Automated high‐throughput electron tomography by pre‐calibration of image shifts

Ziese, Ulrike; Janssen, A.H.; Murk, Jean L.; Geerts, Willie J. C.; Krift, Theo van der; Verkleij, Arie J.; Koster, Abraham J.

doi:10.1046/j.0022-2720.2001.00987.x

Cited by 92 publications

(61 citation statements)

References 43 publications

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“…Automated ET was performed according to previously described basic principles including the predictive tracking method (33,34). Tilt series of plunge-frozen specimens were acquired with an FEI G2 Polara transmission electron microscope operated at 300 keV and equipped with a Gatan GIF 2002 postcolumn energy filter and a 2,048-by-2,048 Multiscan charge-coupled-device (CCD) camera (Gatan, Pleasanton, CA).…”

Section: Methodsmentioning

confidence: 99%

The Nucleocapsid Domain of Gag Is Dispensable for Actin Incorporation into HIV-1 and for Association of Viral Budding Sites with Cortical F-Actin

Stauffer

Rahman

Marco

et al. 2014

J Virol

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Actin and actin-binding proteins are incorporated into HIV-1 particles, and F-actin has been suggested to bind the NC domain in HIV-1 Gag. Furthermore, F-actin has been frequently observed in the vicinity of HIV-1 budding sites by cryo-electron tomography (cET). Filamentous structures emanating from viral buds and suggested to correspond to actin filaments have been observed by atomic force microscopy. To determine whether the NC domain of Gag is required for actin association with viral buds and for actin incorporation into HIV-1, we performed comparative analyses of virus-like particles (VLPs) obtained by expression of wild-type HIV-1 Gag or a Gag variant where the entire NC domain had been replaced by a dimerizing leucine zipper [Gag(LZ)]. The latter protein yielded efficient production of VLPs with near-wild-type assembly kinetics and size and exhibited a regular immature Gag lattice. Typical HIV-1 budding sites were detected by using cET in cells expressing either Gag or Gag(LZ), and no difference was observed regarding the association of buds with the F-actin network. Furthermore, actin was equally incorporated into wild-type HIV-1 and Gag-or Gag(LZ)-derived VLPs, with less actin per particle observed than had been reported previously. Incorporation appeared to correlate with the relative intracellular actin concentration, suggesting an uptake of cytosol rather than a specific recruitment of actin. Thus, the NC domain in HIV-1 Gag does not appear to have a role in actin recruitment or actin incorporation into HIV-1 particles. IMPORTANCEHIV-1 particles bud from the plasma membrane, which is lined by a network of actin filaments. Actin was found to interact with the nucleocapsid domain of the viral structural protein Gag and is incorporated in significant amounts into HIV-1 particles, suggesting that it may play an active role in virus release. Using electron microscopy techniques, we previously observed bundles of actin filaments near HIV-1 buds, often seemingly in contact with the Gag layer. Here, we show that this spatial association is observed independently of the proposed actin-binding domain of HIV-1. The absence of this domain also did not affect actin incorporation and had a minor effect on the viral assembly rate. Furthermore, actin was not enriched in the virus compared to the average levels in the respective producing cell. Our data argue against a specific recruitment of actin to HIV-1 budding sites by the nucleocapsid domain of Gag.

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Section: Methodsmentioning

confidence: 99%

The Nucleocapsid Domain of Gag Is Dispensable for Actin Incorporation into HIV-1 and for Association of Viral Budding Sites with Cortical F-Actin

Stauffer

Rahman

Marco

et al. 2014

J Virol

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“…Colloidal gold particles (10 nm) were added to one side of the grid as markers to align the series of tilted images. Tilt series of representative Golgi stacks were automatically recorded (Ziese et al, 2002) at 200 kV using a Tecnai20 electron microscope (FEI/Philips Electron Optics, Eindhoven, The Netherlands) equipped with a slow-scan CCD camera (TemCam F214, TVIPS [Tietz Video and Image Processing Systems], Gauting, Germany) and a motorized goniometer. Recording was made with Xplore 3D software package; FEI) at a final magnification of 14,000ϫ.…”

Section: Electron Microscopy Electron Tomography and 3d Modelingmentioning

confidence: 99%

Diacylglycerol Is Required for the Formation of COPI Vesicles in the Golgi-to-ER Transport Pathway

Fernández-Ulibarri

Vilella

Lázaro‐Diéguez

et al. 2007

MBoC

103

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Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation. INTRODUCTIONRecent observations from several laboratories indicate that membrane lipids regulate intracellular membrane transport, particularly in distal stages of the secretory pathway. Diacylglycerol (DAG) is a simple and small sized signal-transducing lipid which among other functions is necessary for protein transport from the Golgi complex to the cell surface both in yeast and in mammals. Thus, in budding yeast phosphatidylinositol (PI)-transfer Sec14p protein directly regulates DAG homeostasis in the Golgi complex and protein secretion (Bankaitis et al., 1990;Kearns et al., 1997;Huijbregts et al., 2000). In mammals, the reduction of DAG levels at the Golgi caused by the depletion of Nir2 (a peripheral Golgi protein containing a PI-transfer domain) inhibits post-Golgi protein transport (Litvak et al., 2005). DAG acts in the trans-Golgi network (TGN) as a membrane acceptor for specific proteins such as the protein kinase C (PKC) family member PKD/ PKC (Prestle et al., 1996;Liljedahl et al., 2001;Baron andMalhotra, 2002), and Hmun13 (Speight andSilverman, 2005).PKD together with PKC, the trimeric G-protein subunits ␤/␥ (Díaz Anel and Malhotra, 2005), and phosphatidylinositol-4 kinase III␤ (Hausser et al., 2005) directly participate in the post-Golgi transport of plasma membrane proteins containing basolateral sorting information (Yeaman et al., 2004). On the other hand, Hmun13, through the recruitment of Rab34, participates in the Golgi-lysosome protein trafficking (Speight and Silverman, 2005). DAG also promotes the Golgi membrane targeting and activation of other C1 domain-containing signaling molecules such as other PKC isoforms (PKC, PKC, and PKC␦;Maissel et al., 2006;Lehel et al., 1995; Wang et al., 1999, respectively) and Ras guanosine nucleotide-releasing proteins (RasGRPs;Caloca et al., 2003), whose potential involvement in Golgi-associated transport functions remains unexplored.The aforementioned Go...

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“…Colloidal gold particles (10 nm) were added to one side of the grid to serve as fiducial markers for aligning the series of tilted images. Tilt series of representative Golgi stacks were automatically recorded [Ziese et al, 2002] at 200 kV using a Tecnai20 electron microscope (FEI/Philips Electron Optics, Eindoven, The Netherlands) equipped with a slow-scan CCD camera (TemCam F214, TVIPS GmbH, Germany) and a motorized goniometer. Every specimen was tilted about two orthogonal axes from À658 to þ658 at 18 intervals, resulting in two datasets of 131 high-resolution digital images.…”

Section: Electron Tomography and 3d Modelingmentioning

confidence: 99%

Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra‐Golgi pH

Lázaro‐Diéguez

Jiménez

Barth

et al. 2006

Cell Motil. Cytoskeleton

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Here we examine the contribution of actin dynamics to the architecture and pH of the Golgi complex. To this end, we have used toxins that depolymerize (cytochalasin D, latrunculin B, mycalolide B, and Clostridium botulinum C2 toxin) or stabilize (jasplakinolide) filamentous actin. When various clonal cell lines were examined by epifluorescence microscopy, all of these actin toxins induced compaction of the Golgi complex. However, ultrastructural analysis by transmission electron microscopy and electron tomography/three-dimensional modelling of the Golgi complex showed that F-actin depolymerization first induces perforation/fragmentation and severe swelling of Golgi cisternae, which leads to a completely disorganized structure. In contrast, F-actin stabilization results only in cisternae perforation/fragmentation. Concomitantly to actin depolymerization-induced cisternae swelling and disorganization, the intra-Golgi pH significantly increased. Similar ultrastructural and Golgi pH alkalinization were observed in cells treated with the vacuolar H þ -ATPases inhibitors bafilomycin A1 and concanamycin A. Overall, these results suggest that actin filaments are implicated in the preservation of the flattened shape of Golgi cisternae. This maintenance seems to be mediated by the regulation of the state of F-actin assembly on the Golgi pH homeostasis. Cell Motil. Cytoskeleton 63: 778-791, 2006. '

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Automated high‐throughput electron tomography by pre‐calibration of image shifts

Cited by 92 publications

References 43 publications

The Nucleocapsid Domain of Gag Is Dispensable for Actin Incorporation into HIV-1 and for Association of Viral Budding Sites with Cortical F-Actin

The Nucleocapsid Domain of Gag Is Dispensable for Actin Incorporation into HIV-1 and for Association of Viral Budding Sites with Cortical F-Actin

Diacylglycerol Is Required for the Formation of COPI Vesicles in the Golgi-to-ER Transport Pathway

Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra‐Golgi pH

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