Collective Cell Migration Research Articles

175 Collective cell migration of stratified epithelia in confined microenvironments

175 Collective cell migration of stratified epithelia in confined microenvironments

ARHGAP29 promotes keratinocyte proliferation and migration in vitro and is dispensable for in vivo wound healing.

RhoA GTPases play critical roles in actin cytoskeletal remodeling required for controlling a diverse range of cellular functions including cell proliferation, adhesion, migration and changes in cell shape, all required for cutaneous wound healing. RhoA cycles between an active GTP-bound and an inactive GDP-bound form, a process regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). ARHGAP29 is a GAP expressed in skin keratinocytes and is decreased in the absence of interferon regulator factor 6, a critical regulator of cell proliferation, migration, and wound healing. However, the role for ARHGAP29 in keratinocyte biology is unknown. We generated ARHGAP29 knockdown keratinocyte cell lines and show they displayed increased filamentous actin, phospho-myosin regulatory light chain, cell area and population doubling time. Furthermore, we found that ARHGAP29 knockdown keratinocytes displayed significant delays in scratch wound closure in both single and collective cell migration conditions; these delays were rescued by both adding back ARHGAP29 or adding a ROCK inhibitor to ARHGAP29 knockdown cells. In vivo, however, Arhgap29 heterozygotes or keratinocyte-specific knockouts showed on-time wound healing. These data demonstrate that ARHGAP29 is required for keratinocyte morphology, proliferation and migration in vitro but is dispensable during wound healing in vivo.

Loss of myosin light chain kinase induces the cellular senescence associated secretory phenotype to promote breast epithelial cell migration

Overexpression or activation of oncogenes or loss of tumor-suppressor genes can induce cellular senescence as a defense mechanism against tumor development, thereby maintaining cellular homeostasis. However, cancer cells can circumvent this senescent state and continue to spread. Myosin light chain kinase (MLCK) is downregulated in many breast cancers. Here we report that downregulation of MLCK in normal breast epithelial cells induces a senescence-associated secretory phenotype and stimulates migration. The reduction of MLCK results in increased p21Cip1 expression, dependent on p53 and the AKT-mammalian target of rapamycin pathway. Subsequently, p21Cip1 promotes the secretion of soluble ICAM-1, IL-1α, IL-6 and IL-8, thereby enhancing collective cell migration in a non-cell-autonomous manner. These findings provide new mechanistic insights into the role of MLCK in cellular senescence and cancer progression.

Suppression of Pcdh8/paraxial protocadherin is required for efficient neighbor exchange in morphogenetic cell movement during zebrafish notochord formation

In certain forms of collective cell migration, changes in neighboring cells (neighbor exchange, NE) are essential. In the axial mesoderm in zebrafish, for example, the notochord is established through cell movements known as convergence and extension (C&E), which involves NE. For NE to occur efficiently, the balance between cell-scale and supracellular stresses plays a crucial role, but the molecular basis of how these stresses are controlled remains unclear. In this study, we focused on Pcdh8/Paraxial protocadherin (PAPC), which is specifically suppressed in the region (notochord) where and at the time (early gastrula) when extensive C&E occurs. Forced expression of PAPCΔC (PAPC lacking its intracellular domain) persisted in the developing notochord and resulted in morphogenetic defects in zebrafish. PAPCΔC was found to downregulate NE in the notochord in a homophilic contact-dependent manner. By examining oil droplets inserted between cells, we revealed that while cell-scale stresses were apparently unaffected, the direction of bias in the supracellular stresses was stabilized by the introduction of PAPCΔC in the notochordal region. Taken together, our results suggest that suppression of PAPC in the notochord is required to modify supracellular stresses and provide the conditions in which NE occurs efficiently, thus promoting morphogenetic cell movements.

Dynamic cluster field modeling of collective chemotaxis

Collective migration of eukaryotic cells is often guided by chemotaxis, and is critical in several biological processes, such as cancer metastasis, wound healing, and embryogenesis. Understanding collective chemotaxis has challenged experimental, theoretical and computational scientists because cells can sense very small chemoattractant gradients that are tightly controlled by cell-cell interactions and the regulation of the chemoattractant distribution by the cells. Computational models of collective cell migration that offer a high-fidelity resolution of the cell motion and chemoattractant dynamics in the extracellular space have been limited to a small number of cells. Here, we present Dynamic cluster field modeling (DCF), a novel computational method that enables simulations of collective chemotaxis of cellular systems with O(1000)\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\mathcal {O}}(1000)$$\\end{document} cells and high-resolution transport dynamics of the chemoattractant in the time-evolving extracellular space. We illustrate the efficiency and predictive capabilities of our approach by comparing our numerical simulations with experiments in multiple scenarios that involve chemoattractant secretion and uptake by the migrating cells, cell migration in confined spaces, regulation of the attractant distribution by cell motion, and interactions of the chemoattractant with an enzyme. The proposed algorithm opens new opportunities to address outstanding problems that involve collective cell migration in the central nervous system, immune response and cancer metastasis.

The Drosophila hematopoietic niche assembles through collective cell migration controlled by neighbor tissues and Slit-Robo signaling.

Niches are often found in specific positions in tissues relative to the stem cells they support. Consistency of niche position suggests that placement is important for niche function. However, the complexity of most niches has precluded a thorough understanding of how their proper placement is established. To address this, we investigated the formation of a genetically tractable niche, the Drosophila Posterior Signaling Center (PSC), the assembly of which had not been previously explored. This niche controls hematopoietic progenitors of the lymph gland (LG). PSC cells were previously shown to be specified laterally in the embryo, but ultimately reside dorsally, at the LG posterior. Here, using live-imaging, we show that PSC cells migrate as a tight collective and associate with multiple tissues during their trajectory to the LG posterior. We find that Slit emanating from two extrinsic sources, visceral mesoderm and cardioblasts, is required for the PSC to remain a collective, and for its attachment to cardioblasts during migration. Without proper Slit-Robo signaling, PSC cells disperse, form aberrant contacts, and ultimately fail to reach their stereotypical position near progenitors. Our work characterizes a novel example of niche formation and identifies an extrinsic signaling relay that controls precise niche positioning.

A large reverse-genetic screen identifies numerous regulators of testis nascent myotube collective cell migration and collective organ sculpting.

Collective cell migration is critical for morphogenesis, homeostasis, and wound healing. During development migrating mesenchymal cells form tissues that shape some of the body's organs. We have developed a powerful model for examining this, exploring how Drosophila testis nascent myotubes migrate onto the testis during pupal development, forming the muscles that ensheath it and also creating its characteristic spiral shape. To define genes that regulate this process, we have carried out RNAseq to define the genes expressed in myotubes during migration. Using this dataset, we curated a list of 131 ligands, receptors and cytoskeletal regulators, including all Rho-family GTPase GAPs and GEFs, as candidates. We then used the GAL4/UAS system to express 279 shRNAs targeting these genes, using the muscle specific driver dMef2>GAL4, and examined the adult testis. We identified 29 genes with diverse roles in testis morphogenesis. Some have phenotypes consistent with defects in collective cell migration, while others alter testis shape in different ways, revealing some of the underlying logic of testis morphogenesis. We followed up one of these genes in more detail-that encoding the Rho-family GEF dPix. dPix knockdown leads to a drastic reduction in migration and a substantial loss of muscle coverage. Our data suggest different isoforms of dPix play distinct roles in this process, reveal a role for its protein partner Git. We also explore whether cdc42 activity regulation or cell adhesion are among the dPix mechanisms of action. Together, our RNAseq dataset and genetic analysis will provide an important resource for the community to explore cell migration and organ morphogenesis.

Plexin/Semaphorin Antagonism Orchestrates Collective Cell Migration, Gap Closure and Organ sculpting by Contact-Mesenchymalization.

Cell behavior emerges from the intracellular distribution of properties like protrusion, contractility and adhesion. Thus, characteristic emergent rules of collective migration can arise from cell-cell contacts locally tweaking architecture - orchestrating self-regulation during development, wound healing, and cancer progression. The new Drosophila testis-nascent-myotube-system allows dissection of contact-dependent migration in vivo at high resolution. Here, we describe a process driving gap-closure during migration: Contact-mesenchymalization via the axon guidance factor Plexin A. This is crucial for testis myotubes to migrate as a continuous sheet, allowing normal sculpting-morphogenesis. Cells must stay filopodial and dynamically ECM-tethered near cell-cell contacts to spread while collectively moving. Our data suggest Semaphorin 1B acts as a Plexin A antagonist, fine-tuning activation. Our data reveal a contact-dependent mechanism to maintain sheet-integrity during migration, driving organ-morphogenesis using a highly conserved pathway. This is relevant for understanding mesenchymal organ-sculpting and gap-closure in migratory contexts like angiogenesis.

Angiomotin cleavage promotes leader formation and collective cell migration.

Collective cell migration (CCM) is involved in multiple biological processes, including embryonic morphogenesis, angiogenesis, and cancer invasion. However, the molecular mechanisms underlying CCM, especially leader cell formation, are poorly understood. Here, we show that a signaling pathway regulating angiomotin (AMOT) cleavage plays a role in CCM, using mammalian epithelial cells and mouse models. In a confluent epithelial monolayer, full-length AMOT localizes at cell-cell junctions and limits cell motility. After cleavage, the C-terminal fragment of AMOT (AMOT-CT) translocates to the cell-matrix interface to promote the maturation of focal adhesions (FAs), generate traction force, and induce leader cell formation. Meanwhile, decreased full-length AMOT at cell-cell junctions leads to tissue fluidization and coherent migration of cell collectives. Hence, the cleavage of AMOT serves as a molecular switch to generate polarized contraction, promoting leader cell formation and CCM.

Tao and Rap2l ensure proper Misshapen activation and levels during Drosophila border cell migration.

Collective cell migration is fundamental in development, wound healing, and metastasis. During Drosophila oogenesis, border cells (BCs) migrate collectively inside the egg chamber, controlled by the Ste20-like kinase Misshapen (Msn). Msn coordinates the restriction of protrusion formation and contractile forces within the cluster. Here, we demonstrate that Tao acts as an upstream activator of Msn in BCs. Depleting Tao significantly impedes BC migration, producing a phenotype similar to Msn loss of function. Furthermore, we show that the localization of Msn relies on its citron homology (CNH) domain, which interacts with the small GTPase Rap2l. Rap2l promotes the trafficking of Msn to the endolysosomal pathway. Depleting Rap2l elevates Msn levels by reducing its trafficking into late endosomes and increases overall contractility. These data suggest that Tao promotes Msn activation, while global Msn protein levels are controlled via Rap2l and the endolysosomal degradation pathway. Thus, two mechanisms ensure appropriate Msn levels and activation in BCs.

Notch-1 regulates collective breast cancer cell migration by controlling intercellular junction and cytoskeletal organization.

Pathological observations show that cancer cells frequently invade the surrounding normal tissue in collective rather than individual cell migration. However, general principles governing collective cell migration remain to be discovered. Different from individual cell migration, we demonstrated that the Notch-1-activation reduced collective cells speed and distances. In particular, Notch-1-activation induced cellular cytoskeletal remodelling, strengthened the intercellular junctions and cell-matrix adhesions. Mechanistically, Notch-1 activation prevented the phosphorylation of GSK-3β and the translocation of cytoplasmic free β-catenin to the nucleus, which increased E-cadherin expression and tight intercellular junctions. Moreover, Notch-1 signalling also activated the RhoA/ROCK pathway, promoting reorganization of F-actin and contractile forces produced by myosin. Further, Notch-1 activation increased cell adhesion to the extracellular substrate, which inhibited collective cell migration. These findings highlight that cell adhesions and cell-cell junctions contribute to collective cell migration and provide new insights into mechanisms of the modulation of Notch-1 signalling pathway on cancer cell malignancy.

Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters during collective cell migration of epithelial cells.

Epidermal growth factor receptor ligands (EGFRLs) consist of seven proteins. In stark contrast to the amassed knowledge concerning the epidermal growth factor receptors themselves, the extracellular dynamics of individual EGFRLs remain elusive. Here, employing fluorescent probes and a tool for triggering ectodomain shedding of EGFRLs, we show that EREG, a low-affinity EGFRL, exhibits the most rapid and efficient activation of EGFR in confluent epithelial cells and mouse epidermis. In Madin-Darby canine kidney (MDCK) renal epithelial cells, EGFR- and ERK-activation waves propagate during collective cell migration in an ADAM17 sheddase- and EGFRL-dependent manner. Upon induction of EGFRL shedding, radial ERK activation waves were observed in the surrounding receiver cells. Notably, the low-affinity ligands EREG and AREG mediated faster and broader ERK waves than the high-affinity ligands. The integrity of tight/adherens junctions was essential for the propagation of ERK activation, implying that the tight intercellular spaces prefer the low-affinity EGFRL to the high-affinity ligands for efficient signal transmission. To validate this observation in vivo , we generated EREG-deficient mice expressing the ERK biosensor and found that ERK wave propagation and cell migration were impaired during skin wound repair. In conclusion, we have quantitatively demonstrated the distinctions among EGFRLs in shedding, diffusion, and target cell activation in physiological contexts. Our findings underscore the pivotal role of low-affinity EGFRLs in rapid intercellular signal transmission.

Dysfunction of Calcyphosine-Like gene impairs retinal angiogenesis through the MYC axis and is associated with familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a severe genetic disorder characterized by incomplete vascularization of the peripheral retina and associated symptoms that can lead to vision loss. However, the underlying genetic causes of approximately 50% of FEVR cases remain unknown. Here, we report two heterozygous variants in calcyphosine-like gene (CAPSL) that is associated with FEVR. Both variants exhibited compromised CAPSL protein expression. Vascular endothelial cell (EC)-specific inactivation of Capsl resulted in delayed radial/vertical vascular progression, compromised endothelial proliferation/migration, recapitulating the human FEVR phenotypes. CAPSL-depleted human retinal microvascular endothelial cells (HRECs) exhibited impaired tube formation, decreased cell proliferation, disrupted cell polarity establishment, and filopodia/lamellipodia formation, as well as disrupted collective cell migration. Transcriptomic and proteomic profiling revealed that CAPSL abolition inhibited the MYC signaling axis, in which the expression of core MYC targeted genes were profoundly decreased. Furthermore, a combined analysis of CAPSL-depleted HRECs and c-MYC-depleted human umbilical vein endothelial cells uncovered similar transcription patterns. Collectively, this study reports a novel FEVR-associated candidate gene, CAPSL, which provides valuable information for genetic counseling of FEVR. This study also reveals that compromised CAPSL function may cause FEVR through MYC axis, shedding light on the potential involvement of MYC signaling in the pathogenesis of FEVR.

Glycerol-blended chitosan membranes with directional micro-grooves and reduced stiffness improve Schwann cell wound healing

Regenerative medicine is continuously looking for new natural, biocompatible and possibly biodegradable materials, but also mechanically compliant. Chitosan is emerging as a promising FDA-approved biopolymer for tissue engineering, however, its exploitation in regenerative devices is limited by its brittleness and can be further improved, for example by blending it with other materials or by tuning its superficial microstructure. Here, we developed membranes made of chitosan (Chi) and glycerol, by solvent casting, and micro-patterned them with directional geometries having different levels of axial symmetry. These membranes were characterized by light microscopies, atomic force microscopy (AFM), by thermal, mechanical and degradation assays, and also testedin vitroas scaffolds with Schwann cells (SCs). The glycerol-blended Chi membranes are optimized in terms of mechanical properties, and present a physiological-grade Young's modulus (≈0.7 MPa). The directional topographies are effective in directing cell polarization and migration and in particular are highly performant substrates for collective cell migration. Here, we demonstrate that a combination of a soft compliant biomaterial and a topographical micropatterning can improve the integration of these scaffolds with SCs, a fundamental step in the peripheral nerve regeneration process.

Immunohistochemical Expression of Bcl-2, E-cadherin, CD34 and CD105 in Basaloid Squamous Cell Carcinoma - An In Vitro Study

Abstract Introduction: Basaloid squamous cell carcinoma (BSCC) is a rare aggressive variant of oral squamous cell carcinoma (OSCC) with a high propensity for distant metastasis. In this article, we present clinicopathological and survival data of eight cases of BSCC and further analyse the behaviour of these tumours with the help of E-cadherin, CD34, CD105 and B cell lymphoma-2 (Bcl-2) immunoexpression. Materials and Methods: Histopathologically confirmed cases of BSCC were retrieved from the department archives. Clinicopathological details and survival data of these patients were collected. Immunohistochemical analysis was performed with Bcl-2, E-cadherin, CD34 and CD105 on these cases and compared with different grades of OSCC (well differentiated, moderately differentiated and poorly differentiated). The statistical analysis was done using IBM SPSS software version 23. Results: BSCC was seen commonly in males of age group 49–71 years and predominantly reported in the retromolar trigone. Bcl-2 expression was significantly lower in BSCCs when compared to the conventional OSCC groups (P < 0.05). E-cadherin expression showed no significant difference between BSCC and well-differentiated OSCC group (P = 0.487). The overall mean survival for patients with BSCC was 6.37 months. Discussion: BSCCs of the oral cavity show increased CD105, CD34, E-cadherin and low Bcl-2 labelling. A substantial relationship between the tumour neo-angiogenesis, collective cell migration and apoptotic property could be related to the aggressive nature of this tumour and its poor overall survival rate. BSCCs are common in middle to older aged male and show increased expression of CD105, CD34 and E-cadherin.

Regulation of intercellular viscosity by E-cadherin-dependent phosphorylation of EGFR in collective cell migration

Collective cell migration is crucial in various physiological processes, including wound healing, morphogenesis, and cancer metastasis. Adherens Junctions (AJs) play a pivotal role in regulating cell cohesion and migration dynamics during tissue remodeling. While the role and origin of the junctional mechanical tension at AJs have been extensively studied, the influence of the actin cortex structure and dynamics on junction plasticity remains incompletely understood. Moreover, the mechanisms underlying stress dissipation at junctions are not well elucidated. Here, we found that the ligand-independent phosphorylation of epithelial growth factor receptor (EGFR) downstream of de novo E-cadherin adhesion orchestrates a feedback loop, governing intercellular viscosity via the Rac pathway regulating actin dynamics. Our findings highlight how the E-cadherin-dependent EGFR activity controls the migration mode of collective cell movements independently of intercellular tension. This modulation of effective viscosity coordinates cellular movements within the expanding monolayer, inducing a transition from swirling to laminar flow patterns while maintaining a constant migration front speed. Additionally, we propose a vertex model with adjustable junctional viscosity, capable of replicating all observed cellular flow phenotypes experimentally.

Back Cover Image

ON THE BACK COVER: DA‐Raf inhibits ERK activation, resulting in the suppression of collective cell migration and invasion of KRAS‐mutant cancer cells. In human pancreatic carcinoma MIA PaCa‐2 cells, phospho‐ERK1/2 (magenta) is suppressed by the expression of EGFP‐DA‐Raf (green). EGFP‐DA‐Raf is concentrated on the plasma membrane.Credit: Aoi Matsuda and Takeshi Endo (Chiba University, Japan) image

A Simple Method for Visualization and Quantitative Evaluation of Single and Collective Cell Migration in Bright Field Microscopy Images

Cell migration is observed in various cases such as embryonic and lesion developments. The migration directly influences the phenomena around the migration path. Bright field microscopy, generally used for cell observation, is effective in tracking cell movement, but the detection of the cell outline via image processing methods partially fails. In this study, a simple method, utilizing the intensity fluctuation of the image caused by the passage of a cell as a parameter for evaluation of the cell movement, has been proposed to visualize the region where the cell passed and quantitatively evaluate its frequency by the fluctuation pattern. The map that depicts the activity of cell movement is created, and the geometrical characteristics of each region and fluctuation degree are quantitatively shown. Because detection of the cell outline is not necessary, this method is applicable to collective cells as well as single cells. When this method was applied to the images of hemocytes inHalocynthia roretzi(Ascidiacea), which were composed of single and collective cells and showed complex patterns in movement, the map and quantitative parameters for the fluctuation were successfully obtained. The method can be improved by the calibration of intensity distribution and applied to cells in various species.

Sensory nerves drive migration of dental pulp stem cells via the CGRP-Ramp1 axis in pulp repair

Dental pulp stem cells (DPSCs) are responsible for maintaining pulp structure and function after pulp injury. DPSCs migrate directionally to the injury site before differentiating into odontoblast-like cells, which is a prerequisite and a determinant in pulp repair. Increasing evidence suggests that sensory neuron-stem cell crosstalk is critical for maintaining normal physiological functions, and sensory nerves influence stem cells mainly by neuropeptides. However, the role of sensory nerves on DPSC behaviors after pulp injury is largely unexplored. Here, we find that sensory nerves released significant amounts of calcitonin gene-related peptide (CGRP) near the injury site, acting directly on DPSCs via receptor activity modifying protein 1 (RAMP1) to promote collective migration of DPSCs to the injury site, and ultimately promoting pulp repair. Specifically, sensory denervation leads to poor pulp repair and ectopic mineralization, in parallel with that DPSCs failed to be recruited to the injury site. Furthermore, in vitro evidence shows that sensory nerve-deficient microenvironment suppressed DPSC migration prominently among all related behaviors. Mechanistically, the CGRP-Ramp1 axis between sensory neurons and DPSCs was screened by single-cell RNA-seq analysis and immunohistochemical studies confirmed that the expression of CGRP rather than Ramp1 increases substantially near the damaged site. We further demonstrated that CGRP released by sensory nerves binds the receptor Ramp1 on DPSCs to facilitate cell collective migration by an indirect co-culture system using conditioned medium from trigeminal neurons, CGRP recombinant protein and antagonists BIBN4096. The treatment with exogenous CGRP promoted the recruitment of DPSCs, and ultimately enhanced the quality of pulp repair. Targeting the sensory nerve could therefore provide a new strategy for stem cell-based pulp repair and regeneration.

Substrate stress relaxation regulates monolayer fluidity and leader cell formation for collectively migrating epithelia.

Groups of cells must coordinate their movements in order to sculpt organs during development and maintain tissues. The mechanical properties of the underlying substrate on which cells reside are known to influence key aspects of single and collective cell migration. Despite being a nearly universal feature of biological tissues, the role of viscoelasticity (i.e., fluid-like and solid-like behavior) in collective cell migration is unclear. Using tunable engineered biomaterials, we demonstrate that sheets of epithelial cells display enhanced migration on slower-relaxing (more elastic) substrates relative to faster-relaxing (more viscous) substrates. Building our understanding of tissue-substrate interactions and collective cell dynamics provides insights into approaches for tissue engineering and regenerative medicine, and therapeutic interventions to promote health and treat disease.