GPI-anchored human placental alkaline phosphatase has a nonpolarized distribution on the cell surface of mouse cerebellar granule neurons in vitro.
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.
Kollins
K M
KM
Department of Biology, University of Maryland, College Park 20742-4415, USA.
Powell
S K
SK
Rivas
R J
RJ
D016475:3T3 Cells; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001369:Axons / Q000502:physiology / Q000648:ultrastructure; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D016764:Cell Polarity; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology* / Q000502:physiology; D003712:Dendrites / Q000502:physiology / Q000648:ultrastructure; D005260:Female; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D051379:Mice; D016501:Neurites / Q000502:physiology / Q000648:ultrastructure; D009474:Neurons / Q000166:cytology* / Q000502:physiology*; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D017261:Glycosylphosphatidylinositols; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase