Impact of heating on passive and active biomechanics of suspended cells.

2014·0.77 MB·English

by  Chan, C. J.

Impact of heating on passive and active
biomechanics of suspended cells
C. J. Chan1,2, G. Whyte3, L. Boyde1, G. Salbreux4 and J. Guck1,2
rsfs.royalsocietypublishing.org
1Cavendish Laboratory,Departmentof Physics, UniversityofCambridge, Cambridge, UK
2BiotechnologyCenter,Technische Universita¨tDresden, Dresden,Germany
3DepartmentofPhysicsand Institute ofMedical Biotechnology, Universityof Erlangen-Nuremberg,
Erlangen, Germany
4MaxPlanck Institute for the PhysicsofComplex Systems,Dresden, Germany
Research
Acellisacomplexmaterialwhosemechanicalpropertiesareessentialforits
Citethisarticle:ChanCJ,WhyteG,BoydeL, normalfunctions.Heatingcanhaveadramaticeffectonthesemechanicalprop-
erties, similar to its impact on the dynamics of artificial polymer networks.
SalbreuxG,GuckJ.2014Impactofheatingon
Weinvestigatedsuchmechanicalchangesbytheuseofamicrofluidicoptical
passive and active biomechanics of suspended
stretcher,whichallowedustoprobecellmechanicswhenthecellsweresub-
cells. Interface Focus 4: 20130069.
jected to different heating conditions at different time scales. We find that
http://dx.doi.org/10.1098/rsfs.2013.0069 HL60/S4 myeloid precursorcells becomemechanicallymore compliant and
fluid-likewhensubjectedtoeitherasuddenlaser-inducedtemperatureincrease
orprolongedexposuretohigherambienttemperature.Aboveacriticaltemp-
One contribution of 8 to a Theme Issue
erature of 52+18C,we observedactive cellcontraction,which wasstrongly
‘Nanobiomechanics of living materials’.
correlatedwithcalciuminfluxthroughtemperature-sensitivetransientreceptor
potentialvanilloid2(TRPV2)ionchannels,followedbyasubsequentexpan-
Subject Areas: sion incellvolume. The change frompassive to active cellular response can
biomechanics, biophysics be effectively described by a mechanical model incorporating both active
stressandviscoelasticcomponents.OurworkhighlightstheroleofTRPV2in
regulatingthethermomechanicalresponseofcells.Italsooffersinsightsinto
Keywords:
howcorticaltensionandosmoticpressuregoverncellmechanicsandregulate
cell contraction, calcium, cortical tension, cell-shapechangesinresponsetoheatandmechanicalstress.
myeloid precursor cell, optical stretcher,
cell rheology
1. Introduction
Author for correspondence:
C. J. Chan Cellsexhibitarichdynamicbehaviourwhensubjectedtomechanicalstress.Over
e-mail: [email protected] the years, several theoretical approaches have been developed to describe cell
mechanicalproperties,althoughthereisnounambiguouslyacceptedtheoretical
description as of yet. These approaches range from phenomenological models
thatpicturecellsasasuitablecombinationofspringanddashpotelements[1,2]
to the scale-free power-law models that well characterize the physical stress
responseofalargeclassoflivingcells[3,4].Tothisend,avastarrayofexperimen-
tal techniques have been developed to study cellular mechanics. For example,
magnetictwistingcytometryandatomicforcemicroscopy[5–7]havebeenused
extensively to probe local stress response of adherent cells, while micropipette
aspiration and microplate manipulation have been developed to assess global
mechanicalpropertiesofcells[8–10].Comprehensivereviewsofthedifferenttech-
niquescanbefoundinrecentpublications[11–13].Amongthesetechniques,the
microfluidicopticalstretcherhasemergedasastandardtooltoprobewhole-cell
mechanicsinanon-invasiveway[14],henceofferingpowerfulinsightsintothe
dynamicsofnon-adherentcells,forexampleduringamoeboidmigration[15,16].
Despite a great deal of research on cell mechanics, little is known about the
impact of heating on cells. Previous studies [17–19] showed that both adherent
and suspended cells, such as fibroblasts and leucocytes, soften with increased
temperature.Bycontrast,otherstudies[20]suggestedthatadherentcellsbecome
Electronic supplementary material is available stifferandmoresolid-likewithincreasedtemperature,possiblyowingtoincreased
at http://dx.doi.org/10.1098/rsfs.2013.0069 or contractile activity of molecular motors. The exact mechanisms for such
via http://rsfs.royalsocietypublishing.org.
&
2014TheAuthors.PublishedbytheRoyalSocietyunderthetermsoftheCreativeCommonsAttribution
License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original
authorandsourcearecredited.
temperature-inducedcellstiffeningorsofteningremainelusive. (a) 2
1064nm Hg
In a similar vein, the impact of heating in cell deformation stretch PC CCD fluorescent
studieswithanopticalstretcherhasbeengivenlittleattention. laser lamp rs
fs
Recent studies using fluorescence ratio thermometry [21] .ro
thermal y
showed thatatawavelength of1064nm,whichismostoften chamber res 2 als
uinsgedofforaobpotuictal1t3ra8Cppiwngit,h1inWaofflraascetiropnowoefrawisllecinodnudc.eHaihgehaetr- IRhe laatmerp fibre ends 1h4el8aa0stiennrmg ocietyp
u
wavelengthswillbeabsorbedevenstrongerandleadtomore b
heating,roughlylinearlyproportionaltothechangeinabsorp- glass ofpitbicreasl lishin
tioncoefficientofwater.Furtherworkquantifiedtheimpactof g.o
laser heating on cell viabilityandcalcium influx in an optical res 1 rg
stretcher[22,23].Arecentstudy[24]providedfurtherinsights cofiubprleer In
into how different time scales of heating may elicit different terfa
cellmechanicalresponsesinanopticalstretcher. ce
Thispaperthereforeattemptstoshednewlightonthetopic (b) 106h4ignhm i nltaesnesri btye am Fo
c
u
oftemperature-inducedchangesofcellmechanicalproperties s
4
bymeasuringsuspendedcellsinanopticalstretcherwhensub- :
2
jIenctteegdrattionghethateinmgicartofvluairdioicusoptetimcaplesrtarteutcrehserawndithtima e14s8c0anlems. optical fibre cell optical fibre 01300
6
heatinglaserandaheatingchamberequippedwithanadjusta- optical stress low intensity 9
1480nm laser beam
ble heat source, we observedand characterizedthedegreeof
thermal softening of cells in the passive viscoelastic regime. (c)
Interestingly,wereportstrikingevidenceofactivecellcontrac-
tion whenever the overall temperature, independent of the 2(r +Dr)
0
combinationofchambertemperatureorlaser-inducedtempera-
ture increase, rose above 528C. Such behaviour is correlated
withacalciuminfluxregulatedbytemperature-sensitivetransi-
1.00W (1064nm)
ent receptor potential vanilloid 2 (TRPV2) ion channels. We 2r0
showedthatsucharheologicaltransitionfromthermalsoften-
ingtoactivecellcontractioncouldbeeffectivelymodelledwith
2(r +Dr¢)
a mechanically equivalent circuit. Our findings highlight the 0
0.40W (1064nm)
interestingroleofTRPV2ionchannelsinregulatingcellmech-
anicalpropertiesandcell-shapechangesinresponsetoexternal
heatandmechanicalstress. 1.00W (1064nm)+0.04W (1480nm)
Figure 1. Experimental set-up of a microfluidic optical stretcher. (a) Sche-
matic diagram of a microfluidic flow chamber (phase-contrast image)
2. Experimental methods housed in a heating chamber, with optical fibres (left and right) arranged
perpendicular to the walls of a square glass capillary (top to bottom),
2.1. Optical stretcher set-up
which contains cell suspension. Scale bar is 40mm. (b) The momentum
The principle and set-up of the microfluidic optical stretcher transfer of light to the surface of the cell results in (mainly axial) optically
havebeendescribedextensivelyelsewhere[25,26].Essentially, induced surface stresses, leading to the deformation of the cell along the
the device is a dual beam laser trap capable of trapping and opticalaxis.Inadditiontothetwocounterpropagatingtrappingandstretch-
deforming the cells through optically induced stress acting ing laser beams (1064nm, blue), an additional 1480nm laser beam (red)
on the cell surface. The microfluidic flow chamber, as shown was coupled into one of the optical fibres and co-propagated to irradiate
withaphase-contrastimageinfigure1a,consistsoftwoco-axial and heat the cell with minimal additional power. (c) Left: an HL60 cell
opticalfibresalignedperpendiculartoasquareglasscapillary, with radius r was trapped with a total power of 0.40W in the optical
0
which delivers the cells in suspension. The chamber is then stretcher. Right (top): an increase in the 1064nm laser powers to 1.00W
mounted on an inverted phase-contrast microscope, which is led to a stretching of the cell along the laseraxis, resulting in an increased
attachedto a CCDcamerafor image acquisition.The flow of radius r þDr: The strain g(t) is then given by Dr/r : Right (bottom):
0 0
cellsuspensionwasadjustedthroughtherelativedifferencein applying 40mW of 1480nm heating laser led to a further axial strain
heightsofinletandoutletreservoirsconnectedtothecapillary. r þDr0: Scale bar is 5mm.
0
Theentireset-upwasthenenclosedinaheatingchamberwith
thechambertemperaturecontrolledbytheuseofaninfrared increase scales linearly with absorption coefficient at a parti-
lampandheater.Thisallowedthecellstohavesufficienttime cular wavelength, the about 180 times higher absorption at
(about 30min) to adapt to the new chamber temperature 1480nm compared with 1064nm [27] means that the
beforebeingopticallytrappedanddeformed. 1480nm laser could be used to introduce significant heating
In addition, the output of a laser diode operating at at minimal additional laser power, and consequentlya negli-
l ¼1480nm was spliced into one of the 1064nm optical gible amount of optically induced stress on the cells. In this
0
fibresusingawavelengthmultiplexer,sothatitcanbeoperated way, heating and stretching can be effectively decoupled.
simultaneously with the 1064nm fibre laser. The coupling of In contrast to adjustments of the chamber temperatures,
1480nm single beam to one of the 1064nm dual beams pro- thisapproachallowedustoprobetemperature-dependentcell
vides almost instantaneous (within milliseconds) heating in rheology when the cells were heated over a much shorter
addition to cell deformation (figure 1b). As the temperature time scale. As shown in figure 1c, a cell can be effectively
trappedandstretchedwiththe1064nmstretchlaser,withthe with less than 40nm precision. The percentage change in 3
axial strain readily obtained for further analysis as discussed cell volume was computed from the relative deformations
in§2.2.Furtherapplicationofthe1480nmheatinglaserledto (cellularstrains)alongthecellmajorandminoraxis,assum- rs
fs
a further change in cellular strain, which demonstrates the ing the rotational symmetry of a prolate spheroid, that is, .ro
y
workingprincipleofthe1480nmheatinglaser. V¼ð4/3Þpa2b, where b and a represent the lengths of cells als
o
The laser-induced temperature increase inside the glass along major and minor axis, respectively. For each optical cie
capillarywasmeasuredusingfluorescenceratiothermometry, stretcherexperiment,thenumberofcollectedcellswasn(cid:3)30. typ
u
asdescribedpreviously[21].Themethodisbasedonmeasuring The cellular strain and compliance data are presented as b
lis
thelaser-inducedfluorescenceofahighlytemperature-sensitive mean+s.e.m.Representativestrainandcompliancedatawere hin
dye(RhodamineB)andareferencedye(Rhodamine110)with chosen from two or more independent experiments. In order g.o
temperature-independentfluorescence.Theratiooftheintensi- to correct for different cellular response owing to slight vari- rg
tiesofthetwodyesscaleslinearlywithtemperature.Byvarying ations in cell cycle or nutrient concentration in a particular In
the chamber temperature between 25 and 558C, the intensity batch ofmedium (e.g. HL60 cells have been reported to show terfa
ratio was measured and converted into temperature values decreased strain with increased culture density [14]), data for ce
using the established calibration. This allowed usto obtain a each power were taken over a number of days. To minimize Fo
c
u
precise measurement of the peak temperatures in the optical additional systematic errors, for example changes in cell s
4
trapwhenthe1064nmandthe1480nmlaserswereoperated deformability with post-incubation period [30], cells were :
2
(seeelectronicsupplementarymaterial,figureS1).Theincrease stretched with a random sequence of powers for each exper- 01
3
intemperaturefor1064nmstretchlaserand1480nmheating iment. During stretching, a range of cell sizes were measured 00
6
laser was measured to be 10.58CW21 and 2.28CdBmW21, to ensure the results were representative of the entire popu- 9
respectively,wherethepowerP ,measuredindecibelsof lation. Care was taken to exclude any irregular-shaped cells,
dBmW
powerand the power P , measured in W, are related by the as they introduce unwanted rotations during stretching,
W
equation:P ¼10log (P )þ30. giving rise to ‘false’ deformations. The flow was adjusted and
dBmW 10 W
always made to stop before trapping to minimize rotations
2.2. Imaging and data analysis and wobbling before the start of a stretch. To avoid non-uni-
AcustomwrittenLABVIEWsoftwarewasusedtotrackthetem- form pressure gradient that disturbs the flow, care was taken
poralevolutionofthecelledge(figure1c).Dataformajoraxis toremoveanyairbubblesinthecapillaryandcelldebrisinsus-
deformationr(t)werestoredforeverytime-frame,whiler was pension. The latter was minimized by using rapidly growing
0
evaluatedasthemean lengthofmajoraxisduringtheinitial cells (logarithmic phase) for experiments or centrifuging cells
trapping period. The time-varying axial strain gðtÞ¼Dr/r , beforeexperiment.
0
asdepictedinfigure1c,wasthenevaluatedaccordingly.The
opticallyinducedstresswascomputedbasedonthegeneral- 2.3. Cell preparation
ized Lorenz–Mie model [28]. Once the peak stresss along
0 HL60/S4 myeloid precursor cells were chosen as the model
the laser axis was evaluated, the time-dependent creep
cellsforthisstudy,becausetheynaturallygrowinsuspension,
complianceJ(t)couldbereadilyobtained
whichmeanstheyaremeasuredintheirphysiologicalenviron-
gðtÞ ment in a microfluidic optical stretcher. The cells were
JðtÞ¼ ; ð2:1Þ
s0Fg incubated at 37.58C with 5%carbondioxide level. Cellswere
chosen to be stretched when they were at their logarithmic
whereF isageometricfactorthattakesintoaccountthecell
g
phaseofgrowth,whichoccurredtypically36–48hafterresus-
sizeandstressdistribution[29].
pension.Trypanblueexclusionmethodwasemployedtocheck
Recentstudieshaveshownthatonecanemployeitherthe
forcellviabilitypriortoeveryexperiment.Cellswerekeptincu-
power-law or the mechanical models to extract cell visco-
batedinvialsandallowedtoequilibrateataspecificchamber
elastic parameters in the optical stretcher experiments
temperaturefor20minpriortoopticalstretchingexperiments.
[4,16,30].Theequationforthepower-lawmodelisasfollows:
All opticalstretching experiments were performed within 2h
(cid:2)t(cid:3)b afterthecellsweretakenoutoftheincubator.Forcalciumima-
JðtÞ¼J : ð2:2Þ
0 t0 ging experiments, HL60 cells were loaded with 1mM Fluo-4,
AM (Invitrogen, F14201) and incubated for 20min at 258C.
Here, avalue ofb¼0 gives Hooke’s law whileb¼1 corre-
Subsequently, the AM ester solutions were removed by cen-
sponds to complete viscous behaviour. t ¼1s and J is
0 0
trifugationandcellswereresuspendedinRPMI1640medium
defined as the characteristic inverse Young’s modulus. bis,
orphosphatebufferedsaline(PBS)medium withoutcalcium,
therefore,ameasureofthecellfluiditywhileJ isameasure
0
unlessotherwisestated.ForexperimentsoninhibitingTRPV2
of cell compliance. On the other hand, a simple mechanical
ion channels, cells were measured in 10mM ruthenium red
model, for example the standard linear liquid (SLL) model,
(Sigma-Aldrich,84071)solution.
hasbeen recentlyshown to characterize cell deformation for
blood precursorcells[16].The modelemploysthe following
equation forthecreep compliance:
3. Results
1 1
JðtÞ¼ tþ (1(cid:2)e(cid:2)ðE1=h1Þt); ð2:3Þ
h0 E1 3.1. Cells are more compliant at higher temperatures
whereh describesthesteady-stateviscosityofthemodel,h Toinvestigatetheeffectoncelldeformationasitexperiences
0 1
andE thetransientviscousandelasticresponse,respectively. asuddentemperaturejump,weconductedopticalstretching
1
In estimating changes in cell volume, we note that the experimentsusingthe1480nmlaserset-up,whereaninstan-
tracking of relative cell edge movement can be performed taneous temperature jump within milliseconds was applied
(a) (b) 4
7
6 TscohFagm=be0r=.8205P°aC DTlaser 10 4471..66°°CC DsoTFchga=mb1e.r0=0+P6a°C rs
43.4°C fs
cellular strain (%) 2345 4 44 3 6325....0915°°°°CCCC cellular strain (%) 468 37.4°C v .royalsocietypublish
2 sF =0.64Pa in
1 o g g
0 0 DTchamber=+6°C .org
–1 0 1 2 3 4 5 –1 0 1 2 3 4 5 6 7
time (s) time (s) Inte
(c) 9 TsoFg=1=.0305P°aC (d) 4 rface
7 chamber n=30 Fo
3 cu
major axis s
%) 5 2 4:
cellular strain ( –113–1 0 1 2 3 4 5 6 7 DVV/ (%)–101 20130069
minor axis
–3
–2
–5
time (s)
(e) (f)
0.65
0.15
–1a) 0.60
cell compliance (P 00..0150 bcell fluidity, 000...455505
0 0.40
25 30 35 40 45 50 25 30 35 40 45 50
T (°C) T (°C)
overall overall
Figure 2. Passive viscoelastic cell deformation in response to heating. (a) Cellular strain as a function of time when the cells were stretched at 0.80Pa by the
1064nmlaserandheatedinstantaneouslytohighertemperaturesbythe1480nmheatinglaserwithchambertemperaturekeptat258C.Theredarrowindicates
increasing temperature owing to laser heating. (b) Cellular strain as a function of time when the cells were stretched at 0.64 and 1.00Pa but subjected to an
increaseinthechambertemperature.Thebluearrowsindicateincreasingtemperatureowingtochamberheating.(c)Cellularstrainalongthemajorandminoraxes
asafunctionoftimewhenthecellswerestretchedat1.00Paand358C.(d)Changeincellvolumeafter6sofstretching.DatarepresentcellsstretchedatT ,
overall
528C. Mean change in cell volume¼0.21% with s.e.m.¼0.23%. (e) Cell compliance at 4s into the stretch, J(t¼4s), plotted as afunction of overall temp-
erature (T ¼T þDT ): The line represents a linear fit to the data points, with R2¼0.95. (f ) Cell fluidity,b, plotted as a function of T .
overall chamber laser overall
Legends in (a,b) indicate T . Grey bars along the time axis in (a–c) indicate the period of stretch.
overall
inadditiontothedeformationbythe1064nmstretchlaser,as Poisson ratios very close to 0.5. This suggests that the cell
described in §2.1. Using the calibrated temperature increase volume was conserved, assuming that the cells remained
for heating by the 1480nm laser, we observed an increase rotationally symmetric in the form of a prolate spheroid
in peak cellular strain along the cell’s majoraxis (parallel to during the stretch. Indeed, no significant change in cell
the laser axis) with increased laser heating ðDT Þ, as indi- volumewasobserved(figure2d)aslongastheoveralltemp-
laser
cated by the red arrow in figure 2a. To investigate whether erature stayed below 528C. To compare changes to cell
a similar thermal softening behaviour is observed for cells materialpropertiesatdifferentoveralltemperatures,wecom-
under prolonged exposure to heat, we stretched cells with puted the cell compliance at 4s into the stretch J(t¼4s)
thesameopticalstressbutsubjectedtohigherchambertemp- using equation (2.1), and the results are plotted in figure 2e.
eratures. Figure 2b compares the strain curves for cells Clearly,thecellcomplianceincreasedlinearlywiththeover-
stretched at 0.64 and 1.00Pa, each subjected to an increase alltemperature,suggestingthatthecellcomplianceisonlya
in chamber temperature from 29 to 358C. Clearly, the peak function oftheabsolute overalltemperature,independent of
strain increased with increased chamber temperatures at the time scales involved in the heat treatments. The thermal
both stresses. Further experiments at higher stresses with softening effect is significant, with the cells experiencing a
increased chamber temperatures revealed the same thermal markedfourfoldincreaseincreepcomplianceasthetempera-
softening behaviour (not shown). ture increased from 27 to 478C. The cell fluidity,b, can also
Thetemporalevolutionofcellularstrainalongtheminor be obtained by fitting the creep compliance curves to the
axiswasalsocharacterized(figure2c,opencircles).Thecells power-law model from equation (2.2), and the results are
showedanegativestraininthisdirection,withthecalculated summarizedinfigure2f.Here,weobservedalowvalueofb
(a) 7 soFg=0.80Pa (b) 30 sF =1.80Pa 5
T =25°C o g
6 c5h5am.4b°eCr 25 5582..11°°CC rs
cellular strain (%) 2345 D543T385la...s175e°°°rCCC v cellular strain (%) 112050 Tc4h7am.1b°eCr fs.royalsocietypub
lis
5 h
1 in
g
0 0 .org
–1 0 1 2 3 4 5 –1 0 1 2 3 4 5 6 7
time (s) time (s)
In
(c) 11 soFg=1.30Pa (d) terfa
T =35°C 10 c
chamber e
9 n=36
F
o
%) 7 major axis 8 cus
ellular strain ( 135 DVV/ (%) 46 4:2013006
c 9
–1–1 0 1 2 3 4 5 6 7
minor axis 2
–3
–5
0
time (s)
(e) (f)
35 60
active cell contraction
30 55
25
T(°C)laser 1250 T(°C)overall 4550
D D 40
10
5 35
passive viscoelastic softening
0 30
21 23 25 27 29 31 33 35 37 0 0.5 1.0 1.5 2.0
T (°C) sF (Pa)
chamber o g
Figure3.Activecellcontractioninresponsetoheating.(a)Cellularstrainasafunctionoftimewhenthecellswerestretchedat0.80Paandheatedbythe1480nm
heatinglasertoT above528C.Theredarrowindicatesincreasingtemperatureowingtolaserheating.(b)Cellularstrainasafunctionoftimewhenthecells
overall
werestretchedat1.80PaandsubjectedtohigherchambertemperaturessuchthatT exceeds528C.Thebluearrowindicatesincreasingtemperatureowingto
overall
chamberheating.(c)Cellularstrainalongthemajorandminoraxes,asafunctionoftime,at1.30Paand358C,whichcorrespondedtoT ¼528C.(d)Change
overall
incellvolumeafter6sofstretching.DatarepresentcellsstretchedatT (cid:3)528C.Meanchangeincellvolume¼4.39%withs.e.m.¼0.31%.(e)Schematic
overall
phasediagramshowingcellularresponsewhenthecellsweresubjectedtoanambientcombinationoflaser-inducedtemperatureincreaseðDT Þandchamber
laser
temperature(T ).Triangularsymbolsdenotecellularresponsebythe1480nmheatingat0.80PaatT ¼258C.Circularsymbolsdenotecellularresponse
chamber chamber
whenstretchedandheatedbythe1064nmlaseronly.(f )Schematicphasediagramshowingcellularresponsewhenthecellsweresubjectedtovariousoptically
induced stresses (sF ) at various T . Legends in (a,b) indicate T . Grey bars along the time axis in (a–c) indicate the period of stretch.
0 g overall overall
hovering below 0.45, when the overall temperatures were temperaturesareshowninfigure3a.Theinitialincreaseincel-
below 378C. Above the physiological temperatures, there lular strain at higher temperature (yellow to gold) was
appearstobeanonsetofanonlinearincreaseinthecellfluid- indicativeofthermalsofteningbehaviour,asdiscussedinthe
ity, suggesting some form of a multiphasic response. To previous section, but at even higher temperatures the cells
conclude, in the presence ofhighertemperatures induced by began to exhibit contractile behaviour along the major axis
a sudden temperature jump or prolonged exposure to heat, (orangeandred).Thetransitiontemperaturewasobservedto
the cells were able to adapt and remodel themselves to occurataround 52+18C.To furtherinvestigatewhetherthe
becomemoremechanicallycompliantandfluid-like. observedcontractionwasindeedinducedbyheatandoccurred
only at this particular temperature, we carried out optical
stretching experiments where the temperature increase was
3.2. Cells actively contract above a critical temperature
adjustedthroughraisingthechambertemperature.Asshown
Interestingly, we observed a strange, unexpected cellular in figure 3b, cells showed normal viscoelastic deformation
response at much higher overall temperatures, independent (lightblue)whenfirststretchedat1.80Paatachambertemp-
ofthetimescalesofheattreatments.Inthefirstsetofexper- erature of 238C. When the cells were stretched at the same
iments, we heated up the cells with various powers of stress but at higher chamber temperatures of 29 and 358C
1480nmheatinglaserfor4s,beforestretchingthematacon- (mediumanddarkblue),weobservedaninitialdeformation
stant stress of 0.80Pa. The results of cellular deformation followedbyacontractionalongthecellmajoraxis.Thesame
(along cell major axis) against time at various overall transition to contraction at elevated chamber temperatures
wasalsoobservedforotherstretchpowers.Usingthepredeter- differentmedia,namelyPBSwithorwithoutfreecalciumions 6
mined calibrated temperature increase (10.5+18CW21 for andinRPMI1640mediumenrichedwith0.42mMoffreecal-
1064nm stretch laser), we arrived at the same observation cium ions. The results of the experiments are shown infigure rs
fs
thatcellcontractionoccurredonlywhentheoveralltempera- 4a.When thecellswerestretched with 2.00Paat258C,which .ro
y
ture rose above 52+18C. We note that the results were correspondstoanoveralltemperatureof528C,cellcontraction als
o
striking given that the same active contraction was observed was only observed in the presence of extracellular calcium cie
independent of how the cells were heated, specifically the ions, indicating a crucial role of calcium in cell contraction. typ
u
timescalesinvolved. The difference in refractive index between PBS and RPMI is b
lis
Thetemporalevolutionofcellularstrainalongtheminor small(nPBS¼1.334,nRPMI¼1.338),sothedifferenceinoptically hin
axis (figure 3c, open circles) showed that the negative strain inducedstressonthecellsinbothmediaisnegligible.Further- g.o
ratedidnotextendthroughoutthestretch.Instead,itreached more, the measured osmolalities for PBS with calcium rg
a peak negative strain before assuming a positive rate of (282mOsmkg21 H2O) and without calcium (300mOsmkg21 In
strain. The peak strains along both the major and minor H2O)arecomparable,sothechangeincellularresponseisnot terfa
axisoccurredataroundthesametime(approx.3–4s).Inter- likelytobearesultofachangeinosmoticcondition. ce
estingly,despiteits contractionalongthemajoraxis,thecell Wespeculatedthattheobservedcellularcontractionmust Fo
c
u
expanded in volume. The change in cell volume throughout be attributed to calcium signalling. As such, we performed s
the stretch was significant (4.39+0.31%), as shown in calcium imaging experiments on cells loaded with the Ca2þ 4:
2
figure 3d, which is in contrast to the non-contracting cells dye Fluo-4. Images of the fluorescent cells can be found in 01
3
which underwent normal viscoelastic deformation at lower the electronic supplementary material, figure S2. When we 00
6
overalltemperatures(figure2d). stretched the cells with 2.00Pa at 258C, which corresponds 9
Wefollowedupwithaseriesofexperimentstosystemati- toanoveralltemperatureof528C,weobservedapronounced
cally investigate the cellular response overa range of overall increaseinfluorescenceintensityinthecell(figure4b,blue),
temperatures achieved through various combinations of though no significant change in fluorescence intensity was
laser-induced heating and adjustment of chamber tempera- observed for cells stretched below this critical temperature.
tures. Theresultsare depictedin a schematicphase diagram Thisindicatesthatcell contraction may becausedbycalcium
(figure3e)withthespacespannedbytwoparametersDT influx triggered by heat. A plausible mechanism may be
laser
and T denoting laser-induced temperature increase owing to the activation of temperature-sensitive TRPV2 ion
chamber
and the chamber temperature, respectively. Notably, we channelswhichhavebeenreportedtohavethehighestopening
observedactivecellcontraction(orangesymbols,blueregion) probabilityat approximately528C [31,32]. Expression of such
whenever the overall temperature, independent of proteins in HL60 cells has also been positively confirmed in
the combination of chamber temperature or laser-induced the past [33,34]. To this end, we added 10mM of ruthenium
temperature increase, rose above 528C. On the other hand, red to the cells prior to stretching. Ruthenium red has been
whentheoveralltemperaturewasbelow52+18C(greensym- reported to be an effective blocker of TRPV2 channels [35].
bols, white region), we observed only passive, thermal When stretched with an overall temperature of 528C, we
softeningbehaviourcharacterizedbyanincreaseincellcompli- observed only a low baseline fluorescence in the cell (figure
ance and fluidity with increased overall temperature. We 4b, magenta), indicating that no measurable amount of Ca2þ
thereforetermedthisregionasactivecellcontractiontodistin- entered the cell. Simultaneous imaging of cell deformation
guish this behaviour from the passive viscoelastic softening also showed normal deformation without contraction. This
behaviouratloweroveralltemperatures. suggeststhatTRPV2channelactivationistheprimarypathway
Todeterminewhethermechanicalstressplaysaroleindic- for calcium influx and regulates the thermomechanical
tatingthepeculiarresponseofcellsatelevatedtemperatures, responseofHL60cells.Whenweperformedthesameoptical
wemappedthecellularresponseinanotherschematicphase stretchingexperimentsunderthesameexperimentalconditions
diagram spanned by the overall temperature T and the butincalcium-freePBSmedium,cellsagaindisplayedonlylow
overall
opticallyinducedstresssF .Asshowninfigure3f,thetran- baseline fluorescence throughoutthestretch (similartofigure
0 g
sition from passive viscoelastic deformation to active cell 4b, magenta), indicating that the observed increase in fluor-
contraction at 528C depends onlyon the overall temperature escence intensity was very likely owing to the influx of
butnotthe magnitudeof opticallyinduced stressexertedby extracellularcalciumionsandnotthroughintracellularrelease
the 1064nm stretch laser. This suggests that the observed viainternalstores.
activecontractionwastriggeredbyaheat-sensitivemechanism Apart from driving cell contraction, the influx of calcium
instead of a specific response to a large mechanical stress. ionscouldgenerateanosmoticpotentialthatdrivestheinflow
More specifically, this implies that the cell actively contracts ofwater,leadingtothesubsequentexpansionofcellvolume,
regardless of the amount of axial strain it experiences, as as shown for a representative contracting cell in figure 4c
shown in figure 3a where contraction was seen to occur at (blue).Specifically,therewas atimelagbetween theonsetof
verysmallpeakstrain(4%). calciuminfluxwhichoccurredimmediatelyafterthestretchat
t¼2s and the onset of volume expansion (t¼4s). Interest-
3.3. Temperature-sensitive TRPV2 ion channels regulate ingly,cellstreatedwithrutheniumredshowednocontraction
along the major and minor axes and no significant change
cell mechanical response
incellvolume(magenta)throughoutthestretchperiod.Further
Motivatedbyarecentpaperontheroleofcalciuminregulating analysis for a sample population of cells for both cases is
cellmechanicsatelevatedtemperatures[23],wehypothesized depicted in figure 4d. The contracting cells expanded signifi-
that calcium influx could perhaps lead to enhanced myosin cantly (4.85+0.60%) after the stretch, while no significant
activity and the observed cell contraction above 528C. To test volumechangewasobservedforcellstreatedwithruthenium
this hypothesis, separate experiments were performed in redorimmersed incalcium-free medium(0.56+0.48%). The
(a) (b) 3.5 sF =2.00Pa 7
cellular strain (%) 11111202468024680 sTcohFagm=be2r=.0205P°aC PPRBBPSSM +-I CCaa22++ FFrelative fluorescence intensity (/)o 011223......0505050 Tcohagmber=25°C rcuotnhternoilum red treated rsfs.royalsocietypublishing.org
–1 0 1 2 3 4 5 6 7 –1 0 1 2 3 4 5 6 7 8
time (s) time (s)
In
(c) (d) te
1.05 sF =2.00Pa 12 n=36 n=36 rfac
o g 10 e
1.04 Tchamber=25°C Fo
control 8 cu
1.03 6 s
4
:
1.02 %) 4 20
VV/o 1.01 DVV/ ( 02 13006
1.00 9
–2
0.99 –4
ruthenium red treated
–6
0.98
–1 0 1 2 3 4 5 6 7 –8
time (s)
(e)
T <52°C T >52°C
overall overall
control ruthenium red
m
u
di
e
m
+)
2+Ca( no contnroac ctiaolcni uamlo nign fmluaxjor axis contracctiaolcni uamlo nign fmluaxjor axis no contnroac ctiaolcni uamlo nign fmluaxjor axis
no change in cell volume cell volume increases no change in cell volume
m
u
di
e
m
2+a(–) no contnroac ctiaolcni uamlo nign fmluaxjor axis no contnroac ctiaolcni uamlo nign fmluaxjor axis
C
no change in cell volume no change in cell volume
Figure 4. Mechanismofthermomechanicalresponse.(a)Cellularstrainasafunctionoftimewhenthecellswerestretchedwith2.00Paat258C,inPBS(withand
withoutcalcium)andRPMI.(b)Relativefluorescenceintensity(F/F)forrepresentativecontrolcells(blue)andcellstreatedwithrutheniumred(magenta),asafunction
0
time,whenloadedwithCa2þdyeFluo-4andstretchedincalcium-richPBSatT (cid:3)528C.(c)Changeincellvolumeforarepresentativecontrolcell(blue)andacell
overall
treatedwithrutheniumred(magenta),whenstretchedatT (cid:3)528C.(d)Boxplotsshowingthechangeincellvolumeforcontrolcells(blue)andcellstreatedwith
overall
rutheniumredorincalcium-freemedium(magenta)atT (cid:3)528C.Thetwopopulationsaresignificantlydifferent (p,0.01).(e)Schematicdiagramofcellular
overall
responseindifferentbufferconditionsaboveandbelow528C.Greybarsalongthetimeaxisin(a–c)indicatetheperiodofstretch.
pictureemergingfromourresultsisschematicallysummarized componentinparallelwithanelasticelementE (t)originat-
M
infigure4e.Ourdatasuggestastrongcorrelationbetweenthe ing from the effective cell elasticity generated by the cell
threevariables.Whilethecalciuminflux,throughtheactivation cortical tension T. Indeed, the deformation of the cell from
of TRPV2 ion channels, may directly impact cell contraction itsinitialsphericalshapeleadstoanexpansionofthecellsur-
along the cell major axis, it also correlates with overall cell faceresistedbyitssurfacetension.Assumingthattheoptical
expansioninitsvolume,albeitadelayedresponse.Toconclude, stretcherappliedapressureprofiles(u)¼scos2u,whereuis
0
wehaveidentifiedTRPV2ionchannelsasacrucialplayerinreg- the meridional angle in a polar coordinate where the polar
ulating cell mechanics and cell-shape changes in response to axis is aligned with the laser optical axis, and furthermore
externalheatinthepresenceofextracellularcalciumions. assuming cell volume conservation, one finds an effective
elasticmodulusE ¼ð24/pÞðT/RÞresistingcelldeformation,
M
3.4. Theoretical modelling of thermorheological withRthecellradius.Inaddition,h0representstheeffectivecel-
lular long time-scale viscosity, possibly originating from
behaviour
molecularslipbetweenthetransientcross-linkersinthecytos-
The rheological transition from thermal softening to active keleton, h represents the transient viscous effect possibly
1
cell contraction can be described bya mechanicallyequival- arising from the poroelastic effect [36], while E represents
1
ent model (figure 5a), which essentially consists of a SLL the elastic response within the cytoskeleton. Because E is
M
(a) (b) 8
7
E 36°C
h 1 6 49°C rsfs
0 %) 5 53°C .roy
ain ( 4 alsoc
soFg h1 soFg ular str 3 ietypub
cell 2 lishin
1 g
E .o
M rg
0 1 2 3 4
stretch time (s) In
(c) te
rfa
c
e
SLL+E h (Pas) h (Pas) E (Pa) E (Pa) F
M 0 1 1 M o
c
u
s
36°C 47.6 ± 0.7 23.2 ± 1.0 122.0 ± 3.6 22 ± 0.2 4
:
2
0
49°C 28.9 ± 0.4 12.1 ± 0.6 70.9 ± 2.1 13.4 ± 0.1 13
0
0
E =18.2 ± 0.5 69
53°C 17.4 ± 0.6 16.5 ± 1.1 105.3 ± 9.1 mo
E =2.3 ± 0.1
mt
Figure 5. Mechanically equivalent circuit describing the thermomechanical regulation of HL60 cells. (a) The model consists of a SLL model in parallel with a
dynamicelasticcomponentE arisingfromcellcorticaltension.sF denotestheconstantopticallyinducedstress.(b)Theoreticalcreepresponseofthemechanical
M 0 g
modelpredictingatransitionfrompassiveviscoelasticdeformation(opencircles:experiment;blueline:modelfit)tothermalsoftening(opencircles:experiment;red
line: model fit), followed by active cell contraction above 528C (open circles: experiment; black line: model fit). The legend indicates the overall temperatures.
(c) Various viscoelastic parameters extracted from fitting the mechanical model to the cell deformation curves at low and high temperatures.
associated with the cell cortical tension generated mainly by staticanddynamicterms,respectively.Theexponentgfollows
the myosin II activity, it is expected to be significant at a from I(t), which was found to scale with a power-law form,
longtimescalecomparedtocytoskeletalturnoverevents[37]. I(t)(cid:2)tgwithg¼1.298+0.062. Incorporating this final form
We numerically solved the equations of the mechanical ofE (t),thenumericalsolutionspredictatransitionfroman
M
system under constant external force at temperatures below initial viscoelastic deformation to a subsequent contraction
and above the critical temperatures (see the electronic within a few seconds (figure 5b, black), in agreement with
supplementarymaterial). ourexperimentalobservationsofadelayedresponsebetween
We assumed that at low temperatures when the cells theinitialcalciuminfluxandtheeffectofphosphorylation.Fur-
experienced no calcium influx during the short period of thermore,byperformingagoodfittothestraincurveat538C,
stretch (4s), the effective elastic modulus E remained asshowninfigure5b(blackline),weareabletoextractallthe
M
approximatelyconstantduringthestretch.Inthiscase,thepre- fiveviscoelasticparameters,asshowninfigure5c.
dicted creep response consisted of three regimes: a very fast
elasticresponse,aslowingdownofthedeformationfollowed
byaviscous flow, in linewith the experimentalobservation. 4. Discussion
Anumericalfittothestraincurvesat36and498C,asshown
infigure5b(blueandredlines),allowedustoextractthevar- Our results demonstrate clearly that an increase in ambient
ious viscoelastic parameters as tabulated in figure 5c. Our temperaturesinandaroundasuspendedcellcansignificantly
results predicted two changes to cell material properties at impact its material properties, making it more mechanically
higher temperatures. First, there is a drop in the steady-state compliant and fluid-like. Previous studies [17,19,40,41]
viscosity h, indicative of a more latent fluid-like behaviour observed similar thermal softening behaviour using a
0
at long time scales at highertemperatures.Second, there isa numberoftechniquesonbothadherentandsuspendedcells,
concomitantdecreaseinE ,suggestingthatatahighertemp- suchasfibroblastsandleucocytes.Interestingly,otherstudies
M
erature, the drop in cell compliance is also associated with a indicatedtheoppositeeffectofthermalstiffeningwithadher-
reductionincellcorticaltension. ent cells grown on culture dishes [20,42], possibly owing to
AbovethecriticaltemperaturewhentheTRPV2ionchan- increased contractile activity of molecular motors leading to
nels get activated, we assume that the dominant effect of enhanced cellular prestress and stiffness. This was not the
calcium influx is the formation of calcium–camodulin com- caseinourmeasurements,wherecellsassumedaroundgeo-
plexes which triggersextensive myosinphosphorylation and metry in their naturally suspended state in the absence of
generates large cell cortical tension [38,39]. We assume that stress fibres. The increase in cell compliance with higher
this leads to a time-dependent, effective elastic modulus temperatures has also been confirmed with another recent
E (t) with a temporal profile that is linearly proportional to independentstudyusingtheopticalstretcher[24],wherethe
M
thecalciuminfluxindicatedbytheaveragefluorescenceinten- authors explained the qualitative changein the thermorheol-
sity I(t) (representative curves are depicted in figure 4b). ogy of cells by invoking the concept of time–temperature
Specifically, this leads to E (t)¼aI(t)¼E þE (t/t)g, superposition widely used in polymer physics. Despite the
M mo mt 0
wheret isassumedtobe1sandE andE representthe similarity of our work, we further characterized the changes
0 mo mt
tosuspendedcellfluiditywithtemperaturevariations,which thetransientcalciuminflux,andsuccessfullypredictedatran- 9
is,toourknowledge,thefirstsystematicstudyinthisarea. sitionfrompassivedeformationtoactivecellularcontraction.
Theobservedcreepcomplianceofcellsappearedtoscale Ourresultsalsosuggestedthatintheactiveregime,thecellular rs
fs
linearly with the overall temperatures (figure 2e). Previous response is dominated by the increased myosin activity .ro
y
studies observed an increased dissociation rate of protein owing to calcium influx, rather than the thermal unbinding als
o
cross-linkers, for example a-actinin, with temperature [43]. mechanism of transient cross-linkers, which accounts forcell cie
In vitro studies have shown that such thermal unbinding of softeninginthepassiveregime.Furtherexperimentsoncells typ
u
transient cross-links results in stress relaxation which treated with myosin inhibitors, for example blebbistatin, in b
lis
decreases the static network elasticity and increases the vis- the active regime showed a partial abolishment of the active hin
cous dissipation [44,45]. Based on the affine stretching contraction to the point where the strain rate of the major g.o
model[46],suchmechanismpredictsaninversetemperature axis never became negative (see electronic supplementary rg
dependenceofnetworkelasticityG0 (cid:2)k02/kBTj2l3c,wherek0is material, figure S3), indicating that Ca2þ-activated myosin In
the actin filament bending stiffness, jis the network mesh activity is indeed involved in active cell contraction, though terfa
sizeandlcistheaveragecross-linkerdistance.Thistranslates othermechanism(s)maybeinvolvedaswell.Ourresultsare ce
to a linear temperature dependence of creep compliance, in consistentwithsomeofthelatestfindingsbyGygeretal.[54] Fo
c
u
goodagreementwithourobservation.Theincreaseinviscous wheretheyobservedsimilaractivecontractionsinsuspended s
4
dissipation owing to thermal unbinding of transient cross- epithelialcellstransfectedwithTRPVchannels.Nevertheless, :
2
linkers may also explain the observed changes to the visco- contrary to their report, we observed no negative cellular 01
3
elastic parameters in both models at higher temperatures: strainduringactivecellularcontractionswithinthetimescale 00
6
the increase in cell fluidity b(figure 2f) within the power- of6s.Moreimportantly,whileGygeretal.adoptedaphenom- 9
lawmodel,andthedecreaseinboththesteady-stateviscosity enological mathematical model to account for active cellular
h and the effective elastic modulus E , which origina- contractions, our mechanical model offers a simpler, more
0 M
ted from the cell cortical tension, within the mechanical intuitiveapproach,whichshedslightonhowactiveremodel-
model (figure 5c). At 368C, our theoretical model predicted lingofcellcorticaltensioncanregulatecell-shapechangesin
E (cid:2)22Pa, which is equivalent to a cortical tension responsetoheatandbiochemicalsignals.
M
of 25pNmm21, close to that of blood granulocytes Theobservedtimescaleforanosmoticresponseisinagree-
(30pNmm21) as measured with the micropipette aspiration ment with previous reports on diffusion dynamics of water
technique [47,48]. Furthermore, our theoretical model pre- across cell membranes (diffusion time approx. 0.2s) [55,56].
dicted a drop in cell cortical tension at higher temperatures, Assumingthatthecellvolumechangesonlybythepermeation
consistent with recent experimental studies [40,41,49]. Our ofwaterthroughthecellmembrane,thewaterfluxisgivenby
resultsthereforehighlightthefundamentalrolethatcellcorti- the classical permeation law j¼L rðDP(cid:2)DPÞ, where L
p p p
cal tension plays in resisting cell deformation, the reduction isthepermeationconstantofonepore,r istheporedensity
p
ofwhichleadstoasignificantincreaseinthecellcompliance. in the membrane, DP¼P (cid:2)P isthe hydrostatic pressure
in out
Atsignificantlyhighertemperatures,ourresultsrevealed difference between inside and outside of the cell and DPis
adramaticchangefrompassivedeformationtoanactivecell the osmotic pressure difference between inside and outside
response regulated by the TRPV2, which provides an ion of the cell. At quasi-static equilibrium, the relative change in
channel gating mechanism for cellular entry of calcium cellvolumeisproportionaltotherelativechangeinintracellu-
ions. TRPV2 ion channels have recently been shown to play lar osmolyte concentration [57]. Assuming that the calcium
acrucialroleinearlystepsofphagocytosis[35]andregulat- influxcontributesonlypassivelytothecellosmoticpressure,
ory volume decrease [50]. Our work further suggests that the observed increase in cell volume (approx. 5%) suggests
these temperature-sensitive ion channels help to regulate thattheamountofcalciumenteringthroughcalciumchannels
the cell contractility through coupled dynamics of calcium represents roughly 5% of the total number of osmolytes in
influxandincreasedmyosinactivity.MyosinII,animportant thecell.
molecular motor, is known to playacrucial role in cell con- We rule out other potential mechanisms for cell con-
tractility and shape control [37]. One previous study [51] traction. Firstly, it is likely that an increase in intracellular
observed reversible motor activity without denaturation water content may lead to a potential drop in cell refractive
upon raising the temperature instantaneously to above index, and consequently the optically induced stress, as the
608C. On a similar note, Wetzel et al. [22] showed that latter is proportional to the difference in refractive index
whenthe cellswereexposedto lessthan3s of laserheating betweenthecellandthemedium[14].Thisreductioninopti-
in an optical stretcher with overall temperature above 508C, calstresscouldleadtoadropincelldeformation,andhence
more than 70% of the stretched cells remained viable. We theobserved‘contraction’,whichwouldthenbeunderstood
note that the observed cell contraction was rapid, typically as a reduction in the stretching force. We argue that this
2s following the onset of calcium influx (figure 4b,c). This increase in cell volume was too small to initiate any signifi-
suggests that the signalling mechanism was possibly a first- cant change in overall cell refractive index, as supported by
order event, for example an increase in myosin phosphoryl- Kemper’s study [58] on human pancreas and liver tumour
ation via the calmodulin-myosin light chain kinase (MLCK) cells,whichshowedanapproximate0.36%dropincellrefrac-
pathway, which regulates the assembly of myosin motors tiveindexwith a4%increase incellvolume.Asimilardrop
into contractile filaments, leading to subsequent cell con- in cell refractive index with increase in cell volume has also
tractility [38,39,52,53]. The pathway involves the binding of been reported recently in macrophages [59]. Secondly, we
calmodulintofourcalciumionsandtheMLCKandthetypical exclude the role of stretch-activated ion channels in regulat-
timescalesofthisreactionarelessthanasecond.Wesimulated ing cell contraction, as contraction was only dependent on
thisprocesswithamechanicalmodelwhichincludesadyna- the overall temperature but not the magnitude of optically
mical elastic term E (t), which grows in time in response to induced stress and the resultant peak strain (figure 3f).
M
Specifically,activecellcontractioncanoccuratverylowpeak exhibits more fluid-like behaviour at higher temperatures. 10
strain(4%)whenheatedby1480nmlaser(figure3a).Finally, Our results suggest that in the passive viscoelastic regime
it is plausible that below 52+18C contraction was already below a critical temperature, the rheology of HL60 cells can rs
fs
takingplacebutwassuppressedbydominantpassivedefor- be effectively described by the thermal unbinding mechan- .ro
y
mation, and one could argue that cells may contract given ism of transient cross-links in the actin cortex. Beyond a als
o
sufficient time of observation. We ruled out such a possi- critical temperature of 52+18C, the cells exhibit active con- cie
bility, as we observed no active contraction below 528C, traction along the direction of maximal stress owing to the typ
u
even when the cells were stretched for 30s (data not activation of TRPV2 ion channels, leading to an eventual b
lis
shown). Nevertheless, these mechanisms could still contrib- expansion in overall cell volume. Our work points to an hin
ute or modify the actually observed cell behaviour, but the importantroleofTRPV2ionchannelsandcalciumsignalling g.o
dominanteffectislikelytobetheincreaseinmyosinactivity in the active regulation of cell cortex stiffness and cell-shape rg
in response to the calcium influx through TRPV2 ion changesatelevatedtemperatures. In
channelsabove52+18C. Fundingstatement.TheauthorsaregratefultotheEUMarieCurieInitial terfa
c
TrainingNetworksTRANSPOL(toC.J.C.)andtheEngineeringand e
F
PhysicalSciencesResearchCouncil(toL.B.)forfundingthisproject o
c
5. Conclusion u
through scholarship support. J.G. acknowledges financial support s
via the ‘LightTouch’ Starting Investigator Grant of the European 4:
In this work, we introduced a modified version of a micro- ResearchCouncilandtheHumboldtProfessorshipfromtheAlexan- 20
1
fluidicoptical stretcher with integratedheatingcomponents, der von Humboldt Foundation. The authors also acknowledge 3
0
which allowed us to probe cell mechanics when the cells financial support by the Human Frontier Science Program (to J.G. 06
9
andG.W.)andtheClusterofExcellenceEAMatFAUErlangen-Nur-
were subjected to an extended period of heating or to a
emberg funded by the German Research Foundation (DFG) in the
suddenincreaseintemperature.Wefindthatthecellcompli-
framework of its Excellence Initiative (to G.W.). The HL60/S4 cell
ance of HL60 cells scales linearly with the temperature, line was a generous gift from Don and Ada Olins, University of
independent of the time scales of thermal treatments, and NewEngland.
References
1. Wottawah F,Schinkinger S,Lincoln B, insitu connectivity. Science 266, 1032–1035. deformabilityonpositiononthecell,temperature,
Ananthakrishnan R,RomeykeM,Guck J, Ka¨sJ. (doi:10.1126/science.7973655) andcytochalasin B.Proc. Natl Acad.Sci.USA79,
2005Optical rheologyofbiological cells.Phys. 9. Thoumine O, Ott A,Cardoso O, Meister JJ.1999 5327–5331.(doi:10.1073/pnas.79.17.5327)
Rev.Lett.94,098103.(doi:10.1103/PhysRevLett.94. Microplates: anewtool for manipulation and 18. Lightman M, Kearney E.1973Rheologyof
098103) mechanical perturbation ofindividual cells. leukocytes,leukocyte suspensions, andblood in
2. Kollmannsberger P, FabryB.2010Linearand J. Biochem.Biophys. Methods39, 47–62.(doi:10. leukemia.J.Clin. Invest. 52, 350–358.(doi:10.
nonlinearrheologyoflivingcells.Annu.Rev.Mater. 1016/S0165-022X(98)00052-9) 1172/JCI107191)
Res. 41,75–97. (doi:10.1146/annurev-matsci- 10. DespratN, RichertA,Simeon J, AsnaciosA.2005 19. WilliamsonJ,ShanahanM,HochmuthR.1975The
062910-100351) Creepfunctionofasinglelivingcell.Biophys.J.88, influenceoftemperatureonred cell deformability.
3. TrepatX, Deng L,An SS, NavajasD, Tschumperlin 2224–2233. (doi:10.1529/biophysj.104.050278) Blood46, 611–624.
DJ,Gerthoffer WT, Butler JP,Fredberg JJ.2007 11. HoffmanBD,MassieraG,VanCittersKM,CrockerJC. 20. SunyerR,TrepatX,FredbergJJ,Farre´ R,NavajasD.
Universalphysicalresponsestostretchintheliving 2006The consensus mechanicsofcultured 2009Thetemperaturedependenceofcellmechanics
cell. Nature 447, 592–595. (doi:10.1038/ mammalian cells. Proc. Natl Acad.Sci.USA 103, measuredbyatomicforcemicroscopy.Phys.Biol.6,
nature05824) 10259–10264.(doi:10.1073/pnas.0506130103) 025009.(doi:10.1088/1478-3975/6/2/025009)
4. Maloney JM,LehnhardtE, LongAF, Van Vliet KJ. 12. Mofrad MR, KammRD (eds). 2006Cytoskeletal 21. Ebert S, TravisK, LincolnB, GuckJ. 2007
2013Mechanicalfluidityoffullysuspended mechanics: modelsandmeasurements incell Fluorescenceratiothermometryin amicrofluidic
biological cells. Biophys.J. 105, 1767–1777. mechanics, 1stedn. Cambridge, UK:Cambridge dual-beam lasertrap.Opt. Expr. 15,15493–
(doi:10.1016/j.bpj.2013.08.040) University Press. 15499.(doi:10.1364/OE.15.015493)
5. FabryB,Maksym G,ButlerJ, GlogauerM, Navajas 13. Pullarkat P, FernandezP,Ott A.2007Rheological 22. WetzelF,Ro¨nickeS,Mu¨llerK,GygerM,RoseD,Zink
D, Fredberg J. 2001Scaling themicrorheologyof properties oftheeukaryotic cellcytoskeleton. M,Ka¨s J. 2011Singlecell viabilityand impact of
living cells. Phys.Rev.Lett. 87, 148102.(doi:10. Phys.Rep.449, 29–53.(doi:10.1016/j.physrep. heating bylaserabsorption. Eur. Biophys.J. 40,
1103/PhysRevLett.87.148102) 2007.03.002) 1109–1114.(doi:10.1007/s00249-011-0723-2)
6. AlcarazJ,BuscemiL,GrabulosaM,TrepatX,Fabry 14. Lincoln B.2006Themicrofluidic opticalstretcher. 23. Gyger M, RoseD, Stange R,KiesslingT,Zink M,
B,Farre´R,NavajasD.2003Microrheologyofhuman PhD thesis, Universita¨tLeipzig, Leipzig,Germany. FabryB,Ka¨sJ.2011Calciumimagingintheoptical
lung epithelialcells measured byatomic force 15. Guck J, Lautenschla¨ger F, Paschke S,Beil M. 2010 stretcher. Opt. Expr.19,19212–19222.(doi:10.
microscopy. Biophys.J.84, 2071–2079.(doi:10. Critical review: cellular mechanobiologyand 1364/OE.19.019212)
1016/S0006-3495(03)75014-0) amoeboidmigration.Int.Biol.2,575–583.(doi:10. 24. Kießling TR,Stange R,Ka¨s JA,Fritsch AW. 2013
7. Mahaffy RE,ParkS, GerdeE, Ka¨s J, ShihCK.2004 1039/c0ib00050g) Thermorheologyofliving cells: impact of
Quantitativeanalysisoftheviscoelasticpropertiesof 16. EkpenyongAEetal.2012Viscoelasticpropertiesof temperaturevariations oncell mechanics.New
thinregions offibroblastsusing atomicforce differentiatingbloodcells arefate- and function- J.Phys.15, 045026.(doi:10.1088/1367-2630/15/4/
microscopy. Biophys.J.86, 1777–1793.(doi:10. dependent. PLoSONE 7, e45237.(doi:10.1371/ 045026)
1016/S0006-3495(04)74245-9) journal.pone.0045237) 25. GuckJ,AnanthakrishnanR,MahmoodH,MoonTJ,
8. DischerDE, Mohandas N,Evans E.1994Molecular 17. PetersenN, McconnaugheyWB, Elson EL.1982 CunninghamCC,Ka¨sJ.2001Theopticalstretcher:a
mapsofredcelldeformation:hiddenelasticityand Dependence oflocallymeasured cellular novellasertooltomicromanipulatecells.Biophys.J.