Presentation Title

Functional Comparison of Toxoplasma and Chicken Skeletal Muscle Actins in Motility Assays Using Toxoplasma Myosin

Abstract

Toxoplasma gondii, an obligate intracellular apicomplexan parasite, utilizes gliding motility for active penetration and proactive egress from host cells. Gliding motility is made possible in apicomplexan parasites by use of the myosin motor complex, which is an actin-activated molecular motor. Toxoplasma actin is encoded by the single copy ACT1 gene which is transcribed and translated into a single actin isoform and is inherently unstable in the filamentous form. In T. gondii, globular actin polymerizes to filamentous actin and is then driven rearward by the myosin motor during gliding motility. Prior research has shown differences between Apicomplexan and mammalian actin and myosin. The in vitro motility assay is used to study the Toxoplasma myosin motor complex, and chicken skeletal muscle actin is used in place of Toxoplasma actin. Here, recombinant Toxoplasma actin is expressed, purified, and polymerized in preparation for labeling and use in the in vitro motility assay. Confirmation of Toxoplasma actin expression was achieved by sequencing baculovirus encoding recombinant Toxoplasma actin and western blot analysis. Purification was achieved via nickel affinity chromatography, followed by cycles of polymerization and depolymerization. Preparing stabilized and labeled actin filaments allows for the comparison of the speeds Toxoplasma myosin moves Toxoplasma actin versus mammalian actin.

Primary Faculty Mentor Name

Dr. Gary Ward

Secondary Mentor NetID

akelsen

Secondary Mentor Name

Anne Kelsen

Status

Undergraduate

Student College

College of Agriculture and Life Sciences

Program/Major

Molecular Genetics

Primary Research Category

Biological Sciences

Second College (optional)

Honors College

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Functional Comparison of Toxoplasma and Chicken Skeletal Muscle Actins in Motility Assays Using Toxoplasma Myosin

Toxoplasma gondii, an obligate intracellular apicomplexan parasite, utilizes gliding motility for active penetration and proactive egress from host cells. Gliding motility is made possible in apicomplexan parasites by use of the myosin motor complex, which is an actin-activated molecular motor. Toxoplasma actin is encoded by the single copy ACT1 gene which is transcribed and translated into a single actin isoform and is inherently unstable in the filamentous form. In T. gondii, globular actin polymerizes to filamentous actin and is then driven rearward by the myosin motor during gliding motility. Prior research has shown differences between Apicomplexan and mammalian actin and myosin. The in vitro motility assay is used to study the Toxoplasma myosin motor complex, and chicken skeletal muscle actin is used in place of Toxoplasma actin. Here, recombinant Toxoplasma actin is expressed, purified, and polymerized in preparation for labeling and use in the in vitro motility assay. Confirmation of Toxoplasma actin expression was achieved by sequencing baculovirus encoding recombinant Toxoplasma actin and western blot analysis. Purification was achieved via nickel affinity chromatography, followed by cycles of polymerization and depolymerization. Preparing stabilized and labeled actin filaments allows for the comparison of the speeds Toxoplasma myosin moves Toxoplasma actin versus mammalian actin.