Pocket CLARITY enables distortion-mitigated cardiac microstructural tissue characterization of large-scale specimens

Molecular phenotyping by imaging of intact tissues has been used to reveal 3D molecular and structural coherence in tissue samples using tissue clearing techniques. However, clearing and imaging of cardiac tissue remains challenging for large-scale (>100 mm(3)) specimens due to sample distortion. Thus, directly assessing tissue microstructural geometric properties confounded by distortion such as cardiac helicity has been limited. To combat sample distortion, we developed a passive CLARITY technique (Pocket CLARITY) that utilizes a permeable cotton mesh pocket to encapsulate the sample to clear large-scale cardiac swine samples with minimal tissue deformation and protein loss. Combined with light sheet auto-fluorescent and scattering microscopy, Pocket CLARITY enabled the characterization of myocardial microstructural helicity of cardiac tissue from control, heart failure, and myocardial infarction in swine. Pocket CLARITY revealed with high fidelity that transmural microstructural helicity of the heart is significantly depressed in cardiovascular disease (CVD), thereby revealing new insights at the tissue level associated with impaired cardiac function

High Resolution Optical Tweezers for Biological Studies

In the past decades, numerous single-molecule techniques have been developed to investigate individual bio-molecules and cellular machines. While a lot is known about the structure, localization, and interaction partners of such molecules, much less is known about their mechanical properties. To investigate the weak, non-covalent interactions that give rise to the mechanics of and between proteins, an instrument capable of resolving sub-nanometer displacements and piconewton forces is necessary. One of the most prominent biophysical tool with such capabilities is an optical tweezers. Optical tweezers is a non-invasive all-optical technique in which typically a dielectric microsphere is held by a tightly focused laser beam. This microsphere acts like a microscopic, three-dimensional spring and is used as a handle to study the biological molecule of interest. By interferometric detection methods, the resolution of optical tweezers can be in the picometer range on millisecond time scales. However, on a time scale of seconds—at which many biological reactions take place—instrumental noise such as thermal drift often limits the resolution to a few nanometers. Such a resolution is insufficient to resolve, for example, the ångstrom-level, stepwise translocation of DNA-binding enzymes corresponding to distances between single basepairs of their substrate. To reduce drift and noise, differential measurements, feedback-based drift stabilization techniques, and ‘levitated’ experiments have been developed. Such methods have the drawback of complicated and expensive experimental equipment often coupled to a reduced throughput of experiments due to a complex and serial assembly of the molecular components of the experiments. We developed a high-resolution optical tweezers apparatus capable of resolving distances on the ångstrom-level over a time range of milliseconds to 10s of seconds in surface-coupled assays. Surface-coupled assays allow for a higher throughput because the molecular components are assembled in a parallel fashion on many probes. The high resolution was a collective result of a number of simple, easy-to-implement, and cost-efficient noise reduction solutions. In particular, we reduced thermal drift by implementing a temperature feedback system with millikelvin precision—a convenient solution for biological experiments since it minimizes drift in addition to enabling the control and stabilization of the experiment’s temperature. Furthermore, we found that expanding the laser beam to a size smaller than the objective’s exit pupil optimized the amount of laser power utilized in generating the trapping forces. With lower powers, biological samples are less susceptible to photo-damage or, vice versa, with the same laser power, higher trapping forces can be achieved. With motorized and automated procedures, our instrument is optimized for high-resolution, high-throughput surface-coupled experiments probing the mechanics of individual biomolecules. In the future, the combination of this setup with single-molecule fluorescence, super-resolution microscopy or torque detection will open up new possibilities for investigating the nanomechanics of biomolecules

Processivity of molecular motors under vectorial loads

Molecular motors are cellular machines that drive the spatial organization of the cells by transporting cargos along intracellular filaments. Although the mechanical properties of single molecular motors are relatively well characterized, it remains elusive how the geometry of a load imposed on a motor affects its processivity, i.e., the average distance that a motor moves per interaction with a filament. Here, we theoretically explore this question for a single-kinesin molecular motor by analyzing the load dependence of the stepping and detachment processes. We find that the processivity of the kinesin increases with lowering the load angle between the kinesin and the microtubule filament, due to the deceleration of the detachment rate. When the load angle is large, the processivity is predicted to enhance with accelerating the stepping rate through an optimal distribution of the load over the kinetic transition rates underlying a mechanical step of the motor. These results provide new insights into understanding of the design of potential synthetic biomolecular machines that can travel long distances with high velocities

The Kinetics of Nucleotide Binding to Isolated Chlamydomonas Axonemes Using UV-TIRF Microscopy

Cilia and flagella are long, slender organelles found in many eukaryotic cells, where they have sensory, developmental, and motile functions. All cilia and flagella contain a microtubule-based structure called the axoneme. In motile cilia and flagella, which drive cell locomotion and fluid transport, the axoneme contains, along most of its length, motor proteins from the axonemal dynein family. These motor proteins drive motility by using energy derived from the hydrolysis of ATP to generate a bending wave, which travels down the axoneme. As a first step toward visualizing the ATPase activity of the axonemal dyneins during bending, we have investigated the kinetics of nucleotide binding to axonemes. Using a specially built ultraviolet total internal reflection fluorescence microscope, we found that the fluorescent ATP analog methylanthraniloyl ATP (mantATP), which has been shown to support axonemal motility, binds all along isolated, immobilized axonemes. By studying the recovery of fluorescence after photobleaching, we found that there are three mantATP binding sites: one that bleaches rapidly (time constant ≈ 1.7 s) and recovers slowly (time constant ≈ 44 s), one that bleaches with the same time constant but does not recover, and one that does not bleach. By reducing the dynein content in the axoneme using mutants and salt extraction, we provide evidence that the slow-recovering component, but not the other components, corresponds to axonemal dyneins. The recovery rate of this component, however, is too slow to be consistent with the activation of beating observed at higher mantATP concentrations; this indicates that the dyneins may be inhibited due to their immobilization at the surface. The development of this method is a first step toward direct observation of the traveling wave of dynein activity.ISSN:0006-3495ISSN:1542-008

Nicotinamide adenine dinucleotides and their precursor NMN have no direct effect on microtubule dynamics in purified brain tubulin.

Microtubules are dynamic cytoskeletal polymers that provide mechanical support for cellular structures, and play important roles in cell division, migration, and intracellular transport. Their intrinsic dynamic instability, primarily controlled by polymerization-dependent GTP hydrolysis, allows for rapid rearrangements of microtubule arrays in response to signaling cues. In neurons, increases in intracellular levels of nicotinamide adenine dinucleotide (NAD+) can protect against microtubule loss and axonal degeneration elicited by axonal transection. The protective effects of NAD+ on microtubule loss have been shown to be indirect in some systems, for example through the sirtuin-3 pathway. However, it is still possible that NAD+ and related metabolites have direct effects on microtubule dynamics to promote assembly or inhibit disassembly. To address this question, we reconstituted microtubule dynamics in an in vitro assay with purified bovine brain tubulin and examined the effects of NAD+, NADH, and NMN. We found that the compounds had only small effects on the dynamics at the plus and minus ends of the microtubules. Furthermore, these effects were not statistically significant. Consequently, our data support earlier findings that NADs and their precursors influence microtubule growth through indirect mechanisms

Under-filling trapping objectives optimizes the use of the available laser power in optical tweezers.

For optical tweezers, especially when used in biological studies, optimizing the trapping efficiency reduces photo damage or enables the generation of larger trapping forces. One important, yet not-well understood, tuning parameter is how much the laser beam needs to be expanded before coupling it into the trapping objective. Here, we measured the trap stiffness for 0.5-2 μm-diameter microspheres for various beam expansions. We show that the highest overall trapping efficiency is achieved by slightly under-filling a high-numerical aperture objective when using microspheres with a diameter corresponding to about the trapping-laser wavelength in the medium. The optimal filling ratio for the lateral direction depended on the microsphere size, whereas for the axial direction it was nearly independent. Our findings are in agreement with Mie theory calculations and suggest that apart from the choice of the optimal microsphere size, slightly under-filling the objective is key for the optimal performance of an optical trap

An AAV capsid increases transduction of striatum and a ChAT promoter allows selective cholinergic neuron transduction

Adeno-associated virus (AAV) vectors are currently the most efficient option for intracranial gene therapies to treat neurodegenerative disease. Increased efficacy and safety will depend upon robust and specific expression of therapeutic genes into target cell-types within the human brain. In this study, we set out with two objectives: (1) to identify capsids with broader transduction of the striatum upon intracranial injection in mice and (2) to test a truncated human choline acetyltransferase (ChAT) promoter that would allow efficient and selective transduction of cholinergic neurons. We compared AAV9 and an engineered capsid, AAV-S, to mediate widespread reporter gene expression throughout the striatum. We observed that AAV-S transduced a significantly greater area of the injected hemisphere primarily in the rostral direction compared with AAV9 (CAG promoter). We tested AAV9 vectors packaging a reporter gene expression cassette driven by either the ChAT or CAG promoter. Specificity of transgene expression of ChAT neurons over other cells was 7-fold higher, and efficiency was 3-fold higher for the ChAT promoter compared with the CAG promoter. The AAV-ChAT transgene expression cassette should be a useful tool for the study of cholinergic neurons in mice, and the broader transduction area of AAV-S warrants further evaluation of this capsid