M1-spastin, a neuronal protein, recruits ATL1 to three-way junctions in cultured non-neuronal cells and stimulates ATL1-mediated liposome fusion. (A) COS7 cells were transfected with genes encoding the indicated proteins, and the intracellular localizations of EGFP-ATL1 and myc-tagged M1-spastin were analyzed by fluorescence microscopy. Myc-tagged M1-spastin was visualized using an anti-myc antibody conjugated with Alexa Fluor 594. Scale bar: 5 μm. (B) Donor and acceptor liposomes bearing ATL1 alone, M1-spastin alone, or both ATL1 and M1-spastin at a protein-to-lipid ratio of 1:1,000 were mixed and incubated in the absence or presence of GTP. Fusion is represented as the percentage of total NBD fluorescence. The kinetics graph (left) is representative of three independent results, which are presented as a bar graph (right). Data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. ***P < 0.001, two-way ANOVA with Tukey’s multiple comparisons test. (C) Donor and acceptor liposomes bearing ATL1 alone, ATL1 and M1-spastin, ATL1 and M87-spastin, or ATL1 and M1-spastin (K388R) at a ratio of each protein/lipid of 1:1,000 were mixed and incubated in the absence or presence of GTP. Fusion is represented as the percentage of total NBD fluorescence. Data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. **P < 0.01, two-tailed unpaired Student’s t test. (D) The GTPase activity of ATL1 alone, spastin alone, or ATL1 in the presence of spastin was determined by measuring the release of inorganic phosphate using a Malachite Green Phosphate Assay Kit (Sigma-Aldrich). Data represent the means ± SEM (error bars; n = 3). The difference between samples was not statistically significant (n.s.), two-tailed unpaired Student’s t test.
Figure 4.

M1-spastin, a neuronal protein, recruits ATL1 to three-way junctions in cultured non-neuronal cells and stimulates ATL1-mediated liposome fusion. (A) COS7 cells were transfected with genes encoding the indicated proteins, and the intracellular localizations of EGFP-ATL1 and myc-tagged M1-spastin were analyzed by fluorescence microscopy. Myc-tagged M1-spastin was visualized using an anti-myc antibody conjugated with Alexa Fluor 594. Scale bar: 5 μm. (B) Donor and acceptor liposomes bearing ATL1 alone, M1-spastin alone, or both ATL1 and M1-spastin at a protein-to-lipid ratio of 1:1,000 were mixed and incubated in the absence or presence of GTP. Fusion is represented as the percentage of total NBD fluorescence. The kinetics graph (left) is representative of three independent results, which are presented as a bar graph (right). Data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. ***P < 0.001, two-way ANOVA with Tukey’s multiple comparisons test. (C) Donor and acceptor liposomes bearing ATL1 alone, ATL1 and M1-spastin, ATL1 and M87-spastin, or ATL1 and M1-spastin (K388R) at a ratio of each protein/lipid of 1:1,000 were mixed and incubated in the absence or presence of GTP. Fusion is represented as the percentage of total NBD fluorescence. Data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. **P < 0.01, two-tailed unpaired Student’s t test. (D) The GTPase activity of ATL1 alone, spastin alone, or ATL1 in the presence of spastin was determined by measuring the release of inorganic phosphate using a Malachite Green Phosphate Assay Kit (Sigma-Aldrich). Data represent the means ± SEM (error bars; n = 3). The difference between samples was not statistically significant (n.s.), two-tailed unpaired Student’s t test.

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