John A. Mercer

Myosin Vb Mobilizes Recycling Endosomes and AMPA Receptors for Postsynaptic Plasticity

Learning-related plasticity at excitatory synapses in the mammalian brain requires the trafficking of AMPA receptors and the growth of dendritic spines. However, the mechanisms that couple plasticity stimuli to the trafficking of postsynaptic cargo are poorly understood. Here we demonstrate that myosin Vb (MyoVb), a Ca2+-sensitive motor, conducts spine trafficking during long-term potentiation (LTP) of synaptic strength. Upon activation of NMDA receptors and corresponding Ca2+ influx, MyoVb associates with recycling endosomes (REs), triggering rapid spine recruitment of endosomes and local exocytosis in spines. Disruption of MyoVb or its interaction with the RE adaptor Rab11-FIP2 abolishes LTP-induced exocytosis from REs and prevents both AMPA receptor insertion and spine growth. Furthermore, induction of tight binding of MyoVb to actin using an acute chemical genetic strategy eradicates LTP in hippocampal slices. Thus, Ca2+-activated MyoVb captures and mobilizes REs for AMPA receptor insertion and spine growth, providing a mechanistic link between the induction and expression of postsynaptic plasticity.

A chemical-genetic strategy implicates myosin-1c in adaptation by hair cells

Myosin-1c (also known as myosin-Ibeta) has been proposed to mediate the slow component of adaptation by hair cells, the sensory cells of the inner ear. To test this hypothesis, we mutated tyrosine-61 of myosin-1c to glycine, conferring susceptibility to inhibition by N(6)-modified ADP analogs. We expressed the mutant myosin-1c in utricular hair cells of transgenic mice, delivered an ADP analog through a whole-cell recording pipette, and found that the analog rapidly blocked adaptation to positive and negative deflections in transgenic cells but not in wild-type cells. The speed and specificity of inhibition suggests that myosin-1c participates in adaptation in hair cells.

Class V myosins

1. IntroductionThe myosin family of actin-based molecular mo-tors consists of 15 known classes that are structurallydistinct based on comparisons of the primary struc-ture of the motor domains of the known myosinheavy chain genes [1^3]. Information regarding thefunction and/or biochemical properties of most ofthese myosin classes is sparse relative to the well-characterized class II and class I myosins; neverthe-less, the range of proposed functions for these myo-sins is already remarkably broad [1]. There are anumber of recent reviews that provide an overviewof the rapidly growing myosin gene family [1,4^8].Among the best characterized and functionally di-verse of the recently discovered myosin classes arethe class V myosins, the focus of this review (forother reviews see [9^11]).Myosin-V was initially characterized as an unusualcalmodulin binding protein from brain with a num-ber of myosin-like biochemical properties [12^14].Subsequently, myosin-V heavy chain genes werecloned from mouse, yeast and chicken, thus de¢ningthe ¢fth class of actin-based motors [15^19]. Studiesof the mouse and yeast class V myosins provided the¢rst insights regarding the cellular function of myo-sin-V. Phenotypes of the mutant dilute mouse andthe temperature-sensitive yeast mutant, myo2-66 ledto the hypothesis that class V myosins may functionin cell polarity and membrane tra⁄cking [15,16].Moreover, mutations in the human ortholog of thedilute heavy chain gene cause Griscelli syndrome, arare recessive disease characterized by pigmentarydilution and in most, but not all cases immunode¢-ciency [20^22]. Neurological disorders have also beenreported in Griscelli syndrome patients [22,23]. Sincethese ¢rst studies, a great deal has been learnedabout the biochemistry, biophysics and cellular func-tion of the class V myosins. This review will discussthe emerging evidence that myosin-V is a processiveactin-based motor that has multiple functions in thecell ranging from mRNA transport, cell polarity andmembrane tra⁄cking.There are currently nine complete myosin-V heavychain sequences known (Fig. 1). Analysis of the twocompleted eukaryotic genomes of Saccharomyces ce-revisiae and Caenorhabditis elegans reveals that yeasthave two class V myosins, while C. elegans has asingle class V myosin heavy chain gene. In verte-brates, there are at least three distinct subclasses ofmyosin-V. The three most closely related heavy chainsequences, that of chicken brain myosin-V, the

Absence of the tight junctional protein AF-6 disrupts epithelial cell–cell junctions and cell polarity during mouse development

BACKGROUND The establishment, maintenance and rearrangement of junctions between epithelial cells are extremely important in many developmental, physiological and pathological processes. AF-6 is a putative Ras effector; it is also a component of tight and adherens junctions, and has been shown to bind both Ras and the tight-junction protein ZO-1. In the mouse, AF-6 is encoded by the Af6 gene. As cell-cell junctions are important in morphogenesis, we generated a null mutation in the murine Af6 locus to test the hypothesis that lack of AF-6 function would cause epithelial abnormalities. RESULTS Although cell-cell junctions are thought to be important in early embryogenesis, homozygous mutant embryos were morphologically indistinguishable from wild-type embryos through 6.5 days post coitum (dpc) and were able to establish all three germ layers. The earliest morphological abnormalities were observed in the embryonic ectoderm of mutant embryos at 7.5 dpc. The length of the most apical cell-cell junctions was reduced, and basolateral surfaces of those cells were separated by multiple gaps. Cells of the embryonic ectoderm were less polarized as assessed by histological criteria and lateral localization of an apical marker. Mutant embryos died by 10 dpc, probably as a result of placental failure. CONCLUSIONS AF-6 is a critical regulator of cell-cell junctions during mouse development. The loss of neuroepithelial polarity in mutants is consistent with a loss of efficacy of the cell-cell junctions that have a critical role in establishing apical/basolateral asymmetry.

Fast Adaptation in Vestibular Hair Cells Requires Myosin-1c Activity

In sensory hair cells of the inner ear, mechanical amplification of small stimuli requires fast adaptation, the rapid closing of mechanically activated transduction channels. In frog and mouse vestibular hair cells, we found that the rate of fast adaptation depends on both channel opening and stimulus size and that it is modeled well as a release of a mechanical element in series with the transduction apparatus. To determine whether myosin-1c molecules of the adaptation motor are responsible for the release, we introduced the Y61G mutation into the Myo1c locus and generated mice homozygous for this sensitized allele. Measuring transduction and adaptation in the presence of NMB-ADP, an allele-specific inhibitor, we found that the inhibitor not only blocked slow adaptation, as demonstrated previously in transgenic mice, but also inhibited fast adaptation. These results suggest that mechanical activity of myosin-1c is required for fast adaptation in vestibular hair cells.

Melanophilin, the Product of the Leaden Locus, is Required for Targeting of Myosin-Va to Melanosomes

The formation of complex subcellular organelles requires the coordinated targeting of multiple components. Melanosome biogenesis in mouse melanocytes is an excellent model system for studying the coordinated function of multiple gene products in intracellular trafficking. To begin to order events in melanosome biogenesis and distribution, we employed the classical coat‐color mutants ashen, dilute, and leaden, which affect melanosome distribution, but not melanin synthesis. The loci have been renamed Rab27a, Myo5a, and Mlph for their gene products. While each of the three loci has been shown to be required for melanosome distribution, the point(s) at which each acts is unknown. We have utilized primary melanocytes to examine the interdependencies between rab27a, myosin‐Va, and melanophilin. The localization of rab27a to melanosomes did not require the function of either myosin‐Va or melanophilin, but leaden function was required for the association of myosin‐Va with melanosomes. In leaden melanocytes permeabilized before fixation, myosin‐Va immunoreactivity was greatly attenuated, suggesting that myosin‐Va is free in the cytoplasm. Finally, we have complemented both the leaden and ashen phenotypes by cell fusion and observed redistribution of mature melanosomes in the absence of both protein and melanin synthesis. Together, our data suggest a model for the initial assembly of the machinery required for melanosome distribution.

Engineering of the Myosin-Iβ Nucleotide-binding Pocket to Create Selective Sensitivity to N 6-modified ADP Analogs

Distinguishing the cellular functions carried out by enzymes of highly similar structure would be simplified by the availability of isozyme-selective inhibitors. To determine roles played by individual members of the large myosin superfamily, we designed a mutation in myosins nucleotide-binding pocket that permits binding of adenine nucleotides modified with bulky N 6substituents. Introduction of this mutation, Y61G in rat myosin-Iβ, did not alter the enzymes affinity for ATP or actin and actually increased its ATPase activity and actin-translocation rate. We also synthesized several N 6-modified ADP analogs that should bind to and inhibit mutant, but not wild-type, myosin molecules. Several of these N 6-modified ADP analogs were more than 40-fold more potent at inhibiting ATP hydrolysis by Y61G than wild-type myosin-Iβ; in doing so, these analogs locked Y61G myosin-Iβ tightly to actin.N 6-(2-methylbutyl) ADP abolished actin filament motility mediated by Y61G, but not wild-type, myosin-Iβ. Furthermore, a small fraction of inhibited Y61G molecules was sufficient to block filament motility mediated by mixtures of wild-type and Y61G myosin-Iβ. Introduction of Y61G myosin-Iβ molecules into a cell should permit selective inhibition byN 6-modified ADP analogs of cellular processes dependent on myosin-Iβ.

A molecular genetic linkage map of mouse chromosome 2

Interspecific backcross mice were used to create a molecular genetic linkage map of chromosome 2. Genomic DNAs from N2 progeny were subjected to Southern blot analysis using molecular probes that identified the Abl, Acra, Ass, C5, Cas-1, Fshb, Gcg, Hox-5.1, Jgf-1, Kras-3, Ltk, Pax-1, Prn-p, and Spna-2 loci; these loci were added to the 11 loci previously mapped to the distal region of chromosome 2 in the same interspecific backcross to generate a composite multilocus linkage map. Several loci mapped near, and may be the same as, known mutations. Comparisons between the mouse and the human genomes indicate that mouse chromosome 2 contains regions homologous to at least six human chromosomes. Mouse models for human diseases are discussed.

Myosin-Vb functions as a dynamic tether for peripheral endocytic compartments during transferrin trafficking

BackgroundMyosin-Vb has been shown to be involved in the recycling of diverse proteins in multiple cell types. Studies on transferrin trafficking in HeLa cells using a dominant-negative myosin-Vb tail fragment suggested that myosin-Vb was required for recycling from perinuclear compartments to the plasma membrane. However, chemical-genetic, dominant-negative experiments, in which myosin-Vb was specifically induced to bind to actin, suggested that the initial hypothesis was incorrect both in its site and mode of myosin-Vb action. Instead, the chemical-genetic data suggested that myosin-Vb functions in the actin-rich periphery as a dynamic tether on peripheral endosomes, retarding transferrin transport to perinuclear compartments.ResultsIn this study, we employed both approaches, with the addition of overexpression of full-length wild-type myosin-Vb and switching the order of myosin-Vb inhibition and transferrin loading, to distinguish between these hypotheses. Overexpression of full-length myosin-Vb produced large peripheral endosomes. Chemical-genetic inhibition of myosin-Vb after loading with transferrin did not prevent movement of transferrin from perinuclear compartments; however, virtually all myosin-Vb-decorated particles, including those moving on microtubules, were halted by the inhibition. Overexpression of the myosin-Vb tail caused a less-peripheral distribution of early endosome antigen-1 (EEA1).ConclusionAll results favored the peripheral dynamic tethering hypothesis.

Chemical-genetic inhibition of a sensitized mutant myosin Vb demonstrates a role in peripheral-pericentriolar membrane traffic

Selective, in situ inhibition of individual unconventional myosins is a powerful approach to determine their specific physiological functions. Here, we report the engineering of a myosin Vb mutant that still hydrolyzes ATP, yet is selectively sensitized to an N6-substituted ADP analog that inhibits its activity, causing it to remain tightly bound to actin. Inhibition of the sensitized mutant causes inhibition of accumulation of transferrin in the cytoplasm and increases levels of plasma-membrane transferrin receptor, suggesting that myosin Vb functions in traffic between peripheral and pericentrosomal compartments.