Stemmann.html

As dividing cells undergo mitosis the replicated chromosomes are equally partitioned into two arising daughter cells. The faithful segregation of sister chromatids during anaphase is critical for the homeostasis of a stable complement of chromosomes. Mistakes in sister chromatid separation lead to aneuploidy which in turn gives raise to cancer and often there is a direct correlation between the degree of aneuploidy and the malignancy of tumors. Missing or super-numerous chromosomes caused by errors during meiosis and mitosis also contribute to severe congenital defects like Downís syndrome and spontaneous fetal abortion. Apart from its important clinical implications, the investigation of chromosome segregation is also a means to study the most beautiful and dramatic cell cycle transition that is tractable by light microscopy. The goal of our group is to understand the detailed molecular mechanism of how sister chromatids are segregated into newly forming daughter cells and how this process is regulated to ensure its high accuracy.
Sister chromatid cohesion is mediated by a multi-subunit protein complex, named cohesin. According to the current model, dissolution of cohesion is governed by a cysteine endopeptidase, separase, which clips one of the cohesin subunits, Scc1, thus triggering anaphase. In metaphase and earlier stages of the cell cycle separase is kept inactive by association with its inhibitor, securin. The timely activation of separase at the metaphase-anaphase transition is achieved by the activation of a ubiquitin ligase, APC/C^Cdc20, which mediates the degradation of securin, thereby liberating separase. Considering the importance of anaphase for chromosome stability, it comes as no surprise that this process is highly regulated. A surveillance mechanism (mitotic checkpoint) exists that inhibits APC/C (and hence the onset of anaphase) as long as chromosomes have not achieved a stable bipolar attachment to the microtubules of the mitotic spindle. Only when all chromosomes have aligned on the metaphase plate is the block relieved and securin degraded. Recent evidence indicates that anaphase is additionally regulated by securin-independent mechanisms.
During my postdoctoral work in Marc Kirschner's laboratory we discovered a novel regulation mechanism of vertebrate separase by inhibitory phosphorylation (Stemmann et al. Cell 107, 715-726, 2001). Using Xenopus egg extracts to recapitulate sister chromatid separation in vitro, we demonstrated that high CDC2/cyclinB1 activity inhibits anaphase (see Figure 1) but not securin degradation.

In vitro, inhibition of separase by phosphorylation or by binding to securin occurs independently of each another. We further showed in vivo that phosphorylation of the inhibitory site of separase is quantitative in metaphase and drops as cells undergo anaphase. Based on our findings we proposed that separase activation at the metaphase-anaphase transition in vertebrates requires the removal of both securin and an inhibitory phosphate (see Figure 3).

Securin knock-out mice and human cells that lack securin are viable demonstrating that the regulation of sister chromatid separation is still regulated in the absence of securin. Furthermore, the mitotic checkpoint remains intact in securin-depleted cells as exemplified by the fact that they still arrest at metaphase in response to spindle toxins. Our results provide a possible explanation for these observations. Currently, we are testing in securin knock-out cells whether preventing inhibitory phosphorylation of separase results in increased chromosome missegregation and/or a compromised checkpoint response. The identification of the physiological kinase and phosphatase that act on separase to inhibit and promote, respectively, its activity are other goals of this project.

In the primary structure of separase, the inhibitory phosphorylation occurs far away from the active site. Likewise, there are indications that securin might inhibit separase in a non-competitive manner. Another focus of our current research is the conformation/structure determination of phosphorylated and unphosphorylated separase and of separase in complex with securin. We do this by both analytical ultra-centrifugation and high-resolution electron microscopy on highly purified proteins. Thereby, we hope to gain insights into the inhibition mechanisms that control the activity of this crucial protease.

1) In-depth biochemical characterization of separase to fully understand its multi-layered regulation.

2) Analysis of securin in terms of possible separase-independent functions and its mitosis-specific phosphorylation.

3) Identification and functional analysis of separase substrates other than cohesin.

4) Investigation of the difference between centromere- and arm-specific cohesin.

1) An interesting question that remains to be answered is whether securin and the inhibitory phosphorylation are the only regulatory constraints that act on separase. Active separase has not been characterized in detail and it is possible that posttranslational modifications or co-factors are required for protease activity. We will use the Xenopus- and tissue culture system to isolate and characterize anaphase-specific, active separase.
It has been noted that securin does not only inhibit separase but at the same time has a positive effect on separase activity, too. In flies, for example, loss of securin function results in the same phenotype as does loss of separase function. How securin exerts its positive role on separase is not clear.
Does securin act as a specific chaperone that assists in the proper folding of separase? Is the presence of securin essential to produce separase that can then be activated? Can the activating and inhibitory functions of securin be separated thereby enabling us design a securin deletion mutant that will assist folding without inhibiting separase activity? Or can securin be dissociated from separase under non-denaturing conditions without the need for its degradation in Xenopus extracts?
We will use various recombinant systems to address these exciting questions.

2) Yeast securin blocks mitotic exit, a function that it might fulfill independent of separase. Furthermore, vertebrate securin has been implied to act as a transcriptional activator. From our own work we know that about 50% of securin in HeLaS3 cells is not associated with separase. We will isolate this fraction of securin and identify any associated proteins. This biochemical approach might help to elucidate additional functions of vertebrate securin.
Furthermore, we mapped a phosphorylation site in securin and created a mutant that can no longer be phosphorylated. We are currently using this mutant to examine the role of securin phosphorylation in mitosis.

3) Yeast separase cleaves not only cohesin but also Slk19, a kinetochore protein that is required for spindle stability. Further indications suggest there might be even more substrates of separase in yeast. In vertebrates no substrate other than cohesin is known to date. We will use the technique of in vitro expression cloning (IVEC) to search for novel substrates of vertebrate separase. IVEC is a powerful high-throughput method that was developed in the Kirschner lab and that we adapted to screen for separase substrates (see Figure 4). Our goal is the identification of novel separase targets followed by detailed characterization of their cellular functions.

4) It has been shown that in vertebrates but not yeast most of cohesin dissociates from DNA early in mitosis while a small fraction remains at the centromeres to maintain cohesion until execution of anaphase. What is special about the small subset of cohesin that saves it from the first wave of displacement in prophase? We believe that the answer to this important question lies in the cohesin complex itself, e.g. in the presence of an additional, protecting factor that is characteristic for the centromere-specific cohesin complex. Therefore, we propose to purify metaphase-specific cohesin using isolated metaphase chromosomes as a starting material. To facilitate purification of the scarce complex we will start off with cells that are engineered to overexpress tagged cohesin.

RESEARCH	PUBLICATIONS	PEOPLE
LINKS	CV	OPEN POSITIONS

since 2002	Group Leader	Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Martinsried/München.
01/00-5/02	Postdoctoral Fellow	Department of Cell Biology, Harvard Medical School, Boston, USA, Laboratory of Dr. Marc W. Kirschner
03/99 -12/99	Postdoctoral Fellow	Center of Biochemistry (BZH), University of Heidelberg, Germany, Laboratory of Dr. Johannes Lechner
03/95 - 02/99	Graduate Student	Department of Biochemistry, University of Regensburg, Germany. Laboratory of Dr. Johannes Lechner Title of thesis: The S. cerevisiae CBF3 kinetochore complex? In vitro reconstitution, identification of a new subunit and interaction with other kinetochore proteins.
10/97	Biochemistry Student	Research project at the Department of Biological Sciences, Unsiversity of California, Santa Barbara, USA. Laboratory of Dr. John Carbon
10/89 - 02/95	Biochemistry Student	University of Regensburg, Germany
08/92 - 05/93	Exchange Graduate Student	Washington University, Saint Louis, USA. Laboratory of Dr. Douglas E. Berg