Msps protein is localized to acentrosomal poles to ensure bipolarity of Drosophila meiotic spindles.

The female meiotic spindle is commonly formed in a centrosome-independent manner. Here we report the identification of proteins at acentrosomal poles in the female meiotic spindle of Drosophila. The acentrosomal poles contain at least two proteins, Mini-spindles (Msps) and D-TACC, which are also associated with mitotic centrosomes. These proteins interact with one another and are both required for maintaining the bipolarity of acentrosomal spindles. The polar localization of Msps is dependent on D-TACC and Ncd, a kinesin-like microtubule motor. We propose that the polar localization of Msps mediated by D-TACC and Ncd may be crucial for the stabilization of meiotic spindle bipolarity.

A new genetic method for isolating functionally interacting genes: high plo1(+)-dependent mutants and their suppressors define genes in mitotic and septation pathways in fission yeast.

We describe a general genetic method to identify genes encoding proteins that functionally interact with and/or are good candidates for downstream targets of a particular gene product. The screen identifies mutants whose growth depends on high levels of expression of that gene. We apply this to the plo1(+) gene that encodes a fission yeast homologue of the polo-like kinases. plo1(+) regulates both spindle formation and septation. We have isolated 17 high plo1(+)-dependent (pld) mutants that show defects in mitosis or septation. Three mutants show a mitotic arrest phenotype. Among the 14 pld mutants with septation defects, 12 mapped to known loci: cdc7, cdc15, cdc11 spg1, and sid2. One of the pld mutants, cdc7-PD1, was selected for suppressor analysis. As multicopy suppressors, we isolated four known genes involved in septation in fission yeast: spg1(+), sce3(+), cdc8(+), and rho1(+), and two previously uncharacterized genes, mpd1(+) and mpd2(+). mpd1(+) exhibits high homology to phosphatidylinositol 4-phosphate 5-kinase, while mpd2(+) resembles Saccharomyces cerevisiae SMY2; both proteins are involved in the regulation of actin-mediated processes. As chromosomal suppressors of cdc7-PD1, we isolated mutations of cdc16 that resulted in multiseptation without nuclear division. cdc16(+), dma1(+), byr3(+), byr4(+) and a truncated form of the cdc7 gene were isolated by complementation of one of these cdc16 mutations. These results demonstrate that screening for high dose-dependent mutants and their suppressors is an effective approach to identify functionally interacting genes.

mini spindles: A gene encoding a conserved microtubule-associated protein required for the integrity of the mitotic spindle in Drosophila.

We describe a new Drosophila gene, mini spindles (msps) identified in a cytological screen for mitotic mutant. Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells. Nucleation of microtubules from centrosomes, metaphase alignment of chromosomes, or the focusing of spindle poles appears much less affected. The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast. Consistent with their sequence similarity, Msps protein also associates with microtubules in vitro. In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase. The absence of centrosomal staining in interphase of the cellularized embryos suggests that the interactions between Msps protein and microtubules or centrosomes may be regulated during the cell cycle.

Parameters of growth in primary cultures and cell lines established from Drosophila imaginal discs.

We have further characterised our tissue culture system for the growth in vitro of Drosophila imaginal disc cells, including the culture medium requirements for optimum growth and we have adjusted the protocol recommended for the initiation of cultures. Many imaginal disc fragments become organised into vesicles, and some of these secrete extracellular material into the lumen. Sensory axons differentiate in primary disc cultures, in the absence of bristle formation. The early stages of cell division to form a cell line are recorded.

Neutrophil chemotactic behaviour in patients with early-onset forms of periodontitis (I). Leading front analysis in Boyden chambers.

Despite some reports to the contrary, it is generally assumed that early-onset forms of periodontal disease (including both juvenile and rapidly progressive periodontitis) are associated with a defect in neutrophil (PMN) chemotactic behaviour. Using the Boyden chamber technique and N-formyl-methionyl-leucylphenylalanine (FMLP) to assess locomotion by the leading front method, we have failed to show any evidence for such depressed PMN locomotion. Indeed, when PMN chemotaxis and chemokinesis are considered in response to a range of chemoattractant doses our results indicate significant enhancement of all but random locomotion. When taken together with other studies, our results suggest that PMNs from patients with early-onset periodontitis may show abnormal locomotory behaviour which can either be enhanced or reduced in nature. The extent to which these results reflect in vitro methodology in uncertain and, furthermore, their in vivo significance is unclear.

Neutrophil chemotactic behaviour in patients with early-onset forms of periodontitis (II). Assessment using the under agarose technique.

The locomotory behaviour of peripheral blood neutrophils (PMNs) from patients with juvenile (JP) and rapidly progressive (RPP) forms of early-onset periodontal disease was studied using the under agarose technique and n-formyl-methionyl-leucyl-phenylalanine (FMLP) as the chemotractant. PMNs from experimental patients showed normal random, chemotactic and chemokinetic locomotory behaviour when compared with control subjects. Further investigation of single-cell movements using time-lapse video analysis also failed to show any significant differences in locomotory behaviour between the PMNs of experimental and control individuals. We conclude that differences in technique may account for much of the variation which exists in the literature with respect to PMN locomotion in periodontal disease. In the final analysis, it is difficult to dispute direct observation of moving cells, and using this approach, we have been unable to confirm the presence of any PMN locomotory defect in our series of patients with early-onset periodontal disease.