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Ankita Umapathy, Julie Chung, Madeline Tomlinson, Caleb Lim, David S Williams; Spatiotemporal dynamics of photoreceptor outer segment phagosome ingestion by the retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2021;62(8):2952.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal pigment epithelium (RPE) performs several functions crucial to the maintenance of vision, including the daily phagocytosis of the distal tips of the photoreceptor outer segments (OS). Previous visualization of the phagocytic cup formed by the RPE has been limited to electron microscopy. These static images, though informative, do not accurately capture an inherently transient and dynamic process. We used live imaging, for the first time, to visualize the dynamics of OS ingestion and the transient localization of proteins involved in phagocytosis.
Primary mouse RPE cells were transfected with proteins of interest (Tractin-RFP/GFP, GFP-FBP17, AMPH1-BAR-mCherry, mRFP-RAB5, GFP-RAB7, or LAMP1-YFP), fed labeled isolated OS, and imaged using spinning disk confocal microscopy. Phagosome diameter was measured in cells pre-treated with cytochalasin D (to disrupt actin dynamics) or DMSO vehicle.
Live imaging of RPE cells transfected with Tractin-RFP (actin) and fed labeled OS showed ingestion of OS by apical actin-rich phagocytic cups that bit phagosome-sized pieces. Cytochalasin D pre-treatment provided greater temporal resolution of actin involvement in phagosome scission such as localized increases in actin intensity coinciding with the site and time of scission events, and concentrated bursts of actin polymerization within the cup pushing phagosomes further into the cell. Furthermore, inhibitor-treated cells tended to produce phagosomes with smaller diameters. Membrane curvature-sensing proteins and regulators of actin dynamics, FBP17 and AMPH1-BAR, also localized to the phagocytic cup. Both coincided with highly curved membranes; AMPH1 was found at the rim and base of the cup, and FBP17 was restricted to the rim of the growing (but not regressing) cup. Early and late endosomal markers, RAB5 and RAB7 (but not the lysosomal marker, LAMP1), entered the phagocytic cup, before closure, to interact with incoming phagosomes.
By visualizing actin dynamics during phagocytosis, we show a role for actin in driving phagosome scission, size, and movement into the cell. The spatiotemporal localization of FBP17 and AMPH1-BAR highlight the transient nature of phagocytosis, whereas that of endosomal markers suggests that phagosome maturation may begin earlier than expected. These data provide unprecedented insight into the process of OS phagocytosis.
This is a 2021 ARVO Annual Meeting abstract.
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