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.

Systematic identification of anticancer drug targets reveals a nucleus-to-mitochondria ROS-sensing pathway.

Multiple anticancer drugs have been proposed to cause cell death, in part, by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs, exactly how the resultant ROS function and are sensed is poorly understood. It remains unclear which proteins the ROS modify and their roles in drug sensitivity/resistance. To answer these questions, we examined 11 anticancer drugs with an integrated proteogenomic approach identifying not only many unique targets but also shared ones-including ribosomal components, suggesting common mechanisms by which drugs regulate translation. We focus on CHK1 that we find is a nuclear H2O2 sensor that launches a cellular program to dampen ROS. CHK1 phosphorylates the mitochondrial DNA-binding protein SSBP1 to prevent its mitochondrial localization, which in turn decreases nuclear H2O2. Our results reveal a druggable nucleus-to-mitochondria ROS-sensing pathway-required to resolve nuclear H2O2 accumulation and mediate resistance to platinum-based agents in ovarian cancers.

Cilia function as calcium-mediated mechanosensors that instruct left-right asymmetry.

The breaking of bilateral symmetry in most vertebrates is critically dependent upon the motile cilia of the embryonic left-right organizer (LRO), which generate a directional fluid flow; however, it remains unclear how this flow is sensed. Here, we demonstrated that immotile LRO cilia are mechanosensors for shear force using a methodological pipeline that combines optical tweezers, light sheet microscopy, and deep learning to permit in vivo analyses in zebrafish. Mechanical manipulation of immotile LRO cilia activated intraciliary calcium transients that required the cation channel Polycystin-2. Furthermore, mechanical force applied to LRO cilia was sufficient to rescue and reverse cardiac situs in zebrafish that lack motile cilia. Thus, LRO cilia are mechanosensitive cellular levers that convert biomechanical forces into calcium signals to instruct left-right asymmetry.

Dynamic microtubules slow down during their shrinkage phase.

Microtubules are dynamic polymers that undergo stochastic transitions between growing and shrinking phases. The structural and chemical properties of these phases remain poorly understood. The transition from growth to shrinkage, termed catastrophe, is not a first-order reaction but rather a multistep process whose frequency increases with the growth time: the microtubule ages as the older microtubule tip becomes more unstable. Aging shows that the growing phase is not a single state but comprises several substates of increasing instability. To investigate whether the shrinking phase is also multistate, we characterized the kinetics of microtubule shrinkage following catastrophe using an in vitro reconstitution assay with purified tubulins. We found that the shrinkage speed is highly variable across microtubules and that the shrinkage speed of individual microtubules slows down over time by as much as several fold. The shrinkage slowdown was observed in both fluorescently labeled and unlabeled microtubules as well as in microtubules polymerized from tubulin purified from different species, suggesting that the shrinkage slowdown is a general property of microtubules. These results indicate that microtubule shrinkage, like catastrophe, is time dependent and that the shrinking microtubule tip passes through a succession of states of increasing stability. We hypothesize that the shrinkage slowdown is due to destabilizing events that took place during growth, which led to multistep catastrophe. This suggests that the aging associated with growth is also manifested during shrinkage, with the older, more unstable growing tip being associated with a faster depolymerizing shrinking tip.

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 mm3) 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.

RBPMS2 Is a Myocardial-Enriched Splicing Regulator Required for Cardiac Function.

BACKGROUND: RBPs (RNA-binding proteins) perform indispensable functions in the post-transcriptional regulation of gene expression. Numerous RBPs have been implicated in cardiac development or physiology based on gene knockout studies and the identification of pathogenic RBP gene mutations in monogenic heart disorders. The discovery and characterization of additional RBPs performing indispensable functions in the heart will advance basic and translational cardiovascular research. METHODS: We performed a differential expression screen in zebrafish embryos to identify genes enriched in nkx2.5-positive cardiomyocytes or cardiopharyngeal progenitors compared to nkx2.5-negative cells from the same embryos. We investigated the myocardial-enriched gene RNA-binding protein with multiple splicing (variants) 2 [RBPMS2)] by generating and characterizing rbpms2 knockout zebrafish and human cardiomyocytes derived from RBPMS2-deficient induced pluripotent stem cells. RESULTS: We identified 1848 genes enriched in the nkx2.5-positive population. Among the most highly enriched genes, most with well-established functions in the heart, we discovered the ohnologs rbpms2a and rbpms2b, which encode an evolutionarily conserved RBP. Rbpms2 localizes selectively to cardiomyocytes during zebrafish heart development and strong cardiomyocyte expression persists into adulthood. Rbpms2-deficient embryos suffer from early cardiac dysfunction characterized by reduced ejection fraction. The functional deficit is accompanied by myofibril disarray, altered calcium handling, and differential alternative splicing events in mutant cardiomyocytes. These phenotypes are also observed in RBPMS2-deficient human cardiomyocytes, indicative of conserved molecular and cellular function. RNA-sequencing and comparative analysis of genes mis-spliced in RBPMS2-deficient zebrafish and human cardiomyocytes uncovered a conserved network of 29 ortholog pairs that require RBPMS2 for alternative splicing regulation, including RBFOX2, SLC8A1, and MYBPC3. CONCLUSIONS: Our study identifies RBPMS2 as a conserved regulator of alternative splicing, myofibrillar organization, and calcium handling in zebrafish and human cardiomyocytes.

The force required to remove tubulin from the microtubule lattice by pulling on its α-tubulin C-terminal tail.

Severing enzymes and molecular motors extract tubulin from the walls of microtubules by exerting mechanical force on subunits buried in the lattice. However, how much force is needed to remove tubulin from microtubules is not known, nor is the pathway by which subunits are removed. Using a site-specific functionalization method, we applied forces to the C-terminus of α-tubulin with an optical tweezer and found that a force of ~30 pN is required to extract tubulin from the microtubule wall. Additionally, we discovered that partial unfolding is an intermediate step in tubulin removal. The unfolding and extraction forces are similar to those generated by AAA-unfoldases. Lastly, we show that three kinesin-1 motor proteins can also extract tubulin from the microtubule lattice. Our results provide the first experimental investigation of how tubulin responds to mechanical forces exerted on its α-tubulin C-terminal tail and have implications for the mechanisms of severing enzymes and microtubule stability.

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.

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules.

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and wide-field illumination microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.

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.

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.

Spastin is a dual-function enzyme that severs microtubules and promotes their regrowth to increase the number and mass of microtubules.

The remodeling of the microtubule cytoskeleton underlies dynamic cellular processes, such as mitosis, ciliogenesis, and neuronal morphogenesis. An important class of microtubule remodelers comprises the severases-spastin, katanin, and fidgetin-which cut microtubules into shorter fragments. While severing activity might be expected to break down the microtubule cytoskeleton, inhibiting these enzymes in vivo actually decreases, rather increases, the number of microtubules, suggesting that severases have a nucleation-like activity. To resolve this paradox, we reconstituted Drosophila spastin in a dynamic microtubule assay and discovered that it is a dual-function enzyme. In addition to its ATP-dependent severing activity, spastin is an ATP-independent regulator of microtubule dynamics that slows shrinkage and increases rescue. We observed that spastin accumulates at shrinking ends; this increase in spastin concentration may underlie the increase in rescue frequency and the slowdown in shortening. The changes in microtubule dynamics promote microtubule regrowth so that severed microtubule fragments grow, leading to an increase in the number and mass of microtubules. A mathematical model shows that spastin's effect on microtubule dynamics is essential for this nucleation-like activity: spastin switches microtubules into a state where the net flux of tubulin onto each polymer is positive, leading to the observed exponential increase in microtubule mass. This increase in the microtubule mass accounts for spastin's in vivo phenotypes.

Label-free high-speed wide-field imaging of single microtubules using interference reflection microscopy.

When studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labelling or there is concern that labelling will disrupt function. Commonly used techniques for observing unlabelled microtubules, such as video enhanced differential interference contrast, dark-field and more recently laser-based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark-field is highly sensitive to impurities and optical misalignments. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark-field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has a similar signal-to-noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high-speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the optical quality of interference reflection microscopy falls within the range of other microscope techniques, being inferior to some and superior to others, depending on the metric used and, with minimal microscope modification, can be used to study the dynamics of unlabelled microtubules. LAY DESCRIPTION: The cytoskeleton gives a cell its shape and plays a major role in its movement and division. It's also helps organise the content of cells and is the base for intracellular transport. Important components of the cytoskeleton are microtubules, which are hollow cylindrical beams (25 nm in diameter) that assemble from protein building blocks called tubulin. Deficiencies in microtubules are related to many diseases including cancer and Alzheimer. Given their important role, microtubules are heavily investigated in many laboratories. One way to study microtubules is to isolate them from cells and image them using light microscopy. Over the years a number of imaging techniques have been used. These techniques have a number of drawbacks which are addressed by ongoing efforts which this work is a part of. Here, we present a method based on light interference that produce high quality images of microtubules. The technique is cheap and easy to implement making it accessible to a wide base of researchers.

Kinesin Kip2 enhances microtubule growth in vitro through length-dependent feedback on polymerization and catastrophe.

The size and position of mitotic spindles is determined by the lengths of their constituent microtubules. Regulation of microtubule length requires feedback to set the balance between growth and shrinkage. Whereas negative feedback mechanisms for microtubule length control, based on depolymerizing kinesins and severing proteins, have been studied extensively, positive feedback mechanisms are not known. Here, we report that the budding yeast kinesin Kip2 is a microtubule polymerase and catastrophe inhibitor in vitro that uses its processive motor activity as part of a feedback loop to further promote microtubule growth. Positive feedback arises because longer microtubules bind more motors, which walk to the ends where they reinforce growth and inhibit catastrophe. We propose that positive feedback, common in biochemical pathways to switch between signaling states, can also be used in a mechanical signaling pathway to switch between structural states, in this case between short and long polymers.

Stu2, the budding yeast XMAP215/Dis1 homolog, promotes assembly of yeast microtubules by increasing growth rate and decreasing catastrophe frequency.

Stu2 is the budding yeast member of the XMAP215/Dis1 family of microtubule polymerases. It is essential in cell division, allowing proper spindle orientation and metaphase chromosome alignment, as well as spindle elongation during anaphase. Despite Stu2 having a phenotype that suggests it promotes microtubule growth, like the other members of the XMAP215/Dis1 family, previous studies with purified Stu2 indicate only that it antagonizes microtubule growth. One potential explanation for these contradictory findings is that the assays were performed with mammalian brain tubulin, which may not be the right substrate to test the activity of Stu2 given that yeast and brain tubulins are quite divergent in sequence and that the vertebrate tubulins are subject to many post-translational modifications. To test this possibility, we reconstituted the activity of Stu2 with purified budding yeast tubulin. We found that Stu2 accelerated microtubule growth in yeast tubulin by severalfold, similar to the acceleration reported for XMAP215 in porcine brain tubulin. Furthermore, Stu2 accelerated polymerization in yeast tubulin to a much greater extent than in porcine brain tubulin, and the concentration of Stu2 required to reach 50% maximum activity in yeast tubulin was nearly 2 orders of magnitude lower than that in porcine brain tubulin. We conclude that Stu2 is a microtubule polymerase, like its relatives, and that its activity is considerably higher in yeast tubulin compared with mammalian brain tubulin. The biochemical properties of Stu2 reported here account for many of the phenotypes of Stu2 observed in cells.

Inertial effects of a small Brownian particle cause a colored power spectral density of thermal noise.

The random thermal force acting on Brownian particles is often approximated in Langevin models by a "white-noise" process. However, fluid entrainment results in a frequency dependence of this thermal force giving it a "color." While theoretically well understood, direct experimental evidence for this colored nature of the noise term and how it is influenced by a nearby wall is lacking. Here, we directly measured the color of the thermal noise intensity by tracking a particle strongly confined in an ultrastable optical trap. All our measurements are in quantitative agreement with the theoretical predictions. Since Brownian motion is important for microscopic, in particular, biological systems, the colored nature of the noise and its distance dependence to nearby objects need to be accounted for and may even be utilized for advanced sensor applications.

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.

Optical tweezers with millikelvin precision of temperature-controlled objectives and base-pair resolution.

In optical tweezers, thermal drift is detrimental for high-resolution measurements. In particular, absorption of the trapping laser light by the microscope objective that focuses the beam leads to heating of the objective and subsequent drift. This entails long equilibration times which may limit sensitive biophysical assays. Here, we introduce an objective temperature feedback system for minimizing thermal drift. We measured that the infrared laser heated the objective by 0.7 K per watt of laser power and that the laser focus moved relative to the sample by approximately 1 nm/mK due to thermal expansion of the objective. The feedback stabilized the temperature of the trapping objective with millikelvin precision. This enhanced the long-term temperature stability and significantly reduced the settling time of the instrument to about 100 s after a temperature disturbance while preserving single DNA base-pair resolution of surface-coupled assays. Minimizing systematic temperature changes of the objective and concurrent drift is of interest for other high-resolution microscopy techniques. Furthermore, temperature control is often a desirable parameter in biophysical experiments.