Assembly and turnover of neurofilaments in growing axonal neurites.

Edward F Boumil,Harish Pant,Rishel Vohnoutka,Thomas B Shea,Sangmook Lee

doi:10.1242/bio.028795

Abstract

ABSTRACTNeurofilaments (NFs) are thought to provide stability to the axon. We examined NF dynamics within axonal neurites of NB2a/d1 neuroblastoma by transient transfection with green fluorescent protein-tagged NF-heavy (GFP-H) under the control of a tetracycline-inducible promoter. Immunofluorescent and biochemical analyses demonstrated that GFP-H expressed early during neurite outgrowth associated with a population of centrally-situated, highly-phosphorylated crosslinked NFs along the length of axonal neurites (‘bundled NFs’). By contrast, GFP-H expressed after considerable neurite outgrowth displayed markedly reduced association with bundled NFs and was instead more evenly distributed throughout the axon. This differential localization was maintained for up to 2 weeks in culture. Once considerable neurite outgrowth had progressed, GFP that had previously associated with the NF bundle during early expression was irreversibly depleted by photobleaching. Cessation of expression allowed monitoring of NF turnover. GFP-H associated bundled NFs underwent slower decay than GFP-H associated with surrounding, less-phosphorylated NFs. Notably, GFP associated with bundled NFs underwent similar decay rates within the core and edges of this bundle. These results are consistent with previous demonstration of a resident NF population within axonal neurites, but suggest that this population is more dynamic than previously considered.

Highlights

Neurofilaments (NFs) are heteropolymers composed of proteins, termed NF-light (NF-L), NF-medium (NF-M) and NF-heavy (NF-H) with respect to their molecular masses (Liem et al, 1978) along alpha-internexin and peripherin in central and peripheral neurons, respectively (Yuan et al, 2006a, 2012)
green fluorescent protein-tagged NF-heavy (GFP-H) was associated with filamentous structures along the length of axonal neurites within 24 h, and was retained for many days (Fig. 1A)
To provide control over expression levels, differentiating NB2a/d1 cells were transiently transfected with green fluorescent protein (GFP)-H under the control of a tetinducible promoter, after which expression was induced for 12 h after 24 h of differentiation and in other cells after 72 h of differentiation

Summary

Introduction

Neurofilaments (NFs) are heteropolymers composed of proteins, termed NF-light (NF-L), NF-medium (NF-M) and NF-heavy (NF-H) with respect to their molecular masses (Liem et al, 1978) along alpha-internexin and peripherin in central and peripheral neurons, respectively (Yuan et al, 2006a, 2012). Phosphorylation of the NF-H C-terminal domain protects NFs from proteolysis (Goldstein et al, 1987; Pant, 1988; Greenwood et al, 1993; Rao et al, 2012) and promotes divalent cation-mediated associations between NFs and with other cytoskeletal elements, which generates a cytoskeletal lattice along the length of axons (Gotow and Tanaka, 1994; Gotow et al, 1994; Yabe et al, 2001a,b; Kushkuley et al, 2009) This ‘resident’ or stationary population is thought to be formed and maintained by continuous exchange with more rapidly transporting NFs/NF subunits; a population of highly-phosphorylated NFs provides stability to axons, while a population of more relentlessly transporting, poorly-phosphorylated NFs repairs and regenerates this stationary cytoskeleton (Nixon and Logvinenko, 1986; Rao et al, 2012; Lee and Shea, 2012)

Methods

Results

Conclusion