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Final Report Summary - BIOMIMETIC-MECHANICS (The mechanics and transport of the active cytoskeleton in biomimetic and living cellular systems)

Intracellular transport involves movement of molecules and organelles through the subcellular environment and is critical for proper cell function. It can be driven by molecular motors or by cytoskeletal fluctuations and flow of the cytoplasm. Transport plays an integral role in many cellular functions, including migration and division, which are intimately linked to the metastatic spread of cancer. Transport of intracellular components requires interaction with the cytoskeleton in the bulk cytoplasm as well as the cell cortex. Cells also respond to their surrounding physical environment by changing shape and reorganizing their internal structures via passive and active processes. Thus, the interplay between the cell cortex and the bulk cytoskeleton is a key factor in understanding intracellular transport. However, the cytoskeleton-cortex interaction and its role in cell mechanics is not well understood.
The objective of the project was to characterize the mechanics of the cytoskeleton in biomimetic and living systems. We focused on three main questions: (1) What are the mechanical properties of the actin cytoskeleton under geometric confinement? (2) What is the role of active processes in structural reorganization? (3) How does the active reorganization of a living cell contribute to intracellular transport? The structural reorganization of the actin cytoskeleton was measured via confocal imaging and subcellular transport or flow was quantified by single particle tracking of tracers/organelles. The mechanics of the active cytoskeleton was measured using active microrheology and fluctuation spectroscopy. This study investigated how the active cytoskeleton dictates cell mechanics and its contribution to intracellular transport. The outcomes of this project will lead to a better understanding of how mechanics affects cell function (e.g. oocyte development) as reported in several publications [1-4].
Since the beginning of the project the work performed includes: the development of a theoretical and experimental framework to characterize nonequilibrium activity in living cells and other biomimetic systems; the development of protocols for optical tweezer measurements in biomimetic systems and living cells; force measurement experiments on biomimetic systems, mouse oocytes, and other living cells; and extensive training for Dr. Ahmed in physical, biological, and chemical techniques relevant to the project.
The main results achieved during this project, as reported in publications, are as follows:
1. In somatic cells, the position of the cell centroid is dictated by the centrosome. The centrosome is instrumental in nucleus positioning, the two structures being physically connected. Mouse oocytes have no centrosomes, yet harbour centrally located nuclei. We demonstrate how oocytes define their geometric centre in the absence of centrosomes. Using live imaging of oocytes, knockout for the formin 2 actin nucleator, with off-centred nuclei, together with optical trapping and modelling, we discover an unprecedented mode of nucleus positioning. We document how active diffusion of actin-coated vesicles, driven by myosin Vb, generates a pressure gradient and a propulsion force sufficient to move the oocyte nucleus. It promotes fluidization of the cytoplasm, contributing to nucleus directional movement towards the centre. Our results highlight the potential of active diffusion, a prominent source of intracellular transport, able to move large organelles such as nuclei, providing in vivo evidence of its biological function. [1]
2. Living cells are active mechanical systems that are able to generate forces. Their structure and shape are primarily determined by biopolymer filaments and molecular motors that form the cytoskeleton. Active force generation requires constant consumption of energy to maintain the nonequilibrium activity to drive organization and transport processes necessary for their function. To understand this activity it is necessary to develop new approaches to probe the underlying physical processes. Active cell mechanics incorporates active molecular-scale force generation into the traditional framework of mechanics of materials. This review highlights recent experimental and theoretical developments towards understanding active cell mechanics. We focus primarily on intracellular mechanical measurements and theoretical advances utilizing the Langevin framework. These developing approaches allow a quantitative understanding of nonequilibrium mechanical activity in living cells. [2]
3. Active diffusion of intracellular components is emerging as an important process in cell biology. This process is mediated by complex assemblies of molecular motors and cytoskeletal filaments that drive force generation in the cytoplasm and facilitate enhanced motion. The kinetics of molecular motors have been precisely characterized in-vitro by single molecule approaches, however, their in-vivo behavior has remained elusive. Here, we study the myosin-V driven active diffusion of vesicles in mouse oocytes, where this process plays a key role in nuclear positioning during development, and combine an experimental and theoretical framework to extract molecular-scale force kinetics in-vivo (motor force, power-stroke, and velocity). We find that myosin-V induces rapid kicks of duration τ~300 μs resulting in an average force of F~0.4 pN on vesicles. Our results reveal that measuring in-vivo active fluctuations allows extraction of the underlying molecular motor activity and demonstrates a widely applicable mesoscopic framework to access molecular-scale force kinetics. [3]
4. Living organisms are inherently out-of-equilibrium systems. We employ recent developments in stochastic energetics and rely on a minimal microscopic model to predict the amount of mechanical energy dissipated by such dynamics. Our model includes complex rheological effects and nonequilibrium stochastic forces. By performing active microrheology and tracking micron-sized vesicles in the cytoplasm of living oocytes, we provide unprecedented measurements of the spectrum of dissipated energy. We show that our model is fully consistent with the experimental data, and we use it to offer predictions for the injection and dissipation energy scales involved in active fluctuations. [4]

References: (* equal first-author contribution)
[1] *Almonacid, M. and *Ahmed. W. et al. Active diffusion positions the nucleus in mouse oocytes. Nature Cell Biology 17, 470–479 (2015).
[2] Ahmed, W., Fodor, E. & Betz, T. Active cell mechanics: Measurement and theory. Biochim Biophys Acta 1853, 3083–3094 (2015).
[3] *Ahmed, W., *Fodor, E., *Almonacid, M. et al. Active mechanics reveal molecular-scale force kinetics in living oocytes., (2016) (under review at Biophysical Journal).
[4] *Fodor, E., *Ahmed, W., *Almonacid, M. et al. Nonequilibrium dissipation in living oocytes. Europhysics Letters (2016) (accepted).

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