Fluorescent Protein-Based Autophagy Biosensors

Kim, Heejung

doi:10.3390/ma14113019

Cited by 8 publications

(4 citation statements)

References 162 publications

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“…Employing correlative live-cell and super-resolution imaging (Balint et al, 2013; Verdeny-Vilanova et al, 2017) with sensors for autophagy (Kim and Seong, 2021), mTOR activity (Zhou et al, 2015), and luminal pH (Chin et al, 2021) could reveal more about these relationships. Previous studies in neurons showed that not all LAMP1-positive LELs contain degradative enzymes like cathepsin D. (Cheng et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

Multiplexed DNA-PAINT Imaging of the Heterogeneity of Late Endosome/Lysosome Protein Composition

Bond,

Hugelier,

Xing

et al. 2024

Preprint

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Late endosomes/lysosomes (LELs) are crucial for numerous physiological processes and their dysfunction is linked to many diseases. Proteomic analyses have identified hundreds of LEL proteins, however, whether these proteins are uniformly present on each LEL, or if there are cell-type dependent LEL sub-populations with unique protein compositions is unclear. We employed a quantitative, multiplexed DNA-PAINT super-resolution approach to examine the distribution of six key LEL proteins (LAMP1, LAMP2, CD63, TMEM192, NPC1 and LAMTOR4) on individual LELs. While LAMP1 and LAMP2 were abundant across LELs, marking a common population, most analyzed proteins were associated with specific LEL subpopulations. Our multiplexed imaging approach identified up to eight different LEL subpopulations based on their unique membrane protein composition. Additionally, our analysis of the spatial relationships between these subpopulations and mitochondria revealed a cell-type specific tendency for NPC1-positive LELs to be closely positioned to mitochondria. Our approach will be broadly applicable to determining organelle heterogeneity with single organelle resolution in many biological contexts.

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Section: Discussionmentioning

confidence: 99%

Multiplexed DNA-PAINT Imaging of the Heterogeneity of Late Endosome/Lysosome Protein Composition

Bond,

Hugelier,

Xing

et al. 2024

Preprint

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“…When autophagosomes fuse with lysosomes to become autolysosomes, the pH-sensitive red FP, but not the pH-stable blue FP, decreases its uorescent intensity. Therefore, real-time progression of autophagy can be monitored by measuring the red/blue (R/B) intensity ratios of RB-LC3 in live cells 20,22 .…”

Section: Autophagy Process For Mutant Htt Is Impairedmentioning

confidence: 99%

Distinct recognition of mutant huntingtin aggregates by autophagy receptor SQSTM1/p62 versus optineurin has differential effects on cell survival

Seong,

Kim,

Lee

et al. 2024

Preprint

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Huntington's disease (HD) is a neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the N-terminus of huntingtin (HTT). Mutant HTT (mHTT) undergoes misfolding and tends to aggregate, yet these aggregates are not effectively cleared by autophagy thus contributing to neurotoxicity in HD. The polyQ length of HTT in patients with HD varies from 40 to > 90; however, the precise mechanisms of autophagy dysfunction for mHTT with varying polyQ lengths remain unclear. In this study, we developed new HTT-polyQ aggregation sensors based on bimolecular fluorescence complementation (BiFC) to monitor the real-time aggregation process of mHTT with varying polyQ lengths. Using BiFC-based aggregation sensors, we demonstrated that mHTT aggregation kinetics is faster with a longer polyQ length, suggesting a correlation between polyQ length and the onset age of HD. Interestingly, we discovered that the different aggregation kinetics of mHTT may determine the physical properties of the aggregates: mHTT-polyQ43 forms liquid-like protein condensates, whereas mHTT-polyQ103 generates tightly concentrated aggregates. Furthermore, mHTT aggregates with different physical states were selectively recognized by distinct autophagy receptors, which resulted in differential effects on cell viability. The liquid-like mHTT-polyQ43 condensates were recognized by SQSTM1/p62 but failed to proceed through autophagy thereby facilitating cytotoxicity. In contrast, mHTT-polyQ103 aggregates were selectively recognized by optineurin, which led to autophagic degradation and prolonged cell survival. Therefore, our results suggest that different therapeutic strategies should be considered for the HD patients with different polyQ lengths.

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“…However, these conclusions were based on results from tandem EGFP-mCherry-LC3B experiments, which have technical limitations. For example, EGFP retains some mild fluorescence at a lysosomal pH, and a neutral pH buffer like the common fixative agent paraformaldehyde can restore GFP fluorescence that has been reduced due to lowered pH conditions [ 47 , 48 , 49 , 50 ]. Published guidelines exist for the standardization of methodology in studies of autophagy, with discussion of mitigation strategies [ 51 ].…”

Section: Role Of Vcp In the Cytosolmentioning

confidence: 99%

Valosin-Containing Protein (VCP): A Review of Its Diverse Molecular Functions and Clinical Phenotypes

Pontifex,

Zaman,

Fanganiello

et al. 2024

IJMS

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In this review we examine the functionally diverse ATPase associated with various cellular activities (AAA-ATPase), valosin-containing protein (VCP/p97), its molecular functions, the mutational landscape of VCP and the phenotypic manifestation of VCP disease. VCP is crucial to a multitude of cellular functions including protein quality control, endoplasmic reticulum-associated degradation (ERAD), autophagy, mitophagy, lysophagy, stress granule formation and clearance, DNA replication and mitosis, DNA damage response including nucleotide excision repair, ATM- and ATR-mediated damage response, homologous repair and non-homologous end joining. VCP variants cause multisystem proteinopathy, and pathology can arise in several tissue types such as skeletal muscle, bone, brain, motor neurons, sensory neurons and possibly cardiac muscle, with the disease course being challenging to predict.

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Fluorescent Protein-Based Autophagy Biosensors

Cited by 8 publications

References 162 publications

Multiplexed DNA-PAINT Imaging of the Heterogeneity of Late Endosome/Lysosome Protein Composition

Multiplexed DNA-PAINT Imaging of the Heterogeneity of Late Endosome/Lysosome Protein Composition

Distinct recognition of mutant huntingtin aggregates by autophagy receptor SQSTM1/p62 versus optineurin has differential effects on cell survival

Valosin-Containing Protein (VCP): A Review of Its Diverse Molecular Functions and Clinical Phenotypes

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