Distinct mechanisms regulating mechanical force-induced Ca2+ signals at the plasma membrane and the ER in human MSCs
- University of Illinois, Urbana-Champaign, United States
- Delft University of Technology, Netherlands
- Korea Institute of Science and Technology, Republic of Korea
- University of California, Irvine, United States
- University of Colorado, Boulder, United States
- Howard Hughes Medical Institute, University of Illinois, Urbana-Champaign, United States
- University of California, San Diego, United States
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 …
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 …
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) …
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 …
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 …
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 …
Figure 3
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 …
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), …
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) …
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
