Figure 5. Point mutations in the 15R... | Rockefeller University Press

Figure 5.

$Point mutations in the 15R segment impair MT-bundling activity of gAnkB.(A) MT cosedimentation assay performed with the WT gAnkB 12-aa repeats (15R) or the PSK mutant of gAnkB 12-aa repeats (15R-PSK) with Trx as the control (each at 10 µM and Trx at 30 µM). The SDS-PAGE gel is shown in the left panel, and the percentage of proteins recovered from the MT-binding pellet fractions is shown in the right panel. Mean ± SEM; n = 3. (B) Confocal images of in vitro Taxol-stabilized MTs (contains ∼8% rhodamine-labeled tubulin; red) mixed with WT gAnkB 15R (upper panel, labeled with Alexa Fluor 488; green) or the PSK mutant of gAnkB 15R (lower panel, labeled with Alexa Fluor 488; green). Scale bars, 30 µm. MT filament or bundle width quantification is presented in the right panel. Mean ± SEM; ****P < 0.0001; t test; n = 20 for 15R; and n = 25 for 15R-PSK. (C) Nontransfected astrocytes (left) and astrocytes transfected with gAnkB-WT-halo (middle) or gAnkB-PSK-halo (right) were fixed and stained with anti-tubulin antibody (green) and stained with Janelia Fluor 549 dye (red) after 24 h of expression (scale bars, 20 µm). Dashed lines indicate the borders of the astrocyte. The nuclear region is indicated with N. The histogram of the diameter and the angle of the MT bundle for all three conditions are displayed to the right (for diameter measurement, n = 156 for nontransfected; n = 144 for gAnkB-WT; n = 136 for gAnkB-PSK; for angle measurement, n = 164 for nontransfected; n = 219 for gAnkB-WT; n = 221 for gAnkB-PSK). Data were collected from 10 cells of three biological replicates. The arrowheads indicate the peak of distribution in each experimental group.$

Point mutations in the 15R segment impair MT-bundling activity of gAnkB. (A) MT cosedimentation assay performed with the WT gAnkB 12-aa repeats (15R) or the PSK mutant of gAnkB 12-aa repeats (15R-PSK) with Trx as the control (each at 10 µM and Trx at 30 µM). The SDS-PAGE gel is shown in the left panel, and the percentage of proteins recovered from the MT-binding pellet fractions is shown in the right panel. Mean ± SEM; n = 3. (B) Confocal images of in vitro Taxol-stabilized MTs (contains ∼8% rhodamine-labeled tubulin; red) mixed with WT gAnkB 15R (upper panel, labeled with Alexa Fluor 488; green) or the PSK mutant of gAnkB 15R (lower panel, labeled with Alexa Fluor 488; green). Scale bars, 30 µm. MT filament or bundle width quantification is presented in the right panel. Mean ± SEM; ****P < 0.0001; t test; n = 20 for 15R; and n = 25 for 15R-PSK. (C) Nontransfected astrocytes (left) and astrocytes transfected with gAnkB-WT-halo (middle) or gAnkB-PSK-halo (right) were fixed and stained with anti-tubulin antibody (green) and stained with Janelia Fluor 549 dye (red) after 24 h of expression (scale bars, 20 µm). Dashed lines indicate the borders of the astrocyte. The nuclear region is indicated with N. The histogram of the diameter and the angle of the MT bundle for all three conditions are displayed to the right (for diameter measurement, n = 156 for nontransfected; n = 144 for gAnkB-WT; n = 136 for gAnkB-PSK; for angle measurement, n = 164 for nontransfected; n = 219 for gAnkB-WT; n = 221 for gAnkB-PSK). Data were collected from 10 cells of three biological replicates. The arrowheads indicate the peak of distribution in each experimental group.