LSI Seminar Series: Gary Brouhard, Ph.D., McGill University
Doublecortin regulates neuronal migration by editing the tubulin code
Doublecortin (DCX) is a neuronal microtubule-associated protein (MAP) that binds directly to microtubules via two Doublecortin (DC) domains. The DC domains sense the nucleotide state, longitudinal curvature, and protofilament number of the microtubule lattice, indicating a role in the regulation of microtubule structure in neurons. Mutations in DCX cause lissencephaly and subcortical band heterotopia (also known as double-cortex syndrome) due to impaired neuronal migration. To better understand the role of DCX in neuronal migration, we developed a model system based on induced pluripotent stem cells (iPSCs). We used CRISPR/Cas9 to knock out the Dcx gene in iPSCs and differentiated the cells into cortical neurons. Compared to control neurons, the DCX-KO neurons showed reduced velocities of nuclear movements. The reduced velocities correlated with an increase in the number of neurites early in the neuronal development process, consistent with a neuronal migration phenotype and previous findings in a DCX-KO mouse model.
Neurite branching is regulated by a host of MAPs and other factors, as well as by microtubule polymerization dynamics; however, microtubule dynamics were unchanged in DCX-KO neurons, with similar growth rates, lifetimes, and numbers. Rather, we observe a significant reduction in tubulin polyglutamylation in DCX-KO neurons. Polyglutamylation is usually abundant in neurons and regulates microtubule severing enzymes and intracellular trafficking by molecular motors. Consistently, we observe that lysosomes in DCX-KO neurons show a reduction of their processivity. We propose that the reduction of polyglutamylation leads to increased neurite branching and thus reduced neuronal migration. Our results indicate an unexpected role for DCX in the homeostasis of the tubulin code.
Gary Brouhard is an alumnus of the University of Michigan, where he received a bachelor's degree in chemical engineering and philosophy, as well as a master's degree and Ph.D. in biomedical engineering. He then went onto a postdoctoral fellowship at the Max Planck Institute of Molecular Cell Biology and Genetics before joining the faculty of McGill University.
Brouhard's lab examines the biophysical mechanisms by which cells engineer a wide variety of large-scale structures from proteins — in other words, the molecular basis of morphology. The subject of his current research is the microtubule cytoskeleton. Microtubules are long, slender polymers of the protein tubulin, from which the cell constructs the cytoskeleton, the mitotic spindle, axonemes and neuronal processes. These structures are not static. Rather, microtubules are broken down and rebuilt as the cell grows and changes shape, undergoes differentiation, and progresses through the cell cycle. The aim of his research program is to understand the means by which cells accomplish these remarkable rearrangements of their microtubules.