Map7D2 and Map7D1 facilitate microtubule stabilization through distinct mechanisms in neuronal cells

Abstract

Microtubule (MT) dynamics are modulated through the coordinated action of various MT-associated proteins (MAPs). However, the regulatory mechanisms underlying MT dynamics remain unclear. We show that the MAP7 family protein Map7D2 stabilizes MTs to control cell motility and neurite outgrowth. Map7D2 directly bound to MTs through its N-terminal half and stabilized MTs in vitro. Map7D2 localized prominently to the centrosome and partially on MTs in mouse N1-E115 neuronal cells, which expresses two of the four MAP7 family members, Map7D2 and Map7D1. Map7D2 loss decreased the resistance to the MT-destabilizing agent nocodazole without affecting acetylated/detyrosinated stable MTs, suggesting that Map7D2 stabilizes MTs via direct binding. In addition, Map7D2 loss increased the rate of random cell migration and neurite outgrowth, presumably by disturbing the balance between MT stabilization and destabilization. Map7D1 exhibited similar subcellular localization and gene knockdown phenotypes to Map7D2. However, in contrast to Map7D2, Map7D1 was required for the maintenance of acetylated stable MTs. Taken together, our data suggest that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms in cell motility and neurite outgrowth.

Figure S1.. Sequence alignment among human MAP7 family proteins.

(A) Schematic structures of human MAP7 family proteins, Map7, Map7D1, Map7D2, and Map7D3. (B) Sequence alignment of conserved N-terminal regions in each human MAP7 family protein. (C) Sequence alignment of conserved C-terminal regions in each human MAP7 family protein.

Figure 1.. Tissue distribution of Map7D2.

(A) The full scan image of Northern blot analysis. An RNA blot membrane (CLONTECH) was hybridized with the ³²P-labeled full coding sequences of rMap7D2 according to the manufacturer’s protocol. (B) The full scan image of Immunoblotting analysis. Various tissue lysates (20 μg of protein) were subjected to SDS–PAGE, followed by immunoblotting with the anti-Map7D2 antibody. (C) Expression patterns of Map7D2 in the brain and testis by immunofluorescence. Upper panels, frozen sagittal sections of postnatal day 0 mouse brains were stained with anti-Map7D2 (magenta) and antibodies against mature neuron marker Map2 (green). DNA was labeled with DAPI (gray). For a comparison of signal intensities, images were captured under the same parameters. Contrast-enhanced images of Map7D2 staining were shown in the rightmost column. Lower panels, frozen coronal sections of adult mouse testis were stained with anti-Map7D2 (magenta) and antibodies against Sertoli cell marker Tubb3 (green). DNA was labeled with DAPI (gray). Data information: In (C), scale bars in upper panels or lower panels are represented as 100 or 50 μm, respectively.

Figure S2.. Confirmation for the specificity of anti-Map7D2 antibody.

(A) Confirmation for the specificity of anti-Map7D2 antibody using V5His₆-tagged human Map7 (hMap7) or rat Map7D2 (rMap7D2). An empty vector, hMap7-V5His₆, or rMap7D2-V5His₆ was transfected into HeLa cells, and cell lysates were subjected to SDS–PAGE, followed by immunoblotting with the anti-Map7D2 or anti-V5 antibody. (B) Endogenous expression of the MAP7 family genes in N1-E115 cells. Total RNA was extracted from N1-E115 cells and was subjected to reverse transcription. qPCR was performed with gene-specific primes using the cDNA as a template. Relative target gene mRNA expression was normalized to Gapdh expression. Data are from three independent experiments, and represent the average ± SD. (C) Knockdown of endogenous Map7d2 or Map7d1 in N1-E115 cells. Cells were treated with each siRNA, and cell lysates were subjected to SDS–PAGE, followed by immunoblotting with the anti-Map7D2, anti-Map7, or anti-Clathrin HC antibody.

Figure S3.. Database-based analyses for the expression distribution of Map7d2 in the mouse brain.

The Map7d2 expression distribution in the mouse brain was analyzed using the dataset of RNA-Seq CAGE (left), RNA-Seq (middle), and SILAC (right) datasets in the Expression Atlas (https://www.ebi.ac.uk/gxa/home/).

Figure 2.. Map7D2 has the ability to stabilize MTs.

(A) Schematic structures of hMap7, hMap7D2, and rMap7D2. (B) Co-sedimentation of rMap7D2 with MTs. Left panel, GST-rMap7D2 (34 μg/ml) was mixed with MTs, followed by ultracentrifugation. Comparable amounts of the supernatant and pellet fractions were subjected to SDS–PAGE, followed by CBB protein staining. S, supernatant; P, pellet. Middle panel, various amounts of GST-rMap7D2 were mixed with MTs, followed by ultracentrifugation. Amounts of free and bound GST-rMap7D2 were calculated by determining protein amounts from the supernatant and pellet fractions, respectively, with a densitometer. Right panel, Scatchard analysis. (C) Location of the MT-binding domain. GST-rMap7D2-N (80 μg/ml) or GST-rMap7D2-C (200 μg/ml) was mixed with MTs, followed by ultracentrifugation. Comparable amounts of the supernatant and pellet fractions were subjected to SDS–PAGE, followed by CBB protein staining. S, supernatant; and P, pellet. (D) Turbidity measurement. GST-rMap7D2 was mixed with tubulin. The sample was incubated at 37°C and continuously monitored at 350 nm using a spectrophotometer. (○) without GST-rMap7D2; and (●) with GST-rMap7D2. (E) Immunofluorescent observation. GST-rMap7D2 was incubated for 20 min at 37°C with rhodamine-labeled tubulin. After fixation, the sample was spotted on a slide glass and viewed under a fluorescence microscope. (F) HeLa cells transiently overexpressing Myc-rMap7D2. Myc-rMap7D2 was transfected into HeLa cells, and the cells were then double-stained with anti-Myc and anti–α-tubulin antibodies. Arrowheads show MT bundles. Data information: In (D), *P < 0.003 (the F-test). Scale bars, 50 μm in (E) and 10 μm in (F). Source data are available for this figure.

Figure S4.. Map7 or Map7D2 forms a complex with Kif5b, a member of kinesin-1.

Lysates from HeLa cells co-expressing hMap7-V5His₆ (1), V5His₆-tagged chimeric protein of rMap7D2 N-terminus and hMap7 C-terminus (2), or rMap7D2-V5His₆ (3) with hKif5b-EGFP were immunoprecipitated with an anti-V5 antibody, and the immunoprecipitates were probed with anti-GFP and anti-V5 antibodies. E represents an empty vector control. Map7 has a region on the C-terminus that is required for complex formation with Kif5b, a member of kinesin-1.

Figure S5.. Subcellular localization of Map7D1 in proliferative and differentiated N1-E115 cells.

(A) Neuronal differentiation of N1-E115 cells was induced by decreasing the concentration of fetal bovine serum in the medium to 0.5% fetal bovine serum and adding 1% dimethylsulfoxide. Arrowheads indicate elongated neurites. After 6 h of induction, elongated neurites were observed. (B, C, D) Localization of Map7D1 in interphase (B), mitosis (C), and differentiation state (D) of N1-E115 cells. Cells were double-stained with anti-Map7D1 and anti–α-tubulin antibodies. In (B), the insets show enlarged images of regions indicated by a white box. In (C), the inset shows metaphase cells. In (D), images of differentiated cells were captured by z-sectioning because the focal planes of the centrosome and neurites are different. Each inset shows an enlarged image of the region indicated with a white box at each focal plane. Arrowheads show centrosomal localization of Map7D1. Data information: scale bars, 50 μm in (A) and 10 μm in (B, C, D).

Figure 3.. Subcellular localization of Map7D2 in proliferative and differentiated N1-E115 cells.

(A, B, C) Localization of Map7D2 in interphase (A), mitosis (B), and differentiation state (C) of N1-E115 cells. Cells were double-stained with anti-Map7D2 and anti–α-tubulin antibodies. In (A), the insets show enlarged images of regions indicated by a white box. In (B), the inset shows metaphase cells. In (C), images of differentiated cells were captured by z-sectioning because the focal planes of the centrosome and neurites are different. Each inset shows an enlarged image of the region indicated with a white box at each focal plane. Arrowheads indicate the centrosomal localization of Map7D2. (D) Generation of N1-E115 cells stably expressing EGFP-rMap7D2. To check the expression level of EGFP-rMap7D2, lysates derived from the indicated cells were probed with anti-GFP (top panel) and anti-Map7D2 (middle panel) antibodies. The blot was reprobed for γ-tubulin as a loading control (bottom panel). The amount of endogenous Map7D2 or EGFP-rMap7D2 was normalized to the amount of γ-tubulin, and the value relative to endogenous Map7D2 in the parental control was calculated. (E) Confirmation for subcellular localization of Map7D2 using N1-E115 cells stably expressing EGFP-rMap7D2. Images were captured by z-sectioning. Top panels show images taken with the lower or upper focal plane, and bottom panels show the image reconstructed in the z-axis direction. Arrow head shows centrosomal localization of Map7D2. Data information: scale bars, 10 μm in (A, B, C, E). Source data are available for this figure.

Figure S6.. Generation of Map7d2 knockout N1-E115 cells.

(A) Schematic representation of Map7d2 knockout (Map7d2^−/−) N1-E115 cell generation using the CRISPR-Cas9 technique. (B) Map7d2 knockout was confirmed by immunoblotting. Three independent clones of Map7d2^−/− cells were used in this study. Lysates derived from the indicated cells were probed with anti-Map7D2 antibody. In addition, the effect of Map7d2 knockout on Map7D1 expression was analyzed using the anti-Map7D1 antibody. The blot was reprobed for α-tubulin as a loading control. The expression levels of the indicated proteins in Map7d2^−/− cells were carefully compared with dilution series of wild-type lysates.

Figure 4.. Map7D2 is required for MT stabilization within the cell.

(A) Confirmation of MT stability by low-dose nocodazole treatment in the indicated N1-E115 cells. The cells were treated with or without a low concentration of nocodazole (10 ng/ml) for 1 h and were stained with an anti–α-tubulin antibody. Cells with the elongated MT arrays or the MT shrinkage were counted, and the rate of cells with the elongated MT arrays was calculated. Data are from three independent experiments and represent the average ± SD. (B) Immunofluorescence staining for EB1 and Kif5b in N1-E115 cells treated with each siRNA (Top panels). The insets show enlarged images of regions indicated by a white box. Bottom left panel, the intensities of EB1 at the cell periphery in the indicated cells were measured via ROI analysis (each, n = 30 cells from three independent experiments). Data from Map7d1 or Map7d2 knockdown were shown by normalizing with the control value. Bottom right panel, the intensities of Kif5b at the cell periphery (white box) and the internal region (cyan box) were measured via ROI analysis, and the intensity ratios of cell peripheral-internal Kif5b were calculated (each, n = 30 cells from three independent experiments). Of note, a value of 1 means that Kif5b is distributed throughout the cell, and a value greater or less than 1 means that Kif5b is distributed at the cell periphery or in the internal region, respectively. Data information: In (A), *P < 0.0008 (the t test). Scale bars, 10 μm in (A, B). Source data are available for this figure.

Figure S7.. Map7D2 stabilizes MTs within the cell.

(A) Images of EB1 and Kif5b at the protrusion in N1-E115 cells treated with each siRNA, related to Fig 4A. (B) Schematic representation of neurite outgrowth assay, related to Fig 6D. Data information: Scale bars, 10 μm in (A).

Figure 5.. Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms.

(A) Immunoblot analysis for acetylated (Ace-) and detyrosinated (Detyr-) tubulin in N1-E115 cells treated with each siRNA. Lysates derived from the indicated cells were separated by SDS–PAGE and subjected to immunoblotting with anti-Map7D1, anti-Map7D2, anti–Ace-tubulin, or anti–Detyr-tubulin antibodies. The blot was reprobed for Clathrin heavy chain (HC) or α-tubulin as a loading control. (B) Immunoblot analysis for Ace- and Detyr-tubulins in wild-type and Map7d2^−/− N1-E115 cells. Three independent Map7d2^−/− clones were used in this study. Lysates derived from the indicated cells were separated by SDS–PAGE and were immunoblotted with anti-Map7D1, anti-Map7D2, anti–Ace-tubulin, or anti–Detyr-tubulin antibodies. The blot was reprobed for α-actin or α-tubulin as a loading control. (C) Immunofluorescence staining for α-tubulin, Ace-tubulin, and Map7D1 or Map7D2 in N1-E115 cells treated with each siRNA. For a comparison of signal intensities, images were captured under the same parameters. The insets show enlarged images of regions indicated by a white box. Of note, Ace-tubulin was present predominately around the centrosome in N1-E115 cells as indicated by arrowheads. (C, D) Quantification for immunofluorescence staining shown in (C). Left panels, the intensities of α-tubulin, Ace-tubulin, and Map7D1 around the centrosome in the indicated cells were measured via ROI analysis (control, n = 197 cells; siMap7d2, n = 192 cells from three independent experiments). Right panels, the intensities of α-tubulin, Ace-tubulin, and Map7D2 around the centrosome in the indicated cells were measured by ROI analysis (control, n = 193 cells; siMap7d1, n = 227 cells from three independent experiments). Data information: In (D), the bars of box-and-whisker plots show the 5 and 95 percentiles. *P < 1 × 10⁻¹³; **P < 1 × 10⁻⁸ (the t test). Scale bars, 10 μm in (C) and 5 μm in (D). Source data are available for this figure.

Figure 6.. Map7D2 suppresses random cell migration and neurite outgrowth.

(A) Bright-field images of migrating N1-E115 cells. Arrowheads show lamellipodia formed in the direction of migration. (B) Tracking analysis of random cell migration in the indicated cells. Each color represents the trajectory of 12 randomly selected cells. (C) Velocity and net distance measured in the indicated cells (control: n = 114 cells; siMap7d1: n = 100 cells; siMap7d2: n = 71 cells; siMap7d1/d2: n = 107 cells; Map7d2^−/−: n = 60 cells from three independent experiments). (D) Neurite outgrowth assay in the indicated cells. Neurites and cell bodies were visualized by α-tubulin staining (upper). The neurite outgrowth from each cell was distinguished by acquiring images with Z-sectioning. Data are from three or four independent experiments and represent the average ± SD. (E) Proposed model for the distinct mechanisms of Map7D2 and Map7D1 for MT stabilization. See the Discussion section for further detail. Data information: In (C), the bars of box-and-whisker plots show the 5 and 95 percentiles. *P < 1 × 10⁻⁴; **P < 0.002 (the t test). In (D), *P < 0.002; **P < 0.0002 (the t test). Scale bars, 20 μm in (A, D). Source data are available for this figure.