Kollins K M, Powell S K, Rivas R J
Department of Biology, University of Maryland, College Park 20742-4415, USA.
J Neurobiol. 1999 Apr;39(1):119-41. doi: 10.1002/(sici)1097-4695(199904)39:1<119::aid-neu10>3.0.co;2-m.
The glycosyl phosphatidylinositol (GPI) lipid anchor, which directs GPI-anchored proteins to the apical cell surface in certain polarized epithelial cell types, has been proposed to act as an axonal protein targeting signal in neurons. However, as several GPI-anchored proteins have been found on both the axonal and somatodendritic cell-surface domains of a variety of neuronal cell types, the role of the GPI anchor in protein localization to the axon remains unclear. To begin to address the role of the GPI anchor in neuronal protein localization, we used a replication-incompetent retroviral vector to express a model GPI-anchored protein, human placental alkaline phosphatase (hPLAP), in early postnatal mouse cerebellar granule neurons developing in vitro. Purified granule neurons were cultured in large mitotically active cellular reaggregates to allow retroviral infection of undifferentiated, proliferating granule neuron precursors. To more easily visualize hPLAP localization during the sequence of differentiation of single postmitotic granule neurons, reaggregates were dissociated following infection, plated as high-density monolayers, and maintained for 1-9 days under serum-free culture conditions. As we previously demonstrated for uninfected granule neurons developing in monolayer culture, hPLAP-expressing granule neurons likewise developed in vitro through a series of discrete temporal stages highly similar to those observed in situ. hPLAP-expressing granule neurons first extended either a single neurite or two axonal processes, and subsequently attained a mature, well-polarized morphology consisting of multiple short dendrites and one or two axons that extended up to 3 mm across the culture substratum. hPLAP was expressed uniformly on the entire cell surface at each stage of granule neuron differentiation. Thus, it appears that the GPI anchor is not sufficient to confer axonal localization to an exogenous GPI-anchored protein expressed in a well-polarized primary neuronal cell type in vitro; other signals, such as those present in the extracellular domain of these proteins, may be necessary for the polarized targeting or retention of axon-specific GPI-anchored proteins.
糖基磷脂酰肌醇（GPI）脂质锚定物可将GPI锚定蛋白导向某些极化上皮细胞类型的顶端细胞表面，有人提出它可作为神经元轴突蛋白靶向信号。然而，由于在多种神经元细胞类型的轴突和体树突细胞表面结构域均发现了几种GPI锚定蛋白，GPI锚定物在蛋白定位于轴突中的作用仍不清楚。为了开始探讨GPI锚定物在神经元蛋白定位中的作用，我们使用无复制能力的逆转录病毒载体，在体外发育的新生小鼠小脑颗粒神经元中表达一种模型GPI锚定蛋白，即人胎盘碱性磷酸酶（hPLAP）。纯化的颗粒神经元在有丝分裂活跃的大型细胞团聚体中培养，以使未分化、增殖的颗粒神经元前体受到逆转录病毒感染。为了在单个有丝分裂后颗粒神经元的分化过程中更轻松地观察hPLAP的定位，感染后将团聚体解离，以高密度单层铺板，并在无血清培养条件下维持1 - 9天。正如我们之前在单层培养中发育的未感染颗粒神经元所证明的那样，表达hPLAP的颗粒神经元同样在体外经历了一系列与原位观察到的高度相似的离散时间阶段的发育。表达hPLAP的颗粒神经元首先伸出单个神经突或两个轴突样突起，随后形成成熟的、高度极化的形态，由多个短树突和一两个轴突组成，轴突在培养底物上延伸可达3毫米。在颗粒神经元分化的每个阶段，hPLAP均在整个细胞表面均匀表达。因此，似乎GPI锚定物不足以使体外在高度极化的原代神经元细胞类型中表达的外源性GPI锚定蛋白定位于轴突；其他信号，例如这些蛋白细胞外结构域中存在的信号，可能是轴突特异性GPI锚定蛋白极化靶向或保留所必需的。
