| Abstract No.: | B-B2041 |
| Country: | Canada |
| | |
| Title: | TRAFFICKING DYNAMICS OF NEUROLIGIN-1 AND -2 IN LIVE NEURONS |
| | |
| Authors/Affiliations: | 2 Kim Gerrow*; 2 Hideto Takahasi; 1 Mark Howarth; 1 Alice Ting; 2 Alaa El-Husseini;
1 Massachusetts Institute of Technology, Cambridge, MA, USA; 2 University of British Columbia, Vancouver, BC, Canada
|
| | |
| Content: | Members of the neuroligin family have been shown to modulate certain aspects of excitatory and inhibitory synapse maturation. However, the mechanisms that govern the differential trafficking and retention of neuroligin-1 to glutamatergic synapses and neuroligin-2 to GABAergic synapses remain unclear. In order to monitor the trafficking and recruitment/retention of neuroligin-1 and -2 to synaptic sites, we have utilized a site-specific biotinylation-based approach that allows for the visualization of surface proteins in live neurons with monovalent streptavidin. We generated tagged versions of these proteins containing a 15 amino acid acceptor peptide (AP), which is recognized by a bacterially generated biotin ligase (BirA). Using monovalent streptavidin conjugated with Alexa568, we were able to detect neuroligin-1 and -2 on the surface of live hippocampal neurons and visualize their dynamics using time-lapse imaging. Control measurements showed that the expression levels of neuroligin-1 and -2 were close to endogenous levels, that presynaptic terminals were not significantly enhanced, and targeting to synapses was similar to that of their endogenous counterparts. Our analysis revealed rapid changes in fluorescence intensity of both neuroligin-1 and -2 clusters present at the cell surface. FRAP (fluorescence recovery after photo beaching) analysis showed similar recovery rates for GFP-tagged neuroligins (representative of the total pool) as for AP-tagged neuroligins (representative of the surface pool). The rate of recovery of neuroligin-1 clusters was significantly faster than neuroligin-2 clusters at sites positive for Shank1b. The recovery of neuroligin-2 clusters was depressed at div 14, compared to div 7, however, neuroligin-1 did not show a significant change. The mobility of neuroligin-1 clusters at particular sites was influenced by the expression of specific scaffolding proteins. Exogenous expression of PSD-95, but not Shank1b, increased the recovery time and reduced the mobile pool of neuroligin-1, suggesting that PSD-95 interaction reduces the extent of neuroligin-1 recycling/turnover. Thus the differential mobility of neuroligin molecules as well as association with particular scaffolding proteins may contribute to their retention at particular synaptic sites. |
| | |
| Back |
|
