CellMigration,theCytoskeleton,Chemotaxis,and Haptotaxis

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When,Where,Whydocellsmigrate?

2

1.NeutrophilMigrationtoBattleInfection

2.Development3.WoundHealing4.Disease

WoundHealing

3

Disease

4Jeon etal.2014

BasicMigratoryProcessObservedthroughTime-Lapsemicroscopy

5

CellsconnecttotheECM:ECMàIntegrinàFocal AdhesionàActin

Transmitforceandmovementincellviacytoskeletonandfocaladhesions

6

Tensionistranslatedtobiochemicalinformationatadhesionsites

7Grashoff andHoffmanetal.2010

FRET:Fluorescence(Forster)ResonanceEnergyTransfer

P=ProtrudingR=Retracting

Actinfilaments:doublehelixwith5-9nmdiameter,connecttointegrins

(indirectlyviafocaladhesionproteins)

8

• Eachclassoffilamentsisapolymer:- madeupofsmaller,solublesubunits

• CellsusingATPenergytopolymerizeanddepolymerizemonomerswhenneeded

9

Mena11a

ElectronMicrographviewoftheActincytoskeletoninLamellipodia

MicheleBalsamo&LeslieMebane,Gertler Lab,MIT 10

Catchvs.Slipbonds

11Guo andGuildford,2006

Catch-SlipBonds:Calculatingruptureforceasafunctionofloadingrate

𝜒"

𝑘$

𝑟&

k off(f)= k offo exp(x β f/k BT)

SlipBonds

CharacteristicBondLength

UnloadedDissociationRateConstantRateofapplicationofforce

Let’slookatmovementmoreclosely–howdowemeasure/predict?

SampleMoviesfromPeytonLab

BreastCancerCellsmigratingonabiomaterialCourtesyPeytonLab

12

Howdoesonequantifythismovement?Speed

Displacement

PathLengthstart

finish

t=1

t=2

t=3

Speed(t1 − t2 ) =(x2 − x1)

2 + (y2 − y1)2

(t2 − t1)

x

y

TotalSpeed =Speed

t∑

# time intervals

displacement = (x f − xi )2 + (yf − yi )

2

PathLength = (x2 − x1)2 + (y2 − y1)

2∑13

-8 -7 -6 -5 -4 -3 -2 -1 0 1

-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

MeanSquaredDisplacementanalysisFreediffusion

<r2 >(µ

m)

Time(min)

r2 = 2NDt

Dimension(1,2or3) Diffusioncoefficient

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MigrationisRandomatLongTimepoints,butpersistentatshortintervals

Longertimepoints (min-hr):Celllocomotionobserved

BreastCancerCellsmigratingonabiomaterialCourtesyPeytonLab

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AccountingforthisinMSDanalysisPersistentRandomWalk

( ) ( )PtPePtPStr /22 2 -+-=

16

17

Anomalousdiffusion:Oftenconfined

Ifthereareobstaclesortrapsintheway,diffusionmightbeanomalous(dependsonobstacleconcentration).

Time(min)

r2 = 2NDt

Saxton1994

<r2 >(µ

m)

Anomalousdiffusionexponent

2 2r ND at=

18

Whatcausesdirectedmigration?(Haptotaxis)

Stiff

HighGrowthFactor

Upstreaminshearflow

Duro

Rheo

Chemo

AlongCellTracks Plitho

Soft

LowGrowthFactor

Downstreaminshearflow

SingleCell

Haptokinesis vs Haptotaxis

DiMilla etal.,JCB1993

IncreasingProteinConcentration(FNorCollagenIV)

20

1:StepChangesinStiffness

DISCUSSION

The phenomenon

The most significant finding in this study is that culturedcells can guide their movement by probing the substraterigidity. As the leading edge crosses onto rigid substrates,lamellipodia and lamella expand, leading to directed migra-tion onto the rigid substrate. Conversely, as the leading edgeapproaches the soft side, local retractions take place, caus-ing the cell to change direction.

In addition to substrate rigidity, we have demonstratedthat mechanical input generated by substrate deformationalso regulates the formation and retraction of lamellipodia.This is to be expected in an active sensing system, becausethe force/deformation caused by the external manipulationwill be superimposed on the effects of the cellular probingforces. In all cases cells responded with the formation/expansion of lamellipodia when the substratum was locallypulled outward from the center, and with retraction whenthe substratum was pushed inward. Because fibroblasts ex-

FIGURE 1 Movements of National Institutes of Health 3T3 cells on substrates with a rigidity gradient. Images were recorded with simultaneous phaseand fluorescence illumination. Changes in substrate rigidity can be visualized as changes in the density of embedded fluorescent beads. (a) A cell movedfrom the soft side of the substrate toward the gradient. The cell turned by !90° and moved into the stiff side of the substrate. Note the increase in spreadingarea 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 enteredthe gradient and moved along the boundary. Bar, 40 !m.

Substrate Rigidity Regulates Cell Movement 147

Biophysical Journal 79(1) 144–152

Biophys J.Loetal.(2000)79;144-152

3T3FibroblastsonPAAMigratefromsoft-to-stiffsubstrates

Durotaxis: gradients viaphotomaskpolymerization

22Wong,J.Langmuir,2003

Adaptingmicrofluidicstocreatehaptotaxic gradients

Burdicketal.,Langmuir200423

Durokinesis:BiphasicMigrationDependenceon

SubstrateStiffness

0.4

0.5

0.6

0.7

0.8

1.0 21.6 45.8 51.9 308 P S

Mea

n C

ell S

peed

( µm

/min

)

Young's Modulus (kPa)

**

FN: 0.8 ug/cm2

0.4

0.5

0.6

0.7

0.8

1.0 21.6 45.8 51.9 308 P S

Mea

n C

ell S

peed

( µm

/min

)

Young's Modulus (kPa)

*

*FN: 8 µg/cm2

FN: 0.8 µg/cm2

**

Speed(um/hr)

Substratestiffness

PeytonandPutnam,J.Cell.Phys.200524

• Durokinesis:SMCsmigratefastestonan‘optimallystiff’ substrate

•Actinpolymerizationcontrolledbyadhesiveproteindensityaswell(Haptokinesis).

•Cellsneedstiffersubstratewhenlessfibronectinisattachedtosurfacetomigrateatmaximumcapacity

CytoskeletalAssemblyRegulatedbySubstrateStiffness

PeytonandPutnam,J.Cell.Phys.200525

Chemotaxis:ControllingDirectionofMotilityviaSolubleChemicalCues

26

ChemotacticIndexisameasureofhowefficientlyacellfollowsachemicalgradient

C.I. = Displacement(µm)PathLength(µm)

C.I. =1 C.I. = 0

0 ≤C.I. ≤127

Invitro ChemotaxisBoydenChamber Under-Agarose Assay

Microfluidics

28

Plithotaxis:CellsMigrateintheDirectionoftheGreatestNormalStressandLowestShearStress

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Rheotaxis:CellMigrationUpstreaminShearFlow

30Polacheck etal.2014

Mechanotransduction

• TheabilityofacelltoturnamechanicalcuefromtheECMintoanintracellularsignal– RhoA,pSrc,pAkt

• Andeventuallyintoaphenotypicresponse–Migration,differentiation,shape,growth

Mechanotransduction:CellcantranslateMechanicalInformationfromtheECMtoanintracellularbiochemicalsignal

“Mechanotransduction”

Howdoesthishappen?• Focaladhesions.– Remember,thoseconnectionsbetweenintegrinsandtheactincytoskeletoninacell.

• When,howdofocaladhesionsre-arrangeinresponsetomechanicalforces?

S=structuralP=signaler

SS

S

P

P

P

S

S

S

VibratingCells:Cellswillpullatthesiteofvibration

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0026181#s5

Pullingoncellattachmentpoints:Focaladhesionsarerecruitedtothesiteofstretch

Stretchingtheunderneathsubstrate:Microtubulesassemble(polymerize)whencellisstretched

Putnametal.,JCS,1998

Proposed:Cell-ECMforcebalancethroughF-actinandmicrotubules

• InresponsetoextracellularstretchoranintrinsicECMstiffness,F-actinmicrofilamentsadjustintensionalresistance,andthemicrotubulenetworkadjustsincompressiveresistance.

CourtesyofA.Putnam

Tensegrity:aPhysicalMechanismofMechanotransduction

Cytoskeletonconnectsfromfocaladhesionstonucleus.Forcesatfocaladhesionscanpropogate tochangesinshapeofnucleusà affectstranscriptionregulatorsà geneexpression/phenotype

MigrationThroughSmallChannelsCausesNuclearStrainandRupture

39Denais etal.2016McGregoretal.2016

ModelingofNuclearMechanicsthatLimitCellMotility

40Caoetal.2016

TensionAltersGeneExpression

41Tajiketal.2016

TractionForceMicroscopy:TooltoMeasureCellularForcesExertedonSubstrate

ElastomericPosts

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