| In recent years, it has become apparent that neurotransmitter receptor transport into and out of postsynaptic membranes may be of central importance for synaptic plasticity, which is thought to be the sub-cellular basis for memory formation. This has led to an explosion of research on neurotransmitter receptor transport in dendrites. However, the larger area of protein transport in dendrites remains poorly understood. In order to provide a larger context for understanding neurotransmitter receptor transport, I studied the transport and localization of a model trafficking receptor, TGN38, within dendrites of hippocampal neurons. With immunofluorescence imaging, GFP-TGN38 was found to move rapidly between the dendritic plasma membrane and intracellular membranes, including those of dendritic spines, suggesting that TGN38 may be capable of rapidly altering the protein content of the post-synaptic plasma membrane. These results are consistent with the idea that TGN38 has a role in synaptic plasticity and strengthened the rationale for studying the function of TGN38, which is the subject of chapters 3 and 4. The functional studies focus on two hypotheses for TGN38 function: (1) TGN38 maintains the normal morphology of the trans-Golgi network (TGN). (2) TGN38 is a cargo receptor (i.e., it transports proteins from one subcellular compartment to another). Despite depleting NRK cells of TGN38 by 90–95% with RNA-interference, no gross changes were observed in TGN morphology with fluorescence microscopy and 3-dimensional reconstructions. Thus, normal levels of TGN38 are not required for the maintenance of TGN morphology. Based on these observations, I have suggested an alternative interpretation of data previously interpreted by others as supporting a role for TGN38 in the maintenance of TGN morphology. To identify potential cargo proteins of TGN38 in NRK cells, a functional proteomics screen was developed. One protein, identified with mass spectrometry as glutathione s-transferase pi, was found to be in greater abundance on the surface of TGN38-depleted cells indicating that it may be a cargo of TGN38. However, no gross changes in the surface protein content of the TGN38-depleted cells were found. Altogether, these results are consistent with TGN38 functioning as a cargo receptor and suggest that it may have a role in synaptic plasticity. |
