Differential Contributions of Nonmuscle Myosin II Isoforms and Functional Domains to Stress Fiber Mechanics

Abstract

Synaptic stimulation leads to expansion of the dendritic spine caused by rearrangement of the actin cytoskeleton triggered by phosphorylation of the calcium calmodulin dependent kinase (CaMKII) and by myosin II activity. To study CaMKII-actomyosin interactions we have visualized single GFP-tagged CaM-KII holoenzymes bound to tagRFP-tagged cortical actin in human endothelial cells by total internal reflection fluorescence microscopy. CaMKII phosphorylation mutants were used to examine whether release of CaMKII sequestered actin and myosin II induced F-actin fragmentation can explain the timing and extent of spine expansion. Thus far, we have found (i) Single GFP-CaMKII holoenzymes were resolved; in contrast to the less intense and rapidly mobile single GFP molecules. (ii) The holoenzymes have unimodal intensity distributions with < 30% standard deviation. The constitutively-active CaMKII T286D mutant, the sole exception, gave bimodal distributions with the higher mode six times as intense as the lower one. Subunit stoichiometries were determined by comparison against immobilized GFP single molecule intensities. (iii) CaMKII dwell-time distributions on stress fibers and filopodial structures were characterized. Dissociation rates were also measured by evanescent irradiation of photo-activatable GFP-CaMKII constructs bound to cortical actin (iv) The green / red fluorescence ratio of the actin structures relative to background gave an equilibrium measure of CaMKII binding. Affinity differences for different CaMKII isoforms and different phosphorylation states were estimated from the fluorescence ratios. In addition, single particle tracking was used to measure transient binding to the actin cytoskeleton. (v) We are currently measuring spatial co-operativity in the F-actin CaMKII binding sites and extending the methodology to analyse CaMKII diffusion along dendrites and spines in hippocampal neuron cultures. Our single molecule imaging data reveal spatial and macromolecular heterogeneities that may underlie aspects of the signal transduction response and cytoskeletal dynamics that are important for synaptic plasticity.

Geometry and network connectivity govern the mechanics of stress fibers

“…By contrast, peripherally located SFs such as the ones severed in our experiments are primarily regulated by myosin light chain kinase and are thus expected to be minimally perturbed by ROCK manipulation (17,18). To suppress ROCK activity and dissipate central connections, we treated cells with 5 μM Y-27632.…”

“…We then applied SLA on SFs along the FN-free edge and observed their retraction kinetics. As in our previous studies, we fit each retraction curve to a Kelvin-Voigt viscoelastic cable model described by a time constant (τ), which reflects the SF's effective viscosity/ elasticity ratio, and a plateau retraction distance (L o ), which correlates to the elastic energy dissipated by half of the severed SF (3,17,18) (Fig. 1D) (see SI Text and Fig.…”

“…Immunostaining imaging and SF SLA experiments were performed on a Zeis LSM 510 Meta Confocal microscope equipped with a MaiTai Ti:sapphire femtosecond laser (Spectra Physics) (9,17,18).…”

“…We and others have applied SLA to determine the viscoelastic contraction of sarcomeric structures within SFs (14-16) and tension distribution after SF ablation to focal adhesions (FAs) throughout the cell (9). Moreover, SLA has been used to spatially map SF viscoelastic properties within the cell and associate activities of specific NMMII activators and isoforms to these subpopulations (17,18).…”

Synaptopodin is required for stress fiber and contractomere assembly at the epithelial junction

“…The 3 regimes allow independent regulations and force transmission pathways that are structurally and functionally segregated. Previous studies had shown that myosin IIA structures are primarily controlled by Rho/Rock/MLC whereas myosin IIB structures are mainly regulated by Ca ++ /calmodulin/MLCK (Chang and Kumar, 2015;Kassianidou et al, 2017;Kuragano et al, 2018;Morin et al, 2014;Totsukawa et al, 2004). Phosphorylation studies using purified myosin II indicate that MLCK phosphorylates myosin IIB more efficiently than ROCK, whereas the reverse is true for myosin IIA (Amano et al, 1996;Sandquist et al, 2006).…”