PicoNewton Force Spectroscopy of Live Neuronal Cells using Optical Tweezers

1. PicoNewton Force Spectroscopy of Live Neuronal Cells using Optical Tweezers *Dan Cojoc, Enrico Ferrari, Francesco Di Fato, Rajesh Shahapure, Jumi Laishram, Massimo Righini, ^Enzo Di Fabrizio, Vincent Torre CNR – INFM, Laboratorio Nazionale TASC, Trieste SISSA, Neurobiology sector, Trieste *CBM, Trieste; ^Univ. Magna Grecia, Catanzaro E-mail: cojoc@tasc.infm.it, http://www.tasc-infm.it

2. Outline • Motivation, goal, approach • Force spectroscopy using Optical Tweezers • Force measurements – Results • Conclusions

3. Motivation and goal of our work Key determinant of axonal growth isthe growth cone: "They will adopt pre-determined directions and establish connections with defined neural or extra neural elements ... without deviations or errors, as if guided by an intelligent force ." 1890 RAMON Y CAJAL • Structural elements of the growth cone www.biology.lsa.umich.edu/research/labs/ktosney/

4. Growth cone dynamics Movie available on request Movie available on request Scale Bar = 5μm Acquisition freq= 0.2Hz Time in min.sec Scale bar = 2 μm; Acquisition freq = 0.3Hz

5. Growth cones connection Movie available on request Scale Bar = 3 μm Acquisition freq= 0.2Hz Time in min.sec

6. Goal - Approach Goal: measure the forces exerted by lamellipodia and filopodia Experimental approach • Calibrate the trap by measuring the fluctuations of the bead in trap • Micro beads trapped by IR laser and positioned in front of lamellipodia and/or filopodia • Measure the fluctuations of the bead in the trap, • due to its interaction with the neurite, and convert them into forces. J.L. Goldberg, Genes and Dev. 17 941 (2003)

7. Motivation, goal, approach • Force spectroscopy using Optical Tweezers • Force measurements – Results • Conclusions

8. Optical Tweezers setup Including force spectroscopy and multiple trapping Bead position was determined by back focal plane (BFP) detection: BFP of the condenser was imaged onto a QPD Force = K . Δ X K = stiffness of the trap (spring constant) ΔX = Displacement

9. Trap calibration from the fluctuations of the bead Schematic of a μm bead diffusing in an optical trap Mechanical model of the forces acting on the bead The power spectrum density S( f ) of these fluctuations near the center of an optical trap is approximately Lorentzian (Svoboda and Block, 1994; Gittes and Schmidt, 1997)

10. XY Z Centered Back focal plane interferometry detect the thermal fluctuations of the bead with F. Gittes, Optics Letters, (1998) Displacement from the focus Voltage change on the detector

11. Trap stiffness and detector sensivity Sv(f) - measured power spectrum S(f) - density Lorentzian fit f0 – corner frequency k – trap stifness γ – Stokes drag coefficient of the bead β– detector sensivity S0 – trap stifness PV – plateu of The power spectrum (dotted line) of a trapped 1 μm silica bead acquired at 10 KHz and fitted to a Lorentzian (solid line).

12. Motivation, goal, approach • Force spectroscopy using Optical Tweezers • Force measurements – Results • Conclusions

13. Experimental results Neurons obtained from dorsal root ganglia (DRG), isolated from P0-12 rats and plated on poly-L-lysine-coated glass dishes. 48 hours after incubation in 50 ng/ml of nerve growth factor (NGF). Features of our setup Trap stiffness: 5-100 pN/μm Resolution: ~10nm (1 nm) Force range: 1-25 pN Errors are about 10% (Some) Problems encountered: Stuck beads to the substrate Trapping and calibration close to the substrate (<2 μm ) and at T=37 C Influence of floating particles on the interference pattern Filopodia collisions reveal lower forces than expected ?Tam-Tam !

14. Measurement away from Neuron Measurements done by QPD & Video tracking Overlapped Measurement during collision Criteria to define a collision

15. Results Movie available on request Filopodia 2 minutes event, Fmax= 2pN

16. Results Clicking on !! You might see the lamellipodia taking the bead out from the trap Lamellipodia 2 minutes event, F> 20pN

17. Acquisition rate: 20Hz Scale Bar = 2μm Time in seconds Acquisition rate : 4KHz Subsampeled at : 2KHz Force exerted by Lamellipodia Movie available on request

18. Acquisition rate: 20Hz Scale Bar = 2μm Time in seconds Acquisition rate : 4KHz Subsampeled at : 2KHz Force exerted by Filopodia - Protrusion Movie available on request

19. 2 μm Force exerted by Filopodia - Protrusion

20. Acquisition rate: 20Hz Scale Bar = 2μm Numbers indicate time in seconds Acquisition rate : 4KHz Subsampeled at : 2KHz Force exerted by Filopodia - Lateral collision Movie available on request

21. Multiple beads near Lamellipodia

22. Conclusions • Introduce a method to measure pN forces expressed by filopodia (3 pN) and lamellipodia (more than 20 pN)   PlosOne accepted Sept 2007 • Found that even one neuron is (very) intelligent • Found lot of questions to answer to in the future work Acknowledgments TASC: Enrico Ferrari, Valeria Garbin, Lilit Group SISSA: Vincent Torre, Rajesh Shahapure, Massimo Righi Francesco Difato, Jummi Laishram