CAPNS1 depletion causes accumulation of LC3-positive structures. (A) Control and CAPNS1-depleted U2OS cells were treated with RFP-GFP-LC3 baculovirus reagent. 24 h later, the cells were analysed under a confocal microscope over a 120 min time-lapse experiment. After the first 15 min, 100 nM thapsigargin was added to induce autophagy. The first (T0) and last images (T120) of the experiments are shown. (B) Control and CAPNS1-depleted U2OS cells were treated with commercial RFP-GFP-LC3-expressing baculovirus. 24 h later, the cells were treated with 100 nM thapsigargin or DMSO as control, for 1 h, and then fixed and analysed. Representative confocal microscopy pictures are shown. At least 25 cells were analysed for each replica and the number of yellow dots per cell was counted. The graph reports the mean and s.d. calculated for three independent experiments.

Early endosome dynamics is impaired in CAPNS1-depleted cells. (A) Control and shCAPNS1 U2OS cells were grown on Petri dishes, treated with commercial baculovirus reagent expressing GFP-Rab5 protein and RFP-KDEL. 24 h later, the cells were analysed using a confocal microscope to detect the dynamics of Rab5-positive vesicles. Images were acquired every 2 min over a period of 50 min. The first (T0) and last (T50) pictures of each-time lapse experiment are shown. Scale bars: 20 μm. (B) Corrected total cell fluorescence (CTCF) levels of GFP-Rab5 measured on 100 fixed control and shCAPNS1 cells. The graph indicates the percentage of cells that show a CTFC ≥3×10⁶; the error bars represent s.d. of three different independent experiments; P<0.01.

CAPNS1 depletion perturbs Golgi stack distribution. (A) Control, shCAPNS1 and CAPNS1-rescued U2OS cells were fixed and stained with GM130 antibody. Hoechst dye was used to stain nuclei. A fluorescence microscope was used to acquire pictures; Scale bars: 20 μm. Lower and right panels are magnifications of boxed areas. 100 cells for each sample were considered and the percentage of cells with Golgi stacks surrounding the nucleus was quantified. (B) Means and s.d. of three independent experiments.

Atg9 dynamics is impaired in CAPNS1-depleted cells. (A) ShCAPNS1 and control cells were co-transfected with GFP-Atg9 and HcRed-LC3. 24 h later, the cells were analysed using a confocal microscope. Images were acquired every 2 min over a 50 min time interval. The first (T0) and last (T90) merged pictures of the 2 h time-lapse experiments are shown. Scale bars: 20 μm. Magnifications of boxed areas are shown in the right panels. Arrows indicate ATG9 vesicles adjacent to or fused with LC3 vesicles. (B) Quantification of GFP-Atg9 and HcRed-LC3 colocalization by Pearson's coefficient determination in control and CAPNS1-depleted U2OS cells, before and after thapsigargin treatment. (C) Images sequences (T21, T23, T25) of the GFP-ATG9/HcRed-LC3 time-lapse experiment made using control and CAPNS1-depleted U2OS cells. The arrow indicates one ATG9 vesicle that meets and fuses with a LC3-positive vesicle in control cells. In shCAPNS1 cells, ATG9- and LC3-positive vesicles are more stationary. (D) U2OS cells stably expressing Flag-Atg9 were transfected with a CAPNS1-specific siRNA or a control siRNA. 48 h later, the cells were treated for 1 h with DMSO or 100 nM thapsigargin before lysis and immunoprecipitation with an anti-Flag antibody. The immunoprecipitation products were analysed by western blot to visualize Flag-Atg9, endogenous TfR and Vps34.

Atg9/Bif-1 trafficking from the Golgi is impaired in CAPNS1-depleted cells. (A) Control and shCAPNS1 U2OS cells were transiently co-transfected with AmCyan-Bif-1 and HcRed-LC3. 16 h later, the cells were fixed and analysed by a confocal microscope. Arrows indicate areas of colocalization of AmCyan-Bif-1 and HcRed-LC3. Scale bars: 20 μm. (B) 100 cells for each sample were considered and the number of cells where Bif-1 forms aggregates or single dots was counted. The graph represents the means and s.d. of three independent experiments. (C) U2OS cells stably expressing Flag-Atg9 were transiently silenced with control or CAPNS1-specific siRNA and then transfected with AmCyan-Bif-1. 24 h later the cells were treated for 1 h with or without 100 nM thapsigargin, and then fixed and analysed by immunofluorescence to visualize endogenous GM130, Flag-Atg9 and AmCyan-Bif1. Representative confocal merged images are shown. Scale bars: 20 μm. (D) 50 cells for each sample were considered and the number of cells where Atg9/Bif-1 positive vesicles colocalize with endogenous GM130 was counted. Mean and s.d. of three independent experiments.

The N-terminal region of Bif-1 protein is cleaved by calpain. (A) Schematic representation of the structure of wild-type Bif-1 and mutants. Bif-1 contains two domains involved in membrane binding: H0 and H1, a N-BAR domain and a C-terminal SH3 domain. (B) Bif-1 wt, Bif-1 ΔSH3, Bif-1 SH3 and p50 NF-κB1 were produced as ³⁵S-methionine-labeled proteins by in vitro transcription and translation and incubated for the indicated time intervals with commercial micro-calpain, as previously described (Demarchi et al., 2005). The reactions were then stopped in Laemmli buffer and analysed by SDS-PAGE and autoradiography. Parallel reactions in the presence of 10 μM EGTA were carried out to prove the calcium dependency of the reactions. Arrows indicate calpain cleavage products. (C) Schematic drawing of Bif-1 and its derivative fragments obtained after calpain cleavage and analysed by western blot anti-myc tag and anti-Bif-1 reported on the right side of the panel. The white arrow indicates the wt Bif-1 cleavage product retaining the SH3 domain. The solid arrows indicate lower molecular weight cleavage products lacking the C-terminal SH3 domain.