TRACTION and STRESS MICROSCOPY for CELLS:

Transcription

1 TRACTION and STRESS MICROSCOPY for CELLS: the WOUND HEALING CASE Embryo Physics Course APRIL 2, 2014 Vito Conte, A. Brugués, E. Anon, J.H. Veldhuis, J. Colombelli, J.J. Muñoz, G.W. Brodland, B. Ladoux and Xavier Trepat

2 WHAT IS THE MECHANISM EPITHELIAL WOUND HEALING 0 min DRIVING? AgustíBrugués Ester Añón with Julien Colombelli Benoit Ladoux Xavier Trepat Lifeact 20 μm

3 WHAT IS THE MECHANISM EPITHELIAL WOUND HEALING 12 min DRIVING? Lifeact 20 μm

4 WHAT IS THE MECHANISM EPITHELIAL WOUND HEALING 40 min DRIVING? Lifeact 20 μm

5 WHAT IS THE MECHANISM EPITHELIAL WOUND HEALING 76 min DRIVING? Lifeact 20 μm

6 KEY STRUCTURES lamellipodia contractile actomyosin ring DAPI Actin 20 μm MDCK epithelium

7 TRACTION MICROSCOPY & LASER ABLATION cell epithelium fluorescent beads cell traction forces elastic gel substrate

8 TRACTION MICROSCOPY & LASER ABLATION LASER ABLATION cell epithelium fluorescent beads cell traction forces elastic gel substrate

9 TRACTION MICROSCOPY & LASER ABLATION cell epithelium fluorescent beads cell traction forces elastic gel substrate

10 TRACTION MICROSCOPY & LASER ABLATION cells close the region by grabbing on the substrate substrate deforms and beads displace cell epithelium original bead positions displaced beads cell traction forces elastic gel substrate

11 TRACTION MICROSCOPY & LASER ABLATION cell epithelium cell epithelium is washed away from substrate beads displace back to initial position Trypsin Trypsin original bead positions displaced beads cell traction forces elastic gel substrate

12 TRACTION MICROSCOPY & LASER ABLATION bead displacemtents are measured cell traction forces on the substrate are computed via Inverse Methods bead displacements cell traction forces elastic gel substrate

13 CELL TRACTIONS ON THE SUBSTRATE +150 tractions towards s exterior radial direction 0 tractions towards s interior Lifeact 20 μm -150 (Pa)

14 CELL TRACTIONS ON THE SUBSTRATE +150 tractions towards s exterior radial direction edge 0 tractions towards s interior Lifeact 20 μm -150 (Pa)

15 CELL TRACTIONS ON THE SUBSTRATE 32 min +150 tractions towards s exterior radial direction 0 tractions towards s interior Lifeact 20 μm -150 (Pa)

16 CELL TRACTIONS ON THE SUBSTRATE 40 min +150 tractions towards s exterior radial direction 0 tractions towards s interior Lifeact 20 μm -150 (Pa)

17 CELL TRACTIONS ON THE SUBSTRATE 77 min +150 tractions towards s exterior radial direction 0 tractions towards s interior Lifeact 20 μm -150 (Pa)

18 RADIAL TRACTIONS AVERAGE Lifeact t = 0 min edge centroid average distance 20 μm radial direction

19 RADIAL TRACTIONS AVERAGE Lifeact t = 0 min centroid average distances radial direction

20 RADIAL TRACTIONS AVERAGE Lifeact t = 0 min RADIAL TRACTIONS averages at t = 0 min side epithelium side centroid average distances (Pa) average radial distance from centroid

21 RADIAL TRACTIONS AVERAGE T I M E Lifeact RADIAL TRACTIONS kymograph epithelium 20 μm (Pa) average radial distance from centroid

22 RADIAL TRACTIONS AVERAGE T I M E Lifeact RADIAL TRACTIONS kymograph epithelium 20 μm (Pa) average radial distance from centroid

23 MECHANISM EXPERIMENTAL OUTLINE RADIAL TRACTION kymograph time (min) distance from center (μm) s interior s exterior

24 MECHANISM EXPERIMENTAL OUTLINE DAPI Actin RADIAL TRACTION kymograph edge lamellipodia time (min) distance from center (μm) s interior s exterior

25 DECIPHERING THE MECHANISM CELL CRAWLING ( lamellipodia & filipodia ) RADIAL TRACTION kymograph 20 time (min) distance from center (μm) Omelchenko et al. PNAS (2003) Poujade et al. PNAS (2007) Lee P et al. PLOS Comp. Biol. (2011) etc. s interior s exterior

26 MECHANISM EXPERIMENTAL OUTLINE RADIAL TRACTION kymograph time (min) distance from center (μm) s interior s exterior

27 MECHANISM EXPERIMENTAL OUTLINE EPITHELIAL ACTIN DENSITY kymograph RADIAL TRACTION kymograph max 20 average position of the max actin density 0 time (min) average position of the actomyosin ring distance from center (μm) s interior s exterior

28 MECHANISM EXPERIMENTAL OUTLINE DAPI Actin RADIAL TRACTION kymograph time (min) average position of the actomyosin ring actomyosin ring distance from center (μm) s interior s exterior

29 DECIPHERING THE MECHANISM min top view RADIAL TRACTION kymograph Lifeact substrate lateral view lateral view epithelium imaging cut 4 min time (min) average position of the actomyosin ring distance from center (μm) 32 min 40 min s interior s exterior

30 DECIPHERING THE MECHANISM PURSE STRING ( actomyosin cable contraction ) RADIAL TRACTION kymograph 20 time (min) actomyosin ring distance from center (μm) Martin et al. Nature (1992) Davidson et al. Cell Motil Cytoskeleton (2002) Wood et al. Nature Cell Biology (2004) Tamada et al. Journal Cell Biology (2007) etc. s interior s exterior

31 DECIPHERING THE MECHANISM time (min) RADIAL TRACTION kymograph tractions distance from center (μm) s interior s exterior

32 DECIPHERING THE MECHANISM CELL CRAWLING ( lamellipodia & filipodia ) RADIAL TRACTION kymograph time (min) tractions distance from center (μm) s interior s exterior

33 DECIPHERING THE MECHANISM CELL CRAWLING ( lamellipodia & filopodia ) & PURSE STRING ( actomyosin cable contraction ) RADIAL TRACTION kymograph 20 time (min) tractions distance from center (μm) Anon et al. PNAS (2012) Klarlund PNAS (2012) Abreu-Blanco et al. J. Cell Sci.. (2012) etc. s interior s exterior

34 DECIPHERING THE MECHANISM CELL CRAWLING ( lamellipodia & filopodia ) & PURSE STRING ( actomyosin cable contraction ) RADIAL TRACTION kymograph time (min) tractions distance from center (μm) s interior s exterior

35 DECIPHERING THE MECHANISM PURSE STRING (actomyosin cable contraction ) RADIAL TRACTION kymograph time (min) tractions distance from center (μm) s interior s exterior

36 EXPERIMENTAL VALIDATION of the MECHANISM EGTA CALCIUM CHELATION ( weakened cell-cell junctions ) RADIAL TRACTION kymograph epithelium NO actomyosin ring time (min) actomyosin ring distance from center (μm) phospho-myosin 20 μm

37 EXPERIMENTAL VALIDATION of the MECHANISM EGTA CALCIUM CHELATION ( weakened cell-cell junctions ) RADIAL TRACTION kymograph epithelium NO actomyosin ring time (min) phospho-myosin 20 μm distance from center (μm)

38 EXPERIMENTAL VALIDATION of the MECHANISM time (min) EGTA RADIAL TRACTION kymograph distance from center (μm) time (min) RADIAL TRACTION kymograph distance from center (μm)

39 WE EXPERIMENTALLY QUANTIFIED AND DETAILED MECHANISM A CANDIDATE DRIVING EPITHELIAL WOUND HEALING Lifeact 20 μm CELL CRAWLING + PURSE STRING

40 MECHANICAL VALIDATION of the MECHANISM Wayne Brodland epithelium Jim Veldhuis MULTI-SCALE MODEL for Epithelial Wound Healing

41 MECHANICAL VALIDATION of the MECHANISM actomyosin ring lamellipodia epithelium MULTI-SCALE MODEL for Epithelial Wound Healing

42 MECHANICAL VALIDATION of the MECHANISM actomyosin ring lamellipodia single cell epithelium MULTI-SCALE MODEL for Single Cell

43 MULTI-SCALE MODEL for SINGLE CELL node line CELL UNIT geometry of nodes joined through lines

44 MULTI-SCALE MODEL for SINGLE CELL dashpot CELL CYTOPLASM responds viscously to deformations

45 MULTI-SCALE MODEL for SINGLE CELL constant surface area CELL CYTOPLASM incompressibility

46 MULTI-SCALE MODEL for SINGLE CELL boundary force dipoles constant in time CORTICAL TENSION

47 MULTI-SCALE MODEL for SINGLE CELL localised force dipoles variable in time CORTEX CONTRACTILITY

48 MULTI-SCALE MODEL for SINGLE CELL dashpot low viscous friction at all nodes substrate SUBSTRATE INTERACTION

49 MULTI-SCALE MODEL for SINGLE CELL dashpot high viscous friction at selected sides substrate SUBSTRATE INTERACTION

50 MULTI-SCALE MODEL for SINGLE CELL A A' substrate B B' focal adhesion CRAWLING lamellipodium

51 MULTI-SCALE MODEL for SINGLE CELL fibres A A' substrate fibres B B' focal adhesion CRAWLING lamellipodium

52 PARAMETERS OF THE MODEL actomyosin ring lamellipodia epithelium

53 EPITHELIUM PARAMETERS h citoplasm viscosity c cortical tension no prescriptions at boundary epithelium

54 ACTOMYOSIN RING PARAMETERS m intensity actomyosin ring m 0 t

55 ACTOMYOSIN RING PARAMETERS m intensity actomyosin ring a increase rate m 0 a t

56 ACTOMYOSIN RING PARAMETERS m intensity actomyosin ring a increase rate w ring friction w.r.t. epithelium (i.e. high/low friction ratio) high friction zone

57 ACTOMYOSIN RING PARAMETERS m intensity a increase rate w ring friction w.r.t. epithelium (i.e. high/low friction ratio) SEGMENTED actomyosin ring r density of myosin segments

58 LAMELLIPODIA PARAMETERS l length q direction t duration j strength lamellipodia b distribution p probability of being generated w location weight ( / cryptic ) r reduction factor ( -diameter based ) e cell side selection probability

59 LAMELLIPODIA PARAMETERS VALUES l length q direction l lamellipodia 2.5l cryptic lamellipodia polarization towards s centroid: (d,q ) = (0,0 ) ; (30,±45 ); (60,±180 ); t duration j strength b distribution 3±1 time units ( and cryptic) lamellipodia 25% stronger than cryptic backward distribution p probability of being generated 6 out of cell-sides (randomly) w location weight ( / cryptic ) r reduction factor ( -diameter based ) e cell side selection probability 1 out of 3 generated lamellipodia is cryptic % current length uniform

60 MECHANICAL VALIDATION of the MECHANISM 0 min Lifeact 20 μm 40 min 76 min in vitro RADIAL TRACTION 170 KYMOGRAPH distance from center (μm) time (min) (Pa) 0 min 40 min lamellipodia + actomyosin ring in silico 76 min RADIAL TRACTION 170 KYMOGRAPH distance from center (μm) time (min) (Pa)

61 TANGENTIAL TRACTIONS AVERAGE Lifeact radial direction

62 TANGENTIAL TRACTIONS AVERAGE Lifeact tangential direction time (min) TANGENTIAL TRACTION kymograph distance from center 60 (μm) distance from center (μm)

63 TRACTIONS KYMOGRAPHS time (min) RADIAL TRACTION kymograph distance from center (μm) time (min) TANGENTIAL TRACTION kymograph distance from center 60 (μm) distance from center (μm) CELL CRAWLING PURSE STRING?

64 MECHANICAL VALIDATION of the MECHANISM 0 min Lifeact 0 min 20 μm 40 min 40 min lamellipodia + actomyosin ring 76 min in vitro in silico 76 min TANGENTIAL TRACTIONS 170 KYMOGRAPH distance from center (μm) time (min) WHAT IS MISSING? TANGENTIAL TRACTION 170 KYMOGRAPH distance from center (μm) time (min) (Pa) (Pa)

65 STRESS MICROSCOPY IN THE SUBSTRATE 20 μm epithelium elastic gel substrate

66 STRESS MICROSCOPY IN THE SUBSTRATE epithelium trace gel interface tractions towards s interior elastic gel substrate tractions towards s exterior

67 STRESS MICROSCOPY IN THE SUBSTRATE external forces elastic gel substrate

68 STRESS MICROSCOPY IN THE SUBSTRATE José Muñoz external forces compressive and tensile states an elastic gel of a subject solid material to external forces are fully will described deform and by a undergo physical a quantity state of called compression Stress Tensor tension s (x,y,z) elastic gel substrate

69 STRESS MICROSCOPY IN THE SUBSTRATE finite flement external forces FINITE ELEMENT METHOD Stress Tensor s (x,y,z)

70 AVERAGE NORMAL STRESS IN THE SUBSTRATE AVERAGE COMPRESSION / TENSION (Pa) (Pa) TENSILE TENSILE COMPRESSIVE COMPRESSIVE

71 STRESS MICROSCOPY IN THE SUBSTRATE gel substrate radial stress (Pa) in vitro r y t x Lifeact

72 STRESS MICROSCOPY IN THE SUBSTRATE gel substrate radial stress (Pa) in vitro actomyosin ring segment Lifeact

73 GROUNDED & SEGMENTED ACTOMYOSIN RING edge actomyosin ring segment tangential focal adhesions Talin Lifeact Lifeact

74 GROUNDED & SEGMENTED ACTOMYOSIN RING tangential focal adhesions trace tangential traction dipole gel interface elastic gel substrate

75 GROUNDED & SEGMENTED ACTOMYOSIN RING gel substrate (TOP VIEW) virtual trace in silico tangential forces tangential focal adhesions gel interface elastic gel substrate

76 GROUNDED & SEGMENTED ACTOMYOSIN RING external forces gel interface elastic gel substrate

77 GROUNDED & SEGMENTED ACTOMYOSIN RING finite flement external forces FINITE ELEMENT METHOD Stress Tensor s (x,y,z)

78 GROUNDED & SEGMENTED ACTOMYOSIN RING AVERAGE COMPRESSION / TENSION (Pa) (Pa) TENSILE TENSILE COMPRESSIVE COMPRESSIVE

79 GROUNDED & SEGMENTED ACTOMYOSIN RING AVERAGE COMPRESSION / TENSION (Pa) (Pa) TENSILE TENSILE in silico in vitro COMPRESSIVE COMPRESSIVE

80 GROUNDED & SEGMENTED ACTOMYOSIN RING gel substrate virtual actomyosin ring trace gel substrate tangential stresses (Pa) in silico in vitro tangential traction dipoles Lifeact

81 GROUNDED & SEGMENTED ACTOMYOSIN RING gel substrate virtual actomyosin ring trace gel substrate tangential stresses (Pa) in silico in vitro tangential traction dipoles Lifeact

82 GROUNDED & SEGMENTED ACTOMYOSIN RING gel substrate virtual actomyosin ring trace gel substrate tangential stresses (Pa) in silico in vitro tangential traction dipoles THE ACTOMYOSIN RING IS BASAL, GROUNDED AND SEGMENTED Lifeact

83 GROUNDED & SEGMENTED ACTOMYOSIN RING gel substrate virtual actomyosin ring trace gel substrate tangential stresses (Pa) in silico in vitro tangential traction dipoles gel substrate displacements (μm) Lifeact

84 EPITHELIAL SELF-STEERING SPECULATION gel substrate displacements (μm) Lifeact

85 WHAT IS MISSING? time (min) time (min) RADIAL TRACTION KYMOGRAPH distance from center (μm) -150 (Pa) distance from center (μm) RADIAL TRACTION KYMOGRAPH 150 Lifeact in vitro in silico (Pa) 150 distance from center (μm) TANGENTIAL TRACTION KYMOGRAPH lamellipodia + CONTINUOUS actomyosin ring 0 TANGENTIAL TRACTION KYMOGRAPH distance from center (μm) time (min) time (min)

86 MECHANICAL VALIDATION of the MECHANISM SEGMENTED actomyosin ring lamellipodia epithelium MULTI-SCALE MODEL for Epithelial Wound Healing

87 MECHANICAL VALIDATION of the MECHANISM time (min) time (min) RADIAL TRACTION KYMOGRAPH distance from center (μm) -150 (Pa) distance from center (μm) RADIAL TRACTION KYMOGRAPH 150 Lifeact in vitro in silico (Pa) 150 distance from center (μm) TANGENTIAL TRACTION KYMOGRAPH lamellipodia + SEGMENTED actomyosin ring 0 TANGENTIAL TRACTION KYMOGRAPH distance from center (μm) time (min) time (min)

88 RADIAL CONCLUSIONS in vitro & in silico cell crawling purse string a MECHANISM for WOUND HEALING Lifeact TISSUE DYNAMICS

89 RADIAL CONCLUSIONS in vitro & in silico cell crawling purse string a MECHANISM for WOUND HEALING Lifeact TISSUE DYNAMICS grounded purse string TANGENTIAL

90 ACKNOWLEDGMENTS