Journal: PLoS Pathogens

Article Title: Cellular Mechanisms of Alpha Herpesvirus Egress: Live Cell Fluorescence Microscopy of Pseudorabies Virus Exocytosis

doi: 10.1371/journal.ppat.1004535

Figure Lengend Snippet: Cells were transduced to express mCherry-tagged Rab proteins, infected with PRV 486 expressing gM-pHluorin, and imaged at 4.5–5 hr after PRV infection. (A,D,G) The indicated Rab proteins colocalize with gM-pHluorin particle at the moment of exocytosis (yellow circle). Images correspond to . Scale bars represent 2 µm. (B,E,H) Kymographs of indicated Rab protein (red) and gM-pHluorin (green) fluorescence over time. (C,F,I) Ensemble averages of gM-pHluorin (top, green line) and indicated Rab protein (bottom, red line) relative fluorescence. Shaded area represents standard deviation. (A–C) mCherry-Rab6a. Data represent 37 exocytosis events in 4 independent experiments. (D–F) mCherry-Rab8a. Data represent 41 exocytosis events in 3 independent experiments. (G–I) mCherry-Rab11a. Data represent 34 exocytosis events in 3 independent experiments. (J–K) Rab6a is associated with exocytosis of assembled virions containing capsids. Cells were transduced to express mCherry-Rab6a, and co-infected with PRV 950 and PRV 486. (J) Image is a maximum difference projection corresponding to , depicting virus particle exocytosis events over a 13.7 min time course. Exocytosis events associated with Rab6a (blue) containing gM-pHluorin (green) and capsids (red) are indicated (white circles). Scale bar represents 2 µm. (K) Still images from , depicting a single viral exocytosis event. Images correspond to the boxed area in panel B. Scale bar represents 1 µm.

Article Snippet: Rab8a was obtained from M. Nachury , via Addgene (#24898).

Techniques: Infection, Expressing, Fluorescence, Standard Deviation, Virus

Journal: Human Molecular Genetics

Article Title: LRRK2 phosphorylates membrane-bound Rabs and is activated by GTP-bound Rab7L1 to promote recruitment to the trans-Golgi network

doi: 10.1093/hmg/ddx410

Figure Lengend Snippet: LRRK2 phosphorylation of GTP-bound Rab10 may decrease interaction with the GAP protein AS160. (A) Structural alignment of Rab8-GTP (Green) and Rab8-GDP (magenta). The unstructured switch II motif in the GDP-bound form is shown as dashed line. The Thr72 side chain is shown as sticks. (B) Assessment of purity of recombinant proteins isolated from HEK293 cells, for G2019S-LRRK2, WT-Rab10, and WT-Rab7L1, by SDS-PAGE followed by coomassie staining. All proteins showed >95% purity. (C) Calculated ratios of pRab10 from in vitro kinase assays in the presence of 500 μM of GTP, or GDP, with MLi2 (100 nM), as indicated. (D) Lysates from HEK293 cells co-transfected with LRRK2 and WT, TA, TD-Rab10, as indicated. MLi2 was included at 100 nM as indicated. Representative phos-tag analysis and immunoblots are shown. (E) Live cell imaging of SH-SY5Y cells. Wide-field fluorescence of the eGFP-tagged Rab10 construct is shown on a dark phase-contrast background, 16 h after transfection. (F) Cells over-expressing the Rab10 GAP AS160 or mock (empty vector) controls were lysed and incubated with bead-immobilized WT, TA or TD-Rab10. Complexes were analysed by immunoblots as shown and quantified (G) as column graphs depicting Rab10 interaction with Rab10 variants, relative to WT-Rab10. All data are averaged from three independent experiments or are representative of three independent experiments. Data are means ± SEM; significance assessed by one-way ANOVA with Tukey’s post hoc test. *P < 0.01, **P < 0.001.

Article Snippet: Rab8 constructs are obtained from Addgene (Plasmids 86075, 86076, 86077) courtesy of the Lei Lu laboratory. pEGFP-Rab7L1 plasmids are generated as previously described ( 9 ).

Techniques: Phospho-proteomics, Recombinant, Isolation, SDS Page, Staining, In Vitro, Transfection, Western Blot, Live Cell Imaging, Fluorescence, Construct, Expressing, Plasmid Preparation, Incubation

Journal: iScience

Article Title: Pathogenic LRRK2 regulates centrosome cohesion via Rab10/RILPL1-mediated CDK5RAP2 displacement.

doi: 10.1016/j.isci.2022.104476

Figure Lengend Snippet: Figure 2. LRRK2-mediated centrosomal cohesion deficits depend on both GTP conformation and phosphorylation status of Rab8a (A) Example of HEK293T cells co-transfected with FLAG-tagged wild-type LRRK2 and GFP-tagged Rab8a-Q67L (GTP-trapped) or GFP-tagged Rab8a-Q67L- T72A (GTP-trapped but non-phosphorylatable), and stained with antibody against FLAG (red) and the centrosomal marker pericentrin (pseudocolored blue) and DAPI. Arrows point to centrosomes in transfected cells. Cell boundaries (yellow) are shown and were determined by FLAG staining because of FLAG- tagged wild-type LRRK2 expression (or GFP because of GFP-tagged Rab8a expression for single transfection experiments). Scale bar, 10 mm. (B) Quantification of the split centrosome phenotype in cells expressing the indicated constructs, in either the absence or presence of LRRK2 kinase inhibitor MLi2 (100 nM, 2 h) as indicated. Around 100–150 cells were quantified per condition and experiment. Bars represent mean G SEM (n = two to three in- dependent experiments; wt-LRRK2 versus wt-LRRK2 + Rab8a, p = 0.007; wt-LRRK2 + Rab8a versus wt-LRRK2 + Rab8a + MLi2, p = 0.024; wt-LRRK2 versus wt- LRRK2 + Rab8a-Q67L, p = 0.003; wt-LRRK2 + Rab8a-Q67L versus wt-LRRK2 + Rab8a-Q67L + MLi2, p = 0.026); ***p < 0.005; **p < 0.01; *p < 0.05. (C) Cells were co-transfected with FLAG-tagged wild-type LRRK2 and GFP-tagged Rab8a constructs as indicated, left untreated or incubated with 100 nM MLi2 for 2 h, and extracts blotted for FLAG-tagged LRRK2 (FLAG-LRRK2), FLAG-tagged S935-phosphorylated LRRK2 (FLAG-pS935-LRRK2) as a readout for on-target effect of MLi2, phosphorylated GFP-tagged Rab8a (GFP-pT72-Rab8a), total GFP-tagged Rab8a (GFP-Rab8a), and tubulin or GAPDH as loading controls. (D) HeLa cells were transfected with the indicated GFP-tagged Rab8a constructs, briefly fixed, washed and stained with DAPI. All GFP-tagged Rab8a constructs except the inactive T22N mutant display a localization consistent with their presence in a tubular early recycling compartment. Scale bar, 10 mm.

Article Snippet: pDEST53 GFP-hLRRK2 WT Greggio et al., 2006 Addgene Cat#25044 pDEST53 GFP-hLRRK2 G2019S Greggio et al., 2006 Addgene Cat#25045 pDEST53 GFP-hLRRK2 R1441C Greggio et al., 2006 Addgene Cat#25046 pDEST53 GFP-hLRRK2 Y1699C Greggio et al., 2006 Addgene Cat#25048 pDEST53 GFP-hLRRK2 K1906M Blanca Ramı́rez et al., 2017 N/A pCHMWS 3xflag-hLRRK2 Greggio et al., 2009 Prof. Elisa Greggio pCHMWS 3xflag-hLRRK2 Y1699C Greggio et al., 2009 Prof. Elisa Greggio pGFP-hRab8a Nachury et al., 2007 Addgene Cat#24898 pGFP-hRab8A Q67L Nachury et al., 2007 Addgene Cat#24900 pGFP-hRab8a T22N Nachury et al., 2007 Addgene Cat#24899 pGFP-hRab8a T72A Madero-Pérez et al., 2018 N/A pGFP-hRab8A Q67L-T72A This study N/A GFP-hTBC1D30 Yoshimura et al., 2007 Prof. Francis Barr GFP-hTBC1D4 Yoshimura et al., 2007 Prof. Francis Barr GFP-hTBC1D4 R973K Yoshimura et al., 2007 Prof. Francis Barr GFP-hTBC1D17 Yoshimura et al., 2007 Prof. Francis Barr (Continued on next page) 18 iScience 25, 104476, June 17, 2022

Techniques: Phospho-proteomics, Transfection, Staining, Marker, Expressing, Construct, Incubation, Mutagenesis