Structural analysis of receptors and actin polarity in platelet protrusions.

During activation the platelet cytoskeleton is reorganized, inducing adhesion to the extracellular matrix and cell spreading. These processes are critical for wound healing and clot formation. Initially, this task relies on the formation of strong cellular-extracellular matrix interactions, exposed in subendothelial lesions. Despite the medical relevance of these processes, there is a lack of high-resolution structural information on the platelet cytoskeleton controlling cell spreading and adhesion. Here, we present in situ structural analysis of membrane receptors and the underlying cytoskeleton in platelet protrusions by applying cryoelectron tomography to intact platelets. We utilized three-dimensional averaging procedures to study receptors at the plasma membrane. Analysis of substrate interaction-free receptors yielded one main structural class resolved to 26 Å, resembling the αIIbß3 integrin folded conformation. Furthermore, structural analysis of the actin network in pseudopodia indicates a nonuniform polarity of filaments. This organization would allow generation of the contractile forces required for integrin-mediated cell adhesion.

The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function.

Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane of metazoan cells that represent sites of attachment to - and degradation of - the extracellular matrix. The key proteins in these structures include the actin regulators cortactin and neural Wiskott-Aldrich syndrome protein (N-WASP), the adaptor proteins Tyr kinase substrate with four SH3 domains (TKS4) and Tyr kinase substrate with five SH3 domains (TKS5), and the metalloprotease membrane type 1 matrix metalloprotease (MT1MMP; also known as MMP14). Many cell types can produce these structures, including invasive cancer cells, vascular smooth muscle and endothelial cells, and immune cells such as macrophages and dendritic cells. Recently, progress has been made in our understanding of the regulatory and functional aspects of podosome and invadopodium biology and their role in human disease.

WHIMP links the actin nucleation machinery to Src-family kinase signaling during protrusion and motility.

Cell motility is governed by cooperation between the Arp2/3 complex and nucleation-promoting factors from the Wiskott-Aldrich Syndrome Protein (WASP) family, which together assemble actin filament networks to drive membrane protrusion. Here we identify WHIMP (WAVE Homology In Membrane Protrusions) as a new member of the WASP family. The Whimp gene is encoded on the X chromosome of a subset of mammals, including mice. Murine WHIMP promotes Arp2/3-dependent actin assembly, but is less potent than other nucleation factors. Nevertheless, WHIMP-mediated Arp2/3 activation enhances both plasma membrane ruffling and wound healing migration, whereas WHIMP depletion impairs protrusion and slows motility. WHIMP expression also increases Src-family kinase activity, and WHIMP-induced ruffles contain the additional nucleation-promoting factors WAVE1, WAVE2, and N-WASP, but not JMY or WASH. Perturbing the function of Src-family kinases, WAVE proteins, or Arp2/3 complex inhibits WHIMP-driven ruffling. These results suggest that WHIMP-associated actin assembly plays a direct role in membrane protrusion, but also results in feedback control of tyrosine kinase signaling to modulate the activation of multiple WASP-family members.

PlexinD1 signaling controls morphological changes and migration termination in newborn neurons.

Newborn neurons maintain a very simple, bipolar shape, while they migrate from their birthplace toward their destinations in the brain, where they differentiate into mature neurons with complex dendritic morphologies. Here, we report a mechanism by which the termination of neuronal migration is maintained in the postnatal olfactory bulb (OB). During neuronal deceleration in the OB, newborn neurons transiently extend a protrusion from the proximal part of their leading process in the resting phase, which we refer to as a filopodium-like lateral protrusion (FLP). The FLP formation is induced by PlexinD1 downregulation and local Rac1 activation, which coincide with microtubule reorganization and the pausing of somal translocation. The somal translocation of resting neurons is suppressed by microtubule polymerization within the FLP The timing of neuronal migration termination, controlled by Sema3E-PlexinD1-Rac1 signaling, influences the final positioning, dendritic patterns, and functions of the neurons in the OB These results suggest that PlexinD1 signaling controls FLP formation and the termination of neuronal migration through a precise control of microtubule dynamics.

Regulation of the apical extension morphogenesis tunes the mechanosensory response of microvilliated neurons.

Multiple types of microvilliated sensory cells exhibit an apical extension thought to be instrumental in the detection of sensory cues. The investigation of the mechanisms underlying morphogenesis of sensory apparatus is critical to understand the biology of sensation. Most of what we currently know comes from the study of the hair bundle of the inner ear sensory cells, but morphogenesis and function of other sensory microvilliated apical extensions remain poorly understood. We focused on spinal sensory neurons that contact the cerebrospinal fluid (CSF) through the projection of a microvilliated apical process in the central canal, referred to as cerebrospinal fluid-contacting neurons (CSF-cNs). CSF-cNs respond to pH and osmolarity changes as well as mechanical stimuli associated with changes of flow and tail bending. In vivo time-lapse imaging in zebrafish embryos revealed that CSF-cNs are atypical neurons that do not lose their apical attachment and form a ring of actin at the apical junctional complexes (AJCs) that they retain during differentiation. We show that the actin-based protrusions constituting the microvilliated apical extension arise and elongate from this ring of actin, and we identify candidate molecular factors underlying every step of CSF-cN morphogenesis. We demonstrate that Crumbs 1 (Crb1), Myosin 3b (Myo3b), and Espin orchestrate the morphogenesis of CSF-cN apical extension. Using calcium imaging in crb1 and espin mutants, we further show that the size of the apical extension modulates the amplitude of CSF-cN sensory response to bending of the spinal cord. Based on our results, we propose that the apical actin ring could be a common site of initiation of actin-based protrusions in microvilliated sensory cells. Furthermore, our work provides a set of actors underlying actin-based protrusion elongation shared by different sensory cell types and highlights the critical role of the apical extension shape in sensory detection.

An endocytic-secretory cycle participates in Toxoplasma gondii in motility.

Apicomplexan parasites invade host cells in an active process involving their ability to move by gliding motility. While the acto-myosin system of the parasite plays a crucial role in the formation and release of attachment sites during this process, there are still open questions regarding the involvement of other mechanisms in parasite motility. In many eukaryotes, a secretory-endocytic cycle leads to the recycling of receptors (integrins), necessary to form attachment sites, regulation of surface area during motility, and generation of retrograde membrane flow. Here, we demonstrate that endocytosis operates during gliding motility in Toxoplasma gondii and appears to be crucial for the establishment of retrograde membrane flow, because inhibition of endocytosis blocks retrograde flow and motility. We demonstrate that extracellular parasites can efficiently incorporate exogenous material, such as labelled phospholipids, nanogold particles (NGPs), antibodies, and Concanavalin A (ConA). Using labelled phospholipids, we observed that the endocytic and secretory pathways of the parasite converge, and endocytosed lipids are subsequently secreted, demonstrating the operation of an endocytic-secretory cycle. Together our data consolidate previous findings, and we propose an additional model, working in parallel to the acto-myosin motor, that reconciles parasite motility with observations in other eukaryotes: an apicomplexan fountain-flow-model for parasite motility.

Comparative mapping of crawling-cell morphodynamics in deep learning-based feature space.

Navigation of fast migrating cells such as amoeba Dictyostelium and immune cells are tightly associated with their morphologies that range from steady polarized forms that support high directionality to those more complex and variable when making frequent turns. Model simulations are essential for quantitative understanding of these features and their origins, however systematic comparisons with real data are underdeveloped. Here, by employing deep-learning-based feature extraction combined with phase-field modeling framework, we show that a low dimensional feature space for 2D migrating cell morphologies obtained from the shape stereotype of keratocytes, Dictyostelium and neutrophils can be fully mapped by an interlinked signaling network of cell-polarization and protrusion dynamics. Our analysis links the data-driven shape analysis to the underlying causalities by identifying key parameters critical for migratory morphologies both normal and aberrant under genetic and pharmacological perturbations. The results underscore the importance of deciphering self-organizing states and their interplay when characterizing morphological phenotypes.

Programming cell growth into different cluster shapes using diffusible signals.

Advances in genetic engineering technologies have allowed the construction of artificial genetic circuits, which have been used to generate spatial patterns of differential gene expression. However, the question of how cells can be programmed, and how complex the rules need to be, to achieve a desired tissue morphology has received less attention. Here, we address these questions by developing a mathematical model to study how cells can collectively grow into clusters with different structural morphologies by secreting diffusible signals that can influence cellular growth rates. We formulate how growth regulators can be used to control the formation of cellular protrusions and how the range of achievable structures scales with the number of distinct signals. We show that a single growth inhibitor is insufficient for the formation of multiple protrusions but may be achieved with multiple growth inhibitors, and that other types of signals can regulate the shape of protrusion tips. These examples illustrate how our approach could potentially be used to guide the design of regulatory circuits for achieving a desired target structure.

Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum.

Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I-III. We propose two distinct sporulation strategies used by C. botulinum Groups I-III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum.

Distinct P2Y Receptors Mediate Extension and Retraction of Microglial Processes in Epileptic and Peritumoral Human Tissue.

Microglia exhibit multiple, phenotype-dependent motility patterns often triggered by purinergic stimuli. However, little data exist on motility of human microglia in pathological situations. Here we examine motility of microglia stained with a fluorescent lectin in tissue slices from female and male epileptic patients diagnosed with mesial temporal lobe epilepsy or cortical glioma (peritumoral cortex). Microglial shape varied from ramified to amoeboid cells predominantly in regions of high neuronal loss or closer to a tumor. Live imaging revealed unstimulated or purine-induced microglial motilities, including surveillance movements, membrane ruffling, and process extension or retraction. At different concentrations, ADP triggered opposing motilities. Low doses triggered process extension. It was suppressed by P2Y12 receptor antagonists, which also reduced process length and surveillance movements. Higher purine doses caused process retraction and membrane ruffling, which were blocked by joint application of P2Y1 and P2Y13 receptor antagonists. Purinergic effects on motility were similar for all microglia tested. Both amoeboid and ramified cells from mesial temporal lobe epilepsy or peritumoral cortex tissue expressed P2Y12 receptors. A minority of microglia expressed the adenosine A2A receptor, which has been linked with process withdrawal of rodent cells. Laser-mediated tissue damage let us test the functional significance of these effects. Moderate damage induced microglial process extension, which was blocked by P2Y12 receptor antagonists. Overall, the purine-induced motility of human microglia in epileptic tissue is similar to that of rodent microglia in that the P2Y12 receptor initiates process extension. It differs in that retraction is triggered by joint activation of P2Y1/P2Y13 receptors.SIGNIFICANCE STATEMENT Microglial cells are brain-resident immune cells with multiple functions in healthy or diseased brains. These diverse functions are associated with distinct phenotypes, including different microglial shapes. In the rodent, purinergic signaling is associated with changes in cell shape, such as process extension toward tissue damage. However, there are little data on living human microglia, especially in diseased states. We developed a reliable technique to stain microglia from epileptic and glioma patients to examine responses to purines. Low-intensity purinergic stimuli induced process extension, as in rodents. In contrast, high-intensity stimuli triggered a process withdrawal mediated by both P2Y1 and P2Y13 receptors. P2Y1/P2Y13 receptor activation has not previously been linked to microglial morphological changes.

Diet regulates membrane extension and survival of niche escort cells for germline homeostasis via insulin signaling.

Diet is an important regulator of stem cell homeostasis; however, the underlying mechanisms of this regulation are not fully known. Here, we report that insulin signaling mediates dietary maintenance of Drosophila ovarian germline stem cells (GSCs) by promoting the extension of niche escort cell (EC) membranes to wrap around GSCs. This wrapping may facilitate the delivery of bone morphogenetic protein stemness factors from ECs in the niche to GSCs. In addition to the effects on GSCs, insulin signaling-mediated regulation of EC number and protrusions controls the division and growth of GSC progeny. The effects of insulin signaling on EC membrane extension are, at least in part, driven by enhanced translation of Failed axon connections (Fax) via Ribosomal protein S6 kinase. Fax is a membrane protein that may participate in Abelson tyrosine kinase-regulated cytoskeletal dynamics and is known to be involved in axon bundle formation. Therefore, we conclude that dietary cues stimulate insulin signaling in the niche to regulate EC cellular structure, probably via Fax-dependent cytoskeleton remodeling. This mechanism enhances intercellular contact and facilitates homeostatic interactions between somatic and germline cells in response to diet.

Satellite Glial Cells and Astrocytes, a Comparative Review.

Astroglia are neural cells, heterogeneous in form and function, which act as supportive elements of the central nervous system; astrocytes contribute to all aspects of neural functions in health and disease. Through their highly ramified processes, astrocytes form close physical contacts with synapses and blood vessels, and are integrated into functional syncytia by gap junctions. Astrocytes interact among themselves and with other cells types (e.g., neurons, microglia, blood vessel cells) by an elaborate repertoire of chemical messengers and receptors; astrocytes also influence neural plasticity and synaptic transmission through maintaining homeostasis of neurotransmitters, K+ buffering, synaptic isolation and control over synaptogenesis and synaptic elimination. Satellite glial cells (SGCs) are the most abundant glial cells in sensory ganglia, and are believed to play major roles in sensory functions, but so far research into SGCs attracted relatively little attention. In this review we compare SGCs to astrocytes with the purpose of using the vast knowledge on astrocytes to explore new aspects of SGCs. We survey the main properties of these two cells types and highlight similarities and differences between them. We conclude that despite the much greater diversity in morphology and signaling mechanisms of astrocytes, there are some parallels between them and SGCs. Both types serve as boundary cells, separating different compartments in the nervous system, but much more needs to be learned on this aspect of SGCs. Astrocytes and SGCs employ chemical messengers and calcium waves for intercellular signaling, but their significance is still poorly understood for both cell types. Both types undergo major changes under pathological conditions, which have a protective function, but an also contribute to disease, and chronic pain in particular. The knowledge obtained on astrocytes is likely to benefit future research on SGCs.

Visualizing bleb mass dynamics in single cells using quantitative phase microscopy.

Understanding biological responses to directed energy (DE) is critical to ensure the safety of personnel within the Department of Defense. At the Air Force Research Laboratory, we have developed or adapted advanced optical imaging systems that quantify biophysical responses to DE. One notable cellular response to DE exposure is the formation of blebs, or semi-spherical protrusions of the plasma membrane in living cells. In this work, we demonstrate the capacity of quantitative phase imaging (QPI) to both visualize and quantify the formation of membrane blebs following DE exposure. QPI is an interferometric imaging tool that uses optical path length as a label-free contrast mechanism and is sensitive to the non-aqueous mass density, or dry mass, of living cells. Blebs from both CHO-K1 and U937 cells were generated after exposure to a series of 600 ns, 21.2 kV/cm electric pulses. These blebs were visualized in real time, and their dry mass relative to the rest of the cell body was quantified as a function of time. It is our hope that this system will lead to an improved understanding of both DE-induced and apoptotic blebbing.

Coexistence of Two Chiral Helices Produces Kink Translation in Spiroplasma Swimming.

The mechanism underlying Spiroplasma swimming is an enigma. This small bacterium possesses two helical shapes with opposite-handedness at a time, and the boundary between them, called a kink, travels down, possibly accompanying the dual rotations of these physically connected helical structures, without any rotary motors such as flagella. Although the outline of dynamics and structural basis has been proposed, the underlying cause to explain the kink translation is missing. We here demonstrated that the cell morphology of Spiroplasma eriocheiris was fixed at the right-handed helix after motility was stopped by the addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the preferential state was transformed to the other-handedness by the trigger of light irradiation. This process coupled with the generation and propagation of the artificial kink, presumably without any energy input through biological motors. These findings indicate that the coexistence of two chiral helices is sufficient to propagate the kink and thus to propel the cell body.IMPORTANCE Many swimming bacteria generate a propulsion force by rotating helical filaments like a propeller. However, the nonflagellated bacteria Spiroplasma spp. swim without the use of the appendages. The tiny wall-less bacteria possess two chiral helices at a time, and the boundary called a kink travels down, possibly accompanying the dual rotations of the helices. To solve this enigma, we developed an assay to determine the handedness of the body helices at the single-wind level, and demonstrated that the coexistence of body helices triggers the translation of the kink and that the cell body moves by the resultant cell bend propagation. This finding provides us a totally new aspect of bacterial motility, where the body functions as a transformable screw to propel itself forward.

Hydrodynamics of transient cell-cell contact: The role of membrane permeability and active protrusion length.

In many biological settings, two or more cells come into physical contact to form a cell-cell interface. In some cases, the cell-cell contact must be transient, forming on timescales of seconds. One example is offered by the T cell, an immune cell which must attach to the surface of other cells in order to decipher information about disease. The aspect ratio of these interfaces (tens of nanometers thick and tens of micrometers in diameter) puts them into the thin-layer limit, or "lubrication limit", of fluid dynamics. A key question is how the receptors and ligands on opposing cells come into contact. What are the relative roles of thermal undulations of the plasma membrane and deterministic forces from active filopodia? We use a computational fluid dynamics algorithm capable of simulating 10-nanometer-scale fluid-structure interactions with thermal fluctuations up to seconds- and microns-scales. We use this to simulate two opposing membranes, variously including thermal fluctuations, active forces, and membrane permeability. In some regimes dominated by thermal fluctuations, proximity is a rare event, which we capture by computing mean first-passage times using a Weighted Ensemble rare-event computational method. Our results demonstrate a parameter regime in which the time it takes for an active force to drive local contact actually increases if the cells are being held closer together (e.g., by nonspecific adhesion), a phenomenon we attribute to the thin-layer effect. This leads to an optimal initial cell-cell separation for fastest receptor-ligand binding, which could have relevance for the role of cellular protrusions like microvilli. We reproduce a previous experimental observation that fluctuation spatial scales are largely unaffected, but timescales are dramatically slowed, by the thin-layer effect. We also find that membrane permeability would need to be above physiological levels to abrogate the thin-layer effect.

Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy.

Well-coordinated activation of all cardiomyocytes must occur on every heartbeat. At the cell level, a complex network of sarcolemmal invaginations, called the transverse-axial tubular system (TATS), propagates membrane potential changes to the cell core, ensuring synchronous and uniform excitation-contraction coupling. Although myocardial conduction of excitation has been widely described, the electrical properties of the TATS remain mostly unknown. Here, we exploit the formal analogy between diffusion and electrical conductivity to link the latter with the diffusional properties of TATS. Fluorescence recovery after photobleaching (FRAP) microscopy is used to probe the diffusion properties of TATS in isolated rat cardiomyocytes: A fluorescent dextran inside TATS lumen is photobleached, and signal recovery by diffusion of unbleached dextran from the extracellular space is monitored. We designed a mathematical model to correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the fluorescent molecules. Then, apparent diffusion is linked to electrical conductivity and used to evaluate the efficiency of the passive spread of membrane depolarization along TATS. The method is first validated in cells where most TATS elements are acutely detached by osmotic shock and then applied to probe TATS electrical conductivity in failing heart cells. We find that acute and pathological tubular remodeling significantly affect TATS electrical conductivity. This may explain the occurrence of defects in action potential propagation at the level of single T-tubules, recently observed in diseased cardiomyocytes.

EB1 and cytoplasmic dynein mediate protrusion dynamics for efficient 3-dimensional cell migration.

Microtubules have long been implicated to play an integral role in metastatic disease, for which a critical step is the local invasion of tumor cells into the 3-dimensional (3D) collagen-rich stromal matrix. Here we show that cell migration of human cancer cells uses the dynamic formation of highly branched protrusions that are composed of a microtubule core surrounded by cortical actin, a cytoskeletal organization that is absent in cells on 2-dimensional (2D) substrates. Microtubule plus-end tracking protein End-binding 1 and motor protein dynein subunits light intermediate chain 2 and heavy chain 1, which do not regulate 2D migration, critically modulate 3D migration by affecting RhoA and thus regulate protrusion branching through differential assembly dynamics of microtubules. An important consequence of this observation is that the commonly used cancer drug paclitaxel is 100-fold more effective at blocking migration in a 3D matrix than on a 2D matrix. This work reveals the central role that microtubule dynamics plays in powering cell migration in a more pathologically relevant setting and suggests further testing of therapeutics targeting microtubules to mitigate migration.-Jayatilaka, H., Giri, A., Karl, M., Aifuwa, I., Trenton, N. J., Phillip, J. M., Khatau, S., Wirtz, D. EB1 and cytoplasmic dynein mediate protrusion dynamics for efficient 3-dimensional cell migration.

Morphology of the archaellar motor and associated cytoplasmic cone in Thermococcus kodakaraensis.

Archaeal swimming motility is driven by archaella: rotary motors attached to long extracellular filaments. The structure of these motors, and particularly how they are anchored in the absence of a peptidoglycan cell wall, is unknown. Here, we use electron cryotomography to visualize the archaellar basal body in vivo in Thermococcus kodakaraensis KOD1. Compared to the homologous bacterial type IV pilus (T4P), we observe structural similarities as well as several unique features. While the position of the cytoplasmic ATPase appears conserved, it is not braced by linkages that extend upward through the cell envelope as in the T4P, but rather by cytoplasmic components that attach it to a large conical frustum up to 500 nm in diameter at its base. In addition to anchoring the lophotrichous bundle of archaella, the conical frustum associates with chemosensory arrays and ribosome-excluding material and may function as a polar organizing center for the coccoid cells.

Regulation of ECM degradation and axon guidance by growth cone invadosomes.

Invadopodia and podosomes, collectively referred to as invadosomes, are F-actin-rich basal protrusions of cells that provide sites of attachment to and degradation of the extracellular matrix. Invadosomes promote the invasion of cells, ranging from metastatic cancer cells to immune cells, into tissue. Here, we show that neuronal growth cones form protrusions that share molecular, structural and functional characteristics of invadosomes. Growth cones from all neuron types and species examined, including a variety of human neurons, form invadosomes both in vitro and in vivo. Growth cone invadosomes contain dynamic F-actin and several actin regulatory proteins, as well as Tks5 and matrix metalloproteinases, which locally degrade the matrix. When viewed using three-dimensional super-resolution microscopy, F-actin foci often extended together with microtubules within orthogonal protrusions emanating from the growth cone central domain. Finally, inhibiting the function of Tks5 both reduced matrix degradation in vitro and disrupted motoneuron axons from exiting the spinal cord and extending into the periphery. Taken together, our results suggest that growth cones use invadosomes to target protease activity during axon guidance through tissues.

Fragility of foot process morphology in kidney podocytes arises from chaotic spatial propagation of cytoskeletal instability.

Kidney podocytes' function depends on fingerlike projections (foot processes) that interdigitate with those from neighboring cells to form the glomerular filtration barrier. The integrity of the barrier depends on spatial control of dynamics of actin cytoskeleton in the foot processes. We determined how imbalances in regulation of actin cytoskeletal dynamics could result in pathological morphology. We obtained 3-D electron microscopy images of podocytes and used quantitative features to build dynamical models to investigate how regulation of actin dynamics within foot processes controls local morphology. We find that imbalances in regulation of actin bundling lead to chaotic spatial patterns that could impair the foot process morphology. Simulation results are consistent with experimental observations for cytoskeletal reconfiguration through dysregulated RhoA or Rac1, and they predict compensatory mechanisms for biochemical stability. We conclude that podocyte morphology, optimized for filtration, is intrinsically fragile, whereby local transient biochemical imbalances may lead to permanent morphological changes associated with pathophysiology.