Extension of the endoplasmic reticulum (ER) network into dendritic spines of Purkinje neurons (PNs) is required for cerebellar synaptic plasticity and disrupted in animals with mutations in MYO5A, the gene encoding the class V myosin Myosin Va. Notably, the mechanism ensuring the ERs localization to spines has not been unraveled. While it has been proposed that animal class V myosins localize organelles by tethering them to the actin cytoskeleton, we demonstrate here that Myosin Va is an organelle transporter that pulls ER as cargo into PN spines. We show that the myosin accumulates at the ER tip as the organelle moves into spines, and that the myosins ability to hydrolyze ATP is required for the ERs movement into spines. Importantly, we provide direct proof for Myosin Va driving ER motility, as an attenuation of the myosins capability to move along actin filaments reduces the velocity of ER movement. Thus, we establish for the first time within animal cells that an actin-based motor is moving ER, and we thereby uncover the basis of the mechanism that mediates ER localization to PN spines, a prerequisite for synaptic plasticity. The contractile vacuole (CV) complex in Dictyostelium is a tubulovesicular osmoregulatory organelle that exhibits extensive motility along the actin-rich cortex, providing a useful model for investigating myosin-dependent membrane transport. Here we show that the type V myosin myoJ localizes to CV membranes and is required for efficient osmoregulation, the normal accumulation of CV membranes in the cortex, and the conversion of collapsed bladder membranes into outwardly radiating cortical CV tubules. Complementation of myoJ null cells with mechanochemically compromised versions of myoJ results in predictable changes in the dynamics of these radiating tubules, confirming myoJs role in moving CV membranes along the cortex. MyoJ null cells also exhibit a dramatic concentration of CV membranes around the MTOC. Consistently, we demonstrate that CV membranes also move bi-directionally on microtubules between the cortex and the MTOC. Therefore, myoJ cooperates with plus and minus end-directed microtubule motors to drive the normal distribution and dynamics of the CV complex in Dictyostelium. Bulk solution assays have shown that the isolated CAH3 domain from mouse and Acanthamoeba CARMIL rapidly and potently restores actin polymerization when added to actin filaments previously capped with Capping Protein (CP). To demonstrate this putative uncapping activity directly, we used TIRF microscopy to observe single, CP-capped actin filaments before and after the addition of the CAH3 domain from mouse CARMIL-1 (mCAH3). Addition of mCAH3 rapidly restored the polymerization of individual capped filaments, consistent with uncapping. To verify uncapping, filaments were capped with recombinant mouse CP tagged with monomeric GFP (mGFP-CP). Restoration of polymerization upon mCAH3 addition was immediately preceded by the complete dissociation of mGFP-CP from the filament end, confirming the CAH3-driven uncapping mechanism. Quantitative analyses showed that the percentage of capped filaments that uncapped increased as the concentration of mCAH3 was increased, reaching a maximum of 90% at 250 nM mCAH3. Moreover, the time interval between mCAH3 addition and uncapping decreased as the concentration of mCAH3 increased, with the half-time of CP at the barbed end decreasing from 30 minutes without mCAH3 to 10 seconds with a saturating amount of mCAH3. Finally, using mCAH3 tagged with mGFP, we obtained direct evidence that the complex of CP and mCAH3 has a small but measurable affinity for the barbed end, as inferred from previous studies and kinetic modeling. We conclude that the isolated CAH3 domain of CARMIL, and presumably the intact molecule as well, possesses the ability to uncap CP-capped actin filaments.

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National Heart, Lung, and Blood Institute
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Hammer, John A (2018) Myosin goes for blood. Proc Natl Acad Sci U S A 115:4813-4815
Alexander, Christopher J; Wagner, Wolfgang; Copeland, Neal G et al. (2018) Creation of a myosin Va-TAP tagged mouse and identification of potential myosin Va-interacting proteins in the cerebellum. Cytoskeleton (Hoboken) :
Heimsath Jr, Ernest G; Yim, Yang-In; Mustapha, Mirna et al. (2017) Myosin-X knockout is semi-lethal and demonstrates that myosin-X functions in neural tube closure, pigmentation, hyaloid vasculature regression, and filopodia formation. Sci Rep 7:17354
Bruun, Kyle; Beach, Jordan R; Heissler, Sarah M et al. (2017) Re-evaluating the roles of myosin 18A? and F-actin in determining Golgi morphology. Cytoskeleton (Hoboken) 74:205-218
Burman, Jonathon L; Pickles, Sarah; Wang, Chunxin et al. (2017) Mitochondrial fission facilitates the selective mitophagy of protein aggregates. J Cell Biol 216:3231-3247
Varadarajan, Ramya; Hammer, John A; Rusan, Nasser M (2017) A centrosomal scaffold shows some self-control. J Biol Chem 292:20410-20411
Beach, Jordan R; Bruun, Kyle S; Shao, Lin et al. (2017) Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments. Nat Cell Biol 19:85-93
Beach, Jordan R; Hammer 3rd, John A (2015) Myosin II isoform co-assembly and differential regulation in mammalian systems. Exp Cell Res 334:2-9
Li, Dong; Shao, Lin; Chen, Bi-Chang et al. (2015) ADVANCED IMAGING. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 349:aab3500
Billington, Neil; Beach, Jordan R; Heissler, Sarah M et al. (2015) Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments. Curr Biol 25:942-8

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