We examined the steady-state distribution and axonal transport of neurofilament (NF) subunits within growing axonal neurites of NB2a/d1 cells. Ultrastructural analyses demonstrated a longitudinally oriented "bundle" of closely apposed NFs that was surrounded by more widely spaced individual NFs. NF bundles were recovered during fractionation and could be isolated from individual NFs by sedimentation through sucrose. Immunoreactivity toward the restrictive C-terminal phospho-dependent antibody RT97 was significantly more prominent on bundled than on individual NFs. Microinjected biotinylated NF subunits, GFP-tagged NF subunits expressed after transfection, and radiolabeled endogenous subunits all associated with individual NFs before they associated with bundled NFs. Biotinylated and GFP-tagged NF subunits did not accumulate uniformly along bundled NFs; they initially appeared within the proximal portion of the NF bundle and only subsequently were observed along the entire length of bundled NFs. These findings demonstrate that axonal NFs are not homogeneous but, rather, consist of distinct populations. One of these is characterized by less extensive C-terminal phosphorylation and a relative lack of NF-NF interactions. The other is characterized by more extensive C-terminal NF phosphorylation and increased NF-NF interactions and either undergoes markedly slower axonal transport or does not transport and undergoes turnover via subunit and/or filament exchange with individual NFs. Inhibition of phosphatase activities increased NF-NF interactions within living cells. These findings collectively suggest that C-terminal phosphorylation and NF-NF interactions are responsible for slowing NF axonal transport.

We examined the form(s) in which NF subunits undergo axonal transport. Pulse‐chase radiolabeling analyses with 35S‐methioinine revealed that newly synthesized Triton‐soluble NF subunits accumulated within axonal neurites elaborated by NB2a/d1 neuroblastoma prior to the accumulation of Triton‐insoluble subunits. Gel chromatographic, immunological, ultrastructural, and autoradiographic analyses of Triton‐soluble axonal fractions demonstrated that radiolabeled, Triton‐soluble subunits were associated with NFs. Triton‐soluble, radiolabeled axonal NF subunits were also detected within retinal ganglion cell axons following intravitreal injection of 35S‐methioinine. Microinjected biotinylated subunits were prominent within axonal neurites of NB2a/d1 cells and cultured dorsal root ganglion neurons substantially before they were retained following Triton‐extraction. Prevention of biotinylated subunit, but not dextran tracer, translocation into neurites by nocodazole confirmed that microinjected subunits did not enter axons merely due to diffusion or injection‐based pressure. Immuno‐EM confirmed the association of biotin label with axonal NFs. These findings point towards multiple populations of NF subunits within axons and leave open the possibility that axonal NFs may be more dynamic than previously considered. Cell Motil. Cytoskeleton 40:44–58, 1998. © 1998 Wiley‐Liss, Inc.

Axonal maturation in situ is accompanied by the transition of neurofilaments (NFs) comprised of only NF-M and NF-L to those also containing NF-H. Since NF-H participates in interactions of NFs with each other and with other cytoskeletal constituents, its appearance represents a critical event in the stabilization of axons that accompanies their maturation. Whether this transition is effected by replacement of "doublet" NFs with "triplet" NFs, or by incorporation of NF-H into existing doublet NFs is unclear. To address this issue, we examined the distribution of NF subunit immunoreactivity within axonal cytoskeletons of differentiated NB2a/d1 cell and DRG neurons between days 3-7 of outgrowth. Endogenous immunoreactivity either declined in a proximal-distal gradient or was relatively uniform along axons. This distribution was paralleled by microinjected biotinylated NF-L. By contrast, biotinylated NF-H displayed a bipolar distribution, with immunoreactivity concentrated within the proximal- and distal-most axonal regions. Proximal biotinylated NF-H accumulation paralleled that of endogenous NF immunoreactivity; however, distal-most biotinylated NF-H accumulation dramatically exceeded that of endogenous NFs and microinjected NF-L. This phenomenon was not due to co-polymerization of biotin-H with vimentin or alpha-internexin. This phenomenon declined with continued time in culture. These data suggest that NF-H can incorporate into existing cytoskeletal structures, and therefore suggest that this mechanism accounts for at least a portion of the accumulation of triplet NFs during axonal maturation. Selective NF-H accumulation into existing cytoskeletal structures within the distal-most region may provide de novo cytoskeletal stability for continued axon extension and/or stabilization.

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.

Fertilization of sea urchin eggs results in the rapid polymerization of actin filaments and subsequent formation of a brush border-like cortical cytoskeleton. A 110 × 10(3) Mr (110K) actin binding protein has been purified from extracts of unfertilized Strongylocentrotus purpuratus eggs. Analysis of polymerization kinetics using fluorescence and viscometry assays demonstrated that 110K accelerated the nucleation phase of actin assembly only in the presence of elevated Ca2+. The Ca(2+)-mediated effects were correlated with a decrease in sedimentable polymer and a decrease in average filament length. Addition of Ca2+ to solutions of 110K and F-actin, polymerized in the presence of EGTA, resulted in a precipitous drop in viscosity and the decreased viscosity was fully reversible upon chelation of Ca2+. The Ca2+ threshold for 110K activation was in the 10(−6) to 10(−7) M range. Nucleated assembly experiments using Limulus sperm acrosomal processes demonstrated that egg 110K capped the barbed ends of actin filaments. In the absence of Ca2+, 110K organized actin filaments into bundles at pH values less than 7.4. Anti-egg 110K antibody crossreacted with chicken intestinal epithelial cell villin and anti-porcine villin headpiece monoclonal antibody crossreacted with 110K. Further, 110K possesses an approximately 10 × 10(3) Mr terminal polypeptide segment that is immunologically related to villin headpiece. These studies demonstrate that sea urchin egg 110K is functionally, immunologically and structurally related to villin, an actin binding protein expressed in specific epithelial tissues in vertebrates. Consequently, this finding provides insight into the potential mechanisms that might determine the genesis of the cortical brush border cytoarchitecture in sea urchin eggs and further sheds light on the evolution of the villin protein family.