Date of Degree

2011

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Ray H. Gavin

Committee Members

Shaneen M. Singh

Theodore R. Muth

Chang-Hui Shen

Selwyn A. Williams

Christina King-Smith

Subject Categories

Biology

Abstract

MyTH4 and FERM are conserved tail domains in myosin classes VII, X, XII, XIV, XV and in MyoG. Myo1, a class XIV myosin in Tetrahymena thermophila, contains MyTH4 and FERM. Previous studies have shown that Myo1 localizes to phagosomes, the cytoskeleton, and the macronucleus, and that phagosome trafficking and division of the macronucleus are affected in a MYO1 knockout. To investigate the roles for MyTH4 and FERM in the function of Myo1, GFP-tagged MyTH4, FERM, and truncated FERM were separately overexpressed in Tetrahymena. Actin antibody coprecipitated tubulin, GFP-MyTH4, and GFP-FERM. GFP-MyTH4 and GFP-FERM cosedimented with either exogenous microtubules or exogenous F-actin. GFP-MyTH4 localized to phagosomes and colocalized with antitubulin to intranuclear microtubules. Overexpression of GFP-MyTH4 inhibited the organization of the parallel array of intranuclear microtubules that form prior to division of the macronucleus. Cells that failed to form the parallel array of microtubules did not advance in nuclear division. Overexpression of GFP-MyTH4 did not affect phagosome recycling. Overexpressed GFP-FERM localized to phagosomes, cytoskeleton, and intranuclear puncta and did not affect division of the macronucleus. Overexpression of truncated GFP-FERM did not localize to the cytoskeleton or nucleus and led to the accumulation of phagosomes at the membrane recycling site in the posterior of the cell. It is unlikely that the overexpression phenotypes are nonspecific effects of GFP. Localization of GFP-fusions is consistent with the localization of full-length Myo1, and overexpression phenotypes mimic the knockout phenotype. Furthermore, GFP-MyTH4 from Myo9, another Tetrahymena myosin, did not localize in Tetrahymena thermophila. We conclude that MyTH4 and FERM have overlapping roles as indicated by the interaction with actin and tubulin. However, MyTH4 and FERM appear to have distinct roles in the function of Myo1. MyTH4 affects the organization of microtubules involved in macronuclear division, whereas FERM affects recycling of phagosomes and is required for localization to the cytoskeleton.

Comments

Digital reproduction from the UMI microform.

Included in

Biology Commons

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