Analysis of Golgi Morphology Using Immunofluorescence and CellProfiler Software

Mejia, Isabel; Chen, Yu-Chuan; Díaz, Begoña

doi:10.1007/978-1-0716-2639-9_46

Cited by 3 publications

(3 citation statements)

References 16 publications

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“…ER-Golgi dynamics have been shown to regulate the localization of HS-modifying enzymes 53 . Thus we monitored whether Golgi structure was affected in C3orf58 and SLC39A9 KO cells by using a modified Golgi morphology analysis pipeline 54 ( Supplementary Figure 17 ). Golgi area was increased in both lines in a rescuable manner.…”

Section: Resultsmentioning

confidence: 99%

“…Z-stacks (60 stacks per field, 0.185 μm steps) were acquired using a Nikon A1 confocal microscope mounted with a Plan Apo λ 60x 1.40 objective. Then, a customized CellProfiler pipeline largely inspired from Mejia et al 54 was used to analyze Golgi morphology. Briefly, changes were made to the Mejia et al pipeline so that cell outlines corresponded to nuclei extended by 17 pixels, and those that touched the image border were filtered out from analyses.…”

Section: Methodsmentioning

confidence: 99%

“…Western blot analysis showed a 1.5-fold increase in total NDST1 levels, an observation in line with the RNASeq data (Supplementary Figure 15) suggesting an accumulation of the enzyme and faster migration in lysates from SLC39A9 KO cells, probably due to lesser N-glycosylation 53 (Figure 8A). Beyond expression levels, regulation of HS enzymes trafficking is believed to modulate HS modification 54 . C3orf58 was previously shown to colocalize with COPI vesicles responsible for intra-Golgi trafficking 40 and its overexpression modulates secretory protein transit from the ER to the Golgi 55 .…”

Section: A Tight Balance In Ndst1 Expression Is Needed For Pff Uptake...mentioning

confidence: 99%

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A genome-wide CRISPR/Cas9 screen identifies genes that regulate the cellular uptake of α-synuclein fibrils by modulating heparan sulfate proteoglycans

Vanderperre,

Muraleedharan,

Dorion

et al. 2023

Preprint

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Synucleinopathies are characterized by the accumulation and propagation of α-synuclein (α-syn) aggregates throughout the brain, leading to neuronal dysfunction and death. Understanding how these aggregates propagate from cell to cell in a prion-like fashion thus holds great therapeutic promises. Here, we focused on understanding the cellular processes involved in the entry and accumulation of pathological α-syn aggregates. We used an unbiased FACS-based genome-wide CRISPR/Cas9 knockout (KO) screening to identify genes that regulate the accumulation of α-syn preformed fibrils (PFFs) in cells. We identified key genes and pathways specifically implicated in α-syn PFFs intracellular accumulation, including heparan sulfate proteoglycans (HSPG) biosynthesis and Golgi trafficking. We show that all confirmed hits affect heparan sulfate (HS), a post-translational modification known to act as a receptor for proteinaceous aggregates including of α-syn and tau. Intriguingly, KO ofSLC39A9andC3orf58genes, encoding respectively a Golgi-localized exporter of Zn2+, and the Golgi-localized putative kinase DIPK2A, specifically impaired the uptake of α-syn PFFs uptake but not of tau oligomers, by preventing the binding of PFFs to the cell surface. Mass spectrometry-based analysis of HS chains indicated major defects in HS maturation inSLC39A9andC3orf58KO cells, explaining the cell surface binding deficit. Our findings now clearly establish these two genes as HSPG-modulating factors. Interestingly,C3orf58KO human iPSC-derived microglia exhibited a strong reduction in their ability to internalize α-syn PFFs. Altogether, our data establish HSPGs as major receptors for α-syn PFFs binding on the cell surface and identifies new players in α-syn PFFs cell surface binding and uptake.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: A Tight Balance In Ndst1 Expression Is Needed For Pff Uptake...mentioning

confidence: 99%

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A genome-wide CRISPR/Cas9 screen identifies genes that regulate the cellular uptake of α-synuclein fibrils by modulating heparan sulfate proteoglycans

Vanderperre,

Muraleedharan,

Dorion

et al. 2023

Preprint

View full text Add to dashboard Cite

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Split but merge: Golgi fragmentation in physiological and pathological conditions

Zobaroğlu-Özer,

Bora-Akoğlu

2024

Mol Biol Rep

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Loss of CLN3 in microglia leads to impaired lipid metabolism and myelin turnover

Yasa,

Butz,

Colombo

et al. 2024

Commun Biol

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Loss-of-function mutations in CLN3 cause juvenile Batten disease, featuring neurodegeneration and early-stage neuroinflammation. How loss of CLN3 function leads to early neuroinflammation is not yet understood. Here, we have comprehensively studied microglia from Cln3 ∆ex7/8 mice, a genetically accurate disease model. Loss of CLN3 function in microglia leads to lysosomal storage material accumulation and abnormal morphology of subcellular organelles. Moreover, pathological proteomic signatures are indicative of defects in lysosomal function and abnormal lipid metabolism. Consistent with these findings, CLN3-deficient microglia are unable to efficiently turnover myelin and metabolize the associated lipids, showing defects in lipid droplet formation and cholesterol accumulation. Accordingly, we also observe impaired myelin integrity in aged Cln3 ∆ex7/8 mouse brain. Autophagy inducers and cholesterol-lowering drugs correct the observed microglial phenotypes. Taken together, these data implicate a cell-autonomous defect in CLN3-deficient microglia that impacts their ability to support neuronal cell health, suggesting microglial targeted therapies should be considered for CLN3 disease.

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Analysis of Golgi Morphology Using Immunofluorescence and CellProfiler Software

Cited by 3 publications

References 16 publications

A genome-wide CRISPR/Cas9 screen identifies genes that regulate the cellular uptake of α-synuclein fibrils by modulating heparan sulfate proteoglycans

A genome-wide CRISPR/Cas9 screen identifies genes that regulate the cellular uptake of α-synuclein fibrils by modulating heparan sulfate proteoglycans

Split but merge: Golgi fragmentation in physiological and pathological conditions

Loss of CLN3 in microglia leads to impaired lipid metabolism and myelin turnover

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