The predominant form in which neurofilament subunits undergo axonal transport varies during axonal initiation, elongation, and maturation

The forms in which neurofilament (NF) subunits undergo axonal transport is controversial. Recent studies from have provided real-time visualization of the slow axonal transport of NF subunits by transfecting neuronal cultures with constructs encoding green fluorescent protein (GFP)-conjugated NF-M s ubunits. In our studies in differentiated NB2a/dl cells, the majority NF su bunits underwent transport in the form of punctate NF precursors, while stu dies in cultured neurons have demonstrated transport of NF subunits in pred ominantly filamentous form. Although different constructs were used in thes e studies, transfection of the same cultured neurons with our construct yie lded the filamentous pattern observed by others, while transfection of our cultures with their construct generated punctate structures, confirming tha t the observed differences did not reflect variances in assembly-competence among the constructs. Manipulation of intracellular kinase, phosphatase, a nd protease activities shifted the predominant form of GFP-conjugated subun its between punctate and filamentous, confirming, as shown previously for v imentin, that punctate structures represent precursors for intermediate fil ament formation. Since these prior studies were conducted at markedly diffe ring neuronal differentiation states, we tested the alternate hypothesis th at these differing results reflected developmental alterations in NF dynami cs that accompany various stages of neuritogenesis. We conducted time-cours e analyses of transfected NB2a/dl cells, including monitoring of transfecte d cells over several days, as well as transfecting cells at varying interva ls prior to and following induction of differentiation and axonal neurite o utgrowth. GFP-conjugated subunits were predominantly filamentous during the period of most robust axonal outgrowth and NF accumulation, and presented a mixed profile of punctate and filamentous forms prior to neuritogenesis a nd following the developmental slowing of neurite outgrowth. These analyses demonstrate that NF subunits are capable of undergoing axonal transport in multiple forms, and that the predominant form in which NF subunits undergo axonal transport varies in accord with the rate of axonal elongation and a ccumulation of NFs within developing axons. Cell Motil. Cytoskeleton 48: 61 -83, 2001. (C) 2001 Wiley-Liss, Inc.