Dynamic macrophage “probing” is required for the efficient capture of phagocytic targets

Rather than passively binding ligands via immunoreceptors, macrophages capture particles by repeated extension of actin-rich protrusions

Piecewise-stationary motion modeling and iterative smoothing to track heterogeneous particle motions in dense environments

International audienceOne of the major challenges in multiple particle tracking is the capture of extremely heterogeneous movements of objects in crowded scenes. The presence of numerous assignment candidates in the expected range of particle motion makes the tracking ambiguous and induces false positives. Lowering the ambiguity by reducing the search range, on the other hand, is not an option, as this would increase the rate of false negatives. We propose here a piecewise-stationary motion model (PMM) for the particle transport along an iterative smoother that exploits recursive tracking in multiple rounds in forward and backward temporal directions. By fusing past and future information, our method, termed PMMS, can recover fast transitions from freely or confined diffusive to directed motions with linear time complexity. To avoid false positives we complemented recursive tracking with a robust inline estimator of the search radius for assignment (a.k.a. gating), where past and future information are exploited using only two frames at each optimization step. We demonstrate the improvement of our technique on simulated data – especially the impact of density, variation in frame to frame displacements, and motion switching probability. We evaluated our technique on the 2D particle tracking challenge dataset published by Chenouard et al in 2014. Using high SNR to focus on motion modeling challenges, we show superior performance at high particle density. On biological applications, our algorithm allows us to quantify the extremely small percentage of motor-driven movements of fluorescent particles along microtubules in a dense field of unbound, diffusing particles. We also show with virus imaging that our algorithm can cope with a strong reduction in recording frame rate while keeping the same performance relative to methods relying on fast sampling

S. cerevisiae Chromosomes Biorient via Gradual Resolution of Syntely between S Phase and Anaphase

SummaryFollowing DNA replication, eukaryotic cells must biorient all sister chromatids prior to cohesion cleavage at anaphase. In animal cells, sister chromatids gradually biorient during prometaphase, but current models of mitosis in S. cerevisiae assume that biorientation is established shortly after S phase. This assumption is based on the observation of a bilobed distribution of yeast kinetochores early in mitosis and suggests fundamental differences between yeast mitosis and mitosis in animal cells. By applying super-resolution imaging methods, we show that yeast and animal cells share the key property of gradual and stochastic chromosome biorientation. The characteristic bilobed distribution of yeast kinetochores, hitherto considered synonymous for biorientation, arises from kinetochores in mixed attachment states to microtubules, the length of which discriminates bioriented from syntelic attachments. Our results offer a revised view of mitotic progression in S. cerevisiae that augments the relevance of mechanistic information obtained in this powerful genetic system for mammalian mitosis

Kinetochore alignment within the metaphase plate is regulated by centromere stiffness and microtubule depolymerases

During mitosis in most eukaryotic cells, chromosomes align and form a metaphase plate halfway between the spindle poles, about which they exhibit oscillatory movement. These movements are accompanied by changes in the distance between sister kinetochores, commonly referred to as breathing. We developed a live cell imaging assay combined with computational image analysis to quantify the properties and dynamics of sister kinetochores in three dimensions. We show that baseline oscillation and breathing speeds in late prometaphase and metaphase are set by microtubule depolymerases, whereas oscillation and breathing periods depend on the stiffness of the mechanical linkage between sisters. Metaphase plates become thinner as cells progress toward anaphase as a result of reduced oscillation speed at a relatively constant oscillation period. The progressive slowdown of oscillation speed and its coupling to plate thickness depend nonlinearly on the stiffness of the mechanical linkage between sisters. We propose that metaphase plate formation and thinning require tight control of the state of the mechanical linkage between sisters mediated by centromeric chromatin and cohesion

Tracking heterogeneous particle motions in dense intra-cellular environments

International audienceOne of the major challenges in multiple particle tracking is the capture of heterogeneous movements of objects in crowded scenes. This scenario is particularly prominent in bioimaging, where intracellular structures undergo instantaneous switches between cytoplasmic diffusion and motor-mediated, fast displacements We propose here a piecewise-stationary motion model (PMM) for the particle transport along an iterative smoother that exploits recursive tracking in multiple rounds in forward and backward temporal directions. By fusing past and future information, our method, coined PMMS, can recover fast transitions from freely or confined diffusive to directed motions with linear time complexity. We complemented recursive tracking with a robust inline estimator of the search radius for assignment. We demonstrate the improvement of our technique on simulated data. On biological applications, our algorithm allows us to quantify the extremely small percentage of motor-driven movements of intermediate filament precursor particles along microtubules in a dense field of unbound particles. We also show that our algorithm can cope with a strong reduction in recording frame rate. We will conclude this presentation with preliminary results in dynamic quantification with light-sheet microscopy for 3D subcellular imaging in the context of mitosis. While these techniques allows for complete imaging of rapid intra-cellular phenomenon, new challenges arise to validate and interpret the data