Chapter 7 Monitoring Autophagy in Yeast using FM 4‐64 Fluorescence

Journo, Dikla; Winter, Gal; Abeliovich, Hagai

doi:10.1016/s0076-6879(08)03207-2

Cited by 17 publications

(13 citation statements)

References 13 publications

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“…1 D and E). The delocalized stain and multiple intravacuolar structures within the larger vacuolar space of Δyca1 cells were consistent with the morphology of autophagic bodies (18). The proteomic alterations and accumulation of autophagic bodies in the Δyca1 strain suggested a possible autophagic fate; however, Δyca1 is a viable strain that grows at rates comparable with those of wild type, suggesting that the substantial vacuolation and enrichment of vacuolar peptidases are markers of a limited autophagic process in viable cells.…”

Section: Resultssupporting

confidence: 60%

Metacaspase Yca1 is required for clearance of insoluble protein aggregates

Lee

Brunette

Puente

et al. 2010

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

149

172

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In complex organisms, caspase proteases mediate a variety of cell behaviors, including proliferation, differentiation, and programmed cell death/apoptosis. Structural homologs to the caspase family (termed metacaspases) engage apoptosis in singlecell eukaryotes, yet the molecular mechanisms that contribute to nondeath roles are currently undefined. Here, we report an unexpected role for the Saccharomyces cerevisiae metacaspase Yca1 in protein quality control. Quantitative proteomic analysis of Δyca1 cells identified significant alterations to vacuolar catabolism and stress-response proteins in the absence of induced stress. Yca1 protein complexes are enriched for aggregate-remodeling chaperones that colocalize with Yca1-GFP fusions. Finally, deletion and inactivation mutants of Yca1 accrue protein aggregates and autophagic bodies during log-phase growth. Together, our results show that Yca1 contributes to the fitness and adaptability of growing yeast through an aggregate remodeling activity.nonapoptotic caspase activity | quality control | yeast | prion domain

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Section: Resultssupporting

confidence: 60%

Metacaspase Yca1 is required for clearance of insoluble protein aggregates

Lee

Brunette

Puente

et al. 2010

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

149

172

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“…The fluorescent vital dye FM4-64 has been shown to stain differentially the acidic vacuoles of autophagic yeast cells due to accumulation of membrane material (26). When FM4-64 was added to cultures of SG and CR cells, a pattern of solid vacuoles was observed, similar in appearance to what is observed in nitrogen-starved yeast cells (Fig.…”

Section: Effect Of Ipp1psupporting

confidence: 52%

Inorganic Pyrophosphatase Defects Lead to Cell Cycle Arrest and Autophagic Cell Death through NAD+ Depletion in Fermenting Yeast

Serrano-Bueno

Hernández

López-Lluch

et al. 2013

Journal of Biological Chemistry

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“…Vacuolar membrane specific lipophilic dye, FM 4-64 staining in yeast was done as described in Journo et al, 2008 [42]. Saccharomyces cerevisiae (BY4743, KVY55, and Atg8Δ) yeast cells were cultured to mid exponential phase in YEPD media.…”

Section: Methodsmentioning

confidence: 99%

Latrepirdine (Dimebon™) Enhances Autophagy and Reduces Intracellular GFP-Aβ42 Levels in Yeast

Bharadwaj

Verdile

Barr

et al. 2012

JAD

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Latrepirdine (Dimebon™), an anti-histamine, has shown some benefits in trials of neurodegenerative diseases characterized by accumulation of aggregated or misfolded protein such as Alzheimer’s disease (AD) and has been shown to promote the removal of α-synuclein protein aggregates in vivo. An important pathway for removal of aggregated or misfolded proteins is the autophagy-lysosomal pathway, which has been implicated in AD pathogenesis, and enhancing this pathway has been shown to have therapeutic potential in AD and other proteinopathies. Here we use a yeast model Saccharomyces cerevisiae, to investigate whether latrepirdine can enhance autophagy and reduce levels of Aβ42 aggregates. Latrepirdine was shown to up-regulate yeast vacuolar (lysosomal) activity and promote transport of the autophagic marker (Atg8) to the vacuole. Using an in vitro GFP tagged Aβ yeast expression system, we investigated whether latrepirdine-enhanced autophagy was associated with a reduction in levels of intracellular GFP-Aβ42. GFP-Aβ42 was localized into punctate patterns compared to the diffuse cytosolic pattern of GFP and the GFP-Aβ42 (19:34), which does not aggregate. In the autophagy deficient mutant (Atg8Δ), GFP-Aβ42 showed a more diffuse cytosolic localization, reflecting the inability of this mutant to sequester GFP-Aβ42. Similar to rapamycin, we observed that latrepirdine significantly reduced GFP-Aβ42 in wild-type compared to the Atg8Δ mutant. Further, latrepirdine treatment attenuated Aβ42-induced toxicity in wild-type cells but not in the Atg8Δ mutant. Together, our findings provide evidence for a novel mechanism of action for latrepirdine in inducing autophagy and reducing intracellular levels of GFP-Aβ42.

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Chapter 7 Monitoring Autophagy in Yeast using FM 4‐64 Fluorescence

Cited by 17 publications

References 13 publications

Metacaspase Yca1 is required for clearance of insoluble protein aggregates

Metacaspase Yca1 is required for clearance of insoluble protein aggregates

Inorganic Pyrophosphatase Defects Lead to Cell Cycle Arrest and Autophagic Cell Death through NAD+ Depletion in Fermenting Yeast

Latrepirdine (Dimebon™) Enhances Autophagy and Reduces Intracellular GFP-Aβ42 Levels in Yeast

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