A rapid method for the preparation of ultrapure, functional lysosomes using functionalized superparamagnetic iron oxide nanoparticles

Walker, Mathew; Lloyd-Evans, Emyr

doi:10.1016/bs.mcb.2014.10.019

Cited by 35 publications

(35 citation statements)

References 35 publications

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“…To investigate this issue further specifically in lysosomes, we used a superparamagnetic chromatography isolation procedure (Walker and Lloyd-Evans, 2015) to isolate highly enriched lysosomal preparations from PS1KO and WT cells, comprising 3% of the total cell lysate (protein: protein) and representing an enrichment of the LAMP2 lysosomal marker. Western blot analysis of enriched lysosomes confirmed the purity of lysosome fractions by demonstrating the presence of minimal calnexin and EEA1 and strong enrichment of LC3-II and CatD (Fig 4A).…”

Section: Resultsmentioning

confidence: 99%

Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

et al. 2015

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Summary Presenilin-1 (PS1) deletion or Alzheimer’s Disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in PS1KO cells induces abnormal Ca2+ efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca2+. In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca2+ homeostasis, but correcting lysosomal Ca2+ deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss of function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca2+ homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

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Section: Resultsmentioning

confidence: 99%

Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

et al. 2015

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“…To focus only on the lysosomes, we utilized a well characterized protocol to isolate ultrapure lysosomal fractions using magnetic dextran-coated nanobeads 24 and assessed mTOR distribution in RAW-derived osteoclasts and in non-differentiated RAW264.7 cells in response to starvation conditions. Similar to the fractionation experiments, RAW-derived osteoclast lysosomes were probed for mTOR and showed that there was no discernable difference in mTOR distribution between control and starvation conditions (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…24 . Undifferentiated RAW264.7 cells and RAW264.7-derived osteoclasts, an established in vitro model of osteoclastogenesis, were used in these experiments.…”

Section: Methodsmentioning

confidence: 99%

Activity-independent targeting of mTOR to lysosomes in primary osteoclasts

Wang

Carraro‐Lacroix

Owen

et al. 2017

Sci Rep

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Mammalian target of rapamycin (mTOR) is activated by numerous stimuli, including amino acids and growth factors. This kinase is part of the mTOR complex 1 (mTORC1) which regulates cell proliferation, differentiation, and autophagy. Active mTORC1 is located on lysosomes and has been reported to disassociate from the lysosomal surface in the absence of amino acids. Furthermore, mTORC1 activity has been linked to the vacuolar H+-ATPases (V-ATPases), the proton pumps responsible for lysosomal acidification; however, the exact role of the V-ATPases in mTORC1 signaling is not known. To elucidate the mechanisms involved in mTORC1 regulation by the V-ATPases, we used primary osteoclasts derived from mice carrying a point (R740S) mutation in the a3 subunit of the V-ATPase. In these cells, the mutant protein is expressed but the pump is not functional, resulting in higher lysosomal pH. By analyzing mTOR activation, mTOR/lysosome co-localization, and lysosomal positioning using confocal microscopy, fractionation, and ultrapure lysosomal purification methods, we demonstrate that in primary osteoclasts, mTOR is localized on the lysosomal surface even when mTOR activity is inhibited. Our findings reveal that mTOR targeting to the lysosome in osteoclasts is activity-independent, and that its disassociation from the lysosome during starvation is not universal.

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“…In this work we adopted recently published method for lysosomal isolation using ferrofluid particles (21). Using this method we isolated lysosomal fractions which were positive for LAMP1…”

Section: Lysosomal Isolation From Npc1-null and Chowt Cells Using Thementioning

confidence: 99%

“…Lysosomes were purified according the Walker and Lloyde Evans protocol (21). Briefly, the cells were grown in T75 flasks, they were incubated with 10% ferrofluid solution (superparamagnetic iron oxide nanoparticles, 10 mg/mL of 40 kDa dextran-stabilized magnetite, Liquids Research Ltd, UK.)…”

Section: Magnetic Separation Of Lysosomes Form Whole Cellsmentioning

confidence: 99%

N-glycome of the Lysosomal Glycocalyx is Altered in Niemann-Pick Type C Disease (NPC) Model Cells

Kosicek

Gudelj

Horvatić

et al. 2018

Molecular & Cellular Proteomics

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A rapid method for the preparation of ultrapure, functional lysosomes using functionalized superparamagnetic iron oxide nanoparticles

Cited by 35 publications

References 35 publications

Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

Activity-independent targeting of mTOR to lysosomes in primary osteoclasts

N-glycome of the Lysosomal Glycocalyx is Altered in Niemann-Pick Type C Disease (NPC) Model Cells

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