Abstract
Purpose :
Amino acid homeostasis is crucial for the proper function, structure, and maintenance of the retina and its dysregulation leads to blinding diseases such as retinitis pigmentosa (RP). Cells utilize vesicles as a storage compartment for maintaining amino acid concentration in times of starvation and for trafficking amino acids to subcellular compartments for use. Solute Carrier Family 7 Member 14 (SLC7A14) is a predicted lysosomal cationic amino acid transporter and its mutation and deletion is known to cause RP. However, the molecular mechanisms by which loss of SLC7A14 causes blindness are unknown.
Methods :
We generated a gain-of-function hTERT-RPE1 cell line through lentiviral transduction that stably expresses SLC7A14-FLAG. for fixed imaging. Immunofluorescent staining of SLC7A14-FLAG cells was then performed using antibodies for the FLAG epitope, early endosome antigen 1 (EEA1), and lysosomal-associated membrane protein 1 (LAMP1). Parental hTERT-RPE1 cells were co-transfected with SLC7A14-EGFP and LAMP1-mCherry, and their lysosomes labeled with the small molecule dye SiR-lysosome. Spinning-disc confocal microscopy was used to track the movement of vesicles of cells grown either in complete media or those starved of amino acids and serum.
Results :
SLC7A14-FLAG was observed within intracellular vesicles throughout the cytoplasm, and these vesicles showed an enrichment in the perinuclear region of cells grown in complete media. Vesicles containing SLC7A14-FLAG only occasionally co-localized with lysosomal marker proteins; however, approximately 60% of such vesicles showed colocalization with EEA1. Live-cell microscopy revealed that the spatial organization of SLC7A14-EGFP vesicles was dynamic in response to nutrient availability. Upon amino acid and serum starvation, SLC7A14 -EGFP showed an enhanced perinuclear localization and was recruited to multivesicular bodies containing both LAMP1-mCherry and SiR-Lysosome.
Conclusions :
These findings suggest that SLC7A14 is dynamically recruited to discrete endolysosomal compartments in response to intracellular nutrient demands. Future studies will determine the functional determinants of SLC7A14 compartmentalization and wheter RP-associated mutations alter its vesicular localization and dynamics. Together, these results will provide insight into the function of SLC7A14 in cells and may provide molecular insights into how the loss of SLC7A14 causes blindness.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.