Synchronizing Protein Traffic to the Primary Cilium

Abstract

The primary cilium is able to maintain a specific protein composition, which is critical for its function as a signaling organelle. Here we introduce a system to synchronize biosynthetic trafficking of ciliary proteins that is based on conditional aggregation domains (CADs). This approach enables to create a wave of ciliary proteins that are transported together, which opens novel avenues for visualizing and studying ciliary import mechanisms. By using somatostatin receptor 3 (SSTR3) as model protein we studied intracellular transport and ciliary import with high temporal and spatial resolution in epithelial Madin-Darby canine kidney (MDCK) cells. This yielded the interesting discovery that SSTR3, besides being transported to the primary cilium, is also targeted to the basolateral plasma membrane. In addition, we found a similar behavior for another ciliary protein, nephrocystin-3 (NPHP3), thus suggesting a potential correlation between ciliary and basolateral trafficking. Furthermore, our CAD-based system allowed assembling a large dataset in which apical and basolateral surface SSTR3 signals could be compared to ciliary SSTR3 signals on a single cell level. This enabled to generate novel complementary evidence for the previously proposed lateral import mechanism of SSTR3 into the cilium along the plasma membrane.

Molecular and Cellular Mechanisms Underlying Somatostatin-Based Signaling in Two Model Neural Networks, the Retina and the Hippocampus

“…In the newt retina, SST 3 has been detected in the inner segment of cones and in the connecting cilia of rods, which correspond to the outer photoreceptor segments [78]. This is in agreement with the demonstration that SST 3 is localized in the neuronal cilia in many brain regions, in which it might play a role as chemical sensor [79] or in ciliary trafficking [80]. SST 4 is localized in the ganglion cells in both mouse [60,67] and rat [81] retinas.…”

TBC1D8B Mutations Implicate RAB11-Dependent Vesicular Trafficking in the Pathogenesis of Nephrotic Syndrome

“…We chose MDCK cells that form an epithelial monolayer with apico-basal polarity for this assay because these cells had previously been used for this approach. 56,57 Studying nephrin trafficking this way, we observed no nephrin on the cell surface before the induction of ER release ( Figure 4H, upper panels), indicating successful ER retention. Upon exposure to D/D solubilizer, we observed trafficking of nephrin toward the apical cell surface over time ( Figure 4H, lower panels).…”

“…Quantification of cell surface arrival of nephrin was done as described previously, with a custom-written MATLAB program. 56,57 Immunoblotting, Immunoprecipitation, Pull-Down Assay, and Immunofluorescence Staining Immunoblotting, immunoprecipitation, and immunofluorescence staining were performed as described previously. 58 Briefly, HEK293T were lysed and precleared using rec-Protein A-Sepharose 4B Conjugate (Life Technologies) overnight.…”

“…To study biosynthetic trafficking of nephrin directly, we utilized an approach on the basis of conditional aggregation domains (CAD) that has been used for other proteins. 56,57 Adapting this approach for nephrin, we introduced CADs to the N terminus of nephrin (pCAD4-HA-nephrin-mCherry, Figure 4G). CADs are expected to induce retention of newly synthesized protein within the ER by formation of aggregates.…”

HDAC6 Signaling at Primary Cilia Promotes Proliferation and Restricts Differentiation of Glioma Cells

“…First, we found that EGFP:HDAC6 transfected cells displayed weak immunolabeling for aaTub compared to control EGFP-transfected cells or neighboring untransfected cells ( Figure 7 A–L), a result that validates the expected targeting of aaTub by HDAC6. In control EGFP-transfected cells, cilia were detectable but weakly labeled with aaTub, a possible transfection-related issue that has been shown to affect primary cilia aaTub staining in other studies [ 35 ]. Nevertheless, we did not observe cilia loss on these EGFP:HDAC6-expressing cells.…”