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 Jeon et al. 2014 4
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 Grashoff and Hoffman et al. 2010 P = Protruding R = Retracting 7
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 k off (f) = k o off exp(x β f/k B T) Catch-Slip Bonds: Calculating rupture force as a function of loading rate Guoand Guildford, 2006 χ " k $ r & Characteristic Bond Length Unloaded Dissociation Rate Constant Rate of application of force 11
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 Speed(t 1 t 2 ) = (x 2 x 1 )2 + (y 2 y 1 ) 2 TotalSpeed = Displacement t (t 2 t 1 ) Speed #timeint ervals displacement = (x f x i ) 2 + (y f y i ) 2 t=3 t=2 t=1 x y start Path Length PathLength = (x 2 x 1 ) 2 + (y 2 y 1 ) 2 13
-8-7 -6-5 -4-3 -2-1 0 1 Mean Squared Displacement analysis Free diffusion <r 2 > (µm) Dimension (1, 2 or 3) r 2 = 2NDt Diffusion coefficient -3 Time (min) -2.5-2 -1.5-1 -0.5 0 0.5 1 1.5 2 14
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 2 ( ) ( -t / P t = 2S P t - P + Pe ) 16
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Anomalous diffusion: Often confined If there are obstacles or traps in the way, diffusion might be anomalous (depends on obstacle concentration). <r 2 > (µm) r 2 = 2NDt Anomalous diffusion exponent r 2 = 2 NDt a Time (min) 18 Saxton 1994
What causes directed migration? (Haptotaxis) Soft Stiff Duro Low Growth Factor Single Cell Downstream in shear flow High Growth Factor Along Cell Tracks Upstream in shear flow Chemo Plitho Rheo
Haptokinesis vs Haptotaxis Increasing Protein Concentration (FN or Collagen IV) DiMilla et al., JCB 1993 20
1: Step Changes in Stiffness 3T3 Fibroblasts on PAA Migrate from soft-to-stiff substrates Biophys J. Lo et al. (2000) 79;144-152
Durotaxis: gradients via photomask polymerization Wong, J. Langmuir, 2003 22
Adapting microfluidics to create haptotaxic gradients Burdick et al., Langmuir 2004 23
Durokinesis: Biphasic Migration Dependence on Substrate Stiffness Mean Cell Speed (µm/min) Durokinesis: SMCs migrate fastest on an optimally stiff substrate Actin polymerization controlled by adhesive protein density as well (Haptokinesis). Cells need stiffer substrate when less fibronectin is attached to surface to migrate at maximum capacity 0.8 0.7 0.6 0.5 0.4 Speed (um/hr) FN: 8 µg/cm 2 FN: 0.8 ug/cm 2 FN: 0.8 µg/cm 2 * * * * 1.0 21.6 45.8 51.9 308 PS Substrate stiffness Young's Modulus (kpa) 24 Peyton and Putnam, J. Cell. Phys. 2005
Cytoskeletal Assembly Regulated by Substrate Stiffness Peyton and Putnam, J. Cell. Phys. 2005 25
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( µ m) C.I. = PathLength( µ m) C.I. = 0 C.I. = 1 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 Polacheck et al. 2014 30
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. P P S S S S S=structural P=signaler S P S When, how do focal adhesions re-arrange in response to mechanical forces?
http://www.plosone.org/article/info%3adoi%2f10.1371%2fjournal.pone.0026181#s5 Vibrating Cells: Cells will pull at the site of vibration
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 Cao et al. 2016 40
Tension Alters Gene Expression Tajik et al. 2016 41
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