Article Figures & Data
Figures
- Figure 1.
ch-TOG and SLAIN1/2 are expressed and interact with each other in brain tissue. A, B, D, Western blot analysis of different adult rat tissues (A), mouse brain tissue of different developmental stages (B), and extracts of primary hippocampal neuron culture extracts at different stages of development in vitro (D) using the indicated antibodies. C, Immunoprecipitations (IP) from extracts of rat embryonic cortex or adult brain with either control IgG or SLAIN1/2 antibodies analyzed by Western blotting with the indicated antibodies. E, F, Images of neurons fixed on DIV5 and labeled with the indicated antibodies. The insets show enlargements of the boxed areas, which show a dendritic (E) and an axonal (F) growth cone. In the overlay, EB1 is shown in green and ch-TOG (E) and SLAIN1/2 (F) in red. Endog., Endogenous; T., total.
- Figure 2.
Disruption of the SLAIN-ch-TOG complex in neuronal cells. A, Western blots of extracts of hippocampal neuronal cultures transduced with lentiviruses expressing the indicated shRNAs. SLAIN1+2—simultaneous transduction with shRNAs against SLAIN1 and SLAIN2. B, Quantification of the number of EB1 comets in the cell body per 100 μm2 surface area in control shRNA, ch-TOG shRNA, GFP control, and GFP-SLAIN2–N1-expressing neurons (10–15 cells were analyzed for each condition). Statistically significant differences are indicated (**p < 0.05, ***p < 0.001). Error bars indicate SD. C, D, Images of neurons transfected at DIV1 with indicated constructs, fixed on DIV5, and labeled with antibodies against EB1. Asterisks in C and D indicate transfected cells. Endog., Endogenous.
- Figure 3.
SLAIN-ch-TOG complex controls MT growth in neuronal cells. A, Live images of neurons transfected at DIV1 with mCherry-MT+TIP together with GFP, GFP-SLAIN2–N1, or shRNA constructs against SLAIN1 and SLAIN2 or ch-TOG. Live images were collected at DIV5 with 0.5 s intervals. Single frames and maximum projections of 61 frames (bottom) are shown. B, Kymographs illustrating MT growth using mCherry-MT+TIP in neurons coexpressing mCherry-MT+TIP together with GFP (control), GFP-SLAIN2–N1, SLAIN1+2, or ch-TOG shRNAs. C, Quantification of the catastrophe frequency in control, GFP-SLAIN2–N1-expressing cells, or cells expressing the indicated shRNAs using data shown in A and B. Error bars indicate SEM. Fifty to 200 growth episodes in five to nine cells observed predominantly in the cell body and proximal neurites were analyzed in each condition. Data for GFP control and control shRNA showed no significant differences and were pooled. D, Mean instantaneous growth rates (growth rates measured between two frames acquired with a 0.5 s interval) and histograms of MT growth rate distributions based on the displacement of mCherry-MT+TIP comets in DIV5 neurons cotransfected at DIV1 with GFP (control), GFP-SLAIN2–N1, or the shRNA constructs. Values significantly different from control are indicated by asterisks (**p < 0.05, ***p < 0.001).
- Figure 4.
ch-TOG and SLAIN1/2 are necessary for axon extension. A, Images of neurons transfected at DIV1 with the control GFP construct, fixed at DIV5, and labeled with tau or MAP2 antibodies. B, Images of neurons transfected with the indicated constructs and GFP as a neuronal morphology marker at DIV1 and fixed at DIV5. C, Quantification of the total length of axons and dendrites in DIV5 neurons transfected at DIV1 with the indicated constructs. GFP was used as a morphology marker, and tau and MAP2 staining were used to distinguish axons and dendrites. Nine to 12 cells were analyzed for each condition. Statistically significant differences are indicated (**p < 0.05). Error bars indicate SEM.
