research
 
            


Our research focuses on mitosis and meiosis in higher
eukaryotes. Particularly, we are interested in the
question of
i) how kinesins contribute to chromosome congression in mammalian cells
ii) how anaphase onset is controlled by the ubiquitin/
proteasome system
iii) how cytokinesis is synchronized with chromosome
segregation.

For these studies we combine molecular and cell biological approaches with biochemistry and live-cell microscopy studies.
In addition to siRNAs, we apply cell-permeable small molecule inhibitors to modulate the activity of proteins in living cells, an approach termedchemical biology”.

The function of kinesins in chromosome congression

Kinesins are molecular motor proteins that convert energy
released by ATP-hydrolysis into mechanical force. Recently,
we could show that Kif18A, a kinesin-8 family member, plays
a key role in chromosome congression in mammalian cells.
Kif18A localizes to the plus-ends of kinetochore microtubules
(see Figure 1). Cells depleted of Kif18A show a dramatic delay
in prometaphase characterized by severe chromosome
congression defects. Furthermore, immunofluorescence
studies revealed that spindles are significantly longer
and sister kinetochores are under less tension in Kif18A-
depleted cells. Consistent with these in-vivo observations,
our biochemical studies demonstrated that Kif18A is a slow
plus-end directed kinesin that depolymerizes microtubules
in a length-dependent manner. Thus, Kif18A is a unique
kinesin that integrates both motility and micro tubule depolymerizing activity. Currently we are investigating
how the activity of Kif18A is regulated in time and space.

 
Fig. 1. Kif18A localizes to the plus-ends of kinetochore microtubules.
Immunofluorescence images of HeLa-cells expressing GFP-Kif18A. Kif18A
localizes chromosome proximal to HURP, a Ran-importinβ regulated factor
that stabilizes kinetochore microtubules

 

 

The regulation of anaphase onset in meiosis

In many vertebrates meiosis halts at metaphase of meiosis II,
yielding a fertilizable or so called “mature” egg. On fertilization
the egg quickly enters anaphase and completes  the second
meiotic division to generate a haploid pronucleus that can then
fuse with the male pronucleus to form a diploid zygote. In their
classic study more than three decades ago Masui and Markert
termed the activity that induces the metaphase II arrest
cytostatic factor” (CSF). Since then, CSF has resisted its
biochemical identification. Our research identified XErp1
(Xenopus Emi1-related protein) as the cytostatic factor
essential for metaphase II arrest in mature ocytes.

Biochemical assays revealed that XErp1 prevents anaphase
onset by directly inhibiting the anaphase-promoting complex/
cyclosome (APC), a ubiquitin-ligase complex that targets
cyclin-B1 and securin for proteasome dependent degradation
(Figure 2). Studies addressing the mechanism of XErp1
regulation, identified calcium/calmodulin-dependent kinase II
(CaMKII) and Xenopus Polo-like kinase 1 (Plx1) as the key
components initiating XErp1 destruction upon fertilization.
Specifically, CaMKII activated by the transient calcium wave
triggered by fertilization serves as a priming kinase for Plx1
by creating a docking site for the polo-box domain of Plx1.
Upon binding, Plx1 targets XErp1 for degradation by
phosphorylating a so-called phosphodegron recognized
by the ubiquitin-ligase SCFb-TRCP (Figure 3).
Thus, our studies not only identified XErp1 as the sought-after
cytostatic factor but also revealed how the transient calcium
wave triggers anaphase onset upon fertilization. Further
studies will focus on the function of XErp1 in mitosis in mammalian
cells.

The coordination of cytokinesis with karyokinesis

The genetic integrity of each organism depends on the correct
co- ordination of cytokinesis with karyokinesis (mitosis) in both
time and space. While it is clear that microtubule and actin
associated proteins are key to orchestrate both processes,
the underlying mechanism remains largely elusive. To address
this question, we applied a small molecule screen to identify
compounds that specifically target kinesins involved in cytokinesis.
Two compounds discovered in these screens selectively inhibit
the ATPase activities of Mklp2 and MPP1. In vivo, these compounds
induce binucleated cells consistent with the reported requirement
of Mklp2 and MPP1 for proper cytokinesis in mammalian cells
(Figure 4). Specificity analyses indicate that Mklp2 and MPP1
are the relevant binding partners of the identified compounds
in vivo. Currently, we are applying these compounds in combination
with live-cell microscopy studies to investigate the specific function of
Mklp2 and MPP1 in cytokinesis. In parallel, we are addressing the
mode of action of the identified inhibitors by biochemical analyses.

 

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