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Cell Migration, the Cytoskeleton, Chemotaxis, and Haptotaxis

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Cell Migration, the Cytoskeleton, Chemotaxis, and Haptotaxis 3/9/17 ChE 575 When, Where, Why do cells migrate? 1. Neutrophil Migration to Battle Infection 2. Development 3. Wound Healing 4. Disease 2 Wound Healing 3 Disease 4 Jeon et al. 2014 Basic Migratory Process Observed through Time-Lapse microscopy 5 Cells connect to the ECM: ECMàIntegrinàFocal AdhesionàActin Transmit force and movement in cell via cytoskeleton and focal adhesions 6 Tension is translated to biochemical information at adhesion sites FRET: Fluorescence (Forster) Resonance Energy Transfer P = Protruding 7 Grashoff and Hoffman et al. 2010 R = Retracting Actin filaments: double helix with 5- 9nm diameter, connect to integrins (indirectly via focal adhesion proteins) 8 • Each class of filaments is a polymer: - made up of smaller, soluble subunits • Cells using ATP energy to polymerize and depolymerize monomers when needed 9 Electron Micrograph view of the Actin cytoskeleton in Lamellipodia Mena11a Michele Balsamo & Leslie Mebane, Gertler Lab, MIT 10 Catch vs. Slip bonds Slip Bonds o k off(f) = k off exp(x β f/k BT) Catch-Slip Bonds: Calculating rupture force as a function of loading rate �" Characteristic Bond Length �$ Unloaded Dissociation Rate Constant �& Rate of application of force 11 Guo and Guildford, 2006 Let’s look at movement more closely – how do we measure/predict? Sample Movies from Peyton Lab Breast Cancer Cells migrating on a biomaterial Courtesy Peyton Lab 12 How does one quantify this movement? Speed finish 2 2 (x2 − x1) + (y2 − y1) Speed(t1 − t2 ) = (t2 − t1) y ∑Speed t=3 TotalSpeed = t x #timeint ervals t=2 Displacement t=1 2 2 displacement = (x f − xi ) + (y f − yi ) start Path Length 2 2 PathLength = ∑ (x2 − x1) + (y2 − y1) 13 Mean Squared Displacement analysis Free diffusion Dimension (1, 2 or 3) Diffusion coefficient > (µm) 2 2 r = 2NDt <r -3 Time (min) -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 14 2 -8 -7 -6 -5 -4 -3 -2 -1 0 1 Migration is Random at Long Time points, but persistent at short intervals Longer timepoints (min-hr): Cell locomotion observed Breast Cancer Cells migrating on a biomaterial Courtesy Peyton Lab 15 Accounting for this in MSD analysis Persistent Random Walk r 2 (t) = 2S 2P(t - P + Pe-t / P ) 16 17 Anomalous diffusion: Often confined If there are obstacles or traps in the way, diffusion might be anomalous (depends on obstacle concentration). Anomalous diffusion exponent 2 > (µm) 2 r = 2NDt <r rND2 = 2 t a Time (min) 18 Saxton 1994 What causes directed migration? (Haptotaxis) Soft Stiff Duro Low Growth High Growth Chemo Factor Factor Single Cell Along Cell Tracks Plitho Downstream in Upstream in shear flow shear flow Rheo Haptokinesis vs Haptotaxis Increasing Protein Concentration (FN or Collagen IV) DiMilla et al., JCB 1993 20 1: Step Changes in StiffnessSubstrate Rigidity Regulates Cell Movement 147 3T3 Fibroblasts on PAA Migrate from soft-to-stiff substrates Biophys J. Lo et al. (2000) 79;144-152 FIGURE 1 Movements of National Institutes of Health 3T3 cells on substrates with a rigidity gradient. Images were recorded with simultaneous phase and fluorescence illumination. Changes in substrate rigidity can be visualized as changes in the density of embedded fluorescent beads. (a) A cell moved from the soft side of the substrate toward the gradient. The cell turned by ϳ90° and moved into the stiff side of the substrate. Note the increase in spreading area as the cell passed the boundary. (b) A cell moved from the stiff side of the substrate toward the gradient. The cell changed its direction as it entered the gradient and moved along the boundary. Bar, 40 ␮m. DISCUSSION In addition to substrate rigidity, we have demonstrated that mechanical input generated by substrate deformation The phenomenon also regulates the formation and retraction of lamellipodia. The most significant finding in this study is that cultured This is to be expected in an active sensing system, because cells can guide their movement by probing the substrate the force/deformation caused by the external manipulation rigidity. As the leading edge crosses onto rigid substrates, will be superimposed on the effects of the cellular probing lamellipodia and lamella expand, leading to directed migra- forces. In all cases cells responded with the formation/ tion onto the rigid substrate. Conversely, as the leading edge expansion of lamellipodia when the substratum was locally approaches the soft side, local retractions take place, caus- pulled outward from the center, and with retraction when ing the cell to change direction. the substratum was pushed inward. Because fibroblasts ex- Biophysical Journal 79(1) 144–152 Durotaxis: gradients via photomask polymerization Wong, J. Langmuir, 2003 22 Adapting microfluidics to create haptotaxic gradients 23 Burdick et al., Langmuir 2004 FN: 8 µg/cm2 FN: 0.8 ug/cm2 FN: 0.8 µg/cm2 Durokinesis: 0.8 * ** Biphasic Migration ** 0.7 m/min) m/min) * Dependence on µ µ ) Substrate Stiffness hr 0.6 • Durokinesis: SMCs migrate fastest on an ‘optimally stiff’ substrate Speed (um/ 0.5 •Actin polymerization controlled by adhesive protein density as well Mean Cell Speed ( Mean Cell Speed ( (Haptokinesis). 0.4 •Cells need stiffer substrate when less fibronectin is attached to surface to 1.0 21.6 45.8 51.9 308 PS migrate at maximum capacity Young'sSubstrate stiffness Modulus (kPa) 24 Peyton and Putnam, J. Cell. Phys. 2005 Cytoskeletal Assembly Regulated by Substrate Stiffness 25 Peyton and Putnam, J. Cell. Phys. 2005 Chemotaxis: Controlling Direction of Motility via Soluble Chemical Cues 26 Chemotactic Index is a measure of how efficiently a cell follows a chemical gradient Displacement(µm) C.I. = PathLength(µm) C.I. = 1 C.I. = 0 0 ≤ C.I. ≤1 27 In vitro Chemotaxis Boyden Chamber Under-Agarose Assay Microfluidics 28 Plithotaxis: Cells Migrate in the Direction of the Greatest Normal Stress and Lowest Shear Stress 29 Rheotaxis: Cell Migration Upstream in Shear Flow 30 Polacheck et al. 2014 Mechanotransduction • The ability of a cell to turn a mechanical cue from the ECM into an intracellular signal – RhoA, pSrc, pAkt • And eventually into a phenotypic response – Migration, differentiation, shape, growth Mechanotransduction: Cell can translate Mechanical Information from the ECM to an intracellular biochemical signal “Mechanotransduction” How does this happen? • Focal adhesions. – Remember, those connections between integrins and the actin cytoskeleton in a cell. S P S S=structural P=signaler S P S S P S • When, how do focal adhesions re-arrange in response to mechanical forces? Vibrating Cells: Cells will pull at the site of vibration http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0026181#s5 Pulling on cell attachment points: Focal adhesions are recruited to the site of stretch Stretching the underneath substrate: Microtubules assemble (polymerize) when cell is stretched Putnam et al., JCS, 1998 Proposed: Cell-ECM force balance through F-actin and microtubules Courtesy of A. Putnam • In response to extracellular stretch or an intrinsic ECM stiffness, F-actin microfilaments adjust in tensional resistance, and the microtubule network adjusts in compressive resistance. Tensegrity: a Physical Mechanism of Mechanotransduction Cytoskeleton connects from focal adhesions to nucleus. Forces at focal adhesions can propogate to changes in shape of nucleus à affects transcription regulators à gene expression/phenotype Migration Through Small Channels Causes Nuclear Strain and Rupture Denais et al. 2016 McGregor et al. 2016 39 Modeling of Nuclear Mechanics that Limit Cell Motility 40 Cao et al. 2016 Tension Alters Gene Expression 41 Tajik et al. 2016 Traction Force Microscopy: Tool to Measure Cellular Forces Exerted on Substrate Elastomeric Posts Have a Good Break! • Reminder: You have a paper review on Tuesday after break 44.
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