Migration of Dictyostelium Amoeba: role of Adhesion and Quorum

135 Pages·2015·5.7 MB·English

Migration d’amibe de dictyostelium: rôle de l’adhésion
et de la détection de Quorum
Laurent Golé
To cite this version:
Laurent Golé. Migration d’amibe de dictyostelium: rôle de l’adhésion et de la détection de Quorum.
Autre [cond-mat.other]. Université Claude Bernard - Lyon I, 2011. Français. ￿NNT: 2011LYO10288￿.
￿tel-00846586￿
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Migration of Dictyostelium Amoeba:
Role of Adhesion and Quorum Sensing
Laurent Gole´
LPMCN
Universite´ deLyon
Presente´epourl’obtention dutitre deDocteurdel’Universite´ ClaudeBernardLyon1
Soutenupubliquement le9de´cembre2011
Rapporteurs:
FrancoisAmblard
ClaireWilhelm
Examinateur:
FranzBruckert
PierreCosson
Directeursdethe`se:
Jean-PaulRieu
Charlotte Riviere
Abstract
Thisthesisfocusesontheanalysisoftheroleofadhesionbetweensubstrateandcellandfactorsof
QuorumsensingonthemigrationofDictyosteliumamoeba. Toolstoautomatetherecordingsofvideo
microscopy and image analysis have been developed to work with very large samples of cells and to
quantifycellmigration. Amicrofluidicdeviceforcelldetachmentinhydrodynamicflowcombinedwith
amotorizedstagehasallowedastatisticalstudyofadhesionbutalsothedynamicsofdetachment. The
analysisofthemigrationofDictyosteliuminnonnutritivemediumhighlightstheroleofdensityoncell
differentiationandmigrationcapacity. Weobservethepresenceofamaximumspeedofmigrationafter
6 hours of starvation. We show that the adhesion to glass is twice as low in deprivation buffer as in
thenutrientmedium. Theexperiencesofmigrationingrowthmediumrevealedthepresenceofafactor
ofdetectionofdensitysecretedbythecellsandregulatingtheirrandommigration. Thediffusioncoef-
ficient, thepersistenceofthemovementandmorphologyofcellsvarydependingontheconcentration
of this factor. This factor does not affect cell adhesion but only the dynamics of detachment. Finally,
the testing protocol developed allowed us to make a comparative study of migration by varying other
parameterssuchassurfaceorthechemicalcompositionofexperimentalmedium. Thisworkconcludes
byoutliningthepossibleroleofadhesiontothemigrationofDictyosteliuminnutrientmedium.
Re´sume´
Cettethe`seestcentre´esurl’analyseduroˆledel’adhe´sioncellule-substratetdesfacteursdede´tection
de Quorum sur la migration amibienne de Dictyostelium . Des outils pour automatiser les enreg-
istrementsde vide´omicroscopie et l’analyse d’image onte´te´ de´veloppe´s afin de travailleravec de tre`s
grandse´chantillonsdecellulesetdequantifierlamigrationcellulaire. Undispositifmicrofluidiquede
de´tachement cellulaire sous flux hydrodynamique combine´ une platine motorise´e a permis une e´tude
statistique de l’adhe´sion mais aussi de la dynamique de de´tachement. L’analyse de la migration de
Dictyosteliumenmilieunonnutritifmetene´videnceleroˆledeladensite´ surladiffe´rentiationdescel-
luleset leur capacite´ de migration. Nous observons lapre´sence d’une vitessemaximale de migration
apre`s6hdecarence. Nousmontronsquel’adhe´sionsurverreestdeuxfoisplusfaibleenmilieucarence´
qu’enmilieunutritif. Lesexpe´riencesdemigrationenmilieunutritifontre´ve´le´lapre´senced’unfacteur
de de´tection de densite´ se´cre´te´ par les cellules et re´gulant leur migration ale´atoire. Le coefficient de
diffusion,lapersistancedumouvementetlamorphologiedescellulesvarientenfonctiondelaconcen-
tration de ce facteur. Ce facteur ne modifie pas l’adhe´sion cellulaire mais uniquement la dynamique
dede´tachement. Enfin,leprotocoled’analysede´veloppe´ nousapermisdefaireunee´tudecomparative
delamigrationenfaisantvarierd’autresparame`trestelquelasurfaceoulacompositionchimiquedu
milieuexpe´rimental. Cetravailseconclueenexposantlepossibleroˆledel’adhe´sionsurlamigration
chezDictyosteliumenmilieunutritif.
ii
Contents
Preface v
1 Introduction 1
1.1 Cellmotility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.1 Migrationandadhesioninterplay . . . . . . . . . . . . . . . . . . . . 2
1.1.2 Howdocellsmove . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Mammalianmotilitymachinery . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.1 Thecytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2.1.1 Actin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2.1.2 Microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.2.1.3 Intermediatefilaments . . . . . . . . . . . . . . . . . . . . . 6
1.2.2 FocalAdhesion-RelatedStructures. . . . . . . . . . . . . . . . . . . . 6
1.2.3 Integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.2.4 MyosinII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.3 Differenttypeof2D-cellmigration . . . . . . . . . . . . . . . . . . . . . . . . 11
1.3.1 Fibroblasts-likecellmotility . . . . . . . . . . . . . . . . . . . . . . . 11
1.3.2 Keratocyte-likecellmotility . . . . . . . . . . . . . . . . . . . . . . . 12
1.3.3 Amoeboid-likecellmotility . . . . . . . . . . . . . . . . . . . . . . . 13
1.4 DictyosteliumDiscoideum . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.4.1 DictyosteliumLifeCycle . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.4.2 DictyosteliumMotilityMachinery . . . . . . . . . . . . . . . . . . . . 16
1.4.2.1 Coordinated shape changes and traction forces during Dic-
tyosteliummotility . . . . . . . . . . . . . . . . . . . . . . . 17
1.4.2.2 Actin-based and myosin II-based propulsive forces in mi-
gratingDictyosteliumcells . . . . . . . . . . . . . . . . . . 19
i
CONTENTS
1.4.2.3 ArelationbetweenmigrationefficiencyandadhesioninDic-
tyostelium? . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1.4.2.4 AdhesionproteinsinDictyostelium . . . . . . . . . . . . . . 24
1.4.3 DictyosteliumandQuorumSensing(QS) . . . . . . . . . . . . . . . . 25
2 Protocols 29
2.1 Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.1.1 Strain&culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.1.2 Cellstarvation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.1.3 Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.1.4 ImageProcessing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.1.5 MotilityAssay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.1.6 Measurementofmotionparameters . . . . . . . . . . . . . . . . . . . 33
2.1.7 Statisticalanalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.2 Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.2.1 Devicefabrication . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.2.2 Channelcoating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
2.2.3 Flowcreation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.2.4 Cellinjection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.2.5 Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.2.6 ImageProcessing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.2.7 Cellcountingandwallshearstressreliabilities . . . . . . . . . . . . . 40
3 VegetativecellMigration 43
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.2 Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.2.1 Arandommotionwithpersistence . . . . . . . . . . . . . . . . . . . . 45
3.2.2 Bimodalanalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.2.3 Cellshapeandcellmigrationarecorrelated . . . . . . . . . . . . . . . 52
3.2.4 Cellsmovefasteratlowcelldensity . . . . . . . . . . . . . . . . . . . 53
3.2.5 CellmigrationisregulatedbyaQSfactorsecretedbycells . . . . . . . 55
3.2.6 A simple kinetics model to describe the QSF concentration for each
experimentalsituation . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.2.7 Cellsdonotorganizespatiallyathighdensity. . . . . . . . . . . . . . . 58
ii
CONTENTS
3.3 Comparativemotilitystudy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
3.4.1 PresenceanddetectionofQuorumSensingFactorsQSF . . . . . . . . 62
3.4.2 ConsensualandnovelaspectsofDictyosteliumdynamics. . . . . . . . 64
3.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
4 Cellinthecourseofdevelopment 67
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
4.1.1 cAMPsignalingandrelay . . . . . . . . . . . . . . . . . . . . . . . . 68
4.1.1.1 cAMPexpression,functionandregulation . . . . . . . . . . 68
4.1.1.2 InfluenceofcelldensityoncAMPproduction . . . . . . . . 70
4.1.2 DictyosteliuminthecourseofDevelopment . . . . . . . . . . . . . . . 71
4.1.2.1 Cellvelocityandcellshapes . . . . . . . . . . . . . . . . . 71
4.1.2.2 Tractionforces . . . . . . . . . . . . . . . . . . . . . . . . . 72
4.2 Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4.2.1 Cellmigrationduringdevelopment . . . . . . . . . . . . . . . . . . . 74
4.2.2 Effectofcelldensityduringdevelopment . . . . . . . . . . . . . . . . 76
4.2.3 Evidenceofheterogeneouscellpopulation . . . . . . . . . . . . . . . 77
4.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5 Cellunderhydrodynamicshearstress 85
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
5.1.1 Stateoftheart: celladhesionexperiments . . . . . . . . . . . . . . . . 87
5.2 Resultsanddiscussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
5.2.1 Devicedesignandrationale . . . . . . . . . . . . . . . . . . . . . . . 89
5.2.2 Mathematicaldescriptionofadhesionanddetachment . . . . . . . . . 91
5.2.3 Adhesionthresholdforamoeboidcellsfollowlognormaldistribution . 92
5.2.4 Quantitativecomparisonofdetachmentparameters . . . . . . . . . . . 93
5.2.5 Celltracking,migration,detachmentmode . . . . . . . . . . . . . . . 96
5.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
iii
CONTENTS
6 Conclusion&Perspectives 99
6.1 Developmentofefficienttoolsforstatisticalanalysisofcellmigrationandad-
hesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
6.2 Newaspectofdictycelldynamics . . . . . . . . . . . . . . . . . . . . . . . . 100
6.2.1 RandommigrationofDictyosteliumispurelydiffusive . . . . . . . . . 100
6.2.2 Antagonist effect of cell density on cell migration for vegetative and
starvedcells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
6.2.3 Individual cells do not change their mode of locomotion in the course
ofdevelopment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
6.2.4 Migration-Adhesioninterplay . . . . . . . . . . . . . . . . . . . . . . 103
References 107
Appendix 113
A. MigrationErrorAnalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
B. Adhesionerroranalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
C. Shearstresscalculus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
D. Softlithographyandproductionofamicrosystem . . . . . . . . . . . . . . . . 123
E. MeansquaredDisplacementEquation . . . . . . . . . . . . . . . . . . . . . . 125
F. AdhesioninthecourseofDevelopments: preliminaryresults . . . . . . . . . . 126
iv
Preface
Cell adhesion and migration processes have focused more and more studies during the last
decades. Even if the knowledge of such systems has increased considerably, many questions
remainopen. Inparticular,thepreciseroleofcell-substrateadhesiononmigrationisstillunder
debate in the case of amoeba cells. The initial goal of this work was to bring some answers
ontheinterplaybetweenmigrationandadhesionbyaquantitativeanalysisofcellmotility. At
the beginning of my thesis, it was very difficult to get reproducible results. A large part of
myworkhasthenbeendedicatedtodeveloprigorousmethodologiestoquantifycellmigration
and adhesion. Careful and precise cell culture is the necessary first step of good research in
cellular biophysics. This is particularly important for Dictyostelium , as modifications of cell
phenotype occur rapidly if cell density in cell culture is too high. Also, understanding that
cell density was one of the major parameter regulating migration was a major improvement
in reproducibility of our results. Developing automated statistical analysis was necessary to
perform multiple experiments and gather large amount of data within the same experimental
day. Weusedmicrofluidicdevicetoquantifyadhesion. Agoodcontroloftheexactshearflow
andasufficientlylargestressrangewasnoteasytoobtain. Differentmicrofluidicdesignswere
tested in the course of my thesis. Only the final version is developed in this manuscript. Due
to lack of time, I could not take the full advantages of this device to test all desired experi-
mental parameters. In particular, a systematic study of adhesion during development was not
performed.
Atfirst,weaimedatmeasuringbothadhesionandmigrationwithinthesameexperimental
set-up. However, quantifying cell migration in microfluidic channels is not straightforward
as cell migration can be greatly affected (i) by the small dimensions found in microfluidic
devices leading to a fast concentration increase of molecules secreted by cells if the medium
is not renewed, or (ii) by the mechanical stimulation induced if a high enough shear flow is
applied, leading to bias in cell migration trajectories. Therefore, we have decided to analyze
v
cellmigrationandadhesionseparately(migrationismeasuredinaseparateopendish). Results
obtainedaredevelopedinChapter3,4and5.
Chapter 1 is a description of the biological architecture of cells. It summarizes the main
proteinsinvolvedinthemotilityprocessofeukaryoticcellsandexplainsthecoordinatedsteps
required for cells to move. Moreover, state-of-the-art of Dictyostelium cells migration and
adhesionisgiven.
In chapter 2, we start by the description of the tools developed to quantify cell motility.
The study of Dictyostelium cells motion requires large amount of data and precise statistical
analysis. We define a diffusion coefficient that characterized cell motion. We then, study the
relationship between this parameter and cell shape as well as cell persistence. Dictyostelium
cells motion in nutrient medium and in the course of development is presented in chapter 3
and chapter 4, respectively. We present how the cell density is a key component tuning cell
motion as well as cell morphology in both nutritive and starved condition, with antagonist
effectsthough.
In parallel, we have developed a microfluidic device to quantify cell detachment and to
haveinsightintocelladhesion. Thankstotheparallelizationprovidedbythemicrofluidicset-
updeveloped, itisalsopossibletogetstatisticallyrelevantresultswithinasingleexperiment.
Recording time lapse video of cells submitted to shear flow, we are able to quantify cell ad-
hesion as well as the detachment kinetics by counting cells remaining in the channel at each
successive frame. Results are presented in chapter 5. Finally, by comparing cell motion in
variousenvironmentalconditions(eitherstudyingcellmotionondifferentsubstratesorstudy-
ingcelladhesionmutants),preliminaryconclusionsofourunderstandingofcellmigrationand
adhesioninterplayforamoeboidcellsarediscussedinchapter6.
vi
Chapter 1
Introduction
Cell motility ability requires the coordination of complex mechanisms. After de-
scribing of how migration and adhesion are connected, we will introduce the basic
biological features needed for cell motility. Particularities will be given for differ-
ent type of eukaryotic cells. Then, we will present in detail, Dictyostelium cells, the
modelsystemwechooseinthisthesis. Wewillespeciallydiscussthecellmachinery,
celladhesionandquorumsensinginDictyostelium.
1