Distinct mechanisms regulating mechanical force-induced Ca2+ signals at the plasma membrane and the ER in human MSCs

  1. Tae-Jin Kim
  2. Chirlmin Joo
  3. Jihye Seong
  4. Reza Vafabakhsh
  5. Elliot L Botvinick
  6. Michael W Berns
  7. Amy E Palmer
  8. Ning Wang
  9. Taekjip Ha
  10. Eric Jakobsson
  11. Jie Sun  Is a corresponding author
  12. Yingxiao Wang  Is a corresponding author
  1. University of Illinois, Urbana-Champaign, United States
  2. Delft University of Technology, Netherlands
  3. Korea Institute of Science and Technology, Republic of Korea
  4. University of California, Irvine, United States
  5. University of Colorado, Boulder, United States
  6. Howard Hughes Medical Institute, University of Illinois, Urbana-Champaign, United States
  7. University of California, San Diego, United States
4 figures and 2 videos

Figures

Figure 1 with 2 supplements
Intracellular Ca2+ oscillations in response to mechanical force in HMSCs with Ca2+-free medium.

(A) A schematic drawing of the activation mechanism of the Ca2+ FRET biosensor. (B) Beads coated with Fn or BSA were seeded onto the cell and mechanical force was applied by pulling a Fn-coated bead …

https://doi.org/10.7554/eLife.04876.003
Figure 1—figure supplement 1
Laser-tweezer pulling of a Fn-coated bead on a BAEC in Ca2+-free medium.

Color images (upper panels) represent the YPet/ECFP emission ratio of the cytoplasmic Ca2+ biosensor. The color scale bars represent the range of emission ratio, with cold and hot colors indicating …

https://doi.org/10.7554/eLife.04876.004
Figure 1—figure supplement 2
Mechanical force doesn’t induce any increase in IP3 level.

The time courses represent the YPet/ECFP emission ratio of cytoplasmic Ca2+ in HMSCs pretreated with (A) Thapsigargin (TG, 10 µM), a SERCA pump blocker, or (B) 2-APB (100 µM), an IP3R blocker. (C) …

https://doi.org/10.7554/eLife.04876.005
Figure 2 with 2 supplements
Cytoskeletal support, actomyosin contractility, and TRPM7 channels mediate the force-induced intracellular Ca2+ oscillations.

The time courses represent the YPet/ECFP emission ratio of cytoplasmic Ca2+ in HMSCs in the absence of extracellular Ca2+ when these cells were pretreated with (A) 2 μM Cyto D (n = 8), (B) 1 μM Noc …

https://doi.org/10.7554/eLife.04876.007
Figure 2—figure supplement 1
Stretch-activated or store-operated channels at the plasma membrane mediate force-induced Ca2+ release from ER.

The time courses represent the YPet/ECFP emission ratio of cytoplasmic Ca2+ in HMSCs pretreated with (A) GdCl3 (5 µM), a broad spectrum Ca2+ channel inhibitor, (B) LaCl3 (100 µM), a broad spectrum …

https://doi.org/10.7554/eLife.04876.008
Figure 2—figure supplement 2
TRPM7 channels as well as cytoskeletal support and actomyosin contractility mediate the force-induced intracellular Ca2+ oscillations.

(A) HMSCs expressing Ca2+ biosensor and transfected with non-targeting (NT) or TRPM7 siRNA were immunostained (upper images) or immunoblotted (lower panels) with polyclonal TRPM7 antibody to assess …

https://doi.org/10.7554/eLife.04876.009
The visualization of force-induced Ca2+ release from ER using a FRET-based ER Ca2+ biosensor.

(A) The time course and (B) the color images of YPet/ECFP emission ratio in HMSCs expressing the D3ER biosensor before and after force application. The red arrows indicated episodes of Ca2+ release …

https://doi.org/10.7554/eLife.04876.010
Figure 4 with 1 supplement
Ca2+ influx in response to mechanical force in Ca2+-containing medium. Ca2+ release from ER in all the HMSCs was blocked by pretreatment with 2-APB.

(A, C) Color images represent the YPet/ECFP emission ratio of the cytoplasmic Ca2+ biosensor in control cells treated by 2-APB only (n = 5) or those co-treated by Nifedipine (n = 5), Gd3+ (n = 3), …

https://doi.org/10.7554/eLife.04876.012
Figure 4—figure supplement 1
Src, FAK or PI3K has no effect on the mechanical force-induced Ca2+ signals.

(A) The inhibition of neither Src by PP1 (n = 4), FAK by PF228 (n = 4), nor PI3K by LY294002 (n = 3) abolished the force-induced cytosolic Ca2+ oscillations in HMSCs without extracellular Ca2+. (B) …

https://doi.org/10.7554/eLife.04876.013

Videos

Video 1
A HMSC transfected with cytosolic Ca2+ biosensors before and after mechanical force application by optical laser tweezers on a Fn-coated bead attached to the cell (Duration of Video: 2700 s).
https://doi.org/10.7554/eLife.04876.006
Video 2
A HMSC transfected with ER Ca2+ biosensors before and after mechanical force stimulation by optical laser tweezers on a Fn-coated bead attached to the cell (Duration of Video: 450 s).
https://doi.org/10.7554/eLife.04876.011

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