Endocytic myosin-1 is a force-insensitive, power-generating motor.

Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical properties of the relevant motors have not been investigated. Myosins have diverse mechanochemical activities, ranging from powerful contractility against mechanical loads to force-sensitive anchoring. To better understand the essential molecular contribution of myosin to endocytosis, we studied the in vitro force-dependent kinetics of the Saccharomyces cerevisiae endocytic type I myosin called Myo5, a motor whose role in clathrin-mediated endocytosis has been meticulously studied in vivo. We report that Myo5 is a low-duty-ratio motor that is activated ∼10-fold by phosphorylation and that its working stroke and actin-detachment kinetics are relatively force-insensitive. Strikingly, the in vitro mechanochemistry of Myo5 is more like that of cardiac myosin than that of slow anchoring myosin-1s found on endosomal membranes. We, therefore, propose that Myo5 generates power to augment actin assembly-based forces during endocytosis in cells.

Psychotropic Polypharmacy in the US Pediatric Population: A Methodologic Critique and Commentary.

Background: Psychotropic concomitant medication use for the treatment of youth with emotional and behavioral disorders has grown significantly in the U.S. over the past 25 years. The use of pharmacy claims to analyze these trends requires the following: age of the selected population, overlapping days of use, and precision of the outcome itself. This review will also address the gaps in reporting of pediatric psychotropic polypharmacy. Methods: An electronic literature search was undertaken for the period 2000 through 2020 using keywords such as "pediatric," "concomitant," "polypharmacy," "multiple medications," and "concurrent psychotropic"; Relevant references in textbooks were also used. Only English language and U.S. studies were included, resulting in 35 inter-class studies. Results: Studies were organized into seven groups according to data sources and clinical topics: (1) population surveys; (2a) multi-state publicly insured populations; (2b) single/two state studies; (3) privately insured populations; (4) diagnosed populations; (5) foster care populations; (6) special settings. Across 20 years it is apparent that pediatric psychotropic polypharmacy affects substantially more children and adolescents today than had been the case. As many as 300,000 youth now receive 3 or more classes concomitantly. The duration of concomitant use is relatively long, e.g., 69-89% of annual medicated days. Finally, more adverse event reports were associated with 3-class compared with 2-class drug regimens. Discussion: Factors that contribute to the growth of pediatric psychotropic polypharmacy include: (1) predominance of the biological model in psychiatric practice; (2) invalid assumptions on efficacy of combinations, (3) limited professional awareness of metabolic and neurological adverse drug events, and (4) infrequent use of appropriate deprescribing. Conclusion: A review of publications documenting U.S. pediatric psychotropic polypharmacy written over the last 20 years supports the need to standardize the methodologies used. The design of population-based studies should maximize information on the number of youth receiving regimens of 3-, 4-, and 5 or more concomitant classes and the duration of such use. Next, far more post-marketing research is needed to address the effectiveness, safety and tolerability of complex drug regimens prescribed for youngsters.

Antidepressant Use in Medicaid-Insured Youth: Trends, Covariates, and Future Research Needs.

BACKGROUND: Detailed research on long-term antidepressant (AD) trends within a single large US Medicaid population of youth has not heretofore been reported. METHODS: Administrative claims data for eight annual timepoints across 28 years (1987-2014) were organized for youth (<20 years old) who were continuously enrolled during each study year in a mid-Atlantic state Medicaid program. Total annual AD prevalence and age-, gender-, race-, eligibility group-, and diagnosis-specific prevalence were formed from bivariate analyses; logistic regression assessed the change in use (2007-2014) adjusted for covariates. AD-polypharmacy data were assessed in 2014. RESULTS: The major findings are: 1) AD use in state Medicaid enrollees grew 14-fold between 1987 and 2014. Data from 2014 revealed significantly increased odds of youth with SSRI/SNRI dispensings compared to 2007 (AOR=1.15 95% CI 1.11-1.19), representing 78% of total AD users. 2) Recent AD increases were greatest for 15-19-year olds. 3) AD use in girls passed up AD use in boys for the first time in 2014. 4) In 2014, ADs for foster care (12.7%) were 6 times greater than for their income-eligible Medicaid-counterparts. 5) In 2014, a quarter of AD-medicated youth were diagnosed with a behavior disorder. 6) More than 40 percent of AD medicated youth had >=1 other concomitant psychotropic classes for 60 or more days. CONCLUSIONS: Second-generation antidepressant use in Medicaid-insured youth has increased despite growing questions that pediatric AD benefits may not outweigh harms. These patterns support the call for publicly funded, independent investigator-conducted post-marketing outcomes research.

Overprescribed Medications for US Adults: Four Major Examples.

To understand possible medication overprescribing, it would be important to know which classes are the most prescribed, for which indications, for what duration, and for which age groups. Among the 10 most frequently prescribed medication classes for US adults, four were evaluated for overprescribing, and systematically assessed in relation to their primary indication. The assessment included usage patterns, trends, age of recipients, treatment duration, and benefits versus adverse consequences. The findings in this selective review are supported by an extensive search of the medical literature. The four selected medication categories and their most common indication included opioids for chronic pain, proton pump inhibitors for indigestion, levothyroxine for subclinical hypothyroidism, and antidepressants for subsyndromal levels of depression. These medications, grouped by their most frequent indication along with polypharmacy, have experienced major prescription increases in recent years, particularly among older patients. Most concerning is that they have been frequently prescribed for extended periods, usually with inadequate evidence of benefit. High drug usage patterns can aid in quantifying overprescribing within polypharmacy by age group.

Myosin Va and myosin VI coordinate their steps while engaged in an in vitro tug of war during cargo transport.

Myosin Va (myoV) and myosin VI (myoVI) are processive molecular motors that transport cargo in opposite directions on actin tracks. Because these motors may bind to the same cargo in vivo, we developed an in vitro "tug of war" to characterize the stepping dynamics of single quantum-dot-labeled myoV and myoVI motors linked to a common cargo. MyoV dominates its myoVI partner 79% of the time. Regardless of which motor wins, its stepping rate slows due to the resistive load of the losing motor (myoV, 2.1 pN; myoVI, 1.4 pN). Interestingly, the losing motor steps backward in synchrony with the winning motor. With ADP present, myoVI acts as an anchor to prevent myoV from stepping forward. This model system emphasizes the physical communication between opposing motors bound to a common cargo and highlights the potential for modulating this interaction by changes in the cell's ionic milieu.

Cargo binding induces dimerization of myosin VI.

Although myosin VI has properties that would allow it to function optimally as a dimer, full-length myosin VI exists as a monomer in isolation. Based on the ability of myosin VI monomers to dimerize when held in close proximity, we postulated that cargo binding normally regulates dimerization of myosin VI. We tested this hypothesis by expressing a known dimeric cargo adaptor protein of myosin VI, optineurin, and the myosin VI-binding segment from a monomeric cargo adaptor protein, Dab2. In the presence of these adaptor proteins, full-length myosin VI has ATPase properties of a dimer, appears as a dimer in electron micrographs, and moves processively on actin filaments. The results support a model in which cargo binding exposes internal dimerization sequences within full-length myosin VI. Because, unexpectedly, a monomeric fragment of Dab2 triggers dimerization, it would appear that myosin VI is designed to function as a dimer in cells.

Cross-correlated TIRF/AFM reveals asymmetric distribution of force-generating heads along self-assembled, "synthetic" myosin filaments.

Myosin-II's rod-like tail drives filament assembly with a head arrangement that is often considered to be a symmetric bipole that generates equal and opposite contractile forces on actin. Self-assembled myosin filaments are shown here to be asymmetric in physiological buffer based on cross-correlated images from both atomic force microscopy and total internal reflection fluorescence. Quantitative cross-correlation of these orthogonal methods produces structural information unavailable to either method alone in showing that fluorescence intensity along the filament length is proportional to height. This implies that myosin heads form a shell around the filament axis, consistent with F-actin binding. A motor density of approximately 50-100 heads/micrometer is further estimated but with an average of 32% more motors on one half of any given filament compared to the other, regardless of length. A purely entropic pyramidal lattice model is developed and mapped onto the Dyck paths problem that qualitatively captures this lack of length dependence and the distribution of filament asymmetries. Such strongly asymmetric bipoles are likely to produce an unbalanced contractile force in cells and in actin-myosin gels and thereby contribute to motility as well as cytoskeletal tension.

Contributions from all over: widely distributed residues in thymosin beta-4 affect the kinetics and stability of actin binding.

We have used site-directed mutagenesis to investigate the contributions of widely distributed residues in the thymosin beta-4 (Tbeta4) sequence to the formation and stability of the actin-Tbeta4 complex. Equilibrium and kinetic studies of actin binding were performed by monitoring the change in fluorescence of an N-iodoacetyl-N9-(5-sulfo-1-naphthyl)ethylenediamine (Aedans) fluorophore on actin cysteine-374. We evaluated the contributions of hydrophobic residues throughout Tbeta4, the conserved and variable proline residues, and the conserved lysine residues to the kinetics and thermodynamics of Tbeta4 binding to MgATP-actin monomers. Pro4, Lys18, Lys19, Pro27, Leu28, Pro29, and Ile34 were substituted by alanine residues. All these mutations weaken the affinity of the actin-Tbeta4 complex, but the kinetic basis of the lower stability of the complex varies among the mutants. Our results support a model in which Tbeta4 initially binds actin through an electrostatic interaction, followed by the formation of widely distributed hydrophobic contacts. Several mutants, particularly at proline residues, dissociate much more rapidly than the wild-type complex, but also show small increases in the rate of association. This seeming paradox suggests that conformational searching of Tbeta4, and particularly cis-trans isomerization of proline residues, contributes to the slow association rate constant of Tbeta4, and to the stability of the hydrophobic contacts associated with strong actin binding.

Thymosin beta4 induces a conformational change in actin monomers.

Using fluorescence resonance energy transfer spectroscopy we demonstrate that thymosin beta(4) (tbeta(4)) binding induces spatial rearrangements within the small domain (subdomains 1 and 2) of actin monomers in solution. Tbeta(4) binding increases the distance between probes attached to Gln-41 and Cys-374 of actin by 2 A and decreases the distance between the purine base of bound ATP (epsilonATP) and Lys-61 by 1.9 A, whereas the distance between Cys-374 and Lys-61 is minimally affected. Distance determinations are consistent with tbeta(4) binding being coupled to a rotation of subdomain 2. By differential scanning calorimetry, tbeta(4) binding increases the cooperativity of ATP-actin monomer denaturation, consistent with conformational rearrangements in the tbeta(4)-actin complex. Changes in fluorescence resonance energy transfer are accompanied by marked reduction in solvent accessibility of the probe at Gln-41, suggesting it forms part of the binding interface. Tbeta(4) and cofilin compete for actin binding. Tbeta(4) concentrations that dissociate cofilin from actin do not dissociate the cofilin-DNase I-actin ternary complex, consistent with the DNase binding loop contributing to high-affinity tbeta(4)-binding. Our results favor a model where thymosin binding changes the average orientation of actin subdomain 2. The tbeta(4)-induced conformational change presumably accounts for the reduced rate of amide hydrogen exchange from actin monomers and may contribute to nucleotide-dependent, high affinity binding.

Thymosin beta4 regulation, expression and function in aortic valve interstitial cells.

BACKGROUND AND AIMS OF THE STUDY: Previous research has demonstrated that tenascin-C, an extracellular matrix protein involved in bone development and mineralization, was specifically present in calcified aortic valves, always in association with matrix metalloproteinase (MMP)-2, and was not detectable in non-calcified human aortic valves. The aim of the present study was to identify downstream targets of tenascin-C in aortic valve interstitial cells. METHODS: Subtractive hybridization was performed using sheep aortic valve interstitial cells (SAVIC) grown on a substrate of collagen plus tenascin-C, versus cells grown on type I collagen alone. RESULTS: Subtractive hybridizations revealed that nearly 70% of the clones isolated contained the sequence for thymosin beta-4, an actin-binding protein, also associated with mineralization, regulation of MMPs and inflammation. In cell culture studies, it was shown that both thymosin beta4 and thymosin beta4 sulfoxide are produced by SAVIC. When aortic valve interstitial cells were grown on a substrate of tenascin-C, thymosin beta4 expression was up-regulated. The addition of thymosin beta4 to aortic valve interstitial cell cultures resulted in a re-orientation of cytoskeletal F-actin, and induction of MMP-2. However, thymosin beta4 antisense oligonucleotide transfection did not suppress MMP-2 expression. CONCLUSION: Thymosin beta4 is up-regulated by tenascin-C, and may be involved in the primary initiation of valvular calcification.

Kinetic characterization of the weak binding states of myosin V.

Myosin V is a molecular motor shown to move processively along actin filaments. We investigated the properties of the weak binding states of monomeric myosin V containing a single IQ domain (MV 1IQ) to determine if the affinities of these states are increased as compared to conventional myosin. Further, using a combination of non-hydrolyzable nucleotide analogues and mutations that block ATP hydrolysis, we sought to probe the states that are populated during ATP-induced dissociation of actomyosin. MV 1IQ binds actin with a K(d) = 4 microM in the presence of ATP gamma S at 50 mM KCl, which is 10-20-fold tighter than that of nonprocessive class II myosins. Mutations within the switch II region trapped MV 1IQ in two distinct M.ATP states with very different actin binding affinities (K(d) = 0.2 and 2 microM). Actin binding may change the conformation of the switch II region, suggesting that elements of the nucleotide binding pocket will be in a different conformation when bound to actin than is seen in any of the myosin crystal structures to date.