Automated Deep Lineage Tree Analysis Using a Bayesian Single Cell Tracking Approach

Single-cell methods are beginning to reveal the intrinsic heterogeneity in cell populations, arising from the interplay of deterministic and stochastic processes. However, it remains challenging to quantify single-cell behaviour from time-lapse microscopy data, owing to the difficulty of extracting reliable cell trajectories and lineage information over long time-scales and across several generations. Therefore, we developed a hybrid deep learning and Bayesian cell tracking approach to reconstruct lineage trees from live-cell microscopy data. We implemented a residual U-Net model coupled with a classification CNN to allow accurate instance segmentation of the cell nuclei. To track the cells over time and through cell divisions, we developed a Bayesian cell tracking methodology that uses input features from the images to enable the retrieval of multi-generational lineage information from a corpus of thousands of hours of live-cell imaging data. Using our approach, we extracted 20,000 + fully annotated single-cell trajectories from over 3,500 h of video footage, organised into multi-generational lineage trees spanning up to eight generations and fourth cousin distances. Benchmarking tests, including lineage tree reconstruction assessments, demonstrate that our approach yields high-fidelity results with our data, with minimal requirement for manual curation. To demonstrate the robustness of our minimally supervised cell tracking methodology, we retrieve cell cycle durations and their extended inter- and intra-generational family relationships in 5,000 + fully annotated cell lineages. We observe vanishing cycle duration correlations across ancestral relatives, yet reveal correlated cyclings between cells sharing the same generation in extended lineages. These findings expand the depth and breadth of investigated cell lineage relationships in approximately two orders of magnitude more data than in previous studies of cell cycle heritability, which were reliant on semi-manual lineage data analysis

A phenomenological density-scaling approach to lamellipodial actin dynamics

The integration of protein function studied in vitro in a dynamic system like the cell lamellipodium remains a significant challenge. One reason is the apparent contradictory effects that perturbations of some proteins can have on the overall lamellipodium dynamics, depending on exact conditions. Theoretical modeling offers one approach for understanding the balance between the mechanisms that drive and regulate actin network growth and decay. Most models use a \bottom-up" approach, involving explicitly assembling biochemical components to simulate observable behaviour. Their correctness therefore relies on both the accurate characterisation of all the components and the completeness of the relevant processes involved. To avoid potential pitfalls due to this uncertainty, we used an alternative \top-down" approach, in which measurable features of lamellipodium behaviour, here observed in two different cell types (HL60 and B16-F1), directly inform the development of a simple phenomenological model of lamellipodium dynamics. We show that the kinetics of F-actin association and dissociation scales with the local F-actin density, with no explicit location dependence. This justifies the use of a simplified kinetic model of lamellipodium dynamics that yields predictions testable by pharmacological or genetic intervention. A length-scale parameter (the lamellipodium width), emerges from this analysis as an experimentally accessible probe of network regulatory processes

Cortical cell stiffness is independent of substrate mechanics

Cortical stiffness is an important cellular property that changes during migration, adhesion and growth. Previous atomic force microscopy (AFM) indentation measurements of cells cultured on deformable substrates have suggested that cells adapt their stiffness to that of their surroundings. Here we show that the force applied by AFM to a cell results in a significant deformation of the underlying substrate if this substrate is softer than the cell. This ‘soft substrate effect’ leads to an underestimation of a cell’s elastic modulus when analysing data using a standard Hertz model, as confirmed by finite element modelling and AFM measurements of calibrated polyacrylamide beads, microglial cells and fibroblasts. To account for this substrate deformation, we developed a ‘composite cell–substrate model’. Correcting for the substrate indentation revealed that cortical cell stiffness is largely independent of substrate mechanics, which has major implications for our interpretation of many physiological and pathological processes

Actin cortex architecture regulates cell surface tension

Animal cell shape is largely determined by the cortex, a thin actin network underlying the plasma membrane in which myosin-driven stresses generate contractile tension. Tension gradients result in local contractions and drive cell deformations. Previous cortical tension regulation studies have focused on myosin motors. Here, we show that cortical actin network architecture is equally important. First, we observe that actin cortex thickness and tension are inversely correlated during cell-cycle progression. We then show that the actin filament length regulators CFL1, CAPZB and DIAPH1 regulate mitotic cortex thickness and find that both increasing and decreasing thickness decreases tension in mitosis. This suggests that the mitotic cortex is poised close to a tension maximum. Finally, using a computational model, we identify a physical mechanism by which maximum tension is achieved at intermediate actin filament lengths. Our results indicate that actin network architecture, alongside myosin activity, is key to cell surface tension regulation

Extent of myosin penetration within the actin cortex regulates cell surface mechanics.

In animal cells, shape is mostly determined by the actomyosin cortex, a thin cytoskeletal network underlying the plasma membrane. Myosin motors generate tension in the cortex, and tension gradients result in cellular deformations. As such, many cell morphogenesis studies have focused on the mechanisms controlling myosin activity and recruitment to the cortex. Here, we demonstrate using super-resolution microscopy that myosin does not always overlap with actin at the cortex, but remains restricted towards the cytoplasm in cells with low cortex tension. We propose that this restricted penetration results from steric hindrance, as myosin minifilaments are considerably larger than the cortical actin meshsize. We identify myosin activity and actin network architecture as key regulators of myosin penetration into the cortex, and show that increasing myosin penetration increases cortical tension. Our study reveals that the spatial coordination of myosin and actin at the cortex regulates cell surface mechanics, and unveils an important mechanism whereby myosin size controls its action by limiting minifilament penetration into the cortical actin network. More generally, our findings suggest that protein size could regulate function in dense cytoskeletal structures

Electric Field Exposure Triggers and Guides Formation of Pseudopod-Like Blebs in U937 Monocytes

We describe a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells stimulated by nanosecond pulsed electric field (nsPEF). In contrast to regular, round-shaped blebs, which are often seen in response to cell damage, pseudopod-like blebs (PLBs) formed as longitudinal membrane protrusions toward anode. PLB length could exceed the cell diameter in 2 min of exposure to 60-ns, 10-kV/cm pulses delivered at 10-20 Hz. Both PLBs and round-shaped nsPEF-induced blebs could be efficiently inhibited by partial isosmotic replacement of bath NaCl for a larger solute (sucrose), thereby pointing to the colloid-osmotic water uptake as the principal driving force for bleb formation. In contrast to round-shaped blebs, PLBs retracted within several minutes after exposure. Cells treated with 1 nM of the actin polymerization blocker cytochalasin D were unable to form PLBs and instead produced stationary, spherical blebs with no elongation or retraction capacity. Live cell fluorescent actin tagging showed that during elongation actin promptly entered the PLB interior, forming bleb cortex and scaffold, which was not seen in stationary blebs. Overall, PLB formation was governed by both passive (physicochemical) effects of membrane permeabilization and active cytoskeleton assembly in the living cell. To a certain extent, PLB mimics the membrane extension in the process of cell migration and can be employed as a nonchemical model for studies of cytomechanics, membrane-cytoskeleton interaction and cell motility

L-selectin regulates human neutrophil transendothelial migration

The migration of circulating neutrophils towards damage/infected tissue is absolutely critical to the inflammatory response. L-selectin is a cell adhesion molecule abundantly expressed on circulating neutrophils. For over two decades, neutrophil L-selectin has been assigned the exclusive role of supporting tethering and rolling - the initial stages of the multi-step adhesion cascade. Here, we provide direct evidence for L-selectin contributing to neutrophil transendothelial migration (TEM). We show that L-selectin co-clusters with PECAM-1 - a well-characterised cell adhesion molecule involved in regulating neutrophil TEM. This co-clustering behaviour occurs specifically during TEM, which serves to augment ectodomain shedding of L-selectin and expedite the time taken for TEM (TTT) to complete. Blocking PECAM-1 signalling (through mutation of its cytoplasmic tail), PECAM-1-dependent adhesion, or L-selectin shedding, led to a significant delay in the TTT. Finally, we show that co-clustering of L-selectin with PECAM-1 occurs specifically across TNF-α- but not IL-1β-activated endothelial monolayers - implying unique adhesion interactomes forming in a cytokine-specific manner. To our knowledge, this is the first report to implicate a non-canonical role for L-selectin in regulating neutrophil TEM

Mechanisms of leading edge protrusion in interstitial migration.

While the molecular and biophysical mechanisms underlying cell protrusion on two-dimensional substrates are well understood, our knowledge of the actin structures driving protrusion in three-dimensional environments is poor, despite relevance to inflammation, development and cancer. Here we report that, during chemotactic migration through microchannels with 5 μm × 5 μm cross-sections, HL60 neutrophil-like cells assemble an actin-rich slab filling the whole channel cross-section at their front. This leading edge comprises two distinct F-actin networks: an adherent network that polymerizes perpendicular to cell-wall interfaces and a 'free' network that grows from the free membrane at the cell front. Each network is polymerized by a distinct nucleator and, due to their geometrical arrangement, the networks interact mechanically. On the basis of our experimental data, we propose that, during interstitial migration, medial growth of the adherent network compresses the free network preventing its retrograde movement and enabling new polymerization to be converted into forward protrusion

F-Actin Interactome Reveals Vimentin as a Key Regulator of Actin Organization and Cell Mechanics in Mitosis.

Most metazoan cells entering mitosis undergo characteristic rounding, which is important for accurate spindle positioning and chromosome separation. Rounding is driven by contractile tension generated by myosin motors in the sub-membranous actin cortex. Recent studies highlight that alongside myosin activity, cortical actin organization is a key regulator of cortex tension. Yet, how mitotic actin organization is controlled remains poorly understood. To address this, we characterized the F-actin interactome in spread interphase and round mitotic cells. Using super-resolution microscopy, we then screened for regulators of cortex architecture and identified the intermediate filament vimentin and the actin-vimentin linker plectin as unexpected candidates. We found that vimentin is recruited to the mitotic cortex in a plectin-dependent manner. We then showed that cortical vimentin controls actin network organization and mechanics in mitosis and is required for successful cell division in confinement. Together, our study highlights crucial interactions between cytoskeletal networks during cell division

Poroelastic osmoregulation of living cell volume

Cells maintain their volume through fine intracellular osmolarity regulation. Osmotic challenges drive fluid into or out of cells causing swelling or shrinkage, respectively. The dynamics of cell volume changes depending on the rheology of the cellular constituents and on how fast the fluid permeates through the membrane and cytoplasm. We investigated whether and how poroelasticity can describe volume dynamics in response to osmotic shocks. We exposed cells to osmotic perturbations and used defocusing epifluorescence microscopy on membrane-attached fluorescent nanospheres to track volume dynamics with high spatiotemporal resolution. We found that a poroelastic model that considers both geometrical and pressurization rates captures fluid-cytoskeleton interactions, which are rate-limiting factors in controlling volume changes at short timescales. Linking cellular responses to osmotic shocks and cell mechanics through poroelasticity can predict the cell state in health, disease, or in response to novel therapeutics