Autophagic Vacuoles Produced in Vitro

Fedorko, Martha E.; Hirsch, James G.; Cohn, Zanvil A.

doi:10.1083/jcb.38.2.392

Cited by 113 publications

(18 citation statements)

References 18 publications

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“…7) revealed that the large vacuoles present in the chloroquine-treated cells contained morphologically intact viruses as well as other membranous and amorphous material. The chloroquine-treated cells were highly vacuolated as previously described in several cell systems (Federko et al, 1968;Wibo & Poole, 1974).…”

Section: Effects Of Lysosomotropic Agents On Binding Endocytosis Andmentioning

confidence: 99%

Inhibition of Semliki Forest Virus Penetration by Lysosomotropic Weak Bases

Helenius

Marsh

White

1982

Journal of General Virology

240

157

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SUMMARYThe effect of five lysosomotropic weak bases (chloroquine, amantadine, tributylamine, methylamine and NH4CI) on Semliki Forest virus (SFV) infection has been studied in BHK-21 cells. When present at concentrations equal to or greater than 0-1, 0.5, 2, 15 and 15 mM respectively, the agents inhibited SFV infection by more than 90%. The effect was reversible and involved a process occurring within the first 60 min of virus-cell contact. The agents did not have a direct virucidal effect nor did they affect virus binding to the cells, receptor-mediated endocytosis of prebound virus, intracellular distribution of virus after endocytosis, or the low pH-induced membrane fusion activity of the virus spike glycoproteins. The step blocked by chloroquine and NH4CI occurred intracellularly and was identified as the release of the virus nucleocapsid into the cytoplasm or the uncoating process. On the basis of these results, our previous studies on SFV entry, and the known effects of lipophilic amines on lysosomes, we conclude that the agents affect entry by a common mechanism: they prevent the transfer of the virus nucleocapsid into the cytoplasm by increasing the lysosomal pH above the critical value needed to trigger a low pH-dependent fusion reaction between the membranes of the tysosome and the virus.

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Section: Effects Of Lysosomotropic Agents On Binding Endocytosis Andmentioning

confidence: 99%

Inhibition of Semliki Forest Virus Penetration by Lysosomotropic Weak Bases

Helenius

Marsh

White

1982

Journal of General Virology

240

157

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“…Early studies of autophagy were, therefore, greatly facilitated by treatments that promoted the accumulation of autophagic vacuoles. This could be achieved either by stimulation of autophagic activity, e.g., by glucagon treatment or withdrawal of growth factors like insulin or serum, or amino acid deprivation as discussed above, or by inhibition of late stages of the autophagic-lysosomal flux by lysosomotropic amines 82,[103][104][105] inhibitors of lysosomal proteases, [104][105][106][107][108] or chemicals that disrupt microtubules. 40,41,56,105,109,110 Among the latter, the widely used drug, vinblastine, deserves special comment.…”

Section: O N O T D I S T R I B U T Ementioning

confidence: 99%

Seeing is believing: The impact of electron microscopy on autophagy research

et al. 2011

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Autophagy was first discovered by transmission electron microscopy more than 50 years ago. For decades, electron microscopy was the only way to reliably detect autophagic compartments in cells because no specific protein markers were known. In the 1970s, however, the introduction of biochemical methods enabled quantitative studies of autophagic-lysosomal degradation, and in the 1980s specific biochemical assays for autophagic sequestration became available. Since the identification of autophagy-related genes in the 1990s, combined fluorescence microscopy, biochemical and genetic methods have taken the leading role in autophagy research. However, electron microscopy is still needed to confirm and verify results obtained by other methods, and also to produce novel knowledge that would not be achievable by any other experimental approach. Confocal microscopy, with its ever-improving resolution, is probably the best-suited morphological approach to investigate the dynamic aspects of autophagy. However, for analyzing the ultrastructural details of the many novel organelles and mechanisms involved in specific subtypes of autophagy, the electron microscope is still indispensable. This review will summarize the impact that electron microscopy has had on autophagy research since the discovery of this self-degradation process in the mid-1950s. Astonishingly, some of the "novel" concepts and principles of autophagy, presented in the recent studies, were already proposed several decades ago by the pioneering, accurate and passionate work of virtuoso electron microscopists.

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“…We report in this paper and in the accompanying paper (7) observations of the effects in vitro of chloroquine on cultures of mouse macrophages and L cells. Low concentrations of this drug rapidly induce autophagic vacuole formation in these cells.…”

Section: Introductionmentioning

confidence: 99%

Autophagic Vacuoles Produced in Vitro

Fedorko

Hirsch

Cohn

1968

The Journal of Cell Biology

Self Cite

141

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Mouse macrophages exposed to 30 pzg/ml of chloroquine in vitro develop autophagic vacuoles containing various cytoplasmic components and acid phosphatase. The early toxic vacuoles appear in the perinuclear region within 15 min; on electron microscopy, they show irregular shape, amorphous moderately dense content, apparent double membranes, and in some instances curved thin tubular extensions with a central, dark linear element. Cytoplasmic structures are probably transported into the vacuoles by invagination of the vacuolar membrane. After exposure to chloroquine for 1-4 hr, macrophages display large vacuoles containing degraded cytoplasmic structures, membranous whorls, and amorphous material. When chloroquine is removed by changing the culture medium after 4 hr, the cells survive and 24 hr later they exhibit no abnormality except for large cytoplasmic dense bodies packed with membrane lamellae. During recovery chloroquine disappears from the cells. 24 hr after exposure to chloroquine the macrophages have accumulated less hydrolases than control cells.

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Autophagic Vacuoles Produced in Vitro

Cited by 113 publications

References 18 publications

Inhibition of Semliki Forest Virus Penetration by Lysosomotropic Weak Bases

Inhibition of Semliki Forest Virus Penetration by Lysosomotropic Weak Bases

Seeing is believing: The impact of electron microscopy on autophagy research

Autophagic Vacuoles Produced in Vitro

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