Particle tracking is of key importance for quantitative analysis of intracellular dynamic processes from time-lapse microscopy image data. Since manually detecting and following large numbers of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized, for the first time, an open competition, in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios. Performance was assessed using commonly defined measures. Although no single method performed best across all scenarios, the results revealed clear differences between the various approaches, leading to important practical conclusions for users and developers.

Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1–2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of ∼5×10−3 s−1, corresponding to 8–9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at ∼1,500±700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics.

Although essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was long believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form flat structures that then bend while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical growth laws to study the ultrastructural rearrangements of the clathrin coat during endocytosis in BSC-1 mammalian cells. We confirm that clathrin coats initially grow flat and demonstrate that curvature begins when around 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the notion that BSC-1 mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

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Supplementary data are available at Bioinformatics online.

The role of the intranuclear movement of chromatin in gene expression is not well-understood. Herpes simplex virus forms replication compartments (RCs) in infected cell nuclei as sites of viral DNA replication and late gene transcription. These structures develop from small compartments that grow in size, move, and coalesce. Quantitative analysis of RC trajectories, derived from 4D images, shows that most RCs move by directed motion. Directed movement is impaired in the presence of actin and myosin inhibitors as well as a transcription inhibitor. In addition, RCs coalesce at and reorganize nuclear speckles. Lastly, distinct effects of actin and myosin inhibitors on viral gene expression suggest that RC movement is not required for transcription, but rather, movement results in the bridging of transcriptionally active RCs with nuclear speckles to form structures that enhance export of viral late mRNAs.gene movement | nuclear export M ovement of chromatin and its interactions and location in the nucleus are believed to be important for regulation of gene expression (1). A number of studies have shown an association between the movement of chromosomal domains and changes in transcriptional activity. In yeast, activation of genes is associated with targeting of the gene to the nuclear periphery (2, 3), and this is believed to be because of the linkage of actively transcribed genes to the nuclear pore complex for RNA export (4). Lymphoid cell genes move to positions near centromeric heterochromatin coincident with transcriptional silencing (5). Transcriptional activation of the β-globin locus involves movement of the gene away from centromeric heterochromatin (6). During B-cell development, the IgH and Igκ loci are localized at the nuclear periphery in hematopoietic progenitor cells, but they move internally coincident with transcriptional activation in pro-B cells (7). Similarly, the β-globin gene relocalizes to the nuclear interior as it is activated during murine erythroid differentiation (8). Actively transcribed genes are also thought to be brought into close contact with nuclear speckles (9, 10), sites often regarded as hubs of RNA metabolism. Although these results link gene movement with changes in transcriptional activity, it is unclear whether intranuclear movement of chromatin is the cause or result of changes in transcriptional activity (1). Therefore, it is important to define systems in which the function of chromatin movement can be experimentally assessed.Nuclear forms of actin and myosin have been implicated in mediating long-range directed movement (11-13), but the mechanism by which this putative nucleoskeletal system operates is unknown. In addition to their possible roles in mediating intranuclear movement, there is increasing evidence for roles of nuclear actin and myosin in cellular transcription, chromatin remodeling, and mRNA export (reviewed in ref. 14). How and whether their roles in movement are connected to regulation of gene activity are unclear.Viruses are simple genetic entities and ha...

The existence and nature of an active chromosome segregation apparatus in bacteria has been a long-standing debate. A novel Brownian ratchet-type mechanism of chromosome segregation mediated by the Min system is identified in E. coli.

Pathogens face varying microenvironments in vivo, but suitable experimental systems and analysis tools to dissect how three-dimensional (3D) tissue environments impact pathogen spread are lacking. Here we develop an Integrative method to Study Pathogen spread by Experiment and Computation within Tissue-like 3D cultures (INSPECT-3D), combining quantification of pathogen replication with imaging to study single-cell and cell population dynamics. We apply INSPECT-3D to analyze HIV-1 spread between primary human CD4 T-lymphocytes using collagen as tissue-like 3D-scaffold. Measurements of virus replication, infectivity, diffusion, cellular motility and interactions are combined by mathematical analyses into an integrated spatial infection model to estimate parameters governing HIV-1 spread. This reveals that environmental restrictions limit infection by cell-free virions but promote cell-associated HIV-1 transmission. Experimental validation identifies cell motility and density as essential determinants of efficacy and mode of HIV-1 spread in 3D. INSPECT-3D represents an adaptable method for quantitative time-resolved analyses of 3D pathogen spread.