Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome

doi: 10.1073/pnas.1018823108

Figure Lengend Snippet: Rab8 localizes to developing cilia but departs mature cilia. (A) Rab8 is an early marker of ciliogenesis. GFP-Rab8a localization in cilia was scored in live RPE GFP-Rab8a cells, and Actub-positive cilia were counted in fixed cells (n = 75 cells). (B) Rab8 accumulates in the growing cilia. One hour after serum withdrawal, RPE GFP-Rab8a cells transiently expressing tRFP-Centrin2 for 24 h were imaged every 10 min by SDC microscopy over 6 h with concurrent imaging in both red and green channels. Representative contrast-adjusted images shown are projections of 20 z-sections (step size 0.5 μm). (Bottom Row) 3D surface rendering. (Scale bar: 5 μm.) (Movie S1). (C) Kinetics of ciliogenesis. Keying on the initiation of nascent cilia, GFP-Rab8a levels in 12 developing cilia were measured from projected z-stacks collected as described in B. Regression analysis was performed on raw data (Inset) to determine the time required for GFP fluorescence to reach maximal levels (106 ± 12 min).

Article Snippet: GFP-Rab8a cilia localization was observed live by epifluorescence microscopy or Marianas spinning disk confocal (SDC) microscopy (Intelligent Imaging Innovations) at 40× magnification.

Techniques: Marker, Expressing, Microscopy, Imaging, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome

doi: 10.1073/pnas.1018823108

Figure Lengend Snippet: Serum withdrawal triggers Rabin8 association with vesicles that accumulate dynamically at the centrosome before ciliary membrane assembly. (A) Rabin8 localizes to dynamic centrosomal vesicles following serum withdrawal. Serum-starved (60 min) RPE GFP-Rabin8 cells were fixed and stained with the centriolar marker γ-tubulin (Movie S2). (Scale bar: 2 μm.) (B) Serum starvation causes transient association of Rabin8 with membrane vesicles and accumulation at the centrosome mediated by its COOH-terminal domain. Shown is quantification of GFP-Rabin8 and GFP-Rabin81–316 (ΔC)-positive vesicles and centrosomal accumulation in RPE cells (n = 50) (Fig. S2 A and B). (C) The Rabin8 COOH-terminal domain is required for ciliation. GFP-Rab8a–positive cilia were counted in cells transiently expressing control (tRFP), tRFP-Rabin8, and tRFP-Rabin81–316 for 72 h; serum was removed for the last 24 h. GFP-positive cilia were counted only in tRFP-positive cells (n = 50; P < 0.001). (D) Time course of RPE cells accumulating GFP-Rabin8 at the centrosome after serum removal. Fluorescence signal (black) has been inverted in D and E for better viewing of structures. (Scale bar: 2 μm.) n, nucleus. (E) Rabin8 centrosomal targeting precedes Rab8a ciliary membrane formation and is reduced after ciliogenesis. RPE GFP-Rabin8 cells transiently expressing tRFP-Rab8a for 24 h were imaged live every 30 min after serum withdrawal. GFP and tRFP images were collected separately. (Scale bar: 2 μm.)

Article Snippet: GFP-Rab8a cilia localization was observed live by epifluorescence microscopy or Marianas spinning disk confocal (SDC) microscopy (Intelligent Imaging Innovations) at 40× magnification.

Techniques: Staining, Marker, Expressing, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 1: EPI64A and EPI64B both localize to apical microvilli. (A) Schematic of EPI64A and EPI64B domains with percentage identities and the TBC/RabGAP arginine residue necessary for the RabGAP activity indicated. (B) Western blots with antibodies to EPI64B and tubulin of Jeg-3 cell lysates after treatment with the indicated siRNAs to EPI64B. (C) Western blot with antibodies to EPI64B on cell lysates from several cultured cell lines. (D) SIM microscopy of Jeg-3 cells transfected with GFP-EPI64A (top block of panels) or GFP-EPI64B (bottom block of panels). Cells were transfected to express GFP-tagged constructs (green) and stained for ezrin (red) and actin (blue). Arrows indicate the localization of GFP-EPI64A to the base of microvilli. Scale bars: top panels, 10 µm; bottom panels, 10 µm.(E) Quantitation of GFP- EPI64A and GFP-EPI64B to localize to microvilli in wild-type cells.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Residue, Activity Assay, Western Blot, Cell Culture, Microscopy, Transfection, Blocking Assay, Construct, Staining, Quantitation Assay

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 2: EPI64A and EPI64B can localize to microvilli independently of the scaffolding protein EBP50 (A) Jeg-3 cells transfected with 3xFLAG-EBP50 were co-transfected with either GFP-EPI64A or GFP-EPI64B. The GFP-tagged proteins were immunoprecipitated with GFP-Trap beads and the immunoprecipitates blotted for FLAG and GFP. (B) Confocal imaging of microvillar localization of GFP-EPI-64A and GFP-EPI64A-LA, which cannot bind EBP50, in Jeg-3 cells. Scale bar 10 µm. (C) Western blot of cell lysates of Jeg-3 wild type, or CRISPR-modified EBP50 deletion cell line, blotted for ezrin, EBP50, and tubulin. Scale bar: 10 µm (D) Localization of ezrin and actin in Jeg-3 cells lacking endogenous EBP50. Scale bars: 10 µm. (E) Confocal imaging of GFP-EPI64A or GFP-EPI64B in Jeg-3 cells lacking EBP50. Scale bars: 10 µm.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Scaffolding, Transfection, Immunoprecipitation, Imaging, Western Blot, CRISPR, Modification

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 3: EPI64A contains a localization domain spanning its TBC domain. (A) Schematic of the EPI64 constructs used in B and C and summary of results shown in this figure (B) Confocal imaging of GFP-EPI64A-314-508, which contains the C-terminal -DTYL sequence, in Jeg-3 wild-type cells and Jeg-3 cells lacking EBP50. (C) Confocal images showing the localization of GFP-tagged deletion constructs of GFP-EPI64A. Scale bar 10 µm. (D) Immunolocalization of two HA-tagged constructs, the top one containing the minimal region that localizes to microvilli (HA-EPI64A-61-408) and the bottom one (HA-71-408) that does not localize. Scale bar: 10 µm. (E) GFP-trap pull down: Jeg-3 cells were transfected with either GFP or GFP-Arf6 together with the indicated HA-EPI64A constructs. The GFP or GFP-Arf6 were recovered and analyzed for the presence of the HA-EPI64A constructs by immunoblotting.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Construct, Imaging, Sequencing, Transfection, Western Blot

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 5: Jeg-3 cells lacking EPI64A and EPI64B lack microvilli (A) Western blot with antibodies to EPI64A, EPI64B, and tubulin on whole cell lysates of Jeg-3 cells genetically modified to lack EPI64A, EPI64B, or both proteins (DKO). (B) Confocal imaging showing localization of ezrin and actin in wild-type Jeg-3 cells and the single (A-KO, B-KO) and double knockout cells. Scale bar 10 µm. (C) Quantitation of the percentage of indicated cells stained for ezrin that express surface microvilli. Normal defined >50% coverage of the apical surface with microvilli. One-way analysis of variance gave the indicated p values. (D) EPI64A/B double knockout cells were transfected to express the indicated constructs and the percentage of ezrin-stained cells (total for either untransfected or GFP-expressing for transfected cells) that express normal apical microvilli. One-way analysis of variance gave the indicated p values. (E) Localization of tight junction ZO-1 in wild-type and knockout Jeg-3 cells. Scale bar: 10 µm.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Western Blot, Genetically Modified, Imaging, Double Knockout, Quantitation Assay, Staining, Transfection, Construct, Expressing, Knock-Out

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 6: Dominant negative Rab8A and Rab35A can restore microvilli to EPI64A/B double knockout cells. (A) Wild-type Jeg-3 cells were transfected to express GFP or the indicated GFP-Rab proteins and the percentage of cells (total for either untransfected or GFP-expressing for transfected cells) determined that express apical microvilli. (B) Jeg-3 EPI64A/B double knockout cells were transfected with GFP or the indicated GFP-Rab proteins and the percentage of cells (total for either untransfected or GFP-expressing for transfected cells) expressing microvilli scored. One-way analysis of variance gave the indicated p values.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Dominant Negative Mutation, Double Knockout, Transfection, Expressing

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 7: Caco-2 cells lacking EPI64A and EPI64B have microvilli (A) Western blots of whole cell lysates from Caco-2 BBE1 wild-type, EPI64A, and EPI64B single knockout and double knockout cells blotted for EPI64A, EPI64B, and tubulin. (B) Confocal imaging showing localization of ezrin in the apical region of wild-type and knockout cells. Scale bar: 10 µm. (C) Localization of GFP-EPI64A and GFP-EPI64B expressed in double knockout cells. Scale bar 10 µm.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Western Blot, Knock-Out, Double Knockout, Imaging

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 8: Caco-2 cells lacking EPI64A and EPI64B have aberrant apical junctions (A) Fields of wild-type, EPI64A and EPI64B single knockout and EPI64A/B double knockout cells stained for actin and the tight junction marker ZO-1. The phenotypes seen were variable, so the most wild–type-looking regions of cells are shown (Normal) and contrasted with regions where the normal polygonal organization is disrupted (Severe). Scale bar: 10 µm. (B) Percentage of wild-type and knockout cells in which one or more of its junctions shows a reflex angle (>180°). One-way analysis of variance gave the indicated p values. (C) Example of stellate knockout cell stained for ezrin, myosin IIA, and actin XY-dimensions (top panels) and YZ-dimensions (bottom panel). (D) Localization of vinculin, actin, and myosin IIA in the apical (top panels) and basal (bottom panels) sections of wild-type and double knockout Caco-2 cells. Scale bars 10 µm.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Knock-Out, Double Knockout, Staining, Marker

Journal: eLife

Article Title: Genome-wide screen reveals Rab12 GTPase as a critical activator of Parkinson’s disease-linked LRRK2 kinase

doi: 10.7554/eLife.87098

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pMCB306 GFP-Rab8A , Addgene , RRID: Addgene_198470 , PMID: 29125462.

Techniques: Bioprocessing, Generated, Labeling, Recombinant, Software