Journal: Molecular Cell

Article Title: c-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces

doi: 10.1016/j.molcel.2019.11.006

Figure Lengend Snippet: σ WhiG Activity Is Regulated Post-translationally by a Novel Anti-σ, RsiG (A) Automated western blot analysis showing σ WhiG protein levels throughout development in Difco Nutrient Broth (DNB) medium, generated using the quantitative Wes capillary electrophoresis and blotting system (ProteinSimple, San Jose, CA; ). Equal amounts (2.5 μg) of total protein were loaded for each sample, and σ WhiG was detected with a polyclonal anti-σ WhiG antibody. A single replicate is shown for each strain. (B) Quantification of σ WhiG levels (area under each peak, a.u.). All samples were analyzed in triplicate, and the mean value and its SE are shown for each sample. (C) whiI mRNA abundance in the WT during submerged sporulation, determined by qRT-PCR. Expression values are calculated relative to the hrdB reference, normalized to the value at 10 h. RNA samples were prepared from the same culture as the protein samples analyzed in (A) and (B). All samples were analyzed in triplicate, and the mean value and its SE are shown for each sample. (D) whiI mRNA abundance during submerged sporulation in the rsiG mutant, determined by qRT-PCR. Expression values are calculated relative to the hrdB reference, normalized to the WT value at 10 h. The data for the WT are replotted from (C). All samples were analyzed in triplicate, and the mean value and its SE are shown for each sample. (E) Bacterial two-hybrid analysis to investigate the interaction between σ WhiG and RsiG. The listed pairs of constructs were transferred into the BACTH reporter strain E. coli BTH101 by transformation. The resulting transformants were selected on L Broth agar containing 100 μg/mL carbenicillin and 50 μg/mL kanamycin and incubated at 37°C before single colonies were picked and subjected to β-galactosidase assays. Strains expressing fusions of both adenylate cyclase domains to the leucine zipper domain of GCN4 (zip) served as a positive control, whereas strains expressing fusions of one adenylate cyclase domain to a zip domain and the other to either WhiG or RsiG served as negative controls. Results are the average of three replicate cultures derived from the same single colony. Error bars represent SEM. (F) Co-expression and purification of a soluble σ WhiG -RsiG complex. σ WhiG and RsiG were co-overexpressed in E. coli using pCOLADuet-1, with σ WhiG carrying an N-terminal His tag. Following co-overexpression, the soluble extract was passed over a nickel column, and, after washing, bound proteins were eluted and analyzed on a 12% SDS-polyacrylamide gel. (G) Deletion of rsiG leads to hypersporulation on solid medium. Shown is a scanning electron micrograph of the rsiG mutant imaged after 7 days of growth on Difco Nutrient Agar (DNA) medium. See also Figure S1 and for the effect of deleting rsiG on sporulation in liquid culture.

Article Snippet: Shown is a scanning electron micrograph of the rsiG mutant imaged after 7 days of growth on Difco Nutrient Agar (DNA) medium.

Techniques: Activity Assay, Western Blot, Generated, Electrophoresis, Quantitative RT-PCR, Expressing, Mutagenesis, Construct, Transformation Assay, Incubation, Positive Control, Derivative Assay, Purification, Over Expression, Nickel Column

Journal: Scientific Reports

Article Title: Kinesin 1 regulates cilia length through an interaction with the Bardet-Biedl syndrome related protein CCDC28B

doi: 10.1038/s41598-018-21329-6

Figure Lengend Snippet: KLC1 and KIF5B are found at the base of cilia. Confocal microscopy analysis of hTERT-RPE cells. ( A ) Cells were transfected to overexpress Myc-tagged KLC1. In addition to cytoplasmic aggregates, a pool of Myc-KLC1 (anti-Myc, green) is found at the base of cilia (anti acetylated α-tubulin, red). ( B ) In addition to a diffuse cytoplasmic signal, the anti-KLC1 antibody shows an accumulation of endogenous protein at the base of cilia. ( C ) Similarly, endogenous KIF5B is found in the cytoplasm and also concentrated at the base of cilia. Yellow boxes mark the area that is magnified and showed in panels on the right. DAPI was used to stain nuclei. Scale bars correspond to 10 μm. ( D ) Scanning electron micrographs showing cilia in hTERT-RPE cells transfected with control (S.CTRL), KLC1 (S.KLC1) or KIF5B (S.KIF5B) stealth RNA oligos. Scale bars correspond to 1 μm. ( E , F ) The level of tubulin acetylation in control and KLC1 KD cilia was quantified measuring the fluorescence intensity of the signal obtained using the acetylated α-tubulin antibody (green). The anti-ARL13 (red) signal was used to mark the entire length of the cilium. Scale bars correspond to 2 μm. Each cilium was divided in 10 segments from base to tip (see methods) and the mean intensity was computed. For each experiment (KLC1 KD and control) a ten-point intensity profile was computed by averaging the measure of all cilia in each one of the regions of interest. These profiles are shown, normalized by the measure of its first region of interest. Vertical bars plot the 95% confidence interval about the mean.

Article Snippet: Scale bars correspond to 10 μm. ( D ) Scanning electron micrographs showing cilia in hTERT-RPE cells transfected with control (S.CTRL), KLC1 (S.KLC1) or KIF5B (S.KIF5B) stealth RNA oligos.

Techniques: Confocal Microscopy, Transfection, Staining, Control, Fluorescence