Stem cell asymmetric division requires tight control of spindle orientation. To study this key process, we have recorded Drosophila larval neural stem cells (NBs) engineered to express fluorescent reporters for microtubules, pericentriolar material (PCM), and centrioles. We have found that early in the cell cycle, the two centrosomes become unequal: one organizes an aster that stays near the apical cortex for most of the cell cycle, while the other loses PCM and microtubule-organizing activity, and moves extensively throughout the cell until shortly before mitosis when, located near the basal cortex, it recruits PCM and organizes the second mitotic aster. Upon division, the apical centrosome remains in the stem cell, while the other goes into the differentiating daughter. Apical aster maintenance requires the function of Pins. These results reveal that spindle orientation in Drosophila larval NBs is determined very early in the cell cycle, and is mediated by asymmetric centrosome function.
Purpose. To evaluate the pharmacological activity of insulin-loaded alginate/chitosan nanoparticles following oral dosage in diabetic rats. Methods. Nanoparticles were prepared by ionotropic pre-gelation of an alginate core followed by chitosan polyelectrolyte complexation. In vivo activity was evaluated by measuring the decrease in blood glucose concentrations in streptozotocin induced, diabetic rats after oral administration and flourescein (FITC)-labelled insulin tracked by confocal microscopy. Results. Nanoparticles were negatively charged and had a mean size of 750 nm, suitable for uptake within the gastrointestinal tract due to their nanosize range and mucoadhesive properties. The insulin association efficiency was over 70% and insulin was released in a pH-dependent manner under simulated gastrointestinal conditions. Orally delivered nanoparticles lowered basal serum glucose levels by more than 40% with 50 and 100 IU/kg doses sustaining hypoglycemia for over 18 h. Pharmacological availability was 6.8 and 3.4% for the 50 and 100 IU/kg doses respectively, a significant increase over 1.6%, determined for oral insulin alone in solution and over other related studies at the same dose levels. Confocal microscopic examinations of FITC-labelled insulin nanoparticles showed clear adhesion to rat intestinal epithelium, and internalization of insulin within the intestinal mucosa. Conclusion. The results indicate that the encapsulation of insulin into mucoadhesive nanoparticles was a key factor in the improvement of its oral absorption and oral bioactivity.
Abscission is the last step of cytokinesis that physically separates the cytoplasm of sister cells. As the final stage of cell division, abscission is poorly characterized during animal development. Here, we show that Aurora B and Survivin regulate the number of germ cells in each Drosophila egg chamber by inhibiting abscission during differentiation. This inhibition is mediated by an Aurora B-dependent phosphorylation of Cyclin B, as a phosphomimic form of Cyclin B rescues premature abscission caused by a loss-of-function of Aurora B. We show that Cyclin B localizes at the cytokinesis bridge, where it promotes abscission. We propose that mutual inhibitions between Aurora-B and Cyclin-B regulate the duration of abscission and thereby the number of sister cells in each cyst. Finally, we show that inhibitions of Aurora B and Cdk-1 activity in vertebrate cells also have opposite effects on the timing of abscission, suggesting a possible conservation of these mechanisms.
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.
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.
SUMMARY The actin-binding protein adducin was recently identified as a component of the neuronal subcortical cytoskeleton. Here we analyzed mice lacking adducin to uncover the function of this protein in actin rings. α-adducin knockout mice presented progressive axon enlargement in the spinal cord, optic and sciatic nerve, followed by axon degeneration and loss. Using stimulated emission depletion super-resolution microscopy, we show that a periodic subcortical actin cytoskeleton is assembled in every neuron type inspected including retinal ganglion cells and dorsal root ganglia neurons. In neurons devoid of adducin, the actin ring diameter increased, although the inter-ring periodicity was maintained. In vitro, the actin ring diameter adjusted as axons grew suggesting the lattice is dynamic. Our data support a model in which adducin activity is not essential for actin ring assembly and periodicity, but is necessary to control the diameter of both actin rings and axons, and actin filament growth within rings.
Through mutational analysis in Drosophila, we have identi®ed 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 identi®ed from yeast to man, revealing a novel protein family. Antibodies against Mast and analysis of tissue culture cells expressing an enhanced green¯uorescent protein±Mast fusion protein show that during mitosis, this protein localizes to centrosomes, the mitotic spindle, centromeres and spindle midzone. Microtubulebinding assays indicate that Mast is a microtubuleassociated protein displaying strong af®nity 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.
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