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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.

Stentor coeruleus has long been employed as a model organism to understand unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genes -- many not associated with the oral apparatus -- differentially regulated in phases throughout the regeneration process. We hypothesized that this systemic reorganization and mobilization of cellular resources towards growth of a new oral apparatus will lead to observable changes in movement and behavior corresponding in time to the phases of differential gene expression, thus allowing an assay of functional regeneration based on motility analysis. However, the morphological complexity of Stentor coeruleus meant the development of an assay to capture the statistics and timescale was necessary. Using a custom script to track cells in short videos, we compile statistics over a large population (N(~)100) to show that, upon loss of the OA, Stentor coeruleus initially loses the ability for directed motion, then starting at approximately four hours exhibits a significant drop in speed until approximately eight hours. This assay provides a useful tool for the screening of motility phenotypes, and can be adapted for the investigation of other organisms.

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genes-many not associated with the oral apparatus (OA)-that are differentially regulated in phases throughout the regeneration process. It was hypothesized that this systemic reorganization and mobilization of cellular resources towards growth of a new OA will lead to observable changes in movement and behavior corresponding in time to the phases of differential gene expression. However, the morphological complexity of S. coeruleus necessitated the development of an assay to capture the statistics and timescale. A custom script was used to track cells in short videos, and statistics were compiled over a large population (N ~100). Upon loss of the OA, S. coeruleus initially loses the ability for directed motion; then starting at ~4 h, it exhibits a significant drop in speed until ~8 h. This assay provides a useful tool for the screening of motility phenotypes and can be adapted for the investigation of other organisms.

The phenomenon of ciliary coordination has garnered increasing attention in recent decades and multiple theories have been proposed to explain its occurrence in different biological systems. While hydrodynamic interactions are thought to dictate the large-scale coordinated activity of epithelial cilia for fluid transport, it is rather basal coupling that accounts for synchronous swimming gaits in model microeukaryotes such asChlamydomonas.Unicellular ciliates present a fascinating yet understudied context in which coordination is found to persist in ciliary arrays positioned across millimetre scales on the same cell. Here, we focus on the ciliateStentor coeruleus, chosen for its large size, complex ciliary organization, and capacity for cellular regeneration. These large protists exhibit ciliary differentiation between cortical rows of short body cilia used for swimming, and an anterior ring of longer, fused cilia called the membranellar band (MB). The oral cilia in the MB beat metachronously to produce strong feeding currents. Remarkably, upon injury, the MB can be shed and regenerated de novo. Here, we follow and track this developmental sequence in its entirety to elucidate the emergence of coordinated ciliary beating: from band formation, elongation, curling and final migration towards the cell anterior. We reveal a complex interplay between hydrodynamics and ciliary restructuring inStentor, and highlight for the first time the importance of a ring-like topology for achieving long-range metachronism in ciliated structures.This article is part of the Theo Murphy meeting issue ‘Unity and diversity of cilia in locomotion and transport’.

Quantum dots are fluorescent nanoparticles with narrow-band, size-tunable, and long-lasting emission. Typical formulations used for imaging proteins in cells are hydrodynamically much larger than the protein targets, so it is critical to assess the impact of steric effects deriving from hydrodynamic size. This report analyzes a new class of quantum dots that have been engineered for minimized size specifically for imaging receptors in narrow synaptic junctions between neurons. We use fluorescence correlation spectroscopy and transmission electron microscopy to calculate the contributions of the crystalline core, organic coating, and targeting proteins (streptavidin) to the total hydrodynamic diameter of the probe, using a wide range of core materials with emission spanning 545–705 nm. We find the contributing thickness of standard commercial amphiphilic polymers to be ~8 to ~14 nm, whereas coatings based on the compact ligand HS-(CH(2))(11)(−) (OCH(2)CH(2))(4)-OH contribute ~6 to ~9 nm, reducing the diameter by ~2 to ~5 nm, depending on core size. When the number of streptavidins for protein targeting is minimized, the total diameter can be further reduced by ~5 to ~11 nm, yielding a diameter of 13.8–18.4 nm. These findings explain why access to the narrow synapse derive primarily from the protein functionalization of commercial variants, rather than the organic coating layers. They also explain why those quantum dots with size around 14 nm with only a few streptavidins can access narrow cellular structures for neuronal labeling, whereas those >27 nm and a large number of streptavidins, cannot.

SummaryThe phenomenon of ciliary coordination has garnered increasing attention in recent decades, with multiple theories accounting for its emergence in different contexts. The heterotrich ciliateStentor coeruleusis a unicellular organism which boasts a number of features which present unrivalled opportunities for biophysical studies of cilia coordination. With their cerulean colour and distinctive morphology, these large protists possess a characteristic differentiation between cortical rows of short body cilia used for swimming, and an anterior ring structure of fused oral cilia forming a membranellar band. The oral cilia beat metachronously to produce strong feeding currents. In addition to this complex body plan,Stentorhave remarkable regenerative capabilities. Minute fragments of single cells can over the period of hours or days, regenerate independently into new, proportional individuals. Certain environmental perturbations elicit a unique programmed response known as oral regeneration wherein only the membranellar band is shed and a new, ciliated oral primordium formed on the side of the body. Here, we target oral regeneration induced by sucrose-shock to reveal the complex interplay between ciliary restructuring and hydrodynamics inStentor, which accompanies the complete developmental sequence from band formation, elongation, curling, and migration toward the cell anterior.“When the anterior part is open, one may perceive about its Edges a very lively Motion; and when the Polyps presents itself in a certain manner, it discovers, on either side of these edges of its anterior part, somewhat very much resembling the wheels of a little Mill, that move with great velocity.”A. Trembley F.R.S describing the membranellar band of Stentor, Phil. Soc. Trans. Royal Society (London), 1744.

As resolving power targets have increased with each generation of beamlines commissioned in synchrotron radiation facilities worldwide, diffraction gratings are quickly becoming crucial optical components for meeting performance targets. However, the metrology of variable-line-spacing (VLS) gratings for high resolution beamlines is not widespread; in particular, no metrology facility at any US DOE facility is currently equipped to fully characterize such gratings. To begin to address this issue, the Optics Group at the Advanced Photon Source at Argonne, in collaboration with SOLEIL and with support from Brookhaven National Laboratory (BNL), has developed an alternative beam path addition to the Long Trace Profiler (LTP) at Argonne’s Advanced Photon Source. This significantly expands the functionality of the LTP not only to measure mirrors surface slope profile at normal incidence, but also to characterize the groove density of VLS diffraction gratings in the Littrow incidence up to 79°, which covers virtually all diffraction gratings used at synchrotrons in the first order. The LTP light source is a 20mW HeNe laser, which yields enough signal for diffraction measurements to be performed on low angle blazed gratings optimized for soft X-ray wavelengths. We will present the design of the beam path, technical requirements for the optomechanics, and our data analysis procedure. Finally, we discuss challenges still to be overcome and potential limitations with use of the LTP to perform metrology on diffraction gratings.

We introduce a method for using Fizeau interferometry to measure the intrinsic resolving power of a diffraction grating. This method is more accurate than traditional techniques based on a long-trace profiler (LTP), since it is sensitive to long-distance phase errors not revealed by a d-spacing map. We demonstrate 50,400 resolving power for a mechanically ruled XUV grating from Inprentus, Inc., 12 pages, 6 figures

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.

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (>200 µm), localization precision of 2-3 nm can be achieved.

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a "double decker" microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

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)/O-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.

In neurons, microtubule motor driven transport is crucial for communication between processes and the cell body. Disruptions in transport are associated with a variety of neurodegenerative diseases. Previous studies implicate phosphorylation of serine 176 in kinesin-1 in the impaired axonal transport associated with Huntington's disease. In isolated squid axoplasm, introduction of pathogenic huntingtin protein activates the kinase, JNK3, which specifically phosphorylates kinesin at S176 (Morfini, Nature Neuroscience, 2009). The mechanism by which S176 modification leads to impaired transport is not very well understood. It is not known whether phosphorylation of kinesin alone is sufficient to cause impaired cargo transport. To investigate the effect of residue 176 on kinesin transport, we use optical trapping and single-molecule fluorescence imaging to study purified kinesin. We employ two constructs, S176A and S176D, truncated at residue 888 to remove the autoinhibition domain, resulting in constitutively active motors. There is no significant difference in the processivity, ATPase, or microtubule binding activity between the phosphomimetic S176D construct and the non-phosphorylatable S176A construct. However, we find that S176D has an attenuated stall force (5pN) compared to S176A (7 pN). Furthermore, polystyrene bead cargos coated with dynein and S176D are transported preferentially in the minus direction in comparison with cargos coated with equivalent concentrations of dynein and S176A. We also perform phosphorylation assays using JNK3 on both 888-truncated and full-length wild-type kinesin in which radiolabeling is used to quantify the percentage of protein phosphorylated in the assay. The pattern of stall force attenuation and directional bias observed for the S176D mutant is also observed in JNK3 phosphorylated samples in which 60-80% of the wild type protein has been phosphorylated. These results show that modification of serine 176 alone is sufficient to alter the behavior of kinesin.

Myosin Va is a motor protein responsible for vesicular transport inside eukaryotic cells. Its structure has been well-studied and is known to contain a flexible hinge region approximately half-way between the N-terminal motor domain and C-terminal globular tail. Previous studies have shown two distinct conformations for Myosin Va, one where the hinge remains extended and the protein is active, and one where the hinge allows the protein to fold, allowing auto-inhibition between the globular tail and motor regions. It is assumed, but never shown at the single-molecule level, that Myosin Va actively switches between these conformations at the timescales typical of their runs. Traditional two-dimensional fluorescence tracking techniques cannot adequately capture the conformation change. However, it is possible to track an out-of-focus particle in all three dimensions with a modified form of FIONA microscopy. The point spread function of the out-of-focus particle takes the shape of an Airy disc, with the width of the disc proportional to the distance by which the particle is out of focus. With this technique, the spatial resolution degrades quickly with drift, so we have further modified the microscope with an auto-focus system based on feedback of backscattered signal from a secondary IR laser. We present tracking data of quantum dots bound to full-length Myosin Va with < 5 nm spatial resolution in XY, < 30 nm in Z at a time resolution of 100 ms, which is sufficient to resolve conformation changes.