Date of Award

2018

Document Type

Open Access Dissertation

Department

Biological Sciences

Sub-Department

College of Arts and Sciences

First Advisor

Deanna S. Smith

Second Advisor

Jeff Twiss

Abstract

Cytoplasmic dynein 1 (dynein) is a microtubule motor that plays a role in mitosis, cell migration, and minus-end directed microtubule-based transport. The lissencephaly protein, Lis1, and its binding partner, Ndel1, are critical regulators of cytoplasmic dynein (Niethammer et al., 2000). In humans, haploinsufficiency of Lis1 leads to lissencephaly, a devastating developmental neurological disorder characterized by severe brain malformation, leading to cognitive and motor defects, and progressively worsening seizures (Dobyns et al., 1993). While Lis1 is known to play a role in regulating dynein-dependent functions such as neuronal migration and mitotic spindle orientation during development, the protein is still highly expressed in the adult mouse nervous system, suggesting an important postdevelopmental role in neurons. Indeed, it has been established that Lis1 and Ndel1 regulate dynein-dependent axonal transport in mature neurons (Niethammer et al., 2000; Smith et al., 2000; Pandey and Smith, 2011; Klinman and Holzbaur, 2015). To further elucidate the importance of Lis1 in the adult nervous system, we generated a tamoxifen-inducible Lis1 knockout (KO) mouse to remove the gene post-developmentally. Using an actin promoter to drive expression of a tamoxifen-inducible cre recombinase, homozygous Lis1 KO caused the rapid onset of neurological symptoms, such as hind limb clasping and spinal kyphosis. Administration of tamoxifen resulted in dose-dependent onset of a severe phenotype, which correlated with the extent of recombination observed in the midbrain/hindbrain. Chromatolysis, a sign of axonal dysfunction, was observed in neurons of brainstem cardiorespiratory nuclei of Lis1 KO animals. Additionally, transport defects, axonal swellings, and altered neurofilament distribution were observed in cultured DRG neurons from Lis1 KO mice. Restricting Lis1 KO to cardiomyocytes resulted in no observed symptoms, indicating loss of Lis1 in the heart is not the cause of the Lis1 KO phenotype. Thus my work suggests that Lis1 plays a vital role in autonomic neurons and disrupted axonal transport is the primary cause of the Lis1 KO phenotype.

Dynein has also been shown to interact with adenomatous polyposis coli (APC). This interaction is regulated by insulin signaling, specifically through phosphorylation by glycogen synthase kinase 3 (GSK3) (Gao et al., 2015; Gao et al., 2017). In wild-type (WT) cells, inhibition of GSK3 causes release of dynein from APC, leading to accumulation of dynein at the centrosome. In cells with the multiple intestinal neoplasia (MIN) mutation of the APC gene, GSK3 is unable to regulate the dynein-APC interaction. Using western blot analysis, I found that the insulin-signaling pathway remains functional in MIN cells. Since APC binds to microtubules, which are the tracks for dynein-dependent transport, I looked for changes in overall microtubule morphology and posttranslational modifications. No difference was observed in microtubule morphology, but there was less detyrosinated tubulin in MIN cells. However, this is unlikely to cause the observed phenotype, as dynein motility is decreased on detyrosinated microtubules (McKenney et al., 2016). Finally, using coimmunoprecipitation, I found that dynein interacts with the C-terminus of APC, which is absent in MIN cells, and not the N-terminus. My work indicates that the absence of the C-terminus of APC, and not alterations of the microtubule cytoskeleton or insulin-signaling pathway, is responsible for the inability of GSK3 to regulate dynein in MIN cells.

Rights

© 2018, Timothy Joshua Hines

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