An Arp2/3 Nucleated F-Actin Shell Fragments Nuclear Membranes at Nuclear Envelope Breakdown in Starfish Oocytes.
Mori, M., Somogyi, K., Kondo, H., Monnier, N., Falk, H.J., Machado, P., Bathe, M., Nedelec, F. & Lenart, P.
Curr Biol. 2014 Jun 4. pii: S0960-9822(14)00544-2. doi:10.1016/j.cub.2014.05.019.
Animal cells disassemble and reassemble their nuclear envelopes (NEs) upon each division [1, 2]. Nuclear envelope breakdown (NEBD) serves as a major regulatory mechanism by which mixing of cytoplasmic and nuclear compartments drives the complete reorganization of cellular architecture, committing the cell for division [2, 3]. Breakdown is initiated by phosphorylation-driven partial disassembly of the nuclear pore complexes (NPCs), increasing their permeability but leaving the overall NE structure intact [4-7]. Subsequently, the NE is rapidly broken into membrane fragments, defining the transition from prophase to prometaphase and resulting in complete mixing of cyto- and nucleoplasm [6, 8]. However, the mechanism underlying this rapid NE fragmentation remains largely unknown. Here, we show that NE fragmentation during NEBD in starfish oocytes is driven by an Arp2/3 complex-nucleated F-actin "shell" that transiently polymerizes on the inner surface of the NE. Blocking the formation of this F-actin shell prevents membrane fragmentation and delays entry of large cytoplasmic molecules into the nucleus. We observe spike-like protrusions extending from the F-actin shell that appear to "pierce" the NE during the fragmentation process. Finally, we show that NE fragmentation is essential for successful reproduction, because blocking this process in meiosis leads to formation of aneuploid eggs.
Bayesian approach to MSD-based analysis of particle motion in live cells.
Monnier, N., Guo, S.M., Mori, M., He, J., Lenart, P. & Bathe, M.
Biophys J. 2012 Aug 8;103(3):616-26. doi: 10.1016/j.bpj.2012.06.029.
Quantitative tracking of particle motion using live-cell imaging is a powerful approach to understanding the mechanism of transport of biological molecules, organelles, and cells. However, inferring complex stochastic motion models from single-particle trajectories in an objective manner is nontrivial due to noise from sampling limitations and biological heterogeneity. Here, we present a systematic Bayesian approach to multiple-hypothesis testing of a general set of competing motion models based on particle mean-square displacements that automatically classifies particle motion, properly accounting for sampling limitations and correlated noise while appropriately penalizing model complexity according to Occam's Razor to avoid over-fitting. We test the procedure rigorously using simulated trajectories for which the underlying physical process is known, demonstrating that it chooses the simplest physical model that explains the observed data. Further, we show that computed model probabilities provide a reliability test for the downstream biological interpretation of associated parameter values. We subsequently illustrate the broad utility of the approach by applying it to disparate biological systems including experimental particle trajectories from chromosomes, kinetochores, and membrane receptors undergoing a variety of complex motions. This automated and objective Bayesian framework easily scales to large numbers of particle trajectories, making it ideal for classifying the complex motion of large numbers of single molecules and cells from high-throughput screens, as well as single-cell-, tissue-, and organism-level studies.
Systematic phosphorylation analysis of human mitotic protein complexes.
Hegemann, B., Hutchins, J.R., Hudecz, O., Novatchkova, M., Rameseder, J., Sykora, M.M., Liu, S., Mazanek, M., Lenart, P., Heriche, J.K., Poser, I., Kraut, N., Hyman, A.A., Yaffe, M.B., Mechtler, K. & Peters, J.M.
Sci Signal. 2011 Nov 8;4(198):rs12. doi: 10.1126/scisignal.2001993.
Progression through mitosis depends on a large number of protein complexes that regulate the major structural and physiological changes necessary for faithful chromosome segregation. Most, if not all, of the mitotic processes are regulated by a set of mitotic protein kinases that control protein activity by phosphorylation. Although many mitotic phosphorylation events have been identified in proteome-scale mass spectrometry studies, information on how these phosphorylation sites are distributed within mitotic protein complexes and which kinases generate these phosphorylation sites is largely lacking. We used systematic protein-affinity purification combined with mass spectrometry to identify 1818 phosphorylation sites in more than 100 mitotic protein complexes. In many complexes, the phosphorylation sites were concentrated on a few subunits, suggesting that these subunits serve as "switchboards" to relay the kinase-regulatory signals within the complexes. Consequent bioinformatic analyses identified potential kinase-substrate relationships for most of these sites. In a subsequent in-depth analysis of key mitotic regulatory complexes with the Aurora kinase B (AURKB) inhibitor Hesperadin and a new Polo-like kinase (PLK1) inhibitor, BI 4834, we determined the kinase dependency for 172 phosphorylation sites on 41 proteins. Combination of the results of the cellular studies with Scansite motif prediction enabled us to identify 14 sites on six proteins as direct candidate substrates of AURKB or PLK1.
Bulk cytoplasmic actin and its functions in meiosis and mitosis.
Field, C.M. & Lenart, P.
Curr Biol. 2011 Oct 11;21(19):R825-30.
Discussions of actin cell biology generally focus on the cortex, a thin, actin-rich layer of cytoplasm under the plasma membrane. Here we review the much less studied biology of actin filaments deeper in the cytoplasm and their recently revealed functions in mitosis and meiosis that are most prominent in large oocyte, egg and early embryo cells. The cellular functions of cytoplasmic actin range from the assembly and positioning of meiotic spindles to the prevention of cytoplasmic streaming. We discuss the possible use of evolutionarily conserved mechanisms to nucleate and organize actin filaments to achieve these diverse cellular functions, the cell-cycle regulation of these functions, and the many unanswered questions about this largely unexplored mechanism of cytoplasmic organization.
Intracellular transport by an anchored homogeneously contracting F-actin meshwork.
Mori, M., Monnier, N., Daigle, N., Bathe, M., Ellenberg, J. & Lenart, P.
Curr Biol. 2011 Apr 12;21(7):606-11.
Actin-based contractility orchestrates changes in cell shape underlying cellular functions ranging from division to migration and wound healing. Actin also functions in intracellular transport, with the prevailing view that filamentous actin (F-actin) cables serve as tracks for motor-driven transport of cargo. We recently discovered an alternate mode of intracellular transport in starfish oocytes involving a contractile F-actin meshwork that mediates chromosome congression. The mechanisms by which this meshwork contracts and translates its contractile activity into directional transport of chromosomes remained open questions. Here, we use live-cell imaging with quantitative analysis of chromosome trajectories and meshwork velocities to show that the 3D F-actin meshwork contracts homogeneously and isotropically throughout the nuclear space. Centrifugation experiments reveal that this homogeneous contraction is translated into asymmetric, directional transport by mechanical anchoring of the meshwork to the cell cortex. Finally, by injecting inert particles of different sizes, we show that this directional transport activity is size-selective and transduced to chromosomal cargo at least in part by steric trapping or "sieving." Taken together, these results reveal mechanistic design principles of a novel and potentially versatile mode of intracellular transport based on sieving by an anchored homogeneously contracting F-actin meshwork.
APPL proteins FRET at the BAR: direct observation of APPL1 and APPL2 BAR domain-mediated interactions on cell membranes using FRET microscopy.
Chial, H.J., Lenart, P. & Chen, Y.Q.
PLoS One. 2010 Aug 30;5(8):e12471.
BACKGROUND: Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH) domain, and a C-terminal phosphotyrosine binding (PTB) domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported. METHODOLOGY: Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET) experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species. CONCLUSIONS: All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2) and heterotypic (i.e., APPL1-APPL2) manner on curved cell membranes. Furthermore, the results of our experiments did not show photoconversion of YFP into a CFP-like species following photobleaching, supporting the use of CFP donor/YFP acceptor FRET pairs in acceptor photobleaching studies.
Structure of the anaphase-promoting complex/cyclosome interacting with a mitotic checkpoint complex.
Herzog, F., Primorac, I., Dube, P., Lenart, P., Sander, B., Mechtler, K., Stark, H. & Peters, J.M.
Science. 2009 Mar 13;323(5920):1477-81.
Once all chromosomes are connected to the mitotic spindle (bioriented), anaphase is initiated by the protein ubiquitylation activity of the anaphase-promoting complex/cyclosome (APC/C) and its coactivator Cdc20 (APC/C(Cdc20)). Before chromosome biorientation, anaphase is delayed by a mitotic checkpoint complex (MCC) that inhibits APC/C(Cdc20). We used single-particle electron microscopy to obtain three-dimensional models of human APC/C in various functional states: bound to MCC, to Cdc20, or to neither (apo-APC/C). These experiments revealed that MCC associates with the Cdc20 binding site on APC/C, locks the otherwise flexible APC/C in a "closed" state, and prevents binding and ubiquitylation of a wide range of different APC/C substrates. These observations clarify the structural basis for the inhibition of APC/C by spindle checkpoint proteins.
Polo on the Rise-from Mitotic Entry to Cytokinesis with Plk1.
Petronczki, M., Lenart, P. & Peters, J.M.
Dev Cell. 2008 May;14(5):646-59.
Polo-like kinase 1 (Plk1) is a key regulator of cell division in eukaryotic cells. New techniques, including the application of small-molecule inhibitors, have greatly expanded our knowledge of the functions, targets, and regulation of this key mitotic enzyme. In this review, we focus on how Plk1 is recruited to centrosomes, kinetochores, and the spindle midzone and what the specific tasks of Plk1 at these distinct subcellular structures might be. In particular, we highlight new work on the role of Plk1 in cytokinesis in human cells. Finally, we describe how better understanding of Plk1 functions allows critical evaluation of Plk1 as a potential drug target for cancer therapy.
Sororin is required for stable binding of cohesin to chromatin and for sister chromatid cohesion in interphase.
Schmitz, J., Watrin, E., Lenart, P., Mechtler, K. & Peters, J.M.
Curr Biol. 2007 Apr 3;17(7):630-6. Epub 2007 Mar 8.
Sister chromatid cohesion depends on cohesin [1-3]. Cohesin associates with chromatin dynamically throughout interphase . During DNA replication, cohesin establishes cohesion , and this process coincides with the generation of a cohesin subpopulation that is more stably bound to chromatin . In mitosis, cohesin is removed from chromosomes, enabling sister chromatid separation . How cohesin associates with chromatin and establishes cohesion is poorly understood. By searching for proteins that are associated with chromatin-bound cohesin, we have identified sororin, a protein that was known to be required for cohesion . To obtain further insight into sororin's function, we have addressed when during the cell cycle sororin is required for cohesion. We show that sororin is dispensable for the association of cohesin with chromatin but that sororin is essential for proper cohesion during G2 phase. Like cohesin, sororin is also needed for efficient repair of DNA double-strand breaks in G2. Finally, sororin is required for the presence of normal amounts of the stably chromatin-bound cohesin population in G2. Our data indicate that sororin interacts with chromatin-bound cohesin and functions during the establishment or maintenance of cohesion in S or G2 phase, respectively.
BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo.
Steegmaier, M., Hoffmann, M., Baum, A., Lenart, P., Petronczki, M., Krssak, M., Gurtler, U., Garin-Chesa, P., Lieb, S., Quant, J., Grauert, M., Adolf, G.R., Kraut, N., Peters, J.M. & Rettig, W.J.
Curr Biol. 2007 Feb 20;17(4):316-22. Epub 2007 Feb 8.
Fine-mapping of the cell-division cycle, notably the identification of mitotic kinase signaling pathways, provides novel opportunities for cancer-drug discovery. As a key regulator of multiple steps during mitotic progression across eukaryotic species, the serine/threonine-specific Polo-like kinase 1 (Plk1) is highly expressed in malignant cells and serves as a negative prognostic marker in specific human cancer types . Here, we report the discovery of a potent small-molecule inhibitor of mammalian Plk1, BI 2536, which inhibits Plk1 enzyme activity at low nanomolar concentrations. The compound potently causes a mitotic arrest and induces apoptosis in human cancer cell lines of diverse tissue origin and oncogenome signature. BI 2536 inhibits growth of human tumor xenografts in nude mice and induces regression of large tumors with well-tolerated intravenous dose regimens. In treated tumors, cells arrest in prometaphase, accumulate phosphohistone H3, and contain aberrant mitotic spindles. This mitotic arrest is followed by a surge in apoptosis, detectable by immunohistochemistry and noninvasive optical and magnetic resonance imaging. For addressing the therapeutic potential of Plk1 inhibition, BI 2536 has progressed into clinical studies in patients with locally advanced or metastatic cancers.
The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1.
Lenart, P., Petronczki, M., Steegmaier, M., Di Fiore, B., Lipp, J.J., Hoffmann, M., Rettig, W.J., Kraut, N. & Peters, J.M.
Curr Biol. 2007 Feb 20;17(4):304-15. Epub 2007 Feb 8.
BACKGROUND: The mitotic kinases, Cdk1, Aurora A/B, and Polo-like kinase 1 (Plk1) have been characterized extensively to further understanding of mitotic mechanisms and as potential targets for cancer therapy. Cdk1 and Aurora kinase studies have been facilitated by small-molecule inhibitors, but few if any potent Plk1 inhibitors have been identified. RESULTS: We describe the cellular effects of a novel compound, BI 2536, a potent and selective inhibitor of Plk1. The fact that BI 2536 blocks Plk1 activity fully and instantaneously enabled us to study controversial and unknown functions of Plk1. Cells treated with BI 2536 are delayed in prophase but eventually import Cdk1-cyclin B into the nucleus, enter prometaphase, and degrade cyclin A, although BI 2536 prevents degradation of the APC/C inhibitor Emi1. BI 2536-treated cells lack prophase microtubule asters and thus polymerize mitotic microtubules only after nuclear-envelope breakdown and form monopolar spindles that do not stably attach to kinetochores. Mad2 accumulates at kinetochores, and cells arrest with an activated spindle-assembly checkpoint. BI 2536 prevents Plk1's enrichment at kinetochores and centrosomes, and when added to metaphase cells, it induces detachment of microtubules from kinetochores and leads to spindle collapse. CONCLUSIONS: Our results suggest that Plk1's accumulation at centrosomes and kinetochores depends on its own activity and that this activity is required for maintaining centrosome and kinetochore function. Our data also show that Plk1 is not required for prophase entry, but delays transition to prometaphase, and that Emi1 destruction in prometaphase is not essential for APC/C-mediated cyclin A degradation.
Checkpoint activation: don't get mad too much.
Lenart, P. & Peters, J.M.
Curr Biol. 2006 Jun 6;16(11):R412-4.
The Mad2 protein is required to delay sister chromatid separation until all chromosomes have been aligned on the mitotic spindle. Two recent studies provide new insights into how Mad2 contributes to this remarkable task.
Monitoring the permeability of the nuclear envelope during the cell cycle.
Lenart, P. & Ellenberg, J.
Methods 2006 Jan;38(1):17-24.
In animal organisms the nuclear envelope (NE) disassembles during cell division resulting in complete intermixing of cytoplasmic and nuclear compartments. This leads to the activation of many mitotic enzymes, which were kept away from their substrates or regulators by nuclear or cytoplasmic sequestration in interphase. Nuclear envelope breakdown (NEBD) is thus an essential step of mitotic entry and commits a cell to M-phase. NEBD begins with the partial disassembly of nuclear pore complexes, leading to a limited permeabilization of the NE for molecules up to approximately 40nm diameter. This is followed by the complete disruption of nuclear pores, which causes local fenestration of the double nuclear membrane and subsequently breakdown of the entire NE structure. Here, we describe the use of different sized inert fluorescent tracer molecules to directly visualize these different steps of NEBD in live cells by fluorescence microscopy.
A contractile nuclear actin network drives chromosome congression in oocytes.
Lenart, P., Bacher, C.P., Daigle, N., Hand, A.R., Eils, R., Terasaki, M. & Ellenberg, J.
Nature 2005 Aug 11;436(7052):812-8. Epub 2005 Jul 13.
Chromosome capture by microtubules is widely accepted as the universal mechanism of spindle assembly in dividing cells. However, the observed length of spindle microtubules and computer simulations of spindle assembly predict that chromosome capture is efficient in small cells, but may fail in cells with large nuclear volumes such as animal oocytes. Here we investigate chromosome congression during the first meiotic division in starfish oocytes. We show that microtubules are not sufficient for capturing chromosomes. Instead, chromosome congression requires actin polymerization. After nuclear envelope breakdown, we observe the formation of a filamentous actin mesh in the nuclear region, and find that contraction of this network delivers chromosomes to the microtubule spindle. We show that this mechanism is essential for preventing chromosome loss and aneuploidy of the egg--a leading cause of pregnancy loss and birth defects in humans.
Dynamic targeting of microtubules by TPPP/p25 affects cell survival.
Lehotzky, A., Tirian, L., Tokesi, N., Lenart, P., Szabo, B., Kovacs, J. & Ovadi, J.
J Cell Sci. 2004 Dec 1;117(Pt 25):6249-59.
Recently we identified TPPP/p25 (tubulin polymerization promoting protein/p25) as a brain-specific unstructured protein that induced aberrant microtubule assemblies and ultrastructure in vitro and as a new marker for Parkinson's disease and other synucleopathies. In this paper the structural and functional consequences of TPPP/p25 are characterized to elucidate the relationship between the in vitro and the pathological phenomena. We show that at low expression levels EGFP-TPPP/p25 specifically colocalizes with the microtubule network of HeLa and NRK cells. We found that the colocalization was dynamic (tg=5 seconds by fluorescence recovery after photobleaching) and changed during the phases of mitosis. Time-lapse and immunofluorescence experiments revealed that high levels of EGFP-TPPP/p25 inhibited cell division and promoted cell death. At high expression levels or in the presence of proteosome inhibitor, green fusion protein accumulated around centrosomes forming an aggresome-like structure protruding into the nucleus or a filamentous cage of microtubules surrounding the nucleus. These structures showed high resistance to vinblastin. We propose that a potential function of TPPP/p25 is the stabilization of physiological microtubular ultrastructures, however, its upregulation may directly or indirectly initiate the formation of aberrant protein aggregates such as pathological inclusions.
Dynamics of nuclear pore complex organization through the cell cycle.
Rabut, G., Lenart, P. & Ellenberg, J.
Curr Opin Cell Biol 2004 Jun;16(3):314-21.
In eukaryotic cells, all macromolecules that traffic between the nucleus and the cytoplasm cross the double nuclear membrane through nuclear pore complexes (NPCs). NPCs are elaborate gateways that allow efficient, yet selective, translocation of many different macromolecules. Their protein composition has been elucidated, but how exactly these nucleoporins come together to form the pore is largely unknown. Recent data suggest that NPCs are composed of an extremely stable scaffold on which more dynamic, exchangeable parts are assembled. These could be targets for molecular rearrangements that change nuclear pore transport properties and, ultimately, the state of the cell.
Light microscopy of echinoderm embryos.
Strickland, L., von Dassow, G., Ellenberg, J., Foe, V., Lenart, P. & Burgess, D.
Methods Cell Biol 2004;74:371-409. Europe PMC
Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes.
Lénart, P., Rabut, G., Daigle, N., Hand, A.R., Terasaki, M. & Ellenberg, J.
J Cell Biol 2003 Mar 31;160(7):1055-68.
Breakdown of the nuclear envelope (NE) was analyzed in live starfish oocytes using a size series of fluorescently labeled dextrans, membrane dyes, and GFP-tagged proteins of the nuclear pore complex (NPC) and the nuclear lamina. Permeabilization of the nucleus occurred in two sequential phases. In phase I the NE became increasingly permeable for molecules up to approximately 40 nm in diameter, concurrent with a loss of peripheral nuclear pore components over a time course of 10 min. The NE remained intact on the ultrastructural level during this time. In phase II the NE was completely permeabilized within 35 s. This rapid permeabilization spread as a wave from one epicenter on the animal half across the nuclear surface and allowed free diffusion of particles up to approximately 100 nm in diameter into the nucleus. While the lamina and nuclear membranes appeared intact at the light microscopic level, a fenestration of the NE was clearly visible by electron microscopy in phase II. We conclude that NE breakdown in starfish oocytes is triggered by slow sequential disassembly of the NPCs followed by a rapidly spreading fenestration of the NE caused by the removal of nuclear pores from nuclear membranes still attached to the lamina.
Nuclear envelope dynamics in oocytes: from germinal vesicle breakdown to mitosis.
Lenart, P. & Ellenberg, J.
Curr Opin Cell Biol 2003 Feb;15(1):88-95.
We have recently gained new insight into the mechanisms involved in nuclear envelope breakdown, the irreversible step that commits a cell to the M phase. Results from mammalian cell and starfish oocyte studies suggest that mechanical forces of the cytoskeleton, as well as biochemical disassembly of nuclear envelope protein complexes, play important roles in this process.
Involvement of MAP kinase SIMK and actin cytoskeleton in the regulation of root hair tip growth.
Samaj, J., Ovecka, M., Hlavacka, A., Lecourieux, F., Meskiene, I., Lichtscheidl, I., Lenart, P., Salaj, J., Volkmann, D., Bogre, L., Baluska, F. & Hirt, H.
Cell Biol Int. 2003;27(3):257-9. Europe PMC
Histone H3 phosphorylation during Xenopus oocyte maturation: regulation by the MAP kinase/p90Rsk pathway and uncoupling from DNA condensation.
Schmitt, A., Gutierrez, G.J., Lenart, P., Ellenberg, J. & Nebreda, A.R.
FEBS Lett 2002 May 8;518(1-3):23-8.
Here we show that during the meiotic maturation of Xenopus oocytes, histone H3 becomes phosphorylated on serine-10 at about the time of maturation promoting factor activation and meiosis I entry. However, overexpression of cAMP-dependent protein kinase that blocks entry into M phase, also leads to massive serine-10 phosphorylation of histone H3 in intact Xenopus oocytes but does not cause chromosome condensation. We also show that the phosphorylation of histone H3 during oocyte maturation requires the activation of the mitogen-activated protein kinase/p90Rsk pathway. Our results indicate that in G2-arrested oocytes, which are about to enter M phase, histone H3 phosphorylation is not sufficient for chromosome condensation.