| The cytoskeleton and focal adhesions(FAs)are key subcellular structures that mediate the perception and response of cells to mechanical signal stimulation.The newly discovered FAs protein actopaxin can bind to cytoskeletal actin and paxillin in the "membrane adhesion integrin signaling layer" of FAs through its C-terminal domain.Therefore,it is speculated that actopaxin may be an important link in the intracellular stress transmission and transduction pathway.Due to the lack of effective tools for the direct detection of stress transmission between the cytoskeleton and FAs,the mechanism of actopaxin-mediated stress transfer in cells as well as the functional classification of actopaxin in the specific layer of FAs are still unclear.In this paper,a molecular probe ASSA,which can detect the stress transfer between the cytoskeleton and FAs in living cells,was designed and constructed based on fluorescence resonance energy transfer(FRET)technology.One end of the ASSA probe is the β-actin sequence that can be embedded in the cytoskeleton,the other end is the actopaxin sequence that can be localized to FAs,and the middle is the "ECFP-SS-Ypet" stress detection unit.The specificity of the probe was verified by observing the morphological difference in the expression of the target probe and the control probes in cells,and the generality was verified by transfecting the target probe into different types of cells;Then,the U-2 OS osteosarcoma cells transfected with ASSA target probe were seeded on substrates with different matrix stiffnesses or coated with different materials,and the responses of ASSA probe and their differences were observed;Finally,the si RNA was used to interfere the expression of actopaxin,and the responses of the paxillin stress detection probe DSMP in the cells on substrates coated with different materials were observed,as well as the roles of actopaxin and paxillin in intracellular stress transmission were analyzed.The experimental results show that the ASSA target probe has been successfully constructed.The probe has good localization specificity,multi-cellular versatility and stress sensitivity,and it can realize the visual detection of stress transmission between the cytoskeleton and FAs by observing the change of Fret Ratio;The differences in extracellular matrix(ECM)stiffness and composition led to differences in the magnitude of stress between the cytoskeleton and FAs.On the one hand,the stress decreased with the decrease of the matrix stiffness,but it did not change after the matrix stiffness reached a certain value.On the other hand,the stress between the cytoskeleton and FAs was the smallest under the uncoated condition,the largest under the fibronectin-coated condition,and the middle under the poly-lysine or collagen type I-coated conditions;The difference of ECM composition led to the difference of the force on paxillin.The force on paxillin was smallest under the uncoated and poly-lysine-coated conditions,in the middle under the fibronectin-coated condition,and the largest under the collagen type I-coated condition;Silencing actopaxin had no effect on the force on paxillin under different ECM composition conditions.Based on this,this paper draws the following conclusions.First,the response of the ASSA probe demonstrates a direct stress transfer between the cytoskeleton and FAs;Second,the stiffness and composition of the ECM can regulate the magnitude of the stress between the cytoskeleton and FAs,but the response of the stress between the cytoskeleton and FAs to matrix stiffness is nonlinear;Third,actopaxin is an important mechanotransducer in FAs and may belong to the "mechanotransduction layer" in FAs,which plays a key role in mediating cellular stress transmission and other processes.This paper provides a powerful tool for cell mechanobiology-related research,and also provides a new basis for in-depth understanding of the internal connection between the tumor mechanical behavior signaling pathway and the mechanism of stress transduction and transduction of FAs. |
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