Diurnal changes of lysosomes including ultrastructural changes of phagosomes and acid phosphatase reactions in phagosomes, as well as diurnal biochemical changes in cathepsin D activity, were studied in the retinal pigment epithelium (RPE) of the rabbit. The rabbit was maintained on a natural light-dark cycle over seven days in fall and was sacrificed at various times during the day and night. The number of lysosomes or phagosomes in the RPE was the highest at 1.5 hours after exposure to sunlight (8:00 AM), and thereafter decreased with time. Three types of phagosomes were observed and acid phosphatase reactions were different in each type of phagosome; the fresh phagosomes were negative or positive, lamellar bodies positive, and dense bodies partially positive. The biochemical activity of cathepsin D was the highest at 8:00 AM, and this was consistent with the time of peak in phagocytic activity in the RPE. This report shows that phagocytic activity in the RPE occurred in the early stage after exposure to sunlight, and that fresh phagosomes were sequentially degraded to lamellar or dense bodies. Cathepsin D activity also increased, and this was consistent with the phagocytic activity in the RPE.
Acid Phosphatase/metabolism ; Animals ; Cathepsin D/metabolism ; Cell Count ; Choroid/metabolism/ultrastructure ; Circadian Rhythm/*physiology ; Lysosomes/*metabolism/ultrastructure ; Phagosomes/*metabolism/ultrastructure ; Pigment Epithelium of Eye/*metabolism/ultrastructure ; Rabbits

OBJECTIVETo observe the ultracytochemical localization of H(+)-adenosine triphosphatase (H(+)-ATPase) in the cell organelles.

METHODSThe localization of H(+)-ATPase in the cell organelles was observed in the hepatocytes and renal cells of Wistar rats using routine ultracytochemical methods.

RESULTSH(+)-ATPase activities were observed on the lysosomal membrane and nuclear envelope of the hepatocytes and proximal tubule epithelial cells of the nephron in Wistar rats.

CONCLUSIONThis finding supports the hypothesis that H(+)-ATPase (V-ATPase) is present on the plasma membrane and in the endomembrane system.

Animals ; Cell Membrane ; enzymology ; Hepatocytes ; cytology ; enzymology ; ultrastructure ; Histocytochemistry ; methods ; Kidney ; cytology ; enzymology ; ultrastructure ; Lysosomes ; enzymology ; Male ; Organelles ; enzymology ; Rats ; Rats, Wistar ; Vacuolar Proton-Translocating ATPases ; metabolism

TRPML1 channel is a non-selective group-2 transient receptor potential (TRP) channel with Ca permeability. Located mainly in late endosome and lysosome of all mammalian cell types, TRPML1 is indispensable in the processes of endocytosis, membrane trafficking, and lysosome biogenesis. Mutations of TRPML1 cause a severe lysosomal storage disorder called mucolipidosis type IV (MLIV). In the present study, we determined the cryo-electron microscopy (cryo-EM) structures of Mus musculus TRPML1 (mTRPML1) in lipid nanodiscs and Amphipols. Two distinct states of mTRPML1 in Amphipols are added to the closed state, on which could represent two different confirmations upon activation and regulation. The polycystin-mucolipin domain (PMD) may sense the luminal/extracellular stimuli and undergo a "move upward" motion during endocytosis, thus triggering the overall conformational change in TRPML1. Based on the structural comparisons, we propose TRPML1 is regulated by pH, Ca, and phosphoinositides in a combined manner so as to accommodate the dynamic endocytosis process.
Animals ; Calcium ; metabolism ; Cryoelectron Microscopy ; Endocytosis ; Endosomes ; metabolism ; Gene Expression ; HEK293 Cells ; Humans ; Hydrogen-Ion Concentration ; Lysosomes ; metabolism ; Mice ; Models, Biological ; Mucolipidoses ; genetics ; metabolism ; pathology ; Nanostructures ; chemistry ; ultrastructure ; Phosphatidylinositols ; metabolism ; Transgenes ; Transient Receptor Potential Channels ; chemistry ; genetics ; metabolism

OBJECTIVETo label rat neural stem cells (NSCs) with the complex of Sinerem, the ultrasmall superparamagnetic iron oxide (USPIO), and poly-L-lysine (PLL), and evaluate the feasibility of tracking the labeled cells with magnetic resonance imaging (MRI) in vitro and in vivo.

METHODSSinerem was incubated with PLL to obtain the complex of Sinerem-PLL. The mesenchymal stem cells (MSCs) isolated from the bone marrow of SD rats were cultured and induced to differentiate into the neural stem cells. The second-passage cells were cultured overnight with the Sinerem-PLL complex, after which Prussian blue staining and transmission electron microscopy were performed to observe the nanoparticles in the cytoplasm. Cell apoptosis assay was performed to assess the cell viability 1 day, 1 week, and 2 weeks after the labeling. Cell tracking with 4.7 MR system was carried out in vivo and in vitro using T(2)WI and T(2)*WI sequences.

RESULTSThe NSCs could be effectively labeled with Sinerem-PLL complex with the labeling efficiency exceeding 95%. Prussian blue staining showed numerous blue iron particles in the cytoplasm, and under transmission electron microscope, these particles accumulated in the endosomes/lysosomes. The labeling did not significantly affect the cell viability and proliferation. Remarkable low signal density changes of the labeled cells was seen on T(2)WI and T(2)*WI in vivo and in vitro.

CONCLUSIONNSCs can be effectively labeled with Sinerem-PLL complex, and MRI can be used to track the labeled cells in vivo and in vitro.

Animals ; Cell Differentiation ; Cells, Cultured ; Dextrans ; metabolism ; Endosomes ; metabolism ; Ferrosoferric Oxide ; metabolism ; Lysosomes ; metabolism ; Magnetic Resonance Imaging ; methods ; Magnetite Nanoparticles ; Male ; Mesenchymal Stromal Cells ; cytology ; Microscopy, Electron, Transmission ; Neurons ; cytology ; metabolism ; ultrastructure ; Polylysine ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; metabolism ; ultrastructure ; Time Factors