From a Drosophila mutant screen for defects in the formation of the embryonic neuromuscular junction (NMJ), we have learned that mutations in components of the kinetochore complex are needed for the transformation of a growth cone into a correctly shaped synaptic connection. Loss of these proteins also alters the structure of sensory dendrites. In cultured mammalian neurons, kinetochore proteins also appear to guide development: their knockdown by RNAi causes an excess of filipodia like protrusions to form on hippocampal dendrites. This is a completely novel function for the kinetochore, a protein complex previously known only to function at the centromere of chromosomes where it is required in dividing cells to ?catch? and stabilize spindle microtubules and thereby enable the segregation of chromosomes to the daughter cells. The neuronal phenptypes cannot be explained by defects in chromosome mechanics and therefore we hypothesize a postmitotic function for a ?neuro-kinetochore?, a function that is likely to involve the same core property of the centromeric kinetochore: the ability to bind to the plus-ends of microtubules and stabilize them. We propose to test the hypothesis that a complex very much akin to that found at centromeres will function locally in post-mitotic neurons to assist in the transformation of dynamic growth cone microtubules into stable bundles of synaptic microtubules and similarly to stabilize dendritic microtubules and thereby arrest dendrite growth. The proposal makes use of the advantages of both Drosophila and mammalian systems.
Aim 1 of this proposal therefore seeks to characterize in greater depth the nature of the defects at the embryonic fly NMJ and in hippocampal dendrites.
Aim 2 delves into the mechanism underlying the phenotype by asking whether structure function studies support the hypothesis of a kinetochore-like structure that must bind to microtubules.
Aim 3 focuses on the microtubule cytoskeleton and asks whether there are defects in microtubule dynamics and stabilization in the mutants, and how this novel role for kinetochore proteins fits into our knowledge of the processes that transform growth cones into mature endings and determine the morphology of dendrites.

Public Health Relevance

Correct synapse formation is essential for brain development and defects in that process underlie a host of neurodevelopmental defects and loss of synapses is an early hallmark of neurodegenerative disorders such as Alzheimer's. This proposal seeks to understand the role of kinetochore proteins in the formation of synaptic terminals, dendrites, and dendritic spines. These proteins have never previously been implicated in any process other than cell division and the research, therefore, has the potential to expand our understanding of what shapes neuronal connections.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Method to Extend Research in Time (MERIT) Award (R37)
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Neurodifferentiation, Plasticity, and Regeneration Study Section (NDPR)
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Miller, Daniel L
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Boston Children's Hospital
United States
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