Differential synthesis and cytoskeletal deposition of neurofilament subunits before and during axonal outgrowth in NB2a/d1 cells: Evidence that segregation of phosphorylated subunits within the axonal cytoskeleton involves selective deposition

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Abstract
NB2a/d1 cells constitutively express and extensively phosphorylate neurofilament (NF) triplet proteins. However, only hypophosphorylated NFs are observed within the Triton‐insoluble perikaryal cytoskeletons of undifferentiated and differentiated cells, while phosphorylated NF isoforms accumulate exclusively within the axonal neurites elaborated following treatment with dbcAMP. We examined NF synthesis and distribution of newly synthesized subunits by immunoprecipitation from 35S‐methionine‐radiolabeled undifferentiated and dbcAMP‐treated differentiated cells. Following a 15 min pulse radiolabeling, NF‐H isoforms migrating from approximately 160–200 kDa, NF‐M isoforms migrating from approximately 97 k‐145 Da, and a single 70 kDa NF‐L isoform were readily detectable within Triton‐soluble fractions from both undifferentiated and differentiated cells. During chase analyses in the absence of radiolabel, the entire spectrum of isoforms was present in Triton‐soluble and ‐insoluble fractions from both undifferentiated and differentiated cells. However, differentiated cells displayed a significant increase in radiolabel associated with each subunit and isoform. Normalization of their NF synthesis levels to those of undifferentiated cells revealed that differentiated cells deposited 10‐fold more radiolabeled subunits into the Triton‐insoluble cytoskeleton as compared to undifferentiated cells. Similar levels of radiolabeled subunits were observed throughout the 2 hr period in dbcAMP‐treated cells. By contrast, radiolabeled subunits and isoforms increased in undifferentiated cytoskeletons during the chase period, although final levels remained substantially lower than those observed in cytoskeletons of dbcAMP‐treated cells. These data were considered with respect to potential mechanisms by which the phosphorylated NFs are normally excluded from perikaryal cytoskeletons. The presence of extensively phosphorylated subunits within perikarya indicates the presence of necessary NF kinases. The progressive increase of radiolabeled subunits, including phosphorylated isoforms of NF‐H and NF‐M, within undifferentiated cytoskeletons, argues against selective elimination of phosphorylated NFs from perikaryal cytoskeletons by proteolysis or dephosphorylation as exclusive regulatory mechanisms; if these were the case, overall decreases of radiolabeled subunits (proteolysis), or specific loss of phosphorylated isoforms (dephosphorylation) would have been observed during chase analyses. The increased deposition of NF subunits within cytoskeletons in dbcAMP‐treated cells (which have elaborated axonal neurites), coupled with our previous immunocytochemical observation of segregation of phosphorylated NFs within axonal neurites, suggests that selective assembly is a major control mechanism to maintain normal distribution patterns of phosphorylated NFs. The up‐regulation in NF synthesis observed following dbcAMP treatment is likely to support the increased need for NFs by the developing axonal cytoskeleton.

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