Publication Date

2016-05-11

Document Type

Honors Thesis

Department

Biological Sciences

Keywords

Dynein, Proteins, Yeast, Ensemble, Motor protein, Yeast dynein, Dynein motility, Ensemble function, Chimeric yeast motility

Abstract

Cytoplasmic dynein is a microtubule-associated minus-end directed molecular motor with several functions, including the segregation of chromosomes during mitosis and the transport and distribution of intracellular cargo. A member of the AAA+ ATPase family of proteins, the dynein heavy chain has a complex structure consisting of a ring with 6 AAA+ domains, four of which can bind ATP. A coiled-coil stalk projects from this ring, containing the microtubule binding domain (MTBD) at its end. Two of these heavy chains come together to form the dynein homodimer, which is necessary for motor function and processive motility. The MTBD binds to microtubules, forming the interface between the motor and its track. In vitro experiments aimed at discerning the mechanism of dynein revealed that despite significant structural conservation, yeast and mammalian dynein have different motile properties. The mammalian dynein requires accessory proteins like dynactin and BICD2, to confer processivity, whereas yeast dynein is processive on its own. We hypothesized that the motile differences between dyneins from different species could provide opportunities to reengineer dynein for targeted mechanistic investigations. Specifically, a re-engineered chimeric dynein in which a single subdomain of the yeast dynein is replaced with the equivalent subdomain from mammalian dynein could isolate and elucidate the subdomain’s contributions to the overall motor mechanism. Our initial work focuses on the MTBD as it may directly determine many of the motile behaviors of the motor. To this end, we genetically engineered yeast to express dynein with the endogenous MTBD replaced by the mammalian MTBD. Using TIRF microscopy and single molecule biophysical assays, we characterized the behavior of the motor and found significant differences relating to binding and processivity. We observed that the chimeric motors were not processive as individual motors but were able to walk long distances at speeds faster than native yeast dyneins with the help of a synthetic cargo chassis. Our data showed that altering the microtubule binding domain affected the motility characteristics significantly. We explored the role of ATP in the binding of the chimeric motor and studied the differences between the chimera and the control motor.

Language

English

Comments

77 pages : illustrations (some color). Honors project, Smith College, 2016. Includes bibliographical references (pages 74-77)

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