The induction of bulk autophagy by nitrogen starvation in baker's yeast (S. cerevisiae) involves the upregulation of many autophagy related proteins, including Atg7. One way to investigate the importance of this upregulation is to measure the size and number of autophagosomes formed when insufficient amounts of that protein are available. Atg8 is known to affect autophagosome size, consistent with its role in phagophore expansion. Atg7 is upstream of Atg8, and might therefore be expected to affect only autophagosome size. We used electron microscopy to measure the size and number of autophagosomes formed with limiting amounts of Atg7 and found them to be both smaller and fewer than normal. This suggests that Atg7 may have an Atg8-independent role in autophagosome initiation in addition to its Atg8-dependent role in autophagosome expansion. We also present an improved simulation for estimating original autophagic body number based on the number of cross-sections observed in ultrathin sections.
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http://dx.doi.org/10.1016/j.bbrc.2018.06.056 | DOI Listing |
PLoS Pathog
January 2025
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland United States of America.
Autophagy plays a crucial role in the host response to Mycobacterium tuberculosis (Mtb) infection, yet the dynamics and regulation of autophagy induction on Mtb-containing vacuoles (MCVs) remain only partially understood. We employed time-lapse confocal microscopy to investigate the recruitment of LC3B (LC3), a key autophagy marker, to MCVs at the single cell level with our newly developed workflow for single cell and single MCV tracking and fluorescence quantification. We show that approximately 70% of MCVs exhibited LC3 recruitment but that was lost in about 40% of those MCVs.
View Article and Find Full Text PDFMethods Mol Biol
December 2024
Department of Physiology, University of California, San Francisco, San Francisco, CA, USA.
This chapter describes two imaging-based approaches for examining the localization of bridge-like lipid transfer proteins at membrane contact sites during native biological processes. These approaches use multi-color fluorescence imaging, enabling high spatial and temporal resolution and overcoming the limitations of biochemical methods. The first approach involves immunofluorescence in fixed cells, while the second utilizes time-lapse imaging in live cells.
View Article and Find Full Text PDFFront Pharmacol
November 2024
Department of Emergency, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, China.
Background: Autophagy‒endoplasmic reticulum (ER) stress axis dysregulation is linked to myocardial ischemia‒reperfusion injury (MIRI), which counteracts the benefits of acute myocardial infarction (AMI) reperfusion therapy. Qingre Huoxue decoction (QRHX) improves the short- and long-term prognosis of AMI after percutaneous coronary intervention and alleviates myocardial injury in AMI rats by stimulating autophagy via the PI3K/Akt pathway. We aimed to further explore the efficacy of QRHX in treating MIRI and its regulatory relationship with FAM134B-mediated ER-phagy.
View Article and Find Full Text PDFEur J Med Res
November 2024
Department of Rehabilitation Medicine, Taizhou Municipal Hospital, No.381-1, Zhongshan East Road, Taizhou, 318000, Zhejiang, China.
Background: Post-stroke cognitive impairment (PSCI) severely reduces quality of life of patients with stroke. This study aimed to assess the effects of electroacupuncture (EA) on PSCI and the role of the mTOR/NLRP3-mediated autophagy-inflammatory pathway in this process.
Methods: The rat focal cerebral ischemia model was established using middle cerebral artery occlusion (MCAO).
Biomolecules
September 2024
Key Laboratory for Quality Evaluation of Bulk Herbs of Hunan Province, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, China.
Atherosclerosis is a chronic inflammatory disease characterized by lipid accumulation and foam cell formation in the arterial wall. Promoting macrophage autophagy has emerged as a promising therapeutic strategy against atherosclerosis. Dipsacoside B (DB) is an oleanane-type pentacyclic triterpenoid saponin extracted from with potential anti-atherosclerotic properties.
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