Memory and learning in animals are achieved by neurotransmission at neuron-neuron or neuro-muscular junctions called synapses. These synapses are formed at the end of long cable-like extensions of neurons, called axons. The axonal part of the junction is called the pre-synaptic terminal. It hosts small (~50 nm) vesicles containing neurotransmitters. Some of the vesicles are at the active site close to the wall of the synapse, ready to release their contents. Others are clustered within the synapse as a reserve pool. When the neuron fires, an electric signal, known as the action potential, arrives at the synapse. Some of the vesicles at the active site release their neurotransmitters and stimulate the post synaptic terminal. Thus, a signal is transmitted. New vesicle from the reserve pool join the active site. Clearly, to achieve neurotransmission, neurons must cluster reserve-pool vesicles at the synapse against diffusion, and yet provide them directed mobility to replace the released ones at the active site. In spite of decades of research, the mechanism of this duality remains elusive. This project attempts to resolve this paradox by linking a mechanical property of the axon, namely its contractility or mechanical tension, with vesicle clustering, dynamics and release. Prior work of the PI on embryonic Drosophila (fruit fly) revealed that vesicle clustering at the neuromuscular presynaptic terminal depends on mechanical tension of the axons. The findings of this research will be disseminated to the broader audience by developing a short drama with high school students, in collaboration with a drama teacher, to represent neurotransmission -- with characters mimicking vesicles, ions, actin and synapsin-I. The research will also be integrated with education through involvement of undergraduate students from underrepresented groups in research, exhibition modules at the local Children's Museum, and teaching biophysics to high school teachers.
The project is based on the hypothesis that axons of motor neurons forming neuro-muscular junctions in embryonic flies have an contractile acto-myosin network along their entire length, including the synapse. This force continuity results in a stable F-actin architecture at the synapse. Ion sensitive adhesion proteins, e,g., synapsin I, attach (glue) vesicles to synaptic F-actin, thus clustering and immobilizing them against diffusion. During an action potential, synaptic Calcium ion concentration increases, and the adhesion proteins release the vesicles. They are then moved by motor proteins to and away from the active sites along the F-actin fibers. Thus, synaptic F-actin architecture serves as a scaffold for vesicles to cluster, as well as a double-lane highway for their directed mobility. This hypothesis will be tested by studying the neuro-muscular junction of embryonic Drosophila in three steps. First, to test whether acto-myosin machinery is involved in contractile force generation along the entire length of the axon including synapse. Second, to test whether there exists an F-actin architecture at the synapse stabilized by the axonal contractile force, and whether it serves as a scaffold for the vesicles to adhere, as well as a highway for their transport. Finally, to test whether axonal force modulates neurotransmission. Novel nano-mechanical force sensors, micro-fluidics, high resolution microscopy (Stochastic Optical Reconstruction Microscopy, STORM), nano-probe and cyclic voltammetry will be used to test the hypothesis.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
