scholarly journals Plexin-B2 is a key regulator of cell mechanics during multicellular organization

2019 ◽  
Author(s):  
Chrystian Junqueira Alves ◽  
Rafael Dariolli ◽  
Theodore Hannah ◽  
Robert J. Wiener ◽  
Nicolas Daviaud ◽  
...  
Keyword(s):  
Cell Fate ◽  
Cell Biology ◽  
Embryonic Stem ◽  
Tissue Mechanics ◽  
Cell Stiffness ◽  
Adhesive Properties ◽  
Force Microscopy ◽  
Rac Gtpases ◽  
Tensile Forces ◽  
Tissue Geometry

SUMMARYDuring multicellular organization, individual cells need to constantly respond to environmental cues and adjust contractile and adhesive forces in order to maintain tissue integrity. The signaling pathways linking biochemical cues and tissue mechanics are unclear. Here, we show that Plexin-B2 regulates mechanochemical integration during multicellular organization. In human embryonic stem cells (hESCs), Plexin-B2 controls cell shape and tissue geometry in both 2D epithelial colony and 3D spheroid aggregates by regulating actomyosin contractility and junctional/cell-matrix adhesive properties. Atomic force microscopy (AFM) directly demonstrates that Plexin-B2 modulates cell stiffness in hESC colonies, which in turn impacts cell proliferation and cell fate specification through β-catenin signaling and YAP mechanosensing. YAP also functions as a mechanoregulator downstream of Plexin-B2, thus forming a mechanochemical integrative loop. In human neuroprogenitor cells (hNPCs), Plexin-B2 similarly controls cell stiffness and tensile forces, as revealed by AFM and FRET tension sensor studies. Strikingly, Plexin-B2-deficient hNPCs display accelerated neuronal differentiation. From an organogenesis perspective, Plexin-B2 maintains cytoarchitectural integrity of neuroepithelium, as modeled in cerebral organoids. On a signaling level, Plexin-B2 engages extracellular as well as intracellular Ras-GAP and RBD domains for mechanoregulation through Rap and Rac GTPases. Our data unveil a fundamental function of Plexin-B2 for mechanochemical integration during multicellular organization, and shed light on the principle of force-mediated regulation of stem cell biology and tissue morphogenesis.

scholarly journals Wnt-and Glutamate-receptors orchestrate stem cell dynamics and asymmetric cell division

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Sergi Junyent ◽  
Joshua C Reeves ◽  
James LA Szczerkowski1 ◽  
Clare L Garcin ◽  
Tung-Jui Trieu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Division ◽  
Glutamate Receptors ◽  
Cell Fate ◽  
Cell Biology ◽  
Wnt Pathway ◽  
Asymmetric Cell Division ◽  
Cell Signalling ◽  
Embryonic Stem ◽  
Kainate Receptors

The Wnt-pathway is part of a signalling network that regulates many aspects of cell biology. Recently we discovered crosstalk between AMPA/Kainate-type ionotropic glutamate receptors (iGluRs) and the Wnt-pathway during the initial Wnt3a-interaction at the cytonemes of mouse embryonic stem cells (ESCs). Here, we demonstrate that this crosstalk persists throughout the Wnt3a-response in ESCs. Both AMPA- and Kainate-receptors regulate early Wnt3a-recruitment, dynamics on the cell membrane, and orientation of the spindle towards a Wnt3a-source at mitosis. AMPA-receptors specifically are required for segregating cell fate components during Wnt3a-mediated asymmetric cell division (ACD). Using Wnt-pathway component knockout lines, we determine that Wnt co-receptor Lrp6 has particular functionality over Lrp5 in cytoneme formation, and in facilitating ACD. Both Lrp5 and 6, alongside pathway effector β-catenin act in concert to mediate the positioning of the dynamic interaction with, and spindle orientation to, a localized Wnt3a-source. Wnt-iGluR crosstalk may prove pervasive throughout embryonic and adult stem cell signalling.

scholarly journals Alternative Splicing in Self-Renewal of Embryonic Stem Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Clara Y. Cheong ◽  
Thomas Lufkin
Keyword(s):  
Stem Cells ◽  
Alternative Splicing ◽  
Embryonic Stem Cells ◽  
Cell Fate ◽  
Cell Biology ◽  
Genomic Sequence ◽  
Splice Variants ◽  
Embryonic Stem ◽  
Self Renewal ◽  
Additional Layer

Much of embryonic stem cell biology has focused on transcriptional expression and regulation of genes that could mediate its unique potential in self-renewal or pluripotency. In alignment with our present understanding on the genetic, protein, and epigenetic factors that may direct cell fate, we present a short overview of the often overlooked contribution of alternative splice variants to regulatory diversity. Progressing beyond the limitations of a fixed genomic sequence, alternative splicing offers an additional layer of complexity to produce protein variants that may differ in function and localization that can direct embryonic stem cells to specific differentiation pathways. In light of the number of variants that can be produced at key ES cell genes alone, it is challenging to consider how much more multifaceted transcriptional regulation truly is, and if this can be captured more fully in future works.

scholarly journals Engineered illumination devices for optogenetic control of cellular signaling dynamics

2019 ◽  
Author(s):  
Nicole A. Repina ◽  
Thomas McClave ◽  
Xiaoping Bao ◽  
Ravi S. Kane ◽  
David V. Schaffer
Keyword(s):  
Cell Fate ◽  
High Throughput ◽  
Protein Interactions ◽  
Cell Biology ◽  
Low Cost ◽  
Optical Design ◽  
Embryonic Stem ◽  
Protein Protein Interactions ◽  
Optogenetic Control

ABSTRACTSpatially and temporally varying patterns of morphogen signals during development drive cell fate specification at the proper location and time. However, currentin vitromethods typically do not allow for precise, dynamic, spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics impact cell behavior. Here we show that optogenetic Wnt/β-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns using novel engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). The optical design of LAVA devices was optimized for uniform illumination of multi-well cell culture plates to enable high-throughput, spatiotemporal optogenetic activation of signaling pathways and protein-protein interactions. Using the LAVA devices, variation in light intensity induced a dose-dependent response in optoWnt activation and downstream Brachyury expression in human embryonic stem cells (hESCs). Furthermore, time-varying and spatially localized patterns of light revealed tissue patterning that models embryonic presentation of Wnt signalsin vitro. The engineered LAVA devices thus provide a low-cost, user-friendly method for high-throughput and spatiotemporal optogenetic control of cell signaling for applications in developmental and cell biology.

scholarly journals Regulation of Vascular Smooth Muscle Cell Stiffness and Adhesion by [Ca2+]i: An Atomic Force Microscopy-Based Study

2018 ◽  
Vol 24 (6) ◽  
pp. 708-712 ◽  
Author(s):  
Yi Zhu ◽  
Li He ◽  
Jing Qu ◽  
Yong Zhou
Keyword(s):  
Atomic Force Microscopy ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Actin ◽  
Cell Stiffness ◽  
Adhesive Properties ◽  
Acetoxymethyl Ester ◽  
Altered Expression ◽  
Force Microscopy ◽  
Atomic Force

AbstractThe intracellular concentration of calcium ion ([Ca2+]i) is a critical regulator of cell signaling and contractility of vascular smooth muscle cells (VSMCs). In this study, we employed an atomic force microscopy (AFM) nanoindentation-based approach to investigate the role of [Ca2+]i in regulating the cortical elasticity of rat cremaster VSMCs and the ability of rat VSMCs to adhere to fibronectin (Fn) matrix. Elevation of [Ca2+]i by ionomycin treatment increased rat VSMC stiffness and cell adhesion to Fn-biofunctionalized AFM probes, whereas attenuation of [Ca2+]i by 1,2-Bis (2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) treatment decreased the mechanical and matrix adhesive properties of VSMCs. Furthermore, we found that ionomycin/BAPTA-AM treatments altered expression of α5 integrin subunits and α smooth muscle actin in rat VSMCs. These data suggest that [Ca2+]i regulates VSMC elasticity and adhesion to the extracellular matrix by a potential mechanism involving changing dynamics of the integrin–actin cytoskeleton axis.

scholarly journals Wnt- and Glutamate-receptors orchestrate stem cell dynamics and asymmetric cell division

2020 ◽  
Author(s):  
Sergi Junyent ◽  
Joshua Reeves ◽  
James L. A. Szczerkowski ◽  
Clare L. Garcin ◽  
Tung-Jui Trieu ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Fate ◽  
Cell Biology ◽  
Wnt Pathway ◽  
Asymmetric Cell Division ◽  
Embryonic Stem ◽  
Cell Dynamics ◽  
Pathway Activation ◽  
Wnt Ligands ◽  
Ligand Stabilization

ABSTRACTWnt signalling regulates many aspects of cell biology. Wnt-pathway activation and its downstream effects have been extensively studied, but the dynamic analysis of Wnt-ligands on mammalian cellular membranes is obstructed by difficulties of visualization. We overcome this using microbead-tethered Wnts presented to single embryonic stem cells, which undergo Wnt-mediated asymmetric cell division (ACD). Through live imaging and genetic editing, we show that knockout of Wnt co-receptor Lrp5 promotes cytoneme formation and Wnt-recruitment, which requires Lrp6 and β-catenin. Lrp5 facilitates ligand-retention at the membrane, and alongside Lrp6 mediates Wnt-ligand stabilization and positioning. β-catenin or Wnt co-receptor knockout causes misorientation at mitosis, and all but Lrp5 are required for Wnt-orientated ACD. Surprisingly, ionotropic glutamate receptor (iGluR) activity enables initial Wnt-recruitment, positioning, and ultimately oriented ACD. Uniquely, we have scrutinized the early Wnt ligand-membrane interaction, linking roles of Wnt-pathway components and crosstalk with iGluRs in guiding cell fate determination by oriented ACD.

scholarly journals Intrinsic Mechanical Cues and Their Impact on Stem Cells and Embryogenesis

2021 ◽  
Vol 9 ◽  
Author(s):  
Jonna Petzold ◽  
Eileen Gentleman
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Cell Surface ◽  
Cell Fate ◽  
Cell Biology ◽  
Soft Tissues ◽  
Embryonic Stem ◽  
Focal Adhesions ◽  
Cell Fate Decisions ◽  
Applied Forces

Although understanding how soluble cues direct cellular processes revolutionised the study of cell biology in the second half of the 20th century, over the last two decades, new insights into how mechanical cues similarly impact cell fate decisions has gained momentum. During development, extrinsic cues such as fluid flow, shear stress and compressive forces are essential for normal embryogenesis to proceed. Indeed, both adult and embryonic stem cells can respond to applied forces, but they can also detect intrinsic mechanical cues from their surrounding environment, such as the stiffness of the extracellular matrix, which impacts differentiation and morphogenesis. Cells can detect changes in their mechanical environment using cell surface receptors such as integrins and focal adhesions. Moreover, dynamic rearrangements of the cytoskeleton have been identified as a key means by which forces are transmitted from the extracellular matrix to the cell and vice versa. Although we have some understanding of the downstream mechanisms whereby mechanical cues are translated into changes in cell behaviour, many of the signalling pathways remain to be defined. This review discusses the importance of intrinsic mechanical cues on adult cell fate decisions, the emerging roles of cell surface mechano-sensors and the cytoskeleton in enabling cells to sense its microenvironment, and the role of intracellular signalling in translating mechanical cues into transcriptional outputs. In addition, the contribution of mechanical cues to fundamental processes during embryogenesis such as apical constriction and convergent extension is discussed. The continued development of tools to measure the biomechanical properties of soft tissues in vivo is likely to uncover currently underestimated contributions of these cues to adult stem cell fate decisions and embryogenesis, and may inform on regenerative strategies for tissue repair.

scholarly journals Vitamin C in Stem Cell Biology: Impact on Extracellular Matrix Homeostasis and Epigenetics

2017 ◽  
Vol 2017 ◽  
pp. 1-16 ◽  
Author(s):  
Cristina D'Aniello ◽  
Federica Cermola ◽  
Eduardo Jorge Patriarca ◽  
Gabriella Minchiotti
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Stem Cell ◽  
Vitamin C ◽  
Cell Fate ◽  
Cell Biology ◽  
Embryonic Stem ◽  
Redox Status ◽  
Stem Cell Biology ◽  
Prolyl Hydroxylases

Transcription factors and signaling molecules are well-known regulators of stem cell identity and behavior; however, increasing evidence indicates that environmental cues contribute to this complex network of stimuli, acting as crucial determinants of stem cell fate.L-Ascorbic acid (vitamin C (VitC)) has gained growing interest for its multiple functions and mechanisms of action, contributing to the homeostasis of normal tissues and organs as well as to tissue regeneration. Here, we review the main functions of VitC and its effects on stem cells, focusing on its activity as cofactor of Fe+2/αKG dioxygenases, which regulate the epigenetic signatures, the redox status, and the extracellular matrix (ECM) composition, depending on the enzymes’ subcellular localization. Acting as cofactor of collagen prolyl hydroxylases in the endoplasmic reticulum, VitC regulates ECM/collagen homeostasis and plays a key role in the differentiation of mesenchymal stem cells towards osteoblasts, chondrocytes, and tendons. In the nucleus, VitC enhances the activity of DNA and histone demethylases, improving somatic cell reprogramming and pushing embryonic stem cell towards the naive pluripotent state. The broad spectrum of actions of VitC highlights its relevance for stem cell biology in both physiology and disease.

scholarly journals miR-218 Affects the ECM Composition and Cell Biomechanical Properties of Glioblastoma Cells

2021 ◽  
Author(s):  
Małgorzata Grabowska ◽  
Konrad Kuczyński ◽  
Monika Piwecka ◽  
Alicja Rabiasz ◽  
Joanna Zemła ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Real Time ◽  
Protein Level ◽  
Biomechanical Properties ◽  
Cell Stiffness ◽  
Luciferase Assay ◽  
Adhesive Properties ◽  
Force Microscopy ◽  
Time Migration ◽  
Atomic Force

Abstract BackgroundGlioblastoma (GBM) is the most common malignant brain tumour. GBM cells have ability to infiltrate into the surrounding brain tissue, which results in a significant decrease in the patient’s survival rate. Infiltration is a consequence of the low adhesion and high migration of the tumour cells, two features being associated with the highly remodelled extracellular matrix (ECM). MethodsThe expression profile of miRNAs and mRNAs was analysed by qPCR on 19 GBM tissue samples. Than luciferase assay was performed to confirm interaction between miR-218 and target sequences. Next we checked how supplementation of glioma cell line (U118-MG) with microRNA 218 will change expression pattern of TN-C and SDC both on transcript level and on protein level. Analysis of protein level were made with western-blot technique. Last step in expression profiling of genes connected to cellular motility and adhesion was done with use of qPCR analysis after supplementation with miR-218. To assess the abilities of cells to migrate and proliferate real-time cell culture analysis with use of Incelligence system were utilized. Also this results were backed by classic wound-healing assay. To conclude we used atomic force microscopy (AFM) to measure physical properties such as adhesion and stiffness of cells. This study was supported by microscopy analysis of cytoskeleton changes after supplementation of miR-218.ResultsIn this study, we report that ECM composition is partially regulated at the posttranscriptional level by miRNA. Particularly, we show that miR-218, a well-known miRNA suppressor, is involved in direct regulation of ECM components, tenascin-C (TN-C) and syndecan-2 (SDC-2). We demonstrated that the overexpression of miR-218 reduces the mRNA and protein expression levels of TN-C and SDC-2, and subsequently influences biomechanical properties of GBM cells. Atomic force microscopy (AFM) and real-time migration analysis revealed that miR-218 overexpression impairs the migration potential and enhances the adhesive properties of cells. AFM analysis followed by F-actin staining demonstrated that expression level of miR-218 has an impact on cell stiffness and cytoskeletal reorganization. Global gene expression analysis showed deregulation of a number of genes involved in tumour cell motility and adhesion or ECM remodelling upon miR-218 treatment, suggesting further indirect interactions between the cells and ECM. Conclusion The results demonstrated a direct impact of miR-218 reduction in GBM tumours on the qualitative ECM content, leading to changes in the rigidity of the ECM and GBM cells being conducive to increased invasiveness of GBM.

scholarly journals Stem cell biology and neurodegenerative disease

2004 ◽  
Vol 359 (1445) ◽  
pp. 851-856 ◽  
Author(s):  
R. D. McKay
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Brain Development ◽  
Cell Fate ◽  
Cell Biology ◽  
Es Cells ◽  
Embryonic Stem ◽  
Convincing Evidence ◽  
Stem Cell Biology ◽  
Multipotent Stem Cells

The fundamental basis of our work is that organs are generated by multipotent stem cells, whose properties we must understand to control tissue assembly or repair. Central nervous system (CNS) stem cells are now recognized as a well–defined population of precursors that differentiate into cells that are indisputably neurons and glial cells. Work from our group played an important role in defining stem cells of the CNS. Embryonic stem (ES) cells also differentiate to specific neuron and glial types through defined intermediates that are similar to the cellular precursors that normally occur in brain development. There is convincing evidence that the differentiated progeny of ES cells and CNS stem cells show expected functions of neurons and glia. Recent progress has been made on three fundamental developmental processes: (i) cell cycle control; (ii) the control of cell fate; and (iii) early steps in neural differentiation. In addition, our work on CNS stem cells has developed to a stage where there are clinical implications for Parkinson's and other degenerative disorders. These advances establish that stem cell biology contributes to our understanding of brain development and has great clinical promise.

scholarly journals Cell penetration efficiency analysis of different atomic force microscopy nanoneedles into living cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marcos Penedo ◽  
Tetsuya Shirokawa ◽  
Mohammad Shahidul Alam ◽  
Keisuke Miyazawa ◽  
Takehiko Ichikawa ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Cell Membrane ◽  
Cell Biology ◽  
Cell Damage ◽  
Efficiency Analysis ◽  
Living Cells ◽  
Biomolecular Recognition ◽  
Cell Penetration ◽  
Force Microscopy ◽  
Atomic Force

AbstractOver the last decade, nanoneedle-based systems have demonstrated to be extremely useful in cell biology. They can be used as nanotools for drug delivery, biosensing or biomolecular recognition inside cells; or they can be employed to select and sort in parallel a large number of living cells. When using these nanoprobes, the most important requirement is to minimize the cell damage, reducing the forces and indentation lengths needed to penetrate the cell membrane. This is normally achieved by reducing the diameter of the nanoneedles. However, several studies have shown that nanoneedles with a flat tip display lower penetration forces and indentation lengths. In this work, we have tested different nanoneedle shapes and diameters to reduce the force and the indentation length needed to penetrate the cell membrane, demonstrating that ultra-thin and sharp nanoprobes can further reduce them, consequently minimizing the cell damage.

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