scholarly journals Vimentin fibers orient traction stress

2017 ◽  
Vol 114 (20) ◽  
pp. 5195-5200 ◽  
Author(s):  
Nancy Costigliola ◽  
Liya Ding ◽  
Christoph J. Burckhardt ◽  
Sangyoon J. Han ◽  
Edgar Gutierrez ◽  
...  
Keyword(s):  
Cell Migration ◽  
Single Cell ◽  
Graph Matching ◽  
Single Cells ◽  
Mechanical Strain ◽  
Migration Direction ◽  
Human Foreskin Fibroblasts ◽  
And Migration

The intermediate filament vimentin is required for cells to transition from the epithelial state to the mesenchymal state and migrate as single cells; however, little is known about the specific role of vimentin in the regulation of mesenchymal migration. Vimentin is known to have a significantly greater ability to resist stress without breaking in vitro compared with actin or microtubules, and also to increase cell elasticity in vivo. Therefore, we hypothesized that the presence of vimentin could support the anisotropic mechanical strain of single-cell migration. To study this, we fluorescently labeled vimentin with an mEmerald tag using TALEN genome editing. We observed vimentin architecture in migrating human foreskin fibroblasts and found that network organization varied from long, linear bundles, or “fibers,” to shorter fragments with a mesh-like organization. We developed image analysis tools employing steerable filtering and iterative graph matching to characterize the fibers embedded in the surrounding mesh. Vimentin fibers were aligned with fibroblast branching and migration direction. The presence of the vimentin network was correlated with 10-fold slower local actin retrograde flow rates, as well as spatial homogenization of actin-based forces transmitted to the substrate. Vimentin fibers coaligned with and were required for the anisotropic orientation of traction stresses. These results indicate that the vimentin network acts as a load-bearing superstructure capable of integrating and reorienting actin-based forces. We propose that vimentin's role in cell motility is to govern the alignment of traction stresses that permit single-cell migration.

scholarly journals Self-organized cell migration across scales – from single cell movement to tissue formation

Development ◽  
2021 ◽  
Vol 148 (7) ◽  
pp. dev191767
Author(s):  
Jessica Stock ◽  
Andrea Pauli
Keyword(s):  
Cell Migration ◽  
Multiple Scales ◽  
Single Cells ◽  
Cell Movement ◽  
Biological Response ◽  
Collective Cell Migration ◽  
Theoretical Concepts ◽  
Self Organizing

ABSTRACTSelf-organization is a key feature of many biological and developmental processes, including cell migration. Although cell migration has traditionally been viewed as a biological response to extrinsic signals, advances within the past two decades have highlighted the importance of intrinsic self-organizing properties to direct cell migration on multiple scales. In this Review, we will explore self-organizing mechanisms that lay the foundation for both single and collective cell migration. Based on in vitro and in vivo examples, we will discuss theoretical concepts that underlie the persistent migration of single cells in the absence of directional guidance cues, and the formation of an autonomous cell collective that drives coordinated migration. Finally, we highlight the general implications of self-organizing principles guiding cell migration for biological and medical research.

scholarly journals WDR5 modulates cell motility and morphology and controls nuclear changes induced by a 3D environment

2018 ◽  
Vol 115 (34) ◽  
pp. 8581-8586 ◽  
Author(s):  
Pengbo Wang ◽  
Marcel Dreger ◽  
Elena Madrazo ◽  
Craig J. Williams ◽  
Rafael Samaniego ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Polarity ◽  
Underlying Mechanism ◽  
H3k4 Methylation ◽  
Nuclear Mechanics ◽  
3D Environment ◽  
3D Environments ◽  
And Migration

Cell migration through extracellular matrices requires nuclear deformation, which depends on nuclear stiffness. In turn, chromatin structure contributes to nuclear stiffness, but the mechanosensing pathways regulating chromatin during cell migration remain unclear. Here, we demonstrate that WD repeat domain 5 (WDR5), an essential component of H3K4 methyltransferase complexes, regulates cell polarity, nuclear deformability, and migration of lymphocytes in vitro and in vivo, independent of transcriptional activity, suggesting nongenomic functions for WDR5. Similarly, depletion of RbBP5 (another H3K4 methyltransferase subunit) promotes similar defects. We reveal that a 3D environment increases the H3K4 methylation dependent on WDR5 and results in a globally less compacted chromatin conformation. Further, using atomic force microscopy, nuclear particle tracking, and nuclear swelling experiments, we detect changes in nuclear mechanics that accompany the epigenetic changes induced in 3D conditions. Indeed, nuclei from cells in 3D environments were softer, and thereby more deformable, compared with cells in suspension or cultured in 2D conditions, again dependent on WDR5. Dissecting the underlying mechanism, we determined that actomyosin contractility, through the phosphorylation of myosin by MLCK (myosin light chain kinase), controls the interaction of WDR5 with other components of the methyltransferase complex, which in turn up-regulates H3K4 methylation activation in 3D conditions. Taken together, our findings reveal a nongenomic function for WDR5 in regulating H3K4 methylation induced by 3D environments, physical properties of the nucleus, cell polarity, and cell migratory capacity.

scholarly journals Single Cell RNA-Seq Characterises Pre-Leukemic Transformation Driven By CEBPA N321D in the Hoxb8-FL Cell Line

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3887-3887
Author(s):  
Moosa Qureshi ◽  
Fernando Calero-Nieto ◽  
Iwo Kucinski ◽  
Sarah Kinston ◽  
George Giotopoulos ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Single Cell ◽  
Cell Line ◽  
Single Cells ◽  
Mutant Form ◽  
Rna Seq ◽  
Wild Type ◽  
Leukemic Transformation

Abstract The C/EBPα transcription factor plays a pivotal role in myeloid differentiation and E2F-mediated cell cycle regulation. Although CEBPA mutations are common in acute myeloid leukaemia (AML), little is known regarding pre-leukemic alterations caused by mutated CEBPA. Here, we investigated early events involved in pre-leukemic transformation driven by CEBPA N321D in the LMPP-like cell line Hoxb8-FL (Redecke et al., Nat Methods 2013), which can be maintained in vitro as a self-renewing LMPP population using Flt3L and estradiol, as well as differentiated both in vitro and in vivo into myeloid and lymphoid cell types. Hoxb8-FL cells were retrovirally transduced with Empty Vector (EV), wild-type CEBPA (CEBPA WT) or its N321D mutant form (CEBPA N321D). CEBPA WT-transduced cells showed increased expression of cd11b and SIRPα and downregulation of c-kit, suggesting that wild-type CEBPA was sufficient to promote differentiation even under LMPP growth conditions. Interestingly, we did not observe the same phenotype in CEBPA N321D-transduced cells. Upon withdrawal of estradiol, both EV and CEBPA WT-transduced cells differentiated rapidly into a conventional dendritic cell (cDC) phenotype by day 7 and died within 12 days. By contrast, CEBPA N321D-transduced cells continued to grow for in excess of 56 days, with an initial cDC phenotype but by day 30 demonstrating a plasmacytoid dendritic cell precursor phenotype. CEBPA N321D-transduced cells were morphologically distinct from EV-transduced cells. To test leukemogenic potential in vivo, we performed transplantation experiments in lethally irradiated mice. Serial monitoring of peripheral blood demonstrated that Hoxb8-FL derived cells had disappeared by 4 weeks, and did not reappear. However, at 6 months CEBPA N321D-transduced cells could still be detected in bone marrow in contrast to EV-transduced cells but without any leukemic phenotype. To identify early events involved in pre-leukemic transformation, the differentiation profiles of EV, CEBPA WT and CEBPA N321D-transduced cells were examined with single cell RNA-seq (scRNA-seq). 576 single cells were taken from 3 biological replicates at days 0 and 5 post-differentiation, and analysed using the Automated Single-Cell Analysis Pipeline (Gardeux et al., Bioinformatics 2017). Visualisation by t-SNE (Fig 1) demonstrated: (i) CEBPA WT-transduced cells formed a distinct cluster at day 0 before withdrawal of estradiol; (ii) CEBPA N321D-transduced cells separated from EV and CEBPA WT-transduced cells after 5 days of differentiation, (iii) two subpopulations could be identified within the CEBPA N321D-transduced cells at day 5, with a cluster of five CEBPA N321D-transduced single cells distributed amongst or very close to the day 0 non-differentiated cells. Differential expression analysis identified 224 genes upregulated and 633 genes downregulated specifically in the CEBPA N321D-transduced cells when compared to EV cells after 5 days of differentiation. This gene expression signature revealed that CEBPA N321D-transduced cells switched on a HSC/MEP/CMP transcriptional program and switched off a myeloid dendritic cell program. Finally, in order to further dissect the effect of the N321D mutation, the binding profile of endogenous and CEBPA N321D was compared by ChIP-seq before and after 5 days of differentiation. Integration with scRNA-seq data identified 160 genes specifically downregulated in CEBPA N321D-transduced cells which were associated with the binding of the mutant protein. This list of genes included genes previously implicated in dendritic cell differentiation (such as NOTCH2, JAK2), as well as a number of genes not previously implicated in the evolution of AML, representing potentially novel therapeutic targets. Disclosures No relevant conflicts of interest to declare.

scholarly journals Abi2-Deficient Mice Exhibit Defective Cell Migration, Aberrant Dendritic Spine Morphogenesis, and Deficits in Learning and Memory

2004 ◽  
Vol 24 (24) ◽  
pp. 10905-10922 ◽  
Author(s):  
Matthew Grove ◽  
Galina Demyanenko ◽  
Asier Echarri ◽  
Patricia A. Zipfel ◽  
Marisol E. Quiroz ◽  
...  
Keyword(s):  
Cell Migration ◽  
Dendritic Spine ◽  
Actin Polymerization ◽  
Adherens Junctions ◽  
Actin Dynamics ◽  
Long Term Memory ◽  
Cell Morphogenesis ◽  
And Migration

ABSTRACT The Abl-interactor (Abi) family of adaptor proteins has been linked to signaling pathways involving the Abl tyrosine kinases and the Rac GTPase. Abi proteins localize to sites of actin polymerization in protrusive membrane structures and regulate actin dynamics in vitro. Here we demonstrate that Abi2 modulates cell morphogenesis and migration in vivo. Homozygous deletion of murine abi2 produced abnormal phenotypes in the eye and brain, the tissues with the highest Abi2 expression. In the absence of Abi2, secondary lens fiber orientation and migration were defective in the eye, without detectable defects in proliferation, differentiation, or apoptosis. These phenotypes were consistent with the localization of Abi2 at adherens junctions in the developing lens and at nascent epithelial cell adherens junctions in vitro. Downregulation of Abi expression by RNA interference impaired adherens junction formation and correlated with downregulation of the Wave actin-nucleation promoting factor. Loss of Abi2 also resulted in cell migration defects in the neocortex and hippocampus, abnormal dendritic spine morphology and density, and severe deficits in short- and long-term memory. These findings support a role for Abi2 in the regulation of cytoskeletal dynamics at adherens junctions and dendritic spines, which is critical for intercellular connectivity, cell morphogenesis, and cognitive functions.

scholarly journals Myo1g is required for efficient adhesion and migration of activated B lymphocytes to inguinal lymph nodes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
D. Cruz-Zárate ◽  
O. López-Ortega ◽  
D. A. Girón-Pérez ◽  
A. M. Gonzalez-Suarez ◽  
J. L. García-Cordero ◽  
...  
Keyword(s):  
Cell Migration ◽  
Lymph Node ◽  
B Lymphocytes ◽  
Dynamic Process ◽  
Inguinal Lymph Node ◽  
Cytoskeletal Remodeling ◽  
And Migration

AbstractCell migration is a dynamic process that involves adhesion molecules and the deformation of the moving cell that depends on cytoskeletal remodeling and actin-modulating proteins such as myosins. In this work, we analyzed the role of the class I Myosin-1 g (Myo1g) in migratory processes of LPS + IL-4 activated B lymphocytes in vivo and in vitro. In vivo, the absence of Myo1g reduced homing of activated B lymphocytes into the inguinal lymph node. Using microchannel chambers and morphology analysis, we found that the lack of Myo1g caused adhesion and chemotaxis defects. Additionally, deficiency in Myo1g causes flaws in adopting a migratory morphology. Our results highlight the importance of Myo1g during B cell migration.

scholarly journals Single-cell RNA-seq revealed the role of Alkbh5 in reproduction

2021 ◽  
Author(s):  
Xiaozhong Shen ◽  
Gangcai Xie
Keyword(s):  
Single Cell ◽  
Messenger Rna ◽  
Single Cells ◽  
Rna Seq ◽  
Single Cell Sequencing ◽  
Differential Gene ◽  
Higher Eukaryotes

AbstractN(6)-methyladenosine (m(6)a) is the most common internal modification of messenger RNA (mRNA) in higher eukaryotes. According to previous literature reports, alkbh5, as another demethylase in mammals, can reverse the expression of m(6)a gene in vivo and in vitro. In order to reveal the effect of Alkbh5 deletion on the level of single cells in the testis during spermatogenesis in mice, the data were compared using single-cell sequencing. In this article, we discussed the transcription profile and cell type identification of mouse testis, the expression of mitochondrial and ribosomal genes in mice, the analysis of differential gene expression, and the effects of Alkbh5 deletion, and try to explain the role and influence of Alkbh5 on reproduction at the level of single-cell sequencing.

scholarly journals Injury-Free In Vivo Delivery and Engraftment into the Cornea Endothelium Using Extracellular Matrix Shrink-Wrapped Cells

2021 ◽  
Author(s):  
Rachelle N. Palchesko ◽  
Yiqin Du ◽  
Moira L. Geary ◽  
Santiago Carrasquilla ◽  
Daniel J. Shiwarski ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Single Cells ◽  
Tissue Level ◽  
Cell Junctions ◽  
Small Island ◽  
Corneal Endothelial Cells ◽  
Corneal Blindness

AbstractCell injection has emerged as a widespread approach for therapeutic delivery of healthy cells into diseased and damaged tissues to achieve regeneration. However, cell retention, viability and integration at the injection site has generally been poor, driving the need for improved approaches. Additionally, it is unknown how efficiently single cells can integrate and repair tissue level function. Here we have developed a technique to address these issues by engineering islands of interconnected cells on ECM nanoscaffolds that can be non-destructively released from the surface via thermal dissolution of the underlying thermo-responsive polymer. Upon dissolution of the polymer, the ECM nanoscaffold shrink-wraps around the small island of cells, creating a small patch of cells that maintain their cell-cell junctions and cytoskeletal structure throughout collection, centrifugation and injection that we have termed μMonolayers. These μMonolayers were made with corneal endothelial cells, as a model system, as single cell injections of corneal endothelial cells have been used with some success clinically to treat corneal blindness. In vitro our μMonolayers exhibited increased integration compared to single cells into low density corneal endothelial monolayers and in vivo into the high-density healthy rabbit corneal endothelium. These results indicate that this technique could be used to increase the integration of healthy cells into existing tissues to treat not only corneal blindness, but also other conditions such as cystic fibrosis, myocardial infarction, diabetes, etc.One Sentence SummarySmall monolayers of interconnected endothelial cells are shrinkwrapped in a thin layer of ECM and exhibit enhanced adhesion and integration in vivo compared to single cell suspensions.

scholarly journals Single Cell Clones Derived from a Patient's AML Xenograft Display Genetic and Functional Heterogeneity

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1450-1450
Author(s):  
Christina Maria Zeller ◽  
Daniel Richter ◽  
Binje Vick ◽  
Tobias Herold ◽  
Johannes Bagnoli ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Multiple Hypothesis Testing ◽  
Targeted Sequencing ◽  
Functional Heterogeneity ◽  
Nras Mutation ◽  
In Vivo Experiments ◽  
Cell Clones

Introduction: Acute myeloid leukemia (AML) shows substantial genetic and epigenetic heterogeneity, even within an individual patient. Due to treatment resistance and ability to induce relapse, adverse subclones present a major clinical challenge in determining the patient's prognosis. Here, we aimed at characterizing the genetic and functional heterogeneity within a single AML patient, and at identifying adverse subclones that result in therapy failure or give rise to relapse. Methods: Leukemic cells from an AML patient at first and second relapse were transplanted into immuno-compromised mice to generate patient-derived xenografts (PDX). PDX AML cells allowed serial transplantation and genetic engineering by lentiviruses. To distinguish single cells and generate PDX AML clones derived from a single cell (single cell clones, SCC), cells were transduced with a genetic barcode and transplanted into recipient mice near leukemia initiating cell frequency. Resulting SCC were genetically marked to express recombinant fluorochromes to enable flow cytometry analysis. All SCC were characterized for known subclonal mutations of the AML patient by targeted sequencing. Additionally, transcriptome and methylome analysis were performed by SCRB-seq and methylation array, respectively. Results: We successfully generated thirteen serially transplantable PDX SCC from a single AML patient, expressing combinations of up to four fluorochromes to enable competitive in vitro and in vivo experiments. In targeted sequencing, we found that SCC originated from at least four genetically distinct AML subclones and were distinguished by mutations in KRAS (4/13), NRAS (5/13), EZH2 (2/13) or EZH2 and NRAS (2/13). While the NRAS mutation was detected in a minority of bulk cells over serial passages (<10%) from both the first and second relapse, 50% of SCC carried the NRAS mutation. This indicates that NRAS mutated AML cells have an increased stem cell capacity upon transplantation of low cell numbers. Transcriptome analysis revealed 442 genes as differentially expressed between SCC and up to four biological replicates after adjustment for multiple hypothesis testing. Unsupervised clustering demonstrated a strong correlation of gene expression profiles with the respective genotype. In competitive in vivo experiments, homing capability was comparable between the four genetically distinct subclones. However, 2/2 EZH2-mutated SCC overgrew all other clones showing a clear growth advantage within two weeks of in vivo growth. The EZH2-mutated SCC (2/2) were resistant towards in vivo treatment with Cytarabine, whereas the KRAS (4/4), NRAS (5/5) mutated as well as the EZH2 and NRAS double-mutated SCC (2/2) responded to treatment. Conclusion: Taken together, we experimentally prove the existence of genetically and functionally diverse subclones within an individual AML sample. Our approach allows not only genetic, but also functional in vitro and in vivo characterization of adverse subclones. Our approach can be used to identify novel therapeutic approaches in order to specifically target the most adverse cells within patients' AML sample. Disclosures Metzeler: Celgene: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Otsuka: Honoraria.

scholarly journals Rab5 Mediates Caspase-8–promoted Cell Motility and Metastasis

2010 ◽  
Vol 21 (2) ◽  
pp. 369-376 ◽  
Author(s):  
Vicente A. Torres ◽  
Ainhoa Mielgo ◽  
Simone Barbero ◽  
Ruth Hsiao ◽  
John A. Wilkins ◽  
...  
Keyword(s):  
Cell Migration ◽  
Focal Adhesions ◽  
Caspase 8 ◽  
Environmental Cues ◽  
Dependent Manner ◽  
Cell Responses ◽  
Β1 Integrins ◽  
And Migration

Caspase-8 is a key apical sensory protein that governs cell responses to environmental cues, alternatively promoting apoptosis, proliferation, and cell migration. The proteins responsible for integration of these pathways, however, have remained elusive. Here, we reveal that Rab5 regulates caspase-8–dependent signaling from integrins. Integrin ligation leads to Rab5 activation, association with integrins, and activation of Rac, in a caspase-8–dependent manner. Rab5 activation promotes colocalization and coprecipitation of integrins with caspase-8, concomitant with Rab5 recruitment to integrin-rich regions such as focal adhesions and membrane ruffles. Moreover, caspase-8 expression promotes Rab5-mediated internalization and the recycling of β1 integrins, increasing cell migration independently of caspase catalytic activity. Conversely, Rab5 knockdown prevented caspase-8–mediated integrin signaling for Rac activation, cell migration, and apoptotic signaling, respectively. Similarly, Rab5 was critical for caspase-8–driven cell migration in vivo, because knockdown of Rab5 compromised the ability of caspase-8 to promote metastasis under nonapoptotic conditions. These studies identify Rab5 as a key integrator of caspase-8–mediated signal transduction downstream of integrins, regulating cell survival and migration in vivo and in vitro.

scholarly journals Phosphorylation of actopaxin regulates cell spreading and migration

2004 ◽  
Vol 166 (6) ◽  
pp. 901-912 ◽  
Author(s):  
Dominic M. Clarke ◽  
Michael C. Brown ◽  
David P. LaLonde ◽  
Christopher E. Turner
Keyword(s):  
Cell Migration ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Boyden Chamber ◽  
Extracellular Signal ◽  
U2os Cells ◽  
And Migration ◽  
Mutant Cells

Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration.

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