During cell division, the mitotic spindle segregates the sister chromatids into two nascent cells, such that each daughter cell inherits one complete set of chromosomes. Errors in spindle formation can result in both chromosome missegregation and cytokinesis defects and hence lead to genomic instability. To ensure the correct function of the spindle, the activity and localization of spindle associated proteins has to be tightly regulated in time and space. Reversible phosphorylation has been shown to be one of the key regulatory mechanisms for the organization of the mitotic spindle. The relatively low number of identified in vivo phosphorylation sites of spindle components, however, has hampered functional analysis of regulatory spindle networks. A more complete inventory of the phosphorylation sites of spindle-associated proteins would therefore constitute an important advance. Here, we describe the mass spectrometry-based identification of in vivo phosphorylation sites from purified human mitotic spindles. In total, 736 phosphorylation sites were identified, of which 312 could be attributed to known spindle proteins. Among these are phosphorylation sites that were previously shown to be important for the regulation of spindle-associated proteins. Importantly, this data set also comprises 279 novel phosphorylation sites of known spindle proteins for future functional studies. This inventory of spindle phosphorylation sites should thus make an important contribution to a better understanding of the molecular mechanisms that regulate the formation, function, and integrity of the mitotic spindle. mass spectrometry ͉ mitosis ͉ molecular cell biology ͉ phosphorylation ͉ proteomics A t the transition from interphase to mitosis, the microtubule network undergoes a profound change that culminates in the formation of the spindle apparatus. The mitotic spindle then serves to segregate the chromosomes to opposite poles of the cell and to define the plane of cell division. A large number of proteins associate with this microtubule-based structure and regulate its dynamic formation and function (1, 2). Several spindle proteins, including XKCM1 and XMAP215 family members (3, 4), have been identified that either stabilize or destabilize microtubules by mediating the rapid changes between polymerization and depolymerization (5, 6). These proteins play an important role in spindle formation, as illustrated by the striking change in microtubule half-life from 5-10 min in interphase to less than 1 min in mitosis (7), which results in the short and unstable microtubules characteristic of mitosis (5, 6). Another important group of spindle proteins comprises motors of the kinesin and dynein families that are essential for mitotic progression (5,6,8,9). They push the spindle poles away from each other during early mitosis and play crucial roles in capturing chromosomes and positioning them at the metaphase plate. Subsequently, motors also contribute to central spindle formation and cytokinesis. In addition, numerous structural prote...

The accurate distribution of sister chromatids during cell division is crucial for the generation of two cells with the same complement of genetic information. A highly dynamic microtubule-based structure, the mitotic spindle, carries out the physical separation of the chromosomes to opposite poles of the cells and, moreover, determines the cell division cleavage plane. In animal cells, the spindle comprises microtubules that radiate from the microtubule organizing centers, the centrosomes, and interact with kinetochores on the chromosomes. During mitosis, the two newly forming daughter cells must receive one copy of each chromosome. To accomplish this task, the mitotic spindle pulls sister chromatids toward opposite poles of the dividing cell. This microtubule-based structure comprises dynamic polymers made of ␣␤-tubulin heterodimers, associated with a large variety of microtubuleassociated proteins (1-5). At the transition from interphase to mitosis, the microtubule network undergoes a profound morphological change. In particular, the microtubule organizing centers of animal cells, the centrosomes, position to opposite sides of the nucleus and increase their microtubule nucleation capacity. Following nuclear envelope breakdown, microtubules emanating from the centrosomes capture each chromosome at the kinetochore, a protein complex assembled on centromeric DNA (6). Appropriate bipolar attachment of sister chromatids is monitored by a surveillance mechanism, the spindle checkpoint (7). Once all kinetochores are attached to microtubules emanating from opposite poles, the connection between sister chromatids is severed and chromatids are pulled apart. In addition to its central role in chromosome segregation, the mitotic spindle also determines the positioning and orientation of the cleavage plane. Therefore, proper positioning of the mitotic spindle is of particular importance for asymmetric cell divisions during development (8,9).A large number of proteins associate with the mitotic spindle and regulate its dynamic formation and function. Stabilizing and destabilizing proteins control the high turnover rate of mitotic microtubules, which have a half-life of less than 60 s (3). Furthermore, motor proteins of the kinesin and dynein families play crucial roles in the formation of a bipolar mitotic spindle (10,11). By interacting with microtubules during early mitosis, they push the spindle poles apart, then play important roles in chromosome-capture by microtubules and power chromosome movement throughout mitosis. Finally, the spindle harbors several regulatory proteins, notably protein kinases and phosphatases (12), which coordinate spindle function in time and space.Although our understanding of microtubule dynamics and spindle formation has greatly advanced during the last two decades, the complexity of the spindle continues to hamper its investigation. A more complete inventory of the mitotic spindle may thus contribute to a better understanding of how exactly the spindle is assembled, what role it plays in th...

Metaphase-to-anaphase transition is a fundamental step in cell cycle progression where duplicated sister-chromatids segregate to the future daughter cells. The anaphase-promoting complex/cyclosome (APC/C) is a highly regulated ubiquitin-ligase that triggers anaphase onset and mitotic exit by targeting securin and mitotic cyclins for destruction. It was previously shown that the Xenopus polo-like kinase Plx1 is essential to activate APC/C upon release from cytostatic factor (CSF) arrest in Xenopus egg extract. Although the mechanism by which Plx1 regulates APC/C activation remained unclear, the existence of a putative APC/C inhibitor was postulated whose activity would be neutralized by Plx1 upon CSF release. Here we identify XErp1, a novel Plx1-regulated inhibitor of APC/C activity, and we demonstrate that XErp1 is required to prevent anaphase onset in CSF-arrested Xenopus egg extract. Inactivation of XErp1 leads to premature APC/C activation. Conversely, addition of excess XErp1 to Xenopus egg extract prevents APC/C activation. Plx1 phosphorylates XErp1 in vitro at a site that targets XErp1 for degradation upon CSF release. Thus, our data lead to a model of APC/C activation in Xenopus egg extract in which Plx1 targets the APC/C inhibitor XErp1 for degradation.[Keywords: Cell cycle; anaphase-promoting complex/cyclosome; Xenopus; polo-like kinase; cytostatic factor; mitotic exit] Before fertilization, vertebrate eggs are arrested in metaphase of meiosis II by a biochemical activity that was named cytostatic factor (CSF) in a seminal publication over 30 years ago (Masui and Markert 1971). CSF was defined as an activity present in the cytoplasm of frog eggs arrested in metaphase of meiosis II that when injected into one cell of a two-cell embryo produced a cleavage arrest in the injected cell. Since then, numerous attempts have been made to identify the molecular nature of CSF in vertebrate eggs and to dissect the mechanism underlying the meiotic metaphase arrest. Fertilization of the egg causes a transient rise in free intracellular calcium mediating CSF release and thus anaphase-promoting complex/cyclosome (APC/C) activation. Active APC/C mediates the degradation of securin and cyclin B and thereby promotes anaphase onset (Lorca et al. 1993;Murray 2004). Extract from CSF-arrested Xenopus eggs faithfully recapitulates many morphological and biochemical events associated with release from CSF arrest upon addition of calcium ions.Data from various research groups have firmly established a role for the germ-cell specific c-Mos kinase in establishing CSF activity (Sagata et al. 1989;. c-Mos mediates its effect by activating a mitogen-activated protein kinase (MAPK) cascade (Kosako et al. 1994) leading to the phosphorylation and thereby activation of the protein kinase p90Rsk (Sturgill et al. 1988). Components of the spindle assembly checkpoint seem to be the downstream targets of the c-Mos/ MAPK/p90Rsk pathway, because the checkpoint proteins Mad1, Mad2, and Bub1 are required to mediate the c-Mos-initiated CSF arrest ...

Summary:A fully enzymatic assay is described for the determination of triglycerides. The coupled activities of triacylglycerol acylhydrolase and glycerol kinase result in the formation of glycerol-3-phosphate. The System also contains L-a-glycerol-phosphate oxidase, which produces hydrogen peroxide from glycerol-3-phosphate, and a sensitive chromogenic indicator System, consisting of peroxidase, 4-chlorophenol and 4-aminophenazone. We evaluated this rnethod with respect to kinetics, lineärity, blank rates, precision, accuracy, reagent stability and interfering substances.The accuracy of the triglyceride assay demands that each enzymatic reaction Step be complete and homogeneous. We therefore developed HPTLC-1 ) and HPLC-2 ) methods to monitor the course and completeness of each step. Reagenz zur enzymatischen Bestimmung von Triglyceriden im Serum mit verbesserter lipolytischer WirksamkeitZusammenfassung: Es wird ein vollenzymatischer Test zur Bestimmung Von Triglyceriden beschrieben, dessen Prinzip auf der Freisetzung von Wasserstoffperoxid aus Glycerin-3-phosphat mittels L-a-Glycerinphosphatoxidase in Kombination mit einem empfindlichen Farbindikator-System, bestehend aus Peroxidase, 4-Chlorphenol und 4-Aminöphenazon, beruht. Diese Methode wurde hinsichtlich Reaktionsgeschwindigkeit, Linearitätsbereich, Reagenzleerwert, Präzision, Richtigkeit, Reagenzstabilität und interferierender Substanzen untersucht.Da die Richtigkeit der Bestimmung von Triglyceriden einen vollständigen und eindeutigen Verlauf jedes einzelnen enzymatischen Reaktionsschrittes voraussetzt, wurden zur Verlaufskontrolle der einzelnen Teilreaktiönen HPTLC 1 )-sowie HPLC 2 )-Methpden entwickelt.

Proteomics studies of pathogenic bacteria are an important basis for biomarker discovery and for the development of antimicrobial drugs and vaccines. Especially where vaccines are concerned, it is of great interest to explore which bacterial factors are exposed on the bacterial cell surface and thus can be directly accessed by the immune system. One crucial step in proteomics studies of bacteria is an efficient subfractionation of their cellular compartments. We set out to compare and improve different protocols for the fractionation of proteins from Gram-negative bacteria into outer membrane, cytoplasmic membrane, periplasmic, and cytosolic fractions, with a focus on the outer membrane. Overall, five methods were compared, three methods for the fast isolation of outer membrane proteins and two methods for the fractionation of each cellular compartment, using Escherichia coli BL21 as a model organism. Proteins from the different fractions were prepared for further mass spectrometric analysis by SDS gel electrophoresis and consecutive in-gel tryptic digestion. Most published subfractionation protocols were not explicitly developed for proteomics applications. Thus, we evaluated not only the separation quality of the five methods but also the suitability of the samples for mass spectrometric analysis. We could obtain high quality mass spectrometry data from one-dimensional SDS-PAGE, which greatly reduces experimental time and sample amount compared to two-dimensional electrophoresis methods. We then applied the most specific fractionation technique to different Gram-negative pathogens, showing that it is efficient in separating the subcellular proteomes independent of the species and that it is capable of producing high-quality proteomics data in electrospray ionization mass spectrometry.