mutant rab7 cdna  (Thermo Fisher)

Journal:

Article Title: Dynein-mediated Vesicle Transport Controls Intracellular Salmonella Replication

doi: 10.1091/mbc.E03-08-0614

Figure Lengend Snippet: SCV recruits functional Rab7. (A) Confocal fluorescence analysis of HeLa cells transiently transfected with GFP-Rab7 and infected with S. typhimurium SL1344. The SCV recruited GFP-Rab7 after 30 min of infection. Representative confocal images at 3 h and after o/n culture are shown. The insets in the top panels show a coat of GFP-Rab7 on the SCV that is representative for the accumulation of Rab7 observed at all time points. The majority of the SCV contained elongated bacteria after o/n culture, still colocalizing with GFP-Rab7 (bottom). Left, GFP-Rab7; middle, anti-Salmonella LPS; and right, merge. Bar, 10 μm. (B) Quantification of three experiments for colocalization of the SCV with GFP-Rab7. Values are given as mean percentage of colocalization ± SE. t = 0.5 h is the time point immediately after the 30 min infection (for each time point 100 cells were counted). (C) To determine whether the Rab7 recruited to the SCV is functional, the GTPase cycle of Rab7 was studied using FRAP. Representative examples of GFP-Rab7 cells are shown in a glow-over/under representation. Images are shown before bleach (prebleach), immediately after bleach and at three time points after bleach. The bleached area and the nucleus (N) are indicated. Top, SCV indicated with a rectangle and control late endocytic structures in the circle were bleached. Bottom, noninfected control cell was bleached in the circle. Bar, 10 μm. (D) Representative curves of fluorescent recovery in the bleached spot/area. The fluorescence (percentage) was related to the initial fluorescence set at 100%. t = 0 s is the first image after the bleach. (E) Quantification of the recovery time (t1/2) deduced from the curves including the SE. There is no difference between the t1/2 of SCV and late endocytic structures (control) even within the same cell. (F) Sucrose gradient fractionation profile of vesicles from cells infected with GFP-S. typhimurium SL1344. Lysosomes were detected by β-hexosaminidase activity and mature cathepsin D staining. The density (molar concentration sucrose) of the corresponding fractions measured with a refractomer is indicated (top). The localization of the SCV was determined with anti-GFP antibodies. LAMP-1, Rab7, and RILP localization were analyzed with specific antibodies. Bottom, corresponding Western blots for LAMP-1, RILP, Rab7, and GFP. The localization of the soluble fraction, lysosomes, and the SCV are indicated.

Article Snippet: Wild-type and mutant Rab7 cDNA, a kind gift from P. Chavier ( Meresse et al ., 1995 ; Meresse et al ., 1999a ), were subcloned into pcDNA3 (Invitrogen, Carlsbad, CA) with an N-terminal myc-tag for immunodetection.

Techniques: Functional Assay, Fluorescence, Transfection, Infection, Fractionation, Activity Assay, Staining, Concentration Assay, Western Blot

Journal:

Article Title: Dynein-mediated Vesicle Transport Controls Intracellular Salmonella Replication

doi: 10.1091/mbc.E03-08-0614

Figure Lengend Snippet: Expression of RILP results in a replication block of Salmonella. (A) Confocal analysis of infected cells expressing GFP-Rab7 microinjected with RILP and H2B-GFP. From left to right, GFP-Rab7 and H2B-GFP, Salmonella SL1344 labeled with anti-LPS-Texas Red, RILP, and the merge. Bar, 10 μm. (B) Confocal analysis of cells infected with either SL1344 (top) or the 12023 Salmonella strain (bottom) and microinjected with RILP. Cells microinjected with RILP cDNA (and H2B-GFP cDNA as injection marker) show clustering of late endosomes and lysosomes. In these cells, the elongated bacteria are not formed after o/n incubation (top). Note that elongated bacteria occurred in control cells. Microinjected cells are indicated with an asterisk. Left, H2B-GFP and Salmonella (stained with anti-LPS) are shown. Middle, CD63 staining. Right, merge. Bar, 10 μm. Bottom, cells infected with the Salmonella 12023 strain. Microinjected cells are indicated with an asterisk. Left, Salmonella 12023 strain. Middle, RILP. Right, merge. Bar, 10 μm. (C) Quantification of five individual experiments (n = 30–40 microinjected cells and n = 100 control cells). Left, mean percentage of SCV-containing elongated bacteria or 10–0 single bacteria are indicated ± SE for both RILP and control cells infected with the Salmonella SL1344 strain. Right, SCV containing >30 bacteria/cell or 10–0 single bacteria/cell are indicated ± SE for both RILP and control cells infected with the Salmonella 12023 strain.

Article Snippet: Wild-type and mutant Rab7 cDNA, a kind gift from P. Chavier ( Meresse et al ., 1995 ; Meresse et al ., 1999a ), were subcloned into pcDNA3 (Invitrogen, Carlsbad, CA) with an N-terminal myc-tag for immunodetection.

Techniques: Expressing, Blocking Assay, Infection, Labeling, Injection, Marker, Incubation, Staining