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Biology subjects

Losert, W.

Publications and source records attributed to Losert, W..

At least 19 recordsLinked to original sources

Shear effects in active models of normal and cancer cells

Mechanical properties of biological tissues, driven by passive and active forces, play a vital role in several processes ranging from development to cancer metastasis. However, the dynamical responses of cells in tissues, subject to mechanical deformations such as shear and the associated rheological properties, are not well characterized. Here, we use three-dimensional agent-based models for normal and cancer tissues to investigate their responses to simple shear as a function of cell stiffness and stochastic active forces. In the normal epithelium, with uniform strength of active force, the yield stress as a function of shear rate follows the Herschel-Bulkley form over a range of cell volume fraction. Strikingly, the shear rate dependence and the elasticity-dependent changes in the yield stress fall on master curves upon suitable scaling. To model cancer-like behavior, a certain fraction (Np) of cells was chosen to have enhanced activity and decreased stiffness. As Np increases, the extent of collective cell movement decreases, transitioning from affine (collective) to non-affine (individualistic) movement, a finding that is in accord with imaging experiments. Simulations of a model of a stiff solid tumor, with radius Rs embedded in normal tissue, show that as Rs increases, the yield stress increases. Interestingly, the cells migrate collectively as Rs increases. A Gaussian Mixture Model (GMM) and a mean field theory quantitatively account for the simulation as well as experimental results on cancerous, non-cancerous, and a mixture of these two types. The combined theoretical and experimental study establishes that heterogeneity in stiffness and activity determines non-affine movements in normal and cancer tissues.

biophysics↗

Network-Level Characterization of Spontaneous Calcium Activity in an In-Vitro Alzheimer's Disease Model

The neurodegenerative disorder Alzheimers disease (AD) is widely known for biomarkers such as amyloid beta plaques and tauopathy, as well as functional differences in memory and cognitive ability. Despite this devastating functional impact, a large body of work only focuses on molecular biomarkers of AD. In this study, we investigate collective neural dynamics in vitro and assess how network-level properties differ between a well-established model of familial AD (FAD) and a newly developed in vitro accelerated model (acAD). The new model system reliably develops the key structural characteristics of AD in three weeks, but its calcium dynamics had not been characterized previously. Spontaneous network dynamics influences information processing as part of the internal network state. Here we measure this spontaneous activity of a network of hundreds of cells in each field of view. We find that the FAD model has a larger fraction of hyperactive cells, while the acAD model displays similar characteristics to healthy cells. Additionally, the FAD model has altered cooperation between cells, losing a proportion of highly correlated cellular activities, both for fast and slow coupling among cells. The acAD model is again consistent with healthy networks. Since the acAD model does not show the same spontaneous network dysfunction seen in FAD, it can enable measurements of changes in learning and memory associated with the plasticity, rather than the structure of the internal network state.

biophysics↗

Spatially Distinct Myosin II Architectures Regulate Protrusion Dynamics and Directional Persistence during Immune Cell Migration

Directional persistence is essential for efficient immune cell migration in tissues, yet how cytoskeletal systems stabilize migration in complex three-dimensional environments remains unclear. Using intravital subcellular microscopy and quantitative analysis of membrane dynamics, we identify two spatially distinct architectures of non-muscle myosin II (NMII) that coordinate protrusion dynamics during neutrophil migration. In vivo and in collagen matrices, NMII assembles at the leading edge into lattice-like structures that are structurally and functionally distinct from rear contractile actomyosin bundles. Protrusion-resolved analyses reveal that directional persistence correlates strongly with protrusion lifetime and sustained NMII engagement, with rear NMII load showing the strongest association with protrusion persistence. Strikingly, directional migration is not determined by the abundance of favorable protrusions but by their temporal organization during migration. Pharmacological perturbations that redistribute NMII activity disrupt this temporal organization and alter migration trajectories. Together, these findings reveal that spatially distinct NMII architectures coordinate protrusion dynamics across time to stabilize directional migration in complex environments. HighlightsO_LIMigrating neutrophils assemble spatially distinct myosin II architectures at the leading edge and rear C_LIO_LIProtrusion dynamics and directional persistence are linked to sustained myosin II engagement C_LIO_LIDirectional migration emerges from the temporal organization of protrusion states rather than their abundance C_LI

cell biology↗

Localized ROS Generation with UV light in Differentiating Human Neural Progenitor Cells

Reactive Oxygen Species (ROS) and the associated condition of excess ROS, oxidative stress, has been implicated in a number of diseases including neurodegeneration. However, ROS are also crucial second messengers with beneficial impacts. Within neural cells, ROS signals are known to impact maturation of cells as well as memory and learning. Photobiomodulation (PBM), the use of light to impact cells/tissue, is a promising way to noninvasively modulate ROS. This study investigates the effects of PBM using 370 nm light to increase ROS levels in human neural progenitor cells (hNPC), and study potential impacts on calcium dynamics. We find 370 nm light to be effective at inducing ROS within hNPC. The photoinduction of ROS only impacts ROS levels in illuminated cells, with no measurable signal relay to non-illuminated cells within the acute time period we examined. The increase in ROS generated by our UV light exposure creates elevated basal levels of calcium, but does not impact spontaneous calcium signaling in networks of hNPC cells.

cell biology↗

Nanotopographic Control of Actin Waves and Growth Cone Navigation in Developing Neurons

The development of axons and dendrites (neurites) in a neural circuit relies on the dynamic interplay of cytoskeletal components, especially actin, and the integration of diverse environmental cues. Building on prior findings that actin dynamics can serve as a primary sensor of physical guidance cues, this work investigates the role of nanotopography in modulating and guiding actin waves and neurite-tip dynamics during early neural circuit development. Although actin dynamics is well known to contribute to pathfinding in wide axonal tips, typically referred to as growth cones, we also observe dynamic actin remodeling throughout neurites and at other, narrower, neurite tips. We find that actin-wave speeds do not change significantly in the first two weeks of neurite development on flat substrates, but decrease over the same period in neurites on nanoridges. The ability of nanoridges to guide actin waves and the neurite-tip direction also decreases as neurites mature, both for narrow tips and wide growth cones. This change in responsiveness to physical guidance cues with neuronal maturation may impact the regenerative capacity of developing neural cells that are inserted into mature brains.

biophysics↗

Estradiol alters actin and protrusion dynamics in endometriotic epithelial cells

Estradiol (E2), a sex steroid hormone molecule, plays a key role in regulating the actin and shape dynamics of cells in a multitude of normal and pathophysiological conditions. While cytoskeletal rearrangements, membrane dynamics, and cellular protrusions are intimately involved in cell motility and invasiveness, little is known about the impact of E2 on these processes in estrogen-dependent epithelial cells. In this study, we quantified the impact of E2 on cell shape and actin dynamics in 12Z human endometriotic epithelial cells transfected with LifeAct-GFP and observed with lattice lightsheet microscopy, a new imaging technique fast enough to capture 3D dynamics on second timescales. E2, when applied for 24 hours, significantly decreased cell circularity, solidity, and rate of change of circularity, indicating a transition to a more elongated and less variable morphology. 24-hour E2 treatment also induced the formation of large membrane protrusions reminiscent of invadopodia and led to a more disordered flow of actin within those protrusions. However, these effects were not seen after 15 minutes of E2 treatment, suggesting that longer-term signaling is required to drive these structural changes. Together, these results suggest that E2 modulates actin polymerization and membrane protrusion dynamics in endometriotic epithelial cells and may prime them for cell invasion. This work highlights a role for hormonal signaling in mediating cytoskeletal plasticity and migratory cell phenotypes.

biophysics↗

Engineered micropillars to unveil oligodendrocyte responses to physical cues

Destruction of myelin internodes, oligodendrocyte (OL) apoptosis, and axonal degeneration characterize diseased or aged central nervous systems. While OLs can partially regenerate myelin sheaths, the remyelination process ultimately fails. Tissue mechanical and physical properties, such as stiffness and axonal curvature, play a role in this process. However, the complexity of existing models has hindered studies of OL mechanobiology. Here, a tissue-engineered model is presented to investigate the impact of stiffness and axonal diameter on OL myelination. The model consists of poly(dimethylsiloxane) micropillars with biologically relevant diameters (1-5 {micro}m), tunable rigidity, and amenable for surface functionalization. The optimized method enables the production of high-aspect-ratio, transparent micropillar arrays, in a reproducible and scalable system, serving as surrogate axons. Additionally, new protocols for quantifying myelin formation are introduced, which can be adapted to any myelination studies. Softer micropillars accelerate OL differentiation, while rigid ones promote the maintenance of mature OL states. Wrapping of OLs increased with micropillar diameter on rigid substrates, but not on softer ones, suggesting a complex interplay between curvature and rigidity. These processes involve calcium-sensitive channels, histone deacetylases, and microtubules dynamics. The proposed platform constitutes a versatile and user-friendly system, with applications from fundamental myelin research to drug discovery.

bioengineering↗

Collective Learning in Living Neural Networks Facilitated by Contextual Background Photostimulation

This study explores collective learning in living neural networks, focusing on group-to-group Hebbian learning, i.e., strengthening and weakening of links dependent on the precise timing of their activities. While neuronal plasticity is now well understood for single pairs of neurons, recent research has demonstrated that groups of tens of neurons are required to encode information in mammalian brains. Thus, it is critical to understand how mechanisms of plasticity, in particular spike-timing-dependent plasticity (STDP) operate at the group scale. We find that neuronal groups can reach significant plasticity after only 45 stimuli when a proper tradeoff between pulse duration and photostimulation effectiveness is chosen. Background stimulation, which enhances the reliability of response for the targeted neuronal groups, is necessary for rapid network-level Hebbian learning. By demonstrating enhanced learning in the presence of background activity, this study underscores the highly cooperative character of neurons and the importance of investigating learning, information flow, and memory formation at the network scale.

neuroscience↗

Collective Information and Communication in Morphologically Distinct Astrocytes

The discovery that astrocytes are an active, rather than a passive, component of the brain has ushered in a paradigm shift in thinking about how the brain processes information. Although the mechanisms by which astrocytes integrate information from neurons are still debated, such discourse should not distract from the importance of more completely understanding how astrocytes communicate via signals amongst themselves. This work aims to study how different astrocytes signal within their own networks. We investigate group calcium (Ca2+) dynamics in polygonal, stellate, and reactive astrocytes. These distinct and important astrocyte subtypes are present in the brain to varying degrees at different physiological states. We use an information-theoretic framework to quantify the dynamics embedded in the Ca2+ traces within astrocyte networks; specifically, we employ the Hurst exponent, cross-correlation, mutual information, and partitioned entropy to assess differences in the astrocyte signals across subtypes. To gain insights into the ability of astrocyte networks to respond to changes in the extracellular environment, we probe the networks with perturbations affecting their cytoskeletal dynamics (Latrunculin B) and energetic levels (Adenosine triphosphate). Overall, these three classes of astrocytes behave differently and respond idiosyncratically to their extracellular environment. We find that polygonal astrocytes are not quiescent, stellate astrocytes respond most strongly to ATP, and reactive astrocytes are uniquely perturbed by Latrunculin B. Interestingly, despite these distinct differences in behaviors, we find a uniform speed of information transport regardless of subtype or perturbation; this uniformity is maintained when using both cross-correlation and mutual information to assess this speed. We conclude that the differential ways astrocytes signal within our measured framework yield important insights into how astrocytes communicate and contribute to this pressing issue of understanding astrocyte information processing.

biophysics↗

Development of an Accelerated Cellular Model for Alzheimer's Disease

Alzheimers Disease (AD) is a leading cause of dementia characterized by amyloid plaques and neurofibrillary tangles, and its pathogenesis remains unclear. Current cellular models for AD often require several months to exhibit phenotypic features due to the lack of an aging environment in vitro. Lamin A is a key component of the nuclear lamina. And progerin, a truncated protein resulting from specific lamin A mutations, causes Hutchinson-Gilford Progeria Syndrome (HGPS), a disease that prematurely ages individuals. Studies have reported that lamin A expression is induced in the brains of AD patients, and overlapping cellular phenotypes have been observed between HGPS and AD cells. In this study, we investigated the effects of exogenous progerin expression on neural progenitor cells carrying familial AD mutations (FAD). Within three to four weeks of differentiation, these cells exhibited robust AD phenotypes, including increased tau phosphorylation, amyloid plaque accumulation, and an elevated A{beta}42 to A{beta}40 ratio. Additionally, progerin expression significantly increased AD cellular phenotypes such as cell death and cell cycle re-entry. Our results suggest that progerin expression could be used to create an accelerated model for AD development and drug screening. Significance StatementAlzheimers Disease (AD) contributes to most dementia, while its mechanism is still under investigation. One of the challenges for studying AD is the model issue, including the genetic divergence of animals and human, and the rejuvenation of induced pluripotent stem cells (iPSCs). Progerin is a mutant lamin A found in the accelerated aging disease progeria. There are a lot of molecular similarities between Alzheimers Disease (AD) and progeria. Here, we developed an accelerated 2D/3D cell model system for AD by ectopically expressing progerin in a previously characterized AD cell model carrying familial AD (FAD) mutations. Our study showed that progerin addition could accelerate AD phenotypical progression, including tau phosphorylation and formation of {beta}-amyloid plaques.

cell biology↗

Coupled Biomechanical and Ionic Excitability in Developing Neural Cell Networks

Waves and oscillations play a key role in the flow and processing of information in the brain. Recent work has demonstrated that in addition to electrical activity, biomechanical signaling can also be excitable and thus capable of self-sustaining oscillations and waves. Here we measured the biomechanical dynamics of actin polymerization in neural precursor cells throughout their differentiation into populations of neurons and astrocytes. Fluorescence-based live-cell imaging allowed us to analyze the dynamics of actin in conjunction with the dynamics of calcium signals. Actin dynamics throughout differentiation showed a rhythmic character, localized mostly in processes, with changes in scale associated with differentiation. Furthermore, actin dynamics impact ionic dynamics, with an increase in the frequency of calcium bursts accompanied by a decrease in cell-cell correlations when actin dynamics is inhibited. This impact of cytoskeletal dynamics on cell-cell coupling and ionic neural cell signaling suggests that information flow in the brain may be able to harness both biomechanical and electrical/ionic excitability.

cell biology↗

NeuroWRAP: integrating, validating, and sharing neurodata analysis workflows

Multiphoton calcium imaging is one of the most powerful tools in modern neuroscience. However, multiphoton data require significant pre-processing of images and post-processing of extracted signals. As a result, many algorithms and pipelines have been developed for the analysis of multiphoton data, particularly two-photon imaging data. Most current studies use one of several algorithms and pipelines that are published and publicly available, and add customized upstream and downstream analysis elements to fit the needs of individual researchers. The vast differences in algorithm choices, parameter settings, pipeline composition, and data sources combine to make collaboration difficult, and raise questions about the reproducibility and robustness of experimental results. We present our solution, called NeuroWRAP, which is a tool that wraps multiple published algorithms together, and enables integration of custom algorithms. It enables development of collaborative, shareable custom workflows and reproducible data analysis for multiphoton calcium imaging data enabling easy collaboration between researchers. NeuroWRAP implements an approach to evaluate the sensitivity and robustness of the configured pipelines. When this sensitivity analysis is applied to a crucial step of image analysis, cell segmentation, we find a substantial difference between two popular workflows, CaImAn and Suite2p. NeuroWRAP harnesses this difference by introducing consensus analysis, utilizing two workflows in conjunction to significantly increase the trustworthiness and robustness of cell segmentation results.

neuroscience↗

Propagation dynamics of electrotactic motility in large epithelial cell sheets

Directional migration initiated at the wound edge leads epithelial sheets to migrate in wound healing. How such coherent migration is achieved is not well understood. Here we used electric fields to induce robust migration of sheets of human keratinocytes and developed an in silico model to characterize initiation and propagation of epithelial collective migration. Electric fields initiate increase in migrations directionality and speed at the leading edge. The increases propagate across the epithelial sheets, resulting in directional migration of cell sheets as coherent units. Both the experimental and in silico models demonstrated vector-like integration of the electric and default directional cues at the free edge in space and time. The resultant collective migration is remarkably consistent in experiments and modeling, both qualitatively and quantitatively. The keratinocyte model thus faithfully reflects key features of epithelial migration as a coherent tissue in vivo, e.g. that leading cells lead, and that epithelium maintains cell- cell junction.

cell biology↗

Nanotopography modulates intracellular excitable systems through cytoskeleton actuation

Cellular sensing of most environmental cues involves receptors that affect a signal-transduction excitable network (STEN), which is coupled to a cytoskeletal excitable network (CEN). We show that the mechanism of sensing of nanoridges is fundamentally different. CEN activity occurs preferentially on nanoridges, whereas STEN activity is constrained between nanoridges. In the absence of STEN, waves disappear, but long-lasting F-actin puncta persist along the ridges. When CEN is suppressed, wave propagation is no longer constrained by nanoridges. A computational model reproduces these experimental observations. Our findings indicate that nanotopography is sensed directly by CEN, whereas STEN is only indirectly affected due to a CEN-STEN feedback loop. These results explain why texture sensing is robust, and acts cooperatively with multiple other guidance cues in complex microenvironments. One-Sentence SummaryCells sense nanotopography directly through their cytoskeletal dynamics, in a reversal of traditional signaling pathway hierarchies.

biophysics↗

Enabled primarily controls filopodial morphology, not actin organization, in the TSM1 growth cone in Drosophila

Ena/VASP proteins are processive actin polymerases that are required throughout animal phylogeny for many morphogenetic processes, including axon growth and guidance. Here we use live imaging of morphology and actin organization in the TSM1 axon of the Drosophila wing to dissect the mechanism of Ena action. We find that altering Ena activity has a substantial impact on filopodial morphology in this growth cone, but exerts only modest effects on actin organization. This is in contrast to the main regulator of Ena, Abl tyrosine kinase, which has profound effects on actin and only mild effects on TSM1 growth cone morphology. These data suggest that the primary role of Ena in this axon may be to link actin to morphogenetic processes of the plasma membrane, rather than regulating actin organization itself. These data also suggest that a key role of Ena, acting downstream of Abl, may be to maintain a constant filopodial organization of the growth cone, even as Abl activity varies in response to guidance cues in the environment. Summary statementWe dissect the function of the actin polymerase, Enabled, in axon growth by live-imaging of actin dynamics and axon morphology of the TSM1 neuron in its native environment in vivo.

neuroscience↗

Label-free Cell Tracking Enables Collective Motion Phenotyping in Epithelial Monolayers

Collective cell migration is an umbrella term for a rich variety of cell behaviors, whose distinct character is essential for biological function, notably for cancer metastasis. One essential feature of collective behavior is the motion of cells relative to their immediate neighbors. We introduce an AI-based pipeline to segment and track cell nuclei from phase contrast images. Nuclei segmentation is based on a U-Net convolutional neural network trained on images with nucleus staining. Tracking, based on the Crocker-Grier algorithm, quantifies nuclei movement and allows for robust downstream analysis of collective motion. Since the AI algorithm required no new training data, our approach promises to be applicable to and yield new insights for vast libraries of existing collective motion images. In a systematic analysis of a cell line panel with oncogenic mutations, we find that the collective rearrangement metric, D2min, which reflects non-affine motion, shows promise as an indicator of metastatic potential. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/472148v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@82a35corg.highwire.dtl.DTLVardef@b31442org.highwire.dtl.DTLVardef@f703aborg.highwire.dtl.DTLVardef@1120d98_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIVersatile AI-based algorithm can robustly identify individual cells and track their motion from phase contrast images. C_LIO_LIAnalysis of motion of cells relative to nearby neighbors distinguishes weakly tumorigenic (KRas) and metastatic (KRas/PTEN-/-) cell lines. C_LI

biophysics↗

Decoding natural astrocyte rhythms: dynamic actin waves result from environmental sensing by primary rodent astrocytes

Astrocytes are key regulators of brain homeostasis, an important physiological process that includes but is not limited to buffering of extracellular K+, equilibrating osmotic gradients, regulating pH, uptake of neurotransmitters, and releasing growth factors - all of which are essential for proper cognitive function. While previous studies have revealed how specific molecular components of the astrocytic cytoskeleton affect the efficacy of these homeostatic processes, none have studied how homeostasis is linked to the excitable systems character of the cytoskeleton. As recently discovered, excitability of the actin cytoskeleton manifests in second-scale dynamic fluctuations and acts as a sensor of chemo-physical environmental cues. Here we find that homeostatic regulation may be more active than previously thought, involving the excitable dynamics of actin in certain subcellular regions, especially near the cell boundary. Our results further indicate that actin dynamics concentrates into "hotspot" regions that selectively respond to certain chemo-physical stimuli, specifically the homeostatic challenges of ion or water concentration increases. Substrate topography makes the actin dynamics of astrocytes weaker. Superresolution images demonstrate that surface topography is also associated with predominant perpendicular alignment of actin filaments near the cell boundary whereas flat substrates result in an actin cortex mainly parallel to the cell boundary. Additionally, co-culture with neurons increases both the probability of actin dynamics and the strength of hotspots. The excitable systems character of actin thus makes astrocytes direct participants in neural cell network dynamics.

neuroscience↗

Cortical waves mediate the cellular response to electric fields

Electrotaxis, the directional migration of cells in a constant electric field, is important in regeneration, development, and wound healing. Electrotaxis has a slower response and a smaller dynamic range than guidance by other cues, suggesting that the mechanism of electrotaxis share both similarities and differences with chemical-gradient-sensing pathways. We examined a mechanism centered on the excitable system consisting of cortical waves of biochemical signals coupled to cytoskeletal reorganization, which has been implicated in random cell motility. We use electro-fused giant Dictyostelium discoideum cells to decouple waves from cell motion and employ nanotopographic surfaces to limit wave dimensions and lifetimes. We demonstrate that wave propagation in these cells is guided by electric fields. The wave area and lifetime gradually increase in the first 10 minutes after an electric field is turned on, leading to more abundant and wider protrusions in the cell region nearest the cathode. The wave directions display "U-turn" behavior upon field reversal, and this switch occurs more quickly on nanotopography. Our results suggest that electric fields guide cells by controlling waves of signal transduction and cytoskeletal activity, which underlie cellular protrusions. Whereas surface receptor occupancy triggers both rapid activation and slower polarization of signaling pathways, electric fields appear to act primarily on polarization, explaining why cells respond to electric fields more slowly than to other guidance cues.

cell biology↗