Characterizing the Effects of Post-Translational Modifications on the Kinesin Motor Protein Kif18A During Cell Division
Conference Year
January 2019
Abstract
Eukaryotic cells go through a phase in the cell cycle called mitosis in which the duplicated genetic material of the cell, in the form of chromosomes, is separated equally into two daughter cells. Within mitotic cells, there are motor proteins of the kinesin family that walk along the microtubules of the mitotic spindle and are necessary to faithfully segregate chromosomes and avoid aneuploidy, a characteristic of cancer. A mitotic motor protein in the kinesin-8 family, called Kif18A, localizes to the ends of kinetochore-fibers, bundles of microtubules that attach chromosomes via the kinetochore protein complex to the mitotic spindle. Kif18A accumulates at the plus ends of kinetochore-fibers and is essential for the highly conserved process of chromosomal alignment during metaphase. However, it is not understood how Kif18A’s activity is regulated for this function. Proteomic analyses have identified post-translational modifications (PTMs) of Kif18A; however, nothing is known about the effect of PTMs on Kif18A activity. I have characterized the phosphorylation of Kif18A at S357, a serine residue in an important mechanical element of the protein called the neck linker. I hypothesized that a change in charge near the neck linker will disrupt Kif18A’s localization and function during mitosis by altering ATP-dependent conformational changes within the protein that are required for its movement along microtubules. We created a GFP-tagged phosphomimetic construct of Kif18A (S357D) and a non-phosphorylatable version at the same site (S357A). I found that there was a slight localization defect for the phosphomimetic construct and a small chromosomal alignment defect as measured from immunofluorescence in fixed cells. I also started to track mCherry-tagged kinetochore dynamics in live cells expressing these constructs and found that the alignment defect was even more exaggerated. I created new mCherry-tagged constructs so I could repeat these experiments with a finer kinetochore tracking ability. Future directions of my project include repeating the kinetochore tracking with GFP-tagged kinetochores expressing these mCherry-Kif18A constructs for finer tracking ability and determine the effects of the phosphomimetic on Kif18A processivity using in vitro single molecule TIRF microscopy. These findings demonstrate a role for PTM regulation on Kif18A and lay the groundwork for studying PTMs in kinesin motors.
Primary Faculty Mentor Name
Jason Stumpff
Secondary Mentor Name
Heidi Malaby
Faculty/Staff Collaborators
Heidi Malaby (Post-Doctorate)
Status
Undergraduate
Student College
College of Arts and Sciences
Program/Major
Biochemistry
Primary Research Category
Biological Sciences
Characterizing the Effects of Post-Translational Modifications on the Kinesin Motor Protein Kif18A During Cell Division
Eukaryotic cells go through a phase in the cell cycle called mitosis in which the duplicated genetic material of the cell, in the form of chromosomes, is separated equally into two daughter cells. Within mitotic cells, there are motor proteins of the kinesin family that walk along the microtubules of the mitotic spindle and are necessary to faithfully segregate chromosomes and avoid aneuploidy, a characteristic of cancer. A mitotic motor protein in the kinesin-8 family, called Kif18A, localizes to the ends of kinetochore-fibers, bundles of microtubules that attach chromosomes via the kinetochore protein complex to the mitotic spindle. Kif18A accumulates at the plus ends of kinetochore-fibers and is essential for the highly conserved process of chromosomal alignment during metaphase. However, it is not understood how Kif18A’s activity is regulated for this function. Proteomic analyses have identified post-translational modifications (PTMs) of Kif18A; however, nothing is known about the effect of PTMs on Kif18A activity. I have characterized the phosphorylation of Kif18A at S357, a serine residue in an important mechanical element of the protein called the neck linker. I hypothesized that a change in charge near the neck linker will disrupt Kif18A’s localization and function during mitosis by altering ATP-dependent conformational changes within the protein that are required for its movement along microtubules. We created a GFP-tagged phosphomimetic construct of Kif18A (S357D) and a non-phosphorylatable version at the same site (S357A). I found that there was a slight localization defect for the phosphomimetic construct and a small chromosomal alignment defect as measured from immunofluorescence in fixed cells. I also started to track mCherry-tagged kinetochore dynamics in live cells expressing these constructs and found that the alignment defect was even more exaggerated. I created new mCherry-tagged constructs so I could repeat these experiments with a finer kinetochore tracking ability. Future directions of my project include repeating the kinetochore tracking with GFP-tagged kinetochores expressing these mCherry-Kif18A constructs for finer tracking ability and determine the effects of the phosphomimetic on Kif18A processivity using in vitro single molecule TIRF microscopy. These findings demonstrate a role for PTM regulation on Kif18A and lay the groundwork for studying PTMs in kinesin motors.