ABSTRACT
A key challenge in understanding subcellular organization is quantifying interpretable measurements of intracellular structures with complex multi-piece morphologies in an objective, robust and generalizable manner. Here we introduce a morphology-appropriate representation learning framework that uses 3D rotation invariant autoencoders and point clouds. This framework is used to learn representations of complex multi-piece morphologies that are independent of orientation, compact, and easy to interpret. We apply our framework to intracellular structures with punctate morphologies (e.g. DNA replication foci) and polymorphic morphologies (e.g. nucleoli). We systematically compare our framework to image-based autoencoders across several intracellular structure datasets, including a synthetic dataset with pre-defined rules of organization. We explore the trade-offs in the performance of different models by performing multi-metric benchmarking across efficiency, generative capability, and representation expressivity metrics. We find that our framework, which embraces the underlying morphology of multi-piece structures, facilitates the unsupervised discovery of sub-clusters for each structure. We show how our approach can also be applied to phenotypic profiling using a dataset of nucleolar images following drug perturbations. We implement and provide all representation learning models using CytoDL, a python package for flexible and configurable deep learning experiments.
ABSTRACT
In a Comment to the Editor, Skóra raises a concern that the modeling framework implemented in Garner et al. (Biophysical Journal, 2023) neglects a potentially important term in the Brownian dynamics simulation of diffusion. Omission of this diffusivity gradient term may lead to an underestimation of the mean and overestimation of the variance of the cytoplasmic viscosity. In this response, we directly address this concern by incorporating this term into our model and showing that for this data set, its effect is negligible and does not alter the conclusions of this work.
Subject(s)
Cytoplasm , Diffusion , Cytoplasm/metabolism , Viscosity , Models, Biological , Rheology , NanotechnologyABSTRACT
To investigate the fundamental question of how cellular variations arise across spatiotemporal scales in a population of identical healthy cells, we focused on nuclear growth in hiPS cell colonies as a model system. We generated a 3D timelapse dataset of thousands of nuclei over multiple days, and developed open-source tools for image and data analysis and an interactive timelapse viewer for exploring quantitative features of nuclear size and shape. We performed a data-driven analysis of nuclear growth variations across timescales. We found that individual nuclear volume growth trajectories arise from short timescale variations attributable to their spatiotemporal context within the colony. We identified a strikingly time-invariant volume compensation relationship between nuclear growth duration and starting volume across the population. Notably, we discovered that inheritance plays a crucial role in determining these two key nuclear growth features while other growth features are determined by their spatiotemporal context and are not inherited.
ABSTRACT
Immune cells have intensely physical lifestyles characterized by structural plasticity and force exertion. To investigate whether specific immune functions require stereotyped mechanical outputs, we used super-resolution traction force microscopy to compare the immune synapses formed by cytotoxic T cells with contacts formed by other T cell subsets and by macrophages. T cell synapses were globally compressive, which was fundamentally different from the pulling and pinching associated with macrophage phagocytosis. Spectral decomposition of force exertion patterns from each cell type linked cytotoxicity to compressive strength, local protrusiveness, and the induction of complex, asymmetric topography. These features were validated as cytotoxic drivers by genetic disruption of cytoskeletal regulators, live imaging of synaptic secretion, and in silico analysis of interfacial distortion. Synapse architecture and force exertion were sensitive to target stiffness and size, suggesting that the mechanical potentiation of killing is biophysically adaptive. We conclude that cellular cytotoxicity and, by implication, other effector responses are supported by specialized patterns of efferent force.
Subject(s)
Immunological Synapses , Single-Cell Analysis , Animals , Immunological Synapses/immunology , Mice , T-Lymphocytes, Cytotoxic/immunology , Biomechanical Phenomena/immunology , Cytotoxicity, Immunologic , Macrophages/immunology , Mice, Inbred C57BLABSTRACT
Cell states were traditionally defined by how they looked, where they were located, and what functions they performed. In this post-genomic era, the field is largely focused on a molecular view of cell state. Moving forward, we anticipate that the observables used to define cell states will evolve again as single-cell imaging and analytics are advancing at a breakneck pace via the collection of large-scale, systematic cell image datasets and the application of quantitative image-based data science methods. This is, therefore, a key moment in the arc of cell biological research to develop approaches that integrate the spatiotemporal observables of the physical structure and organization of the cell with molecular observables toward the concept of a holistic cell state. In this perspective, we propose a conceptual framework for holistic cell states and state transitions that is data-driven, practical, and useful to enable integrative analyses and modeling across many data types.
Subject(s)
Single-Cell Analysis , Humans , Single-Cell Analysis/methods , AnimalsABSTRACT
50 years ago, cell biology was a nascent field. Today, it is a vast discipline whose principles and tools are also applied to other disciplines; vice versa, cell biologists are inspired by other fields. So, the question begs: what is cell biology? The answers are as diverse as the people who define it.
ABSTRACT
Neutrophils are the most abundant leukocyte in humans and provide a critical early line of defense as part of our innate immune system. We perform a comprehensive, genome-wide assessment of the molecular factors critical to proliferation, differentiation, and cell migration in a neutrophil-like cell line. Through the development of multiple migration screen strategies, we specifically probe directed (chemotaxis), undirected (chemokinesis), and 3D amoeboid cell migration in these fast-moving cells. We identify a role for mTORC1 signaling in cell differentiation, which influences neutrophil abundance, survival, and migratory behavior. Across our individual migration screens, we identify genes involved in adhesion-dependent and adhesion-independent cell migration, protein trafficking, and regulation of the actomyosin cytoskeleton. This genome-wide screening strategy, therefore, provides an invaluable approach to the study of neutrophils and provides a resource that will inform future studies of cell migration in these and other rapidly migrating cells.
Subject(s)
Leukocytes , Neutrophils , Humans , Cell Differentiation/genetics , Cell Movement/genetics , Actin CytoskeletonABSTRACT
The skin epithelium acts as the barrier between an organism's internal and external environments. In zebrafish and other freshwater organisms, this barrier function requires withstanding a large osmotic gradient across the epidermis. Wounds breach this epithelium, causing a large disruption to the tissue microenvironment due to the mixing of isotonic interstitial fluid with the external hypotonic fresh water. Here, we show that, following acute injury, the larval zebrafish epidermis undergoes a dramatic fissuring process that resembles hydraulic fracturing, driven by the influx of external fluid. After the wound has sealed-preventing efflux of this external fluid-fissuring starts in the basal epidermal layer at the location nearest to the wound and then propagates at a constant rate through the tissue, spanning over 100 µm. During this process, the outermost superficial epidermal layer remains intact. Fissuring is completely inhibited when larvae are wounded in isotonic external media, suggesting that osmotic gradients are required for fissure formation. Additionally, fissuring partially depends on myosin II activity, as myosin II inhibition reduces the distance of fissure propagation away from the wound. During and after fissuring, the basal layer forms large macropinosomes (with cross-sectional areas ranging from 1 to 10 µm2). We conclude that excess external fluid entry through the wound and subsequent closure of the wound through actomyosin purse-string contraction in the superficial cell layer causes fluid pressure buildup in the extracellular space of the zebrafish epidermis. This excess fluid pressure causes tissue to fissure, and eventually the fluid is cleared through macropinocytosis.
Subject(s)
Hydraulic Fracking , Zebrafish , Animals , Wound Healing/physiology , Epidermis , Epidermal Cells , Myosin Type IIABSTRACT
Immune cells live intensely physical lifestyles characterized by structural plasticity, mechanosensitivity, and force exertion. Whether specific immune functions require stereotyped patterns of mechanical output, however, is largely unknown. To address this question, we used super-resolution traction force microscopy to compare cytotoxic T cell immune synapses with contacts formed by other T cell subsets and macrophages. T cell synapses were globally and locally protrusive, which was fundamentally different from the coupled pinching and pulling of macrophage phagocytosis. By spectrally decomposing the force exertion patterns of each cell type, we associated cytotoxicity with compressive strength, local protrusiveness, and the induction of complex, asymmetric interfacial topographies. These features were further validated as cytotoxic drivers by genetic disruption of cytoskeletal regulators, direct imaging of synaptic secretory events, and in silico analysis of interfacial distortion. We conclude that T cell-mediated killing and, by implication, other effector responses are supported by specialized patterns of efferent force.
ABSTRACT
Maintaining persistent migration in complex environments is critical for neutrophils to reach infection sites. Neutrophils avoid getting trapped, even when obstacles split their front into multiple leading edges. How they re-establish polarity to move productively while incorporating receptor inputs under such conditions remains unclear. Here, we challenge chemotaxing HL60 neutrophil-like cells with symmetric bifurcating microfluidic channels to probe cell-intrinsic processes during the resolution of competing fronts. Using supervised statistical learning, we demonstrate that cells commit to one leading edge late in the process, rather than amplifying structural asymmetries or early fluctuations. Using optogenetic tools, we show that receptor inputs only bias the decision similarly late, once mechanical stretching begins to weaken each front. Finally, a retracting edge commits to retraction, with ROCK limiting sensitivity to receptor inputs until the retraction completes. Collectively, our results suggest that cell edges locally adopt highly stable protrusion/retraction programs that are modulated by mechanical feedback.
Subject(s)
Carrier Proteins , Neutrophils , Neutrophils/physiology , Cell Movement/physiologyABSTRACT
The cytoplasm is a complex, crowded, actively driven environment whose biophysical characteristics modulate critical cellular processes such as cytoskeletal dynamics, phase separation, and stem cell fate. Little is known about the variance in these cytoplasmic properties. Here, we employed particle-tracking nanorheology on genetically encoded multimeric 40 nm nanoparticles (GEMs) to measure diffusion within the cytoplasm of individual fission yeast (Schizosaccharomyces pombe) cellscells. We found that the apparent diffusion coefficients of individual GEM particles varied over a 400-fold range, while the differences in average particle diffusivity among individual cells spanned a 10-fold range. To determine the origin of this heterogeneity, we developed a Doppelgänger simulation approach that uses stochastic simulations of GEM diffusion that replicate the experimental statistics on a particle-by-particle basis, such that each experimental track and cell had a one-to-one correspondence with their simulated counterpart. These simulations showed that the large intra- and inter-cellular variations in diffusivity could not be explained by experimental variability but could only be reproduced with stochastic models that assume a wide intra- and inter-cellular variation in cytoplasmic viscosity. The simulation combining intra- and inter-cellular variation in viscosity also predicted weak nonergodicity in GEM diffusion, consistent with the experimental data. To probe the origin of this variation, we found that the variance in GEM diffusivity was largely independent of factors such as temperature, the actin and microtubule cytoskeletons, cell-cyle stage, and spatial locations, but was magnified by hyperosmotic shocks. Taken together, our results provide a striking demonstration that the cytoplasm is not "well-mixed" but represents a highly heterogeneous environment in which subcellular components at the 40 nm size scale experience dramatically different effective viscosities within an individual cell, as well as in different cells in a genetically identical population. These findings carry significant implications for the origins and regulation of biological noise at cellular and subcellular levels.
Subject(s)
Cytoskeleton , Cytoplasm , Diffusion , Cytosol , Computer SimulationABSTRACT
Fish basal epidermal cells, known as keratocytes, are well-suited for cell migration studies. In vitro, isolated keratocytes adopt a stereotyped shape with a large fan-shaped lamellipodium and a nearly spherical cell body. However, in their native in vivo environment, these cells adopt a significantly different shape during their rapid migration toward wounds. Within the epidermis, keratocytes experience two-dimensional (2D) confinement between the outer epidermal cell layer and the basement membrane; these two deformable surfaces constrain keratocyte cell bodies to be flatter in vivo than in isolation. In vivo keratocytes also exhibit a relative elongation of the front-to-back axis and substantially more lamellipodial ruffling, as compared to isolated cells. We have explored the effects of 2D confinement, separated from other in vivo environmental cues, by overlaying isolated cells with an agarose hydrogel with occasional spacers, or with a ceiling made of polydimethylsiloxane (PDMS) elastomer. Under these conditions, isolated keratocytes more closely resemble the in vivo migratory shape phenotype, displaying a flatter apical-basal axis and a longer front-to-back axis than unconfined keratocytes. We propose that 2D confinement contributes to multiple dimensions of in vivo keratocyte shape determination. Further analysis demonstrates that confinement causes a synchronous 20% decrease in both cell speed and volume. Interestingly, we were able to replicate the 20% decrease in speed using a sorbitol hypertonic shock to shrink the cell volume, which did not affect other aspects of cell shape. Collectively, our results suggest that environmentally imposed changes in cell volume may influence cell migration speed, potentially by perturbing physical properties of the cytoplasm.
Subject(s)
Keratinocytes , Animals , Cell Movement , Cytoplasm/metabolism , Cells, CulturedABSTRACT
Phagocytosis requires rapid remodeling of the actin cytoskeleton for extension of membrane protrusions and force generation to ultimately drive the engulfment of targets. The detailed mechanisms of phagocytosis have almost exclusively been studied in immortalized cell lines. Here, we make use of high-resolution imaging and novel biophysical approaches to determine the structural and mechanical features of phagocytosis by primary bone marrow-derived macrophages. We find that the signature behavior of these primary cells is distinct from macrophage-like cell lines; specifically, it is gentle, with only weak target constriction and modest polarization of the F-actin distribution inside the phagocytic cup. We show that long-tailed myosins 1e/f are critical for this organization. Deficiency of myo1e/f causes dramatic shifts in F-actin localization, reducing F-actin at the phagocytic cup base and enhancing F-actin-mediated constriction at the cup rim. Surprisingly, these changes can be almost fully reverted upon inhibition of another myosin motor protein, myosin-II. Hence, we show that the biomechanics and large-scale organization of phagocytic cups is tightly regulated through competing contributions from myosin-Ie/f and myosin-II.
Subject(s)
Actins , Phagocytosis , Actins/metabolism , Constriction , Phagocytosis/physiology , Actin Cytoskeleton/metabolism , Myosin Type II/metabolism , Myosins/metabolism , Macrophages/metabolism , Cytoskeletal Proteins/metabolismABSTRACT
Animal cell migration is predominantly driven by the coordinated, yet stochastic, polymerization of thousands of nanometer-scale actin filaments across micron-scale cell leading edges. It remains unclear how such inherently noisy processes generate robust cellular behavior. We employed high-speed imaging of migrating neutrophil-like HL-60 cells to explore the fine-scale shape fluctuations that emerge and relax throughout the process of leading edge maintenance. We then developed a minimal stochastic model of the leading edge that reproduces this stable relaxation behavior. Remarkably, we find lamellipodial stability naturally emerges from the interplay between branched actin network growth and leading edge shape - with no additional feedback required - based on a synergy between membrane-proximal branching and lateral spreading of filaments. These results thus demonstrate a novel biological noise-suppression mechanism based entirely on system geometry. Furthermore, our model suggests that the Arp2/3-mediated ~70-80° branching angle optimally smooths lamellipodial shape, addressing its long-mysterious conservation from protists to mammals.
In every human cell, there are tens of millions of proteins which work together to control everything from the cell's shape to its behavior. One of the most abundant proteins is actin, which organizes itself into filaments that mechanically support the cell and help it to move. These filaments are very dynamic, with individual actin molecules constantly being added or removed. This allows the cell to build large structures with distinct shapes and properties. Many motile cells, for example, have a structure called a lamellipodium which protrudes at their 'leading edge' and pushes them forward. The lamellipodium has a very robust shape that does not vary much between different cell types, or change significantly as cells migrate. But how the tens of thousands of actin molecules inside the lamellipodium organize themselves into this large, stable structure is not fully understood. To investigate, Garner and Theriot used high-speed video microscopy to track the shape of human cells cultured in the laboratory. As the cells crawled along a glass surface, their leading edge undulated like strings being plucked on a guitar. A computer simulation showed that these ripples can be caused by filaments randomly adding and removing actin molecules. While these random movements could destabilize the structure of the leading edge, the simulation suggests that another aspect of actin filament growth smooths out any fluctuations in the lamellipodium's shape. Actin networks in the lamellipodium have a branched configuration, with new strands emerging off each other at an angle like branches in a tree. Garner and Theriot found that the specific angle in which new filaments are added smooths out the lamellipodium's shape, which may explain why this geometry has persisted throughout evolution. These findings suggest that the way in which actin filaments join together helps to maintain the shape of large cellular structures. In the future, scientists could use this design principle to build molecular machines that can self-organize into microstructures. These engineered constructs could be used to modulate the activity of living cells that have been damaged by disease.
Subject(s)
Actins , Pseudopodia , Actin Cytoskeleton , Animals , Cell Movement , Cytoskeleton , MammalsABSTRACT
To combat infectious diseases, it is important to understand how host cells interact with bacterial pathogens. Signals conveyed from pathogen to host, and vice versa, may be either chemical or mechanical. While the molecular and biochemical basis of host-pathogen interactions has been extensively explored, relatively less is known about mechanical signals and responses in the context of those interactions. Nevertheless, a wide variety of bacterial pathogens appear to have developed mechanisms to alter the cellular biomechanics of their hosts in order to promote their survival and dissemination, and in turn many host responses to infection rely on mechanical alterations in host cells and tissues to limit the spread of infection. In this review, we present recent findings on how mechanical forces generated by host cells can promote or obstruct the dissemination of intracellular bacterial pathogens. In addition, we discuss how in vivo extracellular mechanical signals influence interactions between host cells and intracellular bacterial pathogens. Examples of such signals include shear stresses caused by fluid flow over the surface of cells and variable stiffness of the extracellular matrix on which cells are anchored. We highlight bioengineering-inspired tools and techniques that can be used to measure host cell mechanics during infection. These allow for the interrogation of how mechanical signals can modulate infection alongside biochemical signals. We hope that this review will inspire the microbiology community to embrace those tools in future studies so that host cell biomechanics can be more readily explored in the context of infection studies.
Subject(s)
Extracellular Matrix , Host-Pathogen Interactions , BacteriaABSTRACT
We introduce a framework for end-to-end integrative modeling of 3D single-cell multi-channel fluorescent image data of diverse subcellular structures. We employ stacked conditional ß-variational autoencoders to first learn a latent representation of cell morphology, and then learn a latent representation of subcellular structure localization which is conditioned on the learned cell morphology. Our model is flexible and can be trained on images of arbitrary subcellular structures and at varying degrees of sparsity and reconstruction fidelity. We train our full model on 3D cell image data and explore design trade-offs in the 2D setting. Once trained, our model can be used to predict plausible locations of structures in cells where these structures were not imaged. The trained model can also be used to quantify the variation in the location of subcellular structures by generating plausible instantiations of each structure in arbitrary cell geometries. We apply our trained model to a small drug perturbation screen to demonstrate its applicability to new data. We show how the latent representations of drugged cells differ from unperturbed cells as expected by on-target effects of the drugs.
Subject(s)
Cell Nucleus/physiology , Cell Shape/physiology , Induced Pluripotent Stem Cells/cytology , Intracellular Space , Models, Biological , Cells, Cultured , Computational Biology , Humans , Imaging, Three-Dimensional , Intracellular Space/chemistry , Intracellular Space/metabolism , Intracellular Space/physiology , Microscopy, Fluorescence , Single-Cell AnalysisABSTRACT
To migrate efficiently to target locations, cells must integrate receptor inputs while maintaining polarity: a distinct front that leads and a rear that follows. Here we investigate what is necessary to overwrite pre-existing front-rear polarity in neutrophil-like HL60 cells migrating inside straight microfluidic channels. Using subcellular optogenetic receptor activation, we show that receptor inputs can reorient weakly polarized cells, but the rear of strongly polarized cells is refractory to new inputs. Transient stimulation reveals a multi-step repolarization process, confirming that cell rear sensitivity to receptor input is the primary determinant of large-scale directional reversal. We demonstrate that the RhoA/ROCK/myosin II pathway limits the ability of receptor inputs to signal to Cdc42 and reorient migrating neutrophils. We discover that by tuning the phosphorylation of myosin regulatory light chain we can modulate the activity and localization of myosin II and thus the amenability of the cell rear to 'listen' to receptor inputs and respond to directional reprogramming.
Subject(s)
Cell Movement/physiology , Myosin Type II/metabolism , Neutrophils/physiology , Cell Polarity/physiology , Chemotaxis/physiology , HL-60 Cells , Humans , Myosin Light Chains/metabolism , Phosphorylation , cdc42 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolismABSTRACT
Phagocytosis requires rapid actin reorganization and spatially controlled force generation to ingest targets ranging from pathogens to apoptotic cells. How actomyosin activity directs membrane extensions to engulf such diverse targets remains unclear. Here, we combine lattice light-sheet microscopy (LLSM) with microparticle traction force microscopy (MP-TFM) to quantify actin dynamics and subcellular forces during macrophage phagocytosis. We show that spatially localized forces leading to target constriction are prominent during phagocytosis of antibody-opsonized targets. This constriction is largely driven by Arp2/3-mediated assembly of discrete actin protrusions containing myosin 1e and 1f ('teeth') that appear to be interconnected in a ring-like organization. Contractile myosin-II activity contributes to late-stage phagocytic force generation and progression, supporting a specific role in phagocytic cup closure. Observations of partial target eating attempts and sudden target release via a popping mechanism suggest that constriction may be critical for resolving complex in vivo target encounters. Overall, our findings present a phagocytic cup shaping mechanism that is distinct from cytoskeletal remodeling in 2D cell motility and may contribute to mechanosensing and phagocytic plasticity.
Subject(s)
Macrophages/cytology , Myosin Type II/metabolism , Phagocytosis/physiology , Actins/metabolism , Animals , Bone Marrow Cells , Cytoskeleton , HL-60 Cells , Humans , Mice , Mice, Inbred C57BL , Microscopy/methods , Molecular Imaging/methods , RAW 264.7 Cells , Stem CellsABSTRACT
We performed a comprehensive analysis of the transcriptional changes occurring during human induced pluripotent stem cell (hiPSC) differentiation to cardiomyocytes. Using single cell RNA-seq, we sequenced > 20,000 single cells from 55 independent samples representing two differentiation protocols and multiple hiPSC lines. Samples included experimental replicates ranging from undifferentiated hiPSCs to mixed populations of cells at D90 post-differentiation. Differentiated cell populations clustered by time point, with differential expression analysis revealing markers of cardiomyocyte differentiation and maturation changing from D12 to D90. We next performed a complementary cluster-independent sparse regression analysis to identify and rank genes that best assigned cells to differentiation time points. The two highest ranked genes between D12 and D24 (MYH7 and MYH6) resulted in an accuracy of 0.84, and the three highest ranked genes between D24 and D90 (A2M, H19, IGF2) resulted in an accuracy of 0.94, revealing that low dimensional gene features can identify differentiation or maturation stages in differentiating cardiomyocytes. Expression levels of select genes were validated using RNA FISH. Finally, we interrogated differences in cardiac gene expression resulting from two differentiation protocols, experimental replicates, and three hiPSC lines in the WTC-11 background to identify sources of variation across these experimental variables.
Subject(s)
Biomarkers/metabolism , Cell Differentiation , Gene Expression Regulation , Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Transcriptome , Humans , Induced Pluripotent Stem Cells/cytology , RNA-SeqABSTRACT
In Gram-positive bacteria, a thick cross-linked cell wall separates the membrane from the extracellular space. Some surface-exposed proteins, such as the Listeria monocytogenes actin nucleation-promoting factor ActA, remain associated with the bacterial membrane but somehow thread through tens of nanometres of cell wall to expose their amino terminus to the exterior. Here, we report that entropy enables the translocation of disordered transmembrane proteins through the Gram-positive cell wall. We build a physical model, which predicts that the entropic constraint imposed by a thin periplasm is sufficient to drive the translocation of an intrinsically disordered protein such as ActA across a porous barrier similar to a peptidoglycan cell wall. We experimentally validate our model and show that ActA translocation depends on the cell-envelope dimensions and disordered-protein length, and that translocation is reversible. We also show that disordered regions of eukaryotic proteins can translocate Gram-positive cell walls via entropy. We propose that entropic forces are sufficient to drive the translocation of specific proteins to the outer surface.