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Introduction: The diffusion of cell components such as proteins is crucial to the function of all living cells. The abundance of macromolecules in cells is likely to cause a state of macromolecular crowding, but its effects on the extent of diffusion remain poorly understood., Methods: Here we investigate the diffusion rate in three distinct locations in mesenchymal cell types, namely the open cytoplasm, the stress fibers in the open cytoplasm, and those below the nucleus using three kinds of biologically inert green fluorescent proteins (GFPs), namely a monomer, dimer, and trimer GFP. Fluorescence correlation spectroscopy (FCS) was used to determine the diffusion coefficients., Results: We show that diffusion tends to be lowered on average in stress fibers and is significantly lower in those located below the nucleus. Our data suggest that the diffusive properties of GFPs, and potentially other molecules as well, are hindered by macromolecular crowding. However, although the size dependence on protein diffusion was also studied for monomer, dimer, and trimer GFPs, there was no significant difference in the diffusion rates among the GFPs of these sizes. These results could be attributed to the lack of significant change in protein size among the selected GFP multimers., Conclusion: The data presented here would provide a basis for better understanding of the complex protein diffusion in the nonuniform cytoplasm, shedding light on cellular responses to mechanical stress, their local mechanical properties, and reduced turnover in senescent cells., Competing Interests: Conflict of interestThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© The Author(s) 2024.)

Actin stress fibers (SFs), a contractile apparatus in nonmuscle cells, possess a contractile unit that is apparently similar to the sarcomere of myofibrils in muscles. The function of SFs has thus often been addressed based on well-characterized properties of muscles. However, unlike the fixed number of myosin molecules in myofibrils, the number of nonmuscle myosin II (NMII) within the contractile sarcomeric unit in SFs is quite low and variable for some reason. Here we address what factors may determine the specific number of NMII in SFs. We suggest with a theoretical model that the number lies just in between the function of SFs for bearing cellular tension under static conditions and for promptly disintegrating upon forced cell shortening. We monitored shortening-induced disintegration of SFs in human osteosarcoma U2OS cells expressing mutants of myosin regulatory light chain that virtually regulates the interaction of NMII with actin filaments, and the behaviors observed were indeed consistent with the theoretical consequences. This situation-specific nature of SFs may allow nonmuscle cells to respond adaptively to mechanical stress to circumvent activation of pro-inflammatory signals as previously indicated, i.e., a behavior distinct from that of muscles that are basically specialized for exhibiting contractile activity.

Mechanisms of the assembly of actin stress fibers (SFs) have been extensively studied, while those of the disassembly-particularly cell shortening-induced ones-remain unclear. Here, we show that SFs have helical structures composed of multi-subbundles, and they tend to be delaminated upon cell shortening. Specifically, we observed with atomic force microscopy delamination of helical SFs into their subbundles. We physically caught individual SFs using a pair of glass needles to observe rotational deformations during stretching as well as ATP-driven active contraction, suggesting that they deform in a manner reflecting their intrinsic helical structure. A minimal analytical model was then developed based on the Frenet-Serret formulas with force-strain measurement data to suggest that helical SFs can be delaminated into the constituent subbundles upon axial shortening. Given that SFs are large molecular clusters that bear cellular tension but must promptly disassemble upon loss of the tension, the resulting increase in their surface area due to the shortening-induced delamination may facilitate interaction with surrounding molecules to aid subsequent disintegration. Thus, our results suggest a new mechanism of the disassembly that occurs only in the specific SFs exposed to forced shortening.