Cell Shape Transformation Research Articles

Drivers of Morphogenesis: Curvature Sensor Self-Assembly at the Membrane.

This review examines the relationships between membrane chemistry, curvature-sensing proteins, and cellular morphogenesis. Curvature-sensing proteins are often orders of magnitude smaller than the membrane curvatures they localize to. How are nanometer-scale proteins used to sense micrometer-scale membrane features? Here, we trace the journey of curvature-sensing proteins as they engage with lipid membranes through a combination of electrostatic and hydrophobic interactions. We discuss how curvature sensing hinges on membrane features like lipid charge, packing, and the directionality of membrane curvature. Once bound to the membrane, many curvature sensors undergo self-assembly (i.e., they oligomerize or form higher-order assemblies that are key for initiating and regulating cell shape transformations). Central to these discussions are the micrometer-scale curvature-sensing proteins' septins. By discussing recent literature surrounding septin membrane association, assembly, and their many functions in morphogenesis with support from other well-studied curvature sensors, we aim to synthesize possible mechanisms underlining cell shape sensing.

Mechanical forces across compartments coordinate cell shape and fate transitions to generate tissue architecture

Morphogenesis and cell state transitions must be coordinated in time and space to produce a functional tissue. An excellent paradigm to understand the coupling of these processes is mammalian hair follicle development, which is initiated by the formation of an epithelial invagination—termed placode—that coincides with the emergence of a designated hair follicle stem cell population. The mechanisms directing the deformation of the epithelium, cell state transitions and physical compartmentalization of the placode are unknown. Here we identify a key role for coordinated mechanical forces stemming from contractile, proliferative and proteolytic activities across the epithelial and mesenchymal compartments in generating the placode structure. A ring of fibroblast cells gradually wraps around the placode cells to generate centripetal contractile forces, which, in collaboration with polarized epithelial myosin activity, promote elongation and local tissue thickening. These mechanical stresses further enhance compartmentalization of Sox9 expression to promote stem cell positioning. Subsequently, proteolytic remodelling locally softens the basement membrane to facilitate a release of pressure on the placode, enabling localized cell divisions, tissue fluidification and epithelial invagination into the underlying mesenchyme. Together, our experiments and modelling identify dynamic cell shape transformations and tissue-scale mechanical cooperation as key factors for orchestrating organ formation.

MorphoMacro for in vivo and ex vivo quantitative morphometric analysis of microglia.

Microglia cells dynamically survey the central nervous system microenvironment and, in response to tissue damage inflicted by radiation therapy, disease or infection, undergo morphological and functional changes that culminate in microglia activation. Cell shape transformation can be assessed descriptively or, alternatively, it can be quantified as a continuous variable for parameters including total cell size as well as protrusion length, ramification and complexity. The purpose of the MorphoMacro method is to quantitatively profile multiple and single microglia cells using the available ImageJ platform. This method outlines the required steps and ImageJ plugins to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using the MorphoMacro software plugin for multiparametric and multilevel description of microglia cell morphology in vivo and ex vivo. Overall, the protocol provides a quantitative and comprehensive tool that can be used to identify, stratify, and monitor diverse microglia morphologies in homeostatic, different disease conditions and subsequent therapeutic monitoring.

Hemorheological alterations of red blood cells induced by 450-nm and 520-nm laser radiation.

Proper rheological properties of red blood cells (RBC) including flexibility and aggregability are essential for healthy blood microcirculation. Excessive RBC aggregation has been observed to be associated with many pathological conditions and is crucial in acute circulatory problems. Low-level laser radiation (LLLR) has been found to have positive effects on the rheology of human blood, however, the detailed mechanisms of blood photobiomodulation remains unclear. In this study, utilizing the single-cell technique optical tweezers (OT) and traditional light microscopy, the influence of photobiomodulation of human RBC was examined under different conditions of laser irradiation. The results revealed that high radiant exposure (over 170.5J/cm2 radiant fluence) caused enhanced RBC aggregation and cell shape transformation while the aggregation force between single RBC remained unchanged. LLLR with radiant fluence below 9.5J/cm2 by 450nm wavelength improved the RBC deformability, weakened the strength of cell-cell interaction in the RBC disaggregation process, and showed rejuvenating effects on RBC suspended in a harsh cell environment.

Nonapoptotic role of caspase-3 in regulating Rho1GTPase-mediated morphogenesis of epithelial tubes of Drosophila renal system.

Cells trigger caspase-mediated apoptosis to eliminate themselves from the system when tissue needs to be sculptured, or they detect any abnormality within them, thus preventing irreparable damage to the host. However, nonapoptotic activities of caspases are also involved in many cellular functions. Interestingly, Drosophila Malpighian tubules (MTs) express apoptotic proteins, without succumbing to cell death. We show apoptosis-independent role of executioner caspase-3, Drice, in MT morphogenesis. Drice is required for precise cytoskeleton organization and convergent extension, failing which morphology, size, cell number, and arrangement get affected. Furthermore, characteristic stellate cell shape transformation in MTs is also governed by Drice. Genetic interaction study shows that Drice mediates its action by regulating Rho1GTPase functionally, and localization of polarity protein Disc large. Subsequently, downregulation of Rho1GTPase in Drice mutants significantly rescues the cystic MTs phenotype. The study shows a mechanism by which Drice governs tubulogenesis via Rho1GTPase-mediated coordinated organization of actin cytoskeleton and membrane stabilization. Collectively our findings suggest a nonapoptotic function of caspase-3 in fine-tuning of cellular rearrangement during tubule development, and these results will add to the growing understanding of diverse roles of caspases during its evolution in metazoans.

Robust Regeneration of Adult Electric Pulse Fish and the Formation of Its Electric Organ from Fast Muscle Fibers

In most weakly electric fish species, the cells that make up the electric organ (EO) are called electrocytes (EC) and these derive from a subpopulation of striated muscle fibers that undergo an extreme phenotypic conversion to replace their contractile function for an electrogenic one that fish use to navigate, communicate, mate, and detect prey/predators. Our previous findings using the knifefish Sternopygus macrurus revealed that ECs form from the fusion of fast muscle fiber types during adult tail regeneration. Whether or not this EC formation process is conserved in other highly regenerative electric fish species is the focus of this study. Specifically, we used immunolabeling to begin characterizing the morphology and muscle-protein expression of mature electrocytes in the species Brachyhypopomus pinnicaudatus and the muscle fiber type composition in the distal-most tail surrounding ECs prior to tail amputation. Regeneration of skeletal muscle and EO tissues was studied at different time points post tail cut – 1, 2, 3, 4, and 6 weeks. Our analyses to date show a distinct sexual dimorphism in EC length and cross-sectional area (CSA), with a mean (±SE) CSA of 4718µm2±543 in males (n= 3; triplicates per fish) and 1763µm2±181 in females (n=3; triplicates per fish) and a mean EC length of 19µm±2 in males (n= 3; triplicates per fish) and 13µm±1 in females (n= 3; triplicates per fish). Our immunolabeling studies detected the presence of the “muscle” protein desmin in all adult ECs. In addition, some but not all, ECs contained alpha-actinin, tropomyosin, titin, and sarcomeric myosin heavy chain (MHC) IIb. In B. pinnicaudatus, cell differentiation progressed from proximal to distal and peripheral to central, with muscle fibers being detected prior to ECs at each stage of regeneration studied. Myogenesis was evident by Day 7 with presence of fibers positively labeled with mouse antibody (MF20) against all MHC isoforms (n= 2). In addition, by Day 7, regenerating ECs were also evident in 1-week blastemas (n=2) fish based on the clustering of myofibers stained by cresyl violet and these early ECs were immunolabeled with MF20, cell shape transformation, and medial location relative to the muscle fibers. These data suggest that there are sexual dimorphic differences in the CSAs and lengths of ECs in the EOs of male and female B. pinnicaudatus. In addition, similar to S. macrurus, developing ECs in B. pinnicaudatus are given rise to by their type II fast muscle fiber precursors.

Bacterial cell shape control by nutrient-dependent synthesis of cell division inhibitors

By analyzing cell size and shapes of the bacterium Bacillus subtilis under nutrient perturbations, protein depletion, and antibiotic treatments, we find that cell geometry is extremely robust, reflected in a well-conserved scaling relation between surface area (S) and volume (V), S∼Vγ, with γ=0.85. We develop a molecular model supported by single-cell simulations to predict that the surface-to-volume scaling exponent γ is regulated by nutrient-dependent production of metabolic enzymes that act as cell division inhibitors in bacteria. Using theory that is supported by experimental data, we predict the modes of cell shape transformations in different bacterial species and propose a mechanism of cell shape adaptation to different nutrient perturbations. For organisms with high surface-to-volume scaling exponent γ, such as B. subtilis, cell width is not sensitive to growth-rate changes, whereas organisms with low γ, such as Acinetobacter baumannii, cell shape adapts readily to growth-rate changes.

Shape-Morphing Fibrous Hydrogel/Elastomer Bilayers Fabricated by a Combination of 3D Printing and Melt Electrowriting for Muscle Tissue Regeneration.

This paper reports an approach for the fabrication of shape-changing bilayered scaffolds, which allow the growth of aligned skeletal muscle cells, using a combination of 3D printing of hyaluronic acid hydrogel, melt electrowriting of thermoplastic polycaprolactone-polyurethane elastomer, and shape transformation. The combination of the selected materials and fabrication methods allows a number of important advantages such as biocompatibility, biodegradability, and suitable mechanical properties (elasticity and softness of the fibers) similar to those of important components of extracellular matrix (ECM), which allow proper cell alignment and shape transformation. Myoblasts demonstrate excellent viability on the surface of the shape-changing bilayer, where they occupy space between fibers and align along them, allowing efficient cell patterning inside folded structures. The bilayer scaffold is able to undergo a controlled shape transformation and form multilayer scroll-like structures with cells encapsulated inside. Overall, the importance of this approach is the fabrication of tubular constructs with a patterned interior that can support the proliferation and alignment of muscle cells for muscle tissue regeneration.

Multicompartment Polymer Vesicles with Artificial Organelles for Signal‐Triggered Cascade Reactions Including Cytoskeleton Formation

AbstractOrganelles, i.e., internal subcompartments of cells, are fundamental to spatially separate cellular processes, while controlled intercompartment communication is essential for signal transduction. Furthermore, dynamic remodeling of the cytoskeleton provides the mechanical basis for cell shape transformations and mobility. In a quest to develop cell‐like smart synthetic materials, exhibiting functional flexibility, a self‐assembled vesicular multicompartment system, comprised of a polymeric membrane (giant unilamellar vesicle, GUV) enveloping polymeric artificial organelles (vesicles, nanoparticles), is herein presented. Such multicompartment assemblies respond to an external stimulus that is transduced through a precise sequence. Stimuli‐triggered communication between two types of internal artificial organelles induces and localizes an enzymatic reaction and allows ion‐channel mediated release from storage vacuoles. Moreover, cytoskeleton formation in the GUVs' lumen can be triggered by addition of ionophores and ions. An additional level of control is achieved by signal‐triggered ionophore translocation from organelles to the outer membrane, triggering cytoskeleton formation. This system is further used to study the diffusion of various cytoskeletal drugs across the synthetic outer membrane, demonstrating potential applicability, e.g., anticancer drug screening. Such multicompartment assemblies represent a robust system harboring many different functionalities and are a considerable leap in the application of cell logics to reactive and smart synthetic materials.

Deformation behaviour of stomatocyte, discocyte and echinocyte red blood cell morphologies during optical tweezers stretching.

The red blood cell (RBC) deformability is a critical aspect, and assessing the cell deformation characteristics is essential for better diagnostics of healthy and deteriorating RBCs. There is a need to explore the connection between the cell deformation characteristics, cell morphology, disease states, storage lesion and cell shape-transformation conditions for better diagnostics and treatments. A numerical approach inspired from the previous research for RBC morphology predictions and for analysis of RBC deformations is proposed for the first time, to investigate the deformation characteristics of different RBC morphologies. The present study investigates the deformability characteristics of stomatocyte, discocyte and echinocyte morphologies during optical tweezers stretching and provides the opportunity to study the combined contribution of cytoskeletal spectrin network and the lipid-bilayer during RBC deformation. The proposed numerical approach predicts agreeable deformation characteristics of the healthy discocyte with the analogous experimental observations and is extended to further investigate the deformation characteristics of stomatocyte and echinocyte morphologies. In particular, the computer simulations are performed to investigate the influence of direct stretching forces on different equilibrium cell morphologies on cell spectrin link extensions and cell elongation index, along with a parametric analysis on membrane shear modulus, spectrin link extensibility, bending modulus and RBC membrane-bead contact diameter. The results agree with the experimentally observed stiffer nature of stomatocyte and echinocyte with respect to a healthy discocyte at experimentally determined membrane characteristics and suggest the preservation of relevant morphological characteristics, changes in spectrin link densities and the primary contribution of cytoskeletal spectrin network on deformation behaviour of stomatocyte, discocyte and echinocyte morphologies during optical tweezers stretching deformation. The numerical approach presented here forms the foundation for investigations into deformation characteristics and recoverability of RBCs undergoing storage lesion.

Viscosity-dependent variations in the cell shape and swimming manner of Leptospira.

Spirochaetes are spiral or flat-wave-shaped Gram-negative bacteria that have periplasmic flagella between the peptidoglycan layer and outer membrane. Rotation of the periplasmic flagella transforms the cell body shape periodically, allowing the cell to swim in aqueous environments. Because the virulence of motility-deficient mutants of pathogenic species is drastically attenuated, motility is thought to be an essential virulence factor in spirochaetes. However, it remains unknown how motility practically contributes to the infection process. We show here that the cell body configuration and motility of the zoonotic spirochaete Leptospira changes depending on the viscosity of the medium. Leptospira swim and reverse the swimming direction by transforming the cell body. Motility analysis showed that the frequency of cell shape transformation was increased by increasing the viscosity of the medium. The increased cell body transformation induced highly frequent reversal of the swimming direction. A simple kinetic model based on the experimental results shows that the viscosity-induced increase in reversal limits cell migration, resulting in the accumulation of cells in high-viscosity regions. This behaviour could facilitate the colonization of the spirochaete on host tissues covered with mucosa.

MiR-23a/-24-induced gene silencing results in mesothelial cell integration of pancreatic cancer.

Background:Invasion of the surrounding tissue is part of the metastatic cascade. Here, we examined the invasion of pancreatic ductal adenocarcinoma (PDAC) cells into the mesothelial barrier and identified the related microRNA (miRNA) expression profiles.Methods:The interactions between PDAC cells and mesothelial monolayers were characterised and quantified using a specific time-lapse videomicroscopy assay. Pancreatic ductal adenocarcinoma cells were further evaluated using the adhesion assay, and miRNA, mRNA and protein expressions were determined using microarray, q-RT–PCR and western blots, respectively. These data were correlated with in vivo dissemination scores.Results:Two groups of PDAC cell lines were distinguished by their integration capacity into the mesothelial monolayer using mean elongation factors (MEFs). Adhesion assays showed a concordant relation between adhesive properties and integration capacity. The distant metastases scores were reverse correlated with MEFs. Microarray analysis of these groups revealed that miR-23a and/or miR-24 target for FZD5, HNF1B and/or TMEM92, respectively, and that they are significantly deregulated.Conclusions:MiR-23a and/or miR-24 overexpression leads to gene silencing of FZD5, TMEM92 and/or HNF1B. Their downregulation induces deregulated expression and degradation of E-cadherin and β-catenin causing destabilisation of the cadherin/catenin complex, and altered the expression of Wnt-related genes. We propose a molecular (epi)genetic mechanism by which increased EMT-like cell shape transformation and integration into mesothelial monolayers of PDAC cells can be observed.

The actin-binding ERM protein Moesin binds to and stabilizes microtubules at the cell cortex

Ezrin, Radixin, and Moesin (ERM) proteins play important roles in many cellular processes including cell division. Recent studies have highlighted the implications of their metastatic potential in cancers. ERM's role in these processes is largely attributed to their ability to link actin filaments to the plasma membrane. In this paper, we show that the ERM protein Moesin directly binds to microtubules in vitro and stabilizes microtubules at the cell cortex in vivo. We identified two evolutionarily conserved residues in the FERM (4.1 protein and ERM) domains of ERMs that mediated the association with microtubules. This ERM-microtubule interaction was required for regulating spindle organization in metaphase and cell shape transformation after anaphase onset but was dispensable for bridging actin filaments to the metaphase cortex. These findings provide a molecular framework for understanding the complex functional interplay between the microtubule and actin cytoskeletons mediated by ERM proteins in mitosis and have broad implications in both physiological and pathological processes that require ERMs.

Compression and dilation of the membrane-cortex layer generates rapid changes in cell shape

Rapid changes in cellular morphology require a cell body that is highly flexible yet retains sufficient strength to maintain structural integrity. We present a mechanism that meets both of these requirements. We demonstrate that compression (folding) and subsequent dilation (unfolding) of the coupled plasma membrane-cortex layer generates rapid shape transformations in rounded cells. Two- and three-dimensional live-cell images showed that the cyclic process of membrane-cortex compression and dilation resulted in a traveling wave of cortical actin density. We also demonstrate that the membrane-cortex traveling wave led to amoeboid-like cell migration. The compression-dilation hypothesis offers a mechanism for large-scale cell shape transformations that is complementary to blebbing, where the plasma membrane detaches from the actin cortex and is initially unsupported when the bleb extends as a result of cytosolic pressure. Our findings provide insight into the mechanisms that drive the rapid morphological changes that occur in many physiological contexts, such as amoeboid migration and cytokinesis.

Molecular networks linked by Moesin drive remodeling of the cell cortex during mitosis

The cortical mechanisms that drive the series of mitotic cell shape transformations remain elusive. In this paper, we identify two novel networks that collectively control the dynamic reorganization of the mitotic cortex. We demonstrate that Moesin, an actin/membrane linker, integrates these two networks to synergize the cortical forces that drive mitotic cell shape transformations. We find that the Pp1-87B phosphatase restricts high Moesin activity to early mitosis and down-regulates Moesin at the polar cortex, after anaphase onset. Overactivation of Moesin at the polar cortex impairs cell elongation and thus cytokinesis, whereas a transient recruitment of Moesin is required to retract polar blebs that allow cortical relaxation and dissipation of intracellular pressure. This fine balance of Moesin activity is further adjusted by Skittles and Pten, two enzymes that locally produce phosphoinositol 4,5-bisphosphate and thereby, regulate Moesin cortical association. These complementary pathways provide a spatiotemporal framework to explain how the cell cortex is remodeled throughout cell division.

Electrostatic Bending of Lipid Membranes: How Are Lipid and Electrostatic Properties Interrelated?

Electrostatic modification of lipid headgroups and its effect on membrane curvature are not only relevant in a variety of contexts such as cell shape transformation and membrane tubulation but also are intriguingly implicated in membrane functions. For instance, the gating (open vs closed) properties of mechanosensitive channels can be influenced by membrane curvature and ion valence. However, a full theoretical description of membrane electrostatics is still lacking; in the past, membrane bending has often been considered under a few assumptions about how bending modifies lipid arrangements and surface charges. Here, we present a unified theoretical approach to spontaneous membrane curvature, C(0), in which lipid properties (e.g., packing shape) and electrostatic effects are self-consistently integrated. For the description of electrostatic interactions, especially between a lipid charge and a divalent counterion, we implement the Poisson-Boltzmann (PB) approach by incorporation of finite ionic sizes, so as to capture both lateral and transverse charge correlations on the membrane surface. Our results show that C(0) is sensitive to the way lipid rearrangements and divalent counterions are modeled. Interestingly, it can change its sign in the presence of divalent counterions, thus stabilizing reverse hexagonal (H(II)) phases. Our results show how electrostatic modification of headgroups influences the preferred structure of lipid aggregates (inverted micelles vs bilayers).

Agglutination of like-charged red blood cells induced by binding of β2-glycoprotein I to outer cell surface

Plasma protein-mediated attractive interaction between membranes of red blood cells (RBCs) and phospholipid vesicles was studied. It is shown that β 2 -glycoprotein I ( β 2 -GPI) may induce RBC discocyte–echinocyte–spherocyte shape transformation and subsequent agglutination of RBCs. Based on the observed β 2 -GPI-induced RBC cell shape transformation it is proposed that the hydrophobic portion of β 2 -GPI molecule protrudes into the outer lipid layer of the RBC membrane and increases the area of this layer. It is also suggested that the observed agglutination of RBCs is at least partially driven by an attractive force which is of electrostatic origin and depends on the specific molecular shape and internal charge distribution of membrane-bound β 2 -GPI molecules. The suggested β 2 -GPI-induced attractive electrostatic interaction between like-charged RBC membrane surfaces is qualitatively explained by using a simple mathematical model within the functional density theory of the electric double layer, where the electrostatic attraction between the positively charged part of the first domains of bound β 2 -GPI molecules and negatively charged glycocalyx of the adjacent RBC membrane is taken into account.

P204 Protein Is a Novel Modulator of Ras Activity

The murine p200 family protein, p204, modulates cell proliferation and tissue differentiation. Many of its activities are exerted in the nucleus. However, in cardiac myocytes, p204 accumulated in the cytoplasm. A yeast two-hybrid assay revealed a p204-cytoplasmic Ras protein interaction. This was confirmed (i) by coimmunoprecipitation of p204 with Ras in mouse heart extract and with endogenous or ectopic H-Ras and K-Ras in cell lysates as well as (ii) by binding of purified H-Ras-GTP to purified p204 in vitro. p204 inhibited (i) the cleavage of RasGTP to RasGDP by RasGAP; (ii) the binding to RasGTP of Raf-1, phosphatidylinositol 3-kinase, and Ral-GDS, effectors of Ras signaling; and (iii) activation by the Ras pathway of the phosphorylation and thus activation of downstream targets (e.g. MEK, Akt, and p38 MAPK). Oncogenic Ras expression triggered the phosphorylation and translocation of p204 from the nucleus to the cytoplasm. This is expected to increase the interaction between the two proteins. Translocation triggered by Ras oncoprotein was blocked by the LY294002 inhibitor of phosphatidylinositol 3-kinase. Ras did not promote phosphorylation or translocation to the cytoplasm of mutated p204 in which serine 179 was replaced by alanine. p204 overexpression inhibited the anchorage-independent proliferation of cells expressing Ras(Q61L) oncoprotein. Ras oncoprotein triggered in MEF3T3 cells the rearrangement of the actin cytoskeleton and the enhancement of cell migration through a membrane. Overexpression of p204 inhibited both. Ras oncoprotein or activated, wild-type Ras was described to increase Egr-1 transcription factor expression. We report that a sequence in the gene encoding p204 bound Egr-1, and Egr-1 activated p204 expression. Ras oncoprotein or activated wild-type Ras increased the expression in 3T3 cells of p204 together with that of Egr-1. Furthermore, the activation of expression of a single copy of K-ras oncogene in cultured murine embryonic cells induced the expression of a high level of p204 as well as its distribution between the nuclei and the cytoplasm. Thus, p204 may serve as a negative feedback inhibitor of Ras activity.

3P196 線虫の初期胚を用いた細胞分裂における細胞の形状の実験的・理論的解析(細胞生物学的課題(接着・運動・骨格・伝達・膜),ポスター発表,第45回日本生物物理学会年会)

3P196 線虫の初期胚を用いた細胞分裂における細胞の形状の実験的・理論的解析(細胞生物学的課題(接着・運動・骨格・伝達・膜),ポスター発表,第45回日本生物物理学会年会)

A common biomechanical model for the formation of stationary cell domains and propagating waves in the developing organisms

Many important morphogenetic processes that take place in the development of an animal start from the segregation of a homogeneous layer of cells into a different number of the domains of columnar and flattened cells. In many cases, waves of cell shape transformation travel throughout embryonic tissues. A biomechanical model is presented which embraces both kinds of event. The model is based on the idea of interplay between short- and long-range factors. While the former promote the spreading of a given cell state along a cell row in the recalculation direction, long-range factors are associated with self-generated tensions which, after exceeding a certain threshold, induce active cell extension and hence the rise of tangential pressure. Different kinds of biologically realistic stationary structures, as well as various kinds of the running waves, can be modelled under different parameter values. Moreover, the current model can be coupled with the previous one (Beloussov and Grabovsky, Comput. Methods Biomech. Biomed. Eng., 6: 53–63 (2003)) permitting a common causal chain to be created, moving from the state of an initial homogeneous cell layer towards the complicated shapes of embryonic rudiments.