Selective accumulation of the high molecular weight neurofilament subunit within the distal region of growing axonal neurites

Axonal maturation in situ is accompanied by the transition of neurofilament s (NFs) comprised of only NF-M and NF-L to those also containing NF-H. Sinc e NF-H participates in interactions of NFs with each other and with other c ytoskeletal constituents, its appearance represents a critical event in the stabilization of axons that accompanies their maturation. Whether this tra nsition is effected by replacement of "doublet" NFs with "triplet" NFs, or by incorporation of NF-H into existing doublet NFs is unclear. To address t his issue, we examined the distribution of NF subunit immunoreactivity with in axonal cytoskeletons of differentiated NB2a/d1 cell and DRG neurons betw een days 3-7 of outgrowth. Endogenous immunoreactivity either declined in a proximal-distal gradient or was relatively uniform along axons. This distr ibution was paralleled by microinjected biotinylated NF-L. By contrast, bio tinylated NF-H displayed a bipolar distribution, with immunoreactivity conc entrated within the proximal- and distal-most axonal regions. Proximal biot inylated NF-H accumulation paralleled that of endogenous NF immunoreactivit y; however, distal-most biotinylated NF-H accumulation dramatically exceede d that of endogenous NFs and microinjected NF-L. This phenomenon was not du e to co-polymerization of biotin-H with vimentin or alpha -internexin. This phenomenon declined with continued time in culture. These data suggest tha t NF-H can incorporate into existing cytoskeletal structures, and therefore suggest that this mechanism accounts for at least a portion of the accumul ation of triplet NFs during axonal maturation. Selective NF-H accumulation into existing cytoskeletal structures within the distal-most region may pro vide de novo cytoskeletal stability for continued axon extension and/or sta bilization. (C) 2001 Wiley-Liss, Inc.