Protrusion Force Microscopy

Transcription

1 Protrusion Force Microscopy A. Bouissou & R. Poincloux IPBS, Toulouse A. Labernadie, A. Proag, G. Charrière, I. Maridonneau-Parini IPBS, Toulouse P. Delobelle Femto, Besançon T. Mangeat LBCMCP, Toulouse S. Balor IBCG, Toulouse C. Thibault & C. Vieu LAAS, Toulouse

2 Unroofed macrophage by SEM Podosome architecture in human macrophages Macrophage by TIRF mch-lifeact GFP-Vinculin Linder et Wiesner, Cell. Mol. Life Sci. 2015

3 Podosome functions ECM degradation & mechanosensing F-Actin Gelatin-FITC ECM degradation: V. Le Cabec 100kPa 25kPa 6.5kPa 0.5kPa 100kPa 25kPa 6.5kPa 0.5kPa Mechanosensing: Labernadie et al., Nat. Commun. 2014

4 Formed by: vascular smooth muscle cells invasive cancer cells endothelial cells megakaryocytes dendritic cells macrophages lymphocytes osteoclasts neurons

5 Traction force measurement methods M. Gardel s lab Harris et al. Science 1980 Trichet et al. PNAS 2012 Légant et al. Nat Methods 2010

6 Protrusion force measurement method?

7 Protrusive Force Microscopy (PFM) A novel approach to measure podosome protrusive force Development of an suspended elastic Formvar film Formvar (thickness: nm) EM grid S. Balor, METi & IBCG Differentiation into macrophages Buffy coat M-CSF CD14 + human blood monocytes J7 macrophages Macrophages Topography imaging by Atomic Force Microscopy AFM Labernadie et al., Nat. Commun.2014

8 MICROSCOPIE À FORCE ATOMIQUE: Etude topographique et rhéologique à haute résolution d échantillons biologiques Tête AFM Microscope optique IMAGERIE PAR AFM Topographie de l échantillon Mode contact SPECTROSCOPIE DE FORCE Mesure de rigidité/force d interaction

9 AFM experiment Conditions Living cells: Measures in liquid Temperature controller: 37 C Compliant substrate Plan of an experiment 1. Preparation of the formvar film and thickness measurement of the film 2. Laser alignment 3. Cantilever calibration 4. Mounting samples 5. AFM imaging 6. Image processing 7. Image analysis (Image J macro)

10 1.Sample preparation Macrophages >2h Topographic imaging by Atomic Force Microscopy AFM 10 4 cells in a 10µL droplet Fill the well with medium after 1h of adhesion Labernadie et al., Nat. Commun.2014

11 2.Mounting of the AFM tip µm Choice of the cantilever For contact imaging, only very soft cantilever are used to minimize force Cantilever are chosen with the lowest spring constant (0,01-0,03N/m) MLCT probes : soft Silicon Nitride cantilevers with Silicon Nitride tips are ideal for contact imaging modes and liquid operation.

12 2.Setting up the laser detection system Laser adjustment Mirror adjustment +

13 Spring constant calibration The deflection of cantilever is measured by the reflected laser spot on the photodiode. This vertical deflection value is usually in volts, and is a difference in voltage between the different sections of the photodiode. The conversion of the vertical deflection into units of length to forces requires calibration of the cantilever.

14 Spring constant calibration Standard method to determine: -the sensitivity (conversion into units of length) -A force curve on a hard surface -Looking at the deflection of the cantilever when it is in repulsive contact with the sample -& the spring constant (conversion into units of force) The thermal noise method relies on measuring the thermal fluctuations in the deflection of the cantilever, and using equipartition theorem to relate this to spring constant The resonance must be fit with a Lorentz curve. The fit algorithm has found the resonance frequency of the cantilever and calculated its spring constant k

15 Preparation of grids covered by a film of formvar Formvar (thickness: nm) EM grid S. Balor, METi & IBCG Formvar preparation Formvar thickness measured by AFM movie

16 Data Analysis Obtain podosome force from height image

17 Finite-element simulations to evaluate protrusion forces generated by podosomes d

18 Finite-element modelling (COMSOL) Meshing Deformation is computed on each element from force, then the elements are joined together

19 Finite-element simulations to evaluate protrusion forces generated by podosomes Single podosome: Plate thickness h f : ratio of 80 between lowest and highest h f. Traction ring radius r t : 9-wise variation F p = C h f 3 r t 2 h Core ring radius r p : 1.6- wise variation Distance: 1.6- wise variation Traction ring width w: no variation need to measure! negligible C C 0 E E = 2.3GPa C 0 2.7

20 Measurements of podosome size 10µm 1µm Deconvoluted images of unroofed macrophages on formvar, anti-vinculin and phalloidin, 100x, 1.45 with Thomas Mangeat, LBCMCP

21 F-actin vinculin Measurements of podosome size Proag*, Bouissou* et al. ACS Nano 2015 Fluo. image Edge image r t = 360nm ±15% r c = 140nm ±16% w = 200nm ±12%

22 Force evaluation example Height (nm) h r r Profile abscissa (µm)

23 Force evaluation example

24 Proag et al. ACS Nano 2015

25 Force evaluation F p = C h f 3 r t 2 h C 2.7E Assumptions: r p =150nm, r t =350nm, w=25nm d=2µm h f measured (AFM) F p =10.4nN ±30% (3000 podosomes) Proag et al. ACS Nano 2015