Cell Migration, the Cytoskeleton, Chemotaxis, and Haptotaxis. 3/9/17 ChE 575

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1 Cell Migration, the Cytoskeleton, Chemotaxis, and Haptotaxis 3/9/17 ChE 575

2 When, Where, Why do cells migrate? 1. Neutrophil Migration to Battle Infection 2. Development 3. Wound Healing 4. Disease 2

3 Wound Healing 3

4 Disease Jeon et al

5 Basic Migratory Process Observed through Time-Lapse microscopy 5

6 Cells connect to the ECM: ECMàIntegrinàFocal AdhesionàActin Transmit force and movement in cell via cytoskeleton and focal adhesions 6

7 Tension is translated to biochemical information at adhesion sites FRET: Fluorescence (Forster) Resonance Energy Transfer Grashoff and Hoffman et al P = Protruding R = Retracting 7

8 Actin filaments: double helix with 5-9nm diameter, connect to integrins (indirectly via focal adhesion proteins) 8

9 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

10 Electron Micrograph view of the Actin cytoskeleton in Lamellipodia Mena11a Michele Balsamo & Leslie Mebane, Gertler Lab, MIT 10

11 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

12 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

13 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

14 Mean Squared Displacement analysis Free diffusion <r 2 > (µm) Dimension (1, 2 or 3) r 2 = 2NDt Diffusion coefficient -3 Time (min)

15 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

16 Accounting for this in MSD analysis Persistent Random Walk r 2 2 ( ) ( -t / P t = 2S P t - P + Pe ) 16

17 17

18 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

19 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

20 Haptokinesis vs Haptotaxis Increasing Protein Concentration (FN or Collagen IV) DiMilla et al., JCB

21 1: Step Changes in Stiffness 3T3 Fibroblasts on PAA Migrate from soft-to-stiff substrates Biophys J. Lo et al. (2000) 79;

22 Durotaxis: gradients via photomask polymerization Wong, J. Langmuir,

23 Adapting microfluidics to create haptotaxic gradients Burdick et al., Langmuir

24 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 Speed (um/hr) FN: 8 µg/cm 2 FN: 0.8 ug/cm 2 FN: 0.8 µg/cm 2 * * * * PS Substrate stiffness Young's Modulus (kpa) 24 Peyton and Putnam, J. Cell. Phys. 2005

25 Cytoskeletal Assembly Regulated by Substrate Stiffness Peyton and Putnam, J. Cell. Phys

26 Chemotaxis: Controlling Direction of Motility via Soluble Chemical Cues 26

27 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

28 In vitro Chemotaxis Boyden Chamber Under-Agarose Assay Microfluidics 28

29 Plithotaxis: Cells Migrate in the Direction of the Greatest Normal Stress and Lowest Shear Stress 29

30 Rheotaxis: Cell Migration Upstream in Shear Flow Polacheck et al

31 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

32 Mechanotransduction: Cell can translate Mechanical Information from the ECM to an intracellular biochemical signal Mechanotransduction

33 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?

34 Vibrating Cells: Cells will pull at the site of vibration

35 Pulling on cell attachment points: Focal adhesions are recruited to the site of stretch

36 Stretching the underneath substrate: Microtubules assemble (polymerize) when cell is stretched Putnam et al., JCS, 1998

37 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.

38 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

39 Migration Through Small Channels Causes Nuclear Strain and Rupture Denais et al McGregor et al

40 Modeling of Nuclear Mechanics that Limit Cell Motility Cao et al

41 Tension Alters Gene Expression Tajik et al

42 Traction Force Microscopy: Tool to Measure Cellular Forces Exerted on Substrate

43 Elastomeric Posts

44 Have a Good Break! Reminder: You have a paper review on Tuesday after break 44