Clathrin may not be necessary for synaptic vesicle endocytosis

Last Friday, as part of the biology department’s Spring 2010 Cramer Seminar Series at Dartmouth College, Erik Jorgensen presented his research on clathrin-mediated endocytosis in neuronal synapses.  Jorgensen, an HHMI investigator and biology professor at the University of Utah, studies and performs genetic analysis on the nematode C. elegans.

In neurons, the opening of a voltage-gated calcium channels allows Ca⁺² ions to flow into the axon and leads to the fusion of synaptic vesicles with the plasma membrane.  After neurotransmitters flow outside the cell, the vesicle is recycled or endocytosed at the synapse.  Jorgensen has researched this process and tried to disprove the theory that clathrin is necessary to form a vesicle that travels in the retrograde direction.

Jorgensen presented three important experiments that led to his project.  The “freeze slammer” experiment-done by John Heuser and Tom Reese in the late 1970s-involved attaching a sample of frog neurons to a rod, activating a solonoid to drop the rod, and, right as it was about to touch a copper plate cooled with liquid helium, a stimulating wire would activate the neurons.  This millisecond time frame was frozen, and they observed an electron-dense structure around the synaptic vesicles.

The second experiment determined the proteins in clathrin-coated vesicles with a Western blot, and the third concluded that liposomes could form vesicles in the absence of clathrin.  Jorgensen also described a model proposed by Bruno Ceccarelli called the “kiss-and-run” model, which holds that after releasing neurotransmitters, a synaptic vesicle stays completely intact and recoils back rather than fusing with the membrane.

His study-which assumed the exocytosis-endocytosis model is correct-involved two main areas: the necessity of clathrin and the adaptor protein complex that helps form a vesicle.  The popular model for endocytic vesicle formation is that adaptor proteins on the plasma membrane recruit clathrin coat proteins, the vesicle pinches off with the help of dynamin, and the soccer ball cage-like clathrin array depolymerizes shortly thereafter.

However, Jorgensen tried a temperature-sensitive mutation, and the heat was too extreme for the clathrin triskelion to form.  In the absence of functional clathrin, vesicles still could form, but they had a smaller diameter than those at a more optimal temperature.  He said that clathrin may be involved in the curvature of the vesicle, rather than in its formation; clathrin coats still do form, but they are not necessary to form the vesicle.

Jorgensen also looked at adaptor proteins involved in endocytic vesicle formation, which include the AP2 complex, AP180, and stonin.  In one study, he mutated AP180, which caused larger vesicles to form. By tagging the SNARE protein synaptobrevin with green fluorescent protein (GFP), he determined that AP180 is essential for vesicle formation.

Jorgensen has several goals for the future of synaptic vesicle research.  One of these is to develop  fluorescence electron microscopy in order to tag clathrin with fluorescent proteins, figure out where it is in the cell, and follow its path.  He described a new way of viewing cells called photo-activated localization microscopy (PALM).  In addition, he hopes to determine the null phenotype by targeting the gene that codes for clathrin.