Anti-biotin Antibodies Offer Superior Organelle-specific Labeling of Mitochondria over Avidin or Streptavidin

Hollinshead, Michael; Sanderson, Jeremy; Vaux, David J.

doi:10.1177/002215549704500803

Cited by 74 publications

(66 citation statements)

References 13 publications

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“…4). Hollinshead et al have shown that antibodies against biotin can be used to identify mitochondria by immunofluorescence microscopy in normal rat kidney cells (25), and we obtained similar results with Drosophila DL-1 cells (Fig. 4A).…”

Section: Vol 75 2001supporting

confidence: 86%

Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells

Miller

Schwartz

Ahlquist

2001

J Virol

191

296

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The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection of Drosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses. Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40-to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusvirus-and togavirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.Positive-strand RNA viruses are responsible for a wide range of diseases in humans, animals, and plants. Clinically relevant members of this group cause significant morbidity and mortality and include viruses from the Picornaviridae, Caliciviridae, Togaviridae, and Flaviviridae families. Although these pathogens represent a prominent component of the growing list of emerging and potentially devastating viral diseases (40), current therapies for positive-strand RNA virus infections are limited to a few marginally effective drugs (36). The design and investigation of novel and broadly effective therapies require the identification and characterization of fundamental mechanisms in positive-strand RNA virus replication and pathogenesis, such as replication complex formation.Flock house virus (FHV) and the closely related black beetle virus (BBV) are the best-studied alphanodaviruses in the Nodaviridae family (2). FHV was originally isolated from the grass grub Costelytra zealandica (12, 57) and contains one of the smallest known genomes of any animal RNA virus. The 4.5-kb FHV genome is bipartite, with two capped but nonpolyadenylated RNAs copackaged into a 29-nm nonenveloped virion with an icosahedral (Tϭ3) capsid (56, 57). The larger 3.1-kb RNA species (RNA1) encodes protein A (2, 11), a 112-kDa protein with s...

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Section: Vol 75 2001supporting

confidence: 86%

Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells

Miller

Schwartz

Ahlquist

2001

J Virol

191

296

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“…Likewise, under these conditions, anti-biotin decorated the mitochondrial matrix (Fig. 8D), known to contain various biotinylated proteins (55). However, at 0.015% Triton X-100, no staining of HsMuLK or biotinylated proteins was observed (Fig.…”

Section: Subcellular Localization Of Hscerk and Hsmulkmentioning

confidence: 78%

Further characterization of mammalian ceramide kinase: substrate delivery and (stereo)specificity, tissue distribution, and subcellular localization studies

Overloop

Gijsbers²,

Veldhoven

2006

Journal of Lipid Research

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Recombinant human ceramide kinase (HsCERK) was analyzed with regard to dependence on divalent cations and to substrate delivery, spectrum, specificity, and stereoselectivity. Depending on the chain length of the ceramide, either albumin for short-chain ceramide or a mixed micellar form (octylglucoside/cardiolipin) for long-chain ceramide was preferred for the substrate delivery, the former resulting in higher activities. Bacterially expressed HsCERK was highly dependent on Mg 21 ions, much less on Ca 21 ions. A clear preference for the D-erythro isomer was seen. Various N-acylated amino alcohols were no substrate, but Nhexanoyl-1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol and Ntetradecanoyl-2S-amino-1-butanol were phosphorylated, suggesting that the secondary hydroxy group is not required for recognition. The properties of HsCERK, expressed in CHO cells, were similar to those of the bacterially expressed protein, including the Mg 21 dependence. In mouse, the highest activities were found in testis and cerebellum, and upon subcellular fractionation the activity was recovered mainly in the microsomal fraction. This fits with the plasma membrane localization in CHO cells, which was mediated by the N-terminal putative pleckstrin domain.No evidence for phosphorylation of ceramide by the recently described multiple lipid kinase was found. The latter kinase is localized in the mitochondria, but no firm conclusions with regard to its substrate could be drawn.

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“…The same types of changes were observed when the mitochondria were labeled by a MitoTracker, fluorescently labeled Tim23 (an integral mitochondrial inner membrane protein) and by fluorescently labeled streptavidin, which specifically labels mitochondria by binding to biotinylated proteins of mitochondrial matrix. 21 Mitochondria were labeled by streptavidin in this study because the strengths of streptavidin fluorescent signals did not vary during the incubation time of primary hepatocytes.…”

Section: à12mentioning

confidence: 99%

Preapoptotic cell stress response of primary hepatocytes

et al. 2010

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Primary hepatocytes are an important in vitro model for studying metabolism in man. Caspase-9 and Bcl-2-associated X protein (Bax) are regulators of the apoptotic pathway. Here we report on the translocation of procaspase-9 and Bax from cytoplasm to nuclei as well as on dispersion of mitochondria; these processes occur after isolation of primary hepatocytes. The observed changes appear similar to those at the beginning of apoptosis; however, the isolated hepatocytes are not apoptotic for the following reasons: (1) cells have a normal morphology and function; (2) the mitochondria are energized; (3) there is no apoptosis unless it is induced by, e.g., staurosporine or nodularin. Staurosporine does not trigger apoptosis through activation of caspase-9, as its activity is detected later than that of caspase-3. We propose that the translocation of procaspase-9 and Bax into the nuclei reduces the ability to trigger apoptosis through the intrinsic apoptotic pathway. The shifts of procaspase-9 and Bax are reversible in the absence of the apoptotic trigger; the spontaneous reversion was confirmed experimentally for procaspase-9, whereas Bax shifted from the nuclei to the cytosol and mitochondria after the initiation of apoptosis. To distinguish this process from apoptosis, we call it preapoptotic cell stress response. It shares some features with apoptosis; however, it is reversible and apoptosis has to be induced in addition to this process. Conclusion: Knowledge on preapoptotic cell stress response is important for assessing the quality of the cells used in cell therapies, in regenerative medicine, and of those used for modeling metabolic processes. (HEPATOLOGY 2010;51:2140-2151 C ell cultures, especially those of primary cells, are important models for studying biochemical and physiological phenomena; they are also used in cell therapies and in regenerative medicine. Ideally, the metabolism of isolated cells should not differ from the metabolism within the cells of intact tissues; therefore, the primary cell cultures are thought to be the closest models of in vivo processes. The primary cell cultures of isolated hepatocytes are being used as models to study drug metabolism or drug effects on the liver.1 Nevertheless, some metabolic changes occur upon culturing primary hepatocytes. Expression of cytochromes P450 (CYP) is especially well studied, because of their involvement in drug metabolism. The CYP messenger RNA (mRNA) levels of isolated hepatocytes are similar to those of liver 2 ; however, they decline progressively during the first days in culture.3 Also, there are significant perturbations of genes encoding for antioxidant enzymes, heat shock proteins, nitric oxide synthase, and methionine adenosyltransferase following the isolation and culture of hepatocytes. 2As a consequence, the use of primary cultured hepatocytes in drug metabolism studies is confined to the first days in culture. 4 Although the metabolic state of isolated hepatocytes is extensively studied, there are no data on modulation of apoptotic ma...

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Anti-biotin Antibodies Offer Superior Organelle-specific Labeling of Mitochondria over Avidin or Streptavidin

Cited by 74 publications

References 13 publications

Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells

Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells

Further characterization of mammalian ceramide kinase: substrate delivery and (stereo)specificity, tissue distribution, and subcellular localization studies

Preapoptotic cell stress response of primary hepatocytes

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