Regulation of the transition from vimentin to neurofilaments during neuronal differentiation

Abstract

Vimentin (Vm) is initially expressed by nearly all neuronal precursors in vivo, and is replaced by neurofilaments (NFs) shortly after the immature neurons become post-mitotic. Both Vm and NFs can be transiently detected within the same neurite, and Vm is essential for neuritogenesis at least in culture. How neurons effect the orderly transition from expression of Vm as their predominant intermediate filament to NFs remains unclear. We examined this phenomenon within growing axonal neurites of NB2a/d1 cells. Transfection of cells with a construct expressing Vm conjugated to green fluorescent protein confirmed that axonal transport machinery for Vm persisted following the developmental decrease in Vm, but that the amount undergoing transport decreased in parallel to the observed developmental increase in NF transport. Immunoprecipitation from pulse-chase radiolabeled cells demonstrated transient co-precipitation of newly synthesized NF-H with Vm, followed by increasing co-precipitation with NF-L. Immunofluorescent and immuno-electron microscopic analyses demonstrated that some NF and Vm subunits were incorporated into the same filamentous profiles, but that Vm was excluded from the longitudinally-oriented "bundle" of closely-apposed NFs that accumulates within developing axons and is known to undergo slower turnover than individual NFs. These data collectively suggest that developing neurons are able to replace their Vm-rich cytoskeleton with one rich in NFs simply by down-regulation of Vm expression and upregulation of NFs, coupled with turnover of existing Vm filaments and Vm-NF heteropolymers.

A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells

“…The identification of interlinked peptides between various domains of NFM and VIME suggests that extensive interaction interfaces exist between these two proteins and potentially implicate VIME involvement in the polymerization or regulation of neurofilament proteins in HEK 293 cells. It has been reported that VIME co-localizes with neurofilament proteins dynamically during neuronal differentiation, and its co-purification with NFH and NFL has been observed (49). Given the close relationship among the three neurofilament subunits NFL, NFM, and NFH, our findings corroborate well with the known function of vimentin in the development of neurofilaments.…”

3′ Untranslated Region in a Light Neurofilament (NF-L) mRNA Triggers Aggregation of NF-L and Mutant Superoxide Dismutase 1 Proteins in Neuronal Cells

“…2 A, B) and in Triton X-100-insoluble fractions ( Fig. 2 L), consistent with a filamentous assembly in Neuro2a cells (Yabe et al, 2003). GFP-tagged NF-L protein expressed from transgenes lacking NF-L 3ЈUTR (pGFP/NFϪ3ЈUTR) does not colocalize with endogenous NF-H but is diffusely distributed throughout the perikarya and neurites at low and high levels of transgene expression ( Fig.…”

Neurofilament cross-bridging competes with kinesin-dependent association of neurofilaments with microtubules

“…Cytoskeletons were prepared from sciatic nerve of adult FVB mice of both genders constitutively expressing green fluorescent protein (GFP)-tagged NF-H (Letournel et al, 2006), NB2a/d1 cells constitutively expressing GFP-tagged NF-M (Yabe et al, 2003;Yabe et al, 2001a) or murine spinal cord (which were labelled with rhodamine after harvesting as described below). Cytoskeletons were prepared by sedimentation of homogenates of the above tissues and cells at 15,000 g for 15 minutes in 50 mM Tris (pH 6.8) containing 5 mM EDTA, 1 mM phenylmethanesulphonylfluoride (PMSF) and 50 μg/ml leupeptin as described (Yabe et al, 2001a).…”