iBAQ values were normalized to the abundance of the Htr6\APEX\V5 fusion protein

iBAQ values were normalized to the abundance of the Htr6\APEX\V5 fusion protein. Myosin 5a (Myo5a). Interestingly, Myo5a knockout decreases ciliation while enhanced GSK2239633A levels of Myo5a are observed in cilia upon induction of ciliary disassembly. In summary, we present a novel approach to investigate dynamics of the ciliary membrane proteome in mammalian cells and identify actin\binding proteins as mechanosensitive components of cilia that might have important functions in cilia membrane dynamics. = 7. D Stoichiometry determination of significantly enriched actin filament organization proteins (GO:0007015). Error bars represent SEM. = 7. E Stoichiometry determination of selected significantly enriched intrinsic and extrinsic membrane proteins (GO:0031224; GO:0019898). Error bars represent SEM. = 7. F, G IMCD3 cells were starved for 24 h to induce ciliation and immunostained for (F) Filamin A and Arl13b or (G) alpha\actinin and acetylated tubulin. Each arrow highlights one cilium. To determine major biological processes and functions represented within the cmAPEX proteome, we next analyzed the dataset of 301 ciliary proteins for overrepresentation of Gene Ontology (GO) terms. As expected, GO\term analysis revealed a significant overrepresentation of proteins related to keywords such as cilium, septin complex, and receptor complex (Fisher’s exact test, FDR 0.02). Surprisingly, the GO terms cell\matrix adhesion and actin filament organization were almost equally strong enriched (Fig ?(Fig2B).2B). To extract absolute stoichiometries of the ciliary proteome composition, we used intensity\based absolute quantification (iBAQ), a parameter corresponding to the copy numbers of proteins within a biological sample 35. iBAQ values were normalized to the abundance of the Htr6\APEX\V5 fusion protein. Figure ?Figure2CCE2CCE summarizes the iBAQ values of all proteins belonging to cilium (GO: cilium, primary cilium), proteins involved GSK2239633A in actin filament organization (GO: actin filament organization), and some selected membrane proteins (GO:0019898; GO:0031224), respectively. The most abundant membrane\associated proteins were heat\shock protein GSK2239633A family A (Hsp70) member (Hspa5), beta\catenin (Ctnnb1), rho family, small GTP binding protein (Rac1 and Rac3), cadherins (Cdh1 and Cdh3), coxsackie virus and adenovirus receptor (Cxadr) as well as epidermal growth factor receptor (Egfr). Surprisingly, plenty of proteins involved in actin filament and cytoskeleton organization were enclosed in the APEX\based ciliary membrane\associated proteome. A role for actin dynamics in the process of ciliogenesis and the control of ciliary length has been uncovered during the last decade (reviewed in 5, 36). In brief, ciliogenesis requires migration of Rabbit Polyclonal to LDLRAD2 centrosomes to the cell periphery where the later basal body is anchored to the cortical actin network 37, 38. In addition, cytoplasmic F\actin negatively regulates ciliogenesis and ciliary length while depolymerization of cytoplasmic F\actin induces ciliogenesis and cilia elongation 39. Mechanistically, actin depolymerization leads GSK2239633A to the accumulation of Rab11/Rab8\positive vesicles at the centrosome thereby supporting ciliogenesis 39, 40. In addition, actin depolymerization has been shown to inactivate the hippo downstream effectors YAP and TAZ that promote transcription of negative regulators of ciliogenesis such as AurA and Plk1 41. Thus, although vesicle transport and transcriptional programs might provide some explanations, the detailed mechanisms of how actin dynamics interfere with ciliary dynamics and are still unclear. To confirm the localization of ABPs in cilia, we choose two ABPs with the highest ciliary abundance according to our stoichiometric analyses (Fig ?(Fig2D)2D) and stained ciliated murine wild\type IMCD3 cells with antibodies directed against alpha\actinin and Filamin.