Microtubule-associated protein 1B: a neuronal binding partner for gigaxonin

Abstract

Giant axonal neuropathy (GAN), an autosomal recessive disorder caused by mutations in GAN, is characterized cytopathologically by cytoskeletal abnormality. Based on its sequence, gigaxonin contains an NH2-terminal BTB domain followed by six kelch repeats, which are believed to be important for protein-protein interactions (Adams, J., R. Kelso, and L. Cooley. 2000. Trends Cell Biol. 10:17-24.). Here, we report the identification of a neuronal binding partner of gigaxonin. Results obtained from yeast two-hybrid screening, cotransfections, and coimmunoprecipitations demonstrate that gigaxonin binds directly to microtubule-associated protein (MAP)1B light chain (LC; MAP1B-LC), a protein involved in maintaining the integrity of cytoskeletal structures and promoting neuronal stability. Studies using double immunofluorescent microscopy and ultrastructural analysis revealed physiological colocalization of gigaxonin with MAP1B in neurons. Furthermore, in transfected cells the specific interaction of gigaxonin with MAP1B is shown to enhance the microtubule stability required for axonal transport over long distance. At least two different mutations identified in GAN patients (Bomont, P., L. Cavalier, F. Blondeau, C. Ben Hamida, S. Belal, M. Tazir, E. Demir, H. Topaloglu, R. Korinthenberg, B. Tuysuz, et al. 2000. Nat. Genet. 26:370-374.) lead to loss of gigaxonin-MAP1B-LC interaction. The devastating axonal degeneration and neuronal death found in GAN patients point to the importance of gigaxonin for neuronal survival. Our findings may provide important insights into the pathogenesis of neurodegenerative disorders related to cytoskeletal abnormalities.

Figure 1.

Protein expression of gigaxonin. Proteins isolated from mouse tissues were analyzed by immunoblot with rabbit antigigaxonin (lanes 1–5) or mouse anti-HA (Covance) (lanes 6 and 7). The single band of ∼70 kD (lanes 1–4), which is absent in the untransfected COS-7 cells (lane 5), indicates the full-length gigaxonin protein. The 40- and 28-kD bands from transfected COS-7 cells represent the COOH-terminal domain (Gig-C, lane 6) and NH₂-terminal domain (Gig-N, lane 7), respectively. Migration of protein standard (Amersham Biosciences) is indicated at left.

Figure 2.

Gigaxonin associates with MAP1B-LC on microtubules. The expression constructs of HA-Gig-full and flag-MAP1B-LC were cotransfected into COS-7 cells (A–D). The cells were subjected to double immunofluorescence as described previously (Yang et al., 1996). Antibodies are indicated in each panel: (A) Gig-full, (mouse anti-HA); (B) MAP1B-LC (rabbit anti-flag; Sigma-Aldrich). Note that gigaxonin displayed a network array that coaligned with MAP1B-LC in cotransfected cells (C), and a diffuse accumulation in cytoplasm in single transfected cells (D). Bar, 12 μm. (E) The cotransfected cells were processed for coIP using anti-flag (MAP1B-LC) and immunoblotted with anti-HA (gigaxonin, Gig-F). Note the specific band of Gig-F present in cotransfection lane (lane 5) but absent in the single transfections of MAP1B-LC (lane 4) or Gig-F (lane 3). The total cell lysates from Gig-F single transfection without IP (lane 1) and untransfected COS-7 (lane 2) served as controls in this assay.

Figure 3.

Gigaxonin physiologically colocalizes with MAP1B in neurons. (A–C) The cultured mouse DRG neurons were subjected to double immunofluorescence using anti–mouse MAP1B-LC (Sigma-Aldrich) (A, green) and rabbit antigigaxonin (B, red). Arrows denote colocalizations on cytoskeletal structures. Insets in A, B, and C show higher magnifications of the colocalization areas in the white boxes. (D) For double immuno EM, sciatic nerve samples were colabeled with rabbit antigigaxonin and mouse anti–MAP1B-LC followed by gold-conjugated secondary antibodies against mouse (small particles) and rabbit (large particles). The large particles represent gigaxonin, and the small particles identify MAP1B. Arrows identify colocalizations. The samples labeled with only secondary antibodies were used as negative control (E). Bar: (A–C) 20 μm; (D and E) 200 nm.

Figure 4.

The kelch repeat domain of gigaxonin binds to the COOH terminus of MAP1B-LC. The cells were cotransfected for 30 h with flag-MAP1B-LC and HA-Gig-C (A and B), or flag-MAP1B-LC-CT and HA-Gig-C (C and D), or flag-MAP1B-LC and MAP1B-HC-myc (E), or flag-MAP1B-LC, MAP1B-HC-myc, and HA-Gig-full (F–H). (A–D) Mouse anti-HA (Gig-C; B and D, red) and rabbit anti-flag (MAP1B-LC or MAP1B-LC-CT; A and C, green). Note that the Gig-C colocalized with MAP1B-LC-CT. (E and F) Sheep antitubulin (Cytoskeleton Inc.; E, red); mouse anti-myc (CLONTECH Laboratories, Inc.; E and F, green); rabbit anti-flag (F, red) and rabbit anti-myc (G, green); mouse anti-HA (H, red). Bar, 12 μm. The diagram in I indicates that the kelch repeat domain of gigaxonin interacts with the COOH terminus of MAP1B-LC.

Figure 5.

The gigaxonin–MAP1B-LC interaction enhances microtubule stability. Both transfected and untransfected cells were double stained using antibodies indicated in each frame. After treatment with colchicine, the microtubules were seen to be depolymerized within 15 min in untransfected (A) and within 60 min in flag-MAP1B-LC single transfected cells (B). In contrast, the intact network could still be found in 20% of HA-Gig-full/flag-MAP1B-LC cotransfected cells after 2 h treatment (C and D). Double immunostaining reveals that the HA-Gig-G293X (mutant gigaxonin, red) lost association with MAP1B-LC (E, green) and HA-Gig-G293X/flag-MAP1B-LC displayed no enhancement of microtubule stability (F). Bar, 12 μm.