Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks.

Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the dynamics of these 3D, dense networks calls for optical sectioning, which is often associated with fluorescence confocal microscopes. However, recent developments in light-sheet fluorescence microscopy (LSFM) have established it as a cost-effective and, at times, superior alternative. To make LSFM accessible to cytoskeleton researchers less familiar with optics, we present a step-by-step beginner's guide to building a versatile light-sheet fluorescence microscope from off-the-shelf components. To enable sample mounting with traditional slide samples, this LSFM follows the single-objective light-sheet (SOLS) design, which utilizes a single objective for both the excitation and emission collection. We describe the function of each component of the SOLS in sufficient detail to allow readers to modify the instrumentation and design it to fit their specific needs. Finally, we demonstrate the use of this custom SOLS instrument by visualizing asters in kinesin-driven microtubule networks.

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics.

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces using motor proteins such as myosin II and kinesin to drive key processes such as migration, cytokinesis, adhesion, and mechanosensing. While actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay with myosin and kinesin activity is still nascent. This work describes how to engineer tunable three-dimensional composite networks of co-entangled actin filaments and microtubules that undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers. Protocols for fluorescence labeling of the microtubules and actin filaments to most effectively visualize composite restructuring and motion using multi-spectral confocal imaging are also detailed. Finally, the results of data analysis methods that can be used to quantitatively characterize non-equilibrium structure, dynamics, and mechanics are presented. Recreating and investigating this tunable biomimetic platform provides valuable insight into how coupled motor activity, composite mechanics, and filament dynamics can lead to myriad cellular processes from mitosis to polarization to mechano-sensation.

Motor-Driven Restructuring of Cytoskeleton Composites Leads to Tunable Time-Varying Elasticity.

The composite cytoskeleton, comprising interacting networks of semiflexible actin and rigid microtubules, generates forces and restructures by using motor proteins such as myosins to enable key processes including cell motility and mitosis. Yet, how motor-driven activity alters the mechanics of cytoskeleton composites remains an open challenge. Here, we perform optical tweezers microrheology and confocal imaging of composites with varying actin-tubulin molar percentages (25-75, 50-50, and 75-25), driven by light-activated myosin II motors, to show that motor activity increases the elastic plateau modulus by over 2 orders of magnitude by active restructuring of both actin and microtubules that persists for hours after motor activation has ceased. Nonlinear microrheology measurements show that motor-driven restructuring increases the force response and stiffness and suppresses actin bending. The 50-50 composite exhibits the most dramatic mechanical response to motor activity due to the synergistic effects of added stiffness from the microtubules and sufficient motor substrate for pronounced activity.