List of participants
Anna Akhmanova, Brian Burke (organisateur), Karen Carniol, Valérie Doye (organisateur), Vincent Galy, Edgar Gomes, Pierre Gönczy, Rebecca Heald, Alexey Khodjakov, Megan C. King, Ulrike Kutay (organisateur), Jan Lammerding, Helder Maiato, Matthieu Piel, Norma Beatriz Romero, Daniel Sampaio Osorio, Daniel (Dan) Starr, Marvin Tanenbaum, Richard Vallee
by Brian Burke
11 – 16 April , 2011
The spring of 2011 saw the gathering of 19 scientists from Europe, N. America and Asia to discuss emerging developments in the role of nucleo-cytoskeletal coupling in both health and disease. Following a welcome and introduction by Valérie Doye, the scientific proceedings commenced with a talk by Richard Vallee (Columbia University, New York NY) on the role of nuclear migration during brain development. He described how nuclear envelope-associated cytoplasmic dynein, and its regulator Lis1, along with kinesin-3, are involved in the oscillations of nuclei between apical and basal cytoplasmic domains in radial glial cells. The cell cycle-dependent nuclear oscillations are essential for the asymmetric division of these cells and the generation of neurons that migrate out to populate the neocortex. Neuronal migration itself is characterized by the process of nucleokinesis, whereby the centrosome migrates forward continuously with the leading neurite. The nucleus, in contrast, advances intermittently, a process that is in part dependent upon dynein and Lis1. Indeed mutations in Lis1 are associated with inhibition of neuronal migration leading to lissencephaly, a severe neuro-developmental disorder. The theme of nuclear positioning during development was continued by Daniel Starr (University of California, Davis). He described how inner nuclear membrane (INM) SUN domain proteins function as trans-lumenal tethers for outer nuclear membrane (ONM) KASH domain proteins, which in turn may interact with cytoskeletal components. These SUN-KASH pairs are referred to as LINC complexes (LInker of the Nucleoskeleton and Cytoskeleton). In hypodermal cells of C. elegans, appropriate nuclear positioning requires the SUN protein Unc-84 and the KASH protein Unc-83. The latter functions as an ONM adaptor for both Kinesin-1 and cytoplasmic dynein. It is Kinesin-1 that provides the major force for nuclear migration and positioning within the hypodermal cells. Dynein’s role, in contrast, appears to be as a modulator, functioning as a brake on kinesin-dependent nuclear movement. Mammalian LINC complex components were summarized by the next speaker, Brian Burke (IMB, Singapore). He described two new mammalian KASH domain proteins, nesprin4 and Dalek. The former is a Kinesin-1 binding protein that is localized to the ONM of certain epithelial cells. Inappropriate expression of nesprin4 causes cell polarization and separation of the nucleus from the centrosome. Dalek is a dynein binding protein that is expressed in meiotic cells. It was suggested that this protein may have a role in chromosome movements during meiotic prophase. The next session focused on the structure and function of LINC complexes. Ulrike Kutay (ETH, Zurich) described experiments that defined sorting determinants involved in the targeting of mammalian Sun2 to the INM. She was able to conclude that the nucleoplasmic domain of Sun2 likely passes through the peripheral channels of nuclear pore complexes (NPCs) en route to the INM. The NPC subunit, Nup188, was shown to function as a gatekeeper between the ONM and INM for Sun2 translocation. Furthermore, she presented biochemical and structural data defining molecular requirements for SUN-KASH interactions. The next talk by Megan King (Yale University, New Haven CT) explored LINC complex function in the fission yeast, Schizosaccharomyces pombe. Taking advantage of both genetic and cell biological approaches she has been able to define LINC complex components involved in chromatin organization. In particular she described possible roles for these components in the repair of DNA double stranded breaks. To complete this session on LINC complex function Jan Lammerding (Harvard Medical School and Brigham and Women’s Hospital, Boston MA) described biomechanical approaches to investigate the role of LINC complexes in nucleocytoplasmic coupling. He used dominant negative SUN and KASH protein mutants to disrupt LINC complex function. In this way he was able to provide a direct demonstration that LINC complexes help to propagate mechanical forces throughout the cell and are important for both cell polarization and migration.
The second day of the meeting saw the emphasis shift to an examination of the role of nuclear envelope proteins in myofibre formation and muscle disease. Norma Beatriz Romero (Institut de Myologie, Paris) described a new form of congenital muscular dystrophy characterized by highly abnormal myofibre nuclei featuring gross changes in heterochromatin distribution, unusual nuclear inclusions and nuclear fragmentation. While there were no changes in NE proteins, such as A-type lamins and emerin, defects which have been linked to muscular disease, biopsies from dystrophic muscle revealed a loss of Sun1 expression. The implication is that LINC complex defects might be associated with muscular dystrophy. Edgar Gomes (Institut de Myologie, Paris) was exploring the role of nuclear positioning in muscle disease. In mature myofibers, the nuclei are positioned in the periphery of the fiber, with some nuclei clustered at the neuromuscular synapse. The finding that nuclei in myofibers are mis-positioned in some muscular dystrophies such as centro-nuclear myopathies, and during muscle regeneration, suggests a role for nuclear positioning in muscle fiber function. The issue of how nuclear position is established in muscle cells and the molecular mechanisms that are responsible, has not been addressed. He described how different cytoskeletal elements, as well as regulatory molecules, are involved in nuclear movement during myofiber formation, both in normal and in pathological situations. Daniel Osorio (Institut de Myologie, Paris) returned to the theme of nuclear positioning during cell migration. In migrating 3T3 cells the nucleus is positioned to the rear of the centrosome, a process that involves the actin cytoskeleton. He described an RNA interference screen which identified nuclear membrane proteins that are required for efficient reorientation of the nucleus relative to centrosome. One of those was Samp1, the mammalian homologue of fission yeast Ima1, a LINC complex-associated inner nuclear membrane protein. Down regulation of these proteins results in inhibition of migration in classic wound healing assays. This concluded the day’s scientific sessions. A free afternoon in the village of Toutour or a hike in the nearby Gorges du Verdon allowed the participants time to ponder the talks so far.
The next morning began with talks describing the mechanisms involved in the recruitment of motor proteins to the nuclear envelope. Anna Akhmanova (Erasmus University, Rotterdam) described how the coiled-coil protein BicD2 functions as an adapter between RanBP2 (also known as Nup358), a NPC component, and dynein/dynactin in late G2. Depletion of BicD2 by RNA interference results in a failure to recruit dynein to the NE and separation of the centrosome(s) from the nucleus. Marvin Tanenbaum (University of Utrecht) expanded on the role of motor proteins in centrosome separation during late G2 and mitosis. The kinesin-5 family member, Eg5 normally drives centrosome separation by its action on overlapping microtubules. However, in the absence of Eg5 two other motors drive this process. The first is Kif15, a plus-end directed kinesin of the kinesin-12 family, which acts specifically after nuclear envelope breakdown to push centrosomes apart. The second motor is NE-associated cytoplasmic dynein, which in contrast to Kif15 is minus-end directed and acts specifically before nuclear envelope breakdown to promote centrosome separation. In addition to RanBP2-associated BicD2, there is a second system for recruiting dynein to the NE in late G2. Valérie Doye (Institut Jacques Monod, Paris) described how Nup133 serves to anchor dynein/dynactin to NPCs. The N-terminal domain (NTD) of Nup133 functions as a binding site for CENPF which in turn anchors NudE/L. The latter interacts with the dynein regulatory complex, dynactin. In the absence of the Nup133 NTD, dynein no longer concentrates at the NE. At the same time, there is a loss of centrosome tethering at the NE and a concomitant increase in monopolar spindle formation. These talks clearly highlight a role for NPC proteins in early mitotic progression. This notion was expanded upon in the subsequent session. Using a live assay amenable to high temporal and spatial resolution, Pierre Gönczy (École Polytechnique Fédérale de Lausanne) described an RNAi-based modifier screen for novel regulators of mitotic entry in C. elegans embryos. The nucleoporin NPP-3, the worm homologue of mammalian Nup205, was identified as a negative regulator of mitotic entry. He found that NPP-3 protein is lost at the end of prophase in the vicinity of the two centrosomes, and that such loss depends on the activity of the Aurora-A kinase AIR-1. These and other findings suggest a working model in which AIR-1 is needed, directly or indirectly, for the local loss of NPP-3, and thus promotes the onset of mitosis. Vincent Galy (Université Pierre et Marie Curie-CNRS, Paris) described studies on NPP-21 the C. elegans homologous of mammalian Tpr, a component of the filamentous basket-like structure on the nuclear face of NPCs. During mitosis NPP-21 co-localizes with the spindle. During early development it plays an as yet ill-defined role in nuclear/centrosome positioning. Down-regulation of NPP-21 results in increased cell cycle time, an affect that can be partially abrogated by depletion of MAD2, a spindle assembly checkpoint protein. The final talk in this session was provided by Matthieu Piel (Institut Curie, Paris) who presented some striking observations on the affects of restricting cell movement in both two and three dimensions. He showed that dendritic cells when confined in 7µm wide micro-channels could traverse constrictions as narrow as 2µm and up to 15µm in length. This required remarkable nuclear deformation. In other experiments, limiting cell height without constraining their ability to spread resulted in the appearance of mitotic abnormalities. At 5µm there was an increase in the number of tripolar spindles. At 3µm, highly aberrant metaphase cells were observed in which the spindle poles were seen to split leading to multipolar mitoses.
The final session of the meeting was devoted largely to the process of spindle assembly. Alexey Khodjakov (Wadsworth Center, Albany NY) discussed the mechanisms by which microtubules become associated with kinetochores. This is generally considered to occur by way of a “search and capture” process. This mechanism is, however, intrinsically inefficient. He described how “search and capture” can be rendered remarkably effective by optimizing chromosome geometry at nuclear envelope breakdown and by allowing lateral interactions between microtubules and kinetochores prior to anaphase. Rebecca Heald (University of California, Berkeley) spoke about work in her lab investigating how cellular architecture and organelle size are adjusted to different cell sizes and developmental states. By comparing nuclear and spindle assembly in Xenopus laevis extracts to those of the smaller, related frog Xenopus tropicalis, her lab has been able to establish a system to explore intrinsic scaling mechanisms of the microtubule-based spindle and the nucleus. Both computational modeling and experimental approaches are utilized to identify the responsible scaling factors, and to test and whether organelle scaling activities are also present in smaller cells of developing embryos. The final talk of the meeting was delivered by Helder Maiato (Universidade do Porto), who described the control of spindle elongation during anaphase B. In particular he focused on the role of the Aurora B kinase. This protein is a component of the chromosome passenger complex, which relocates from kinetochores to the spindle midzone at anaphase onset. He suggested that it is the dissipation of a gradient of Aurora B activity that limits spindle elongation. Furthermore, only as the chromatids move beyond the influence of Aurora B (i.e. beyond some limiting activity threshold) can nuclear envelope reformation commence.
The venue at Les Treilles with its intimate setting and outstanding staff provided for an exceptionally engaging meeting. Discussions within the scientific sessions were both lively and enlightening, involving all of the participants, including Karen Carniol, our nineteenth invitee who was representing the journal Cell. I think it is fair to say, that after four days of talks, all of the participants returned to their homes with new insight into how the interplay between the nuclear envelope and cytoskeleton impacts multiple aspects of cellular physiology and how this can go awry in a variety of human diseases.