Developmental expression of Mover protein in brain.

<p>Rat brain homogenates from the indicated developmental stages between embryonic day 14 (E14) and adult were separated by molecular weight via SDS-PAGE and analyzed by Western blot using affinity-purified Mover antibodies. Tubulin and synaptophysin served as controls. N = 2 experiments.</p

Mover does not dissociate from synaptic vesicles in response to depolarization.

<p>(A) Assay of glutamate release from synaptosomes using the fluorescence-based NADPH assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063474#pone.0063474-Nicholls1" target="_blank">[26]</a> to verify that SVs in synaptosomes undergo calcium-dependent fusion and exocytosis to release glutamate. Depolarization with 50 mM KCl induces glutamate release in the presence of CaCl2, but not of EGTA. (B) Synaptosomal preparations were incubated for 10 min at 37°C in control conditions, in the presence of 1 mM EGTA to chelate calcium, in 1 µM okadaic acid to phosphorylate proteins, or in depolarizing conditions. Following treatment, each synaptosomal fraction was further fractionated to obtain a crude SV fraction. Equal volumes of the crude SV fractions were then subjected to Western blot analysis to test for Mover and synapsin protein levels associated with vesicles. Mover did not dissociate from vesicles in response to depolarization, whereas synapsin did. N = 2 experiments.</p

Mover associates with synaptic vesicle fractions in a membrane floatation assay.

<p>SV fractions (LP2) with and without the addition of 1% Triton X-100 to solubilize membranes were overlaid with a 25–52.5% continuous sucrose gradient and centrifuged. Without detergent, proteins associated with membranes float upwards in the gradient. With the addition of 1% Triton, only proteins attached to detergent-resistant raft-like SV membranes, such as synaptophysin, float into the gradient. Floating of Mover when membranes are intact, and co-floating of Mover with synaptophysin in 1% Triton X-100 treated SV membranes, indicate that Mover is a membrane-associated protein. N = 3 experiments.</p

Dephosphorylated Mover dissociates from synaptic vesicles.

<p>To analyze a potential influence of phosphorylation on the localization of Mover to SV membranes, SVs were treated with lambda-protein phosphatase and then analyzed for protein content by Western blot. Dephosphorylation caused a shift of Mover immunoreactivity from the pellet to the supernatant, while the immunoreactivity for synaptobrevin and synapsin was unchanged (N = 2 experiments).</p

Mover is present in phosphorylated and non-phosphorylated forms on synaptic vesicles.

<p>(A) Crude synaptosomal fractions were treated with the lambda-protein phosphatase, with and without the addition of the phosphatase inhibitor sodium orthovanadate. In control conditions and in the presence of the phosphatase inhibitor, the anti-phospho-Mover antibody detects a protein band with identical molecular weight to that of non-phosphorylated Mover. This phospho-Mover protein band is reduced in intensity following phosphatase treatment (n = 3 experiments). (B) Phosphorylated Mover, similar to total Mover, is predominantly found in the SV fraction LP2. Indicated fractions are homogenate (H), crude synaptosomal fraction (P2) and corresponding supernatant (S2), synaptosomal fraction (LP1) and corresponding supernatant (LS1), SVs (LP2) and supernatant (LS2), synaptosomal membranes (SPM) and synaptosomal cytosol (Scyt). N = 2 experiments.</p

Mover is a homomeric protein.

<p>(A) Yeast 2-hybrid assay using full length Mover (full-l.) as bait. A plasmid containing only the LexA interaction domain acted as a control. Beta-galactosidase expression was only induced if full-length Mover was used as both prey (rows) and bait (columns). All deletion constructs used as prey exhibited no interaction with full-length Mover. (B) Immunoprecipitation of GFP-Mover with Mover-myc co-expressed in HEK293 cells. Sepharose-coupled antibodies against the myc epitope were used to pull down protein complexes. As in the yeast 2-hybrid assay, only full-length GFP-Mover was pulled down from the HEK cell extracts, whereas GFP and GFP-Mover deletion constructs were not. N = 2 experiments.</p

Localization of Mover deletion constructs in Vero cells.

<p>Upon expression in Vero cells, all variants of GFP-Mover carrying a deletion were diffusely distributed. Immunostaining, Phalloidin staining and line scan analysis were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063474#pone-0063474-g009" target="_blank">Figure 9</a>. The line scan fluorescence profiles indicate that all constructs were characterized by a gradual decline of fluorescence towards the cell periphery. Scale bars are 10 µm. Boxes represent twofold magnification. N = 3 independent cultures.</p

Mover is peripherally associated with synaptic vesicle membranes.

<p>Carbonate stripping by treatment of SVs with 100 mM sodium carbonate was used to determine if Mover is integrally or peripherally associated with SV membranes. Synapsin, a peripheral synaptic-vesicle membrane protein served as a positive control and was partially removed from SVs by carbonate treatment, while synaptobrevin2 (syb2), an integral SV protein, was not. Rab3a is membrane-associated but is resistant to carbonate stripping <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063474#pone.0063474-FischervonMollard1" target="_blank">[30]</a>. Mover was partially removed from SV membranes by carbonate stripping, at similar levels as synapsin, indicating that it is a peripheral SV membrane associated protein. N = 2 experiments.</p

Mutations of N-terminal cysteines within Pex11pβ do not affect peroxisome membrane elongation.

<p>COS-7 cells were transfected with Pex11pβ-Myc (<b>A–C</b>), Pex11pβ-Myc^C18S-C25S (<b>D–F</b>) and Pex11pβ-Myc^{C18S-C25S-C85S} (<b>G–I</b>), and were processed for immunofluorescence microscopy 24 h after transfection using anti-Myc (<b>A, D, G</b>) and anti-Pex14p (<b>B, E, H</b>) antibodies. (<b>J</b>) Quantitative evaluation of peroxisome morphology. Data are from 3 independent experiments and are presented as means ± S.D. Bars, 20 µm.</p

Homomeric interaction of full length Mover in Vero cells.

<p>(A–C) Mover-myc, Mover-GFP and GFP-Mover, each representing full-length versions of Mover, are either diffusely distributed or – in the case of GFP-Mover - as uniformly distributed aggregates, presumably due to some degree of homomerization. Rhodamine-Phalloidin, which stains F-actin associated with cytoplasmic actin-bundles as well as F-actin associated with the sub-plasmalemmal cell cortex, was used to delineate the cell periphery (red color in the merged images and the graphs). (D–F) Upon co-expression with the palmitoylated construct palm-Mover-flag, the constructs are recruited to the plasma membrane. Constructs were immunostained using antibodies against the tags, i.e. myc and GFP (green color in the merged images and the graphs) and flag (red color in the merged images and the graphs). The merged images also show DAPI staining in blue. For line scan analysis (right panels) a bar-shaped region of interest was placed in the image as shown (white bar, representing 5 µm×0.83 µm), and the average fluorescence occurring along its length was plotted in the graph, where 0 µm denotes the end of the bar placed in the extracellular area. Diffusely distributed constructs were characterized by a gradual decline of fluorecence from the cell interior towards the cell periphery (A–C), recruited constructs were characterized by a peak of fluorescence in the cell periphery (D–F). Scale bars are 10 µm. Zooms represent twofold magnification. N = 3 independent cultures.</p