Most domain components are independently transported in the anterograde direction. (A) NF186-mKate2 and a series of EGFP-tagged constructs are expressed diffusely along axons in neuron-only cultures (upper row) and targeted to their appropriate domains (i.e., nodes, paranodes, internodes) in myelinating cocultures of neurons and Schwann cells (+SC, middle and lower rows). Nodes in the last two columns were identified by NF186 staining (red in the Caspr-EGFP cocultures and white in the Necl1-EGFP cocultures), and paranodes were identified by Caspr staining (except in the Caspr-EGFP cocultures); myelin segments were identified by MBP staining (MBP, blue). Bar: 5 µm. (B) Still images of domain components transported in the anterograde direction are shown. NF186-mKate2 (red) is compared with other components labeled with EGFP. Examples of vesicles containing EGFP-positive components are indicated by arrows; yellow indicates cotransport with mKate2 and white for EGFP alone. Two vesicles (a and b) that are colabeled for NF186 and NrCAM are indicated in the top panels. Inset shows higher power image of vesicle indicated by the arrows. Time of imaging (seconds) is indicated in each panel. (C) Kymographs of anterogradely transported vesicles indicated by the arrows in the panels in A are shown. Trajectories of each vesicle are circled (yellow for cotransport, white for EGFP alone) and are numbered in temporal sequence. Dotted blue arrows indicate the direction of anterogradely transported vesicles. (D) Quantification of vesicles that colabeled for NF186 and the indicated EGFP-positive component versus the total number of anterogradely transported NF186 vesicles (average ± SEM) is shown. The numbers of axons counted are NrCAM, n = 14; Na_vβ2, n = 14; Na_vβ1, n = 19; KCNQ3, n = 10; Caspr, n = 16; and Necl1, n = 13. NF186 and NrCAM are uniformly cotransported, whereas other components exhibit negligible cotransport.