Biomedical subjects

Jan Ellenberg

Publications and source records attributed to Jan Ellenberg.

At least 19 recordsLinked to original sources

PubMed2007

Measuring structural dynamics of chromosomes in living cells by fluorescence microscopy.

Mitotic and meiotic chromosomes are the compact packages that faithfully transport the genetic and epigenetic information to the following cell generations. How chromatin dynamically cycles between the decompacted interphase state that supports transcription and replication and the compacted state required for chromosome segregation is not understood. To address this long-standing problem, the structure of chromatin should ideally be studied in the physiological context of intact cells and organisms. We discuss here, the contributions that live-cell imaging can and has made to the study of mitotic chromosome compaction and highlight the power and limitations of this approach. We review methodologies used and suggest that combinatorial approaches and developing new imaging technologies will be key to shedding light on this long-standing question in cell biology.

Cells↗

PubMed2006

Live-cell imaging reveals a stable cohesin-chromatin interaction after but not before DNA replication.

Cohesin is a multisubunit protein complex that links sister chromatids from replication until segregation. The lack of obvious cohesin-targeting-specific sequences on DNA, as well as cohesin's molecular arrangement as a large ring, has led to the working hypothesis that cohesin acts as a direct topological linker. To preserve the identity of sister chromatids, such a linkage would need to stably persist throughout the entire S and G2 phases of the cell cycle. Unexpectedly, cohesin binds chromatin already in telophase, and a large fraction dissociates from chromosomes during prophase in a phosphorylation-dependent manner, whereas initiation of anaphase requires proteolytic cleavage of only a small fraction of cohesin. These observations raised the question of how and when cohesin interacts with chromatin during the cell cycle. Here, we report a cell-cycle dependence in the stability of cohesin binding to chromatin. Using photobleaching and quantitative live-cell imaging, we identified several cohesin pools with different chromatin binding stabilities. Although all chromatin bound cohesin dissociated after a mean residence time of less than 25 min before replication, about one-third of cohesin was bound much more stably after S phase and persisted until metaphase, consistent with long-lived links mediating cohesion between sister chromatids.

Cell Cycle↗

PubMed2006

High-throughput fluorescence microscopy for systems biology.

In this post-genomic era, we need to define gene function on a genome-wide scale for model organisms and humans. The fundamental unit of biological processes is the cell. Among the most powerful tools to assay such processes in the physiological context of intact living cells are fluorescence microscopy and related imaging techniques. To enable these techniques to be applied to functional genomics experiments, fluorescence microscopy is making the transition to a quantitative and high-throughput technology.

Animals↗

PubMed2006

Resolution of chiasmata in oocytes requires separase-mediated proteolysis.

In yeast, resolution of chiasmata in meiosis I requires proteolytic cleavage along chromosome arms of cohesin's Rec8 subunit by separase. Since activation of separase by the anaphase-promoting complex (APC/C) is supposedly not required for meiosis I in Xenopus oocytes, it has been suggested that animal cells might resolve chiasmata by a separase-independent mechanism related to the so-called "prophase pathway" that removes cohesin from chromosome arms during mitosis. By expressing Cre recombinase from a zona pellucida promoter, we have deleted a floxed allele of separase specifically in mouse oocytes. This prevents removal of Rec8 from chromosome arms and resolution of chiasmata. It also hinders extrusion of the first polar body (PBE) and causes female sterility. mRNA encoding wild-type but not catalytically inactive separase restores chiasma resolution. Both types of mRNA restore PBE. Proteolytic activity of separase is therefore essential for Rec8's removal from chromosome arms and for chiasma resolution but not for PBE.

Animals↗

PubMed2006

Nuclear actin: a lack of export allows formation of filaments.

Actin has been found in nuclei of many cell types, but little is known about its form and function. A recent study has shown that a lack of specific export allows actin to accumulate in the nucleus, where it forms a network of actin filaments that may be required to stabilize the giant nucleus of the Xenopus oocyte.

Actins↗

PubMed2006

NuSAP, a mitotic RanGTP target that stabilizes and cross-links microtubules.

Nucleolar and spindle-associated protein (NuSAP) was recently identified as a microtubule- and chromatin-binding protein in vertebrates that is nuclear during interphase. Small interfering RNA-mediated depletion of NuSAP resulted in aberrant spindle formation, missegregation of chromosomes, and ultimately blocked cell proliferation. We show here that NuSAP is enriched on chromatin-proximal microtubules at meiotic spindles in Xenopus oocytes. When added at higher than physiological levels to Xenopus egg extract, NuSAP induces extensive bundling of spindle microtubules and causes bundled microtubules within spindle-like structures to become longer. In vitro reconstitution experiments reveal two direct effects of NuSAP on microtubules: first, it can efficiently stabilize microtubules against depolymerization, and second, it can cross-link large numbers of microtubules into aster-like structures, thick fibers, and networks. With defined components we show that the activity of NuSAP is differentially regulated by Importin (Imp) alpha, Impbeta, and Imp7. While Impalpha and Imp7 appear to block the microtubule-stabilizing activity of NuSAP, Impbeta specifically suppresses aspects of the cross-linking activity of NuSAP. We propose that to achieve full NuSAP functionality at the spindle, all three importins must be dissociated by RanGTP. Once activated, NuSAP may aid to maintain spindle integrity by stabilizing and cross-linking microtubules around chromatin.

Animals↗

PubMed2006

Condensin I stabilizes chromosomes mechanically through a dynamic interaction in live cells.

BACKGROUND: Restructuring chromatin into morphologically distinct chromosomes is essential for cell division, but the molecular mechanisms underlying this process are poorly understood. Condensin complexes have been proposed as key factors, although controversial conclusions about their contribution to chromosome structure were reached by different experimental approaches in fixed cells or cell extracts. Their function under physiological conditions still needs to be defined. RESULTS: Here, we investigated the specific functions of condensin I and II in live cells by fluorescence microscopy and RNAi depletion. Photobleaching and quantitative time-lapse imaging showed that GFP-tagged condensin II bound stably to chromosomes throughout mitosis. By contrast, the canonical condensin I interacted dynamically with chromatin after completion of prophase compaction, reaching steady-state levels on chromosomes before congression. In condensin I-depleted cells, compaction was normal, but chromosomes were mechanically labile and unable to withstand spindle forces during alignment. However, normal levels of condensin II were not required for chromosome stability. CONCLUSIONS: We conclude that while condensin I seems dispensable for normal chromosome compaction, its dynamic binding after nuclear envelope breakdown locks already condensed chromatin in a rigid state required for mechanically stable spindle attachment.

Adenosine Triphosphatases↗

PubMed2006

Monitoring the permeability of the nuclear envelope during the cell cycle.

In animal organisms the nuclear envelope (NE) dis-assembles 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 40 nm 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.

Animals↗

PubMed2006

Minimizing the risk of reporting false positives in large-scale RNAi screens.

Large-scale RNA interference (RNAi)-based analyses, very much as other 'omic' approaches, have inherent rates of false positives and negatives. The variability in the standards of care applied to validate results from these studies, if left unchecked, could eventually begin to undermine the credibility of RNAi as a powerful functional approach. This Commentary is an invitation to an open discussion started among various users of RNAi to set forth accepted standards that would insure the quality and accuracy of information in the large datasets coming out of genome-scale screens.

Databases, Genetic↗

PubMed2006

High-throughput RNAi screening by time-lapse imaging of live human cells.

RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.

Automation↗

PubMed2006

Fluorophores for live cell imaging of AGT fusion proteins across the visible spectrum.

O6-alkylguanine-DNA alkyltransferase (AGT) fusion proteins can be specifically and covalently labeled with fluorescent O6-benzylguanine (O6-BG) derivatives for multicolor live cell imaging approaches. Here, we characterize several new BG fluorophores suitable for in vivo AGT labeling that display fluorescence emission maxima covering the visible spectrum from 472 to 673 nm, thereby extending the spectral limits set by fluorescent proteins. We show that the photostability of the cell-permeable dyes BG Rhodamine Green (BG505) and CP tetramethylrhodamine (CP-TMR) is in the range of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP), and that BG diethylaminomethyl coumarin (BGDEAC), a derivative of coumarin, is even more stable than enhanced cyan fluorescent protein (ECFP). Due to the increasing number of new BG derivatives with interesting fluorescence properties, such as far-red emission, fluorescence labeling of AGT fusion proteins is becoming a versatile alternative to existing live cell imaging approaches.

Animals↗

PubMed2005

Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins.

Quantitative characterization of protein interactions under physiological conditions is vital for systems biology. Fluorescence photobleaching/activation experiments of GFP-tagged proteins are frequently used for this purpose, but robust analysis methods to extract physicochemical parameters from such data are lacking. Here, we implemented a reaction-diffusion model to determine the contributions of protein interaction and diffusion on fluorescence redistribution. The model was validated and applied to five chromatin-interacting proteins probed by photoactivation in living cells. We found that very transient interactions are common for chromatin proteins. Their observed mobility was limited by the amount of free protein available for diffusion but not by the short residence time of the bound proteins. Individual proteins thus locally scan chromatin for binding sites, rather than diffusing globally before rebinding at random nuclear positions. By taking the real cellular geometry and the inhomogeneous distribution of binding sites into account, our model provides a general framework to analyze the mobility of fluorescently tagged factors. Furthermore, it defines the experimental limitations of fluorescence perturbation experiments and highlights the need for complementary methods to measure transient biochemical interactions in living cells.

Active Transport, Cell Nucleus↗

PubMed2005

A contractile nuclear actin network drives chromosome congression in oocytes.

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.

Actins↗

PubMed2005

FRET analyses of the U2AF complex localize the U2AF35/U2AF65 interaction in vivo and reveal a novel self-interaction of U2AF35.

We have analyzed the interaction between the U2AF subunits U2AF35 and U2AF65 in vivo using fluorescence resonance energy transfer (FRET) microscopy. U2 snRNP Auxiliary Factor (U2AF) is an essential pre-mRNA splicing factor complex, comprising 35-kDa (U2AF35) and 65-kDa (U2AF65) subunits. U2AF65 interacts directly with the polypyrimidine tract and promotes binding of U2 snRNP to the pre-mRNA branchpoint, while U2AF35 associates with the conserved AG dinucleotide at the 3' end of the intron and has multiple functions in the splicing process. Using two different approaches for measuring FRET, we have identified and spatially localized sites of direct interaction between U2AF35 and U2AF65 in vivo in live cell nuclei. While U2AF is thought to function as a heterodimeric complex, the FRET data have also revealed a novel U2AF35 self-interaction in vivo, which is confirmed in vitro using biochemical assays. These results suggest that the stoichiometry of the U2AF complex may, at least in part, differ in vivo from the expected heterodimeric complex. The data show that FRET studies offer a valuable approach for probing interactions between pre-mRNA splicing factors in vivo.

Bacterial Proteins↗

PubMed2004

Distinct functions of condensin I and II in mitotic chromosome assembly.

Condensin is a protein complex associated with mitotic chromosomes that has been implicated in chromosome condensation. In vertebrates, two types of condensin complexes have recently been identified, called condensin I and II. Here, we show that in mammalian cells condensin II associates with chromatin in prophase, in contrast to condensin I which is cytoplasmic and can thus interact with chromosomes only after nuclear envelope breakdown. RNA interference experiments in conjunction with imaging of live and fixed cells revealed that condensin II is required for chromosome condensation in early prophase, whereas condensin I appears to be dispensable at this stage. By contrast, condensin I is required for the complete dissociation of cohesin from chromosome arms, for chromosome shortening and for normal timing of progression through prometaphase and metaphase, whereas normal condensin II levels are dispensable for these processes. After depletion of both condensin complexes, the onset of chromosome condensation is delayed until the end of prophase, but is then initiated rapidly before nuclear envelope breakdown. These results reveal that condensin II and I associate with chromosomes sequentially and have distinct functions in mitotic chromosome assembly.

Cell Cycle Proteins↗

PubMed2004

LAP2alpha and BAF transiently localize to telomeres and specific regions on chromatin during nuclear assembly.

Lamina-associated polypeptide (LAP) 2alpha is a LEM (lamina-associated polypeptide emerin MAN1) family protein associated with nucleoplasmic A-type lamins and chromatin. Using live cell imaging and fluorescence microscopy we demonstrate that LAP2alpha was mostly cytoplasmic in metaphase and associated with telomeres in anaphase. Telomeric LAP2alpha clusters grew in size, formed 'core' structures on chromatin adjacent to the spindle in telophase, and translocated to the nucleoplasm in G1 phase. A subfraction of lamin C and emerin followed LAP2alpha to the core region early on, whereas LAP2beta, lamin B receptor and lamin B initially bound to more peripheral regions of chromatin, before they spread to core structures with different kinetics. Furthermore, the DNA-crosslinking protein barrier-to-autointegration factor (BAF) bound to LAP2alpha in vitro and in mitotic extracts, and subfractions of BAF relocalized to core structures with LAP2alpha. We propose that LAP2alpha and a subfraction of BAF form defined complexes in chromatin core regions and may be involved in chromatin reorganization during early stages of nuclear assembly.

Animals↗

PubMed2004

Mapping the dynamic organization of the nuclear pore complex inside single living cells.

Most cellular activities are executed by multi-protein complexes that form the basic functional modules of their molecular machinery. Proteomic approaches can provide an evermore detailed picture of their composition, but do not reveal how these machines are organized dynamically to accomplish their biological function. Here, we present a method to determine the dissociation rates of protein subunits from complexes that have a traceable localization inside single living cells. As a case study, we systematically analysed the dynamic organization of vertebrate nuclear pore complexes (NPCs), large supramolecular complexes of about 30 different polypeptides. NPC components exhibited a wide range of residence times covering five orders of magnitude from seconds to days. We found the central parts of the NPC to be very stable, consistent with a function as a structural scaffold, whereas more peripheral components exhibited more dynamic behaviour, suggesting adaptor as well as regulatory functions. The presented strategy can be applied to many multi-protein complexes and will help to characterize the dynamic behaviour of complex networks of proteins in live cells.

Cells, Cultured↗

PubMed2004

Hypophosphorylated SR splicing factors transiently localize around active nucleolar organizing regions in telophase daughter nuclei.

Upon completion of mitosis, daughter nuclei assemble all of the organelles necessary for the implementation of nuclear functions. We found that upon entry into daughter nuclei, snRNPs and SR proteins do not immediately colocalize in nuclear speckles. SR proteins accumulated in patches around active nucleolar organizing regions (NORs) that we refer to as NOR-associated patches (NAPs), whereas snRNPs were enriched at other nuclear regions. NAPs formed transiently, persisting for 15-20 min before dissipating as nuclear speckles began to form in G1. In the absence of RNA polymerase II transcription, NAPs increased in size and persisted for at least 2 h, with delayed localization of SR proteins to nuclear speckles. In addition, SR proteins in NAPs are hypophosphorylated, and the SR protein kinase Clk/STY colocalizes with SR proteins in NAPs, suggesting that phosphorylation releases SR proteins from NAPs and their initial target is transcription sites. This work demonstrates a previously unrecognized role of NAPs in splicing factor trafficking and nuclear speckle biogenesis.

Cell Nucleolus↗