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Lecture+13-16+Cell+migration - Cell Migration 1 Molecular...

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Unformatted text preview: Cell Migration 1. Molecular machinery 2. Basic steps of cell migration 3. Measurement of traction force 4. Regulation of cell migration by 4. environmental cues 5. Quantitative analysis of migration Example 1: Angiogenesis and Metastasis Cell Cell Migration Cell migration is involved in many physiological and pathological processes, e.g., wound healing, tissue repair, angiogenesis, immune responses. Cell migration is a mechanical process that involves chemical and mechanical signal transd transduction. The cytoskeleton, focal adhesion The focal adhesion proteins and signaling molecules needs to be coordinated to drive cell migration. Example 3 Endothelial Cell Migration during Wound Repair http://www.youtube.com/watch?v=aKBZbxBnpGM Example 2: A Neutrophil Chasing a Bacterium http://www.youtube.com/watch?v=I_xh-bkiv_c Directional migration, similar to “particle diffusion” Example Example 4: Tracking Individual Cell Migration 1. Molecular machinery Cytoskeleton actin microtubule intermediate filament Rho GTPases Focal adhesions “Random walk” Actin Filaments Actin Filaments Lamellipodia and filopodia 8 nm-wide helix Actin Thin and flexible filaments that form intracellular network to provide mechanical support to the plasma membrane etc. Assembly; crosslinking; anchorage crosslinking; Actin Actin in Lamellipodia of Fish Keratocytes Actin Polymerization during Cell Migration Actin Structure in the Establishment of Cell Polarity Actin Polymerization in Filopodia Microtubules Microtubules Microtubules Heterodimers form for 25 nm hollow tube Regulate cell polarity and intracellular transport Intermediate Filaments MICROTUBULES Diameter appearance Component polypeptide MW 4 types of fibrous proteins: keratins, vimentin/desmin/GFAP, neurofilaments, nuclear lamins Support cell and nucleus structure Bound nucleotide MICROFILAMENTS 25 nm 7 nm INTERMEDIATE FILAMENTS 10 nm hollow tubes composed of 13 protofilaments helical filaments rope-like filaments composed of 8 protofilaments I: keratins II: vimentin desmin GFAPs III: neurofilaments IV: nuclear lamins nuclear lamins tubulin globular dimer ( and ) actin globular monomer 50 kDa 43 kDa 40 – 130 kDa GTP (two per dimer) ATP (1:1) none Rho Rho Family GTPases Rho family GTPases belongs to Ras superfamily Rho family GTPases (e superfamily. Rho family GTPases (e.g., Rho, Rac and Cdc42), which function as binary switches that cycle between an active GTP-bound form and an inactive GDP-bound form, have distinct functions in regulating the actin-based cytoskeletal structure. Effects of Rho GTPases on Actin Structure Rho Rho Family GTPases Regulate Actin Filament Organization Cdc42 Rac Rho Lamellipodia filopodia Actin Filaments Focal Complexes Focal Adhesions Provide Anchorage for Actin Filaments Focal Focal Adhesions Actin 2. Basic steps of cell migration Tensin Vinculin -Actinin Talin Paxillin FAK Basal Membrane Integrins Extracellular Matrix Adhesion Strength and Cell Migration Balanced attachment at front and detachment at rear 60 Migration Speed (m/hr) Five Steps of Cell Mi Migration Cell migration requires migration the attachment at front and detachment at rear 50 40 30 20 10 0 0.1 1 10 100 2 Fibronectin (g/cm ) Difficult to release adhesions atat the front of the cell Not enough stable attachment the rear of the cell Adhesion force and Traction Force 3. 3. Measurement of adhesion force and traction force Adhesion force: overall adhesion strength Traction force: subcellular pulling force on ECM exerted by the cell Cell Focal Adhesion Nucleus Extracellular matrix Measuring Adhesion Forces Determine the level of shear stress to detach group of cells detach a group of cells Parallel flow Centrifugal force Determine the force needed to detach single cell Micropipette Other cytodetacher Flow Chamber for Cell Detachment Inflow Outflow Chamber base Gasket Glass slide with cultured cells Flow Flow Chamber for Cell Detachment Inflow Negative pressure Outflow Shear Stress Solve the equation for 2D laminar flow Micropipette Detachment Glass micropipette Cell du dy viscosity Measuring Traction Forces Using Silicone Rubber as Substrate to Measure Traction Forces Using silicone rubber (wrinkle) Using microfibricated cantilever Using polyacrylamide gel embedded with beads Limited spatial resolution and complex relationship between wrinkles and forces Microfabricated Microfabricated cantilever Galbraith et al., PNAS 94: 9114-8, 1997 Determination of Young’s Modulus of Elastic Substrate Young’s modulus: modulus: Traction stress: stress: Y= stress/strain =(F/A) / (∆l/l) Distribution of Substrate Deformation (Displacement of Beads) T= Y * ∆x/x Elastic polymer Fluorescence beads t=0 vs. t=30 min t=30 min, cell Pelham et al., MBC 10:935-45, 1999 4. Regulation of cell migration by environmental cues NCB 2001 3(5) Cellular interactions with fluid phase Chemotaxis Mechanotaxis Cellular interactions with solid phase Cell migration is regulated by environmental cues: • Cellular interactions with fluid phase • Cellular interactions with solid phase (ECM) • Cellular interactions with other cells Geometry and textures (topography) Pattern of adhesiveness Mechanical properties of the substratum Cellular interactions with other cells Cell migration in 3D matrix Extracellular matrix Cellular Cellular Interactions with Fluid Phase during Cell Migration Diffusible molecules, e.g.,Growth factors, cytokines Chemotaxis The directional translocation of cells in a concentration gradient of some chemoattractant or chemorepellent substance substance Mechanical forces Chemoattractants e.g. growth factors, cytokines, matrix cytokines, matrix molecules, peptides. Cell Nucleus Extracellular matrix Extracellular matrix Chemokinesis Molecular Dynamics of Focal Adhesions Stimulated by Serum A kinesis in which the stimulating scalar property is the concentration of some chemical Extracellular matrix Time interval between frames: 10 min Examples Examples of Chemotaxis: The penetration of neutrophils through blood vessel walls Examples of Chemotaxis (chemotropism): Capillary sprouts and nerve axon extension Lumen EC SMC Surrounding tissue Sources of infection Quantification of Chemotaxis: Boyden chamber Quantification of Chemotaxis: Micropatterning •Seed cells on the upper surface of the porous membrane membrane •Add chemoattractants in the lower reservoir •Count cells on the lower surface of the membrane Cells Porous membrane Chemoattractants Jeon et al., (2002) Nat Biotech 826-830 Neutrophil chemotaxis in response to the gradients of IL-8 Fluid Shear Stress Shear Stress y u(y) du dy viscosity The Microscopy System for Studying Cell Migration under Flow Shear Stress Enhances EC Wound Healing Time (hr) 37ºC Static Control Laminar Flow 0 Image Analysis 5% CO2 Pump Cells in flow chamber Image Acquisition 7 Reservoir Microscope CCD camera or Scanner 21 Hsu et al., (2001) BBRC 285: 751-759 Shear stress induces directional migration in the flow direction Effect of Shear Stress on Cell Migration C A Laminar Flow Static † Migration Speed (m/hr) 30 30 m 20 m † 20 † B † 10 † Flow D 0 30 m Time Course of Shear Stress-Induced EC Migration (the directional migration in the flow direction) B A Vy (m/10 min) Serum Shear Stress 10 5 0 -5 -10 0 0.5 1 -10 -5 0 5 10 Vx (m/10 min) (hr) Li et al. (2002) PNAS 99:3546-3551 Flow Migration Rate (m/10 min) Shear Stress Induces “Mechanotaxis” Control 20 m 7 6 5 4 3 2 1 0 -1 Vx Vy 0 0.5 1 1.5 Time (hr) 2 Mechanical Force Regulates Molecular Dynamics of Focal Adhesions During EC Migration under Flow the Dynamics of Focal Adhesions (FAs) Mechanical forces Cell Nucleus Focal Adhesion Adhesion Extracellular matrix Time interval between frames: 2 min Shear stress enhances the speed of cell migration Cellular Interactions with Solid Phase 8 Geometry and textures 7 Speed (m/10 min) 6 Shear 5 Mechanical properties of the substratum 3 2 1 0 0.1 Pattern of adhesiveness Static 4 1 10 Fibronectin (g/cm2) 100 Contact Guidance The directional translocation of cells in response to some anisotropic property of the substratum (e.g., shape texture aligned fibrillar matrices) shape, texture, aligned fibrillar matrices) Effect of Aligned Fibrillar Matrices •Aligned by cells •Aligned by mechanical forces •Aligned by magnetic fields Extracellular matrix By influencing the orientation of focal adhesions and actin filaments Shape Shape effect A cylindrical glass fiber up to 200 m in diameter can still constrain the cell migration to be parallel with the fiber axis. Groove Effect Parallel groves as small as 100 nm in depth can induce guidance responses guidance responses The patterned topography becomes less effective for directional migration as the repeating space increases to the scale bigger than a cell Haptotaxis Haptotaxis The tendency of cells to translocate unidirectionally up a steep gradient of increasing adhesiveness of the substratum Extracellular matrix Cell migration towards the area with higher matrix density Effect of Mechanical Properties of Substratum Pattern Pattern of Adhesiveness Cells migrate towards more rigid surface Adhesive strips become less effective in eliciting guidance as the repeat spacing decreases Biophy J.(2000) 79:144-52 CellCell-Cell Contact Modulates Cell Migration ThreeThree-dimensional (3D) Matrix Collagen Polymer Hydroxyapatite Quantification of migration Time lapse microscopy End-point measurement Hsu et al., (2001) BBRC 285: 751-759 Cell Migration in 3-D Matrix 3- Cell Migration in Three-dimensional (3D) ThreeMatrix (compared with 2D) Decrease of stress fibers fib Higher ligand availability Higher resistance Deformation ability is important Contact dependent proteolysis dependent proteolysis and matrix remodeling Quantitative Analysis of Cell Migration Single cell Speed Mathematical Model of Cell Migration Mi Direction Directionality : Net path length / Total path length Persistence time Net path Total path Quantitative Analysis of Cell Migration Whole populations J = - c1 (dN/dx) + c2 (da/dx) N + ... random motility chemotaxis Wound J: cell flux c1cell random motility coefficient N: cell number cell number c2 chemotaxis coefficient a: chemoattractant concentration x Net Migration Distance ...
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