Mian Horvath, Annie DeHart, and Lauren Sauers
Biology
Dr. Karen Greif

Effects of Synaptotagmin-1 on Filopodia Formation During Neuronal Development

While neuronal networks are the basis for most functions of many organisms, we have a limited understanding of their mechanisms. Within each neuronal network are thousands of axons, each of which produces many branches. While the lengthening of axons is well characterized, much less is known about the mechanisms behind axonal branching. In Dr. Greif’s lab, we will investigate the potential role a protein called Synaptotagmin-1 (syt1) may play in axonal branching of developing eight-day-old embryonic chick forebrain neurons.

Syt1 is a widely characterized Ca2+-binding protein responsible for vesicular exocytosis at the synapses of neurons. At presynaptic terminals, syt1 works as a calcium sensor within a SNARE complex, which expedites the merging of vesicles to the plasma membrane, and subsequently the release in neurotransmitters, in response to elevated Ca2+ levels. Our working hypothesis is that, in a similar fashion, syt1 merges vesicles to the plasma membrane of a developing neuron’s axon, adding additional membrane to allow for branching. Previous research has provided evidence that a surplus of syt1 leads to increases in the number of axonal branches and filopodia formed during development, while deficits in the protein result in a decrease in such branching. This evidence suggests that the syt1 produced during early development regulates the formation of new branches and filopodia. Filopodia are small extensions along the axon from which branches develop; they are distinguishable by a purely actin cytoskeleton, while branches incorporate microtubules.

To confirm our hypothesis, our experiments will test how overexpression of wild-type syt1 affects axonal branching and filopodia development as compared with a mutant form of sty1, which has impaired Ca2+-binding abilities. We will replicate this experiment with in ovo electroporation to simulate conditions in vitro. After using electroporation to incorporate our plasmids, filopodia formation and syt1 expression will be analyzed using immunocytochemistry techniques and fluorescence microscopy. Depending on the results of the initial experiments, they may be followed by rescue experiments. If the expression of mutated syt1 negatively affects filopodia development, the rescue experiments will investigate whether and how function may be restored to the mutated syt1.