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.

Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit.

The trimeric influenza virus polymerase, comprising subunits PA, PB1 and PB2, is responsible for transcription and replication of the segmented viral RNA genome. Using a novel library-based screening technique called expression of soluble proteins by random incremental truncation (ESPRIT), we identified an independently folded C-terminal domain from PB2 and determined its solution structure by NMR. Using green fluorescent protein fusions, we show that both the domain and the full-length PB2 subunit are efficiently imported into the nucleus dependent on a previously overlooked bipartite nuclear localization sequence (NLS). The crystal structure of the domain complexed with human importin alpha5 shows how the last 20 residues unfold to permit binding to the import factor. The domain contains three surface residues implicated in adaptation from avian to mammalian hosts. One of these tethers the NLS-containing peptide to the core of the domain in the unbound state.

Nuclear import and assembly of influenza A virus RNA polymerase studied in live cells by fluorescence cross-correlation spectroscopy.

Intracellular transport and assembly of the subunits of the heterotrimeric RNA-dependent RNA polymerase constitute a key component of the replication cycle of influenza virus. Recent results suggest that efficient polymerase assembly is a limiting factor in the viability of reassortant viruses. The mechanism of nuclear import and assembly of the three polymerase subunits, PB1, PB2, and PA, is still controversial, yet it is clearly of great significance in understanding the emergence of new strains with pandemic potential. In this study, we systematically investigated the interactions between the polymerase subunits and their localization in living cells by fluorescence cross-correlation spectroscopy (FCCS) and quantitative confocal microscopy. We could show that PB1 and PA form a dimer in the cytoplasm, which is imported into the nucleus separately from PB2. Once in the nucleus, the PB1/PA dimer associates with PB2 to form the trimeric polymerase. Photon-counting histogram analysis revealed that trimeric polymerase complexes can form higher-order oligomers in the nucleus. We furthermore demonstrate that impairing the nuclear import of PB2 by mutating its nuclear localization signal leads to abnormal formation of the trimeric polymerase in the cytoplasm. Taken together, our results demonstrate which of the previously discussed influenza virus polymerase transport models operates in live cells. Our study sheds light on the interplay between the nuclear import of the subunits and the assembly of the influenza virus polymerase and provides a methodological framework to analyze the effects of different host range mutations in the future.

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.

Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes.

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.

Quantitative kinetic analysis of nucleolar breakdown and reassembly during mitosis in live human cells.

One of the great mysteries of the nucleolus surrounds its disappearance during mitosis and subsequent reassembly at late mitosis. Here, the relative dynamics of nucleolar disassembly and reformation were dissected using quantitative 4D microscopy with fluorescent protein-tagged proteins in human stable cell lines. The data provide a novel insight into the fates of the three distinct nucleolar subcompartments and their associated protein machineries in a single dividing cell. Before the onset of nuclear envelope (NE) breakdown, nucleolar disassembly started with the loss of RNA polymerase I subunits from the fibrillar centers. Dissociation of proteins from the other subcompartments occurred with faster kinetics but commenced later, coincident with the process of NE breakdown. The reformation pathway also follows a reproducible and defined temporal sequence but the order of reassembly is shown not to be dictated by the order in which individual nucleolar components reaccumulate within the nucleus after mitosis.

Gain of CTCF-Anchored Chromatin Loops Marks the Exit from Naive Pluripotency.

The genome of pluripotent stem cells adopts a unique three-dimensional architecture featuring weakly condensed heterochromatin and large nucleosome-free regions. Yet, it is unknown whether structural loops and contact domains display characteristics that distinguish embryonic stem cells (ESCs) from differentiated cell types. We used genome-wide chromosome conformation capture and super-resolution imaging to determine nuclear organization in mouse ESC and neural stem cell (NSC) derivatives. We found that loss of pluripotency is accompanied by widespread gain of structural loops. This general architectural change correlates with enhanced binding of CTCF and cohesins and more pronounced insulation of contacts across chromatin boundaries in lineage-committed cells. Reprogramming NSCs to pluripotency restores the unique features of ESC domain topology. Domains defined by the anchors of loops established upon differentiation are enriched for developmental genes. Chromatin loop formation is a pervasive structural alteration to the genome that accompanies exit from pluripotency and delineates the spatial segregation of developmentally regulated genes.

A predictable ligand regulated expression strategy for stably integrated transgenes in mammalian cells in culture.

Several strategies for regulated stable transgene expression in mammalian cells have been described. These strategies have different strengths and weaknesses, however they all share a common problem, namely predictability in application. Here we address this problem using the leading strategy for ligand inducible transgene expression, the tetracycline repressor system. Initially, we found the best stable clone out of 48 examined showed only 6-fold inducibility. Hence we looked for additions and modifications that improve the chances of a successful outcome. We document three important aspects; first, use of a mammalian codon-optimized tetracycline repressor gene; second, addition of a steroid hormone receptor ligand binding domain to the tetracycline repressor-virion protein 16 fusion protein activator; third, flanking the tet-operator/transgene cassette with insulator elements from the chicken beta-globin locus. By inclusion of these three design features, 18/18 clones showed low basal and highly inducible (>50 x) expression.

Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging.

Much of life's essential molecular machinery consists of large protein assemblies that currently pose challenges for structure determination. A prominent example is the nuclear pore complex (NPC), for which the organization of its individual components remains unknown. By combining stochastic super-resolution microscopy, to directly resolve the ringlike structure of the NPC, with single particle averaging, to use information from thousands of pores, we determined the average positions of fluorescent molecular labels in the NPC with a precision well below 1 nanometer. Applying this approach systematically to the largest building block of the NPC, the Nup107-160 subcomplex, we assessed the structure of the NPC scaffold. Thus, light microscopy can be used to study the molecular organization of large protein complexes in situ in whole cells.

Intracellular transport by an anchored homogeneously contracting F-actin meshwork.

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.

A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells.

In mammals, silencing of one of the two X chromosomes in female cells compensates for the different number of X chromosomes between the sexes. The noncoding Xist RNA initiates X chromosome inactivation. Xist spreads from its transcription site over the X chromosome territory and triggers the formation of a repressive chromatin domain. To understand localization of Xist over one X chromosome we aimed to develop a system for investigating Xist in living cells. Here we report successful visualization of transgenically expressed MS2-tagged Xist in mouse embryonic stem cells. Imaging of Xist during an entire cell cycle shows that Xist spreads from a single point to a steady state when the chromosome is covered with a constant amount of Xist. Photobleaching experiments of the established Xist cluster indicate that chromosome-bound Xist is dynamic and turns over on the fully Xist covered chromosome. It appears that in interphase the loss of bound Xist and newly produced Xist are in equilibrium. We also show that the turnover of bound Xist requires transcription, and Xist binding becomes stable when transcription is inhibited. Our data reveal a strategy for visualizing Xist and indicate that spreading over the chromosome might involve dynamic binding and displacement.

Normative data for the pyramids and palm trees test in the Quebec-French population.

Semantic memory tests assess long-term memory for facts, objects, and concepts as well as words and their meaning. Since it holds culturally shared information, the development of normative data adjusted to the cultural and linguistic reality of the target population is of particular importance. The present study aimed to establish normative data for the Pyramids and Palm Trees Test, a commonly used test of semantic memory, in the French-Quebec population. The normative sample consisted of 214 healthy French-speaking adults and elderly persons from various regions of the province of Quebec. The effects of participants' age, gender, and education level on test performance were assessed. Results indicated that participants' level of education and age, but not sex, were found to be significantly associated with performance on this test. Normative data are presented as means and standard deviations. Overall, the present norms are consistent with those of previous studies with Spanish samples.

LambdaN-GFP: an RNA reporter system for live-cell imaging.

We describe a GFP-based RNA reporter system (lambdaN-GFP) to visualize RNA molecules in live mammalian cells. It consists of GFP fused to an arginine-rich peptide derived from the phage lambda N protein, lambdaN22, which binds a unique minimal RNA motif and can be used to tag any RNA molecule. LambdaN-GFP uses a small and easy to engineer RNA tag, reducing the likelihood of perturbing the function of the tagged RNA molecule.

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.

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.

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.

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.

Automatic identification of subcellular phenotypes on human cell arrays.

Light microscopic analysis of cell morphology provides a high-content readout of cell function and protein localization. Cell arrays and microwell transfection assays on cultured cells have made cell phenotype analysis accessible to high-throughput experiments. Both the localization of each protein in the proteome and the effect of RNAi knock-down of individual genes on cell morphology can be assayed by manual inspection of microscopic images. However, the use of morphological readouts for functional genomics requires fast and automatic identification of complex cellular phenotypes. Here, we present a fully automated platform for high-throughput cell phenotype screening combining human live cell arrays, screening microscopy, and machine-learning-based classification methods. Efficiency of this platform is demonstrated by classification of eleven subcellular patterns marked by GFP-tagged proteins. Our classification method can be adapted to virtually any microscopic assay based on cell morphology, opening a wide range of applications including large-scale RNAi screening in human cells.