The Myosin Va Head Domain Binds to the Neurofilament-L Rod and Modulates Endoplasmic Reticulum (ER) Content and Distribution within Axons
Figure 3
The N-terminal rod domain of NF-L binds to the motor domain of Myo Va.
(A) Schematic representation of NF-L subunit domains and the deletions. Amino acids are numbered according to [31]. See materials and methods for construction of NF-L domain deletions. B. The amino-terminal motor domain of Myo Va (41–326) binds to the N-terminal rod domain of NF-L. Myo Va constructs were MBP-tagged while NF-L was myc-tagged. Co-immunoprecipitation of NF-L deletion constructs with Myo Va motor domain (41–326). Myo Va was incubated without NF-L (lane 1), with full length NF-L 1-543 (lane 2); NF-L 1-243 (lane 3); NF-L 94-243 (lane 4) and NF-L 1-93 (lane 5) were immunoprecipitated with Myc antibody (9E10), IPs and Sups (10%) were immunoblotted with anti-MBP or anti-Myc antibodies. The molecular weights of NF-L 1-543, 1-243, 94-243 and 1-93 are 68, 27, 20 and 17-kDa respectively. (C) Magnesium ions potentiate Myo Va and NF-L binding. Myo Va motor domain (41–326) was incubated with NF-L (Full length) and immunoprecipitated with α-Myc antibody. The resulting IPs and 5% of sups were immunoblotted with α-MBP-HRP or α-Myc Abs. Addition of magnesium (5 mM) increased binding (see the graph in 3C) while addition of magnesium and calcium (5 mM) further increased the binding of NF-L to Myo Va (compare lane 6 with 5 and 7). Arrows indicate the position of Myo Va motor domain (41–326) of 75-kDa protein. The *-indicates proteolytic fragments of 41–326 construct generated with incubation of calcium and magnesium in both IP and supernatant fractions in lanes 4&5 while these are seen only in IP fractions of lanes 6&7. (D). Myo Va head domain binds to different IF family members. Cytoskeletal preparations from brain (lane 1), sciatic nerve (lane 2), spinal cord (lane 3), purified NF-L (lane 4), purified actin (lane 5), purified vimentin (lane 6), purified desmin (lane 7) and fractionated keratin from human skin (lane 8) were separated on gels and transferred to membranes. Blot overlay assays were performed on membranes with MBP-Myo Va (41–326) protein and immunoblotted with α-MBP-HRP antibody. Anti-MBP: α-MBP; anti-NF-L: α-NF-L and anti-myc: α-myc. (E) Schematic representation of NF-L and Actin binding sites on Myo Va head domain (1-752). The amino acids are numbered according to [6]. (F). Actin activates Myo Va-ATPase activity while NF-L does not. Myo Va was fractionated from adult mouse brain according to [26] (P2 fraction enriched in vesicular Myo Va) and incubated with either actin or NF-L and assayed for ATPase activity. Addition of actin to P2 fraction activated Myo Va-ATPase activity while addition of NF-L did not. *p<0.02. Error bars represent SEM in all experiments.