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Myo1e is a nonmuscle motor protein enriched in podocytes. Mutations inlt;igt;MYO1Elt;/igt; are associated with steroid-resistant nephrotic syndrome (SRNS). Most of thelt;igt;MYO1Elt;/igt; variants identified by genomic sequencing have not been functionally characterized. Here, we set out to analyze two mutations in the Myo1e motor domain, T119I and D388H, which were selected on the basis of protein sequence conservation.EGFP-tagged humanlt;igt;Myo1elt;/igt; constructs were delivered into the Myo1e-KO mouse podocyte-derived cellslt;igt;vialt;/igt; adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncatedlt;igt;Myo1elt;/igt; constructs were expressed using the baculovirus expression system and used to measure Myo1e ATPase and motor activitylt;igt;in vitrolt;/igt;.Both mutants were expressed as full-length proteins in the Myo1e-KO cells. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs). In contrast, D388H variant localization was similar to that of WT. The rate of dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting this mutation affects Myo1e interactions with binding partners. ATPase activity and ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting findings from cell-based experiments.T119I and D388H mutations are deleterious to Myo1e functions. The experimental approaches used in this study can be applied to future characterization of novellt;igt;MYO1Elt;/igt; variants associated with SRNS.

Class III myosins are actin-based motors proposed to transport cargo to the distal tips of stereocilia in the inner ear hairs cells and/or to participate in stereocilia length regulation, which is especially important during development. Mutations in the MYO3A gene are associated with delayed onset deafness. A previous study demonstrated that L697W, a dominant deafness mutation, disrupts MYO3A ATPase and motor properties but does not impair its ability to localize to the tips of actin protrusions. In the current study, we characterized the transient kinetic mechanism of the L697W motor ATPase cycle. Our kinetic analysis demonstrates that the mutation slows the ADP release and ATP hydrolysis steps, which results in a slight reduction in the duty ratio and slows detachment kinetics. Fluorescence recovery after photobleaching (FRAP) of filopodia tip localized L697W and WT MYO3A in COS-7 cells revealed that the mutant does not alter turnover or average intensity at the actin protrusion tips. We demonstrate that the mutation slows filopodia extension velocity in COS-7 cells which correlates with its 2-fold slower in vitro actin gliding velocity. Overall, this work allowed us to propose a model for how the motor properties of MYO3A are crucial for facilitating actin protrusion length regulation.

Myo1e is a non-muscle motor protein enriched in the podocyte foot processes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Here, we set out to differentiate between the pathogenic and neutral MYO1E variants identified in SRNS patients by exome sequencing. Based on protein sequence conservation and structural predictions, two mutations in the motor domain, T119I and D388H, were selected for this study. EGFP-tagged Myo1e constructs were delivered into the Myo1e-KO podocytes via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculoviral expression system and used to measure Myo1e ATPase and motor activity in vitro. Both mutants were expressed as full-length proteins in the Myo1e-KO podocytes. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs) in podocytes. In contrast, the D388H variant localization was similar to the WT. Surprisingly, the dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting that this mutation also affects Myo1e activity. The ATPase activity and the ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting the findings from cell-based experiments. The experimental pipeline developed in this study allowed us to determine that the T119I and D388H mutations appear to be pathogenic and gain additional knowledge in the Myo1e role in podocytes. This workflow can be applied to the future characterization of novel MYO1E variants associated with SRNS.

A micro-electromechanical resonator, consisting of a $$125 \times 125\,\upmu \hbox {m}^2$$ center plate suspended $$2\,\upmu \hbox {m}$$ above a substrate, was immersed in $$^4$$ He along with a quartz tuning fork. The tuning fork was placed 5 mm above the resonator and was used to generate turbulence. The damping of the resonator was investigated in the presence of turbulence generated by the tuning fork. We observe a velocity, $$v_c \simeq 5\,\hbox {mm}\,\hbox {s}^{-1}$$ , above which the damping on the resonator is drastically reduced for all tuning fork velocities studied. We attribute this change in damping to the shedding and capture of vortices.

We present an analytical calculation of the response of a driven Duffing oscillator to low-frequency fluctuations in the resonance frequency and damping. We find that fluctuations in these parameters manifest themselves distinctively, allowing them to be distinguished. In the strongly nonlinear regime, amplitude and phase noise due to resonance frequency fluctuations and amplitude noise due to damping fluctuations are strongly attenuated, while the transduction of damping fluctuations into phase noise remains of order $1$. We show that this can be seen by comparing the relative strengths of the amplitude fluctuations to the fluctuations in the quadrature components, and suggest that this provides a means to determine the source of low-frequency noise in a driven Duffing oscillator., Comment: 5 pages, 3 figures, accepted for publication in Physica D

We report a comprehensive investigation of the effects of quantum turbulence and quantized vorticity in superfluid $^{4}\mathrm{He}$ on the motion of a microelectromechanical systems (MEMS) resonator. We find that the MEMS is uniquely sensitive to quantum turbulence present in the fluid. To generate turbulence in the fluid, a quartz tuning fork (TF) is placed in proximity to the MEMS and driven at a large amplitude. We observe that at low velocity, the MEMS is damped by the turbulence, and that above a critical velocity, ${v}_{c}\ensuremath{\simeq}5\phantom{\rule{4pt}{0ex}}\mathrm{mm}\phantom{\rule{0.28em}{0ex}}{\mathrm{s}}^{\ensuremath{-}1}$, the turbulent damping is greatly reduced. We find that above ${v}_{c}$, the damping of the MEMS is reduced further for increasing velocity, indicating a velocity dependent coupling between the surface of the MEMS and the quantized vortices constituting the turbulence. We propose a model of the interaction between vortices in the fluid and the surface of the MEMS. The sensitivity of these devices to a small number of vortices and the almost unlimited customization of MEMS open the door to a more complete understanding of the interaction between quantized vortices and oscillating structures, which in turn provides a new route for the investigation of the dynamics of single vortices.

Myosins are molecular motors that use a conserved ATPase cycle to generate force. We investigated two mutations in the converter domain of myosin V (R712G and F750L) to examine how altering specific structural transitions in the motor ATPase cycle can impair myosin mechanochemistry. The corresponding mutations in the human β-cardiac myosin gene are associated with hypertrophic and dilated cardiomyopathy, respectively. Despite similar steady-state actin-activated ATPase and unloaded in vitro motility-sliding velocities, both R712G and F750L were less able to overcome frictional loads measured in the loaded motility assay. Transient kinetic analysis and stopped-flow FRET demonstrated that the R712G mutation slowed the maximum ATP hydrolysis and recovery-stroke rate constants, whereas the F750L mutation enhanced these steps. In both mutants, the fast and slow power-stroke as well as actin-activated phosphate release rate constants were not significantly different from WT. Time-resolved FRET experiments revealed that R712G and F750L populate the pre- and post-power-stroke states with similar FRET distance and distance distribution profiles. The R712G mutant increased the mole fraction in the post-power-stroke conformation in the strong actin-binding states, whereas the F750L decreased this population in the actomyosin ADP state. We conclude that mutations in key allosteric pathways can shift the equilibrium and/or alter the activation energy associated with key structural transitions without altering the overall conformation of the pre- and post-power-stroke states. Thus, therapies designed to alter the transition between structural states may be able to rescue the impaired motor function induced by disease mutations.

Actin and myosin play important roles in many devastating diseases and thus are attractive targets for small-molecule therapy. In this issue of JBC, Guhathakurta et al. have developed a high-throughput screening assay to find small molecules that interfere with the actomyosin interaction. They utilized time-resolved FRET (TR-FRET) and a unique donor–acceptor pair (filamentous actin and a peptide that binds near the myosin-binding site on actin) to find novel molecules that interfere with the actomyosin ATPase and alter the structure of actin filaments. These findings demonstrate the power and potential of high-throughput TR-FRET in monitoring molecular interactions.

Whole-exome sequencing of samples from affected members of two unrelated families with late-onset non-syndromic hearing loss revealed a novel mutation (c.2090 T > G; NM_017433) in MYO3A. The mutation was confirmed in 36 affected individuals, showing autosomal dominant inheritance. The mutation alters a single residue (L697W or p.Leu697Trp) in the motor domain of the stereocilia protein MYO3A, leading to a reduction in ATPase activity, motility, and an increase in actin affinity. MYO3A-L697W showed reduced filopodial actin protrusion initiation in COS7 cells, and a predominant tipward accumulation at filopodia and stereocilia when coexpressed with wild-type MYO3A and espin-1, an actin-regulatory MYO3A cargo. The combined higher actin affinity and duty ratio of the mutant myosin cause increased retention time at stereocilia tips, resulting in the displacement of the wild-type MYO3A protein, which may impact cargo transport, stereocilia length, and mechanotransduction. The dominant negative effect of the altered myosin function explains the dominant inheritance of deafness.

This article summarizes a presentation given by Kurt Blythe and Jennifer Solomon at the 28th annual North Carolina Serials Conference in 2019. Blythe and Solomon were brought on to lead a project t...

Developmental processes that control root system architecture are critical for soil exploration by plants, allowing for uptake of water and nutrients. Conversion of the auxin precursor indole-3-butyric acid (IBA) to active auxin (indole-3-acetic acid; IAA) modulates lateral root formation. However, mechanisms governing IBA-to-IAA conversion have yet to be elucidated. We identified TRANSPORTER OF IBA1 (TOB1) as a vacuolar IBA transporter that limits lateral root formation. Moreover, TOB1, which is transcriptionally regulated by the phytohormone cytokinin, is necessary for the ability of cytokinin to exert inhibitory effects on lateral root production. The increased production of lateral roots in tob1 mutants, TOB1 transport of IBA into the vacuole, and cytokinin-regulated TOB1 expression provide a mechanism linking cytokinin signaling and IBA contribution to the auxin pool to tune root system architecture.

The implementation of Resource Description and Access (RDA) in 2013 or after will have a powerful impact on the skill set required of new library and information science professionals. This article chronicles the development of an RDA “boot camp” at UNC–Chapel Hill's School of Information and Library Science. Curriculum for the three-hour camp included a review of relevant theoretical frameworks and a hands-on exercise creating RDA records. Findings from a post–boot camp survey point to areas for further growth in cataloging and metadata course development and suggest that students are eager for more practical experience with emerging schema.

Cardiac troponin I (cTnI) has a unique N-terminal extension that plays a role in modifying the calcium regulation of cardiac muscle contraction. Restrictive cleavage of the N-terminal extension of cTnI occurs under stress conditions as a physiological adaptation. Recent studies have shown that in comparison with controls, transgenic mouse cardiac myofibrils containing cTnI lacking the N-terminal extension (cTnI-ND) had a lower sensitivity to calcium activation of ATPase, resulting in enhanced ventricular relaxation and cardiac function. To investigate which step(s) of the ATPase cycle is regulated by the N-terminal extension of cTnI, here we studied the calcium dependence of cardiac myosin II ATPase kinetics in isolated cardiac myofibrils. ATP binding and ADP dissociation rates were measured by using stopped-flow spectrofluorimetry with mant-dATP and mant-dADP, respectively. We found that the second-order mant-dATP binding rate of cTnI-ND mouse cardiac myofibrils was 3-fold faster than that of wild-type myofibrils at low Ca(2+) concentrations. The ADP dissociation rate of cTnI-ND myofibrils was positively dependent on calcium concentration, while the wild-type controls were not significantly affected. These data from experiments using native cardiac myofibrils under physiological conditions indicate that modification of the N-terminal extension of cTnI plays a role in the calcium regulation of the kinetics of actomyosin ATPase.

Class 5 myosins are actin-based molecular motors implicated in intracellular cargo transport. In vertebrates, myosin 5 (Myo5) consists of three isoforms, named myosin 5a, 5b, and 5c. The ability of myosin V molecules to move continuously along actin filaments (i.e. processivity) is required for efficient cargo transport in cells. This physical property has been well addressed in Myo5a and 5b, which demonstrate processive movement as a single molecule. In contrast, Myo5c shows no processivity as a single molecule, although it is found in cargo and is believed to participate in cargo transports. This raises the possibility that multiple Myo5c molecules should transport cargos. Recent studies have shown that Myo5c binds to Zymogen granule (ZGs) in pancreatic cells and transports ZGs to cell periphery. Purified ZGs was investigated the movement manner in vitro. The velocity and run-length of ZGs along actin track was measured (99 nm/sec and 1255 nm, respectively). We identified that the movement manner of ZGs move in rolling manner instead of gliding along actin tracks. Mathematical model of multiple Myo5c motors ensemble applied to understand the movement behavior vs. number of Myo5c on the vesicle.