Umeda, K. | bioRxiv Science

bioRxiv2023

Evolutionarily acquired activity-dependent transformation of the CaMKII holoenzyme

Ca2+/calmodulin-dependent protein kinase II (CaMKII) has long been central in synaptic plasticity research. CaMKII is a dodecameric serine/threonine kinase that has been essentially conserved across metazoans for over a million years. While the mechanisms of CaMKII activation are well studied, its "behavior" at the molecular level has remained unobserved. Here, high-speed atomic force microscopy was used to visualize the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII in various states at nanometer resolution. Among the species, rat CaMKII underwent internal kinase domain aggregation in an activity-dependent manner and showed a higher tolerance to dephosphorylation by phosphatase. Our findings suggest that mammalian CaMKII has evolutionarily acquired a new structural form and a tolerance to phosphatase to maintain robust CaMKII activity for proper neuronal function. One-Sentence SummaryHigh-speed atomic force microscopy reveals the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII

neuroscience↗

bioRxiv2022

Activity-dependent glassy cell mechanics : Mechanical properties measured with active microrheology

Active microrheology was conducted in living cells by applying an optical-trapping force to vigorously-fluctuating tracer beads with feedback-tracking technology. The complex shear viscoelastic modulus G({omega}) = G'({omega}) - iG''({omega}) was measured in HeLa cells in an epithelial-like confluent monolayer. We found that G({omega}) {propto} (-i{omega})1/2 over a wide range of frequencies (1 Hz <{omega} /2{pi}<10 kHz). Actin disruption and cell-cycle progression from G1 to S and G2 phases only had a limited effect on G({omega}) in living cells. On the other hand, G ({omega}) was found to be dependent on cell metabolism; ATP-depleted cells showed an increased elastic modulus G'({omega}) at low frequencies, giving rise to a constant plateau such that G({omega}) = G0 + A(-i{omega})1/2. Both the plateau and the additional frequency dependency {propto} (-i{omega})1/2 of ATP-depleted cells are consistent with a rheological response typical of colloidal jamming. On the other hand, the plateau G0 disappeared in ordinary metabolically active cells, implying that living cells fluidize their internal states such that they approach the critical jamming point. Statement of SignificanceIntracellular mechanical properties were measured using optical-trap-based microrheology. Despite expectations to the contrary, shear viscoelasticity was hardly affected by reorganization of cytoskeletal structures during cell-cycle progression (G1 to S and G2 phases), nor by artificial disruption of the actin cytoskeleton induced by chemical inhibitors. Rather, the mechanics of cell interiors is governed by the glassy cytoplasm. Cells depleted of ATP solidified, whereas living cells that maintained metabolic activities were more fluid-like. Instead of a completely fluid response, however, we observed a characteristic power-law viscoelasticity G({omega}) {propto} (-i{omega})1/2 over the whole range of frequencies measured. Based on our current understanding of jamming rheology, we discuss how cells fluidize their internal state in a way that pushes the system towards the critical jamming transition.

biophysics↗