Laurence Haren

Plk1-Dependent Recruitment of γ-Tubulin Complexes to Mitotic Centrosomes Involves Multiple PCM Components

The nucleation of microtubules requires protein complexes containing γ-tubulin, which are present in the cytoplasm and associate with the centrosome and with the mitotic spindle. We have previously shown that these interactions require the γ-tubulin targeting factor GCP-WD/NEDD1, which has an essential role in spindle formation. The recruitment of additional γ-tubulin to the centrosomes occurs during centrosome maturation at the G2/M transition and is regulated by the mitotic kinase Plk1. However, the molecular details of this important pathway are unknown and a Plk1 substrate that controls γ-tubulin recruitment has not been identified. Here we show that Plk1 associates with GCP-WD in mitosis and Plk1 activity contributes to phosphorylation of GCP-WD. Plk1 depletion or inhibition prevents accumulation of GCP-WD at mitotic centrosomes, but GCP-WD mutants that are defective in Plk1-binding and -phosphorylation still accumulate at mitotic centrosomes and recruit γ-tubulin. Moreover, Plk1 also controls the recruitment of other PCM proteins implicated in centrosomal γ-tubulin attachment (Cep192/hSPD2, pericentrin, Cep215/Cdk5Rap2). Our results support a model in which Plk1-dependent recruitment of γ-tubulin to mitotic centrosomes is regulated upstream of GCP-WD, involves multiple PCM proteins and therefore potentially multiple Plk1 substrates.

A lateral belt of cortical LGN and NuMA guides mitotic spindle movements and planar division in neuroepithelial cells

Knockdown or mislocalization of LGN complex components disrupts the stereotypic biphasic spindle movements regulating planar cell division and neuroepithelial structure in chick embryos.

Cell and molecular biology of spindle poles and NuMA

Mitotic and meiotic cells contain a bipolar spindle apparatus of microtubules and associated proteins. To arrange microtubules into focused spindle poles, different mechanisms are used by various organisms. Principally, two major pathways have been characterized: nucleation and anchorage of microtubules at preexisting centers such as centrosomes or spindle pole bodies, or microtubule growth off the surface of chromosomes, followed by sorting and focusing into spindle poles. These two mechanisms can even be found in cells of the same organism: whereas most somatic animal cells utilize the centrosome as an organizing center for spindle microtubules, female meiotic cells build an acentriolar spindle apparatus. Most interestingly, the molecular components that drive acentriolar spindle pole formation are also present in cells containing centrosomes. They include microtubule-dependent motor proteins and a variety of structural proteins that regulate microtubule orientation, anchoring, and stability. The first of these spindle pole proteins, NuMA, had already been identified more than 20 years ago. In addition, several new proteins have been characterized more recently. This review discusses their role during spindle formation and their regulation in the cell cycle.

IS911-mediated intramolecular transposition is naturally temperature sensitive

It is shown here that the bacterial insertion sequence IS911 exhibits a temperature‐sensitive transposition phenotype. Previous results have demonstrated that elevated levels of the IS911 transposase OrfAB generate significant quantities of a figure‐eight form, created by cleavage and circularization of one of the transposon strands, and of an excised circular form, in which both transposon strands have been circularized. We show here that the level of both types of molecule observed in vivo was greatly reduced at 42°C compared with 37°C. On the other hand, reducing the temperature to 30°C resulted in a significant increase in production. Transposition activity at this temperature was sufficiently high to permit detection in vivo of an excised circular form of a defective single IS911 chromosomal copy when OrfAB is supplied in trans. A similar temperature–activity profile is observed for a cell‐free reaction that uses partially purified OrfAB and generates the figure‐eight form uniquely. Moreover, two point mutants of OrfAB were obtained, which render the reactions partially temperature resistant both in vivo and in vitro. These results suggest that some property of transposase itself is sensitive to elevated temperatures.

Stability of the small γ-tubulin complex requires HCA66, a protein of the centrosome and the nucleolus

To investigate changes at the centrosome during the cell cycle, we analyzed the composition of the pericentriolar material from unsynchronized and S-phase-arrested cells by gel electrophoresis and mass spectrometry. We identified HCA66, a protein that localizes to the centrosome from S-phase to mitosis and to the nucleolus throughout interphase. Silencing of HCA66 expression resulted in failure of centrosome duplication and in the formation of monopolar spindles, reminiscent of the phenotype observed after γ-tubulin silencing. Immunofluorescence microscopy showed that proteins of the γ-tubulin ring complex were absent from the centrosome in these monopolar spindles. Immunoblotting revealed reduced protein levels of all components of the γ-tubulin small complex (γ-tubulin, GCP2, and GCP3) in HCA66-depleted cells. By contrast, the levels of γ-tubulin ring complex proteins such as GCP4 and GCP-WD/NEDD1 were unaffected. We propose that HCA66 is a novel regulator of γ-tubulin function that plays a role in stabilizing components of the γ-tubulin small complex, which is in turn essential for assembling the larger γ-tubulin ring complex.

IS911 transposon circles give rise to linear forms that can undergo integration in vitro

High levels of expression of the transposase OrfAB of bacterial insertion sequence IS911 leads to the formation of excised transposon circles, in which the two abutted ends are separated by 3 bp. Initially, OrfAB catalyses only single‐strand cleavage at one 3′ transposon end and strand transfer of that end to the other. It is believed that this molecule, in which both transposon ends are held together in a single‐strand bridge, is then converted to the circular form by the action of host factors. The transposon circles can be integrated efficiently into an appropriate target in vivo and in vitro in the presence of OrfAB and a second IS911 protein OrfA. In the results reported here, we have identified linear transposon forms in vivo from a transposon present in a plasmid, raising the possibility that IS911 can also transpose using a cut‐and‐paste mechanism. However, the linear species appeared not to be derived directly from the plasmid‐based copy by direct double‐strand cleavages at both ends, but from preformed excised transposon circles. This was confirmed further by the observation that OrfAB can cleave a cloned circle junction both in vivo and in vitro by two single‐strand cleavages at the 3′ transposon ends to generate a linear transposon form with a 3′‐OH and a three‐nucleotide 5′ overhang at the ends. Moreover, while significantly less efficient than the transposon circle, a precleaved linear transposon underwent detectable levels of integration in vitro. The possible role of such molecules in the IS911 transposition pathway is discussed.

The centrosome protein NEDD1 as a potential pharmacological target to induce cell cycle arrest

BackgroundNEDD1 is a protein that binds to the gamma-tubulin ring complex, a multiprotein complex at the centrosome and at the mitotic spindle that mediates the nucleation of microtubules.ResultsWe show that NEDD1 is expressed at comparable levels in a variety of tumor-derived cell lines and untransformed cells. We demonstrate that silencing of NEDD1 expression by treatment with siRNA has differential effects on cells, depending on their status of p53 expression: p53-positive cells arrest in G1, whereas p53-negative cells arrest in mitosis with predominantly aberrant monopolar spindles. However, both p53-positive and -negative cells arrest in mitosis if treated with low doses of siRNA against NEDD1 combined with low doses of the inhibitor BI2536 against the mitotic kinase Plk1. Simultaneous reduction of NEDD1 levels and inhibition of Plk1 act in a synergistic manner, by potentiating the anti-mitotic activity of each treatment.ConclusionWe propose that NEDD1 may be a promising target for controlling cell proliferation, in particular if targeted in combination with Plk1 inhibitors.

Spindle assembly defects leading to the formation of a monopolar mitotic apparatus

Mitotic spindle formation in animal cells involves microtubule nucleation from two centrosomes that are positioned at opposite sides of the nucleus. Microtubules are captured by the kinetochores and stabilized. In addition, microtubules can be nucleated independently of the centrosome and stabilized by a gradient of Ran—GTP, surrounding the mitotic chromatin. Complex regulation ensures the formation of a bipolar apparatus, involving motor proteins and controlled polymerization and depolymerization of microtubule ends. The bipolar apparatus is, in turn, responsible for faithful chromosome segregation. During recent years, a variety of experiments has indicated that defects in specific motor proteins, centrosome proteins, kinases and other proteins can induce the assembly of aberrant spindles with a monopolar morphology or with poorly separated poles. Induction of monopolar spindles may be a useful strategy for cancer therapy, since ensuing aberrant mitotic exit will usually lead to cell death. In this review, we will discuss the various underlying molecular mechanisms that may be responsible for monopolar spindle formation.

γ-Tubulin Ring Complexes and EB1 play antagonistic roles in microtubule dynamics and spindle positioning

γ‐Tubulin is critical for microtubule (MT) assembly and organization. In metazoa, this protein acts in multiprotein complexes called γ‐Tubulin Ring Complexes (γ‐TuRCs). While the subunits that constitute γ‐Tubulin Small Complexes (γ‐TuSCs), the core of the MT nucleation machinery, are essential, mutation of γ‐TuRC‐specific proteins in Drosophila causes sterility and morphological abnormalities via hitherto unidentified mechanisms. Here, we demonstrate a role of γ‐TuRCs in controlling spindle orientation independent of MT nucleation activity, both in cultured cells and in vivo, and examine a potential function for γ‐TuRCs on astral MTs. γ‐TuRCs locate along the length of astral MTs, and depletion of γ‐TuRC‐specific proteins increases MT dynamics and causes the plus‐end tracking protein EB1 to redistribute along MTs. Moreover, suppression of MT dynamics through drug treatment or EB1 down‐regulation rescues spindle orientation defects induced by γ‐TuRC depletion. Therefore, we propose a role for γ‐TuRCs in regulating spindle positioning by controlling the stability of astral MTs.

08-P014 Control of mitotic spindle rotation and planar divisions in chick neuroepithelial cells

During development of the vertebrate CNS, a precise control of planar orientation of the mitotic spindle of dividing neuroepithelial progenitors regulates the equal partitioning of apical attachment sites. This is necessary to maintain sister cells within the neuroepithelial structure. We have previously demonstrated an essential role for the G-protein regulator LGN in this process. Here we use three-dimensional time lapse imaging of dividing cells in the chick neuroepithelium and describe that the mitotic spindle follows a stereotypical biphasic rotation movement, which first drives the spindle to align with the neuroepithelial surface, then maintains it in this plane while leaving it free to revolve around the apico-basal axis of the cell. We show that Gai subunits, LGN and the dynein/dynactin interactor NuMA localize as nesting ring-shaped cortical domains on the lateral membrane of dividing cells. Gai-GDP recruits LGN, which in turn recruits NuMA to the cell cortex. LGN functions as a Gai-GDP sensor that changes conformation and is able to recruit NuMA only above a certain threshold of cortical Gai-GDP concentration. In absence of these molecules, spindle rotation is lost or erratic and the plane of division is randomized. We propose that a gradient of Gai-GDP is formed within the overall homogeneous cortical Gai distribution. A peak of Gai-GDP on the lateral cell cortex is used as a cue to define the progressively narrower rings of Gai, Gai-GDP/LGN and Gai-GDP/LGN/NuMA complexes on the lateral cell cortex. The latter domain controls astral microtubule stabilization to orients the spindle.