Supplementary MaterialsS1 Fig: EHBP-1 deficient N-terminal C2-like or central CH domain didn’t save the intestinal vacuole (bigger endosome) phenotype. indicate EHBP-1(NT-C2)-GFP and EHBP-1(CH)-GFP tagged puncta in the intestinal cells. Arrows reveal EHBP-1(CH)-GFP tagged intestinal vacuoles. (E-F’) Intestinal manifestation of CH-CC fragment (EHBP-1(NT-C2)) disrupted recycling cargo hTAC-GFP tubular endosomal localization. (G-G’) hTAC-GFP misplaced tubular endosomal localization and gathered on punctate constructions upon the knockdown of PPK-1. (H-I) Manifestation of CH-CC fragment triggered intracellular accumulation of recycling cargo hTAC-GFP about enlarged vacuoles and endosomes. Arrows reveal hTAC-GFP tagged intestinal vacuoles. Size bars stand for 10 m.(TIF) pgen.1006093.s002.tif (2.0M) GUID:?266611FC-D3A5-437B-A587-32B44A93EE3D S3 Fig: Association of EHBP-1 fragments with RAB-10 and ARF-6 tagged endosomes. Colocalization pictures are from confocal picture stacks obtained in intestinal epithelial cells of intact living pets. (A-A”) EHBP-1(NT-C2)-GFP colocalizes Rabbit Polyclonal to IP3R1 (phospho-Ser1764) with recycling endosome marker ARF-6-RFP on punctate constructions. (B-B”) EHBP-1(NT-C2)-GFP also colocalizes on punctate endosomes with RFP-RAB-10. (C-C”) EHBP-1(CH)-GFP colocalizes with ARF-6-RFP on endosomal puncta. (D-D”) EHBP-1(CH)-GFP displayed colocalization with RFP-RAB-10 on basolateral endosomes. (E-E”) ARF-6-RFP colocalizes with EHBP-1(CC)-GFP on basolateral puncta. (F-F”) RAB-10 colocalizes well with EHBP-1(CC)-GFP on medial puncta. Size bars represent 10 m.(TIF) pgen.1006093.s003.tif (1.8M) GUID:?8BD64472-3A9E-477C-A458-59E202D2B65C S4 Fig: The EHBP-1 NT-C2 domain is Gadodiamide not associated with PI(3)P enriched membranes in the intestine. (A-B”) Colocalization images from intact living animals are presented. EHBP-1(NT-C2)-GFP did not colocalize with PI(3)P biosensor RFP-2xFYVE in intestinal cells. (C) Liposome co-sedimentation assay was performed in the presence of liposomes including 0% PI (Control), 5% PI, 5% PI(4)P or 5% PI(4,5)P2. Liposomes had been incubated with 3ug GST as indicated. (D-E) Vacuole phenotype can’t be rescued Gadodiamide by manifestation of EHBP-1-GFP including NT-C2 domain fundamental motif mutations. Size bars stand for 10 m.(TIF) pgen.1006093.s004.tif (3.1M) GUID:?65F6D25D-ED91-4805-A715-3150821E3B37 S5 Fig: PH(PLC)-GFP tagged basolateral endosomal tubules requires intact F-actin and microtubule cytoskeletons. (A-A’) PH(PLC)-GFP brands tubular endosomes after shot of control DMSO. (B-B’) After LatB treatment, PH(PLC)-GFP tagged tubular meshwork was disrupted, and PH(PLC)-GFP puncta quantity improved by ~34%. (C-C’) Gadodiamide Nocodazole (Noc) treatment also disrupted the PH(PLC)-GFP tagged tubular network. (D) PH(PLC)-GFP tagged puncta quantity (structure count number) within device area was quantified. Mistake pubs are SEM (n = 18, 6 pets of every treatment had been sampled in three different device parts of each intestine described with a 100 x 100 (pixel2) package positioned randomly). Asterisks reveal Gadodiamide significant variations in the one-tailed College students t-test (**p 0.01). Size bars stand for 10 m.(TIF) pgen.1006093.s005.tif (1.0M) GUID:?8F243C69-3635-4236-8522-E6145B80DE28 S6 Fig: Recycling transport of hTAC-GFP via tubular endosomal networks requires the cytoskeleton. (A) In pets injected with DMSO, hTAC-GFP primarily localized to tubular and Gadodiamide punctate endosomes. (B) After treatment with G-actin sequestering agent latrunculin B (LatB), hTAC-GFP accumulated in enlarged medial structures. The hTAC-GFP labeled tubular network was disrupted and hTAC-GFP positive structure number decreased significantly (~45%). (C) Microtubule-depolymerizing drug nocodazole (Noc) treatment also disrupted the hTAC-GFP labeled tubular network and caused accumulation of hTAC-GFP. (D) Total fluorescence area of hTAC-GFP signal within unit region was quantified. Error bars are SEM (n = 18 each, 6 animals of each treatment sampled in three different regions of each intestine defined by a 100 x 100 (pixel2) box positioned at random). Asterisks indicate significant differences in the one-tailed Students t-test (**p 0.01, *** p 0.001). Scale bar represents 10 m. (E-E”) EHBP-1-RFP and EMTB-GFP partially overlap on tubular and punctate structures. (F-F”) EHBP-1(CH)-GFP colocalizes with actin marker Lifeact-RFP on sparse medial puncta. Arrows indicate endosomes labeled by both EHBP-1(CH)-GFP and Lifeact-RFP. (G-G”) EHBP-1(CH-CC)-GFP overlaps well with Lifeact-RFP on basolateral punctate structures. Arrowheads indicate endosomes labeled by both EHBP-1(CH-CC)-GFP and Lifeact-RFP. Scale bars represent 10 m.(TIF) pgen.1006093.s006.tif (2.9M) GUID:?00175C3A-660D-431F-827D-A495E655CB03 S7 Fig: The CH-CC fragment has a comparable level of actin filament co-sedimentation to the CH domain. (A) Compared with GST-CH in Fig 5AC5C, GST-CH-CC displayed a similar actin filament co-sedimentation level. P/S ratio (pellet/supernatant) was quantified in (B). Samples were analyzed by SDS-PAGE and coomassie blue stain. (C) The hUtrophin actin binding domain (aa1-261) co-sediments with actin filaments mutant animals, the ARF-6-RFP labeled tubular meshwork was disrupted. Arrowheads indicate.