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Reducing Myosin II and ATP-Dependent Mechanical Activity Increases Order and Stability of Intracellular Organelles.

ABSTRACT: Organization of intracellular content is affected by multiple simultaneous processes, including diffusion in a viscoelastic and structured environment, intracellular mechanical work and vibrations. The combined effects of these processes on intracellular organization are complex and remain poorly understood. Here, we studied the organization and dynamics of a free Ca⁺⁺ probe as a small and mobile tracer in live T cells. Ca⁺⁺, highlighted by Fluo-4, is localized in intracellular organelles. Inhibiting intracellular mechanical work by myosin II through blebbistatin treatment increased cellular dis-homogeneity of Ca⁺⁺-rich features in length scale < 1.1 μm. We detected a similar effect in cells imaged by label-free bright-field (BF) microscopy, in mitochondria-highlighted cells and in ATP-depleted cells. Blebbistatin treatment also reduced the dynamics of the Ca⁺⁺-rich features and generated prominent negative temporal correlations in their signals. Following Guggenberger et al. and numerical simulations, we suggest that diffusion in the viscoelastic and confined medium of intracellular organelles may promote spatial dis-homogeneity and stability of their content. This may be revealed only after inhibiting intracellular mechanical work and related cell vibrations. Our described mechanisms may allow the cell to control its organization via balancing its viscoelasticity and mechanical activity, with implications to cell physiology in health and disease.

SUBMITTER: Wohl I

PROVIDER: S-EPMC8508806 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all levels of Ca(2+)-mediated activation in demembranated cardiac muscle. In the current study we hypothesized that cellular [dATP] could be increased by viral-mediated overexpression of the ribonucleotide reductase (Rrm1 and Rrm2) complex, which would increase contractility of adult rat cardiomyocytes. Cell length and ratiometric (Fura2) Ca(2+) fluorescence were monitored by video microscopy. At 0.5Hz stimulation, the extent of shortening was increased ~40% and maximal rate of shortening was increased ~80% in cardiomyocytes overexpressing Rrm1+Rrm2 as compared to non-transduced cardiomyocytes. The maximal rate of relaxation was also increased ~150% with Rrm1+Rrm2 overexpression, resulting in decreased time to 50% relaxation over non-transduced cardiomyocytes. These differences were even more dramatic when compared to cardiomyocytes expressing GFP-only. Interestingly, Rrm1+Rrm2 overexpression had no effect on minimal or maximal intracellular [Ca(2+)], indicating increased contractility is primarily due to increased myofilament activity without altering Ca(2+) release from the sarcoplasmic reticulum. Additionally, functional potentiation was maintained with Rrm1+Rrm2 overexpression as stimulation frequency was increased (1Hz and 2Hz). HPLC analysis indicated cellular [dATP] was increased by approximately 10-fold following transduction, becoming ~1.5% of the adenine nucleotide pool. Furthermore, 2% dATP was sufficient to significantly increase crossbridge binding and contractile force during sub-maximal Ca(2+) activation in demembranated cardiac muscle. These experiments demonstrate the feasibility of directly targeting the actin-myosin chemomechanical crossbridge cycle to enhance cardiac contractility and relaxation without affecting minimal or maximal Ca(2+). This article is part of a Special issue entitled "Possible Editorial".

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Reducing Myosin II and ATP-Dependent Mechanical Activity Increases Order and Stability of Intracellular Organelles.

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