HVEM tomography of the trans-Golgi network: structural insights and identification of a lace-like vesicle coat.

Ladinsky, Mark S.; Kremer, James R.; Furcinitti, Paul S.; McIntosh, J. Richard; Howell, Kathryn E.

doi:10.1083/jcb.127.1.29

Cited by 221 publications

(164 citation statements)

References 32 publications

(42 reference statements)

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“…Tomography is a useful method for looking at complex 3-D structures such as the centrosome (Moritz et al, 1995), the Golgi apparatus (Ladinsky et al, 1994(Ladinsky et al, , 1999, kinetochores (McEwen et al, 1993), and mitochondria (Mannella, 1997;Perkins et al, 1997). The technique enables one to examine 3-D image data at a resolution better than that obtained by serial sections and to slice through one organelle in many orientations, permitting a better understanding of the geometry of a cellular subsystem.…”

Section: Discussionmentioning

confidence: 99%

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the YeastSaccharomyces cerevisiae

O’Toole¹,

Winey

McIntosh³

1999

MBoC

266

267

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The spindle pole body (SPB) is the major microtubule-organizing center of budding yeast and is the functional equivalent of the centrosome in higher eukaryotic cells. We used fast-frozen, freeze-substituted cells in conjunction with high-voltage electron tomography to study the fine structure of the SPB and the events of early spindle formation. Individual structures were imaged at 5-10 nm resolution in three dimensions, significantly better than can be achieved by serial section electron microscopy. The SPB is organized in distinct but coupled layers, two of which show ordered two-dimensional packing. The SPB central plaque is anchored in the nuclear envelope with hook-like structures. The minus ends of nuclear microtubules (MTs) are capped and are tethered to the SPB inner plaque, whereas the majority of MT plus ends show a distinct flaring. Unbudded cells containing a single SPB retain 16 MTs, enough to attach to each of the expected 16 chromosomes. Their median length is ϳ150 nm. MTs growing from duplicated but not separated SPBs have a median length of ϳ130 nm and interdigitate over the bridge that connects the SPBs. As a bipolar spindle is formed, the median MT length increases to ϳ300 nm and then decreases to ϳ30 nm in late anaphase. Three-dimensional models confirm that there is no conventional metaphase and that anaphase A occurs. These studies complement and extend what is known about the three-dimensional structure of the yeast mitotic spindle and further our understanding of the organization of the SPB in intact cells. INTRODUCTIONMicrotubule-organizing centers (MTOCs) nucleate and anchor microtubules (MTs) during cell differentiation and division (for review, see Brinkley, 1985;Rose et al., 1993;Kellogg et al., 1994;Pereira and Schiebel, 1997). Although MTOCs vary widely in structure, their functions are largely conserved. In metazoa, the principal MTOC is the centrosome, which organizes both the interphase MTs and those of the mitotic spindle. In the yeast Saccharomyces cerevisiae, this role is served by the spindle pole body (SPB), a complex and dynamic organelle that undergoes significant structural changes during the yeast cell cycle (for review, see Winey and Byers, 1993;Kilmartin, 1994;Snyder, 1994). Nuclear MTs, which form the meiotic or mitotic spindles, attach to an inner plaque of the SPB, whereas cytoplasmic MTs, important for nuclear movements and position, are attached to an outer plaque (Moens and Rapport, 1971;Byers and Goetsch, 1974, 1975;Rabinow and Marak, 1966). Recent studies of isolated SPBs by cryomicroscopy and electron tomography have shown that SPBs are organized from six major layers, including a central crystalline core that contains the SPC42 gene product (Bullitt et al., 1997).Duplication of the SPB during the cell cycle begins with the formation of a "satellite" on the cytoplasmic face of the half-bridge, a specialized region of the nuclear envelope that lies immediately adjacent to the existing SPB. By processes not yet described in detail, the satellite develops into a...

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Section: Discussionmentioning

confidence: 99%

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the YeastSaccharomyces cerevisiae

O’Toole¹,

Winey

McIntosh³

1999

MBoC

266

267

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“…Dual-axis tomographic reconstruction was carried out with the IMOD software package as described previously (25)(26)(27). Dualaxis tomograms from serial sections were aligned and combined using the methods previously described (43)(44)(45). A surface mesh was generated to fit the modeled contours.…”

Section: Methodsmentioning

confidence: 99%

CD4⁺T-cell synapses involve multiple distinct stages

Ueda

Morphew

McIntosh

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

114

109

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One very striking feature of T-cell recognition is the formation of an immunological synapse between a T cell and a cell that it is recognizing. Formation of this complex structure correlates with cytotoxicity in the case of killer (largely CD8 + ) T-cell activity, or robust cytokine release and proliferation in the case of the much longer lived synapses formed by helper (CD4 + ) T cells. Here we have used electron microscopy and 3D tomography to characterize the synapses of antigen-specific CD4 + T cells recognizing B cells and dendritic cells at different time points. We show that there are at least four distinct stages in synapse formation, proceeding over several hours, including an initial stage involving invasive T-cell pseudopodia that penetrate deeply into the antigen-presenting cell, almost to the nuclear envelope. This must involve considerable force and may serve to widen the search for potential ligands on the surface of the cell being recognized. We also show that centrioles and the Golgi complex are always located immediately beneath the synapse and that centrioles are significantly shifted toward the late contact zone with either B lymphocytes or bone marrow-derived dendritic cells such as antigenpresenting cells, and that there are dynamic, stage-dependent changes in the organization of microtubules beneath the synapse. These data reinforce and extend previous data on cytotoxic T cells that one of the principal functions of the immunological synapse is to facilitate cytokine secretion into the synaptic cleft, as well as provide important insights into the overall dynamics of this phenomenon. microtubule organizing center | centrosome A prominent feature of many T-lymphocyte interactions with other cells on which it recognizes a particular antigen is the formation of an immunological synapse (IS). The formation of this structure correlates with robust signaling, lineage commitment, and fate determination of T cells (1-6) and the directed secretion of cytokines and/or cytotoxic molecules. There is a wholesale reorganization of the T cell's cytoskeleton, surface molecules, and organelles, and the loss of polarity regulators or guidance cues that negatively affect T-cell activation (7,8). Whereas a great deal has been learned about the dynamics of cell-surface and signaling molecules within this interface (9, 10), much less is known about changes within the cell, especially in the long-lived helper T cell, namely in antigen-presenting cell (APC) interactions, which can last for 6 or more h and need continuous T-cell receptor (TCR) engagement (11). Here electron microscopic studies are particularly valuable for their very high resolution, especially with the power of 3D tomography and highpressure freezing, which preserves cell structure more faithfully. Although a number of high-resolution electron microscopy studies of T cell-APC interactions have been reported (3,(12)(13)(14)(15), these involve studies of cytotoxic T or natural killer cells and their targets for only the first few minutes of CD4 + ...

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“…When combined with high-voltage electron microscopy or serial section approaches, electron tomography can be used to derive 3D reconstructions of relatively large expanses of cellular and subcellular processes (Ladinsky et al, 1994(Ladinsky et al, , 1999Marsh et al, 2001;Martone et al, 2000;Soto et al, 1994;Wilson et al, 1992). The breadth of these reconstructions is approaching that of light microscopy, while still revealing the exquisite, high-resolution detail typically found in the best electron micrographs of ultrathin sections.…”

Section: Introductionmentioning

confidence: 99%

A cell-centered database for electron tomographic data

Martone

Gupta

Wong

et al. 2002

Journal of Structural Biology

106

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HVEM tomography of the trans-Golgi network: structural insights and identification of a lace-like vesicle coat.

Cited by 221 publications

References 32 publications

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the YeastSaccharomyces cerevisiae

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the YeastSaccharomyces cerevisiae

CD4⁺T-cell synapses involve multiple distinct stages

A cell-centered database for electron tomographic data

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HVEM tomography of the trans-Golgi network: structural insights and identification of a lace-like vesicle coat.

Cited by 221 publications

References 32 publications

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the YeastSaccharomyces cerevisiae

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the YeastSaccharomyces cerevisiae

CD4+T-cell synapses involve multiple distinct stages

A cell-centered database for electron tomographic data

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Product

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CD4⁺T-cell synapses involve multiple distinct stages