William J. Godinez

Objective comparison of particle tracking methods

Particle tracking is of key importance for quantitative analysis of intracellular dynamic processes from time-lapse microscopy image data. Because 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 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 notable practical conclusions for users and developers.

Dynamics of HIV-1 assembly and release

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.

Deterministic and probabilistic approaches for tracking virus particles in time-lapse fluorescence microscopy image sequences

Modern developments in time-lapse fluorescence microscopy enable the observation of a variety of processes exhibited by viruses. The dynamic nature of these processes requires the tracking of viruses over time to explore spatial-temporal relationships. In this work, we developed deterministic and probabilistic approaches for multiple virus tracking in multi-channel fluorescence microscopy images. The deterministic approaches follow a traditional two-step paradigm comprising particle localization based on either the spot-enhancing filter or 2D Gaussian fitting, as well as motion correspondence based on a global nearest neighbor scheme. Our probabilistic approaches are based on particle filters. We describe approaches based on a mixture of particle filters and based on independent particle filters. For the latter, we have developed a penalization strategy that prevents the problem of filter coalescence (merging) in cases where objects lie in close proximity. A quantitative comparison based on synthetic image sequences is carried out to evaluate the performance of our approaches. In total, eight different tracking approaches have been evaluated. We have also applied these approaches to real microscopy images of HIV-1 particles and have compared the tracking results with ground truth obtained from manual tracking. It turns out that the probabilistic approaches based on independent particle filters are superior to the deterministic schemes as well as to the approaches based on a mixture of particle filters.

Automatic analysis of dividing cells in live cell movies to detect mitotic delays and correlate phenotypes in time

Live-cell imaging allows detailed dynamic cellular phenotyping for cell biology and, in combination with small molecule or drug libraries, for high-content screening. Fully automated analysis of live cell movies has been hampered by the lack of computational approaches that allow tracking and recognition of individual cell fates over time in a precise manner. Here, we present a fully automated approach to analyze time-lapse movies of dividing cells. Our method dynamically categorizes cells into seven phases of the cell cycle and five aberrant morphological phenotypes over time. It reliably tracks cells and their progeny and can thus measure the length of mitotic phases and detect cause and effect if mitosis goes awry. We applied our computational scheme to annotate mitotic phenotypes induced by RNAi gene knockdown of CKAP5 (also known as ch-TOG) or by treatment with the drug nocodazole. Our approach can be readily applied to comparable assays aiming at uncovering the dynamic cause of cell division phenotypes.

Herpesviral replication compartments move and coalesce at nuclear speckles to enhance export of viral late mRNA

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.

Automated analysis of the mitotic phases of human cells in 3d fluorescence microscopy image sequences

The evaluation of fluorescence microscopy images acquired in high-throughput cell phenotype screens constitutes a substantial bottleneck and motivates the development of automated image analysis methods. Here we introduce a computational scheme to process 3D multi-cell time-lapse images as they are produced in large-scale RNAi experiments. We describe an approach to automatically segment, track, and classify cell nuclei into different mitotic phases. This enables automated analysis of the duration of single phases of the cell life cycle and thus the identification of cell cultures that show an abnormal mitotic behavior. Our scheme proves a high accuracy, suggesting a promising future for automating the evaluation of high-throughput experiments.

Chromosome segregation by the Escherichia coli Min system

The mechanisms underlying chromosome segregation in prokaryotes remain a subject of debate and no unifying view has yet emerged. Given that the initial disentanglement of duplicated chromosomes could be achieved by purely entropic forces, even the requirement of an active prokaryotic segregation machinery has been questioned. Using computer simulations, we show that entropic forces alone are not sufficient to achieve and maintain full separation of chromosomes. This is, however, possible by assuming repeated binding of chromosomes along a gradient of membrane‐associated tethering sites toward the poles. We propose that, in Escherichia coli, such a gradient of membrane tethering sites may be provided by the oscillatory Min system, otherwise known for its role in selecting the cell division site. Consistent with this hypothesis, we demonstrate that MinD binds to DNA and tethers it to the membrane in an ATP‐dependent manner. Taken together, our combined theoretical and experimental results suggest the existence of a novel mechanism of chromosome segregation based on the Min system, further highlighting the importance of active segregation of chromosomes in prokaryotic cell biology.

Visualizing fusion of pseudotyped HIV-1 particles in real time by live cell microscopy.

BackgroundMost retroviruses enter their host cells by fusing the viral envelope with the plasma membrane. Although the protein machinery promoting fusion has been characterized extensively, the dynamics of the process are largely unknown.ResultsWe generated human immunodeficiency virus-1 (HIV-1) particles pseudotyped with the envelope (Env) protein of ecotropic murine leukemia virus eMLV to study retrovirus entry at the plasma membrane using live-cell microscopy. This Env protein mediates highly efficient pH independent fusion at the cell surface and can be functionally tagged with a fluorescent protein. To detect fusion events, double labeled particles carrying one fluorophor in Env and the other in the matrix (MA) domain of Gag were generated and characterized. Fusion events were defined as loss of Env signal after virus-cell contact. Single particle tracking of >20,000 individual traces in two color channels recorded 28 events of color separation, where particles lost the Env protein, with the MA layer remaining stable at least for a short period. Fourty-five events were detected where both colors were lost simultaneously. Importantly, the first type of event was never observed when particles were pseudotyped with a non-fusogenic Env.ConclusionThese results reveal rapid retroviral fusion at the plasma membrane and permit studies of the immediate post-fusion events.

Xenopus cytoplasmic linker–associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules

The importance of microtubules (MTs) in axon outgrowth is studied using automated tracking of MTs and actin in live neurons. MT advance in the growth cone and axon outgrowth are correlated, and the MT-binding protein XCLASP1 promotes both. In addition to regulation of MTs, XCLASP1 is necessary for protrusive actin architecture in lamellipodia.

Tracking and Quantitative Analysis of Dynamic Movements of Cells and Particles

Understanding complex cellular processes requires investigating the underlying mechanisms within a spatiotemporal context. Although cellular processes are dynamic in nature, most studies in molecular cell biology are based on fixed specimens, for example, using immunocytochemistry or fluorescence in situ hybridization (FISH). However, breakthroughs in fluorescence microscopy imaging techniques, in particular, the discovery of green fluorescent protein (GFP) and its spectral variants, have facilitated the study of a wide range of dynamic processes by allowing nondestructive labeling of target structures in living cells. In addition, the tremendous improvements in spatial and temporal resolution of light microscopes now allow cellular processes to be analyzed in unprecedented detail. These state-of-the-art imaging technologies, however, provide a huge amount of digital image data. To cope with the enormous amount of image data and to extract reproducible as well as quantitative information, computer-based image analysis is required. In this article, we describe methods for computer-based analysis of multidimensional live cell microscopy images and their application to study the dynamics of cells and particles. First, we sketch a general workflow for quantitative analysis of live cell images. Then, we detail computational methods for automatic image analysis comprising image preprocessing, segmentation, registration, tracking, and classification. We conclude with a discussion of quantitative analysis and systems biology.