Cell Migration in Three Dimensions

Description

This detailed collection serves as a unique and excellent collection of state-of-the-art methods and protocols to interrogate cell migration in a wide variety of different contexts and model organisms, as well as advanced image analysis and quantitative assessment of a diverse array of parameters related to cell migration. The book focuses on the cell biology of cell migration, developmental model systems to assess cell migration during morphogenesis, cell migration in cancers and the tumor micro-environment, as well as blood vessel formation and interactions. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.  Authoritative and practical, Cell Migration in Three Dimensions provides a solid foundation for scientists of different disciplines to investigate cell migration in biological processes.  Chapters 7, 12, 16, 17, 19, 22, and 24 are available open access under a Creative Commons Attribution 4.0 International License via link.springer.com. Preface Contents Contributors Chapter 1: Cell Migration in Three Dimensions 1 Cell Migration in Health and Disease 2 Regulation of Cell Migration 3 Model Systems to Investigate Cell Migration 4 Developments and Advances in the Cell Migration Field References Part I: The Cell Biology of Cell Migration Chapter 2: Use of Ecto-Tagged Integrins to Monitor Integrin Exocytosis and Endocytosis 1 Introduction 2 Materials 2.1 Cells 2.2 Plasmids 2.3 Culture Media 2.4 Plasticware 2.5 Chloroalkane Halo Ligands 2.6 Buffers and Reagents 2.7 Microscopy Equipment 3 Methods 3.1 Design and Generation of Ecto-Tagged Integrins 3.2 Generation of Stable Cell Lines Expressing Ecto-Tagged β1 Integrins 3.2.1 Production of Lentiviral Particles Driving Expression of Ecto-Tagged β1 Integrin 3.2.2 Replacing Endogenous β1 Integrins with Ecto-Tagged β1 Integrins in Murine Fibroblasts 3.2.3 Replacing Endogenous β1 Integrins with Ecto-Tagged β1 Integrins in Human Cell Lines 3.3 Validation of Ecto-Tagged Integrin Function 3.4 Imaging Exocytosis of Ecto-Tagged Integrins 3.4.1 Imaging Exocytosis of pHluorin-β1 Integrins by Live TIRFM 3.4.2 Imaging Newly Exocytosed Ecto-Halo-β1 Integrins 3.5 Using Ecto-Halo-β1 Integrins to Investigate Integrin Endocytosis and Recycling 3.5.1 Labeling Surface and Intracellular Pools of Ecto-Halo-β1 Integrins with Distinct Halo Ligands and Following Exchange 3.5.2 Imaging Endocytosis of Ecto-Halo-β1 Integrins Using HILO-TIRF 3.5.3 Imaging the Recycling of Ecto-Halo-β1 Integrins Using Reducible Halo Ligands 4 Notes References Chapter 3: Probing the ER-Focal Adhesion Link During Cell Migration 1 Introduction 2 Materials 2.1 Cell Culture and Transfection 2.2 Microscopy and Image Analysis 3 Methods 3.1 Migration Assay 3.2 Live-Cell Imaging 3.3 Image Analysis 4 Notes References Chapter 4: Mapping the Localization of Proteins Within Filopodia Using FiloMap 1 Introduction 2 Materials 3 Methods 3.1 Measuring Line Intensity Profiles in Fiji 3.1.1 Installing FiloMap in Fiji 3.1.2 Analyzing Images in Fiji 3.1.3 Your Result Folder 3.2 Combining Filopodium Intensity Profiles in R 3.2.1 Preparing RStudio Desktop to Use FiloMap 3.2.2 Combining Filopodia in R 3.2.3 Your Result Folder 3.3 Compiling Multiple Filopodia Maps to Create a Heatmap 3.3.1 Combining Multiple Filopodia Maps 3.3.2 Creating a Heatmap in R 4 Notes References Chapter 5: Extended Methods for 2D Confinement 1 Introduction 2 Materials 2.1 Nanogrooved Surfaces 2.1.1 Nanogroove Fabrication 2.1.2 Surface Treatment 2.1.3 Cell Confinement 2.2 Collagen Inclusion 2.2.1 Surface Treatment 2.2.2 Preparation of Collagen Gels 2.2.3 Gel Reticulation and Confinement 2.3 Fixation under Confinement 2.3.1 Preparation of Confinement Rings 2.3.2 Surface Treatment 2.3.3 Preparation of Agarose Gels 2.3.4 Agarose Cell Confinement 2.3.5 Cell Fixation and Removal of Gel 3 Methods 3.1 Cell Confinement Combined with Nanogrooved Surfaces 3.1.1 Fabrication of the Nanogrooved Coverslips (Fig. 2E) 3.1.2 Surface Treatment 3.1.3 Cell Confinement 3.2 Dynamic Cell Confinement Combined with Collagen Inclusion 3.2.1 Surface Treatment 3.2.2 Preparation of the Collagen Gel Solution 3.2.3 Gel Reticulation and Confinement (Fig. 3a) 3.3 Cell Fixation Under Agarose Confinement 3.3.1 Preparation of 3D Printed Confinement Rings 3.3.2 Surface Treatment 3.3.3 Preparation of Agarose Gels (Fig. 4b-c) 3.3.4 Agarose Cell Confinement (Fig. 4b-c) 3.3.5 Cell Fixation and Removal of Agarose Gel 4 Notes References Chapter 6: Visualization of Exosome Release and Uptake During Cell Migration Using the Live Imaging Reporter pHluorin_M153R-CD... 1 Introduction 2 Materials 2.1 Generating Stable Cell Lines 2.2 Live Cell Imaging 2.3 Image Analyses 3 Methods 3.1 Viral Particle Production 3.2 Target Cell Infection 3.3 Observation of Exosome Secretion/Deposition During Cell Migration 3.4 Quantitation of Exosome Secretion/Deposition 3.5 Observation of MVB Trafficking and Fusion During Cell Migration 3.6 Image Analysis of Exosome Secretion Movies 3.7 Observation of Exosome Uptake During Cell Migration 3.8 Quantitation of Exosome Uptake During Cell Migration from Images 4 Notes References Chapter 7: Approaches to Determine Nuclear Shape in Cells During Migration Through Collagen Matrices 1 Introduction 2 Materials 2.1 Embedding of Single Cells 2.2 Fixation and Labeling 2.3 Imaging and Analysis 3 Methods 3.1 Embedding of Cells in Rat Tail Collagen 3.2 Fixation and Labeling (Optional) 3.3 Imaging 3.4 Fiji-Based Image Segmentation and Quantification of Single Images 3.5 Analysis of Nuclear Shapes 3.6 Fiji-Based Image Segmentation and Quantification of Image Sequences 3.7 Analysis of Nuclear Shape Changes over Time 4 Notes References Part II: Developmental Model Systems to Assess Cell Migration During Morphogenesis Chapter 8: Dissecting Collective Cell Behavior in Migrating Testis Myotubes in Drosophila 1 Introduction 2 Materials 2.1 Culture Media 2.1.1 Preparing Agarose Dishes 2.2 Collecting and Timing Prepupae 2.3 Dissecting Testes 2.3.1 Mounting for Long-Term Imaging 2.3.2 Mounting for Short-Term Imaging 2.4 Live-Cell Imaging and Laser Ablation 2.5 Preparation and Staining of Testes 2.5.1 Preparation of Pupal Testes 2.5.2 Preparation of Adult Testes 2.5.3 Fixation and Staining 3 Methods 3.1 Preparing Agarose Dishes 3.2 Collecting and Timing Prepupae 3.3 Dissecting Testes from 33-h APF Drosophila Pupae 3.3.1 Mounting for Long-Term Imaging (>2 h, 25x Magnification) 3.3.2 Mounting for Short-Term Imaging and Laser Cell Ablation (2 h, 25x Magnification) 3.4.2 Short-Term Imaging (