Sorting and directed transport of membrane proteins during development of hippocampal neurons in culture

Abstract

Hippocampal neurons in culture develop morphological polarity in a sequential pattern; axons form before dendrites. Molecular differences, particularly those of membrane proteins, underlie the functional polarity of these domains, yet little is known about the temporal relationship between membrane protein polarization and morphological polarization. We took advantage of viral expression systems to determine when during development the polarization of membrane proteins arises. All markers were unpolarized in neurons before axonogenesis. In neurons with a morphologically distinguishable axon, even on the first day in culture, both axonal and dendritic proteins were polarized. The degree of polarization at these early stages was somewhat less than in mature cells and varied from cell to cell. The cellular mechanism responsible for the polarization of the dendritic marker protein transferrin receptor (TfR) in mature cells centers on directed transport to the dendritic domain. To examine the relationship between cell surface polarization and transport, we assessed the selectivity of transport by live cell imaging. TfR-green fluorescent proteincontaining vesicles were already preferentially transported into dendrites at 2 days, the earliest time point we could measure. The selectivity of transport also varied somewhat among cells, and the amount of TfR-green fluorescent protein fluorescence on intracellular structures within the axon correlated with the amount of cell surface expression. This observation implies that selective microtubule-based transport is the primary mechanism that underlies the polarization of TfR on the cell surface. By 5 days in culture, the extent of polarization on the cell surface and the selectivity of transport reached mature levels.

The PDZ-binding domain is essential for the dendritic targeting of 5-HT2A serotonin receptors in cortical pyramidal neurons in vitro

“…By 5 days in culture, pyramidal neurons have clearly established polarity in that axons and dendrites become distinguishable both morphologically and immunocytochemically (Silverman et al, 2001). An axon assumes the shape of a thin and elongated process, whereas a dendrite possesses a thicker and relatively shorter process.…”

A Functional Green Fluorescent Protein-tagged Erythropoietin Receptor Despite Physical Separation of JAK2 Binding Site and Tyrosine Residues

“…Previous studies in other receptor systems have been limited to the analysis of C-terminally GFP-tagged receptors (28,29). However, the EpoR that contains GFP fused to the C terminus (EpoR-GFP4) is expressed at reduced levels and has a decreased capacity to promote the formation of CFU-E colonies.…”

Heterologous expression of the invertebrate FMRFamide–gated sodium channel as a mechanism to selectively activate mammalian neurons

“…Therefore, the functional expression of HaFaNaC in the axons would increase the utility of HaFaNaC as an activator of specific subtypes of neurons in the mammalian CNS. In the future, this might be accomplished by modifying the sequence of HaFaNaC to contain sequences of amino acids known to target the expression of voltage-dependent ion channels to axons of mammalian neurons (Garrido et al, 2001;Silverman et al, 2001;Garrido et al, 2003;Gu et al, 2003;Sampo et al, 2003;Rivera et al, 2005;Arnold, 2006;Chung et al, 2006;Gu et al, 2006;Pan et al, 2006).…”