Neurofilaments are space-filling cytoskeletal polymers that are transported into axons where they accumulate during development to expand axon caliber. We previously described novel severing and end-to-end annealing mechanisms in neurons that alter neurofilament length. To explore the functional significance of neurofilament length, we developed a long-term multi-field time-lapse method to track the movement of fluorescently tagged neurofilaments in axons of cultured neurons for up to 30 minutes. All filaments moved rapidly, but long filaments paused and reversed more often, resulting in little net movement, whereas short filaments moved persistently for long distances, pausing and reversing less often. Long filaments severed more frequently, generating shorter filaments, and short filaments annealed more frequently, generating longer filaments. Thus, neurofilament length is regulated by a dynamic cycle of severing and annealing and this influences neurofilament transport. Site-directed mutagenesis to mimic phosphorylation at four known phosphorylation sites in the head domain of neurofilament protein L generated shorter neurofilaments that moved more frequently. A non-phosphorylatable mutant had the opposite effect. Treatment of cultured neurons with activators of protein kinase A, which phosphorylates three of these sites, increased neurofilament severing. This effect was blocked by the non-phosphorylatable mutant. We propose that focal destabilization of intermediate filaments by N-terminal phosphorylation of their constituent polypeptides at specific locations along their length may be a general enzymatic mechanism for severing this class of cytoskeletal polymers. Our data suggest a novel mechanism for the control of neurofilament transport and accumulation in axons based on regulation of neurofilament polymer length.

中文翻译：

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通过聚合物切断和退火的动态循环调节神经丝长度和运输

神经丝是填充空间的细胞骨架聚合物，它们被运输到轴突中，在发育过程中积累以扩大轴突口径。我们之前描述了改变神经丝长度的神经元中新的切断和端到端退火机制。为了探索神经丝长度的功能意义，我们开发了一种长期多场延时方法来跟踪荧光标记神经丝在培养神经元轴突中的运动长达 30 分钟。所有细丝都快速移动，但长丝更频繁地暂停和反转，导致很少的净运动，而短丝则持续长距离移动，暂停和反转的频率较低。长丝更频繁地切断，产生更短的丝，而短丝更频繁地退火，产生更长的丝。因此，神经丝长度受切断和退火的动态循环调节，这会影响神经丝运输。定点诱变模拟神经丝蛋白 L 头部结构域中四个已知磷酸化位点的磷酸化，产生更短的移动更频繁的神经丝。不可磷酸化的突变体具有相反的效果。用蛋白激酶 A 激活剂处理培养的神经元，使这些位点中的三个磷酸化，增加了神经丝的切断。这种作用被不可磷酸化的突变体阻断。我们提出，通过在沿其长度的特定位置对其组成多肽的 N 端磷酸化，中间丝的局灶性不稳定可能是用于切断此类细胞骨架聚合物的一般酶促机制。