Fig. 4: Proximity labeling through APEX2-Cep131 identifies multiple FGM components.
From: Fibrogranular materials function as organizers to ensure the fidelity of multiciliary assembly
a Cep131 colocalized with Pcm1 in FGMs. mEPCs fixed at day 3 postserum starvation were immunostained and imaged by 3D-SIM. Cep152-labeled deuterosomes served as MCC marker. The magnified insets show details for cytoplasmic (top) and perinuclear (bottom) FGMs. b APEX2-Cep131 induced biotinylation of FGM proteins. mEPCs infected with adenovirus at day −1 to express APEX2-Cep131 were either mock-treated (upper panels) or treated with biotin-phenol and H2O2 to trigger the proximity labeling reaction (lower panels) at day 3, followed by fluorescent staining and 3D-SIM. Biotinylated proteins were visualized with Alexa Fluor 546-labeled streptavidin30. c FGM candidate proteins identified by differential mass spectrometric analysis. APEX2-Cep131-expressing mEPCs were transfected respectively with Ctrl-i and Pcm-i1, followed by the proximity labeling with biotin-phenol. The biotinylated proteins were precipitated using streptavidin resin and subjected to shotgun mass spectrometry. The pie chart indicates protein compositions according to subcellular localizations. The table lists peptide spectrum matches (PSM) of Pcm1 and seven newly identified FGM proteins in the control (Ctrl-i) and Pcm1-depleted mEPC samples. d Subcellular localization of the indicated FGM components with Pcm1 in mTECs at day 3. Insets are magnified images of typical FGM cores. e Immuno-EM confirmed FGM localization of the indicated proteins in mTECs at day 3. Cep135 and Cep215 were immune-labeled with 10-nm gold particles, whereas Rootletin and Pcnt by 5-nm gold particles. Arrows indicate loose FGMs. FGM cores are encircled. Arrowheads point to typical procentriole-deuterosome complexes. Immuno-EM experiments were performed once. f Expression pattern of the FGM proteins. mTECs collected at the indicated ALI days were used for immunoblotting. Deup1 served as deuterosome marker. Gapdh served as loading control. Two biologically independent experiments were performed and images from the same experiment were presented. Source data are provided in the Source data file.
