Department Affiliation: Ph.D. Candidate in Cell Biology and Anatomy

Education: Licenciado/Universidad Austral de Chile

Telephone Number: 410-955-2337

Fax Number: 410-955-1013

Email Address: rbustos@jhmi.edu

            Synapsin I is abundant in neural tissues.  Its phosphorylation is thought to regulate synaptic vesicle exocytosis in the pre-synaptic terminal by mediating vesicle tethering to the cytoskeleton.  Using anti-synapsin antibodies, we detected an 85 kDa protein in liver cells and identified it as synapsin I.  Like brain synapsin I, non-neuronal synapsin I is phosphorylated in vitro by protein kinase A and yields identical ³²P-peptide maps after limited proteolysis.  We also detected synapsin I mRNA in liver by northern blot analysis.  These results indicate that synapsin I’s expression is more widespread than previously thought.  Immunofluorescence analysis of several non-neuronal cell lines localizes synapsin I to a vesicular compartment adjacent to trans-elements of the Golgi complex, which is also labeled with antibodies against myosin II; no sub-plasma membrane synapsin I is evident.  We conclude that synapsin I is present in epithelial cells and is associated with a trans-Golgi network-derived compartment; this localization suggests that it plays a role in modulating post-TGN trafficking pathways. (JCellSci 114, 3695-3704 (2001).

3D Reconstruction of NRK cell Golgi stained with antibodies to Synapsin I (green) and TGN38(red)

Trafficking of the Wilson Disease Protein in Hepatocytes

The Wilson Disease Protein (Wilson Protein) is a P-type copper-transporting ATPase expressed predominantly in liver and absent in most other tissues. It transfers cytoplasmic copper into the hepatocyte secretory pathway, where copper is delivered to copper-requiring enzymes.  The Wilson Protein also plays a role in excretion of excess copper into bile. In Wilson disease, the failure to correctly dispose of copper by the hepatocyte leads to copper accumulation in the liver parenchyma, and liver disease.  It has been postulated that high copper levels induce the trafficking of the Wilson Protein from the Golgi region, where it normally resides, to the apical region of the hepatocyte, where biliary copper excretion takes place.  The Wilson Protein might be using a little-described direct trafficking pathway from the TGN to the hepatocyte apical plasma membrane (PM). We investigated the trafficking pathway of the Wilson Protein in hepatocytes by characterizing the membrane compartments where the protein resides under basal and high copper levels.  Using a one-step sucrose gradient Golgi-floatation procedure we detected the Wilson Protein population resident in Golgi membranes, and a larger pool of Wilson Protein in unidentified membranes.  The Golgi-resident pool decreased 5-10 fold in response to copper. Using sucrose gradient fractionation of apical and basolateral PM domains, we detected Wilson Protein present exclusively at the apical PM, in both copper-treated and control animals.  We conclude that the Wilson Protein is present in both the Golgi and apical PM, and traffics out of the Golgi in response to high copper levels, possibly to the apical PM or to an unidentified compartment.   Further isolation and characterization of membrane compartments using organelle immunoisolation will help to elucidate the molecular players involved in the trafficking of Wilson Protein. (MolBiolCell 13:S, 502a).