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.
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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NB2a/d1 细胞轴突生长之前和期间神经丝亚基的差异合成和细胞骨架沉积：轴突细胞骨架内磷酸化亚基分离涉及选择性沉积的证据。
DOI：
10.1002/jnr.490400211
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Shea,TB
中科院分区：
文献类型：
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作者：
Shea,TB
NB 2a/d1细胞组成性表达并广泛磷酸化神经丝（NF）三联体蛋白。然而，仅在未分化和分化细胞的Triton不溶的核周细胞骨架内观察到低磷酸化的NF，而磷酸化的NF亚型仅在用dbcAMP处理后产生的轴突神经突内积累。我们从35 S-蛋氨酸-放射性标记的未分化和dbcAMP处理的分化细胞中通过免疫沉淀检测了NF合成和新合成亚基的分布。在15分钟脉冲放射性标记后，在未分化和分化细胞的Triton可溶性组分中可容易地检测到从约160-200 kDa迁移的NF-H亚型、从约97 k-145 Da迁移的NF-M亚型和单个70 kDa NF-L亚型。在不存在放射性标记的情况下进行追踪分析期间，未分化和分化细胞的Triton可溶性和不溶性组分中均存在整个亚型谱。然而，分化的细胞显示与每个亚基和亚型相关的放射性标记显着增加。将它们的NF合成水平标准化为未分化细胞的NF合成水平，发现与未分化细胞相比，分化细胞将10倍多的放射性标记亚基沉积到Triton不溶性细胞骨架中。在dbcAMP处理的细胞中，在整个2小时期间观察到相似水平的放射性标记亚基。相比之下，放射性标记的亚基和亚型增加未分化的细胞骨架在追逐期间，虽然最终水平仍然大大低于观察到的dbcAMP处理cells.These数据被认为是关于潜在的机制，磷酸化的NF通常被排除在核周细胞骨架。胞体内广泛磷酸化亚基的存在表明存在必要的NF激酶。在未分化的细胞骨架中，放射性标记的亚基，包括NF-H和NF-M的磷酸化亚型的逐渐增加，反对通过蛋白水解或去磷酸化作为唯一的调节机制从核周细胞骨架中选择性消除磷酸化NF;如果是这种情况，放射性标记亚基的总体减少在chase分析期间将观察到磷酸化同种型的特异性损失（蛋白水解）或特异性损失（去磷酸化）。在dbcAMP处理的细胞（其具有精细的轴突突起）中，细胞骨架内NF亚基的沉积增加，加上我们先前对轴突突起内磷酸化NF分离的免疫细胞化学观察，表明选择性组装是维持磷酸化NF正常分布模式的主要控制机制。dbcAMP处理后观察到的NF合成的上调可能支持发育中的轴突细胞骨架对NF的需求增加。© 1995 Wiley利斯公司
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 from35S‐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. © 1995 Wiley‐Liss, Inc.