Kazushi Ito

Actin stress fibers are at a tipping point between conventional shortening and rapid disassembly at physiological levels of MgATP

Stress fibers (SFs) composed of nonmuscle actin and myosin II play critical roles in various cellular functions such as structural remodeling in response to changes in cell stress or strain. Previous studies report that SFs rapidly disassemble upon loss of tension caused by reduced myosin activity or sudden cell shortening, but the mechanism is unclear. Here, we showed that Rho-kinase inhibition with Y-27632 led to detachment of intact actin filaments from the SFs rather than depolymerization. Loss of tension may allow SFs to shorten via MgATP-driven cross-bridge cycling, thus we investigated the effects of MgATP concentration on SF shortening and stability. We performed the experiments using extracted SFs to allow control over MgATP concentration. SF contraction and disassembly rates each increased with increasing MgATP concentration. SFs transitioned from conventional SF shortening to rapid disassembly as MgATP concentration increases from 2 to 5mM, which is within the physiological range of intracellular MgATP concentrations. Thus, we submit that SFs in intact cells are inherently on the verge of disassembly, which is likely due to the small number of actomyosin cross-bridges in SFs compared to those found in relatively stable myofibrils. Given that recent studies have revealed that loss of resistive force against myosin II could lower the fraction of the MgATPase cycle time that the myosin head is attached to actin (i.e., the duty ratio), binding of cytoplasmic levels of MgATP to myosin II may be sufficient to cause the disassembly of unloaded SFs. The present study thus describes a putative mechanism for rapid SF disassembly caused by decreased myosin activity or sudden cell shortening.

Synthesis of mesoporous silica with different pore sizes for cellulase immobilization: pure physical adsorption

To discuss the physical adsorption mechanism of the adsorption process of cellulase, a commercial enzyme cocktail sourced from Acremonium was immobilized in mesoporous silica with various pore sizes by pure physical adsorption in this study. Mesoporous silica materials with 17.6 nm and 3.8 nm pore sizes (hereafter referred to as MS-17.6 nm and MS-3.8 nm, respectively) were synthesized in the manner of a seeded-growth method. Other available mesoporous silica materials denoted as H-32 and diatomite were also used as sorbents. Then, the sorbents were characterized via small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), the Barrett–Emmett–Teller (BET) method and the Barrett–Joyner–Halenda (BJH) method to confirm their mesostructure. Furthermore, the adsorption abilities of different sorbents and enzymatic activities of immobilized cellulase were studied. The adsorption amounts exhibited a clear correlation with the pore size of the sorbents; i.e., the adsorption amount of MS-17.6 nm (410 mg g−1) with the pore size similar to the long axes of cellulase molecules was higher than that of MS-3.8 nm (315 mg g−1) with the pore size approximated to the short axes of cellulase (which was realized at 50 °C). Besides, the adsorption behavior of diatomite (with a pore size of about 200 nm) revealed a periodicity because the pore size was significantly larger than cellulase molecules. Moreover, the pore size was suggested to be a critical factor for the enzymatic activity of cellulase. When the average pore size of MS-3.8 nm just matched the short axes of cellulase molecules, immobilized cellulase preserved the active sites of cellulase intactly and showed the best activity (i.e. 63.3% of free cellulase activity at 50 °C). Consequently, the pore size of the sorbents had a significant influence on cellulase immobilization.