Nuclear Volume Dynamics under Confinement
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Cellular response of HeLa cells in terms of the decay time of nuclear volume loss under pressure was checked using a dynamical confiner. The double exponential decay fit of the data revealed two timescales involved in the phenomenon which are being investigated in perspective.
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Nuclear Volume Dynamics under Confinement
- 1. Nuclear volume dynamics under confinement Aditya S Kulkarni AIV Master M1 (2018 - 19) CRI, Paris PI - Matthieu Piel Co-supervisors : Damien Cuvelier Nishit Srivastava UMR144 – Equipe Piel
- 2. OUTLINE ● Introduction ● Question ● Methods ● Results ● Conclusion ● Perspective ● Acknowledgements
- 3. Cell deformation - highly dynamic process Introduction Questions Methods Results Conclusion Perspective Acknowledgements A cell life under confinement, Matthieu Piel - www.cnrs.fr Cell migration through tissues Hawa-Racine Thiam et al, Nature Comm. 2016
- 4. UMR144 - Equipe Piel Introduction Questions Methods Results Conclusion Perspective Acknowledgements 8.144 μm 1.498 μm Hawa-Racine Thiam et al, Nature Comm. 2016 8.144 μm 1.498 μm 10 μm Liu et al, Cell, 2015 h = 3 μm
- 5. Cell migrating through microchannel Raab et al, Science, 2016 Estimated ΔV/V = 30-40% Introduction Questions Methods Results Conclusion Perspective Acknowledgements 15 μm Volume = Area x height (Cuvelier D) 2 μm
- 6. Questions How does cell deformation affect the loss of volume of the nucleus ? Introduction Questions Methods Results Conclusion Perspective Acknowledgements
- 7. Dynamic cell confiner Introduction Questions Methods Results Conclusion Perspective Acknowledgements Berre et al, Integrative Biology, 2012 Before confinement Confinement at fixed height
- 8. Dynamic cell confiner Introduction Questions Methods Results Conclusion Perspective Acknowledgements Berre et al, Integrative Biology, 2012 Before confinement Confinement at fixed height
- 9. Dynamic Confinement of HeLa cells and Hydrogel balls Introduction Questions Methods Results Conclusion Perspective Acknowledgements 100 μm 8 & 35 kPa poly-acrylamide hydrogels h = 7.6 μm t = 301 sec, 1 frame/sec HeLa cells - Hoechst stained nuclei (NucBlue) 10 μm t = 301 sec, 1 frame/sec h = 7.6 μm
- 10. Living cells Cell type – HeLa Hoechst stain (Nucblue) Introduction Questions Methods Results Conclusion Perspective Acknowledgements 10 μm 10 μm Not confined Not confined 10 μm h = 7.6 μm Initial volume 4OX - DAPI - 358 nm4OX - BF Volume ? Height ?
- 11. Method for nuclear volume estimation Introduction Questions Methods Results Conclusion Perspective Acknowledgements h = 7.6 μm 10 μm h = 7.6 μm 10 μm h = 7.6 μm 10 μm BLEACH CORRECTION MASK AUTO THRESHOLD
- 12. Method for nuclear volume estimation Introduction Questions Methods Results Conclusion Perspective Acknowledgements Volume = Area x (height) = (π R2 )h10 μm h = 7.6 μm 10 μm h = 7.6 μm R0 >> h MASK OTSU AREA VOLUME
- 13. Nuclear volume Analysis Confinement height – 7.6 μm, 1 frame/sec, 5 mins Introduction Questions Methods Results Conclusion Perspective Acknowledgements y0 – Final hydrogel volume A1 + A2 – Volume decrease in total y0 + A1 + A2 – Initial hydrogel volume t1 – Characteristic decay time 1 of volume decrease t2 – Characteristic decay time 2 of volume decrease 10 μm t = 301 sec, 1 frame/sec h = 7.6 μm
- 14. Nuclear volume Analysis Confinement height – 7.6 μm, 1 frame/sec, 5 mins Introduction Questions Methods Results Conclusion Perspective Acknowledgements y0 – Final hydrogel volume A1 + A2 – Volume decrease in total y0 + A1 + A2 – Initial hydrogel volume t1 – Characteristic decay time 1 of volume decrease t2 – Characteristic decay time 2 of volume decrease 10 μm t = 301 sec, 1 frame/sec h = 7.6 μm
- 15. Confinement height – 6 μm, 1 frame/sec, 5 mins 10 μm t = 301 sec, 1 frame/sec h = 6 μm Introduction Questions Methods Results Conclusion Perspective Acknowledgements h = 6 μm Confinement height – 3 μm, 1 frame/sec, 5 mins t = 301 sec, 1 frame/sec h = 3 μm 10 μm
- 16. Preparing hydrogel balls - 8 & 35 kPa Acrylamide + BisAcrylamide + Irgacure + Sucrose + Water Oil + Surfactant (Span80) SHAKE Introduction Questions Methods Results Conclusion Perspective Acknowledgements Oil in water emulsion Micelles
- 17. liquid UV GEL ξ(Ε) Centrifugation in PBS Ready to squeeze Introduction Questions Methods Results Conclusion Perspective Acknowledgements 100 μm h = 7.6 μm t = 301 sec, 1 frame/sec
- 18. R0 >> h Volume = h π R2 h = 7.6 μm, 5.9 μm Confinement of hydrogel balls 2R Introduction Questions Methods Results Conclusion Perspective Acknowledgements Top view Side view100 μm h = 7.6 μm t = 301 sec, 1 frame/sec
- 19. Measurement of volume loss y0 – Final hydrogel volume A1 + A2 – Volume decrease in total y0 + A1 + A2 – Initial hydrogel volume t1 – Characteristic decay time 1 of volume decrease t2 – Characteristic decay time 2 of volume decrease Introduction Questions Methods Results Conclusion Perspective Acknowledgements 100 μm h = 7.6 μm t = 301 sec, 1 frame/sec
- 20. Results Introduction Questions Methods Results Conclusion Perspective Acknowledgements Size - Volume lost
- 21. Results Introduction Questions Methods Results Conclusion Perspective Acknowledgements Height - Volume lost
- 22. Conclusion ● Both hydrogel balls and nuclei of HeLa cells lose volume in a double exponential decay manner under confinement. ● There are two timescales (t1 and t2) associated with the volume loss. ● Larger the size of the hydrogel ball, the more volume is lost during compression. ● Relation between the confinement height placed on the cell and the volume lost is not clear (not enough data). Introduction Questions Methods Results Conclusion Perspective Acknowledgements
- 23. What is t1 and t2 ? Before After compression t1t1 t2 t2 t2 t2 t1 – characteristic decay time of water efflux t2 – characteristic decay time due to (?) Introduction Questions Methods Results Conclusion Perspective Acknowledgements 2 responses of cells, friction, mechanical response, is the height not stable ?
- 24. Perspective ❖ More data ❖ Find out what t2 is ❖ Test drugs affecting actin, myosin Introduction Questions Methods Results Conclusion Perspective Acknowledgements
- 25. Acknowledgements Matthieu Piel (PI) Damien Cuvelier (Co-supervisor) Nishit Srivastav (Co-supervisor) Nicolas Carpi(Lab manager) Juanma Guilherme Nader Alice Williart Pablo Saez Aastha Mathur Larisa Venkova Matthieu Deygas Pierre Récho Henrietta Lacks Pierre Gilles de Genes CRI Introduction Questions Methods Results Conclusion Perspective Acknowledgements
- 26. THANK YOU
- 27. Supplementary Introduction Questions Methods Results Summary Perspective Acknowledgements
- 28. Supplementary Introduction Questions Methods Results Summary Perspective Acknowledgements
- 29. Supplementary Introduction Questions Methods Results Summary Perspective Acknowledgements