Claudio E. Sunkel

The 55 kd regulatory subunit of Drosophila protein phosphatase 2A is required for anaphase.

The gene encoding the Drosophila protein phosphatase 2A 55 kd regulatory subunit (PR55) is located at 85F and directs the synthesis of differentially spliced transcripts. Maternal RNAs are present at very high levels in early embryos and decline around cellularization. Zygotic transcripts are present mainly in the developing embryonic nervous system and gonads. Transcripts are uniformly distributed in third instar larval discs and testes and at lower levels in the proliferative centers of the brain. Mutations in abnormal anaphase resolution (aar) are rescued by the wild-type gene for PR55. aar mutants display intact lagging chromatids that have undergone separation from their sisters, but that remain at the position formerly occupied by the metaphase plate, as well as anaphase figures that show bridging chromatin having two centromeric regions.

Gamma-tubulin is required for the structure and function of the microtubule organizing centre in Drosophila neuroblasts.

We report that in Drosophila, gamma‐tubulin is required for the structure as well as the function of microtubule organizing centres (MTOCs). This conclusion is based on the identification and phenotypic characterization of a mutant allele of the gamma‐tubulin gene located at region 23C of the polytene chromosome map. This mutation, which we have called gamma‐tub23CPl, is caused by the insertion of a P‐element within the 5′ untranslated leader of the gamma‐tubulin transcript. Northern and Western analysis show that gamma‐tub23CPl is either a null or a very severe hypomorph as no gamma‐tubulin mRNA or protein can be detected in mutant individuals. Visualization of DNA, MTOCs and microtubules by confocal laser scanning microscopy of cells from individuals homozygous for gamma‐tub23CPl reveals a series of phenotypic abnormalities. Some of these are similar to those observed after disruption of gamma‐tubulin function in other organisms, including mitotic arrest and a dramatic decrease in the number of microtubules, but, in addition, we have observed that mutation in this gene also results in highly abnormal MTOCs which show a variety of shapes and sizes which we never observed in wild type cells. These results show that gamma‐tubulin is required for both structural and functional roles in the MTOCs.

A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis

BACKGROUND Faithful segregation of the genome during mitosis requires interphase chromatin to be condensed into well-defined chromosomes. Chromosome condensation involves a multiprotein complex known as condensin that associates with chromatin early in prophase. Until now, genetic analysis of SMC subunits of the condensin complex in higher eukaryotic cells has not been performed, and consequently the detailed contribution of different subunits to the formation of mitotic chromosome morphology is poorly understood. RESULTS We show that the SMC4 subunit of condensin is encoded by the essential gluon locus in Drosophila. DmSMC4 contains all the conserved domains present in other members of the structural-maintenance-of-chromosomes protein family. DmSMC4 is both nuclear and cytoplasmic during interphase, concentrates on chromatin during prophase, and localizes to the axial chromosome core at metaphase and anaphase. During decondensation in telophase, most of the DmSMC4 leaves the chromosomes. An examination of gluon mutations indicates that SMC4 is required for chromosome condensation and segregation during different developmental stages. A detailed analysis of mitotic chromosome structure in mutant cells indicates that although the longitudinal axis can be shortened normally, sister chromatid resolution is strikingly disrupted. This phenotype then leads to severe chromosome segregation defects, chromosome breakage, and apoptosis. CONCLUSIONS Our results demonstrate that SMC4 is critically important for the resolution of sister chromatids during mitosis prior to anaphase onset.

Human CLASP1 Is an Outer Kinetochore Component that Regulates Spindle Microtubule Dynamics

One of the most intriguing aspects of mitosis is the ability of kinetochores to hold onto plus ends of microtubules that are actively gaining or losing tubulin subunits. Here, we show that CLASP1, a microtubule-associated protein, localizes preferentially near the plus ends of growing spindle microtubules and is also a component of a kinetochore region that we term the outer corona. A truncated form of CLASP1 lacking the kinetochore binding domain behaves as a dominant negative, leading to the formation of radial arrays of microtubule bundles that are highly resistant to depolymerization. Microinjection of CLASP1-specific antibodies suppresses microtubule dynamics at kinetochores and throughout the spindle, resulting in the formation of monopolar asters with chromosomes buried in the interior. Incubation with microtubule-stabilizing drugs rescues the kinetochore association with microtubule plus ends at the periphery of the asters. Our data suggest that CLASP1 is required at kinetochores for attached microtubules to exhibit normal dynamic behavior.

Condensin-dependent localisation of topoisomerase II to an axial chromosomal structure is required for sister chromatid resolution during mitosis.

Assembly of compact mitotic chromosomes and resolution of sister chromatids are two essential processes for the correct segregation of the genome during mitosis. Condensin, a five-subunit protein complex, is thought to be required for chromosome condensation. However, recent genetic analysis suggests that condensin is only essential to resolve sister chromatids. To study further the function of condensin we have depleted DmSMC4, a subunit of the complex, from Drosophila S2 cells by dsRNA-mediated interference. Cells lacking DmSMC4 assemble short mitotic chromosomes with unresolved sister chromatids where Barren, a non-SMC subunit of the complex is unable to localise. Topoisomerase II, however, binds mitotic chromatin after depletion of DmSMC4 but it is no longer confined to a central axial structure and becomes diffusely distributed all over the chromatin. Furthermore, cell extracts from DmSMC4 dsRNA-treated cells show significantly reduced topoisomerase II-dependent DNA decatenation activity in vitro. Nevertheless, DmSMC4-depleted chromosomes have centromeres and kinetochores that are able to segregate, although sister chromatid arms form extensive chromatin bridges during anaphase. These chromatin bridges do not result from inappropriate maintenance of sister chromatid cohesion by DRAD21, a subunit of the cohesin complex. Moreover, depletion of DmSMC4 prevents premature sister chromatid separation, caused by removal of DRAD21, allowing cells to exit mitosis with chromatin bridges. Our results suggest that condensin is required so that an axial chromatid structure can be organised where topoisomerase II can effectively promote sister chromatid resolution.

MAST/Orbit has a role in microtubule–kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity

Multiple asters (MAST)/Orbit is a member of a new family of nonmotor microtubule-associated proteins that has been previously shown to be required for the organization of the mitotic spindle. Here we provide evidence that MAST/Orbit is required for functional kinetochore attachment, chromosome congression, and the maintenance of spindle bipolarity. In vivo analysis of Drosophila mast mutant embryos undergoing early mitotic divisions revealed that chromosomes are unable to reach a stable metaphase alignment and that bipolar spindles collapse as centrosomes move progressively closer toward the cell center and eventually organize into a monopolar configuration. Similarly, soon after depletion of MAST/Orbit in Drosophila S2 cells by double-stranded RNA interference, cells are unable to form a metaphase plate and instead assemble monopolar spindles with chromosomes localized close to the center of the aster. In these cells, kinetochores either fail to achieve end-on attachment or are associated with short microtubules. Remarkably, when microtubule dynamics is suppressed in MAST-depleted cells, chromosomes localize at the periphery of the monopolar aster associated with the plus ends of well-defined microtubule bundles. Furthermore, in these cells, dynein and ZW10 accumulate at kinetochores and fail to transfer to microtubules. However, loss of MAST/Orbit does not affect the kinetochore localization of D-CLIP-190. Together, these results strongly support the conclusion that MAST/Orbit is required for microtubules to form functional attachments to kinetochores and to maintain spindle bipolarity.

Different spindle checkpoint proteins monitor microtubule attachment and tension at kinetochores in Drosophila cells.

The spindle assembly checkpoint detects errors in kinetochore attachment to the spindle including insufficient microtubule occupancy and absence of tension across bi-oriented kinetochore pairs. Here, we analyse how the kinetochore localization of the Drosophila spindle checkpoint proteins Bub1, Mad2, Bub3 and BubR1, behave in response to alterations in microtubule binding or tension. To analyse the behaviour in the absence of tension, we treated S2 cells with low doses of taxol to disrupt microtubule dynamics and tension, but not kinetochore-microtubule occupancy. Under these conditions, we found that Mad2 and Bub1 do not accumulate at metaphase kinetochores whereas BubR1 does. Consistently, in mono-oriented chromosomes, both kinetochores accumulate BubR1 whereas Bub1 and Mad2 only localize at the unattached kinetochore. To study the effect of tension we analysed the kinetochore localization of spindle checkpoint proteins in relation to tension-sensitive kinetochore phosphorylation recognised by the 3F3/2 antibody. Using detergent-extracted S2 cells as a system in which kinetochore phosphorylation can be easily manipulated, we observed that BubR1 and Bub3 accumulation at kinetochores is dependent on the presence of phosphorylated 3F3/2 epitopes. However, Bub1 and Mad2 localize at kinetochores regardless of the 3F3/2 phosphorylation state. Altogether, our results suggest that spindle checkpoint proteins sense distinct aspects of kinetochore interaction with the spindle, with Mad2 and Bub1 monitoring microtubule occupancy while BubR1 and Bub3 monitor tension across attached kinetochores.

Mast, a conserved microtubule‐associated protein required for bipolar mitotic spindle organization

Through mutational analysis in Drosophila, we have identified the gene multiple asters (mast), which encodes a new 165 kDa protein. mast mutant neuroblasts are highly polyploid and show severe mitotic abnormalities including the formation of mono‐ and multi‐polar spindles organized by an irregular number of microtubule‐organizing centres of abnormal size and shape. The mast gene product is evolutionarily conserved since homologues were identified from yeast to man, revealing a novel protein family. Antibodies against Mast and analysis of tissue culture cells expressing an enhanced green fluorescent protein–Mast fusion protein show that during mitosis, this protein localizes to centrosomes, the mitotic spindle, centromeres and spindle midzone. Microtubule‐binding assays indicate that Mast is a microtubule‐associated protein displaying strong affinity for polymerized microtubules. The defects observed in the mutant alleles and the intracellular localization of the protein suggest that Mast plays an essential role in centrosome separation and organization of the bipolar mitotic spindle.

Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator

A putative spindle matrix has been hypothesized to mediate chromosome motion, but its existence and functionality remain controversial. In this report, we show that Megator (Mtor), the Drosophila melanogaster counterpart of the human nuclear pore complex protein translocated promoter region (Tpr), and the spindle assembly checkpoint (SAC) protein Mad2 form a conserved complex that localizes to a nuclear derived spindle matrix in living cells. Fluorescence recovery after photobleaching experiments supports that Mtor is retained around spindle microtubules, where it shows distinct dynamic properties. Mtor/Tpr promotes the recruitment of Mad2 and Mps1 but not Mad1 to unattached kinetochores (KTs), mediating normal mitotic duration and SAC response. At anaphase, Mtor plays a role in spindle elongation, thereby affecting normal chromosome movement. We propose that Mtor/Tpr functions as a spatial regulator of the SAC, which ensures the efficient recruitment of Mad2 to unattached KTs at the onset of mitosis and proper spindle maturation, whereas enrichment of Mad2 in a spindle matrix helps confine the action of a diffusible “wait anaphase” signal to the vicinity of the spindle.

Brista: a gene involved in the specification and differentiation of distal cephalic and thoracic structures in Drosophila melanogaster

SummaryA gene Brista has been identified in chromosome 2R, in the region 60D11-E4, in which mutations cause homoeotic transformation of distal antennal structures to distal leg derivatives, and in which certain alleles also lead to upsets in the formation of distal elements of the legs. This gene is haploinsufficient for the homoeotic phenotype. Several putative null and two hypomorphic alleles have been recovered. The effects of exposure to the non-permissive temperature of a temperature-sensitive allele are cummulative and depend upon the length of the exposure during the period of antennal cell proliferation. It is suggested that this gene contributes to the stability of the state of determination in distal domain of the antennal and leg discs, and its relationship to other genes with similar mutant phenotype is discussed.