Visualizing Cytoplasmic Flow During Single-cell Wound Healing in <em>Stentor coeruleus</em>

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

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 ...

MARINE BIOLOGICAL LABORATORY

University of Illinois Urbana-Champaign

University of Illinois at Urbana-Champaign

Scripps College

Claremont McKenna College

Pitzer College

Traditional microscopy techniques are limited by the wave-like characteristics of light, which dictate that about 250 nm (or roughly half the wavelength of the light) is the smallest distance by which two identical objects can be separated while still being able to distinguish between them. Since most biological molecules are much smaller than this limit, traditional light microscopes are generally not sufficient for single-molecule biological studies. Fluorescence Imaging with One Nanometer Accuracy (FIONA) is a technique that makes possible localization of an object to approximately one nanometer. The FIONA technique is simple in concept; it is built upon the idea that, if enough photons are collected, one can find the exact center of a fluorophore's emission to within a single nanometer and track its motion with a very high level of precision. The center can be localized to approximately (λ/2)/Ö-N, where λ is the wavelength of the light and N is the number of photons collected. When N  =  10,000, FIONA achieves an accuracy of 1-2 nm, assuming the background is sufficiently low. FIONA, thus, works best with the use of high-quality dyes and fluorescence stabilization buffers, sensitive detection methods, and special microscopy techniques to reduce background fluorescence. FIONA is particularly well suited to the study of molecular motors, which are enzymes that couple ATP hydrolysis to conformational change and motion. In this chapter, we discuss the practical application of FIONA to molecular motors or other enzymes in biological systems.

Fluorescence imaging with one nanometer accuracy: in vitro and in vivo studies of molecular motors.

Disruptions in microtubule motor transport are associated with a variety of neurodegenerative diseases. Post-translational modification of the cargo-binding domain of the light and heavy chains of kinesin has been shown to regulate transport, but less is known about how modifications of the motor domain affect transport. Here we report on the effects of phosphorylation of a mammalian kinesin motor domain by the kinase JNK3 at a conserved serine residue (Ser-175 in the B isoform and Ser-176 in the A and C isoforms). Phosphorylation of this residue has been implicated in Huntington disease, but the mechanism by which Ser-175 phosphorylation affects transport is unclear. The ATPase, microtubule-binding affinity, and processivity are unchanged between a phosphomimetic S175D and a nonphosphorylatable S175A construct. However, we find that application of force differentiates between the two. Placement of negative charge at Ser-175, through phosphorylation or mutation, leads to a lower stall force and decreased velocity under a load of 1 piconewton or greater. Sedimentation velocity experiments also show that addition of a negative charge at Ser-175 favors the autoinhibited conformation of kinesin. These observations imply that when cargo is transported by both dynein and phosphorylated kinesin, a common occurrence in the cell, there may be a bias that favors motion toward the minus-end of microtubules. Such bias could be used to tune transport in healthy cells when properly regulated but contribute to a disease state when misregulated.

Motor domain phosphorylation modulates kinesin-1 transport.

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.

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

The cytoskeleton-a composite network of biopolymers, molecular motors, and associated binding proteins-is a paradigmatic example of active matter. Particle transport through the cytoskeleton can range from anomalous and heterogeneous subdiffusion to superdiffusion and advection. Yet, recapitulating and understanding these properties-ubiquitous to the cytoskeleton and other out-of-equilibrium soft matter systems-remains challenging. Here, we combine light sheet microscopy with differential dynamic microscopy and single-particle tracking to elucidate anomalous and advective transport in actomyosin-microtubule composites. We show that particles exhibit multi-mode transport that transitions from pronounced subdiffusion to superdiffusion at tunable crossover timescales. Surprisingly, while higher actomyosin content increases the range of timescales over which transport is superdiffusive, it also markedly increases the degree of subdiffusion at short timescales and generally slows transport. Corresponding displacement distributions display unique combinations of non-Gaussianity, asymmetry, and non-zero modes, indicative of directed advection coupled with caged diffusion and hopping. At larger spatiotemporal scales, particles in active composites exhibit superdiffusive dynamics with scaling exponents that are robust to changing actomyosin fractions, in contrast to normal, yet faster, diffusion in networks without actomyosin. Our specific results shed important new light on the interplay between non-equilibrium processes, crowding and heterogeneity in active cytoskeletal systems. More generally, our approach is broadly applicable to active matter systems to elucidate transport and dynamics across scales.

Motor-driven advection competes with crowding to drive spatiotemporally heterogeneous transport in cytoskeleton composites.

Janet Sheung

Physiology Course

MARINE BIOLOGICAL LABORATORY

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

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

Janet Sheung

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Physiology Course

MARINE BIOLOGICAL LABORATORY

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

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

Physiology Course