TRPML Subfamily of Endolysosomal Channels
Bruno Gasnier, Michael X. Zhu in Ion and Molecule Transport in Lysosomes, 2020
The commonly studied invertebrates, Drosophila melanogaster and Caenorhabditis elegans, possess single TRPML encoding genes – trpml and coelomocyte uptake defective-5 (cup-5), respectively (Fares and Greenwald, 2001; Venkatachalam et al., 2008). The worm cup-5 mutants exhibit maternal-effect embryonic lethality and endolysosomal accumulation. Flies lacking trpml exhibit defects in completion of autophagy with a concomitant build-up of endolysosomes in a wide range of tissues, high rates of pupal lethality, age-dependent neurodegeneration, and locomotor impairment. The MLIV flies also exhibit defects in glutamatergic synapse development and neurotransmission (Venkatachalam et al., 2008; Wong et al., 2015). Interestingly, the neurological phenotypes in the MLIV flies are a result of a complex interplay between neurons and phagocytic cells (Venkatachalam et al., 2008). First, cell autonomous endolysosomal defects in neurons led to accumulation of damaged mitochondria and diminished cell viability. A secondary, non-cell autonomous effect in phagocytic cells such as glia was triggered by the dying neurons and led to neuroinflammation. Remarkably, reintroduction of wild-type trpml in only the phagocytic cells was sufficient to significantly delay the locomotor defects and attendant lethality. These findings led to the intriguing proposal that bone marrow transplantation to introduce functional phagocytic cells such as microglia in patients lacking TRPML1 could delay the onset of MLIV – a concept that was successfully validated in a mouse model of MLIV (Walker and Montell, 2016).
Cationic polystyrene nanoparticle and the sea urchin immune system: biocorona formation, cell toxicity, and multixenobiotic resistance phenotype
Published in Nanotoxicology, 2018
L. F. Marques-Santos, G. Grassi, E. Bergami, C. Faleri, T. Balbi, A. Salis, G. Damonte, L. Canesi, I. Corsi
NRRT assay was conducted using coelomocyte monolayers, by placing 200 mL of coelomocytes suspension collected from three sample replicates (PS-NH2 and control ones) in a glass slide and incubated in a humid chamber for 1 h in the dark at 18 °C. This method allows in maintaining a good preservation of cellular morphology for optical microscopy. Cells were then incubated for 30 min at 18 °C with 100 μL of NR dye solution (final concentration 40 μg mL−1 from a stock solution of 40 mg mL−1 NR in DMSO). Excess of dye was removed using filtered NSW and slides sealed with a coverslip. Slides were checked every 15 min under an optical microscope (Olympus U25ND6, Tokyo, Japan) and the percentage of cells showing loss of the NR dye from lysosomes was counted. An average of 100 cells was observed at each time-point. The end-point of the assay is defined as the time at which 50% of the cells show signs of lysosomal leaking (cytosol becoming red and cells rounded-shaped). For each treatment and controls, three replicates were run in parallel.