Methods for monitoring lysosomal morphology

Kilpatrick, Bethan S.; Eden, Emily R.; Hockey, Leanne N.; Futter, Clare E.; Patel, Sandip

doi:10.1016/bs.mcb.2014.10.018

Cited by 19 publications

(22 citation statements)

References 64 publications

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“…Following transfection, cells were seeded at 6.7×10 4 per 20 mm glass well in 35 mm dishes (MatTek, Ashland, MA) and incubated for 24-48 hours before imaging. Lysosomes in BS-C-1 cells were labeled as described in (Humphries et al, 2011, Kilpatrick et al, 2015, Bright et al, 2005. Briefly, cells were incubated with 0.5 mg/ml Dextran Alexa 488, 10000 MW (Molecular Probes, Eugene, OR) for 3-4 hours followed by 16 hours of chasing.…”

Section: A Model Of Lysosomal Clusteringmentioning

confidence: 99%

Whole-cell scale dynamic organization of lysosomes revealed by spatial statistical analysis

Raghavan

Kiselyov

et al. 2017

Preprint

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SummaryIn eukaryotic cells, lysosomes are distributed in the cytoplasm as individual membranebound compartments to degrade macromolecules and to control cellular metabolism. A fundamental yet unanswered question is whether and, if so, how individual lysosomes are spatially organized so that their functions can be coordinated and integrated to meet changing needs of cells. To address this question, we analyze their collective behavior in cultured cells using spatial statistical techniques. We find that in single cells, lysosomes maintain nonrandom, stable, yet distinct spatial distributions, which are mediated by the coordinated effects of the cytoskeleton and lysosomal biogenesis on different lysosomal subpopulations. Furthermore, we find that throughout the intracellular space, lysosomes form dynamic clusters that substantially increase their interactions with endosomes. Together, our findings reveal the spatial organization of lysosomes at the whole-cell scale and provide new insights into how organelle interactions are mediated and regulated over the entire intracellular space.peer-reviewed)

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Section: A Model Of Lysosomal Clusteringmentioning

confidence: 99%

Whole-cell scale dynamic organization of lysosomes revealed by spatial statistical analysis

Raghavan

Kiselyov

et al. 2017

Preprint

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“…Lysosomes are membrane bound organelles containing acid hydrolases involved in catabolism of intracellular biomolecules and organelles as well as of extracellular components delivered by, respectively, autophagy and the endosomal pathways [15][16][17][18].…”

Section: Introductionmentioning

confidence: 99%

PLA2G4A/cPLA2-mediated lysosomal membrane damage leads to inhibition of autophagy and neurodegeneration after brain trauma

et al. 2019

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Lysosomal membrane permeabilization (LMP) is observed under many pathological conditions, leading to cellular dysfunction and death. However, the mechanisms by which lysosomal membranes become leaky in vivo are not clear. Our data demonstrate that LMP occurs in neurons following controlled cortical impact induced (CCI) traumatic brain injury (TBI) in mice, leading to impaired macroautophagy (autophagy) and neuronal cell death. Comparison of LC-MS/MS lysosomal membrane lipid profiles from TBI and sham animals suggested a role for PLA2G4A/cPLA2 (phospholipase A2, group IVA [cytosolic, calcium-dependent]) in TBIinduced LMP. Activation of PLA2G4A caused LMP and inhibition of autophagy flux in cell lines and primary neurons. In vivo pharmacological inhibition of PLA2G4A attenuated TBI-induced LMP, as well as subsequent impairment of autophagy and neuronal loss, and was associated with improved neurological outcomes. Inhibition of PLA2G4A in vitro limited amyloid-β-induced LMP and inhibition of autophagy. Together, our data indicate that PLA2G4A-mediated lysosomal membrane damage is involved in neuronal cell death following CCI-induced TBI and potentially in other neurodegenerative disorders.

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“…The single Gaussian intensity distributions of both fluorophores overlapped significantly, with centers that colocalized with diffraction-limited resolution. This result suggests that the nanotubes localized to the same region of endolysosomal organelles as dextran, which is known to remain in the endolysosomal lumen 44 .…”

Section: Uptake and Localization Of Dna-swcnts To The Endolysosomal Lmentioning

confidence: 78%

“…Concomitantly, the nIR emission from the nanotubes localized to the same regions of the cell as the visible emission from Lysotracker, further supporting the endolysosomal localization of the nanotubes ( Figure S14). We next assessed the localization of the nanotubes within the lumen of individual endolysosomal organelles, by pulsing the cells with fluorescent (TMR) 10,000 MW dextran, a polymer which accumulates in the endolysosomal lumen and does not degrade 44 . Following overnight incubation, the cells were maintained in dextran-free media for 3 hours, before Alexa-647 labeled nanotube complexes were introduced to the cell media for 30 minutes and then washed away.…”

Section: Uptake and Localization Of Dna-swcnts To The Endolysosomal Lmentioning

confidence: 99%

“…17). To assess the localization of the nanotubes within individual LE/Ly, we pulsed the cells with fluorescent (TMR) 10,000 MW dextran, a polymer which accumulates in the lysosomal lumen and does not degrade 31 . Following overnight incubation, the cells were maintained in dextranfree media for 3 hours, before Alexa-647 labeled nanotube complexes were introduced to the cell media for 30 minutes.…”

Section: Localization and Biocompatibility In The Endolysosomal Lumenmentioning

confidence: 99%

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A carbon nanotube optical reporter maps endolysosomal lipid flux

Jena

Roxbury

Galassi

et al. 2017

Preprint

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Lipid accumulation within the lumen of endolysosomal vesicles is observed in various pathologies including atherosclerosis, liver disease, neurological disorders, lysosomal storage disorders, and cancer. Current methods cannot measure lipid flux specifically within the lysosomal lumen of live cells. We developed an optical reporter, composed of a photoluminescent carbon nanotube of a single chirality, that responds to lipid accumulation via modulation of the nanotube's optical band gap. The engineered nanomaterial, composed of short, singlestranded DNA and a single nanotube chirality, localizes exclusively to the lumen of endolysosomal organelles without adversely affecting cell viability or proliferation or organelle morphology, integrity, or function. The emission wavelength of the reporter can be spatially resolved from within the endolysosomal lumen to generate quantitative maps of lipid content in live cells. Endolysosomal lipid accumulation in cell lines, an example of drug-induced phospholipidosis, was observed for multiple drugs in macrophages, and measurements of patient-derived Niemann−Pick type C fibroblasts identified lipid accumulation and phenotypic reversal of this lysosomal storage disease. Single-cell measurements using the reporter discerned subcellular differences in equilibrium lipid content, illuminating significant intracellular heterogeneity among endolysosomal organelles of differentiating bone-marrowderived monocytes. Single-cell kinetics of lipoprotein-derived cholesterol accumulation within macrophages revealed rates that differed among cells by an order of magnitude. This carbon nanotube optical reporter of endolysosomal lipid content in live cells confers additional capabilities for drug development processes and the investigation of lipid-linked diseases.

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Methods for monitoring lysosomal morphology

Cited by 19 publications

References 64 publications

Whole-cell scale dynamic organization of lysosomes revealed by spatial statistical analysis

Whole-cell scale dynamic organization of lysosomes revealed by spatial statistical analysis

PLA2G4A/cPLA2-mediated lysosomal membrane damage leads to inhibition of autophagy and neurodegeneration after brain trauma

A carbon nanotube optical reporter maps endolysosomal lipid flux

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