Elena Rebollo

Asterless is a centriolar protein required for centrosome function and embryo development in Drosophila

BACKGROUND Centrosomes, the major organizers of the microtubule network in most animal cells, are composed of centrioles embedded in a web of pericentriolar material (PCM). Recruitment and stabilization of PCM on the centrosome is a centriole-dependent function. Compared to the considerable number of PCM proteins known, the molecular characterization of centrioles is still very limited. Only a few centriolar proteins have been identified so far in Drosophila, most related to centriole duplication. RESULTS We have cloned asterless (asl) and found that it encodes a 120 kD highly coiled-coil protein that is a constitutive pancentriolar and basal body component. Loss of asl function impedes the stabilization/maintenance of PCM at the centrosome. In embryos deficient for Asl, development is arrested right after fertilization. Asl shares significant homology with Cep 152, a protein described as a component of the human centrosome for which no functional data is yet available. CONCLUSIONS The cloning of asl offers new insight into the molecular composition of Drosophila centrioles and a possible model for the role of its human homolog. In addition, the phenotype of asl-deficient flies reveals that a functional centrosome is required for Drosophila embryo development.

Spermatocyte cytokinesis requires rapid membrane addition mediated by ARF6 on central spindle recycling endosomes

The dramatic cell shape changes during cytokinesis require the interplay between microtubules and the actomyosin contractile ring, and addition of membrane to the plasma membrane. Numerous membrane-trafficking components localize to the central spindle during cytokinesis, but it is still unclear how this machinery is targeted there and how membrane trafficking is coordinated with cleavage furrow ingression. Here we use an arf6 null mutant to show that the endosomal GTPase ARF6 is required for cytokinesis in Drosophila spermatocytes. ARF6 is enriched on recycling endosomes at the central spindle, but it is required neither for central spindle nor actomyosin contractile ring assembly, nor for targeting of recycling endosomes to the central spindle. However, in arf6 mutants the cleavage furrow regresses because of a failure in rapid membrane addition to the plasma membrane. We propose that ARF6 promotes rapid recycling of endosomal membrane stores during cytokinesis, which is critical for rapid cleavage furrow ingression.

Contribution of noncentrosomal microtubules to spindle assembly in Drosophila spermatocytes.

Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes.

Visualizing the spindle checkpoint in Drosophila spermatocytes

The spindle assembly checkpoint detects defects in spindle structure or in the alignment of the chromosomes on the metaphase plate and delays the onset of anaphase until defects are corrected. Thus far, the evidence regarding the presence of a spindle checkpoint during meiosis in male Drosophila has been indirect and contradictory. On the one hand, chromosomes without pairing partners do not prevent meiosis progression. On the other hand, some conserved components of the spindle checkpoint machinery are expressed in these cells and behave as their homologue proteins do in systems with an active spindle checkpoint. To establish whether the spindle checkpoint is active in Drosophila spermatocytes we have followed meiosis progression by time‐lapse microscopy under conditions where the checkpoint is likely to be activated. We have found that the presence of a relatively high number of misaligned chromosomes or a severe disruption of the meiotic spindle results in a significant delay in the time of entry into anaphase. These observations provide the first direct evidence substantiating the activity of a meiotic spindle checkpoint in male Drosophila.

Cdc37 is essential for chromosome segregation and cytokinesis in higher eukaryotes.

Cdc37 has been shown to be required for the activity and stability of protein kinases that regulate different stages of cell cycle progression. However, little is known so far regarding interactions of Cdc37 with kinases that play a role in cell division. Here we show that the loss of function of Cdc37 in Drosophila leads to defects in mitosis and male meiosis, and that these phenotypes closely resemble those brought about by the inactivation of Aurora B. We provide evidence that Aurora B interacts with and requires the Cdc37/Hsp90 complex for its stability. We conclude that the Cdc37/Hsp90 complex modulates the function of Aurora B and that most of the phenotypes brought about by the loss of Cdc37 function can be explained by the inactivation of this kinase. These observations substantiate the role of Cdc37 as an upstream regulatory element of key cell cycle kinases.

Spindle alignment is achieved without rotation after the first cell cycle in Drosophila embryonic neuroblasts

Spindle alignment along the apicobasal polarity axis is mandatory for proper self-renewing asymmetric division in Drosophila neuroblasts (NBs). In embryonic NBs, spindles have been reported to assemble orthogonally to the polarity axis and later rotate to align with it. In larval NBs, spindles assemble directly aligned with the axis owing to the differential spatiotemporal control of the microtubule organising activity of their centrosomes. We have recorded embryonic NBs that express centrosome and microtubule reporters, from delamination up to the fourth cell cycle, by two-photon confocal microscopy, and have found that the switch between these two spindle orientation modes occurs in the second cell cycle of the NB, the first that follows delamination. Therefore, predetermined spindle orientation is not restricted to larval NBs. On the contrary, it actually applies to all but the first cell cycle of embryonic NBs.

Organized microtubule arrays in γ-tubulin-depleted Drosophila spermatocytes

To assess the role of gamma-tubulin in spindle assembly in vivo, we have followed meiosis progression by immunofluorescence and time-lapse video microscopy in gammaTub23C(PI) mutant spermatocytes. We have found that centrosomes associate with large numbers of astral microtubules even though gamma-tubulin is severely depleted; bipolar meiotic spindles are never assembled; and later in meiosis, the microtubules get organized into a conical structure that is never observed in wild-type cells. Several lines of evidence suggest that these cones may be related to wild-type central spindles. First, they are assembled midway through meiosis and elongate during anaphase. Second, they are constricted during late meiosis, giving rise to a pointed end similar to those that form in each half of the wild-type spindle midzone. Third, Klp3A and Polo, two markers of the wild-type central spindle are also found around the pointed end of the mutant cones. Finally, ectopic cytokinesis furrows are often formed at the distal end of the cone. Our results suggest that microtubule polymerization or stabilization from the centrosome may be possible in a gamma-tubulin-independent manner in Drosophila spermatocytes. However, gamma-tubulin seems to be essential for spindle assembly in these cells. Finally, our results show that at least part of the central spindle and constriction-ring assembly machinery can operate on microtubule bundles that are not organized as bipolar spindles.

Drosophila dd4 mutants reveal that gammaTuRC is required to maintain juxtaposed half spindles in spermatocytes.

The weak spindle integrity checkpoint in Drosophila spermatocytes has revealed a novel function of the γ-tubulin ring complex (γTuRC) in maintaining spindle bipolarity throughout meiosis. Bipolar and bi-astral spindles could form in Drosophila mutants for dd4, the gene encoding the 91 kDa subunit of γTuRC. However, these spindles collapsed around metaphase and began to elongate as if attempting anaphase B. The microtubules of the collapsing spindle folded back on themselves, their putative plus ends forming the focused apexes of biconical figures. Cells with such spindles were unable to undergo cytokinesis. A second type of spindle, monopolar hemi-spindles, also formed as a result of either spindle collapse at an earlier stage or failure of centrosome separation. Multiple centrosome-like bodies at the foci of hemi-spindles nucleated robust asters of microtubules in the absence of detectable γ-tubulin. Time-lapse imaging revealed these to be intermediates that developed into cones, structures that also had putative plus ends of microtubules focused at their tips. Unlike biconical figures, however, cones seemed to contain a central spindle-like structure at their apexes and undergo cytokinesis. We conclude that spermatocytes do not need astral microtubules nucleated by opposite poles to intersect in order to form a central spindle and a cleavage furrow.

Time-Lapse Imaging of Embryonic Neural Stem Cell Division in Drosophila by Two-Photon Microscopy

This unit describes a protocol for live imaging of Drosophila embryonic neural stem cells using two-photon microscopy. Compared to traditional one-photon confocal imaging, this technique renders higher-resolution optical sections from deeper within the embryo. It is ideally suited to following embryonic neuroblasts located underneath the neuroepithelial cell layer for several rounds of cell division.