scholarly journals Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

2017 ◽  
Vol 216 (12) ◽  
pp. 4123-4139 ◽  
Author(s):  
Wei-Ke Ji ◽  
Rajarshi Chakrabarti ◽  
Xintao Fan ◽  
Lori Schoenfeld ◽  
Stefan Strack ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Actin Polymerization ◽  
Mitochondrial Division ◽  
Multiple Factors ◽  
Guanosine Triphosphatase

Drp1 is a dynamin guanosine triphosphatase important for mitochondrial and peroxisomal division. Drp1 oligomerization and mitochondrial recruitment are regulated by multiple factors, including interaction with mitochondrial receptors such as Mff, MiD49, MiD51, and Fis. In addition, both endoplasmic reticulum (ER) and actin filaments play positive roles in mitochondrial division, but mechanisms for their roles are poorly defined. Here, we find that a population of Drp1 oligomers is associated with ER in mammalian cells and is distinct from mitochondrial or peroxisomal Drp1 populations. Subpopulations of Mff and Fis1, which are tail-anchored proteins, also localize to ER. Drp1 oligomers assemble on ER, from which they can transfer to mitochondria. Suppression of Mff or inhibition of actin polymerization through the formin INF2 significantly reduces all Drp1 oligomer populations (mitochondrial, peroxisomal, and ER bound) and mitochondrial division, whereas Mff targeting to ER has a stimulatory effect on division. Our results suggest that ER can function as a platform for Drp1 oligomerization, and that ER-associated Drp1 contributes to mitochondrial division.

scholarly journals Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

2017 ◽  
Author(s):  
Wei-Ke Ji ◽  
Rajarshi Chakrabarti ◽  
Xintao Fan ◽  
Lori Schoenfeld ◽  
Stefan Strack ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Actin Polymerization ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Outer Mitochondrial Membrane ◽  
Mitochondrial Division ◽  
Knock Out ◽  
Multiple Factors ◽  
Latrunculin A

AbstractDrpl is a dynamin GTPase important for mitochondrial and peroxisomal division. Drp1 oligomerization and mitochondrial recruitment are regulated by multiple factors, including interaction with mitochondrial receptors such as Mff, MiD49, MiD51 and Fis. In addition, both endoplasmic reticulum (ER) and actin filaments play positive roles in mitochondrial division, but mechanisms for their roles are poorly defined. Here, we find that a population of Drp1 oligomers is ER-associated in mammalian cells, and is distinct from mitochondrial or peroxisomal Drp1 populations. Sub-populations of Mff and Fis1, which are tail-anchored proteins, also localize to ER. Drp1 oligomers assemble on ER, from which they can transfer to mitochondria. Suppression of Mff or inhibition of actin polymerization through the formin INF2 significantly reduces all Drp1 oligomer populations (mitochondrial, peroxisomal, ER-bound) and mitochondrial division, while Mff targeting to ER has a stimulatory effect on division. Our results suggest that ER can function as a platform for Drp1 oligomerization, and that ER-associated Drp1 contributes to mitochondrial division.SummaryAssembly of the dynamin GTPase Drp1 into constriction-competent oligomers is a key event in mitochondrial division. Here, Ji et al show that Drp1 oligomerization can occur on endoplasmic reticulum through an ER-bound population of the tail-anchored protein Mff.Abbreviations used in this paper: Drp1, dynamin-related protein 1; Fis1, mitochondrial fission 1 protein; INF2, inverted formin 2; KD, siRNA-mediated knock down; KI, CRISPR-mediated knock in; KO, CRISPR-mediated knock out; LatA, Latrunculin A; MDV, mitochondrially-derived vesicle; Mff, mitochondrial fission factor; MiD49 and MiD51, mitochondrial dynamics protein of 49 and 51 kDa; OMM, outer mitochondrial membrane; TA, tail-anchored.

scholarly journals ER–mitochondria contacts: Actin dynamics at the ER control mitochondrial fission via calcium release

2017 ◽  
Vol 217 (1) ◽  
pp. 15-17 ◽  
Author(s):  
Janos Steffen ◽  
Carla M. Koehler
Keyword(s):  
Endoplasmic Reticulum ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Calcium Uptake ◽  
Calcium Release ◽  
Mitochondrial Fission ◽  
Actin Dynamics ◽  
Mitochondrial Division ◽  
Cell Biol ◽  
Important Player

The formin-like protein INF2 is an important player in the polymerization of actin filaments. In this issue, Chakrabarti et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201709111) demonstrate that INF2 mediates actin polymerization at the endoplasmic reticulum (ER), resulting in increased ER–mitochondria contacts, calcium uptake by mitochondria, and mitochondrial division.

scholarly journals Actin enhances ER-mitochondrial calcium transfer and IMM constriction during mitochondrial division

2017 ◽  
Author(s):  
Rajarshi Chakrabarti ◽  
Wei-Ke Ji ◽  
Radu V. Stan ◽  
Jaime de Juan Sanz ◽  
Timothy A. Ryan ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Actin Polymerization ◽  
Mitochondrial Calcium ◽  
Mitochondrial Membranes ◽  
Independent Manner ◽  
Mitochondrial Division ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Transport Chain

SummaryMitochondrial division requires division of both the inner and outer mitochondrial membranes (IMM and OMM, respectively). Interaction with endoplasmic reticulum (ER) promotes OMM division by recruitment of the dynamin Drp1, but effects on IMM division are not well characterized. We previously showed that actin polymerization through the ER-bound formin INF2 stimulates Drp1 recruitment in mammalian cells. Here, we show that INF2-mediated actin polymerization stimulates a second mitochondrial response independent of Drp1: a rise in mitochondrial matrix calcium through the mitochondrial calcium uniporter. ER stores supply the increased mitochondrial calcium, and the role of actin is to increase ER-mitochondria contact. Myosin IIA is also required for this mitochondrial calcium increase. Elevated mitochondrial calcium in turn activates IMM constriction in a Drp1-independent manner. IMM constriction requires electron transport chain activity. IMM division precedes OMM division. These results demonstrate that actin polymerization independently stimulates the dynamics of both membranes during mitochondrial division: IMM through increased matrix calcium, and OMM through Drp1 recruitment.

scholarly journals A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Uri Manor ◽  
Sadie Bartholomew ◽  
Gonen Golani ◽  
Eric Christenson ◽  
Michael Kozlov ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Assembly ◽  
Mitochondrial Division

Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.

scholarly journals A role for cofilin and LIM kinase in Listeria-induced phagocytosis

2001 ◽  
Vol 155 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Hélène Bierne ◽  
Edith Gouin ◽  
Pascal Roux ◽  
Pico Caroni ◽  
Helen L. Yin ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Actin Polymerization ◽  
Essential Feature ◽  
Dominant Negative ◽  
Comet Tail ◽  
Lim Kinase ◽  
Induced Membrane ◽  
Membrane Ruffling

The pathogenic bacterium Listeria monocytogenes is able to invade nonphagocytic cells, an essential feature for its pathogenicity. This induced phagocytosis process requires tightly regulated steps of actin polymerization and depolymerization. Here, we investigated how interactions of the invasion protein InlB with mammalian cells control the cytoskeleton during Listeria internalization. By fluorescence microscopy and transfection experiments, we show that the actin-nucleating Arp2/3 complex, the GTPase Rac, LIM kinase (LIMK), and cofilin are key proteins in InlB-induced phagocytosis. Overexpression of LIMK1, which has been shown to phosphorylate and inactivate cofilin, induces accumulation of F-actin beneath entering particles and inhibits internalization. Conversely, inhibition of LIMK's activity by expressing a dominant negative construct, LIMK1−, or expression of the constitutively active S3A cofilin mutant induces loss of actin filaments at the phagocytic cup and also inhibits phagocytosis. Interestingly, those constructs similarly affect other actin-based phenomenons, such as InlB-induced membrane ruffling or Listeria comet tail formations. Thus, our data provide evidence for a control of phagocytosis by both activation and deactivation of cofilin. We propose a model in which cofilin is involved in the formation and disruption of the phagocytic cup as a result of its local progressive enrichment.

scholarly journals INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division

2017 ◽  
Vol 217 (1) ◽  
pp. 251-268 ◽  
Author(s):  
Rajarshi Chakrabarti ◽  
Wei-Ke Ji ◽  
Radu V. Stan ◽  
Jaime de Juan Sanz ◽  
Timothy A. Ryan ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Actin Polymerization ◽  
Calcium Uptake ◽  
Mitochondrial Calcium ◽  
Independent Manner ◽  
Mitochondrial Division ◽  
Calcium Uniporter ◽  
Transport Chain ◽  
Mitochondrial Calcium Uptake

Mitochondrial division requires division of both the inner and outer mitochondrial membranes (IMM and OMM, respectively). Interaction with endoplasmic reticulum (ER) promotes OMM division by recruitment of the dynamin Drp1, but effects on IMM division are not well characterized. We previously showed that actin polymerization through ER-bound inverted formin 2 (INF2) stimulates Drp1 recruitment in mammalian cells. Here, we show that INF2-mediated actin polymerization stimulates a second mitochondrial response independent of Drp1: a rise in mitochondrial matrix calcium through the mitochondrial calcium uniporter. ER stores supply the increased mitochondrial calcium, and the role of actin is to increase ER–mitochondria contact. Myosin IIA is also required for this mitochondrial calcium increase. Elevated mitochondrial calcium in turn activates IMM constriction in a Drp1-independent manner. IMM constriction requires electron transport chain activity. IMM division precedes OMM division. These results demonstrate that actin polymerization independently stimulates the dynamics of both membranes during mitochondrial division: IMM through increased matrix calcium, and OMM through Drp1 recruitment.

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

2019 ◽  
Vol 476 (21) ◽  
pp. 3241-3260
Author(s):  
Sindhu Wisesa ◽  
Yasunori Yamamoto ◽  
Toshiaki Sakisaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Transmembrane Domains ◽  
Domain Family ◽  
Coiled Coil Domain ◽  
U2os Cells ◽  
Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

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