Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila

Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin''s spindle association, and this in turn dictates γ-tubulin distribution and spindle density. Polo''s negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells. Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.     

Gap junctions in spermatocyte cysts of Drosophila hydei testis

Gap junctions between cyst cells and adjacent spermatocytes have been found in Drosophila hydei by means of the freeze-etching technique. The existence of these cellular junctions suggests that these structures could serve to exchange information between the two types of cells.     

Centrosome hyperamplification with the formation of multiple asters and programmed chromosome inactivation in aberrant spermatocytes during male meiosis in Acricotopus

In the germ line of the midge Acricotopus lucidus, an unequal chromosome segregation occurs in the last gonial mitosis prior to meiosis. This results in one daughter cell receiving only somatic chromosomes (Ss), whereas the other cell is given all the so-called germ line limited chromosomes (Ks) in addition to the Ss. The cytokinesis following this differential mitosis is incomplete and the daughter cells remain connected by a permanent cytoplasmic bridge. The cell with the Ss and Ks develops into a primary oocyte or spermatocyte, whereas the cell containing only Ss differentiates as a nurse cell in the female or as an aberrant spermatocyte in the male. When the primary spermatocyte enters meiosis, the Ss in the connected aberrant spermatocyte undergo chromosome condensation but the aberrant spermatocyte remains undivided, with the condensed metaphase status and inactivation of the Ss persisting during both meiotic divisions. These events indicate a programmed inactivation of all chromosomes in the aberrant spermatocyte at the beginning of meiosis. The alterations in the microtubule arrangements and of the distribution of mitochondria in the spermatocytes during meiosis have been followed via live-cell fluorescence labelling with the TubulinTracker and MitoTracker reagents and by transmission electron microscopy. The observations reveal a hyperamplification of the centrosomes and the formation of tetrapolar asters in the non-dividing aberrant spermatocytes containing the condensed Ss. The programmed inactivation of the Ss in the aberrant spermatocyte is suggested to have developed during evolution to inhibit the entry of the aberrant spermatocytes into meiosis, thereby preventing the formation of sperms containing only Ss but no Ks.     

Actin and myosin inhibitors block elongation of kinetochore fibre stubs in metaphase crane-fly spermatocytes

Summary. We used an ultraviolet microbeam to cut individual kinetochore spindle fibres in metaphase crane-fly spermatocytes. We then followed the growth of the “kinetochore stubs”, the remnants of kinetochore fibres that remain attached to kinetochores. Kinetochore stubs elongate with constant velocity by adding tubulin subunits at the kinetochore, and thus elongation is related to tubulin flux in the kinetochore microtubules. Stub elongation was blocked by cytochalasin D and latrunculin A, actin inhibitors, and by butanedione monoxime, a myosin inhibitor. We conclude that actin and myosin are involved in generating elongation and thus in producing tubulin flux in kinetochore microtubules. We suggest that actin and myosin act in concert with a spindle matrix to propel kinetochore fibres poleward, thereby causing stub elongation and generating anaphase chromosome movement in nonirradiated cells. Correspondence: A. Forer, Biology Department, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.     

Redundant mechanisms for anaphase chromosome movements: crane-fly spermatocyte spindles normally use actin filaments but also can function without them

Summary. Actin inhibitors block or slow anaphase chromosome movements in crane-fly spermatocytes, but stopping of movement is only temporary; we assumed that cells adapt to loss of actin by switching to mechanism(s) involving only microtubules. To test this, we produced actin-filament-free spindles: we added latrunculin B during prometaphase, 9–80 min before anaphase, after which chromosomes generally moved normally during anaphase. We confirmed the absence of actin filaments by staining with fluorescent phalloidin and by showing that cytochalasin D had no effect on chromosome movement. Thus, actin filaments are involved in normal anaphase movements, but in vivo, spindles nonetheless can function normally without them. We tested whether chromosome movements in actin-filament-free spindles arise via microtubules by challenging such spindles with anti-myosin drugs. Y-27632 and BDM (2,3-butanedione monoxime), inhibitors that affect myosin at different regulatory levels, blocked chromosome movement in normal spindles and in actin-filament-free spindles. We tested whether BDM has side effects on microtubule motors. BDM had no effect on ciliary and sperm motility or on ATPase activity of isolated ciliary axonemes, and thus it does not directly block dynein. Nor does it block kinesin, assayed by a microtubule sliding assay. BDM could conceivably indirectly affect these microtubule motors, though it is unlikely that it would have the same side effect on the motors as Y-27632. Since BDM and Y-27632 both affect chromosome movement in the same way, it would seem that both affect spindle myosin; this suggests that spindle myosin interacts with kinetochore microtubules, either directly or via an intermediate component. Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s00709-005-0094-6 Correspondence and reprints: Biology Department, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.     

A protein associated with the manchette during rat spermiogenesis is encoded by a gene of the TBP-1-like subfamily with highly conserved ATPase and protease domains

We have used a rat pachytene spermatocyte cDNA expression library to clone TBP-1 (for tat-binding protein-1; designated rat testis TBP-1 [rtTBP-1]), a new member of the family of putative ATPases associated with the 26S proteasome complex. The 1.63 kb rtTBP-1 cDNA encodes a 49 kDa protein with 99% amino acid identity to human TBP-1 protein. rtTBP-1 protein contains a heptad repeat of six leucine-type zipper fingers at the amino terminal end and highly conserved ATPase and DNA/RNA helicase motifs towards the carboxyl terminal region. Chromatofocusing fractionation of rat testis sucrose extracts demonstrates that the encoded product, recognized by an antiserum raised to the first 196 amino acids of human TBP-1, consists of a protein triplet with a molecular mass range of 52-48 kDa and acidic pI (5.0–5.9). An identical immunoreactive triplet was detected by immunoblotting in extracts of fractionated pachytene spermatocytes, round spermatids and epididymal sperm. In situ hybridization using digoxigenin-labeled antisense RNA probes shows a predominant distribution of specific mRNA in the seminiferous epithelial region occupied by elongating spermatids and primary spermatocytes. Indirect immunofluorescence and immunogold electron microscopy studies show that rtTBP-1 immunoreactive sites colocalize with α-tubulin-decorated manchettes of elongating spermatids. In addition, rtTBP-1 immunoreactivity was detected in fibrillar and granular cytoplasmic bodies typically observed in spermatocytes and spermatids as well as in association with paraaxonemal mitochondria and outer dense fibers of the developing spermatid tail. Results of this study indicate that rtTBP-1 is a member of the highly evolutionary conserved TBP-1-like subfamily of putative ATPases, sharing regions of identity—including ATP-binding sites—with several subunits of the 26S proteasome, known to be involved in the ATP-dependent degradation of ubiquitin-conjugated proteins. Mol. Reprod. Dev. 48:77–89, 1997. © 1997 Wiley-Liss, Inc.     

The role of myosin phosphorylation in anaphase chromosome movement

This work deals with the role of myosin phosphorylation in anaphase chromosome movement. Y27632 and ML7 block two different pathways for phosphorylation of the myosin regulatory light chain (MRLC). Both stopped or slowed chromosome movement when added to anaphase crane-fly spermatocytes. To confirm that the effects of the pharmacological agents were on the presumed targets, we studied cells stained with antibodies against mono- or bi-phosphorylated myosin. For all chromosomes whose movements were affected by a drug, the corresponding spindle fibres of the affected chromosomes had reduced levels of 1P- and 2P-myosin. Thus the drugs acted on the presumed target and myosin phosphorylation is involved in anaphase force production.Calyculin A, an inhibitor of MRLC dephosphorylation, reversed and accelerated the altered movements caused by Y27632 and ML-7, suggesting that another phosphorylation pathway is involved in phosphorylation of spindle myosin. Staurosporine, a more general phosphorylation inhibitor, also reduced the levels of MRLC phosphorylation and caused anaphase chromosomes to stop or slow. The effects of staurosporine on chromosome movements were not reversed by Calyculin A, confirming that another phosphorylation pathway is involved in phosphorylation of spindle myosin.     

XMR, a dual location protein in the XY pair and in its associated nucleolus in mouse spermatocytes

Xlr and Xmr are sex-specific genes which are expressed during the meiotic prophase I in the mouse. In spermatocytes, XMR concentrates on the asynapsed regions of the XY chromosomes, suggesting that XMR plays a role in sex chromosome condensation and silencing. The present study shows that in the mouse, XMR also concentrates in the nucleolus which is closely associated with the XY chromosome pair. In this species, the formation of a large fibrillo-granular nucleolus signals the activation of the ribosomal genes, but release of pre-ribosomal particles is inhibited. Using laser confocal microscopy we characterized the distribution of XMR in the XY body relative to the XY chromatin and the nucleolus. Immunoelectron microscopy showed that XMR concentrates in the fibrillo-granular component and the granular component (GC) of the nucleolus. In (T[X;16]16H) mouse spermatocytes, the nucleolus displays little or no activity and does not associate with the XY pair. XMR concentrated only on the XY chromosomes in (T[X;16]16H) mouse spermatocytes. These data suggest that XMR could play a role both in the XY pair and the nucleolus associated to the sex chromosomes.