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Himanshu Malhotra, Peter D Calvert; sptPALM-based trafficking analysis of GPCRs in primary cilia. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3908 – A0110.
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© ARVO (1962-2015); The Authors (2016-present)
Super resolution microcopy techniques have pushed the limits of optical imaging to unprecedented spatial resolution. A frontier in super-resolution microscopy is its utilization in live cells or organisms. A few studies have examined the trafficking of GPCRs in living primary cilia using sparse individual molecule labeling, e.g., with Qdots. However, many questions remain regarding GPCR movement in primary cilia as well as photoreceptor ciliary outer segment that this approach cannot adequately address due to the low numbers of GPCRs observed. We thus are developing live cell sptPALM (single particle tracking photoactivation localization microscopy) approaches for mapping the motion of hundreds to thousands of individual GPCRs within a single cilium.
Dynamics of the GPCR, somatostatin receptor 3 (SSTR3), was examined in hTERT-RPE1 cell primary cilia by sptPALM. hTERT-RPE1 cells stably expressing SSTR3-mEos4b were generated using lentiviral transduction. Upon starvation, hTERT-RPE1 cells expressing SSTR3-mEos4b generates pocketed cilia where SSTR3-mEos4b is enriched. sptPALM imaging was performed using a Nikon microscope equipped with an iLAS2 ring TIRF illuminator. Sparse photoswitching of mEos4b specifically in the cilium was achieved by dim 405nm laser focused to the diffraction limit and positioned by galvanometer mirrors. Activated molecules were excited with widefield 561nm illumination and videos were acquired on an EMCCD camera at 50 frames per second. Videos were analyzed using TrackMate (Fiji) and MATLAB scripts developed in-house
Hundreds to thousands of individual GPCR molecules were tracked within single cilia using sptPALM. Various parameters of these movements were mapped onto each cilium. Analysis of mean squared displacement as a function of time revealed that GPCR dynamics acquired using sptPALM corresponded to previous results with Qdots. Diffusion coefficients segregated into at least two groups when analyzed by histogram, agreeing with our previous work suggesting that ciliary membranes possess F-actin delimited corrals.
We show that our sptPALM approach is capable of tracking the dynamic of thousands of GPCRs in a single cilium within minutes, at high temporal and spatial resolution. Thus, sptPALM will allow us to map the biophysical properties of membranes and proteins of primary cilia, and photoreceptors, with unprecedented spatial resolution and high throughput.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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