Abstract
Purpose :
Photoreceptor outer segments are modified primary cilia containing hundreds of membranous discs each containing hundreds of thousands of the light-sensitive GPCR, rhodopsin. While the dense packing of rhodopsin into outer segment discs is essential for vision, the mechanism of its delivery remains poorly understood. Additionally, aberration in the trafficking of rhodopsin remains one of the major causes of retinal degeneration, through mechanisms that remain opaque. Here we continue to develop super-resolution live cell imaging approaches to directly evaluate rhodopsin transport mechanisms.
Methods :
We assembled a 3D single particle tracking PALM microscope (3D sptPALM) to quantify the transport of GPCRs expressed in living cells. Super-resolution mapping of molecular position was achieved with a 100x 1.49NA TIRF objective (Nikon) which allowed 20nm resolution in the imaging plane (x,y). Movement in z (the direction of light propagation) was quantified by point spread function remodeling using a Double Helix® phase mask, allowing ~25nm resolution. GPCRs possessing a myc tag on the N-terminus and either mEOS4b or EGFP appended to their C-terminus were expressed in cultured hTERT-RPE-1 or IMCD3 cells under the CMV promotor. Tracking was effected by either sparse activation of mEOS4b with a focused 405nm diode laser or by sparse antibody-mediated labeling with Qdots. Rhodopsins in live RDS-/- mouse retina were labeled with Qdots using antibody 4D2. Tracking analysis was done using FIJI and MATLAB.
Results :
High density 3D mapping of SSTR3 dynamics within single cilia showed that SSTR3 transport is driven primarily by diffusion within and between membrane corrals. Endogenous rhodopsin in RDS-/- photoreceptor cilia primarily exhibited random walk diffusion, with occasional processive movement.
Conclusions :
We generated high density maps and quantified the movement of thousands of GPCRs within single cilia using 3D spt-PALM microscopy. Our results confirmed our previous observations that corralled diffusion is the primary mechanism of SSTR3 transport within primary cilia (Lee et al. JCB). We also achieved, for the first time, super-resolution single molecule imaging of rhodopsin transport in living photoreceptors. Diffusion and processive movement rates in photoreceptor cilia were consistent with previous studies of GPCR movement in primary cilia.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.