Biophysics of Molecules

Transcription

1 Biophysics of Molecules Concepts of cell adhesion and cellular mechanostransduction part II Dr. Carsten Grashoff MPI of Biochemistry

2 Measuring mechanotransduction The problem We need to know which molecules are exposed to mechanical forces within the cells. How can we measure mechanical forces in cells? 2

3 Traction force microscopy nano-pillars from Fu et al., Nat Methods, traction force microscopy is used to determine how much force is exerted by the cell onto an extracellular substrate - nano-pillars of known size and elasticity can be used to determine traction forces microscopically 3

4 Traction force microscopy micro-beads - micro-beads embedded in polyacrylamid-gels of known elastic modulues can be similarly used to measure traction forces with a light microscope 4

5 Methods to determine mechanical parameters experimentally in cells 5

6 Measuring forces across molecules - single-molecule atomic force microscopy is used to measure mechanical forces across distinct proteins - but: we want to measure mechanical forces in cells! from Fisher et al., 1999, Trends in Biochemical Sciences 6

7 Mechanical aspects of cell migration FAs disassemble FAs grow 7

8 Traction forces during cell migration in protrusions: - FAs grow - traction forces high? Beningo et al., JCB, 2001 in retracting areas: - FAs disassemble - traction forces high 8

9 The mechanical cell migration paradox 9

10 Fluorescence resonance energy transfer 10

11 Principle of a FRET-based tension sensor - the linker peptide has to be short (< 5 nm long), elastic and responsive to pn forces 11

12 Remember entropic elastomers? 12

13 A genetically encoded flagelliform sensor flagelliform (spider silk protein) from Becker et al., Nat Mater, 2003 from Grashoff et al., Nature,

14 Single molecule calibration with TJ Ha, University of Illinois, USA 14

15 First application: a vinculin tension sensor - the biosensor cdna is transfected (or infected) and the protein is expressed by the cell but we need controls: Vinculin - a zero-force control that can not be under force - a wildtype vinculin control that behaves as the endogenous protein 15

16 Generating a suitable model system - knockout cells (cells with no endogenous vinculin) should be used - cells are isolated from genetically modified mice and immortalized - the vinculin gene is deleted in vitro - the biosensor is transiently or stably expressed in knockout cells 16

17 Tension sensor evaluation in cells The sensor is stably expressed displays normal localization staining and shows normal FA dynamics Western Blotting FRAP 17

18 Measuring vinculin forces within cells Fluorescence lifetime microcopy 18

19 Forces across vinculin in migrating cells from Grashoff et al., Nature, no differences in FRET of the zero-force control - low FRET (high force) in protrusions - high FRET (low force) in retracting areas 19

20 Automatic quantification of FRET in FAs - due to use of endogenous vinculin levels and small FRET changes experimental data are noisy - using custom-written software individual FAs are automatically segmented and averaged to boost statistical power 20

21 Automatic quantification in dynamic FAs 21

22 Statistical evaluation of data high forces across vinculin in assembling FAs low forces across vinculin in disassembling FAs 22

23 Does vinculin regulate cell migration? traction force high FA stability low vinculin forces low traction force high FA stability high vinculin forces high Hypothesis: Vinculin stabilizes FAs under force 23

24 Testing the hypothesis increases contractility of cells myosin II active RhoA Vinculin does stabilize FAs under force! 24

25 Metavinculin is a muscle-specific isoform altered actin binding normal hypertrophic normal hypertrophic - mice with a metavinculin deletion in the heart muscle develop hypertrophic hearts (cardiomyopathy) and die of heart failure 25

26 Metavinculin mutations lead to disorders Mutations in cardiomyopathy patients: - (Meta)Vinculin L277M - Metavinculin A934V - Metavinculin Δ954 - Metavinculin R975W metavinculin-specific mutations in patients suffering from cardiomyopathy Next question: Do mutations in metavinculin effect the ability of the protein to bear (and transduce) mechanical force? 26

27 Improving the tension sensor technique Matthias Rief Lab (TU Munich, Germany) 27

28 Summary force measurments - there are many biophysical techniques to investigate mechanical properties of cells: - traction force microscopy (using nao-pillars or micro-beads) - atomic force microscopy - single-molecule atomic force microscopy - micro-rheology, etc. - a FRET-based sensor can be used to visualize and measure forces across proteins in cells - vinculin is exposed to mechanical forces of a few pn - vinculin is exposed high force in assembling FAs but is not bearing forces in disassembling FAs 28

29 Mechanobiology is interdisciplinary 29