Integrated intracellular organization and its variations in human iPS cells.

Understanding how a subset of expressed genes dictates cellular phenotype is a considerable challenge owing to the large numbers of molecules involved, their combinatorics and the plethora of cellular behaviours that they determine1,2. Here we reduced this complexity by focusing on cellular organization-a key readout and driver of cell behaviour3,4-at the level of major cellular structures that represent distinct organelles and functional machines, and generated the WTC-11 hiPSC Single-Cell Image Dataset v1, which contains more than 200,000 live cells in 3D, spanning 25 key cellular structures. The scale and quality of this dataset permitted the creation of a generalizable analysis framework to convert raw image data of cells and their structures into dimensionally reduced, quantitative measurements that can be interpreted by humans, and to facilitate data exploration. This framework embraces the vast cell-to-cell variability that is observed within a normal population, facilitates the integration of cell-by-cell structural data and allows quantitative analyses of distinct, separable aspects of organization within and across different cell populations. We found that the integrated intracellular organization of interphase cells was robust to the wide range of variation in cell shape in the population; that the average locations of some structures became polarized in cells at the edges of colonies while maintaining the 'wiring' of their interactions with other structures; and that, by contrast, changes in the location of structures during early mitotic reorganization were accompanied by changes in their wiring.

A mechanism for sarcomere breathing: volume change and advective flow within the myofilament lattice.

During muscle contraction, myosin motors anchored to thick filaments bind to and slide actin thin filaments. These motors rely on energy derived from ATP, supplied, in part, by diffusion from the sarcoplasm to the interior of the lattice of actin and myosin filaments. The radial spacing of filaments in this lattice may change or remain constant during contraction. If the lattice is isovolumetric, it must expand when the muscle shortens. If, however, the spacing is constant or has a different pattern of axial and radial motion, then the lattice changes volume during contraction, driving fluid motion and assisting in the transport of molecules between the contractile lattice and the surrounding intracellular space. We first create an advective-diffusive-reaction flow model and show that the flow into and out of the sarcomere lattice would be significant in the absence of lattice expansion. Advective transport coupled to diffusion has the potential to substantially enhance metabolite exchange within the crowded sarcomere. Using time-resolved x-ray diffraction of contracting muscle, we next show that the contractile lattice is neither isovolumetric nor constant in spacing. Instead, lattice spacing is time varying, depends on activation, and can manifest as an effective time-varying Poisson ratio. The resulting fluid flow in the sarcomere lattice of synchronous insect flight muscles is even greater than expected for constant lattice spacing conditions. Lattice spacing depends on a variety of factors that produce radial force, including cross-bridges, titin-like molecules, and other structural proteins. Volume change and advective transport varies with the phase of muscle stimulation during periodic contraction but remains significant at all conditions. Although varying in magnitude, advective transport will occur in all cases in which the sarcomere is not isovolumetric. Akin to "breathing," advective-diffusive transport in sarcomeres is sufficient to promote metabolite exchange and may play a role in the regulation of contraction itself.

In vivo X-ray diffraction and simultaneous EMG reveal the time course of myofilament lattice dilation and filament stretch.

Muscle function within an organism depends on the feedback between molecular and meter-scale processes. Although the motions of muscle's contractile machinery are well described in isolated preparations, only a handful of experiments have documented the kinematics of the lattice occurring when multi-scale interactions are fully intact. We used time-resolved X-ray diffraction to record the kinematics of the myofilament lattice within a normal operating context: the tethered flight of Manduca sexta As the primary flight muscles of M.sexta are synchronous, we used these results to reveal the timing of in vivo cross-bridge recruitment, which occurred 24 ms (s.d. 26) following activation. In addition, the thick filaments stretched an average of 0.75% (s.d. 0.32) and thin filaments stretched 1.11% (s.d. 0.65). In contrast to other in vivo preparations, lattice spacing changed an average of 2.72% (s.d. 1.47). Lattice dilation of this magnitude significantly affects shortening velocity and force generation, and filament stretching tunes force generation. While the kinematics were consistent within individual trials, there was extensive variation between trials. Using a mechanism-free machine learning model we searched for patterns within and across trials. Although lattice kinematics were predictable within trials, the model could not create predictions across trials. This indicates that the variability we see across trials may be explained by latent variables occurring in this naturally functioning system. The diverse kinematic combinations we documented mirror muscle's adaptability and may facilitate its robust function in unpredictable conditions.

A Bayesian framework for the detection of diffusive heterogeneity.

Cells are crowded and spatially heterogeneous, complicating the transport of organelles, proteins and other substrates. One aspect of this complex physical environment, the mobility of passively transported substrates, can be quantitatively characterized by the diffusion coefficient: a descriptor of how rapidly substrates will diffuse in the cell, dependent on their size and effective local viscosity. The spatial dependence of diffusivity is challenging to quantitatively characterize, because temporally and spatially finite observations offer limited information about a spatially varying stochastic process. We present a Bayesian framework that estimates diffusion coefficients from single particle trajectories, and predicts our ability to distinguish differences in diffusion coefficient estimates, conditional on how much they differ and the amount of data collected. This framework is packaged into a public software repository, including a tutorial Jupyter notebook demonstrating implementation of our method for diffusivity estimation, analysis of sources of uncertainty estimation, and visualization of all results. This estimation and uncertainty analysis allows our framework to be used as a guide in experimental design of diffusivity assays.

The bacterial replisome has factory-like localization.

DNA replication is essential to cellular proliferation. The cellular-scale organization of the replication machinery (replisome) and the replicating chromosome has remained controversial. Two competing models describe the replication process: In the track model, the replisomes translocate along the DNA like a train on a track. Alternately, in the factory model, the replisomes form a stationary complex through which the DNA is pulled. We summarize the evidence for each model and discuss a number of confounding aspects that complicate interpretation of the observations. We advocate a factory-like model for bacterial replication where the replisomes form a relatively stationary and weakly associated complex that can transiently separate.

Probing bacterial cell biology using image cytometry.

Advances in automated fluorescence microscopy have made snapshot and time-lapse imaging of bacterial cells commonplace, yet fundamental challenges remain in analysis. The vast quantity of data collected in high-throughput experiments requires a fast and reliable automated method to analyze fluorescence intensity and localization, cell morphology and proliferation as well as other descriptors. Inspired by effective yet tractable methods of population-level analysis using flow cytometry, we have developed a framework and tools for facilitating analogous analyses in image cytometry. These tools can both visualize and gate (generate subpopulations) more than 70 cell descriptors, including cell size, age and fluorescence. The method is well suited to multi-well imaging, analysis of bacterial cultures with high cell density (thousands of cells per frame) and complete cell cycle imaging. We give a brief description of the analysis of four distinct applications to emphasize the broad applicability of the tool.

Escherichia coli Chromosomal Loci Segregate from Midcell with Universal Dynamics.

The structure of the Escherichia coli chromosome is inherently dynamic over the duration of the cell cycle. Genetic loci undergo both stochastic motion around their initial positions and directed motion to opposite poles of the rod-shaped cell during segregation. We developed a quantitative method to characterize cell-cycle dynamics of the E. coli chromosome to probe the chromosomal steady-state mobility and segregation process. By tracking fluorescently labeled chromosomal loci in thousands of cells throughout the entire cell cycle, our method allows for the statistical analysis of locus position and motion, the step-size distribution for movement during segregation, and the locus drift velocity. The robust statistics of our detailed analysis of the wild-type E. coli nucleoid allow us to observe loci moving toward midcell before segregation occurs, consistent with a replication factory model. Then, as segregation initiates, we perform a detailed characterization of the average segregation velocity of loci. Contrary to origin-centric models of segregation, which predict distinct dynamics for oriC-proximal versus oriC-distal loci, we find that the dynamics of loci were universal and independent of genetic position.