Liste des participants :
Valérie Doye, Jan Ellenberg (organisateur), Martin Hetzer, Tim Hunt, Alexey Khodjakov, Ulrike Kutay, Edward Lemke, Iain Mattaj (organisateur), Andrea Musacchio, Siegfried Musser, François Nedelec, Jan-Michael Peters, Thomas U. Schwartz, Tomoyuki Tanaka, Karsten Weis, Susan Wente, Howard J. Worman, Rick Wozniak
Compte rendu :
by Iain Mattaj
25 – 30 mai 2009
This seminar brought together an international group of renowned scientists working at the intersection of nuclear organization and transport with cell division control. The meeting was organized into six thematic sessions that consisted of presentation of new unpublished results and extensive discussion of these new findings.
The first session opened with two talks describing genetic screens carried out in yeast designed to identify new mutants defective in nuclear envelope (NE) assembly (Susan Wente) or in nuclear pore complex (NPC) formation and/or function (Valerie Doye). Susan Wente (Vanderbilt, USA) described evidence that implicates the endoplasmic reticulum coat proteins (Rtn1, Yop1) in NPC insertion into the NE, then went on to discuss genetic interactions between NPC components and certain chromatin remodeling factors that she suggested might reflect physical interactions between chromatin and NPCs that help organize the yeast nucleus. Valerie Doye (Institut Jacques Monod, France) described POM33, a new transmembrane (TM) nucleoporin in yeast and only the 4th such protein discovered. Aside from exhibiting genetic interactions with other TM and soluble nucleoporin mutants, cells lacking POM33 are unable to efficiently deliver new NPCs into the NE of the daughter bud nucleus. Ulrike Kutay (ETH Zurich, Switzerland) continued with a presentation on the role of protein kinases in NPC disassembly at mitosis. Her group has identified many sites of mitotic phosphorylation on nucleoporins and is making significant progress in identifying both the kinases involved and assigning functional roles to specific phosphorylation events. Rick Wozniak (U Alberta, Canada) has long been interested in the connections between the NPC, kinetochore formation and the spindle assembly checkpoint. In his talk he described the roles of the checkpoint protein Mad1 in targeting Xpo1/Crm1 to the kinetochore, and thus in chromosome segregation, and in altering the capacity of the NPC to permit Kap121-dependent nuclear import during mitosis.
Session 2 focused on the kinetochore, the structure assembled on chromosomes that is responsible for their attachment to the mitotic spindle. Tomo Tanaka (U Dundee, UK) discussed his recent findings on centromere reactivation in budding yeast, where he monitored the polarity of microtubules nucleated from the centromere by live cell imaging. Based on the observation that markers for the growing end of the microtubule faced away from the kinetochore the proposed model is that the stable minus of microtubules is stabilized at the kinetochore. Andrea Musacchio (EIO Milan, Italy) discussed a model for the function of the spindle assembly checkpoint (SAC) based on structural analysis of its proteins and biochemical experiments. The model proposes that microtubule attachment is stabilized by physically removing phosphorylation sites of the Ndc80 complex in the outer kinetochore out of molecular reach of the kinase Aurora B, which resides in the inner kinetochore. Thus checkpoint satisfaction would not rely on stretching between sister kinetochores but rather on molecular conformational changes within one kinetochore and thus uncouple the checkpoints on both sister kinetochores. Next, Alexey Khodjakov (Albany, USA) discussed his results about kinetochore function in mammalian cells that had been artificially induced to enter mitosis prematurely. This was followed by an intense discussion on the molecular mechanism of the SAC, a topic picked up again in the general discussion session that concluded the meeting.
Session 3 focused largely on interactions between NE and NPC components and nucleoplasmic or cytoplasmic structures. Martin Hetzer (Salk Institute, USA) utilized Drosophila salivary gland polytene chromosomes to enable visualization of interactions between nucleoporins and sites of transcription. His first conclusion was that these interactions, which have been described previously in other cell types, are happening intranuclearly, rather than at the NPCs. In some cases, the presence of nucleoporins precedes that of polymerase, and is required for transcription to occur. However, the situation is complex and there seem to be differences both between nucleoporins and between transcription sites. Ulrike Kutay (ETH Zurich, Switzerland) gave a short presentation on the targeting of the inner nuclear membrane SUN 2 protein, then went on to describe unexpected roles for SUN proteins in mitotic spindle organization. Valerie Doye (Institut Jacques Monod, France) described a chain of interactions from the nucleoporins Nup 107/Nup133 over the kinetochore components CENP-F/NudE/NudEL to dynein/dynactin that appear to be transiently required for tethering centrosomes to the NE during mitotic prophase. Howard Worman (Columbia U, USA) discussed evidence, coming from his analysis of mutant forms of lamin that are found in cardiomyopathy or lipodystrophy patients, for a different set of interactions that he proposed involve the lamins, SUN proteins and Nesprins, and thus span the NE, that are required for centrosome-NE interactions during interphase. He also described upregulation of various MAP kinases in cells from Lamin mutant cardiomyopathy patients, and evidence from mouse models that this upregulation may indeed be relevant for the disease phenotype.
Session 4 highlighted novel approaches to understand the structure and function of protein complexes with the example of the nuclear pore complex. Edward Lemke (EMBL, Germany) discussed methods to engineer proteins with artificial amino acids for site specific labeling with fluorescent dyes or photocleavable moieties. Siegfried Musser (Texas A&M, USA) then showed how such labeled proteins can be used to follow the transport and interactions of single protein complexes through the nuclear pore using single molecule fluorescence imaging. This was followed by Thomas Schwartz (MIT, USA) who discussed his recent structural work on nuclear pore protein complexes and a model for how these complexes could be arranged molecularly to form this very large channel in the nuclear envelope. The discussion centered around how broadly applicable these methods are to other protein complexes and when we will have a complete understanding of nuclear pore atomic structure and transport cycles.
Session 5 was centered on chromatin. Karsten Weis (UC Berkeley, USA) discussed novel results in budding yeast addressing recent findings that some genes move to the nuclear periphery upon activation. In contrast to what was previously thought, he presented evidence that movement to the periphery is not required for gene activation, but rather locates inducible genes into an environment where they can be rapidly shut off again. This discussion reconciled conflicting models in the field, according to which the nuclear periphery is alternatively a repressive or permissive environment for gene activation. The theme of gene regulation was continued by Jan-Michael Peters, who discussed data from mammalian cells where his group mapped all binding sites of the cohesin complexes by chromatin immunoprecipitation and compared to the sites of the protein CTCF in involved in transcriptional control as an isolator. A surprising number of sites were specifically occupied by both proteins. Removal of cohesin by RNA interference compromised the ability of CTCF to insulate enhancers from distal genes. Although still preliminary, these results raised the exciting possibility that the ring like cohesin complexes may serve as “clamps” to organize interphase chromatin and help factors like CTCF to isolate gene regulatory regions such as enhancers from distal genes. The chromatin session was concluded by François Nedelec, who presented data on microarrays of chromatin beads, which allowed him to measure the effect of chromatin size on spindle assembly in high throughput in Xenopus egg extracts.
The final, 6th session consisted of talks on mitosis and meiosis. Tim Hunt (CRUK LRI, UK) discussed the thorny issue of assigning functions to specific phosphatases in mitotic progression, in a talk that recalled the difficulties associated with assigning roles to mitotic kinases described by Ulrike Kutay in Session 1. Persistence and determination are going to be necessary to unravel the complexities of mitotic regulation by reversible mitotic phosphorylation but these two talks showed that the situation is far from hopeless. The two organizers, Jan Ellenberg and Iain Mattaj (EMBL, Germany) then provided the last two presentations. Jan Ellenberg discussed their recent advances in tracking the dynamics of meiotic chromosome in living mouse oocytes to understand the mechanism of homologous chromosome segregation. He showed evidence that incorrect attachments of meiotic chromosomes that later have to be corrected appears to be the rule rather than the exception in mammalian meiosis. Iain Mattaj talked about the role of Lipin in NE breakdown. Lipin dephosphorylates phospholipids to produce di-acyl glycerol (DAG), raising the question of whether Lipin acts in NE breakdown via DAG-based signal transduction or by the production of NE membrane lipids. The fact that Lipin mutants fail to break down the nuclear lamina provides some support for the first hypothesis. He then showed data on a group of prokaryotic proteins, found in Planctomycetes, which resemble eukaryotic endomembrane coat proteins. Immuno-EM localization suggests that these proteins are also membrane associated in Planctomycetes. Since this prokaryotic Genus is compartmentalized, this has implications for the evolution of the endomembrane system, including the NE.
The formal sessions were extended by many questions and much discussion, and the meeting was wrapped up by a final discussion in which several of the earlier topics were re-examined. The many connections between components of the nuclear periphery and cell cycle events provided fertile ground for exchanges between the representatives of the two poles of the meeting, and the unique atmosphere of Les Treilles ensured that these discussions were lively, fruitful and long.