Differential Distribution of Dynamin Isoforms in Mammalian Cells

Cao, Hong; García, Fernando; McNiven, Mark A.

doi:10.1091/mbc.9.9.2595

Cited by 370 publications

(426 citation statements)

References 52 publications

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“…Human dynamin 3 (splice form abb 31 , residues and indicated mutants of this construct were expressed from pProEx-HTb (Invitrogen) as Nterminal His 6 -tag fusion followed by a tobacco etch virus (TEV) cleavage site. The crystallized construct contained the K361S mutation.…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of the dynamin tetramer

Reubold

Faelber

Plattner

et al. 2015

Nature

122

160

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The mechano-chemical protein dynamin is the prototype of the dynamin superfamily of large GTPases, which shape and remodel membranes in diverse cellular processes 1 .Dynamin forms predominantly tetramers in the cytosol, which oligomerize at the neck of clathrin-coated vesicles to mediate constriction and subsequent scission of the membrane 1 . Previous studies have described the architecture of dynamin dimers 2,3 , but the molecular determinants for dynamin assembly and its regulation have remained unclear. Here, we present the crystal structure of the nucleotide-free dynamin tetramer.Combining structural data with mutational studies, oligomerization measurements and molecular dynamics simulations, we suggest a mechanism of how oligomerization of dynamin is linked to the release of intramolecular auto-inhibitory interactions. We elucidate how mutations that interfere with tetramer formation and auto-inhibition can lead to the congenital diseases Charcot-Marie-Tooth neuropathy (CMT) 4 and centronuclear myopathy (CNM) 5 , respectively. Strikingly, the bent shape of the tetramer explains how dynamin assembles into a right-handed helical oligomer of defined diameter, which has direct implications for its function in membrane constriction.The three highly conserved vertebrate isoforms of dynamin contain five distinct domains (Extended Data Fig. 1a): an N-terminal GTPase (G) domain mediating nucleotide binding and hydrolysis, a bundle signaling element (BSE), a stalk, a pleckstrin homology (PH) domain involved in lipid binding, and a proline rich domain (PRD) mediating interaction with BAR-and SH3-domain containing scaffolding proteins 6 . To exert its function in clathrinmediated endocytosis (CME), dynamin assembles via the stalks into a helical array surrounding the necks of invaginating clathrin-coated pits (CCP) 7,8 . Dimerization of GTPbound G domains from neighboring helical rungs induces GTP hydrolysis 9 . The ensuing conformational changes are thought to be transmitted from the G domain via the BSE to the stalk resulting in a sliding motion of adjacent helix rungs, concomitant helix constriction 10 , and eventually membrane scission. The inherent tendency to form large assemblies at high protein concentrations has hampered crystallization of dynamin in the past. The use of non-3 oligomerizing mutants led to crystal structures of dynamin 1 2,3 . However, the postulated higher-order assembly interface was not resolved in these structures leaving the oligomerization mechanism unaddressed.We reasoned that an alternative assembly-affecting mutation, such as K361S in dynamin 3 11 , may disturb the oligomerization interface to a lesser extent than the previously used mutants. We obtained crystals of nucleotide-free dynamin 3-K361S lacking the PRD (dynamin 3(∆PRD)-K361S) that diffracted to 3.7 Å (Methods, Extended Data Fig. 1, Extended Data Table 1). Indeed, the asymmetric unit of the crystal lattice contained a dynamin tetramer that did not form the filamentous superstructures seen for dynamin 1 2,3 .The dynamin tetr...

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Section: Methodsmentioning

confidence: 99%

Crystal structure of the dynamin tetramer

Reubold

Faelber

Plattner

et al. 2015

Nature

122

160

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“…The plasmid encoding dynamin-2-GFP (the dynamin isoform aa) was a gift from Mark A. McNiven (Mayo Clinic, Rochester, MN) (Cao et al, 1998). Plasmid encoding td-Tomato-LCa ) was a gift from Tomás Kirchausen (Harvard Medical School, Children's Hospital and Immune Disease Institute, Boston, MA).…”

Section: Plasmidsmentioning

confidence: 99%

Endosomal clathrin drives actin accumulation at the immunological synapse

Calabia-Linares

Robles‐Valero

Fuente

et al. 2011

Journal of Cell Science

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SummaryAntigen-specific cognate interaction of T lymphocytes with antigen-presenting cells (APCs) drives major morphological and functional changes in T cells, including actin rearrangements at the immune synapse (IS) formed at the cell-cell contact area. Here we show, using cell lines as well as primary cells, that clathrin, a protein involved in endocytic processes, drives actin accumulation at the IS. Clathrin is recruited towards the IS with parallel kinetics to that of actin. Knockdown of clathrin prevents accumulation of actin and proteins involved in actin polymerization, such as dynamin-2, the Arp2/3 complex and CD2AP at the IS. The clathrin pool involved in actin accumulation at the IS is linked to multivesicular bodies that polarize to the cell-cell contact zone, but not to plasma membrane or Golgi complex. These data underscore the role of clathrin as a platform for the recruitment of proteins that promote actin polymerization at the interface of T cells and APCs.

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“…In mammals, dynamin is encoded by 3 genes (32): Dnm1, Dnm2, and Dnm3. Dynamin 1 is highly expressed in the nervous system, dynamin 2 is ubiquitous in all tissues, and dynamin 3 is found primarily in neuronal tissues and at low levels in testis (35,36). At nerve terminals, dynamin 1 and 3 contribute to synaptic vesicle reformation and neurotransmission (36)(37)(38)(39)(40); synapses lacking dynamin 1 (and dynamin 3) have fewer vesicles and abundant clathrin-coated profiles (CCPs) accumulated on the PM (36,38,40).…”

Section: Introductionmentioning

confidence: 99%