scholarly journals Membrane Dynamics at the Nuclear Exchange Junction during Early Mating (One to Four Hours) in the Ciliate Tetrahymena thermophila

2014 ◽  
Vol 14 (2) ◽  
pp. 116-127 ◽  
Author(s):  
Eric S. Cole ◽  
Thomas H. Giddings ◽  
Courtney Ozzello ◽  
Mark Winey ◽  
Eileen O'Toole ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Extracellular Space ◽  
Tetrahymena Thermophila ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Membrane Dynamics ◽  
Mating Partner ◽  
Signaling Mechanism ◽  
Content Type ◽  
Nuclear Exchange

ABSTRACT Using serial-section transmission electron microscopy and three-dimensional (3D) electron tomography, we characterized membrane dynamics that accompany the construction of a nuclear exchange junction between mating cells in the ciliate Tetrahymena thermophila . Our methods revealed a number of previously unknown features. (i) Membrane fusion is initiated by the extension of hundreds of 50-nm-diameter protrusions from the plasma membrane. These protrusions extend from both mating cells across the intercellular space to fuse with membrane of the mating partner. (ii) During this process, small membrane-bound vesicles or tubules are shed from the plasma membrane and into the extracellular space within the junction. The resultant vesicle-filled pockets within the extracellular space are referred to as junction lumens. (iii) As junction lumens fill with extracellular microvesicles and swell, the plasma membrane limiting these swellings undergoes another deformation, pinching off vesicle-filled vacuoles into the cytoplasm (reclamation). (iv) These structures (resembling multivesicular bodies) seem to associate with autophagosomes abundant near the exchange junction. We propose a model characterizing the membrane-remodeling events that establish cytoplasmic continuity between mating Tetrahymena cells. We also discuss the possible role of nonvesicular lipid transport in conditioning the exchange junction lipid environment. Finally, we raise the possibility of an intercellular signaling mechanism involving microvesicle shedding and uptake.

scholarly journals Cellular electron tomography of the apical complex in the apicomplexan parasite Eimeria tenella shows a highly organised gateway for regulated secretion.

2021 ◽  
Author(s):  
Alana Burrell ◽  
Virginia Marugan-Hernandez ◽  
Flavia Moreira-Leite ◽  
David J P Ferguson ◽  
Fiona M Tomley ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Electron Tomography ◽  
Parasitophorous Vacuole ◽  
Three Dimensional ◽  
Eimeria Tenella ◽  
Structural Explanation ◽  
Apicomplexan Parasites ◽  
Host Cytoplasm ◽  
Fibre Number ◽  
Apical Complex

The apical complex of apicomplexan parasites is essential for host cell invasion and intracellular survival and as the site of regulated exocytosis from specialised secretory organelles called rhoptries and micronemes. Despite its importance, there is little data on the three-dimensional organisation and quantification of these organelles within the apical complex or how they are trafficked to this specialised region of plasma membrane for exocytosis. In coccidian apicomplexans there is an additional tubulin-containing hollow barrel structure, the conoid, which provides a structural gateway for this specialised secretion. Using a combination of cellular electron tomography and serial block face-scanning electron microscopy (SBF-SEM) we have reconstructed the entire apical end of Eimeria tenella sporozoites. We discovered that conoid fibre number varied, but there was a fixed spacing between fibres, leading to conoids of different sizes. Associated apical structures varied in size to accommodate a larger or smaller conoid diameter. However, the number of subpellicular microtubules on the apical polar ring surrounding the conoid did not vary, suggesting a control of apical complex size. We quantified the number and location of rhoptries and micronemes within cells and show a highly organised gateway for trafficking and docking of rhoptries, micronemes and vesicles within the conoid around a set of intra-conoidal microtubules. Finally, we provide ultrastructural evidence for fusion of rhoptries directly through the parasite plasma membrane early in infection and the presence of a pore in the parasitophorous vacuole membrane, providing a structural explanation for how rhoptry proteins (ROPs) may be trafficked between the parasite and the host cytoplasm

scholarly journals A 3D analysis of yeast ER structure reveals how ER domains are organized by membrane curvature

2011 ◽  
Vol 193 (2) ◽  
pp. 333-346 ◽  
Author(s):  
Matt West ◽  
Nesia Zurek ◽  
Andreas Hoenger ◽  
Gia K. Voeltz
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Membrane Curvature ◽  
3D Analysis ◽  
Wild Type ◽  
Nanometer Resolution ◽  
Ribosome Density ◽  
Multiple Stages

We analyzed the structure of yeast endoplasmic reticulum (ER) during six sequential stages of budding by electron tomography to reveal a three-dimensional portrait of ER organization during inheritance at a nanometer resolution. We have determined the distribution, dimensions, and ribosome densities of structurally distinct but continuous ER domains during multiple stages of budding with and without the tubule-shaping proteins, reticulons (Rtns) and Yop1. In wild-type cells, the peripheral ER contains cytoplasmic cisternae, many tubules, and a large plasma membrane (PM)–associated ER domain that consists of both tubules and fenestrated cisternae. In the absence of Rtn/Yop1, all three domains lose membrane curvature, ER ribosome density changes, and the amount of PM-associated ER increases dramatically. Deletion of Rtns/Yop1 does not, however, prevent bloated ER tubules from being pulled from the mother cisterna into the bud and strongly suggests that Rtns/Yop1 stabilize/maintain rather than generate membrane curvature at all peripheral ER domains in yeast.

scholarly journals Organization of Organelles within Hyphae of Ashbya gossypii Revealed by Electron Tomography

2013 ◽  
Vol 12 (11) ◽  
pp. 1423-1432 ◽  
Author(s):  
Romain Gibeaux ◽  
Dominic Hoepfner ◽  
Ivan Schlatter ◽  
Claude Antony ◽  
Peter Philippsen
Keyword(s):  
High Resolution ◽  
Large Scale ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Multivesicular Bodies ◽  
Ashbya Gossypii ◽  
Space Filling ◽  
Content Type ◽  
Hyphal Tip ◽  
Dense Vesicles

ABSTRACT Ashbya gossypii grows as multinucleated and constantly elongating hyphae. Nuclei are in continuous forward and backward motion, also move during mitosis, and frequently bypass each other. Whereas these nuclear movements are well documented, comparatively little is known about the density and morphology of organelles which very likely influence these movements. To understand the three-dimensional subcellular organization of hyphae at high resolution, we performed large-scale electron tomography of the tip regions in A. gossypii . Here, we present a comprehensive space-filling model in which most membrane-limited organelles including nuclei, mitochondria, endosomes, multivesicular bodies, vacuoles, autophagosomes, peroxisomes, and vesicles are modeled. Nuclei revealed different morphologies and protrusions filled by the nucleolus. Mitochondria are very abundant and form a tubular network with a polarized spherical fraction. The organelles of the degradative pathways show a clustered organization. By analyzing vesicle-like bodies, we identified three size classes of electron-dense vesicles (∼200, ∼150, and ∼100 nm) homogeneously distributed in the cytoplasm which most likely represent peroxisomes. Finally, coated and uncoated vesicles with approximately 40-nm diameters show a polarized distribution toward the hyphal tip with the coated vesicles preferentially localizing at the hyphal periphery.

scholarly journals Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography

2006 ◽  
Vol 174 (6) ◽  
pp. 851-862 ◽  
Author(s):  
Nobuhiro Morone ◽  
Takahiro Fujiwara ◽  
Kotono Murase ◽  
Rinshi S. Kasai ◽  
Hiroshi Ike ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
High Speed ◽  
Plasma Membranes ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Membrane Skeleton ◽  
Single Particle Tracking ◽  
Coated Pits ◽  
Cytoplasmic Surface

Three-dimensional images of the undercoat structure on the cytoplasmic surface of the upper cell membrane of normal rat kidney fibroblast (NRK) cells and fetal rat skin keratinocytes were reconstructed by electron tomography, with 0.85-nm–thick consecutive sections made ∼100 nm from the cytoplasmic surface using rapidly frozen, deeply etched, platinum-replicated plasma membranes. The membrane skeleton (MSK) primarily consists of actin filaments and associated proteins. The MSK covers the entire cytoplasmic surface and is closely linked to clathrin-coated pits and caveolae. The actin filaments that are closely apposed to the cytoplasmic surface of the plasma membrane (within 10.2 nm) are likely to form the boundaries of the membrane compartments responsible for the temporary confinement of membrane molecules, thus partitioning the plasma membrane with regard to their lateral diffusion. The distribution of the MSK mesh size as determined by electron tomography and that of the compartment size as determined from high speed single-particle tracking of phospholipid diffusion agree well in both cell types, supporting the MSK fence and MSK-anchored protein picket models.

Cryo-Electron Tomography of Isolated Triad Junctions from Skeletal Muscle

2001 ◽  
Vol 7 (S2) ◽  
pp. 94-95 ◽  
Author(s):  
C.-E. Hsieh ◽  
M. Marko ◽  
B.K. Rath ◽  
S. Fleischer ◽  
T. Wagenknecht
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Electron Tomography ◽  
Mass Density ◽  
Tomographic Reconstruction ◽  
Three Dimensional ◽  
Cryo Electron Tomography ◽  
Junctional Complexes ◽  
Triad Junction ◽  
Triad Junctions

In skeletal muscle, depolarization of the plasma membrane, which is initiated at the neuromuscular junction, is transduced to a rise in cytoplasmic calcium at specialized structures known as triad junctions (TJs). TJs occur in the myofiber’s interior at regions near the z-lines, where transversely oriented tubular invaginations of the plasma membrane (T-tubules) form junctions with two elements of the sarcoplasmic reticulum (SR). Isolation of membrane fractions that are enriched in junctional complexes and which retain function has been reported.Figure 1 shows a region of an electron micrograph containing an isolated TJ in the frozen-hydrated state. in the orientation shown, two SR-derived vesicles sandwich a flattened vesicle derived from the T-tubule. The junctional regions contain a complex distribution of density, presumably due to proteins that are known to be present in TJs. Electron tomography offers the means to determine the three-dimensional mass density from such micrographs, which would greatly aid in their interpretation. Only recently has the automated data collection technology for determining tomograms of non-stained, frozen-hydrated specimens become available. Here we describe the first tomographic reconstruction of a frozen-hydrated triad junction by automated electron tomography.

scholarly journals Roles of Ras1 Membrane Localization during Candida albicans Hyphal Growth and Farnesol Response

2011 ◽  
Vol 10 (11) ◽  
pp. 1473-1484 ◽  
Author(s):  
Amy E. Piispanen ◽  
Ophelie Bonnefoi ◽  
Sarah Carden ◽  
Aurelie Deveau ◽  
Martine Bassilana ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Hyphal Growth ◽  
Membrane Dynamics ◽  
Environmental Cues ◽  
Content Type ◽  
Lipid Modifications ◽  
Ras Gtpases

ABSTRACTMany Ras GTPases localize to membranes via C-terminal farnesylation and palmitoylation, and localization regulates function. InCandida albicans, a fungal pathogen of humans, Ras1 links environmental cues to morphogenesis. Here, we report the localization and membrane dynamics of Ras1, and we characterize the roles of conserved C-terminal cysteine residues, C287 and C288, which are predicted sites of palmitoylation and farnesylation, respectively. GFP-Ras1 is localized uniformly to plasma membranes in both yeast and hyphae, yet Ras1 plasma membrane mobility was reduced in hyphae compared to that in yeast. Ras1-C288S was mislocalized to the cytoplasm and could not support hyphal development. Ras1-C287S was present primarily on endomembranes, and strains expressingras1-C287Swere delayed or defective in hyphal induction depending on the medium used. Cells bearing constitutively activated Ras1-C287S or Ras1-C288S, due to a G13V substitution, showed increased filamentation, suggesting that lipid modifications are differentially important for Ras1 activation and effector interactions. TheC. albicansautoregulatory molecule, farnesol, inhibits Ras1 signaling through adenylate cyclase and bears structural similarities to the farnesyl molecule that modifies Ras1. At lower concentrations of farnesol, hyphal growth was inhibited but Ras1 plasma membrane association was not altered; higher concentrations of farnesol led to mislocalization of Ras1 and another G protein, Rac1. Furthermore, farnesol inhibited hyphal growth mediated by cytosolic Ras1-C288SG13V, suggesting that farnesol does not act through mechanisms that depend on Ras1 farnesylation. Our findings imply that Ras1 is farnesylated and palmitoylated, and that the Ras1 stimulation of adenylate cyclase-dependent phenotypes can occur in the absence of these lipid modifications.

scholarly journals Three-dimensional architecture of extended synaptotagmin-mediated endoplasmic reticulum–plasma membrane contact sites

2015 ◽  
Vol 112 (16) ◽  
pp. E2004-E2013 ◽  
Author(s):  
Rubén Fernández-Busnadiego ◽  
Yasunori Saheki ◽  
Pietro De Camilli
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Mammalian Cells ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Lipid Binding ◽  
Dense Layer ◽  
Lipid Transfer ◽  
C2 Domains ◽  
Membrane Contact Sites

The close apposition between the endoplasmic reticulum (ER) and the plasma membrane (PM) plays important roles in Ca2+ homeostasis, signaling, and lipid metabolism. The extended synaptotagmins (E-Syts; tricalbins in yeast) are ER-anchored proteins that mediate the tethering of the ER to the PM and are thought to mediate lipid transfer between the two membranes. E-Syt cytoplasmic domains comprise a synaptotagmin-like mitochondrial-lipid–binding protein (SMP) domain followed by five C2 domains in E-Syt1 and three C2 domains in E-Syt2/3. Here, we used cryo-electron tomography to study the 3D architecture of E-Syt–mediated ER–PM contacts at molecular resolution. In vitrified frozen-hydrated mammalian cells overexpressing individual E-Syts, in which E-Syt–dependent contacts were by far the predominant contacts, ER–PM distance (19–22 nm) correlated with the amino acid length of the cytosolic region of E-Syts (i.e., the number of C2 domains). Elevation of cytosolic Ca2+ shortened the ER–PM distance at E-Syt1–dependent contacts sites. E-Syt–mediated contacts displayed a characteristic electron-dense layer between the ER and the PM. These features were strikingly different from those observed in cells exposed to conditions that induce contacts mediated by the stromal interaction molecule 1 (STIM1) and the Ca2+ channel Orai1 as well as store operated Ca2+ entry. In these cells the gap between the ER and the PM was spanned by filamentous structures perpendicular to the membranes. Our results define specific ultrastructural features of E-Syt–dependent ER–PM contacts and reveal their structural plasticity, which may impact on the cross-talk between the ER and the PM and the functions of E-Syts in lipid transport between the two bilayers.

Sign in / Sign up

Export Citation Format

Share Document