Journal: Molecular Biology of the Cell

Article Title: The Rab21-GEF activity of Varp, but not its Rab32/38 effector function, is required for dendrite formation in melanocytes

doi: 10.1091/mbc.E11-04-0324

Figure Lengend Snippet: Rab21 and Rab32/38 are present in distinct membrane compartments in melanocytes. Immunoaffinity purification of Rab32-containing vesicles/organelles from crude membrane fractions of B16-F1 cells was performed by using anti-Rab32 IgG (lane 3) or control rabbit IgG (lane 2) as described in Materials and Methods . The Rab32-containing membrane compartment was analyzed by 10% SDS–PAGE, followed by immunoblotting with anti-Varp antibody (0.4 μg/ml), anti-tyrosinase antibody (0.3 μg/ml), anti-Rab32 antibody (1.5 μg/ml), anti-Rab38 antibody (0.9 μg/ml), anti-Rab21 antibody (0.2 μg/ml), anti-EEA1 antibody (1/1000 dilution), and anti-GM130 antibody (0.3 μg/ml). Note that Rab21 was not copurified with the Rab32-membrane compartment, which clearly includes Rab38, tyrosinase, and Varp but not EEA1 or GM130. Input means 1% volume of the crude membrane fractions used for immunoaffinity purification. The positions of the molecular mass markers (in kilodaltons) are shown on the left.

Article Snippet: Anti-EEA1 mouse monoclonal antibody and anti-GM130 mouse monoclonal antibody were from Cell Signaling Technology (Danvers, MA) and BD Biosciences (San Jose, CA), respectively.

Techniques: Membrane, Immunoaffinity Purification, Control, SDS Page, Western Blot

Journal: The Journal of Cell Biology

Article Title: The somatodendritic endosomal regulator NEEP21 facilitates axonal targeting of L1/NgCAM

doi: 10.1083/jcb.200707143

Figure Lengend Snippet: Colocalization of endocytosed NgCAM with markers for early and late endosomes. (A) NgCAM-expressing neurons were incubated for 20 min with anti-NgCAM antibodies, which were detected after permeabilization with an Alexa 488–goat anti–mouse antibody, whereas EEs were detected with a polyclonal anti-EEA1 antibody (red). Yellow arrowheads indicate examples of colocalizing puncta. (B) NgCAM-expressing neurons were incubated with anti-NgCAM antibody for 30 min at 16°C and washed, and internalized NgCAM antibody was chased at 37°C for 1 h. Surface NgCAM was detected before permeabilization with a cy5–goat anti–mouse secondary antibody (blue). After permeabilization, endocytosed NgCAM antibodies were detected with Alexa 568–goat anti–mouse antibody (red) and LEs/lysosomes were detected with a polyclonal anti-lgp120 antibody (green). A single confocal section is shown. The boxed regions shown in A and B are magnified sections of dendrites for easier comparison of colabeling. (C) Quantification of colocalization of EEA1 and NgCAM endocytosed for 20 min and of lgp120 and NgCAM endocytosed and chased for 60 min. For each puncta, the ratio of intensities of endocytosed NgCAM to either EEA1 (left) or lgp120 (right) was determined as described in Materials and methods. For EEA1 colocalization, 560 endosomes were scored. For lgp120 colocalization, 497 endosomes were scored. (D) Basic organization of endosomes, adapted from fibroblasts. Three distinct pathways are indicated by the arrows: degradative cargo follows the red arrows, somatodendritically recycling cargo follows the green arrows, and transcytosing cargo follows the blue arrows. Cargo enters via several pathways in small carriers vesicles (ECV) that fuse with existing EEs. EEs contain a vacuolar portion as well as tubular extensions. The tubular extensions accumulate recycling cargoes and bud off to transport recycling cargos back to the plasma membrane either directly or via the RE. Endosomal carrier vesicles (ECVs) can be spherical or elongated and serve as transport carriers between compartments. The vacuolar portions of the EE accumulate internal vesicles and mature into MVBs. MVBs are transport carriers that carry cargo to LE/lysosomes (LE/lys) for degradation (red). Some MVBs might not be predegradative but be capable of recycling. Cargo destined for either axons or dendrites are sorted in the RE and transported to their respective final destination from there.

Article Snippet: Rabbit anti-EEA1 antibody was obtained from Chemicon and Texas red–rat Tf was obtained from Jackson ImmunoResearch Laboratories.

Techniques: Expressing, Incubation

Journal: The Journal of Cell Biology

Article Title: The somatodendritic endosomal regulator NEEP21 facilitates axonal targeting of L1/NgCAM

doi: 10.1083/jcb.200707143

Figure Lengend Snippet: Endocytosed NgCAM traverses the NEEP21-positive EE. (A) Neurons were cotransfected with NgCAM and either dominant-negative Ti-VAMP (left) or anti-sense NEEP21 (right). GFP was expressed as a control. 18 h after transfection, surface NgCAM was detected with immunostaining. Coexpression of AS-NEEP21 but not Ti-VAMP-DN led to a significant decrease in the A/D PI (see Materials and methods). Error bars indicate SEM; **, statistical significance from GFP controls at P < 0.001. n = 4 independent experiments, scoring 20–25 cells per experiment for each condition. (B) NgCAM-expressing neurons were allowed to endocytose anti-NgCAM antibodies for 20 min before fixation. Endocytosed NgCAM was detected with a red secondary antibody, whereas endogenous NEEP21 was detected with a rabbit anti-NEEP21 antibody (green). Precise overlap of NgCAM and NEEP21 is observed (colocalization appears yellow). Single channels as well as overlaid channels are shown for the boxed area. A single confocal section is shown. Arrowheads indicate dendrites. (C) Extent of overlap was scored for cells loaded with anti-NgCAM antibodies for 20 min without a chase (left; t = 0) and after a 1-h chase (right; t = 60). Extent of overlap was binned into strong colocalization, NEEP only, and NgCAM only. NgCAM showed high colocalization (black) at t = 0, which diminished with time (right), whereas the no colocalization categories NEEP only and NgCAM only increased with time. (D) The EE populations enriched in EEA1 (red) or in NEEP21 (green) show poor colocalization. Single channel panels are shown on the left.

Article Snippet: Rabbit anti-EEA1 antibody was obtained from Chemicon and Texas red–rat Tf was obtained from Jackson ImmunoResearch Laboratories.

Techniques: Dominant Negative Mutation, Transfection, Immunostaining, Expressing

Journal: The Journal of Cell Biology

Article Title: The somatodendritic endosomal regulator NEEP21 facilitates axonal targeting of L1/NgCAM

doi: 10.1083/jcb.200707143

Figure Lengend Snippet: Dynamic behavior of NEEP21-GFP and endocytosed NgCAM in endosomes. (A and B) Live imaging of endocytosed NgCAM (red) and NEEP21-GFP (green). A portion of a proximal dendrite is shown (A). Images were captured every 2 s as indicated above each panel. Frames not shown displayed no movements. The trajectories for all twenty frames (0–38 s) are displayed in B for the three compartments marked by arrowheads, arrows, and asterisks in A. The starting point at 0 s is indicated. The NEEP21-containing and the NgCAM-containing compartments (marked with arrows/arrowheads) show movements, whereas the compartment containing both NEEP21 and endocytosed NgCAM (marked with an asterisk) does not move. See Video 6 (available at http://www.jcb.org/cgi/content/full/jcb.200707143/DC1 ). (C) Quantification of all scored endosomes ( n = 443) in the 1-min imaging period. (D) Quantification of moving endosomes ( n = 79; colors as defined in C). (E) Model of endosomal compartments involved in NgCAM transcytosis. Tf and NgCAM (aquamarine arrows) enter EEA1-positive EEs (orange) in the somatodendritic domain as well as NEEP21-positive EEs (purple), from where they are transported to the somatodendritic RE. Tf (green arrows) but not NgCAM (blue arrows) recycles to the somatodendritic surface from the EE and the RE. NgCAM (blue), however, sorts away from Tf into putative “transcytotic REs” (red) and travels anterogradely up the axon in small carriers. Large stationary endosomes along the axon (blue) accumulate endocytosed NgCAM and might provide intermediary stopover points into which motile carriers fuse and from which new motile carriers are generated. When NEEP21 is down-regulated by antisense, NgCAM is missorted, presumably in the NEEP21 endosome, toward the somatodendritic surface as well as to lysosomes (gray arrows; LE/lys). Tf, however, is not missorted to the axon.

Article Snippet: Rabbit anti-EEA1 antibody was obtained from Chemicon and Texas red–rat Tf was obtained from Jackson ImmunoResearch Laboratories.

Techniques: Imaging, Generated

Journal: Toxins

Article Title: The Clostridium botulinum C2 Toxin Subunit C2IIa Delivers Enzymes with Positively Charged N-Termini into the Cytosol of Target Cells

doi: 10.3390/toxins15060390

Figure Lengend Snippet: Endocytosis and cytosolic release of His-tagged cargo proteins depends on the uptake and translocation via C2IIa. ( a ) HeLa cells were treated as indicated with 25 nM C2IIa, 250 nM His_DTA, 5 µM Quina, 100 nM BafA1, or left untreated (NC). Representative pictures after 24 h incubation time are depicted. Scale bar corresponds to 100 µm. ( b ) HeLa cells were treated as indicated with the same concentrations as in ( a ). Pictures were taken after 24 h, and the percentage of rounded cells was quantified. Values are given as mean ± SD ( n = 3) of triplicates from one representative experiment out of three replicates. ( c ) Relative viability (% of NC) of the cells from ( b ) measured via MTS assay. Values are given as mean ± SD ( n = 3) of triplicates from one representative experiment out of three replicates. ( b , c ) Statistical analysis was performed compared to the treatment with His_DTA/C2IIa by using non-parametric one-way ANOVA in combination with Dunnett’s correction for multiple comparison as described under in (*** p < 0.001). ( d ) HeLa cells were treated for 30 min with 250 nM His_eGFP_DTA or a combination of 25 nM C2IIa and 250 nM His_eGFP_DTA. STED super-resolution microscopy was performed, and representative micrographs are depicted together with three magnified areas below the image (white squares indicate the respective regions in the main image). eGFP-signals are shown in green, while EEA1-signals are depicted in red, thus overlapping signals appear in yellow. Scale bars correspond to 5 µm for the main images or 300 nm for the magnified areas. ( e ) Cells were treated as in ( d ), with 25 nM C2IIa, or left untreated (NC). The eGFP-signals, recorded via STED microscopy, were quantified (Material and Methods ) and depicted in a column diagram. Values are given as mean ± SD ( n = 5) from five individual experiments. Statistical analysis was performed compared to the treatment with His_eGFP_DTA by using non-parametric one-way ANOVA in combination with Dunnett’s correction for multiple comparison as described under in (ns p ≥ 0.05, *** p < 0.001).

Article Snippet: For immunohistological staining, the cells were incubated at 4 °C overnight with 1 μg/mL of primary rabbit anti-EEA1 antibody (Thermo Scientific, Waltham, MA, USA) and 0.5 μg/mL Atto594-conjugated GFP-booster nanobody (Chromotek, Planegg Germany) in 1:10 diluted permeabilization/blocking solution.

Techniques: Translocation Assay, Incubation, MTS Assay, Comparison, Super-Resolution Microscopy, Microscopy