Thomas U. Mayer

Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis

We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1). MKlp2 is a target for Plk1, and phosphorylated MKlp2 binds to the polo box domain of Plk1. Plk1 also binds directly to microtubules and targets to the central spindle via its polo box domain, and this interaction controls the activity of Plk1 toward MKlp2. An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells. We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.

Dissecting cellular processes using small molecules : identification of colchicine-like, taxol-like and other small molecules that perturb mitosis

BACKGROUNDnUnderstanding the molecular mechanisms of complex cellular processes requires unbiased means to identify and to alter conditionally gene products that function in a pathway of interest. Although random mutagenesis and screening (forward genetics) provide a useful means to this end, the complexity of the genome, long generation time and redundancy of gene function have limited their use with mammalian systems. We sought to develop an analogous process using small molecules to modulate conditionally the function of proteins. We hoped to identify simultaneously small molecules that may serve as leads for the development of therapeutically useful agents.nnnRESULTSnWe report the results of a high-throughput, phenotype-based screen for identifying cell-permeable small molecules that affect mitosis of mammalian cells. The predominant class of compounds that emerged directly alters the stability of microtubules in the mitotic spindle. Although many of these compounds show the colchicine-like property of destabilizing microtubules, one member shows the taxol-like property of stabilizing microtubules. Another class of compounds alters chromosome segregation by novel mechanisms that do not involve direct interactions with microtubules.nnnCONCLUSIONSnThe identification of structurally diverse small molecules that affect the mammalian mitotic machinery from a large library of synthetic compounds illustrates the use of chemical genetics in dissecting an essential cellular pathway. This screen identified five compounds that affect mitosis without directly targeting microtubules. Understanding the mechanism of action of these compounds, along with future screening efforts, promises to help elucidate the molecular mechanisms involved in chromosome segregation during mitosis.

Calcium triggers exit from meiosis II by targeting the APC/C inhibitor XErp1 for degradation.

Vertebrate eggs awaiting fertilization are arrested at metaphase of meiosis II by a biochemical activity termed cytostatic factor (CSF). This activity inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that triggers anaphase onset and mitotic/meiotic exit by targeting securin and M-phase cyclins for destruction. On fertilization a transient rise in free intracellular calcium causes release from CSF arrest and thus APC/C activation. Although it has previously been shown that calcium induces the release of APC/C from CSF inhibition through calmodulin-dependent protein kinase II (CaMKII), the relevant substrates of this kinase have not been identified. Recently, we characterized XErp1 (Emi2), an inhibitor of the APC/C and key component of CSF activity in Xenopus egg extract. Here we show that calcium-activated CaMKII triggers exit from meiosis II by sensitizing the APC/C inhibitor XErp1 for polo-like kinase 1 (Plx1)-dependent degradation. Phosphorylation of XErp1 by CaMKII leads to the recruitment of Plx1 that in turn triggers the destruction of XErp1 by phosphorylating a site known to serve as a phosphorylation-dependent degradation signal. These results provide a molecular explanation for how the fertilization-induced calcium increase triggers exit from meiosis II.

The human kinesin Kif18A is a motile microtubule depolymerase essential for chromosome congression

BACKGROUNDnThe accurate alignment of chromosomes at the spindle equator is fundamental for the equal distribution of the genome in mitosis and thus for the genetic integrity of eukaryotes. Although it is well established that chromosome movements are coupled to microtubule dynamics, the underlying mechanism is not well understood.nnnRESULTSnBy combining RNAi-depletion experiments with in vitro biochemical assays, we demonstrate that the human kinesin Kif18A is a motile microtubule depolymerase essential for chromosome congression in mammalian tissue culture cells. We show that in vitro Kif18A is a slow plus-end-directed kinesin that possesses microtubule depolymerizing activity. Notably, Kif18A like its yeast ortholog Kip3p depolymerizes longer microtubules more quickly than shorter ones. In vivo, Kif18A accumulates in mitosis where it localizes close to the plus ends of kinetochore microtubules. The depletion of Kif18A induces aberrantly long mitotic spindles and loss of tension across sister kinetochores, resulting in the activation of the Mad2-dependent spindle-assembly checkpoint. Live-cell microscopy studies revealed that in Kif18A-depleted cells, chromosomes move at reduced speed and completely fail to align at the spindle equator.nnnCONCLUSIONSnThese studies identify Kif18A as a dual-functional kinesin and a key component of chromosome congression in mammalian cells.

Evidence for a satellite secretory pathway in neuronal dendritic spines

Long-term information storage within the brain requires the synthesis of new proteins and their use in synapse-specific modifications [1]. Recently, we demonstrated that translation sites for the local synthesis of integral membrane and secretory proteins occur within distal dendritic spines [2]. It remains unresolved, however, whether a complete secretory pathway, including Golgi and trans Golgi network-like membranes, exists near synapses for the local transport and processing of newly synthesized proteins. Here, we report evidence of a satellite secretory pathway in distal dendritic spines and distal dendrites of the mammalian brain. Membranes analogous to early (RER and ERGIC), middle (Golgi cisternae), and late (TGN) secretory pathway compartments are present within dendritic spines and in distal dendrites. Local synthesis, processing, and transport of newly translated integral membrane and secretory proteins may thus provide the molecular basis for synapse-specific modifications during long-term information storage in the brain.

Chemical genetics: tailoring tools for cell biology

Chemical genetics is a research approach that uses small molecules as probes to study protein functions in cells or whole organisms. Here, I review the parallels between classical genetic and chemical-genetic approaches and discuss the merits of small molecules to dissect dynamic cellular processes. I then consider the pros and cons of different screening approaches and specify strategies aimed at identifying and validating cellular target proteins. Finally, I highlight the impact of chemical genetics on our current understanding of cell biology and its potential for the future.

Cytostatic factor: an activity that puts the cell cycle on hold

Fertilization is the fundamental process in which two gametes - sperm and oocyte - fuse to generate a zygote that will form a new multicellular organism. In most vertebrates, oocytes await fertilization while arrested at metaphase of meiosis II. This resting state can be stable for many hours and depends on a cytoplasmic activity termed cytostatic factor (CSF). Recently, members of the novel Emi/Erp family of proteins have been put forward as important components of CSF. These proteins inhibit the anaphase-promoting complex/cyclosome (APC/C), which acts at the very core of the cell cycle regulatory machinery. Initially, Xenopus early mitotic inhibitor 1 (Emi1) was proposed to be a component of CSF, but newer work suggests that a structural relative, Emi-related protein 1 (Erp1/Emi2), is essential for maintenance of CSF arrest in Xenopus. Most importantly, studies on Erp1/Emi2 regulation have led to a detailed molecular understanding of the Ca2+-mediated release from CSF arrest that occurs upon fertilization.

Natural Product‐Guided Synthesis of a Spiroacetal Collection Reveals Modulators of Tubulin Cytoskeleton Integrity

The spiro[5.5]ketal moiety forms the underlying structural skeleton of numerous biologically active natural products. Since simplified but characteristic spiroketals derived from the parent natural products retain biological activity, the spiro[5.5]ketal unit can be regarded as a biologically prevalidated framework for the development of natural product-derived compound collections. We report an enantioselective synthesis of spiro[5.5]ketals on solid support. The reaction sequence employs asymmetric boron enolate aldol reactions with the enolate bound to the polymer or in solution as the key enantiodifferentiating step. It proceeds in up to 12 steps on solid support, makes the desired spiroketals available in high overall yields and with high stereoselectivities and is amenable to structural variation of the products. The small spiroketal collection synthesized contains phosphatase inhibitors and compounds that modulate the formation of the tubulin cytoskeleton in human cancer cells without directly targeting microtubules. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Synthesis and Biological Evaluation of Novel Eg5 Inhibitors

Human Eg5 is a mitotic kinesin that is essential for bipolar spindle formation and maintenance during mitosis. Recently, the discovery of compounds that inhibit Eg5 and cause mitotic arrest has attracted great interest, due to their potential use as the next generation of antimitotics. Here, we present the synthesis and biological investigation of 3,4‐dihydrophenylquinazoline‐2(1H)‐thiones as selective and potent Eg5 inhibitors.

High-content screening and profiling of drug activity in an automated centrosome-duplication assay

Maintenance of centrosome number is essential for cell‐cycle progression and genomic stability, but investigation of this regulation has been limited by assay difficulty. We present a fully automated image‐based centrosome‐duplication assay that is accurate and robust enough for both careful cell‐biology studies and high‐throughput screening, and employ this assay in a series of chemical‐genetic studies. We observe that a simple cytometric profiling strategy, which is based on organelle size, groups compounds with similar mechanisms of action; this suggests a simple strategy for excluding compounds that undesirably target such activities as protein synthesis and microtubule dynamics. Screening a library of compounds of known activity, we found unexpected effects on centrosome duplication by a number of drugs, most notably isoform‐specific protein kinase C inhibitors and retinoic acid receptor agonists. From a 16u2009320‐member library of uncharacterized small molecules, we identified five potent centrosome‐duplication inhibitors that do not target microtubule dynamics or protein synthesis. The analysis methodology reported here is directly relevant to studies of centrosome regulation in a variety of systems and is adaptable to a wide range of other biological problems.