Investigating the role of nemitin in neuronal microtubule organization [electronic resource]

Neurons depends on a highly regulated microtubule network to transport essential cargo throughout the cell and establish synaptic connections. Impairments in the microtubule network are a characteristic of several neurodegenerative conditions. However, our understanding of how microtubule networks are generated, maintained, and organized within the neuron is limited. Elucidation of how these processes normally function can help us understand the pathological mechanisms behind neurodegenerative diseases. Here, I identify the protein nemitin as a necessary microtubule organizing protein in both dividing cells and in neurons. Our lab previously described nemitin as a neuron enriched, microtubule interacting protein. To investigate nemitin's neuronal function, we generated a nemitin knockout mouse. Surprisingly, I found that nemitin knockout results in immediate embryo lethality. Consistent with this phenotype, nemitin localizes to microtubule organizing centers (MTOCs) in dividing cells, and loss of nemitin results in a lack of spindle microtubule organization. This phenotypes was verified in cultured Cos7 cells, where loss of nemitin via siRNA leads to microtubule disorganization and eventual cell division failure. Interestingly, I find that nemitin also colocalizes with the microtubule minus end marker Camsap2, a key regulator of acentrosomal microtubule organization in differentiated cells, and that these nemitin-labeled acentrosomal sites are capable of seeding microtubule growth. These new findings led me to speculate that in neurons, nemitin may have a similar role in organizing microtubules. To determine nemitin's functional role in neurons specifically, I used a conditional knockout strategy to knock out neuronal nemitin in developing mice. I found that neuronal knockout of nemitin is perinatal lethal and results in a "small head" phenotype. To explore whether this is due to impairment in the microtubule network, I assessed nemitin's role in neuronal microtubule organization in cultured mouse neurons and found that loss of nemitin -- either with siRNA or conditional knockout -- results in impaired neuronal morphology. To explore potential mechanisms, I investigated nemitin's relationship with Camsap2 and found that nemitin is critical for Camsap2 localization to acentrosomal microtubule minus ends in neurons, and that nemitin interacts with Camsap2 within amino acids 172 to 280. Lastly, I speculate on the underlying mechanism underlying nemitin's function. I propose that nemitin acts an essential adaptor between microtubule nucleation and stabilization and recruits Camsap2 to acentrosomal microtubules. Preliminary evidence is consistent with this model, but future experimentation is needed to understand the underlying mechanisms. By identifying nemitin as a new, key regulator of microtubule generation in cell division and in neurodevelopment, this work has broad significance to research fields from developmental biology to neuroscience.

Advisor: Yang Yanmin
Goodman Miriam Beth advisor. Thesis advisor
Lin Michael Z. advisor. Thesis advisor
Lu Bingwei