Joël Beaudouin

Cyclic, Proteasome-Mediated Turnover of Unliganded and Liganded ERα on Responsive Promoters Is an Integral Feature of Estrogen Signaling

We present an integrated model of hERalpha-mediated transcription where both unliganded and liganded receptors cycle on estrogen-responsive promoters. Using ChIP, FRAP, and biochemical analysis we evaluate hERalpha at several points in these cycles, establishing the ubiquitination status and subnuclear distribution of hERalpha, its mobility, the kinetics of transcriptional activation, and the cyclic recruitment of E3 ligases and the 19S regulatory component of the proteasome. These experiments, together with an evaluation of the inhibition of transcription and proteasome action, demonstrate that proteasome-mediated degradation and hERalpha-mediated transactivation are inherently linked and act to continuously turn over hERalpha on responsive promoters. Cyclic turnover of hERalpha permits continuous responses to changes in the concentration of estradiol.

Nuclear Envelope Breakdown Proceeds by Microtubule-Induced Tearing of the Lamina

The mechanism of nuclear envelope breakdown (NEBD) was investigated in live cells. Early spindle microtubules caused folds and invaginations in the NE up to one hour prior to NEBD, creating mechanical tension in the nuclear lamina. The first gap in the NE appeared before lamin B depolymerization, at the site of maximal tension, by a tearing mechanism. Gap formation relaxed this tension and dramatically accelerated the rate of chromosome condensation. The hole produced in the NE then rapidly expanded over the nuclear surface. NE fragments remaining on chromosomes were removed toward the centrosomes in a microtubule-dependent manner, suggesting a mechanism mediated by a minus-end-directed motor.

Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin

The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin‐interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target‐search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance.

Global Chromosome Positions Are Transmitted through Mitosis in Mammalian Cells

We investigated positioning of chromosomes during the cell cycle in live mammalian cells with a combined experimental and computational approach. By non-invasive labeling of chromosome subsets and tracking by 4D imaging, we could show that no global rearrangements occurred in interphase. Using the same assay, we also observed a striking order of chromosomes throughout mitosis. By contrast, our computer simulation based on stochastic movements of individual chromosomes predicted randomization of chromosome order in mitosis. In vivo, a quantitative assay for single chromosome positioning during mitosis revealed strong similarities between daughter and mother cells. These results demonstrate that global chromosome positions are heritable through the cell cycle in mammalian cells. Based on tracking of labeled chromosomes and centromeres during chromosome segregation and experimental perturbations of chromosomal order, we propose that chromosome specific timing of sister chromatid separation transmits chromosomal positions from one cell generation to the next.

NuSAP, a novel microtubule-associated protein involved in mitotic spindle organization

Here, we report on the identification of nucleolar spindle–associated protein (NuSAP), a novel 55-kD vertebrate protein with selective expression in proliferating cells. Its mRNA and protein levels peak at the transition of G2 to mitosis and abruptly decline after cell division. Microscopic analysis of both fixed and live mammalian cells showed that NuSAP is primarily nucleolar in interphase, and localizes prominently to central spindle microtubules during mitosis. Direct interaction of NuSAP with microtubules was demonstrated in vitro. Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain. In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis. In addition, many NuSAP-depleted interphase cells had deformed nuclei. Both overexpression and knockdown of NuSAP impaired cell proliferation. These results suggest a crucial role for NuSAP in spindle microtubule organization.

Dynamics within the CD95 death-inducing signaling complex decide life and death of cells

This study explores the dilemma in cellular signaling that triggering of CD95 (Fas/APO‐1) in some situations results in cell death and in others leads to the activation of NF‐κB. We established an integrated kinetic mathematical model for CD95‐mediated apoptotic and NF‐κB signaling. Systematic model reduction resulted in a surprisingly simple model well approximating experimentally observed dynamics. The model postulates a new link between c‐FLIPL cleavage in the death‐inducing signaling complex (DISC) and the NF‐κB pathway. We validated experimentally that CD95 stimulation resulted in an interaction of p43‐FLIP with the IKK complex followed by its activation. Furthermore, we showed that the apoptotic and NF‐κB pathways diverge already at the DISC. Model and experimental analysis of DISC formation showed that a subtle balance of c‐FLIPL and procaspase‐8 determines life/death decisions in a nonlinear manner. We present an integrated model describing the complex dynamics of CD95‐mediated apoptosis and NF‐κB signaling.

Calcium Rises Locally Trigger Focal Adhesion Disassembly and Enhance Residency of Focal Adhesion Kinase at Focal Adhesions

Focal adhesion kinase (FAK) activity and Ca2+ signaling led to a turnover of focal adhesions (FAs) required for cell spreading and migration. We used yellow Cameleon-2 (Ycam), a fluorescent protein-based Ca2+ sensor fused to FAK or to a FAK-related non-kinase domain, to measure simultaneously local Ca2+ variations at FA sites and FA dynamics. Discrete subcellular Ca2+ oscillators initiate both propagating and abortive Ca2+ waves in migrating U87 astrocytoma cells. Ca2+-dependent FA disassembly occurs when the Ca2+ wave reaches individual FAs, indicating that local but not global Ca2+ increases trigger FA disassembly. An unexpectedly rapid flux of FAK between cytosolic and FA compartments was revealed by fluorescence recovery after photobleaching studies. The FAK-Ycam recovery half-time (17 s) at FAs was slowed (to 29 s) by Ca2+ elevation. FAK-related non-kinase domain-Ycam had a faster, Ca2+-insensitive recovery half-time (11 s), which is consistent with the effect of Ca2+ on FAK-Ycam dynamics not being due to a general modification of the dynamics of FA components. Because FAK association at FAs was prolonged by Ca2+ and FAK autophosphorylation was correlated to intracellular Ca2+ levels, we propose that local Ca2+ elevations increase the residency of FAK at FAs, possibly by means of tyrosine phosphorylation of FAK, thereby leading to increased activation of its effectors involved in FA disassembly.

Model-based dissection of CD95 signaling dynamics reveals both a pro- and antiapoptotic role of c-FLIPL

A systems biology–based approach shows that life and death decisions for cells depend on the stoichiometry of c-FLIP isoforms.

Four-dimensional imaging and quantitative reconstruction to analyse complex spatiotemporal processes in live cells

Live-cell imaging technology using fluorescent proteins (green fluorescent protein and its homologues) has revolutionized the study of cellular dynamics. But tools that can quantitatively analyse complex spatiotemporal processes in live cells remain lacking. Here we describe a new technique — fast multi-colour four-dimensional imaging combined with automated and quantitative time-space reconstruction — to fill this gap. As a proof of principle, we apply this method to study the re-formation of the nuclear envelope in live cells. Four-dimensional imaging of three spectrally distinct fluorescent proteins is used to simultaneously visualize three different cellular compartments at high speed and with high spatial resolution. The highly complex data, comprising several thousand images from a single cell, were quantitatively reconstructed in time–space by software developed in-house. This analysis reveals quantitative and qualitative insights into the highly ordered topology of nuclear envelope formation, in correlation with chromatin expansion — results that would have been impossible to achieve by manual inspection alone. Our new technique will greatly facilitate study of the highly ordered dynamic architecture of eukaryotic cells.

Ribonucleoprotein-dependent localization of the yeast class V myosin Myo4p

Class V myosins are motor proteins with functions in vesicle transport, organelle segregation, and RNA localization. Although they have been extensively studied, only little is known about the regulation of their spatial distribution. Here we demonstrate that a GFP fusion protein of the budding yeast class V myosin Myo4p accumulates at the bud cortex and is a component of highly dynamic cortical particles. Bud-specific enrichment depends on Myo4ps association with its cargo, a ribonucleoprotein complex containing the RNA-binding protein She2p. Cortical accumulation of Myo4p at the bud tip can be explained by a transient retention mechanism that requires SHE2 and, apparently, localized mRNAs bound to She2p. A mutant She2 protein that is unable to recognize its cognate target mRNA, ASH1, fails to localize Myo4p. Mutant She2p accumulates inside the nucleus, indicating that She2p shuttles between the nucleus and cytoplasm and is exported in an RNA-dependent manner. Consistently, inhibition of nuclear mRNA export results in nuclear accumulation of She2p and cytoplasmic Myo4p mislocalization. Loss of She2p can be complemented by direct targeting of a heterologous lacZ mRNA to a complex of Myo4p and its associated adaptor She3p, suggesting that She2ps function in Myo4p targeting is to link an mRNA to the motor complex.