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Targeting and Delivery of Heterotrimeric G‐proteins to Primary Cilia
Author(s) -
Edwards Clare,
Lambert Nevin
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.665.15
Subject(s) - cilium , heterotrimeric g protein , intraflagellar transport , microbiology and biotechnology , g protein coupled receptor , compartment (ship) , membrane protein , biology , g protein , receptor , fluorescence microscope , transport protein , chemistry , membrane , signal transduction , fluorescence , biochemistry , gene , oceanography , flagellum , physics , quantum mechanics , geology
Primary cilia are fingerlike extensions of the plasma membrane important in cellular signaling and implicated in many disease processes. While much is known about localization and targeting of G‐protein coupled receptors (GPCRs) to cilia, little has been elucidated about the localization and targeting of heterotrimeric G‐proteins that reside downstream. This study aimed to determine if G‐proteins are targeted to the primary ciliary compartment and whether or not they possess the ability to move from the extaciliary membrane into this regulated space. Utilizing confocal microscopy, we imaged Gαi1‐Venus and SSTR3‐mTurq2 expression (a ciliary marker protein) in the primary cilia of NIH3t3 cells and quantified this expression as mean fluorescence in Imagej. Ciliary and membrane fluorescence were compared, and Fluorescence Recovery After Photo bleaching (FRAP) was completed to monitor movement of Gαi1‐V and SSTR3‐mTurq2. A student t‐test revealed that mean fluorescence of Gαi1‐V in the ciliary compartment (n=10, M=77.1, SD=45.8) is not significantly greater than extraciliary membrane fluorescence (n=10, M=75.4, SD=43.2); t(18)=0.08, p<0.05. Additionally, the difference between ciliary Gαi1‐V (n=10, M=77.1, SD=45.8) and membrane Gαi1‐V expression (n=10, M=75.4, SD=43.2) is significantly lower than the difference between expression of SSTR3‐mturq2 in the cilia (n=10, M=189.8, SD=71.5) and on the membrane (n=10, M=48.5, SD=30.1); t(18)=5.31, p<0.05. FRAP analysis of Gαi1‐V (n=6) returned a half recovery time of 12.8 ± 8.24 seconds and a mean mobile fraction of 0.60 ± 0.10, significantly higher than the mobile fraction of SSTR3‐mTurq2 (n=2, MF of 0.04 ± 0.014, t1/2 of 21.6 ± 15.3 seconds); t(6)=4.51, p<0.05. Our data indicate that Gαi1‐V expression is not significantly elevated in the primary cilia, and targeting of heterotrimeric G‐proteins to this space is unlikely. However, there is no evidence of exclusion of Gαi1‐V from the ciliary compartment as expression is visible and able to recover from photo bleaching in a time comparable to diffusion. Support or Funding Information Medical Scholars Program at Medical College of Georgia.