Quick and Mild Isolation of Intact Lysosomes Using Magnetic–Plasmonic Hybrid Nanoparticles

Abstract

Rapid and efficient isolation of intact lysosomes is necessary to study their functions and metabolites by proteomic analysis. We developed a swift and robust nanoparticle-based magnetic separation method in which magnetic− plasmonic hybrid nanoparticles (MPNPs) conjugated with amino dextran (aDxt) were targeted to the lumen of lysosomes via the endocytosis pathway. For well-directed magnetic separation of the lysosomes, it is important to trace the intracellular trafficking of the aDxt-conjugated MPNPs (aDxt-MPNPs) in the endocytosis pathway. Therefore, we analyzed the intracellular transport process of the aDxt-MPNPs by investigating the time-dependent colocalization of plasmonic scattering of aDxt-MPNPs and immunostained marker proteins of organelles using the threshold Manders' colocalization coefficient (R t ). Detailed analysis of time variations of R t for early and late endosomes and lysosomes allowed us to derive the transport kinetics of aDxt-MPNPs in a cell. After confirming the incubation time required for sufficient accumulation of aDxt-MPNPs in lysosomes, the lysosomes were magnetically isolated as intact as possible. By varying the elapsed time from homogenization to complete isolation of lysosomes (t delay ) and temperature (T), the influences of t delay and T on the protein composition of the lysosomes were investigated by polyacrylamide gel electrophoresis and amino acid analysis. We found that the intactness of lysosomes could become impaired quite quickly, and to isolate lysosomes as intact as possible with high purity, t delay = 30 min and T = 4 °C were optimal settings.

Macrophage ALDH2 (Aldehyde Dehydrogenase 2) Stabilizing Rac2 Is Required for Efferocytosis Internalization and Reduction of Atherosclerosis Development

“…Colocalization of Rac2 and ALDH2 was analyzed with the plug in coloc2 for image J and Manders’ Colocalization Coefficients were used to estimate colocalization as previously described. 21 , 22 …”

“…Colocalization of Rac2 and ALDH2 was analyzed with the plug in coloc2 for image J and Manders' Colocalization Coefficients were used to estimate colocalization as previously described. 21,22 Live-Cell Imaging System Primary peritoneal macrophages were placed onto a glass-bottom culture plate and stained with CellTracker Deep Red dye (Invitrogen, C34565). Primary VSMCs were stained with CellTracker Green CMFDA dye (Invitrogen, C2925) and incubated with 1 μmol/L staurosporine for 6 hours.…”

Optogenetic Calcium Ion Influx in Myoblasts and Myotubes by Near-Infrared Light Using Upconversion Nanoparticles

“…UCNPs were encapsulated in phospholipid micelles to make them water-dispersible . Specifically, micelles were composed of biotin-DOPE and DOPE-PEG350 at a molar ratio of 4:1 (Scheme S1 in the Supporting Information).…”

Dysregulation of organelle membrane contact sites in neurological diseases

“…Early in the study of MCS, traditional fractionation methods were adapted to enrich membranes involved in MCS (Vance, 1990). More recently, methods have emerged to isolate intact organelles, such as by immunopurification (Abu-Remaileh et al, 2017;Chen et al, 2017) or nanoparticle-based magnetic separation (Le et al, 2022). In vitro reconstitution of interactions between isolated organelles may help to elucidate key regulators and functions of MCS without the confounder of compensatory homeostatic pathways.…”