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The cell cortex is a dynamic assembly formed by the plasma membrane and underlying cytoskeleton. As the main determinant of cell shape, the cortex ensures its integrity during passive and active deformations by adapting cytoskeleton topologies through yet poorly understood mechanisms. The spectrin meshwork ensures such adaptation in erythrocytes and neurons by adopting different organizations. Erythrocytes rely on triangular-like lattices of spectrin tetramers, whereas in neurons they are organized in parallel, periodic arrays. Since spectrin is ubiquitously expressed, we exploited Expansion Microscopy to discover that, in fibroblasts, distinct meshwork densities co-exist. Through biophysical measurements and computational modeling, we show that the non-polarized spectrin meshwork, with the intervention of actomyosin, can dynamically transition into polarized clusters fenced by actin stress fibers that resemble periodic arrays as found in neurons. Clusters experience lower mechanical stress and turnover, despite displaying an extension close to the tetramer contour length. Our study sheds light on the adaptive properties of spectrin, which participates in the protection of the cell cortex by varying its densities in response to key mechanical features.

Background: Changes in epithelial cell shape reflects optimal cell packing and the minimization of surface free energy, but also cell–cell interactions, cell proliferation, and cytoskeletal rearrangements. Results: Here, we studied the structure of the rat pleura in the first 15 days after birth. After pleural isolation and image segmentation, the analysis demonstrated a progression of epithelial order from postnatal day 1 (P1) to P15. The cells with the largest surface area and greatest shape variability were observed at P1. In contrast, the cells with the smallest surface area and most shape consistency were observed at P15. A comparison of polygonal cell geometries demonstrated progressive optimization with an increase in the number of hexagons (six‐sided) as well as five‐sided and seven‐sided polygons. Analysis of the epithelial organization with Voronoi tessellations and graphlet motif frequencies demonstrated a developmental path strikingly distinct from mathematical and natural reference paths. Graph Theory analysis of cell connectivity demonstrated a progressive decrease in network heterogeneity and clustering coefficient from P1 to P15. Conclusions: We conclude that the rat pleura undergoes a striking change in pleural structure from P1 to P15. Further, a geometric and network‐based approach can provide a quantitative characterization of these developmental changes. Key Findings: Evolving topological order of the pleura within the first two weeks after birth:decrease in shape variabilityincrease in 6‐sided polygonsdecrease in network heterogeneity [ABSTRACT FROM AUTHOR]

At low pH, red blood cells (RBCs) are unable to maintain their disc shape and become stomatocytes. At the same time, at pH 5.0 of a sulphate medium, under conditions of mechanical mixing of the cell suspension, RBCs are partially retransformed into discoid forms, whereas in a chloride medium (pH 5.0) this transformation is observed to a high degree. This indicates that the chloride-binding sites of RBC membranes may be an additional link in the regulation of the shape of RBC under conditions of mechanical action on the cell suspension. The work investigated the change in the RBC shape at a normal pH value (7,4) due to the replacement of Na2SO 4 in the medium with NaCl and recording an increase in the intensity of fluctuations in optical density (OD), as an indicator of an increase in the level of discoid cells (normocytes) in a stirred cell suspension. It has been 3 established that in a medium containing Na2SO4 (110 mmol/l), RBCs are transformed into small stomatocytes within ~20 s. With an increase in NaCl concentration in the range of 15-105 mmol/l, an increase in the level of discoid cells is noted. Increasing the NaCl concentration from 105 to 150 mmol/l, on the contrary, causes a decrease in the level of discoid cells. With the exception of mechanical mixing in a medium with NaCl concentrations of 30-90 mmol/l, RBCs are morphologically stomatocytes. Whereas in a medium containing 150 mmol/l NaCl, RBC are represented by disc-echinocytes and echinocytes. The results obtained show that mechanical mixing of the cell suspension promotes the retransformation of stomatocytic RBCs into discoid forms. The weakening of the retransforming efficiency of mixing with an increase in the NaCl concentration in the medium from 105 to 150 mmol/l and the development of echinocytosis at 150 mmol/l NaCl indicates the existence of 2 types of sites for chloride. Сhloride binding to type 1, at a NaCl concentration in the medium of 15-105 mmol/l, leads to the establishment of discoid forms of RBC. Chloride binding to type 2 sites, at a NaCl concentration in the medium of 105-150 mmol/l, leads to the development of echinocytosis. The retransforming property of mechanical stirring may be associated with a change in the degree of chloride binding to these types of sites. [ABSTRACT FROM AUTHOR]

Controlling cell-substrate interactions via the microstructural characteristics of biomaterials offers an advantageous path for modulating cell dynamics, mechanosensing, and migration, as well as for designing immune-modulating implants, all without the drawbacks of chemical-based triggers. Specifically, recent in vivo studies have suggested that a porous implants microscale curvature landscape can significantly impact cell behavior and ultimately the immune response. To investigate such cell-substrate interactions, we utilized a 3D computational model incorporating the minimum necessary physics of cell migration and cell-substrate interactions needed to replicate known in vitro behaviors. This model specifically incorporates the effect of membrane tension, which was found to be necessary to replicate in vitro cell behavior on curved surfaces. Our simulated substrates represent two classes of porous materials recently used in implant studies, which have markedly different microscale curvature distributions and pore geometries. We found distinct differences between the overall migration behaviors, shapes, and actin polymerization dynamics of cells interacting with the two substrates. These differences were correlated to the shape energy of the cells as they interacted with the porous substrates, in effect interpreting substrate topography as an energetic landscape interrogated by cells. Our results demonstrate that microscale curvature directly influences cell shape and migration and, therefore, is likely to influence cell behavior. This supports further investigation of the relationship between the surface topography of implanted materials and the characteristic immune response, a complete understanding of which would broadly advance principles of biomaterial design.

Abstract Mesotrypsin, encoded by the PRSS3 gene, is a distinctive trypsin isoform renowned for its exceptional resistance to traditional trypsin inhibitors and unique substrate specificity. Within the skin epidermis, this protein primarily expresses in the upper layers of the stratified epidermis and plays a crucial role in processing pro-filaggrin (Pro-FLG). Although prior studies have partially elucidated its functions using primary cultured keratinocytes, challenges persist due to these cells' differentiation-activated cell death program. In the present study, HaCaT keratinocytes, characterized by minimal endogenous mesotrypsin expression and sustained proliferation in differentiated states, were utilized to further scrutinize the function of mesotrypsin. Despite the ready degradation of the intact form of active mesotrypsin in these cells, fusion with Venus, flanked by a peptide linker, enables evasion from the protein elimination machinery, thus facilitating activation of the Pro-FLG processing system. Inducing Venus-mesotrypsin expression in the cells resulted in a flattened phenotype and reduced proliferative capacity. Moreover, these cells displayed altered F-actin assembly, enhanced E-cadherin adhesive activity, and facilitated tight junction formation without overtly influencing epidermal differentiation. These findings underscore mesotrypsin's potentially pivotal role in shaping the characteristic cellular morphology of upper epidermal layers.

At present, selective laser melting (SLM) 3D printing technology can accurately control the internal pore structure and complex cell shape. Three types of reticulated meshes with cubic, G7 and composite structure cell shapes were fabricated by the SLM 3D printing technology using Ti–6Al–4V alloy powders. The bone stresses around the implant and the stresses in the implant were analyzed by ANSYS finite element software, which comprehensively evaluated the effect of porous dental implants with different spatial porosity characteristics on osseointegration. The results show that porous dental implants with composite structure of pore characteristics have improved mechanical and biological properties and can better promote the growth and integration of bone tissue. [ABSTRACT FROM AUTHOR]

Metabolites have to diffuse within the sub-cellular compartments they occupy to specific locations where enzymes are, so reactions could occur. Conventional flux balance analysis (FBA), a method based on linear programming that is commonly used to model metabolism, implicitly assumes that all enzymatic reactions are not diffusion-limited though that may not always be the case. In this work, we have developed a spatial method that implements FBA on a grid-based system, to enable the exploration of diffusion effects on metabolism. Specifically, the method discretises a living cell into a two-dimensional grid, represents the metabolic reactions in each grid element as well as the diffusion of metabolites to and from neighbouring elements, and simulates the system as a single linear programming problem. We varied the number of rows and columns in the grid to simulate different cell shapes, and the method was able to capture diffusion effects at different shapes. We then used the method to simulate heterogeneous enzyme distribution, which suggested a theoretical effect on variability at the population level. We propose the use of this method, and its future extensions, to explore how spatiotemporal organisation of sub-cellular compartments and the molecules within could affect cell behaviour. [ABSTRACT FROM AUTHOR]

ABSTRACT Within the eukaryotic cell, the actin cytoskeleton is a crucial structural framework that maintains cellular form, regulates cell movement and division, and facilitates the internal transportation of proteins and organelles. External cues induce alterations in the actin cytoskeleton primarily through the activation of Rho GTPases, which then bind to a diverse array of effector proteins to promote the local assembly or disassembly of actin. We have harnessed the extensively studied functions of RhoA in the dynamics of the actin cytoskeleton to craft a practical series for Stage 2 Biology students. This series not only imparts essential tissue culture laboratory skills but also reinforces them through repetition. These activities are presented in a scenario designed for students to explore the function of a hypothetical RhoA family member. Students produce slides from transfected cells, undertake fluorescence microscopy, process the images using ImageJ, and compile their findings in a comprehensive scientific report. The composition of the report requires independent acquisition of new knowledge and synoptic learning. According to student feedback, this early experience greatly aids in solidifying and honing the skills required to report on more extensive and intricate research projects, such as capstone projects.

The protein crescentin is required for the crescent shape of the freshwater bacterium Caulobacter crescentus (vibrioides). Crescentin forms a filamentous structure on the inner, concave side of the curved cells. It shares features with eukaryotic intermediate filament (IF) proteins, including the formation of static filaments based on long and parallel coiled coils, the protein's length, structural roles in cell and organelle shape determination and the presence of a coiled coil discontinuity called the "stutter." Here, we have used electron cryomicroscopy (cryo-EM) to determine the structure of the full-length protein and its filament, exploiting a crescentin-specific nanobody. The filament is formed by two strands, related by twofold symmetry, that each consist of two dimers, resulting in an octameric assembly. Crescentin subunits form longitudinal contacts head-to-head and tail-to-tail, making the entire filament non-polar. Using in vivo site-directed cysteine cross-linking, we demonstrated that contacts observed in the in vitro filament structure exist in cells. Electron cryotomography (cryo-ET) of cells expressing crescentin showed filaments on the concave side of the curved cells, close to the inner membrane, where they form a band. When comparing with current models of IF proteins and their filaments, which are also built from parallel coiled coil dimers and lack overall polarity, it emerges that IF proteins form head-to-tail longitudinal contacts in contrast to crescentin and hence several inter-dimer contacts in IFs have no equivalents in crescentin filaments. Our work supports the idea that intermediate filament-like proteins achieve their shared polymerization and mechanical properties through a variety of filament architectures. [ABSTRACT FROM AUTHOR]

The spheroidal shape of the eye lens is crucial for precise light focusing onto the retina. This shape is determined by concentrically aligned, convexly elongated lens fiber cells along the anterior and posterior axis of the lens. Upon differentiation at the lens equator, the fiber cells increase in height as their apical and basal tips migrate towards the anterior and posterior poles, respectively. The forces driving this elongation and migration remain unclear. We found that, in the mouse lens, membrane protrusions or lamellipodia are observed only in the maturing fibers undergoing cell curve conversion, indicating that lamellipodium formation is not the primary driver of earlier fiber migration. We demonstrated that elevated levels of fibroblast growth factor (FGF) suppressed the extension of Racdependent protrusions, suggesting changes in the activity of FGF controlling Rac activity, switching to lamellipodium-driven migration. Inhibitors of ROCK, myosin and actin reduced the height of both early and later fibers, indicating that elongation of these fibers relies on actomyosin contractility. Consistent with this, active RhoA was detected throughout these fibers. Given that FGF promotes fiber elongation, we propose that it does so through regulation of Rho activity. [ABSTRACT FROM AUTHOR]

There is growing evidence that bacterial morphology is closely related to their lifestyle. The helical Helicobacter pylori relies on its unique shape for survival and efficient colonization of the human stomach. Yet, they have been observed to transform into another distinctive morphology, the spherical coccoid. Despite being hypothesized to be involved in the persistence and transmission of this species, years of effort in deciphering the roles of the coccoid form remain fruitless since contrasting observations regarding its lifestyle were reported. Here, we discuss the two forms of H. pylori with a focus on the coccoid form, the molecular mechanism behind its morphological transformation, and experimental approaches to further develop our understanding of this phenomenon. We also propose a putative mechanism of the coccoid formation in H. pylori through induction of a type‐I toxin‐antitoxin (TA) system recently shown to influence the morphology of this species. [ABSTRACT FROM AUTHOR]

Introduction: The last decade has led to rapid developments and increased usage of computational tools at the single-cell level. However, our knowledge remains limited in how extracellular cues alter quantitative macrophage morphology and how such morphological changes can be used to predict macrophage phenotype as well as cytokine content at the single-cell level. Methods: Using an artificial intelligence (AI) based approach, this study determined whether (i) accurate macrophage classification and (ii) prediction of intracellular IL-10 at the single-cell level was possible, using only morphological features as predictors for AI. Using a quantitative panel of shape descriptors, our study assessed image-based original and synthetic single-cell data in two different datasets in which CD14+ monocyte-derived macrophages generated from human peripheral blood monocytes were initially primed with GM-CSF or M-CSF followed by polarization with specific stimuli in the presence/ absence of continuous GM-CSF or M-CSF. Specifically, M0, M1 (GM-CSF-M1, TNFα/IFNγ-M1, GM-CSF/TNFα/IFNγ-M1) and M2 (M-CSF-M2, IL-4-M2a, M-CSF/ IL-4-M2a, IL-10-M2c, M-CSF/IL-10-M2c) macrophages were examined. Results: Phenotypes were confirmed by ELISA and immunostaining of CD markers. Variations of polarization techniques significantly changed multiple macrophage morphological features, demonstrating that macrophage morphology is a highly sensitive, dynamic marker of phenotype. Using original and synthetic single-cell data, cell morphology alone yielded an accuracy of 93% for the classification of 6 different human macrophage phenotypes (with continuous GM-CSF or M-CSF). A similarly high phenotype classification accuracy of 95% was reached with data generated with different stimuli (discontinuous GM-CSF or M-CSF) and measured at a different time point. These comparably high accuracies clearly validated the here chosen AI-based approach. Quantitative morphology also allowed prediction of intracellular IL-10 with 95% accuracy using only original data. Discussion: Thus, image-based machine learning using morphology-based features not only (i) classified M0, M1 and M2 macrophages but also (ii) classified M2a and M2c subtypes and (iii) predicted intracellular IL-10 at the single-cell level among six phenotypes. This simple approach can be used as a general strategy not only for macrophage phenotyping but also for prediction of IL-10 content of any IL-10 producing cell, which can help improve our understanding of cytokine biology at the single-cell level. [ABSTRACT FROM AUTHOR]

Premise: Leaf epidermal cell morphology is closely tied to the evolutionary history of plants and their growth environments and is therefore of interest to many plant biologists. However, cell measurement can be time consuming and restrictive with current methods. CuticleTrace is a suite of Fiji and R‐based functions that streamlines and automates the segmentation and measurement of epidermal pavement cells across a wide range of cell morphologies and image qualities. Methods and Results: We evaluated CuticleTrace‐generated measurements against those from alternate automated methods and expert and undergraduate hand tracings across a taxonomically diverse 50‐image data set of variable image qualities. We observed ~93% statistical agreement between CuticleTrace and expert hand‐traced measurements, outperforming alternate methods. Conclusions: CuticleTrace is a broadly applicable, modular, and customizable tool that integrates data visualization and cell shape measurement with image segmentation, lowering the barrier to high‐throughput studies of epidermal morphology by vastly decreasing the labor investment required to generate high‐quality cell shape data sets. [ABSTRACT FROM AUTHOR]

Tissue swelling represents an early sign of osteoarthritis, reflecting osmolarity changes from iso‐ to hypo‐osmotic in the diseased joints. Increased tissue hydration may drive cell swelling. The opposing cartilages in a joint may swell differently, thereby predisposing the more swollen cartilage and cells to mechanical injuries. However, our understanding of the tissue–cell interdependence in osmotically loaded joints is limited as tissue and cell swellings have been studied separately. Here, we measured tissue and cell responses of opposing patellar (PAT) and femoral groove (FG) cartilages in lapine knees exposed to an extreme hypo‐osmotic challenge. We found that the tissue matrix and most cells swelled during the hypo‐osmotic challenge, but to a different extent (tissue: <3%, cells: 11%–15%). Swelling‐induced tissue strains were anisotropic, showing 2%–4% stretch and 1%–2% compression along the first and third principal directions, respectively. These strains were amplified by 5–8 times in the cells. Interestingly, the first principal strains of tissue and cells occurred in different directions (60–61° for tissue vs. 8–13° for cells), suggesting different mechanisms causing volume expansion in the tissue and the cells. Instead of the continuous swelling observed in the tissue matrix, >88% of cells underwent regulatory volume decrease to return to their pre‐osmotic challenge volumes. Cell shapes changed in the early phase of swelling but stayed constant thereafter. Kinematic changes to tissue and cells were larger for PAT cartilage than for FG cartilage. We conclude that the swelling‐induced deformation of tissue and cells is anisotropic. Cells actively restored volume independent of the surrounding tissues and seemed to prioritize volume restoration over shape restoration. Our findings shed light on tissue–cell interdependence in changing osmotic environments that is crucial for cell mechano‐transduction in swollen/diseased tissues. [ABSTRACT FROM AUTHOR]

The mesophyll cells of grass leaves, such as rice, are traditionally viewed as displaying a relatively uniform pattern, in contrast to the clear distinctions of palisade and spongy layers in typical eudicot leaves. This quantitative analysis of mesophyll cell size and shape in rice leaves reveals that there is an inherent pattern in which cells in the middle layer of the mesophyll are larger and less circular and have a distinct orientation of their long axis compared to mesophyll cells in other layers. Moreover, this pattern was observed in a range of rice cultivars and species. The significance of this pattern with relation to potential photosynthetic function and the implication of the widespread use of middle layer mesophyll cells as typical of the rice leaf have been investigated and discussed. [ABSTRACT FROM AUTHOR]

Mesothelial cells have been shown to have remarkable plasticity towards mesenchymal cell types during development and in disease situations. Here, we have characterized the potential of mesothelial cells to undergo changes toward perivascular cells using an in vitro angiogenesis assay. We demonstrate that GFP-labeled mesothelial cells (GFP-MCs) aligned closely and specifically with endothelial networks formed when human dermal microvascular endothelial cells (HDMECs) were cultured in the presence of VEGF-A165 on normal human dermal fibroblasts (NHDFs) for a 7-day period. The co-culture with GFP-MCs had a positive effect on branch point formation indicating that the cells supported endothelial tube formation. We interrogated the molecular response of the GFP-MCs to the angiogenic co-culture by qRT-PCR and found that the pericyte marker Ng2 was upregulated when the cells were co-cultured with HDMECs on NHDFs, indicating a change towards a perivascular phenotype. When GFP-MCs were cultured on the NHDF feeder layer, they upregulated the epithelial–mesenchymal transition marker Zeb1 and lost their circularity while increasing their size, indicating a change to a more migratory cell type. We analyzed the pericyte-like behavior of the GFP-MCs in a 3D cardiac microtissue (spheroid) with cardiomyocytes, cardiac fibroblasts and cardiac endothelial cells where the mesothelial cells showed alignment with the endothelial cells. These results indicate that mesothelial cells have the potential to adopt a perivascular phenotype and associate with endothelial cells to potentially support angiogenesis. [ABSTRACT FROM AUTHOR]

Haloferax volcanii and other Haloarchaea can be pleomorphic, adopting different shapes, which vary with growth stages. Several studies have shown that H. volcanii cell shape is sensitive to various external factors including growth media and physical environment. In addition, several studies have noticed that the presence of a recombinant plasmid in the cells is also a factor impacting H. volcanii cell shape, notably by favoring the development of rods in early stages of growth. Here we investigated the reasons for this phenomenon by first studying the impact of auxotrophic mutations on cell shape in strains that are commonly used as genetic backgrounds for selection during strain engineering (namely: H26, H53, H77, H98, and H729) and secondly, by studying the effect of the presence of different plasmids containing selection markers on the cell shape of these strains. Our study showed that most of these auxotrophic strains have variation in cell shape parameters including length, aspect ratio, area and circularity and that the plasmid presence is impacting these parameters too. Our results indicated that ΔhdrB strains and hdrB selection markers have the most influence on H. volcanii cell shape, in addition to the sole presence of a plasmid. Finally, we discuss limitations in studying cell shape in H. volcanii and make recommendations based on our results for improving reproducibility of such studies. [ABSTRACT FROM AUTHOR]

Abstract Premise Leaf epidermal cell morphology is closely tied to the evolutionary history of plants and their growth environments and is therefore of interest to many plant biologists. However, cell measurement can be time consuming and restrictive with current methods. CuticleTrace is a suite of Fiji and R‐based functions that streamlines and automates the segmentation and measurement of epidermal pavement cells across a wide range of cell morphologies and image qualities. Methods and Results We evaluated CuticleTrace‐generated measurements against those from alternate automated methods and expert and undergraduate hand tracings across a taxonomically diverse 50‐image data set of variable image qualities. We observed ~93% statistical agreement between CuticleTrace and expert hand‐traced measurements, outperforming alternate methods. Conclusions CuticleTrace is a broadly applicable, modular, and customizable tool that integrates data visualization and cell shape measurement with image segmentation, lowering the barrier to high‐throughput studies of epidermal morphology by vastly decreasing the labor investment required to generate high‐quality cell shape data sets.

Bactofilins have emerged as a widespread family of cytoskeletal proteins with important roles in bacterial morphogenesis, but their precise mode of action is still incompletely understood. In this study, we identify the bactofilin cytoskeleton as a key regulator of cell growth in the stalked budding alphaproteobacterium Hyphomonas neptunium. We show that, in this species, bactofilin polymers localize dynamically to the stalk base and the bud neck, with their absence leading to unconstrained growth of the stalk and bud compartments, indicating a central role in the spatial regulation of cell wall biosynthesis. Database searches reveal that bactofilin genes are often clustered with genes for cell wall hydrolases of the M23 peptidase family, suggesting a functional connection between these two types of proteins. In support of this notion, we find that the H. neptunium M23 peptidase homolog LmdC interacts directly with bactofilin in vitro and is required for proper cell shape in vivo. Complementary studies in the spiral-shaped alphaproteobacterium Rhodospirillum rubrum again reveal a close association of its bactofilin and LmdC homologs, which co-localize at the inner curve of the cell, modulating the degree of cell curvature. Collectively, these findings demonstrate that bactofilins and M23 peptidases form a conserved functional module that promotes local changes in the mode of cell wall biosynthesis, thereby driving cell shape determination in morphologically complex bacteria.

IntroductionThe last decade has led to rapid developments and increased usage of computational tools at the single-cell level. However, our knowledge remains limited in how extracellular cues alter quantitative macrophage morphology and how such morphological changes can be used to predict macrophage phenotype as well as cytokine content at the single-cell level.MethodsUsing an artificial intelligence (AI) based approach, this study determined whether (i) accurate macrophage classification and (ii) prediction of intracellular IL-10 at the single-cell level was possible, using only morphological features as predictors for AI. Using a quantitative panel of shape descriptors, our study assessed image-based original and synthetic single-cell data in two different datasets in which CD14+ monocyte-derived macrophages generated from human peripheral blood monocytes were initially primed with GM-CSF or M-CSF followed by polarization with specific stimuli in the presence/absence of continuous GM-CSF or M-CSF. Specifically, M0, M1 (GM-CSF-M1, TNFα/IFNγ-M1, GM-CSF/TNFα/IFNγ-M1) and M2 (M-CSF-M2, IL-4-M2a, M-CSF/IL-4-M2a, IL-10-M2c, M-CSF/IL-10-M2c) macrophages were examined.ResultsPhenotypes were confirmed by ELISA and immunostaining of CD markers. Variations of polarization techniques significantly changed multiple macrophage morphological features, demonstrating that macrophage morphology is a highly sensitive, dynamic marker of phenotype. Using original and synthetic single-cell data, cell morphology alone yielded an accuracy of 93% for the classification of 6 different human macrophage phenotypes (with continuous GM-CSF or M-CSF). A similarly high phenotype classification accuracy of 95% was reached with data generated with different stimuli (discontinuous GM-CSF or M-CSF) and measured at a different time point. These comparably high accuracies clearly validated the here chosen AI-based approach. Quantitative morphology also allowed prediction of intracellular IL-10 with 95% accuracy using only original data.DiscussionThus, image-based machine learning using morphology-based features not only (i) classified M0, M1 and M2 macrophages but also (ii) classified M2a and M2c subtypes and (iii) predicted intracellular IL-10 at the single-cell level among six phenotypes. This simple approach can be used as a general strategy not only for macrophage phenotyping but also for prediction of IL-10 content of any IL-10 producing cell, which can help improve our understanding of cytokine biology at the single-cell level.

TMEM132D is a human gene identified with multiple risk alleles for panic disorders, anxiety and major depressive disorders. Defining a conserved family of transmembrane proteins, TMEM132D and its homologs are still of unknown molecular functions. By generating loss-of-function mutants of the sole TMEM132 ortholog in C. elegans, we identify abnormal morphologic phenotypes in the dopaminergic PDE neurons. Using a yeast two-hybrid screen, we find that NAP1 directly interacts with the cytoplasmic domain of human TMEM132D, and mutations in C. elegans tmem-132 that disrupt interaction with NAP1 cause similar morphologic defects in the PDE neurons. NAP1 is a component of the WAVE regulatory complex (WRC) that controls F-actin cytoskeletal dynamics. Decreasing activity of WRC rescues the PDE defects in tmem-132 mutants, whereas gain-of-function of TMEM132D in mammalian cells inhibits WRC, leading to decreased abundance of select WRC components, impaired actin nucleation and cell motility. We propose that metazoan TMEM132 family proteins play evolutionarily conserved roles in regulating NAP1 protein homologs to restrict inappropriate WRC activity, cytoskeletal and morphologic changes in the cell.

How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. Here we investigate how the WAVE complex organizes sheet-like lamellipodia. Using super-resolution microscopy, we find that the WAVE complex forms actin-independent 230-nm-wide rings that localize to regions of saddle membrane curvature. This pattern of enrichment could explain several emergent cell behaviors, such as expanding and self-straightening lamellipodia and the ability of endothelial cells to recognize and seal transcellular holes. The WAVE complex recruits IRSp53 to sites of saddle curvature but does not depend on IRSp53 for its own localization. Although the WAVE complex stimulates actin nucleation via the Arp2/3 complex, sheet-like protrusions are still observed in ARP2-null, but not WAVE complex-null, cells. Therefore, the WAVE complex has additional roles in cell morphogenesis beyond Arp2/3 complex activation. Our work defines organizing principles of the WAVE complex lamellipodial template and suggests how feedback between cell shape and actin regulators instructs cell morphogenesis.

Abstract The mesophyll cells of grass leaves, such as rice, are traditionally viewed as displaying a relatively uniform pattern, in contrast to the clear distinctions of palisade and spongy layers in typical eudicot leaves. This quantitative analysis of mesophyll cell size and shape in rice leaves reveals that there is an inherent pattern in which cells in the middle layer of the mesophyll are larger and less circular and have a distinct orientation of their long axis compared to mesophyll cells in other layers. Moreover, this pattern was observed in a range of rice cultivars and species. The significance of this pattern with relation to potential photosynthetic function and the implication of the widespread use of middle layer mesophyll cells as typical of the rice leaf have been investigated and discussed.

ABSTRACTIn the microbial world, cell size and shape impact physiology, but students struggle to visualize spatial relationships between cells and macromolecules. In prokaryotic cells, cell size is limited by reliance on diffusion for nutrient uptake and the transport of nutrients within the cell. Cells must also meet a minimum size threshold to accommodate essential cellular components such as ribosomes and DNA. Using 3D printing allows for the creation of custom models that can be influential teaching tools in the biology classroom. This lesson uses 3D cell models to teach students enrolled in an introductory microbiology course about bacterial cell size and the biological importance of surface-area-to-volume ratio. During the lesson, students interact with 3D cell models and discuss a series of questions in small groups. Student learning was assessed using quantitative and qualitative student response data collected pre- and post-lesson. Student achievement of learning objectives, and their confidence in their knowledge of these concepts, improved post-lesson, and these gains were statistically significant. Our findings suggest that interacting with 3D-printed cell models improves student understanding about bacterial cell size and diffusion.

This review describes the upstream‐directed movement in the small parasitic bacterium Mycoplasma. Many Mycoplasma species exhibit gliding motility, a form of biological motion over surfaces without the aid of general surface appendages such as flagella. The gliding motility is characterized by a constant unidirectional movement without changes in direction or backward motion. Unlike flagellated bacteria, Mycoplasma lacks the general chemotactic signaling system to control their moving direction. Therefore, the physiological role of directionless travel in Mycoplasma gliding remains unclear. Recently, high‐precision measurements under an optical microscope have revealed that three species of Mycoplasma exhibited rheotaxis, that is, the direction of gliding motility is lead upstream by the water flow. This intriguing response appears to be optimized for the flow patterns encountered at host surfaces. This review provides a comprehensive overview of the morphology, behavior, and habitat of Mycoplasma gliding, and discusses the possibility that the rheotaxis is ubiquitous among them. [ABSTRACT FROM AUTHOR]

Abstract Peptidoglycan for elongation in Escherichia coli is synthesized by the Rod complex, which includes RodZ. Although various mutant strains of the Rod complex have been isolated, the relationship between the activity of the Rod complex and the overall physical and chemical structures of the peptidoglycan have not been reported. We constructed a RodZ mutant, termed RMR, and analyzed the growth rate, morphology, and other characteristics of cells producing the Rod complexes containing RMR. The growth and morphology of RMR cells were abnormal, and we isolated suppressor mutants from RMR cells. Most of the suppressor mutations were found in components of the Rod complex, suggesting that these suppressor mutations increase the integrity and/or the activity of the Rod complex. We purified peptidoglycan from wild‐type, RMR, and suppressor mutant cells and observed their structures in detail. We found that the peptidoglycan purified from RMR cells had many large holes and different compositions of muropeptides from those of WT cells. The Rod complex may be a determinant not only for the whole shape of peptidoglycan but also for its highly dense structure to support the mechanical strength of the cell wall.

Despite lignin's global abundance and its use in biomedical studies, our understanding of how lignin regulates disease through modulation of cell morphology and associated phenotype of human cells is unknown. We combined an automated high-throughput image cell segmentation technique for quantitatively measuring a panel of cell shape descriptors, droplet digital Polymerase Chain Reaction for absolute quantification of gene expression and multivariate data analyses to determine whether lignin could therapeutically modulate the cell morphology and phenotype of inflamed, degenerating diseased human cells (osteoarthritic (OA) chondrocytes) towards a healthier cell morphology and phenotype. Lignin dose-dependently modified all aspects of cell morphology and ameliorated the diseased shape of OA chondrocytes by inducing a less fibroblastic healthier cell shape, which correlated with the downregulation of collagen 1A2 (COL1A2, a major fibrosis-inducing gene), upregulation of collagen 2A1 (COL2A1, a healthy extracellular matrix-inducing gene) and downregulation of interleukin-6 (IL-6, a chronic inflammatory cytokine). This is the first study to show that lignin can therapeutically target cell morphology and change a diseased cells' function towards a healthier cell shape and phenotype. This opens up novel opportunities for exploiting lignin in modulation of disease, tissue degeneration, fibrosis, inflammation and regenerative medical implants for therapeutically targeting cell function and outcome. [ABSTRACT FROM AUTHOR]

The molecular links between tissue-level morphogenesis and the differentiation of cell lineages in the pancreas remain elusive despite a decade of studies. We previously showed that in pancreas both processes depend on proper lumenogenesis. The Rab GTPase Rab11 is essential for epithelial lumen formation in vitro , however few studies have addressed its functions in vivo and none have tested its requirement in pancreas. Here, we show that Rab11 is critical for proper pancreas development. Co-deletion of the Rab11 isoforms Rab11A and Rab11B in the developing pancreatic epithelium (Rab11pancDKO) results in ∼50% neonatal lethality and surviving adult Rab11 pancDKO mice exhibit defective endocrine function. Loss of both Rab11A and Rab11B in the embryonic pancreas results in morphogenetic defects of the epithelium, including defective lumen formation and lumen interconnection. In contrast to wildtype cells, Rab11pancDKO cells initiate the formation of multiple ectopic lumens, resulting in a failure to coordinate a single apical membrane initiation site (AMIS) between groups of cells. This results in an inability to form ducts with continuous lumens. Here, we show that these defects are due to failures in vesicle trafficking, as apical and junctional components remain trapped within Rab11 pancDKO cells. Together, these observations suggest that Rab11 directly regulates epithelial lumen formation and morphogenesis. Our report links intracellular trafficking to organ morphogenesis in vivo and presents a novel framework for decoding pancreatic development. [Display omitted] • Rab11Af/f;Rab11B−/-;Pdx1-Cre pancreas displays disruption of epithelial organization and reduction of endocrine cell mass. • Loss of Rab11 results in disruption of pancreatic lumen continuity due to a failure of lumen formation. • Epithelial cells lacking Rab11 display abnormal polarity. [ABSTRACT FROM AUTHOR]

Mechanotransduction is a molecular process by which cells translate physical stimuli exerted by the external environment into biochemical pathways to orchestrate the cellular shape and function. Even with the advancements in the field, the molecular events leading to the signal cascade are still unclear. The current biotechnology of tissue engineering offers the opportunity to study in vitro the effect of the physical stimuli exerted by biomaterial on stem cells and the mechanotransduction pathway involved in the process. Here, we cultured multipotent human mesenchymal/stromal cells (hMSCs) isolated from bone marrow (hBM-MSCs) and adipose tissue (hASCs) on films of poly(butylene 1,4-cyclohexane dicarboxylate) (PBCE) and a PBCE-based copolymer containing 50 mol% of butylene diglycolate co-units (BDG50), to intentionally tune the surface hydrophilicity and the stiffness (PBCE = 560 Mpa; BDG50 = 94 MPa). We demonstrated the activated distinctive mechanotransduction pathways, resulting in the acquisition of an elongated shape in hBM-MSCs on the BDG50 film and in maintaining the canonical morphology on the PBCE film. Notably, hASCs acquired a new, elongated morphology on both the PBCE and BDG50 films. We found that these events were mainly due to the differences in the expression of Cofilin1, Vimentin, Filamin A, and Talin, which established highly sensitive machinery by which, rather than hASCs, hBM-MSCs distinguished PBCE from BDG50 films. [ABSTRACT FROM AUTHOR]

YAP/TAZ is a master regulator of mechanotransduction whose functions rely on translocation from the cytoplasm to the nucleus in response to diverse physical cues. Substrate stiffness, substrate dimensionality, and cell shape are all input signals for YAP/TAZ, and through this pathway, regulate critical cellular functions and tissue homeostasis. Yet, the relative contributions of each biophysical signal and the mechanisms by which they synergistically regulate YAP/TAZ in realistic tissue microenvironments that provide multiplexed input signals remain unclear. For example, in simple two-dimensional culture, YAP/TAZ nuclear localization correlates strongly with substrate stiffness, while in three-dimensional (3D) environments, YAP/TAZ translocation can increase with stiffness, decrease with stiffness, or remain unchanged. Here, we develop a spatial model of YAP/TAZ translocation to enable quantitative analysis of the relationships between substrate stiffness, substrate dimensionality, and cell shape. Our model couples cytosolic stiffness to nuclear mechanics to replicate existing experimental trends, and extends beyond current data to predict that increasing substrate activation area through changes in culture dimensionality, while conserving cell volume, forces distinct shape changes that result in nonlinear effect on YAP/TAZ nuclear localization. Moreover, differences in substrate activation area versus total membrane area can account for counterintuitive trends in YAP/TAZ nuclear localization in 3D culture. Based on this multiscale investigation of the different system features of YAP/TAZ nuclear translocation, we predict that how a cell reads its environment is a complex information transfer function of multiple mechanical and biochemical factors. These predictions reveal a few design principles of cellular and tissue engineering for YAP/TAZ mechanotransduction.

A subset of Spitz tumors harbor fusions of NTRK3 with ETV6, MYO5A, and MYH9. We evaluated a series of 22 melanocytic tumors in which an NTRK3 fusion was identified as part of the diagnostic workup. Tumors in which NTRK3 was fused to ETV6 occurred in younger patients were predominantly composed of epithelioid melanocytes and were classified by their histopathologic features as Spitz tumors. In contrast, those in which NTRK3 was fused to MYO5A were predominantly composed of spindled melanocytes arrayed in fascicles with neuroid features such as pseudo-Verocay bodies. To further investigate the effects of the fusion kinases ETV6-NTRK3 and MYO5A-NTRK3 in melanocytes, we expressed them in immortalized melanocytes and determined their subcellular localization by immunofluorescence. ETV6-NTRK3 was localized to the nucleus and diffusely within the cytoplasm and caused melanocytes to adopt an epithelioid cytomorphology. In contrast, MYO5A-NTRK3, appeared excluded from the nucleus of melanocytes, was localized to dendrites, and resulted in a highly dendritic cytomorphology. Our findings indicate that ETV6-NTRK3 and MYO5A-NTRK3 have distinct subcellular localizations and effects on cellular morphology.

Engineered culture substrates have proven invaluable for investigating the role of cell and extracellular matrix geometry in governing cell behavior. While the mechanisms relating geometry to phenotype are complex, it is clear that the actin cytoskeleton plays a key role in integrating geometric inputs and transducing these cues into intracellular signals that drive downstream biology. Here, we review recent progress in elucidating the role of the cell and matrix geometry in regulating actin cytoskeletal architecture and mechanics. We address new developments in traditional two-dimensional culture paradigms and discuss efforts to extend these advances to three-dimensional systems, ranging from nanotextured surfaces to microtopographical systems (e.g. channels) to fully three-dimensional matrices.

Haloferax volcanii and other Haloarchaea can be pleomorphic, adopting different shapes, which vary with growth stages. Several studies have shown that H. volcanii cell shape is sensitive to various external factors including growth media and physical environment. In addition, several studies have noticed that the presence of a recombinant plasmid in the cells is also a factor impacting H. volcanii cell shape, notably by favoring the development of rods in early stages of growth. Here we investigated the reasons for this phenomenon by first studying the impact of auxotrophic mutations on cell shape in strains that are commonly used as genetic backgrounds for selection during strain engineering (namely: H26, H53, H77, H98, and H729) and secondly, by studying the effect of the presence of different plasmids containing selection markers on the cell shape of these strains. Our study showed that most of these auxotrophic strains have variation in cell shape parameters including length, aspect ratio, area and circularity and that the plasmid presence is impacting these parameters too. Our results indicated that ΔhdrB strains and hdrB selection markers have the most influence on H. volcanii cell shape, in addition to the sole presence of a plasmid. Finally, we discuss limitations in studying cell shape in H. volcanii and make recommendations based on our results for improving reproducibility of such studies.

Key message: GhIQD21, a cotton IQ67-domain protein, interacts with GhCaM7 and alters organ shape in Arabidopsis by modulating microtubule stability. Calcium ion (Ca2+) and the calcium sensor calmodulin play crucial roles in the growth and development of plants. GhCaM7, a calmodulin in upland cotton (Gossypium hirsutum L.), is highly expressed in cotton fiber cells during the rapid elongation period and plays an important role in fiber cell development. In this study, we screened for GhCaM7-interacting proteins and identified GhIQD21, which contains a typical IQ67-domain. GhIQD21 was preferentially expressed at the fiber rapid elongation stage, and the protein localized to microtubules (MTs). Ectopic expression of GhIQD21 in Arabidopsis resulted in shorter leaves, petals, siliques, and plant height, thicker inflorescences, and more trichomes when compared with wild type (WT). Further investigation indicated that the morphogenesis of leaf epidermal cells and silique cells was altered. There was less consistency in the orientation of cortical microtubules in cotyledon and hypocotyl epidermal cells. Furthermore, compared with WT, transgenic seedling hypocotyls were more sensitive to oryzalin, a MT depolymerization drug. These results indicated that GhIQD21 is a GhCaM7-interacting protein located in MTs and that it plays a role in plant growth and potentially cotton fiber development. This study provides a foundation for further studies of the function and regulatory mechanism of GhIQD21 in fiber cell development. [ABSTRACT FROM AUTHOR]

Additive manufacturing (3D printing) and computer-aided design (CAD) still have limited uptake in biomedical and bioengineering research and education, despite the significant potential of these technologies. The utility of organ-scale 3D-printed models of living structures is widely appreciated, while the workflows for microscopy data translation into tactile accessible replicas are not well developed yet. Here, we demonstrate an accessible and reproducible CAD-based methodology for generating 3D-printed scalable models of human cells cultured in vitro and imaged using conventional scanning confocal microscopy with fused deposition modeling (FDM) 3D printing. We termed this technology CiTo-3DP (Cells-in-Touch for 3D Printing). As a proof-of-concept, we created dismountable CiTo-3DP models of human epithelial, mesenchymal, and neural cells by using selectively stained nuclei and cytoskeletal components. We also provide educational and research context for the presented cellular models. In the future, the CiTo-3DP approach can be adapted to different imaging and 3D printing modalities and comprehensively present various cell types, subcellular structures, and extracellular matrices. The resulting CAD and 3D printed models could be used for a broad spectrum of education and research applications. [ABSTRACT FROM AUTHOR]

Blood flow in the microcirculatory system is crucially affected by intrinsic red blood cell (RBC) properties, such as their deformability. In the smallest vessels of this network, RBCs adapt their shapes to the flow conditions. Although it is known that the age of RBCs modifies their physical properties, such as increased cytosol viscosity and altered viscoelastic membrane properties, the evolution of their shape-adapting abilities during senescence remains unclear. In this study, we investigated the effect of RBC properties on the microcapillary in vitro flow behavior and their characteristic shapes in microfluidic channels. For this, we fractioned RBCs from healthy donors according to their age. Moreover, the membranes of fresh RBCs were chemically rigidified using diamide to study the effect of isolated graded-membrane rigidity. Our results show that a fraction of stable, asymmetric, off-centered slipper-like cells at high velocities decreases with increasing age or diamide concentration. However, while old cells form an enhanced number of stable symmetric croissants at the channel centerline, this shape class is suppressed for purely rigidified cells with diamide. Our study provides further knowledge about the distinct effects of age-related changes of intrinsic cell properties on the single-cell flow behavior of RBCs in confined flows due to inter-cellular age-related cell heterogeneity. [ABSTRACT FROM AUTHOR]

Cells and their surrounding extracellular matrix (ECM) are engaged in dynamic reciprocity to maintain tissue homeostasis: cells deposit ECM, which in turn presents the signals that define cell identity. This loop of phenotype is obvious for biochemical signals, such as collagens, which are produced by and presented to cells, but the role of biomechanical signals is also increasingly recognised. In addition, cell shape goes hand in hand with cell function and tissue homeostasis. Aberrant cell shape and ECM is seen in pathological conditions, and control of cell shape in micro-fabricated platforms disclose the causal relationship between cell shape and cell function, often mediated by mechanotransduction. In this manuscript, we discuss the loop of phenotype for tendon tissue homeostasis. We describe cell shape and ECM organization in normal and diseased tissue, how ECM composition influences tenocyte shape, and how that leads to the activation of signal transduction pathways and ECM deposition. We further describe the use of technologies to control cell shape to elucidate the link between cell shape and its phenotypical markers and focus on the causal role of cell shape in the loop of phenotype. The dynamic reciprocity between cells and their surrounding extracellular matrix (ECM) influences biomechanical and biochemical properties of ECM as well as cell function through activation of signal transduction pathways that regulate gene and protein expression. We refer to this reciprocity as Loop of Phenotype and it has been studied and demonstrated extensively by using micro-fabricated platforms to manipulate cell shape and cell fate. In this manuscript, we discuss this concept in tendon tissue homeostasis by giving examples in healthy and pathological tenson tissue. Furthermore, we elaborate this by showing how biomaterials are used to feed this reciprocity to manipulate cell shape and function. Finally, we elucidate the link between cell shape and its phenotypical markers and focus on the activation of signal transduction pathways and ECM deposition. [Display omitted] [ABSTRACT FROM AUTHOR]

Introduction: The size and shape of neurons are important features indicating aging and the pathology of neurodegenerative diseases. Despite the significant advances of optical microscopy, quantitative analysis of the neuronal features in the human brain remains largely incomplete. Traditional histology on thin slices bears tremendous distortions in three-dimensional reconstruction, the magnitude of which are often greater than the structure of interest. Recently development of tissue clearing techniques enable the whole brain to be analyzed in small animals; however, the application in the human remains challenging. Methods: In this study, we present a label-free quantitative optical coherence microscopy (OCM) technique to obtain the morphological parameters of neurons in human entorhinal cortex (EC). OCM uses the intrinsic back-scattering property of tissue to identify individual neurons in 3D. The area, length, width, and orientation of individual neurons are quantified and compared between layer II and III in EC. Results: The high-resolution mapping of neuron size, shape, and orientation shows significant differences between layer II and III neurons in EC. The results are validated by standard Nissl staining of the same samples. Discussion: The quantitative OCM technique in our study offers a new solution to analyze variety of neurons and their organizations in the human brain, which opens new insights in advancing our understanding of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Flower color not only determines the quality and commercial value of ornamental plants, but it also plays a vital role in ecological processes such as pollinator attraction. This study aimed to investigate the relationship between flower color and the cellular physicochemical factors of Bletilla striata. The color space values of 21 samples were initially determined, followed by a selection of five samples with significant color differences for testing cell shape, total flavonoid content (ranging from 1.86 to 5.42 mg/g), total anthocyanin content (ranging from 0.52 to 292.62 (A530 − 0.25 ∗ A657)/g), cell pH (varying between 5.03 and 5.74), and metal ion content (including Al3+, Ca2+, Fe3+, K+, Mg2+, Na+, P5+, Zn2+, Mo6+, Cu2+, and Mn2+). The flowers of Bletilla were predominantly purple, pink, and yellow; and distributed in quadrants I, II, and IV on the a* and b* rectangular coordinate. The a* value was identified as the primary color indicator for this species. Total anthocyanin content and Zn2+ showed a significant positive correlation with a*, while Al3+, Ca2+, Fe3+, Mg2+, Na+, and Mn2+ demonstrated negative correlations. Cell shape, flavonoid content, and pH had nonsignificant correlations with a*. In conclusion, the total anthocyanin content and metal ions play crucial roles in determining the flower color of B. striata, which can have implications for pollinator attraction. Future research should focus on understanding the complex interactions between these factors to develop novel ornamental plant varieties with desirable flower colors and enhanced ecological functions. [ABSTRACT FROM AUTHOR]

Epithelial tubules form critical structures in lung, kidney and vascular tissues. However, the processes that control their morphogenesis and physiological expansion and contraction are not well understood. Here we examine the dynamic remodeling of epithelial tubes in vivo using a novel model system: the extracorporeal vasculature of Botryllus schlosseri, in which the disruption of the basement membrane triggers rapid, massive vascular retraction without loss of barrier function. We developed and implemented 3-D image analysis and virtual reconstruction tools to characterize the cellular morphology of the vascular wall in unmanipulated vessels and during retraction. In both control and regressed conditions, cells within the vascular wall were planar polarized, with an integrin- and curvature-dependent axial elongation of cells and a robust circumferential alignment of actin bundles. Surprisingly, we found no measurable differences in morphology between normal and retracting vessels under ECM disruption. However, inhibition of integrin signaling through FAK inhibition caused disruption of cellular actin organization. Our results demonstrate that epithelial tubes can maintain tissue organization even during extreme remodeling events, but that the robust response to mechanical signals – such as the response to loss of vascular tension after ECM disruption - requires functional force sensing machinery via integrin signaling.

Understanding the mechanisms of cell-to-cell communication is one of the fundamental questions in biology and medicine. In particular, long-range signalling where cells communicate over several cell diameters is vital during development and homeostasis. The major morphogens, their receptors and intracellular signalling cascades have largely been identified; however, there is a gap in our knowledge of how such signalling factors are propagated over a long distance. In addition to the diffusion-based propagation model, new modalities of disseminating signalling molecules have been identified. It has been shown that cells can communicate with direct contact through long, thin cellular protrusions between signal sending and receiving cells at a distance. Recent studies have revealed a type of cellular protrusion termed 'airinemes' in zebrafish pigment cell types. They share similarities with previously reported cellular protrusions; however, they also exhibit distinct morphology and features. Airinemes are indispensable for pigment pattern development by mediating long-distance Delta-Notch signalling between different pigment cell types. Notably, airineme-mediated signalling is dependent on skin-resident macrophages. Key findings of airineme-mediated intercellular signalling in pattern development, their interplay with macrophages and their implications for the understanding of cellular protrusion-mediated intercellular communication will be discussed.

The mechanical phenotype of a cell is an inherent biophysical marker of its state and function, with many applications in basic and applied biological research. Microfluidics-based methods have enabled single-cell mechanophenotyping at throughputs comparable to those of flow cytometry. Here, we present a standardized cross-laboratory study comparing three microfluidics-based approaches for measuring cell mechanical phenotype: constriction-based deformability cytometry (cDC), shear flow deformability cytometry (sDC) and extensional flow deformability cytometry (xDC). All three methods detect cell deformability changes induced by exposure to altered osmolarity. However, a dose-dependent deformability increase upon latrunculin B-induced actin disassembly was detected only with cDC and sDC, which suggests that when exposing cells to the higher strain rate imposed by xDC, cellular components other than the actin cytoskeleton dominate the response. The direct comparison presented here furthers our understanding of the applicability of the different deformability cytometry methods and provides context for the interpretation of deformability measurements performed using different platforms.

The shape of the cell is connected to its function; however, we do not fully understand underlying mechanisms by which global shape regulates a cell's functional capabilities. Using theory, experiments and simulation, we investigated how physiologically relevant cell shape changes affect subcellular organization, and consequently intracellular signaling, to control information flow needed for phenotypic function. Vascular smooth muscle cells going from a proliferative and motile circular shape to a contractile fusiform shape show changes in the location of the sarcoplasmic reticulum, inter-organelle distances, and differential distribution of receptors in the plasma membrane. These factors together lead to the modulation of signals transduced by the M3 muscarinic receptor/Gq/PLCβ pathway at the plasma membrane, amplifying Ca2+ dynamics in the cytoplasm, and the nucleus resulting in phenotypic changes, as determined by increased activity of myosin light chain kinase in the cytoplasm and enhanced nuclear localization of the transcription factor NFAT. Taken together, our observations show a systems level phenomenon whereby global cell shape affects subcellular organization to modulate signaling that enables phenotypic changes.

Alveolar formation increases the surface area for gas-exchange and is key to the physiological function of the lung. Alveolar epithelial cells, myofibroblasts and endothelial cells undergo coordinated morphogenesis to generate epithelial folds (secondary septa) to form alveoli. A mechanistic understanding of alveologenesis remains incomplete. We found that the planar cell polarity (PCP) pathway is required in alveolar epithelial cells and myofibroblasts for alveologenesis in mammals. Our studies uncovered a Wnt5a-Ror2-Vangl2 cascade that endows cellular properties and novel mechanisms of alveologenesis. This includes PDGF secretion from alveolar type I and type II cells, cell shape changes of type I cells and migration of myofibroblasts. All these cellular properties are conferred by changes in the cytoskeleton and represent a new facet of PCP function. These results extend our current model of PCP signaling from polarizing a field of epithelial cells to conferring new properties at subcellular levels to regulate collective cell behavior.