Assembly and turnover of neurofilaments in growing axonal neurites

Abstract

Neurofilaments (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.

Keywords: Axogenesis; Axon; Axonal transport; Cytoskeleton; Neurofilament.

Fig. 1.

Experimental outline and validation of tet-inducible expression system. (A) Representative NB2a/d1 cells at day 3 and 12 after initiation of dbcAMP-induced differentiation, transiently transfected with GFP-H on day 2 after initiation of differentiation. Insets show higher-magnification views of regions of axonal neurites indicated by arrows. Note association of GFP with filamentous structures along the length of axonal neurites along with saturation of the soma. (B) Timeline of induction and cessation of GFP-H expression under control of the tet-inducible promoter by addition of doxycycline (dox) for 12 h after 24 h or 72 h of differentiation (‘Early-On’ and ‘Late-On’, respectively). Additional cultures did not receive dox (‘Leak’) or received dox for 1 week (‘Always-On’). (C) Quantification of GFP under the conditions described in panel B at 24 and 72 h after transfection (n=total of >1000 cells from three separate experiments; >65 cells/condition/time point). *P<0.01. (D) Quantification of GFP within total axonal neurites, or three segments of equivalent length (proximal, central and distal) under Early-On and Late-On conditions as described in B at 24 and 72 h after transfection (n=39 total cells).

Fig. 2.

Differential distribution of GFP under Early-On and Late-On conditions. (A) Representative phase-contrast and corresponding epifluorescent images of Early-On and Late-On cells at the indicated day after induction of differentiation. Arrows in epifluorescent images denote the distribution of GFP; arrows in corresponding phase-contrast images denote the diameter of the axonal neurite. Larger arrows in the merged images also denote the region from which the higher-magnification image was derived. By days 7-14, axonal neurites were often too long for single micrographs at 100×. The ‘Early-On’ axonal neurite micrographs presented for these times in Fig. 2 are composites of two micrographs. Asterisks in the respective phase-contrast images indicate where composite images were merged. (B) Higher-magnification images from representative cells at day 4 after induction of differentiation. Note differential distribution of GFP within the centrally-situated NF bundle and was instead confined to the area surrounding the bundle (‘surround’). Arrows in the phase-contrast image denote the diameter of the axonal neurite. (C) Upper graphs present quantification of distribution of GFP in Early-On and Late-On axonal neurites from multiple cells at the indicated day after induction of differentiation. Lower graphs present the rate of decline of GFP from the surround and from the bundle at days 2 and 7 after induction of differentiation (n=total 96 cells from four separate experiments; P<0.01 for Early-On day 4 versus Late-On day 4 at both 24 and 48 h).

Fig. 3.

Differential distribution of GFP under Early-On and Late-On conditions. (A) Representative phase-contrast and epifluorescent image of the central region of an axonal neurite. Arrows in the phase-contrast image denote the diameter of the axonal neurite. The accompanying graphs present the relative distribution of GFP across the lateral profile (axonal neurite lateral profile), the distribution of GFP across the bundle (bundle profile) and the distribution of GFP within the bundle following binning as described in the Materials and Methods to normalize distribution among multiple profiles. (B) Representative images and quantification of the distribution of GFP and total phospho-H as revealed by SMI-31 immunoreactivity in central segments of axonal neurites as described in A. Arrows denote the region from which respective lateral profiles were generated. Lower graphs present binned distribution from bundles from 53 of such cells. (C) Representative deconvoluted Z-stacks depicting the distribution of GFP and SMI-31 in Early-On and Late-On axonal neurites after 24 h of respective GFP-H expression.

Fig. 4.

Fractionation and differential centrifugation methodology. (A) Fractionation and differential centrifugation protocol to separate axonal neurites from soma, Triton-soluble material and Triton-insoluble cytoskeletons, and bundles from individual NFs from cytoskeletons of axonal neurites as described in the Materials and Methods (Shea et al., 1993). The accompanying nitrocellulose replicas present distribution of total NFs (R39), non-phosphorylated NF-H and M (SMI-32), phosphorylated NF-H and M (SMI-31, RT97), total (DM1A) and acetylated (6-11B) tubulin, MAP2, tau (Tau46) and GAPDH as indicated. (B) Phase-contrast and epifluorescent images of fractions during the above procedure as indicated. Arrows denote NF bundles recovered in the sucrose pellet; not all bundles are indicated. The accompanying graph presents quantification of the diameter of bundles before and following isolation as indicated.

Fig. 5.

Biochemical analyses of GFP-H distribution following differential expression. Nitrocellulose replicas probed as indicated of fractions from Early-On and Late-On cells harvested at 24 and 72 h after expression as indicated, along with uninduced cells harvested at the same time (‘Corresponding Leak’). The accompanying graphs present quantification of the distribution of GFP-H and phospho-NF (RT97) among fractions from three experiments, and the ratio of distribution in Early-On versus Late-On cells as indicated (*P<0.05).

Fig. 6.

Distribution of GFP within bundles along the length of axonal neurites. (A) Quantification of axonal neurite length during dbcAMP-induced outgrowth. The timing of induction of GFP-H expression under Early-On and Late-On conditions is indicated. (B) Phase-contrast image of representative axonal neurite from day 7 after induction of differentiation, depicting segmentation of axonal neurites, excluding the hillock and growth cone, into three equivalent segments (proximal, central and distal as indicated). Arrows denote the diameter of the axonal neurite. (C) Quantification of distribution of GFP and phospho-H (RT97 in lateral profiles of bundles generated as described for Fig. 3, for proximal, central and distal segments as described in B from a total of 198 cells. *P<0.05, **P<0.01.

Fig. 7.

Differential recovery of GFP within bundles and surrounding regions following photobleaching. (A) Representative images of an Early-On cell 72 h after expression of GFP-H before, during, immediately after, and 12 h after photobleaching as described in the Materials and Methods. A region with a varicosity was selected to demonstrate more clearly recovery within the surround but not the bundle. Arrows in phase-contrast images denote the diameter of the axonal neurite. (B,C) Quantification of GFP in multiple unbleached and photobleached axonal neurites of the type presented in panel A. (n=5 cells for each condition; *P<0.05, **P<0.01).

Fig. 8.

Turnover of GFP-H and radiolabeled endogenous NF-H. (A) Quantification of total GFP and GFP within the surround and bundle in Early-On cells. (B) Autoradiographs of NF-L within the retina (R) and 1.1 mm segments of optic axons at the indicated days following injection of ³⁵S-methionine into the vitreous humor as described in the Materials and Methods. The accompanying graph presents quantification of radiolabeled NF-L from the above autoradiographs. (C) Quantification of the total decline and the rate of decline/day for GFP in NB2a/d1 cells and radiolabeled NFs in optic axons over the indicated intervals after expression or injection of radiolabel, respectively (n=72 cells, >10/time point, from three separate experiments). (D) Representative images of axonal neurites of Early-On cells at the indicated days after induction of differentiation. Arrows in phase-contrast images denote the diameter of the axonal neurite. The accompanying graphs present distribution of GFP across lateral profiles of bundles from central segment of multiple axonal neurites generated as described for Fig. 3 and in the Materials and Methods, and the rate of decline of GFP within the center, interior and edge, calculated as described in the Materials and Methods. (E) Autoradiographs of Triton-soluble (sol) and -insoluble (insol) fractions of differentiated NB2a/d1 cells at the indicated intervals after pulse-labeling with 35S-methionine as described in the Materials and Methods. The accompanying graphs present the relative intensity of nonphosphorylated (160 kDa) and extensively phosphorylated (200 kDa) at the indicated intervals after radiolabeling. (F) Autoradiographs of radiolabeled NFs from cells subjected to fractionation and sedimentation over sucrose as described in Fig. 4 at the indicated intervals (chase) following pulse-labeling with ³⁵S-methionine as described in the Materials and Methods.