Dendritic membrane traffic
The dendritic tree comprises one of the most complex subdomain structures of the plasma membrane and very little is known about trafficking pathways involved in the formation and maintainance of this structure. We are focussing on the transport of neurotransmitter receptors as one of the most prominent protein classes in dendrites. These receptors are composed of several subunits and vary in subunit compositon throughout the dendritic tree. One possibility is that their differences in structure result in differences in synaptic plasticity. To analyze how membrane traffic is linked to synaptic plasticity, we are studying the selective transport of GFP-tagged subunits of the GABA A receptor to postsynapes by multi-color imaging (Fig. 1) in combination with biochemical approaches. One interesting observation is that the differential trafficking of selected subunits depends on the developmental stage of hippocampal neurons. We are currently addressing the correlative transport dynamics, the site of integration into the plasma membrane and the endocytosis rate of the respective receptor subunits during neuronal development.
|Figure 1. Expression of GABA-A receptor subunit a1 fused to EYFP in primary hippocampal neurons cultured for 4 days (A) or 10 days (B). Arrowheads, receptor clusters; asterisk, soma; bar. 10µm.|
Biogenesis and transport of secretory granules
Secretory granules (SGs) store neuropeptides and release them upon stimulus-dependent, regulated exocytosis. SGs are formed as short-lived vesicular intermediates at the trans-Golgi network. These intermediates undergo a complex and poorly understood maturation process resulting in mature SGs. A major objective of our laboratory has been to study the sorting of neuropeptides into SGs. This led to the identification of a specific sorting signal. To gain insight into the transport dynamics of newly formed SGs, we express GFP fusion proteins to specifically label SGs in living cells. This facilitates the study of budding, transport and docking of SGs in real time. One of our surprising findings is that SGs from neuro-endocrine PC12 cells possess a dual transport system: after microtuble-dependent delivery to the cell periphery, SGs exhibit a myosin-dependent transport leading to their restriction and even dispersal in the F-actin rich cortex (Fig. 2, ref 3). Using live cell imaging in combination with biochemical approaches, the recruitment and regulation of the respective motor proteins is under investigation. One emphasis is to analyze whether myosins play a role in in the maturation of SGs. Furthermore we are interested in analyzing the mechanism of axon-specific targeting of SGs in polarized neurons.
|Figure 2. Scheme illustrating the role of myosin Va in the transport of secretory granules. TGN, trans-Golgi network; MT, microtubule; PM, plasmamembrane.|
Tunneling nanotubes, a new route for cell-to-cell communication
Recently, we have discovered that cells are connected by thin membrane tubes (Fig. 3, ref. 4).These tubes, referred to as tunneling nanotubes (TNTs), mediate membrane continuity between connected cells and lead to complex cellular networks. TNTs were shown to accomplish the selective uni-directional transfer of endosome-like organelles as well as, on a limited scale, of membrane components and cytoplasmic molecules. The data suggest a new biological principle of cell-to-cell communication based on membrane continuity and intercellular transfer of cellular components like organelles and signaling molecules. It now emerges that TNT-based communication is a widespread mechanism throughout the animal kingdom which includes cellular differentiation, proliferation and development of diseases (ref 5). Based on these findings, our research is focusing on the characterization of the structure and function of TNTs in various cell systems. This includes primary cultures of astrocytes and neurons.
|Figure 3. Scanning electron micrograph showing a thin membrane tube (referred to as tunneling nanotube, TNT) connecting two cultured PC12 cells. Bar, 10 mm.|