Publications by authors named "John A Hammer"

78 Publications

MYO10 drives genomic instability and inflammation in cancer.

Sci Adv 2021 Sep 15;7(38):eabg6908. Epub 2021 Sep 15.

Department of Pharmacology, Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.

[Figure: see text].
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http://dx.doi.org/10.1126/sciadv.abg6908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8443186PMC
September 2021

Filopodia powered by class x myosin promote fusion of mammalian myoblasts.

Elife 2021 09 14;10. Epub 2021 Sep 14.

Department of Pharmacology & Therapeutics, University of Florida College of Medicine, Gainesville, United States.

Skeletal muscle fibers are multinucleated cellular giants formed by the fusion of mononuclear myoblasts. Several molecules involved in myoblast fusion have been discovered, and finger-like projections coincident with myoblast fusion have also been implicated in the fusion process. The role of these cellular projections in muscle cell fusion was investigated herein. We demonstrate that these projections are filopodia generated by class X myosin (Myo10), an unconventional myosin motor protein specialized for filopodia. We further show that Myo10 is highly expressed by differentiating myoblasts, and Myo10 ablation inhibits both filopodia formation and myoblast fusion in vitro. In vivo, Myo10 labels regenerating muscle fibers associated with Duchenne muscular dystrophy and acute muscle injury. In mice, conditional loss of from muscle-resident stem cells, known as satellite cells, severely impairs postnatal muscle regeneration. Furthermore, the muscle fusion proteins Myomaker and Myomixer are detected in myoblast filopodia. These data demonstrate that Myo10-driven filopodia facilitate multinucleated mammalian muscle formation.
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http://dx.doi.org/10.7554/eLife.72419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8500716PMC
September 2021

ZEISS Airyscan: Optimizing Usage for Fast, Gentle, Super-Resolution Imaging.

Methods Mol Biol 2021 ;2304:111-130

Cell and Developmental Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA.

The Zeiss Airyscan microscope transforms a diffraction-limited, point-scanning confocal microscope into a super-resolution microscope using a specialized 32-channel Airyscan detector. By improving resolution twofold and signal-to-noise ratio eightfold relative to conventional confocal microscopes while retaining confocal functionality, the Airyscan microscope has become a very popular super-resolution imaging tool for cell biologists. In this chapter, we describe the fundamentals of Airyscan imaging, with the aim of helping the reader determine the proper acquisition settings for different types of experiments, optimize imaging conditions, and process the raw Airyscan images to obtain final images with the best quality. We also provide some tips, tricks, and best practices for Airyscan imaging. Of note, while our focus is on the Airyscan function of this microscope rather than its conventional confocal function, the Airyscan unit comes as an add-on to the conventional Zeiss laser scanning confocal microscope. This protocol is for the first generation Airyscan Zeiss 800 series microscope.
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http://dx.doi.org/10.1007/978-1-0716-1402-0_5DOI Listing
August 2021

Myosin 18Aα targets the guanine nucleotide exchange factor β-Pix to the dendritic spines of cerebellar Purkinje neurons and promotes spine maturation.

FASEB J 2021 01;35(1):e21092

Molecular Cell Biology Laboratory, Cell and Developmental Biology Center, NHLBI, NIH, Bethesda, MD, USA.

Myosin 18Aα is a myosin 2-like protein containing unique N- and C-terminal protein interaction domains that co-assembles with myosin 2. One protein known to bind to myosin 18Aα is β-Pix, a guanine nucleotide exchange factor (GEF) for Rac1 and Cdc42 that has been shown to promote dendritic spine maturation by activating the assembly of actin and myosin filaments in spines. Here, we show that myosin 18A⍺ concentrates in the spines of cerebellar Purkinje neurons via co-assembly with myosin 2 and through an actin binding site in its N-terminal extension. miRNA-mediated knockdown of myosin 18A⍺ results in a significant defect in spine maturation that is rescued by an RNAi-immune version of myosin 18A⍺. Importantly, β-Pix co-localizes with myosin 18A⍺ in spines, and its spine localization is lost upon myosin 18A⍺ knockdown or when its myosin 18A⍺ binding site is deleted. Finally, we show that the spines of myosin 18A⍺ knockdown Purkinje neurons contain significantly less F-actin and myosin 2. Together, these data argue that mixed filaments of myosin 2 and myosin 18A⍺ form a complex with β-Pix in Purkinje neuron spines that promotes spine maturation by enhancing the assembly of actin and myosin filaments downstream of β-Pix's GEF activity.
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http://dx.doi.org/10.1096/fj.202001449RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8357457PMC
January 2021

Myosin 10 Regulates Invasion, Mitosis, and Metabolic Signaling in Glioblastoma.

iScience 2020 Dec 13;23(12):101802. Epub 2020 Nov 13.

Department of Cancer Biology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224, USA.

Invasion and proliferation are defining phenotypes of cancer, and in glioblastoma blocking one stimulates the other, implying that effective therapy must inhibit both, ideally through a single target that is also dispensable for normal tissue function. The molecular motor myosin 10 meets these criteria. Myosin 10 knockout mice can survive to adulthood, implying that normal cells can compensate for its loss; its deletion impairs invasion, slows proliferation, and prolongs survival in murine models of glioblastoma. Myosin 10 deletion also enhances tumor dependency on the DNA damage and the metabolic stress responses and induces synthetic lethality when combined with inhibitors of these processes. Our results thus demonstrate that targeting myosin 10 is active against glioblastoma by itself, synergizes with other clinically available therapeutics, may have acceptable side effects in normal tissues, and has potential as a heretofore unexplored therapeutic approach for this disease.
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http://dx.doi.org/10.1016/j.isci.2020.101802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702012PMC
December 2020

Endoplasmic reticulum visits highly active spines and prevents runaway potentiation of synapses.

Nat Commun 2020 10 8;11(1):5083. Epub 2020 Oct 8.

Institute for Synaptic Plasticity, Center for Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

In hippocampal pyramidal cells, a small subset of dendritic spines contain endoplasmic reticulum (ER). In large spines, ER frequently forms a spine apparatus, while smaller spines contain just a single tubule of smooth ER. Here we show that the ER visits dendritic spines in a non-random manner, targeting spines during periods of high synaptic activity. When we blocked ER motility using a dominant negative approach against myosin V, spine synapses became stronger compared to controls. We were not able to further potentiate these maxed-out synapses, but long-term depression (LTD) was readily induced by low-frequency stimulation. We conclude that the brief ER visits to active spines have the important function of preventing runaway potentiation of individual spine synapses, keeping most of them at an intermediate strength level from which both long-term potentiation (LTP) and LTD are possible.
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http://dx.doi.org/10.1038/s41467-020-18889-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546627PMC
October 2020

The role of actin and myosin in antigen extraction by B lymphocytes.

Semin Cell Dev Biol 2020 06 18;102:90-104. Epub 2019 Dec 18.

Cell and Developmental Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA. Electronic address:

B cells must extract antigens attached to the surface of antigen presenting cells to generate high-affinity antibodies. Antigen extraction requires force, and recent studies have implicated actomyosin-dependent pulling forces generated within the B cell as the major driver of antigen extraction. These actomyosin-dependent pulling forces also serve to test the affinity of the B cell antigen receptor for antigen prior to antigen extraction. Such affinity discrimination is central to the process of antibody affinity maturation. Here we review the evidence that actomyosin-dependent pulling forces generated within the B cell promote affinity discrimination and power antigen extraction. Our take on these critical B cell functions is influenced significantly by the recent identification of formin-generated, myosin-rich, concentric actin arcs in the medial portion of the T cell immune synapse, as B cells appear to contain a similar contractile actomyosin structure.
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http://dx.doi.org/10.1016/j.semcdb.2019.10.017DOI Listing
June 2020

Pulling in new directions: Myosin 2, Piezo, and metabolism.

F1000Res 2019 22;8. Epub 2019 Aug 22.

Cell and Molecular Physiology, Loyola University Chicago, Stritch School of Medicine, Center for Translational Research and Education, Maywood, IL, USA.

Myosin 2 plays a central role in numerous, fundamental, actin-based biological processes, including cell migration, cell division, and the adhesion of cells to substrates and other cells. Here, we highlight recent studies in which the forces created by actomyosin 2 have been shown to also impact tension-sensitive ion channels and cell metabolism.
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http://dx.doi.org/10.12688/f1000research.18856.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713065PMC
May 2020

Myosin V regulates synaptopodin clustering and localization in the dendrites of hippocampal neurons.

J Cell Sci 2019 08 22;132(16). Epub 2019 Aug 22.

DFG Emmy Noether Group 'Neuronal Protein Transport', Center for Molecular Neurobiology, ZMNH, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany

The spine apparatus (SA) is an endoplasmic reticulum-related organelle that is present in a subset of dendritic spines in cortical and pyramidal neurons, and plays an important role in Ca homeostasis and dendritic spine plasticity. The protein synaptopodin is essential for the formation of the SA and is widely used as a maker for this organelle. However, it is still unclear which factors contribute to its localization at selected synapses, and how it triggers local SA formation. In this study, we characterized development, localization and mobility of synaptopodin clusters in hippocampal primary neurons, as well as the molecular dynamics within these clusters. Interestingly, synaptopodin at the shaft-associated clusters is less dynamic than at spinous clusters. We identify the actin-based motor proteins myosin V (herein referring to both the myosin Va and Vb forms) and VI as novel interaction partners of synaptopodin, and demonstrate that myosin V is important for the formation and/or maintenance of the SA. We found no evidence of active microtubule-based transport of synaptopodin. Instead, new clusters emerge inside spines, which we interpret as the SA being assembled on-site.
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http://dx.doi.org/10.1242/jcs.230177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737913PMC
August 2019

Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation.

Elife 2019 06 3;8. Epub 2019 Jun 3.

Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.

When B cells encounter antigens on the surface of an antigen-presenting cell (APC), B cell receptors (BCRs) are gathered into microclusters that recruit signaling enzymes. These microclusters then move centripetally and coalesce into the central supramolecular activation cluster of an immune synapse. The mechanisms controlling BCR organization during immune synapse formation, and how this impacts BCR signaling, are not fully understood. We show that this coalescence of BCR microclusters depends on the actin-related protein 2/3 (Arp2/3) complex, which nucleates branched actin networks. Moreover, in murine B cells, this dynamic spatial reorganization of BCR microclusters amplifies proximal BCR signaling reactions and enhances the ability of membrane-associated antigens to induce transcriptional responses and proliferation. Our finding that Arp2/3 complex activity is important for B cell responses to spatially restricted membrane-bound antigens, but not for soluble antigens, highlights a critical role for Arp2/3 complex-dependent actin remodeling in B cell responses to APC-bound antigens.
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http://dx.doi.org/10.7554/eLife.44574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591008PMC
June 2019

An Improved Method for Differentiating Mouse Embryonic Stem Cells into Cerebellar Purkinje Neurons.

Cerebellum 2019 Jun;18(3):406-421

Cell and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.

While mixed primary cerebellar cultures prepared from embryonic tissue have proven valuable for dissecting structure-function relationships in cerebellar Purkinje neurons (PNs), this technique is technically challenging and often yields few cells. Recently, mouse embryonic stem cells (mESCs) have been successfully differentiated into PNs, although the published methods are very challenging as well. The focus of this study was to simplify the differentiation of mESCs into PNs. Using a recently described neural differentiation media, we generate monolayers of neural progenitor cells from mESCs and differentiate them into PN precursors using specific extrinsic factors. These PN precursors are then differentiated into mature PNs by co-culturing them with granule neuron (GN) precursors also derived from neural progenitors using different extrinsic factors. The morphology of mESC-derived PNs is indistinguishable from PNs grown in primary culture in terms of gross morphology, spine length, and spine density. Furthermore, mESC-derived PNs express Calbindin D28K, IP3R1, IRBIT, PLCβ4, PSD93, and myosin IIB-B2, all of which are either PN-specific or highly expressed in PNs. Moreover, we show that mESC-derived PNs form synapses with GN-like cells as in primary culture, express proteins driven by the PN-specific promoter Pcp2/L7, and exhibit the defect in spine ER inheritance seen in PNs isolated from dilute-lethal (myosin Va-null) mice when expressing a Pcp2/L7-driven miRNA directed against myosin Va. Finally, we define a novel extracellular matrix formulation that reproducibly yields monolayer cultures conducive for high-resolution imaging. Our improved method for differentiating mESCs into PNs should facilitate the dissection of molecular mechanisms and disease phenotypes in PNs.
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http://dx.doi.org/10.1007/s12311-019-1007-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8788662PMC
June 2019

Origin, Organization, Dynamics, and Function of Actin and Actomyosin Networks at the T Cell Immunological Synapse.

Annu Rev Immunol 2019 04 21;37:201-224. Epub 2018 Dec 21.

Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA; email:

The engagement of a T cell with an antigen-presenting cell (APC) or activating surface results in the formation within the T cell of several distinct actin and actomyosin networks. These networks reside largely within a narrow zone immediately under the T cell's plasma membrane at its site of contact with the APC or activating surface, i.e., at the immunological synapse. Here we review the origin, organization, dynamics, and function of these synapse-associated actin and actomyosin networks. Importantly, recent insights into the nature of these actin-based cytoskeletal structures were made possible in several cases by advances in light microscopy.
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http://dx.doi.org/10.1146/annurev-immunol-042718-041341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8343269PMC
April 2019

The Structure of Melanoregulin Reveals a Role for Cholesterol Recognition in the Protein's Ability to Promote Dynein Function.

Structure 2018 10 30;26(10):1373-1383.e4. Epub 2018 Aug 30.

Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

Melanoregulin (Mreg) is a small, highly charged, multiply palmitoylated protein present on the membrane of melanosomes. Mreg is implicated in the transfer of melanosomes from melanocytes to keratinocytes, and in promoting the microtubule minus end-directed transport of these organelles. The possible molecular function of Mreg was identified by solving its structure using nuclear magnetic resonance (NMR) spectroscopy. Mreg contains six α helices forming a fishhook-like fold in which positive and negative charges occupy opposite sides of the protein's surface and sandwich a putative, cholesterol recognition sequence (CRAC motif). Mreg containing a point mutation within its CRAC motif still targets to late endosomes/lysosomes, but no longer promotes their microtubule minus end-directed transport. Moreover, wild-type Mreg does not promote the microtubule minus end-directed transport of late endosomes/lysosomes in cells transiently depleted of cholesterol. Finally, reversing the charge of three clustered acidic residues partially inhibits Mreg's ability to drive these organelles to microtubule minus ends.
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http://dx.doi.org/10.1016/j.str.2018.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170685PMC
October 2018

Creation of a myosin Va-TAP-tagged mouse and identification of potential myosin Va-interacting proteins in the cerebellum.

Cytoskeleton (Hoboken) 2018 09;75(9):395-409

Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland.

The actin-based motor myosin Va transports numerous cargos, including the smooth endoplasmic reticulum (SER) in cerebellar Purkinje neurons (PNs) and melanosomes in melanocytes. Identifying proteins that interact with this myosin is key to understanding its cellular functions. Toward that end, we used recombineering to insert via homologous recombination a tandem affinity purification (TAP) tag composed of the immunoglobulin G-binding domain of protein A, a tobacco etch virus cleavage site, and a FLAG tag into the mouse MYO5A locus immediately after the initiation codon. Importantly, we provide evidence that the TAP-tagged version of myosin Va (TAP-MyoVa) functions normally in terms of SER transport in PNs and melanosome positioning in melanocytes. Given this and other evidence that TAP-MyoVa is fully functional, we purified it together with associated proteins directly from juvenile mouse cerebella and subjected the samples to mass spectroscopic analyses. As expected, known myosin Va-binding partners like dynein light chain were identified. Importantly, numerous novel interacting proteins were also tentatively identified, including guanine nucleotide-binding protein G(o) subunit alpha (Gnao1), a biomarker for schizophrenia. Consistently, an antibody to Gnao1 immunoprecipitates myosin Va, and Gnao1's localization to PN dendritic spines depends on myosin Va. The mouse model created here should facilitate the identification of novel myosin Va-binding partners, which in turn should advance our understanding of the roles played by this important myosin in vivo.
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http://dx.doi.org/10.1002/cm.21474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8365777PMC
September 2018

Myosin goes for blood.

Authors:
John A Hammer

Proc Natl Acad Sci U S A 2018 05 24;115(19):4813-4815. Epub 2018 Apr 24.

Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892

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http://dx.doi.org/10.1073/pnas.1805253115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5949016PMC
May 2018

Immunology: Is Actin at the Lytic Synapse a Friend or a Foe?

Authors:
John A Hammer

Curr Biol 2018 02;28(4):R155-R157

Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Building 50, Room 2306, 9000 Rockville Pike, Bethesda, MD 20892, USA. Electronic address:

Cytotoxic T cells and natural killer cells defend us against disease by secreting lytic granules. Whether actin facilitates or thwarts lytic granule secretion has been an open question. Recent results now indicate that the answer depends on the maturation stage of the immune cell-target cell contact.
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http://dx.doi.org/10.1016/j.cub.2018.01.013DOI Listing
February 2018

A centrosomal scaffold shows some self-control.

J Biol Chem 2017 12;292(50):20410-20411

From the Cell Biology and Physiology Center, NHLBI, National Institutes of Health, Bethesda Maryland 20892

The scaffolding protein AKAP350A is known to localize to the centrosome and the Golgi, but the molecular details of its function at the centrosome remain elusive. Using structure-function analyses, protein interaction assays, and super-resolution microscopy, Kolobova now identify AKAP350A's specific location and protein partners at the centrosome. The authors further define an autoregulatory mechanism that likely controls AKAP350A's ability to nucleate microtubule growth.
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http://dx.doi.org/10.1074/jbc.H117.806018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733580PMC
December 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 2017 12 11;7(1):17354. Epub 2017 Dec 11.

Department of Cell Biology and Physiology and Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.

Myosin-X (Myo10) is an unconventional myosin best known for its striking localization to the tips of filopodia. Despite the broad expression of Myo10 in vertebrate tissues, its functions at the organismal level remain largely unknown. We report here the generation of KO-first (Myo10 ), floxed (Myo10 ), and KO mice (Myo10 ). Complete knockout of Myo10 is semi-lethal, with over half of homozygous KO embryos exhibiting exencephaly, a severe defect in neural tube closure. All Myo10 KO mice that survive birth exhibit a white belly spot, all have persistent fetal vasculature in the eye, and ~50% have webbed digits. Myo10 KO mice that survive birth can breed and produce litters of KO embryos, demonstrating that Myo10 is not absolutely essential for mitosis, meiosis, adult survival, or fertility. KO-first mice and an independent spontaneous deletion (Myo10 ) exhibit the same core phenotypes. During retinal angiogenesis, KO mice exhibit a ~50% decrease in endothelial filopodia, demonstrating that Myo10 is required to form normal numbers of filopodia in vivo. The Myo10 mice generated here demonstrate that Myo10 has important functions in mammalian development and provide key tools for defining the functions of Myo10 in vivo.
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http://dx.doi.org/10.1038/s41598-017-17638-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5725431PMC
December 2017

Mitochondrial fission facilitates the selective mitophagy of protein aggregates.

J Cell Biol 2017 10 11;216(10):3231-3247. Epub 2017 Sep 11.

Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD

Within the mitochondrial matrix, protein aggregation activates the mitochondrial unfolded protein response and PINK1-Parkin-mediated mitophagy to mitigate proteotoxicity. We explore how autophagy eliminates protein aggregates from within mitochondria and the role of mitochondrial fission in mitophagy. We show that PINK1 recruits Parkin onto mitochondrial subdomains after actinonin-induced mitochondrial proteotoxicity and that PINK1 recruits Parkin proximal to focal misfolded aggregates of the mitochondrial-localized mutant ornithine transcarbamylase (ΔOTC). Parkin colocalizes on polarized mitochondria harboring misfolded proteins in foci with ubiquitin, optineurin, and LC3. Although inhibiting Drp1-mediated mitochondrial fission suppresses the segregation of mitochondrial subdomains containing ΔOTC, it does not decrease the rate of ΔOTC clearance. Instead, loss of Drp1 enhances the recruitment of Parkin to fused mitochondrial networks and the rate of mitophagy as well as decreases the selectivity for ΔOTC during mitophagy. These results are consistent with a new model that, instead of promoting mitophagy, fission protects healthy mitochondrial domains from elimination by unchecked PINK1-Parkin activity.
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http://dx.doi.org/10.1083/jcb.201612106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626535PMC
October 2017

Contractile actomyosin arcs promote the activation of primary mouse T cells in a ligand-dependent manner.

PLoS One 2017 17;12(8):e0183174. Epub 2017 Aug 17.

Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, United States of America.

Mechano-transduction is an emerging but still poorly understood component of T cell activation. Here we investigated the ligand-dependent contribution made by contractile actomyosin arcs populating the peripheral supramolecular activation cluster (pSMAC) region of the immunological synapse (IS) to T cell receptor (TCR) microcluster transport and proximal signaling in primary mouse T cells. Using super resolution microscopy, OT1-CD8+ mouse T cells, and two ovalbumin (OVA) peptides with different affinities for the TCR, we show that the generation of organized actomyosin arcs depends on ligand potency and the ability of myosin 2 to contract actin filaments. While weak ligands induce disorganized actomyosin arcs, strong ligands result in organized actomyosin arcs that correlate well with tension-sensitive CasL phosphorylation and the accumulation of ligands at the IS center. Blocking myosin 2 contractility greatly reduces the difference in the extent of Src and LAT phosphorylation observed between the strong and the weak ligand, arguing that myosin 2-dependent force generation within actin arcs contributes to ligand discrimination. Together, our data are consistent with the idea that actomyosin arcs in the pSMAC region of the IS promote a mechano-chemical feedback mechanism that amplifies the accumulation of critical signaling molecules at the IS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183174PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5560663PMC
October 2017

Re-evaluating the roles of myosin 18Aα and F-actin in determining Golgi morphology.

Cytoskeleton (Hoboken) 2017 May 10;74(5):205-218. Epub 2017 May 10.

Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA.

The peri-centrosomal localization and morphology of the Golgi apparatus depends largely on the microtubule cytoskeleton and the microtubule motor protein dynein. Recent studies proposed that myosin 18Aα (M18Aα) also contributes to Golgi morphology by binding the Golgi protein GOLPH3 and walking along adjacent actin filaments to stretch the Golgi into its classic ribbon structure. Biochemical analyses have shown, however, that M18A is not an actin-activated ATPase and lacks motor activity. Our goal, therefore, was to define the precise molecular mechanism by which M18Aα determines Golgi morphology. We show that purified M18Aα remains inactive in the presence of GOLPH3, arguing against the Golgi-specific activation of the myosin. Using M18A-specific antibodies and expression of GFP-tagged M18Aα, we find no evidence that it localizes to the Golgi. Moreover, several cell lines with reduced or eliminated M18Aα expression exhibited normal Golgi morphology. Interestingly, actin filament disassembly resulted in a marked reduction in lateral stretching of the Golgi in both control and M18Aα-deficient cells. Importantly, this reduction was accompanied by an expansion of the Golgi in the vertical direction, vertical movement of the centrosome, and increases in the height of both the nucleus and the cell. Collectively, our data indicate that M18Aα does not localize to the Golgi or play a significant role in determining its morphology, and suggest that global F-actin disassembly alters Golgi morphology indirectly by altering cell shape.
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http://dx.doi.org/10.1002/cm.21364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8579491PMC
May 2017

Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments.

Nat Cell Biol 2017 02 23;19(2):85-93. Epub 2017 Jan 23.

Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health Bethesda, Maryland 20892, USA.

The cellular mechanisms governing non-muscle myosin II (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamellae, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibres, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibres to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.
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http://dx.doi.org/10.1038/ncb3463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5308804PMC
February 2017

Formin-generated actomyosin arcs propel T cell receptor microcluster movement at the immune synapse.

J Cell Biol 2016 Nov 31;215(3):383-399. Epub 2016 Oct 31.

Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892

Actin assembly and inward flow in the plane of the immunological synapse (IS) drives the centralization of T cell receptor microclusters (TCR MCs) and the integrin leukocyte functional antigen 1 (LFA-1). Using structured-illumination microscopy (SIM), we show that actin arcs populating the medial, lamella-like region of the IS arise from linear actin filaments generated by one or more formins present at the IS distal edge. After traversing the outer, Arp2/3-generated, lamellipodia-like region of the IS, these linear filaments are organized by myosin II into antiparallel concentric arcs. Three-dimensional SIM shows that active LFA-1 often aligns with arcs, whereas TCR MCs commonly reside between arcs, and total internal reflection fluorescence SIM shows TCR MCs being swept inward by arcs. Consistently, disrupting actin arc formation via formin inhibition results in less centralized TCR MCs, missegregated integrin clusters, decreased T-B cell adhesion, and diminished TCR signaling. Together, our results define the origin, organization, and functional significance of a major actomyosin contractile structure at the IS that directly propels TCR MC transport.
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http://dx.doi.org/10.1083/jcb.201603080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5100289PMC
November 2016

V-1 regulates capping protein activity in vivo.

Proc Natl Acad Sci U S A 2016 10 10;113(43):E6610-E6619. Epub 2016 Oct 10.

Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892;

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."
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http://dx.doi.org/10.1073/pnas.1605350113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087048PMC
October 2016

ADVANCED IMAGING. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics.

Science 2015 Aug;349(6251):aab3500

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.

Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.
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http://dx.doi.org/10.1126/science.aab3500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4659358PMC
August 2015

Cooperative interactions of LPPR family members in membrane localization and alteration of cellular morphology.

J Cell Sci 2015 Sep 16;128(17):3210-22. Epub 2015 Jul 16.

Developmental Neurobiology Section, Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA

The lipid phosphate phosphatase-related proteins (LPPRs), also known as plasticity-related genes (PRGs), are classified as a new brain-enriched subclass of the lipid phosphate phosphatase (LPP) superfamily. They induce membrane protrusions, neurite outgrowth or dendritic spine formation in cell lines and primary neurons. However, the exact roles of LPPRs and the mechanisms underlying their effects are not certain. Here, we present the results of a large-scale proteome analysis to determine LPPR1-interacting proteins using co-immunoprecipitation coupled to mass spectrometry. We identified putative LPPR1-binding proteins involved in various biological processes. Most interestingly, we identified the interaction of LPPR1 with its family member LPPR3, LPPR4 and LPPR5. Their interactions were characterized by co-immunoprecipitation and colocalization analysis using confocal and super-resolution microscopy. Moreover, co-expressing two LPPR members mutually elevated their protein levels, facilitated their plasma membrane localization and resulted in an increased induction of membrane protrusions as well as the phosphorylation of S6 ribosomal protein. Taken together, we revealed a new functional cooperation between LPPR family members and discovered for the first time that LPPRs likely exert their function through forming complex with its family members.
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http://dx.doi.org/10.1242/jcs.169789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4582190PMC
September 2015

Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments.

Curr Biol 2015 Mar 5;25(7):942-8. Epub 2015 Mar 5.

Laboratory of Molecular Physiology, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892-8015, USA. Electronic address:

Class-18 myosins are most closely related to conventional class-2 nonmuscle myosins (NM2). Surprisingly, the purified head domains of Drosophila, mouse, and human myosin 18A (M18A) lack actin-activated ATPase activity and the ability to translocate actin filaments, suggesting that the functions of M18A in vivo do not depend on intrinsic motor activity. M18A has the longest coiled coil of any myosin outside of the class-2 myosins, suggesting that it might form bipolar filaments similar to conventional myosins. To address this possibility, we expressed and purified full-length mouse M18A using the baculovirus/Sf9 system. M18A did not form large bipolar filaments under any of the conditions tested. Instead, M18A formed an ∼ 65-nm-long bipolar structure with two heads at each end. Importantly, when NM2 was polymerized in the presence of M18A, the two myosins formed mixed bipolar filaments, as evidenced by cosedimentation, electron microscopy, and single-molecule imaging. Moreover, super-resolution imaging of NM2 and M18A using fluorescently tagged proteins and immunostaining of endogenous proteins showed that NM2 and M18A are present together within individual filaments inside living cells. Together, our in vitro and live-cell imaging data argue strongly that M18A coassembles with NM2 into mixed bipolar filaments. M18A could regulate the biophysical properties of these filaments and, by virtue of its extra N- and C-terminal domains, determine the localization and/or molecular interactions of the filaments. Given the numerous, fundamental cellular and developmental roles attributed to NM2, our results have far-reaching biological implications.
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http://dx.doi.org/10.1016/j.cub.2015.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8760901PMC
March 2015

Myosin II isoform co-assembly and differential regulation in mammalian systems.

Exp Cell Res 2015 May 2;334(1):2-9. Epub 2015 Feb 2.

Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

Non-muscle myosin 2 (NM2) is a major force-producing, actin-based motor in mammalian non-muscle cells, where it plays important roles in a broad range of fundamental biological processes, including cytokinesis, cell migration, and epithelial barrier function. This breadth of function at the tissue and cellular levels suggests extensive diversity and differential regulation of NM2 bipolar filaments, the major, if not sole, functional form of NM2s in vivo. Previous in vitro, cellular and animal studies indicate that some of this diversity is supported by the existence of multiple NM2 isoforms. Moreover, two recent studies have shown that these isoforms can co-assemble to form heterotypic filaments, further expanding functional diversity. In addition to isoform co-assembly, cells may differentially regulate NM2 function via isoform-specific expression, RLC phosphorylation, MHC phosphorylation or regulation via binding partners. Here, we provide a brief summary of NM2 filament assembly, summarize the recent findings regarding NM2 isoform co-assembly, consider the mechanisms cells might utilize to differentially regulate NM2 isoforms, and review the data available to support these mechanisms.
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http://dx.doi.org/10.1016/j.yexcr.2015.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433797PMC
May 2015

Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.

Science 2014 Oct 23;346(6208):1257998. Epub 2014 Oct 23.

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.
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http://dx.doi.org/10.1126/science.1257998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336192PMC
October 2014
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