Quantitative 3D analysis of complex single border cell behaviors in coordinated collective cell migration

During embryonic development, tissues undergo major rearrangements that lead to germ layer positioning, patterning, and organ morphogenesis. Often these morphogenetic movements are accomplished by the coordinated and cooperative migration of the constituent cells, referred to as collective cell migration. The molecular and biomechanical mechanisms underlying collective migration of developing tissues have been investigated in a variety of models, including border cell migration, tracheal branching, blood vessel sprouting, and the migration of the lateral line primordium, neural crest cells, or head mesendoderm. Here we review recent advances in understanding collective migration in these developmental models, focusing on the interaction between cells and guidance cues presented by the microenvironment and on the role of cell–cell adhesion in mechanical and behavioral coupling of cells within the collective.

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Tousled-like kinase regulates cytokine-mediated communication between cooperating cell types during collective border cell migration

Molecular Biology of the Cell ◽

10.1091/mbc.e15-05-0327 ◽

2016 ◽

Vol 27 (1) ◽

pp. 12-19 ◽

Cited By ~ 7

Author(s):

Wenjuan Xiang ◽

Dabing Zhang ◽

Denise J. Montell

Keyword(s):

Cell Migration ◽

Cell Fate ◽

Molecular Mechanisms ◽

Metastatic Cancer ◽

Cell Types ◽

Collective Cell Migration ◽

Border Cells ◽

Border Cell ◽

Stat Signaling ◽

Border Cell Migration

Collective cell migration is emerging as a major contributor to normal development and disease. Collective movement of border cells in the Drosophila ovary requires cooperation between two distinct cell types: four to six migratory cells surrounding two immotile cells called polar cells. Polar cells secrete a cytokine, Unpaired (Upd), which activates JAK/STAT signaling in neighboring cells, stimulating their motility. Without Upd, migration fails, causing sterility. Ectopic Upd expression is sufficient to stimulate motility in otherwise immobile cells. Thus regulation of Upd is key. Here we report a limited RNAi screen for nuclear proteins required for border cell migration, which revealed that the gene encoding Tousled-like kinase (Tlk) is required in polar cells for Upd expression without affecting polar cell fate. In the absence of Tlk, fewer border cells are recruited and motility is impaired, similar to inhibition of JAK/STAT signaling. We further show that Tlk in polar cells is required for JAK/STAT activation in border cells. Genetic interactions further confirmed Tlk as a new regulator of Upd/JAK/STAT signaling. These findings shed light on the molecular mechanisms regulating the cooperation of motile and nonmotile cells during collective invasion, a phenomenon that may also drive metastatic cancer.

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Effect of Geometric Curvature on Collective Cell Migration in Tortuous Microchannel Devices

Micromachines ◽

10.3390/mi11070659 ◽

2020 ◽

Vol 11 (7) ◽

pp. 659

Author(s):

Mazlee Bin Mazalan ◽

Mohamad Anis Bin Ramlan ◽

Jennifer Hyunjong Shin ◽

Toshiro Ohashi

Keyword(s):

Tissue Engineering ◽

Cell Migration ◽

Underlying Mechanism ◽

Collective Cell Migration ◽

Tissue Scaffold ◽

Cell Behaviors ◽

Mechanical Signals ◽

Geometrical Constraints ◽

Engineered Tissue ◽

Microfabrication Technology

Collective cell migration is an essential phenomenon in many naturally occurring pathophysiological processes, as well as in tissue engineering applications. Cells in tissues and organs are known to sense chemical and mechanical signals from the microenvironment and collectively respond to these signals. For the last few decades, the effects of chemical signals such as growth factors and therapeutic agents on collective cell behaviors in the context of tissue engineering have been extensively studied, whereas those of the mechanical cues have only recently been investigated. The mechanical signals can be presented to the constituent cells in different forms, including topography, substrate stiffness, and geometrical constraint. With the recent advancement in microfabrication technology, researchers have gained the ability to manipulate the geometrical constraints by creating 3D structures to mimic the tissue microenvironment. In this study, we simulate the pore curvature as presented to the cells within 3D-engineered tissue-scaffolds by developing a device that features tortuous microchannels with geometric variations. We show that both cells at the front and rear respond to the varying radii of curvature and channel amplitude by altering the collective migratory behavior, including cell velocity, morphology, and turning angle. These findings provide insights into adaptive migration modes of collective cells to better understand the underlying mechanism of cell migration for optimization of the engineered tissue-scaffold design.

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A Drosophila RNAi screen reveals conserved glioblastoma-related adhesion genes that regulate collective cell migration

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab356 ◽

2021 ◽

Author(s):

Nirupama Kotian ◽

Katie M Troike ◽

Kristen N Curran ◽

Justin D Lathia ◽

Jocelyn A McDonald

Keyword(s):

Cell Migration ◽

Cell Adhesion ◽

Cell Invasion ◽

Brain Parenchyma ◽

Cell Junction ◽

Collective Cell Migration ◽

Border Cells ◽

Bioinformatics Analyses ◽

Border Cell ◽

Matrix Cell

Abstract Migrating cell collectives are key to embryonic development but also contribute to invasion and metastasis of a variety of cancers. Cell collectives can invade deep into tissues, leading to tumor progression and resistance to therapies. Collective cell invasion is also observed in the lethal brain tumor glioblastoma, which infiltrates the surrounding brain parenchyma leading to tumor growth and poor patient outcomes. Drosophila border cells, which migrate as a small cell cluster in the developing ovary, are a well-studied and genetically accessible model used to identify general mechanisms that control collective cell migration within native tissue environments. Most cell collectives remain cohesive through a variety of cell-cell adhesion proteins during their migration through tissues and organs. In this study, we first identified cell adhesion, cell matrix, cell junction, and associated regulatory genes that are expressed in human brain tumors. We performed RNAi knockdown of the Drosophila orthologs in border cells to evaluate if migration and/or cohesion of the cluster was impaired. From this screen, we identified eight adhesion-related genes that disrupted border cell collective migration upon RNAi knockdown. Bioinformatics analyses further demonstrated that subsets of the orthologous genes were elevated in the margin and invasive edge of human glioblastoma patient tumors. These data together show that conserved cell adhesion and adhesion regulatory proteins with potential roles in tumor invasion also modulate collective cell migration. This dual screening approach for adhesion genes linked to glioblastoma and border cell migration thus may reveal conserved mechanisms that drive collective tumor cell invasion.

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Fascin limits Myosin activity within Drosophila border cells to control substrate stiffness and promote migration

10.1101/2021.04.27.441651 ◽

2021 ◽

Author(s):

Maureen C. Lamb ◽

Chathuri P. Kaluarachchi ◽

Thiranjeewa I. Lansakara ◽

Yiling Lan ◽

Alexei V. Tivanski ◽

...

Keyword(s):

Cell Migration ◽

Nurse Cell ◽

Cancer Metastasis ◽

Critical Role ◽

Substrate Stiffness ◽

Cell Stiffness ◽

Collective Cell Migration ◽

Border Cells ◽

Border Cell ◽

Border Cell Migration

AbstractA key regulator of collective cell migrations, which drive development and cancer metastasis, is substrate stiffness. Increased substrate stiffness promotes migration and is controlled by Myosin. Using Drosophila border cell migration as a model of collective cell migration, we identify, for the first time, that the actin bundling protein Fascin limits Myosin activity in vivo. Loss of Fascin results in: increased activated Myosin on the border cells and their substrate, the nurse cells; decreased border cell Myosin dynamics; and increased nurse cell stiffness as measured by atomic force microscopy. Reducing Myosin restores on-time border cell migration in fascin mutant follicles. Further, Fascin’s actin bundling activity is required to limit Myosin activation. Surprisingly, we find that Fascin regulates Myosin activity in the border cells to control nurse cell stiffness to promote migration. Thus, these data shift the paradigm from a substrate stiffness-centric model of regulating migration, to uncover that collectively migrating cells play a critical role in controlling the mechanical properties of their substrate in order to promote their own migration. This new means of mechanical regulation of migration is likely conserved across contexts and organisms, as Fascin and Myosin are common regulators of cell migration.

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Src42A required for collective border cell migration in vivo

10.1101/186049 ◽

2017 ◽

Cited By ~ 1

Author(s):

Yasmin Sallak ◽

Alba Yurani Torres ◽

Hongyan Yin ◽

Denise Montell

Keyword(s):

Cell Migration ◽

Cell Junctions ◽

Collective Cell Migration ◽

Border Cells ◽

Border Cell ◽

Border Cell Migration ◽

And Migration ◽

Drosophila Ovary ◽

E Cadherin

AbstractThe tyrosine kinase Src is over-expressed in numerous human cancers and is associated with poor prognosis. While Src has been extensively studied, its contributions to collective cell migration in vivo remain incompletely understood. Here we show that Src42A, but not Src64, is required for the specification and migration of the border cells in the Drosophila ovary, a well-developed and genetically tractable in vivo cell migration model. We found active Src42A enriched at border cell/nurse cell interfaces, where E-cadherin is less abundant, and depleted from border cell/border cell and border cell/polar cell junctions where E-cadherin is more stable, whereas total Src42A protein co-localizes with E-cadherin. Over-expression of wild type Src42A mislocalized Src activity and prevented border cell migration. Constitutively active or kinase dead forms of Src42A also impeded border cells. These findings establish border cells as a model for investigating the mechanisms of action of Src in cooperative, collective, cell-on-cell migration in vivo.

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Drosophila USP22/non-stop regulates the Hippo pathway to polarise the actin cytoskeleton during collective border cell migration

10.1101/2020.07.02.177170 ◽

2020 ◽

Author(s):

Hammed Badmos ◽

Neville Cobbe ◽

Amy Campbell ◽

Daimark Bennett

Keyword(s):

Cell Migration ◽

Actin Cytoskeleton ◽

Hippo Pathway ◽

Collective Cell Migration ◽

Deubiquitinating Enzymes ◽

Border Cell ◽

Border Cell Migration ◽

Actin Protrusions ◽

The Hippo Pathway

Polarisation of the actin cytoskeleton is vital for the collective migration of cells in vivo. During invasive border cell migration in Drosophila, actin polarisation is directly controlled by Hippo pathway components, which reside at contacts between border cells in the cluster. Here we identify, in a genetic screen for deubiquitinating enzymes involved in border cell migration, an essential role for non-stop/USP22 in the expression of Hippo pathway components expanded and merlin; loss of non-stop function consequently leads to a redistribution of F-actin and the polarity determinant Crumbs, loss of polarised actin protrusions and premature tumbling of the border cell cluster. Non-stop is a component of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional coactivator complex, but SAGA’s histone acetyltransferase module, which does not bind to expanded or merlin, is dispensable for migration. Taken together, our results uncover novel roles for SAGA-independent non-stop/USP22 in Hippo-mediated collective cell migration, which may help guide studies in other systems where USP22 is necessary for cell motility and invasion.

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Protein phosphatase 1 activity controls a balance between collective and single cell modes of migration

eLife ◽

10.7554/elife.52979 ◽

2020 ◽

Vol 9 ◽

Author(s):

Yujun Chen ◽

Nirupama Kotian ◽

George Aranjuez ◽

Lin Chen ◽

C Luke Messer ◽

...

Keyword(s):

Cell Migration ◽

Single Cell ◽

Protein Phosphatase ◽

Single Cells ◽

Cell Junctions ◽

Protein Phosphatase 1 ◽

Collective Cell Migration ◽

Border Cells ◽

Border Cell ◽

And Migration

Collective cell migration is central to many developmental and pathological processes. However, the mechanisms that keep cell collectives together and coordinate movement of multiple cells are poorly understood. Using the Drosophila border cell migration model, we find that Protein phosphatase 1 (Pp1) activity controls collective cell cohesion and migration. Inhibition of Pp1 causes border cells to round up, dissociate, and move as single cells with altered motility. We present evidence that Pp1 promotes proper levels of cadherin-catenin complex proteins at cell-cell junctions within the cluster to keep border cells together. Pp1 further restricts actomyosin contractility to the cluster periphery rather than at individual internal border cell contacts. We show that the myosin phosphatase Pp1 complex, which inhibits non-muscle myosin-II (Myo-II) activity, coordinates border cell shape and cluster cohesion. Given the high conservation of Pp1 complexes, this study identifies Pp1 as a major regulator of collective versus single cell migration.

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Three-Dimensional Label-Free Imaging and Quantification of Migrating Cells during Wound Healing

10.1101/2020.07.24.219774 ◽

2020 ◽

Author(s):

A. J. Lee ◽

H. Hugonnet ◽

W.S. Park ◽

Y.K. Park

Keyword(s):

Wound Healing ◽

Cell Migration ◽

High Resolution ◽

Three Dimensional ◽

Collective Cell Migration ◽

3D Analysis ◽

Two Dimensional ◽

Label Free ◽

Wound Healing Assay ◽

Migrating Cells

ABSTRACTThe wound healing assay provides essential information about collective cell migration and cell-to-cell interactions. It is a simple, effective, and widely used tool for observing the effect of numerous chemical treatments on wound healing speed. To perform and analyze a wound healing assay, various imaging techniques have been utilized. However, image acquisition and analysis are often limited in two-dimensional space or require the use of exogenous labeling agents. Here, we present a method for imaging large-scale wound healing assays in a label-free and volumetric manner using optical diffraction tomography (ODT). We performed quantitative high-resolution three-dimensional (3D) analysis of cell migration over a long period without difficulties such as photobleaching or phototoxicity. ODT enables the reconstruction of the refractive index (RI) tomogram of unlabeled cells, which provides both structural and biochemical information about the individual cell at subcellular resolution. Stitching multiple RI tomograms enables long-term (24 h) and large field-of-view imaging (> 800 × 400 μm2) with a lateral resolution of 110 nm. We demonstrated the thickness changes of leading cells and studied the effects of cytochalasin D. The 3D RI tomogram also revealed increased RI values in leading cells compared to lagging cells, suggesting the formation of a highly concentrated subcellular structure.STATEMENT OF SIGNIFICANCEThe wound healing assay is a simple but effective tool for studying collective cell migration (CCM) that is widely used in biophysical studies and high-throughput screening. However, conventional imaging and analysis methods only address two-dimensional properties in a wound healing assay, such as gap closure rate. This is unfortunate because biological cells are complex 3D structures, and their dynamics provide significant information about cell physiology. Here, we presented three-dimensional (3D) label-free imaging for wound healing assays and investigated the 3D dynamics of CCM. High-resolution subcellular structures as well as their collective dynamics were imaged and analyzed quantitatively. Our label-free quantitative 3D analysis method provides a unique opportunity to study the behavior of migrating cells during the wound healing process.

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