleation at the centrosome.     

Microtubule dynamics in neuronal morphogenesis

Microtubules (MTs) are essential for neuronal morphogenesis in the developing brain. The MT cytoskeleton provides physical support to shape the fine structure of neuronal processes. MT-based motors play important roles in nucleokinesis, process formation and retraction. Regulation of MT stability downstream of extracellular cues is proposed to be critical for axonogenesis. Axons and dendrites exhibit different patterns of MT organization, underlying the divergent functions of these processes. Centrosomal positioning has drawn the attention of researchers because it is a major clue to understanding neuronal MT organization. In this review, we focus on how recent advances in live imaging have revealed the dynamics of MT organization and centrosome positioning during neural development.     

The genetic control of plant mitochondrial morphology and dynamics

Little is known about the genetic control of mitochondrial morphology and dynamics in higher plants. We used a genetic screen involving fluorescence microscopic analysis of ethyl methane sulphonate (EMS)-mutated Arabidopsis thaliana seedlings expressing GFP targeted to mitochondria to isolate eight mutants displaying distinct perturbations of the normal mitochondrial morphology or distribution. We describe five mutants with distinct and unique mitochondrial phenotypes, which map to five different loci, not previously implicated in mitochondrial behaviour in plants. We have used a combination of forward and reverse genetics to identify one of the genes, friendly mitochondria (FMT), a homologue of the CluA gene of Dictyostelium discoideum, which is involved in the correct distribution of mitochondria in the cell. The five mutants constitute a powerful resource to aid our understanding of mitochondrial dynamics in higher plants.     

Microtubule nucleation and organization in dendrites

Dendrite branching is an essential process for building complex nervous systems. It determines the number, distribution and integration of inputs into a neuron, and is regulated to create the diverse dendrite arbor branching patterns characteristic of different neuron types. The microtubule cytoskeleton is critical to provide structure and exert force during dendrite branching. It also supports the functional requirements of dendrites, reflected by differential microtubule architectural organization between neuron types, illustrated here for sensory neurons. Both anterograde and retrograde microtubule polymerization occur within growing dendrites, and recent studies indicate that branching is enhanced by anterograde microtubule polymerization events in nascent branches. The polarities of microtubule polymerization events are regulated by the position and orientation of microtubule nucleation events in the dendrite arbor. Golgi outposts are a primary microtubule nucleation center in dendrites and share common nucleation machinery with the centrosome. In addition, pre-existing dendrite microtubules may act as nucleation sites. We discuss how balancing the activities of distinct nucleation machineries within the growing dendrite can alter microtubule polymerization polarity and dendrite branching, and how regulating this balance can generate neuron type-specific morphologies.     

Importin α Partitioning to the Plasma Membrane Regulates Intracellular Scaling

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CYLD coordinates with EB1 to regulate microtubule dynamics and cell migration

Cylindromatosis (CYLD), a deubiquitinase involved in inflammation and tumorigenesis via the modulation of cell signaling, has recently been identified as a critical regulator of microtubule dynamics. CYLD has also been shown to stimulate cell migration and thereby contribute to normal physiological processes. However, it remains elusive how the regulation of microtubule dynamic properties by CYLD is connected to its role in mediating cell migration. In this study, we performed yeast 2-hybrid screening with CYLD as bait and identified 7 CYLD-interacting proteins, including end-binding protein 1 (EB1). The CYLD–EB1 interaction was confirmed both in cells and in vitro, and these 2 proteins colocalized at the plus ends of microtubules. Interestingly, the association of CYLD with EB1 was significantly increased upon the stimulation of cell migration. CYLD coordinated with EB1 to orchestrate tail retraction, centrosome reorientation, and leading-edge microtubule stabilization in migratory cells. In addition, CYLD acted in concert with EB1 to regulate microtubule assembly in vitro, microtubule nucleation at the centrosome, and microtubule growth at the cell periphery. These data provide mechanistic insights into the actions of CYLD in the regulation of microtubule dynamics and cell migration. These findings also support the notion that coordinated actions of microtubule-binding proteins are critical for microtubule-mediated cellular events.