The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly

Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1–GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions

Septin-dependent compartmentalization of the endoplasmic reticulum during yeast polarized growth

Polarized cells frequently use diffusion barriers to separate plasma membrane domains. It is unknown whether diffusion barriers also compartmentalize intracellular organelles. We used photobleaching techniques to characterize protein diffusion in the yeast endoplasmic reticulum (ER). Although a soluble protein diffused rapidly throughout the ER lumen, diffusion of ER membrane proteins was restricted at the bud neck. Ultrastructural studies and fluorescence microscopy revealed the presence of a ring of smooth ER at the bud neck. This ER domain and the restriction of diffusion for ER membrane proteins through the bud neck depended on septin function. The membrane-associated protein Bud6 localized to the bud neck in a septin-dependent manner and was required to restrict the diffusion of ER membrane proteins. Our results indicate that Bud6 acts downstream of septins to assemble a fence in the ER membrane at the bud neck. Thus, in polarized yeast cells, diffusion barriers compartmentalize the ER and the plasma membrane along parallel lines

Rab4b Is a Small GTPase Involved in the Control of the Glucose Transporter GLUT4 Localization in Adipocyte

Endosomal small GTPases of the Rab family, among them Rab4a, play an essential role in the control of the glucose transporter GLUT4 trafficking, which is essential for insulin-mediated glucose uptake. We found that adipocytes also expressed Rab4b and we observed a consistent decrease in the expression of Rab4b mRNA in human and mice adipose tissue in obese diabetic states. These results led us to study this poorly characterized Rab member and its potential role in glucose transport.We used 3T3-L1 adipocytes to study by imaging approaches the localization of Rab4b and to determine the consequence of its down regulation on glucose uptake and endogenous GLUT4 location. We found that Rab4b was localized in endosomal structures in preadipocytes whereas in adipocytes it was localized in GLUT4 and in VAMP2-positive compartments, and also in endosomal compartments containing the transferrin receptor (TfR). When Rab4b expression was decreased with specific siRNAs by two fold, an extent similar to its decrease in obese diabetic subjects, we observed a small increase (25%) in basal deoxyglucose uptake and a more sustained increase (40%) in presence of submaximal and maximal insulin concentrations. This increase occurred without any change in GLUT4 and GLUT1 expression levels and in the insulin signaling pathways. Concomitantly, GLUT4 but not TfR amounts were increased at the plasma membrane of basal and insulin-stimulated adipocytes. GLUT4 seemed to be targeted towards its non-endosomal sequestration compartment.Taken our results together, we conclude that Rab4b is a new important player in the control of GLUT4 trafficking in adipocytes and speculate that difference in its expression in obese diabetic states could act as a compensatory effect to minimize the glucose transport defect in their adipocytes

The dynamic localization and novel functions of the Ipl1/Aurora protein kinase

La ségrégation exacte des chromosomes est un événement indispensable pour une division cellulaire fidèle. Des anormalités dans la ségrégation des chromosomes résultent en une instabilité génomique et une aneuploïdie et sont corrélées avec le cancer. Le laboratoire étudie la ségrégation des chromosomes chez la levure de bière Saccharomyces cerevisiae. Étant donné que ce processus est hautement conservé parmi les eucaryotes, des études chez la levure vont fournir une compréhension fondamentale de ce mécanisme. Ipl1p est, chez la levure, le membre de la famille hautement conservée de protéine kinase : Ipl1/Aurora qui joue un rôle dans la ségrégation des chromosomes, le point de contrôle du fuseau mitotique et la cytocinèse. De plus, plusieurs études ont démontré que les homologues humains de la protéine kinase Aurora sont des oncogènes. Des anormalités dans la régulation de la protéine kinase Ipl1/Aurora mènent à l’aneuploïdie qui résultant en une instabilité génomique. Il est, par conséquent, important de mieux comprendre la régulation et les rôles de cette protéine kinase dans le but d’élucider des détails à propos des mécanismes qui mènent à l’instabilité génomique. Afin d’en apprendre plus sur la régulation et sur la fonction d’Ipl1p, j’ai décidé d’examiner soigneusement la localisation d’Ipl1p. J’ai trouvé qu’Ipl1p se trouve aux kinétochores de G1 à la métaphase. Ensuite, la protéine quitte les kinétochores et se déplace sur le fuseau mitotique. Elle est probablement transportée des kinétochores au fuseau sur les microtubules. Ipl1p s’accumule au milieu du fuseau mitotique où la protéine régule la dépolymérisation du fuseau mitotique. Je propose qu’Ipl1p est un régulateur général des extrémités positives des microtubules. Finalement, dans une autre étude, j’ai trouvé que des mutations dans Ipl1p et le point de contrôle du fuseau mitotique permettent la séparation des chromatides et l’élongation du fuseau mitotique en l’absence de la fonction du complexe promouvant l’anaphase. Les données présentées ici suggèrent que le point de contrôle du fuseau mitotique peut directement inhiber la protéase Esp1p.Proper chromosome segregation is a crucial event for accurate cell division. Defects in chromosome segregation lead to genetic instability and aneuploidy and are correlated with cancer. The laboratory studies chromosome segregation in the budding yeast Saccharomyces cerevisiae. Since this process is highly conserved among eukaryotes, studies in yeast will provide fundamental understanding of this mechanism. Ipl1p is the budding yeast member of the highly conserved family of Ipl1/Aurora protein kinases, which play a role in chromosome segregation, the spindle checkpoint and cytokinesis. In addition, a number of studies have demonstrated that the human Aurora protein kinases are oncogenes. Defects in the regulation of the Ipl1/Aurora kinases leads to aneuploidy resulting in genomic instability. It is therefore important to better understand the regulation and functions of this protein kinase in order to elucidate details about the mechanisms that lead to genomic instability. In an attempt to learn more about Ipl1p regulation and functions, I decided to examine Ipl1p localization carefully. I found that Ipl1p localizes to kinetochores from G1 to metaphase. Ipl1p then leaves the kinetochores and transfers onto the whole spindle. It is probably transported away from the kinetochores on microtubules. Ipl1p then accumulates at the spindle midzone where it regulates spindle disassembly. I propose that Ipl1p is a general regulator of microtubules plus ends. Finally, in a different study, I found that mutations in Ipl1p and the spindle checkpoint allow sister chromatid separation and spindle elongation in the absence of the APC (anaphase promoting complex) function. Data presented here suggest that the spindle checkpoint may directly inhibit the Esp1p protease

Bioinformatic and Comparative Localization of Rab Proteins Reveals Functional Insights into the Uncharacterized GTPases Ypt10p and Ypt11p

A striking characteristic of a Rab protein is its steady-state localization to the cytosolic surface of a particular subcellular membrane. In this study, we have undertaken a combined bioinformatic and experimental approach to examine the evolutionary conservation of Rab protein localization. A comprehensive primary sequence classification shows that 10 out of the 11 Rab proteins identified in the yeast (Saccharomyces cerevisiae) genome can be grouped within a major subclass, each comprising multiple Rab orthologs from diverse species. We compared the locations of individual yeast Rab proteins with their localizations following ectopic expression in mammalian cells. Our results suggest that green fluorescent protein-tagged Rab proteins maintain localizations across large evolutionary distances and that the major known player in the Rab localization pathway, mammalian Rab-GDI, is able to function in yeast. These findings enable us to provide insight into novel gene functions and classify the uncharacterized Rab proteins Ypt10p (YBR264C) as being involved in endocytic function and Ypt11p (YNL304W) as being localized to the endoplasmic reticulum, where we demonstrate it is required for organelle inheritance

The protein phosphatase 2A B'-regulatory subunit par1p is implicated in regulation of the S. pombe septation initiation network.

In order to identify regulators of the Schizosaccharomyces pombe septation initiation network (SIN), which signals the onset of cell division, we have isolated extragenic suppressors of mutations in the GTPase spg1p, which is a central element in this pathway. One of these encodes the protein phosphatase 2A (PP2A) B'-regulatory subunit par1p. Loss of par1p function rescues mutants in cdc11, cdc7, and spg1, but no other SIN mutants. Our data suggest that PP2A-par1p acts as a negative regulator of SIN signalling