The life cycle of the social amoeba Dictyostelium discoideum alternates between a unicellular growth phase and a multicellular developmental phase, and exemplifies the importance of cell-substrate adhesion in a wide variety of biological processes. During the vegetative growth stage, Dictyostelium amoebae adhere to surfaces to achieve motility, and to bacteria for eventual engulfment. Central to these adhesive processes is the requirement for transmembrane adhesion receptors that mediate interactions with extracellular surfaces.
While no definitive vegetative-stage adhesion receptor has been determined in Dictyostelium, one of the candidate proteins, SadA, is especially intriguing. SadA is a putative nine-pass transmembrane protein containing three EGF-like repeat domains in its major extracellular region; its carboxy terminal, cytoplasmic tail is similar in length to other adhesion mediating proteins, and is a potential target for phosphorylation. Cells harboring a disrupted sadA gene are deficient in adhesion to growth surfaces and bacteria, and exhibit a cytokinesis defect concomitant with a disrupted F-actin cytoskeleton. Further, sadA is only expressed during the vegetative stage of the amoeba's life cycle. Collectively, these data demonstrate the importance for SadA in mediating substrate adhesion and actin cytoskeleton integrity. How SadA performs these functions is unknown, but elucidating its mechanism is of great interest.
The primary focus of my doctoral thesis research was to design and perform experiments to address the hypothesis that SadA serves as a link between extracellular surfaces and the inside of a cell via the F-actin cytoskeleton, and whose function is regulated by post-translational modification. Results of this research have led to three fundamental discoveries concerning SadA function: (1) the cytoplasmic, carboxy terminal tail is functionally important and is a probable target for phosphorylation, (2) SadA associates with the F-actin, a connection due in part to its interaction with the actin bundling protein cortexillin I, and (3) the interaction between SadA and cortexillin I is regulated by phosphorylation events in the tail. Further, preliminary data from TIRF-M imaging reveals that SadA associates into dynamic structures (i.e., foci) at cell-surface apposed regions. Finally, results from expression profiling experiments indicate that substrate adhesion, or lack thereof, impacts gene expression.
|Advisor:||Chisholm, Rex L.|
|Commitee:||Gottardi Yajnik, Cara, Jones, Jonathan C., Rice, Sarah E.|
|Department:||Integrated Graduate Program in the Life Sciences|
|School Location:||United States -- Illinois|
|Source:||DAI-B 72/12, Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology|
|Keywords:||Cell substrate adhesion, Dictyostelium discoideum|
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