Cell Shape Changes Research Articles

Air–liquid interface cultures trigger a metabolic shift in intestinal epithelial cells (IPEC-1)

An improved oxygen availability in air–liquid interface (ALI) cultures of enterocytes of the small intestine seems to be primarily responsible for morphological, metabolic, and functional changes. Intestinal porcine epithelial cells 1 (IPEC-1) are less investigated and are rarely used as model for intestinal barrier but showed a profound change of cell shape during ALI cultivation. We aim to answer the following question: Are the observed morphological effects accompanied by changes in metabolic function? A microarray analysis of submerged culture (SMC) and ALI cultures identified 830 significantly regulated genes. Subsequent functional clustering revealed alterations in 31 pathways, with the highest number of regulated genes in metabolic pathways, carbon metabolism, glycolysis, and hypoxia-inducible factor (HIF) signaling. Furthermore, HIF-1α as a mediator of a metabolic switch between glycolysis and oxidative phosphorylation showed a trend of increased mRNA levels in ALI in contrast to a reduced nuclear HIF-1α content in the nucleus. Candidate genes of oxidative phosphorylation such as a mitochondrial marker exhibited enhanced mRNA levels, which was confirmed by western blot analysis. Cytochrome C oxidase (COX) subunit 5B protein was decreased in ALI, although mRNA level was increased. The oxidation of ferrocytochrome C to ferricytochrome C was used for detection of cytochrome C oxidase activity of isolated mitochondria and resulted in a trend of higher activity in ALI. Furthermore, quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. To evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenized cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In contrast, blocking with 2-desoxy-d-glucose (2DG) significantly reduced ATP content in ALI and SMC. These results indicate a metabolic shift in IPEC-1 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis.

Cytochalasin B Influences Cytoskeletal Organization and Osteogenic Potential of Human Wharton's Jelly Mesenchymal Stem Cells.

Among perinatal stem cells of the umbilical cord, human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) are of great interest for cell-based therapy approaches in regenerative medicine, showing some advantages over other MSCs. In fact, hWJ-MSCs, placed between embryonic and adult MSCs, are not tumorigenic and are harvested with few ethical concerns. Furthermore, these cells can be easily cultured in vitro, maintaining both stem properties and a high proliferative rate for several passages, as well as trilineage capacity of differentiation. Recently, it has been demonstrated that cytoskeletal organization influences stem cell biology. Among molecules able to modulate its dynamics, Cytochalasin B (CB), a cyto-permeable mycotoxin, influences actin microfilament polymerization, thus affecting several cell properties, such as the ability of MSCs to differentiate towards a specific commitment. Here, we investigated for the first time the effects of a 24 h-treatment with CB at different concentrations (0.1-3 μM) on hWJ-MSCs. CB influenced the cytoskeletal organization in a dose-dependent manner, inducing changes in cell number, proliferation, shape, and nanomechanical properties, thus promoting the osteogenic commitment of hWJ-MSCs, as confirmed by the expression analysis of osteogenic/autophagy markers.

Immunomodulatory Effects of Bacterial Toll-like Receptor Ligands on the Phenotype and Function of Milk Immune Cells in Dromedary Camel

(1) Toll-like receptors (TLR) are a family of pattern recognition receptors that sense distinct molecular patterns of microbial origin. Although the immune cell composition of camel milk has been recently described, host-pathogen interaction studies in the camel mammary gland are still scarce. The present study aimed to use a whole milk stimulation assay for investigating the modulatory effect of selected Toll-like receptor (TLR) ligands on the phenotype and function of milk immune cells. (2) Methods-camel milk samples (n = 7) were stimulated in vitro with the TLR4 ligand LPS or the TLR2/1 ligand Pam3CSK4, and separated milk cells were evaluated for stimulation-induced shape change, the expression of cell surface markers, phagocytosis, apoptosis, ROS production, and NETosis. Stimulation with PMA was used as a control stimulation. (3) Results-all stimulants induced shape change in milk cells, change in the expression of several cell markers, and increased cell apoptosis and NETosis. In addition, stimulation with Pam3CSK4 and PMA was associated with enhanced ROS production, while only PMA stimulation resulted in enhanced bacterial phagocytosis by milk immune cells. (4) Conclusions-our data indicates selective modulating effects of the TLR ligands LPS and Pam3CSK4 on camel milk phagocytes. These results may have implications for the use of synthetic TLR agonists as immunomodulatory adjuvants of the immune response to intra-mammary vaccines against mastitis pathogens.

Mechanism of cis-Nerolidol-Induced Bladder Carcinoma Cell Death.

Nerolidol is a naturally occurring sesquiterpene alcohol with multiple properties, including antioxidant, antibacterial, and antiparasitic activities. A few studies investigating the antitumor properties of nerolidol have shown positive results in both cell culture and mouse models. In this study, we investigated the antitumor mechanism of cis-nerolidol in bladder carcinoma cell lines. The results of our experiments on two bladder carcinoma cell lines revealed that nerolidol inhibited cell proliferation and induced two distinct cell death pathways. We confirmed that cis-nerolidol induces DNA damage and ER stress. A mechanistic study identified a common cAMP, Ca2+, and MAPK axis involved in signal propagation and amplification, leading to ER stress. Inhibition of any part of this signaling cascade prevented both cell death pathways. The two cell death mechanisms can be distinguished by the involvement of caspases. The early occurring cell death pathway is characterized by membrane blebbing and cell swelling followed by membrane rupture, which can be prevented by the inhibition of caspase activation. In the late cell death pathway, which was found to be caspase-independent, cytoplasmic vacuolization and changes in cell shape were observed. cis-Nerolidol shows promising antitumor activity through an unorthodox mechanism of action that could help target resistant forms of malignancies, such as bladder cancer.

Ascidian gastrulation and blebbing activity of isolated endoderm blastomeres

Gastrulation is the first dynamic cell movement during embryogenesis. Endoderm and mesoderm cells are internalized into embryos during this process. Ascidian embryos provide a simple system for studying gastrulation in chordates. Gastrulation starts in spherical late 64-cell embryos with 10 endoderm blastomeres. The mechanisms of gastrulation in ascidians have been investigated, and a two-step model has been proposed. The first step involves apical constriction of endoderm cells, followed by apicobasal shortening in the second step. In this study, isolated ascidian endoderm progenitor cells displayed dynamic blebbing activity at the gastrula stage, although such a dynamic cell-shape change was not recognized in toto. Blebbing is often observed in migrating animal cells. In ascidians, endoderm cells displayed blebbing activity, while mesoderm and ectoderm cells did not. The timing of blebbing of isolated endoderm cells coincided with that of cell invagination. The constriction rate of apical surfaces correlated with the intensity of blebbing activity in each endoderm-lineage cell. Fibroblast growth factor (FGF) signaling was both necessary and sufficient for inducing blebbing activity, independent of cell fate specification. In contrast, the timing of initiation of blebbing and intensity of blebbing response to FGF signaling were controlled by intrinsic cellular factors. It is likely that the difference in intensity of blebbing activity between the anterior A-line and posterior B-line cells could account for the anteroposterior difference in the steepness of the archenteron wall. Inhibition of zygotic transcription, FGF signaling, and Rho kinase, all of which suppressed blebbing activity, resulted in incomplete apical constriction and failure of the eventual formation of cup-shaped gastrulae. Blebbing activity was involved in the progression and maintenance of apical constriction, but not in apicobasal shortening in whole embryos. Apical constriction is mediated by distinct blebbing-dependent and blebbing-independent mechanisms. Surface tension and consequent membrane contraction may not be the sole mechanical force for apical constriction and formation of cup-shaped gastrulae. The present study reveals the hidden cellular potential of endodermal cells during gastrulation and discusses the possible roles of blebbing in the invagination process.

Wnt4 and ephrinB2 instruct apical constriction via Dishevelled and non-canonical signaling

Apical constriction is a cell shape change critical to vertebrate neural tube closure, and the contractile force required for this process is generated by actin-myosin networks. The signaling cue that instructs this process has remained elusive. Here, we identify Wnt4 and the transmembrane ephrinB2 protein as playing an instructive role in neural tube closure as members of a signaling complex we termed WERDS (Wnt4, EphrinB2, Ror2, Dishevelled (Dsh2), and Shroom3). Disruption of function or interaction among members of the WERDS complex results in defects of apical constriction and neural tube closure. The mechanism of action involves an interaction of ephrinB2 with the Dsh2 scaffold protein that enhances the formation of the WERDS complex, which in turn, activates Rho-associated kinase to induce apical constriction. Moreover, the ephrinB2/Dsh2 interaction promotes non-canonical Wnt signaling and shows how cross-talk between two major signal transduction pathways, Eph/ephrin and Wnt, coordinate morphogenesis of the neural tube.

The centralspindlin complex regulates cytokinesis and morphogenesis in the C. elegans spermatheca.

Organ morphogenesis needs orchestration of a series of cellular events, including cell division, cell shape change, cell rearrangement and cell death. Cytokinesis, the final step of cell division, is involved in the control of organ size, shape and function. Mechanistically, it is unclear how the molecules involved in cytokinesis regulate organ size and shape. Here, we demonstrate that the centralspindlin complex coordinates cell division and epithelial morphogenesis by regulating cytokinesis. Loss of the centralspindlin components CYK-4 and ZEN-4 disrupts cell division, resulting in altered cell arrangement and malformation of the Caenorhabditis elegans spermatheca. Further investigation revealed that most spermathecal cells undergo nuclear division without completion of cytokinesis. Germline mutant-based analyses suggest that CYK-4 regulates cytokinesis of spermathecal cells in a GTPase activator activity-independent manner. Spermathecal morphology defects can be enhanced by double knockdown of rho-1 and cyk-4, and partially suppressed by double knockdown of cdc-42 and cyk-4. Thus, the centralspindlin components CYK-4 and ZEN-4, together with RHO-1 and CDC-42, are central players of a signaling network that guides spermathecal morphogenesis by enabling completion of cytokinesis.

Modulation of self-organizing circuits at deforming membranes by intracellular and extracellular factors.

Mechanical forces exerted to the plasma membrane induce cell shape changes. These transient shape changes trigger, among others, enrichment of curvature-sensitive molecules at deforming membrane sites. Strikingly, some curvature-sensing molecules not only detect membrane deformation but can also alter the amplitude of forces that caused to shape changes in the first place. This dual ability of sensing and inducing membrane deformation leads to the formation of curvature-dependent self-organizing signaling circuits. How these cell-autonomous circuits are affected by auxiliary parameters from inside and outside of the cell has remained largely elusive. Here, we explore how such factors modulate self-organization atthe micro-scale and its emerging properties at the macroscale.

High glucose causes developmental abnormalities in neuroepithelial cysts with actin and HK1 distribution changes.

It has been reported that the offspring of diabetic pregnant women have an increased risk for neural tube defects. Previous studies in animal models suggested that high glucose induces cell apoptosis and epigenetic changes in the developing neural tube. However, effects on other cellular aspects such as the cell shape changes were not fully investigated. Actin dynamics plays essential roles in cell shape change. Disruption on actin dynamics is known to cause neural tube defects. In the present study, we used a 3D neuroepithelial cyst model and a rosette model, both cultured from human embryonic stem cells, to study the cellular effects caused by high glucose. By using these models, we observed couple of new changes besides increased apoptosis. First, we observed that high glucose disturbed the distribution of pH3 positive cells in the neuroepithelial cysts. Secondly, we found that high glucose exposure caused a relatively smaller actin inner boundary enclosed area, which was unlikely due to osmolarity changes. We further investigated key glucose metabolic enzymes in our models and the results showed that the distribution of hexokinase1 (HK1) was affected by high glucose. We observed that hexokinase1 has an apical-basal polarized distribution and is highest next to actin at the boundaries. hexokinase1 was more diffused and distributed less polarized under high glucose condition. Together, our observations broadened the cellular effects that may be caused by high glucose in the developing neural tube, especially in the secondary neurulation process.

Photoablation at single cell resolution and its application in the Drosophila epidermis and peripheral nervous system.

Tissues contain diverse cell populations that, together, make up physiologically functional units. A remarkable example is the animal epidermis, where neuronal and non-neuronal cells intermingle to allow somatosensory perception. In the peripheral nervous system (PNS), the tight association between heterogenous cell types poses challenges when the structural and physiological contributions of neuronal and surrounding cells need to be dissected with suitable precision. When genetic tools for cell-specific, spatiotemporally controlled gene expression are not available, targeted cell ablation represents a considerable obstacle. Here, we describe an efficient method to overcome this limitation and demonstrate its application to the study of the differentiating Drosophila epidermis and PNS. This methodology relies on the use of near infrared (NIR) femtosecond (fs) laser pulses for ablation of the desired cells at the desired time. We show how to confine the photodamage to the targeted cell to induce its death, without harming neighbouring tissues or structures. We validated our approach in the Drosophila PNS by studying the responses of photo-ablated neurons, non-neuronal cells, and the surrounding epidermis. Diverse cellular behaviours including cell extrusion, cell rearrangements and cell shape changes can be monitored in vivo immediately after damage, as well as for several hours post-ablation with high optical resolution using confocal microscopy. This methodology provides a flexible tool to ablate individual cells with high precision and study morphological responses to cell loss in targeted areas or neighbouring structures. We anticipate that this protocol can be easily adapted to other model systems and tissues.

Exploration of cytotoxic potential and tubulin polymerization inhibition activity of cis-stilbene-1,2,3-triazole congeners.

To scrutinize cis-stilbene based molecules with potential anticancer and tubulin polymerization inhibition activity, a new series of cis-stilbene-1,2,3-triazole congeners was designed and synthesized via a click chemistry protocol. The cytotoxicity of these compounds 9a-j and 10a-j was screened against lung, breast, skin and colorectal cancer cell lines. Based on the results of MTT assay, we further evaluated the selectivity index of the most active compound 9j (IC50 3.25 ± 1.04 μM on HCT-116) by comparing its IC50 value (72.24 ± 1.20 μM) to that of the normal human cell line. Further, to confirm apoptotic cell death, cell morphology and staining studies (AO/EB, DAPI and Annexin V/PI) were carried out. The outcomes of studies showed apoptotic features like change in cell shape, cornering of nuclei, micronuclei formation, fragmented, bright, horseshoe-shaped nuclei, etc. Moreover, active compound 9j displayed G2/M phase cell cycle arrest with significant tubulin polymerization inhibition activity with an IC50 value of 4.51 μM. Additionally, in silico ADMET, molecular docking and molecular dynamic studies of 9j with 3E22 protein proved the binding of the compound at the colchicine binding site of tubulin.

Single-Cell Confinement Methods to Study Plant Cytoskeleton.

Progress in cytoskeletal research in animal systems has been accompanied by the development of single-cell systems (e.g., fibroblasts in culture). Single-cell systems exist for plant research, but the presence of a cell wall hinders the possibility to relate cytoskeleton dynamics to changes in cell shape or in mechanical stress pattern. Here we present two protocols to confine wall-less plant protoplasts in microwells with defined geometries. Either protocol might be more or less adapted to the question at hand. For instance, when using microwells made of agarose, the composition of the well can be easily modified to analyze the impact of biochemical cues. When using microwells in a stiff polymer (NOA73), protoplasts can be pressurized, and the wall of the well can be coated with cell wall components. Using both protocols, we could analyze microtubule and actin dynamics in vivo while also revealing the relative contribution of geometry and stress in their self-organization.

ATP accumulation in early resting cyst formation towards cryptobiosis in Colpoda Cucullus

Resting cyst formation is a crucial process of cryptobiosis in protists. In colpodid ciliates, cyst formation is accompanied by large-scale morphological changes such as changes of cell shape, resorption of cilia, and formation of a cyst wall; additionally, the cell cycle is arrested. These changes provide acquired tolerance against environmental stresses. During cyst formation, mitochondrial membrane potential is reduced and the level of the ATP synthase beta chain is suppressed, strongly indicating that metabolism has ceased. Here, however, we show that ATP levels are elevated during the initial phases of encystment implying that metabolism may not be completely suppressed. This finding suggests another aspect of resting cyst formation that is not applicable to cryptobiosis.

Systematic Analysis of Smooth Muscle and Cartilage Ring Formation during Mouse Tracheal Tubulogenesis.

The trachea tube is the exclusive route to allow gas exchange between the external environment and the lungs. Recent studies have shown the critical role of mesenchymal cells in tracheal tubulogenesis. Improved methods for studying the dynamics of the tracheal mesenchyme development are needed to investigate the cellular and molecular mechanisms during tracheal tubulogenesis. Here, we describe a detailed protocol for a systematic analysis of tracheal tube development to enable observing tracheal smooth muscle (SM) and cartilage ring formation. We describe immunostaining, confocal and stereomicroscopy imaging, and quantitative methods to study the process of tracheal SM and cartilage ring development, including SM cell alignment, polarization, and changes in cell shape as well as mesenchymal condensation. The technologies and approaches described here not only improve analysis of the patterning of the developing trachea but also help uncover the mechanisms underlying airway disease. This protocol also provides a useful technique to analyze cell organization, polarity, and nuclear shape in other organ systems.

Live Imaging of Nurse Cell Behavior in Late Stages of Drosophila Oogenesis.

Drosophila oogenesis is a powerful and tractable model for studies of cell and developmental biology due to the multitude of well-characterized events in both germline and somatic cells, the ease of genetic manipulation in fruit flies, and the large number of egg chambers produced by each fly. Recent improvements in live imaging and ex vivo culturing protocols have enabled researchers to conduct more detailed, longer-term studies of egg chamber development, enabling insights into fundamental biological processes. Here, we present a protocol for dissection, culturing, and imaging of late-stage egg chambers to study intercellular and directional cytoplasmic flow during "nurse cell dumping." This critical developmental process towards the latter stages of oogenesis (stages 10b/11) results in rapid growth of the oocyte and shrinkage of the nurse cells and is accompanied by dynamic changes in cell shape. We also describe a procedure to record high-time-resolution movies of the flow of unlabeled cytoplasmic contents within nurse cells and through cytoplasmic bridges in the nurse cell cluster using reflection microscopy, and we describe two ways to analyze data from nurse cell dumping.

Live-Imaging Analysis of Epithelial Zippering During Mouse Neural Tube Closure.

Zippering is a phenomenon of tissue morphogenesis whereby fusion between opposing epithelia progresses unidirectionally over significant distances, similar to the travel of a zip fastener, to ultimately ensure closure of an opening. A comparable process can be observed during Drosophila dorsal closure and mammalian wound healing, while zippering is employed by numerous organs such as the optic fissure, palatal shelves, tracheoesophageal foregut, and presumptive genitalia to mediate tissue sealing during normal embryonic development. Particularly striking is zippering propagation during neural tube morphogenesis, where the fusion point travels extensively along the embryonic axis to ensure closure of the neural tube. Advances in time-lapse microscopy and culture conditions have opened the opportunity for successful imaging of whole-mouse embryo development over time, providing insights into the precise cellular behavior underlying zippering propagation. Studies in mouse and the ascidian Ciona have revealed the fine-tuned cell shape changes and junction remodeling which occur at the site of zippering during neural tube morphogenesis. Here, we describe a step-by-step method for imaging at single-cell resolution the process of zippering and tissue remodeling which occurs during closure of the spinal neural tube in mouse. We also provide instructions and suggestions for quantitative morphometric analysis of cell behavior during zippering progression. This procedure can be further combined with genetic mutant models (e.g., knockouts), offering the possibility of studying the dynamics of tissue fusion and zippering propagation, which underlie a wide range of open neural tube defects.

Active nematics with deformable particles.

The hydrodynamic theory of active nematics has been often used to describe the spatio-temporal dynamics of cell flows and motile topological defects within soft confluent tissues. Those theories, however, often rely on the assumption that tissues consist of cells with a fixed, anisotropic shape and do not resolve dynamical cell shape changes due to flow gradients. In this paper we extend the continuum theory of active nematics to include cell shape deformability. We find that circular cells in tissues must generate sufficient active stress to overcome an elastic barrier to deforming their shape in order to drive tissue-scale flows. Above this threshold the systems enter a dynamical steady-state with regions of elongated cells and strong flows coexisting with quiescent regions of isotropic cells.

Tailup expression in Drosophila larval and adult cardiac valve cells.

In Drosophila larvae, the direction of blood flow within the heart tube, as well as the diastolic filling of the posterior heart chamber, is regulated by a single cardiac valve. This valve is sufficient to close the heart tube at the junction of the ventricle and the aorta and is formed by only two cells; both are integral parts of the heart tube. The valve cells regulate hemolymph flow by oscillating between a spherical and a flattened cell shape during heartbeats. At the spherical stage, the opposing valve cells close the heart lumen. The dynamic cell shape changes of valve cells are supported by a dense, criss-cross orientation of myofibrils and the presence of the valvosomal compartment, a large intracellular cavity. Both structures are essential for the valve cells' function. In a screen for factors specifically expressed in cardiac valve cells, we identified the transcription factor Tailup. Knockdown of tailup causes abnormal orientation and differentiation of cardiac muscle fibers in the larval aorta and inhibits the formation of the ventral longitudinal muscle layer located underneath the heart tube in the adult fly and affects myofibrillar orientation of valve cells. Furthermore, we have identified regulatory sequences of tup that control the expression of tailup in the larval and adult valve cells.

Preprint Highlight: Zasp52 strengthens whole embryo tissue integrity through supracellular actomyosin networks.

Tissue folding requires proper coordination between constricting cells and their neighbors that are not undergoing cell shape changes. Many studies have addressed how different cell populations contribute to tissue rearrangements but few have shown how forces are then restrained to specific compartments. Here, the authors show that Zasp52 acts as a coordinator between junctional actomyosin in different tissues during embryogenesis in Drosophila melanogaster. Zasp52 reinforces actomyosin supracellular cables at tissue boundaries, balancing force distribution and allowing cell shape changes to occur. This model explains how force propagation is restrained to a given tissue through the action of actomyosin cables and Zasp52, which create boundaries that mechanically isolate tissues as they are being rearranged. Given the evolutionary conservation of Zasp52, it may also perform similar roles during development in other animals. This preprint has been assigned the following badges: New Hypothesis, Cross-Validation, Open Data. Read the preprint on bioRxiv ( Ashour et al., 2022 ): https://doi.org/10.1101/2022.10.11.511783 .

Epithelial morphogenesis in the Drosophila egg chamber requires Parvin and ILK.

Integrins are the major family of transmembrane proteins that mediate cell-matrix adhesion and have a critical role in epithelial morphogenesis. Integrin function largely depends on the indirect connection of the integrin cytoplasmic tail to the actin cytoskeleton through an intracellular protein network, the integrin adhesome. What is currently unknown is the role of individual integrin adhesome components in epithelia dynamic reorganization. Drosophila egg chamber consists of the oocyte encircled by a monolayer of somatic follicle epithelial cells that undergo specific cell shape changes. Egg chamber morphogenesis depends on a developmental array of cell-cell and cell-matrix signalling events. Recent elegant work on the role of integrins in the Drosophila egg chamber has indicated their essential role in the early stages of oogenesis when the pre-follicle cells assemble into the follicle epithelium. Here, we have focused on the functional requirement of two key integrin adhesome components, Parvin and Integrin-Linked Kinase (ILK). Both proteins are expressed in the developing ovary from pupae to the adult stage and display enriched expression in terminal filament and stalk cells, while their genetic removal from early germaria results in severe disruption of the subsequent oogenesis, leading to female sterility. Combining genetic mosaic analysis of available null alleles for both Parvin and Ilk with conditional rescue utilizing the UAS/Gal4 system, we found that Parvin and ILK are required in pre-follicle cells for germline cyst encapsulation and stalk cell morphogenesis. Collectively, we have uncovered novel developmental functions for both Parvin and ILK, which closely synergize with integrins in epithelia.