Green Fluorescent Technology: The molecular biological approach to visualizing the dynamic polymerization and organization of microtubules in the fungus Rhizopus (Zygomycota)
Altamirano, Christopher and Roberson, Robert W.
School of Life Sciences, Arizona State University
Of the four phyla of true fungi, Chytridomycota, Ascomycota, Basidiomycota, and Zygomycota, very little is known regarding hyphal cell growth in the latter. The cytoplasmic organization in hyphae of the Zygomycota is considered less evolved than in the Ascomycota and Basidiomycota, in that they lack cellular cross walls (i.e., sepatations) and do not produce a prominent apical body (i.e., Spitzenkörper). Members of the genus Rhizopus impact humans in a number of ways, though the most important is the fact that some species cause disease in humans (i.e., zygomycosis). Rhizopus oryzae, a common bread mold, was chosen for this study due to its nonpathogenic nature as well as its fast growing hyphae, making it a desirable model organism to study polarized hyphal growth in the Zygomycota. The Spitzenkörper, a common cytoplasmic structure at the hyphal tips of Ascomycota and Basidiomycota, is a non-membrane bound structure in which a mass of secretory vesicles, cytoskeletal elements and signaling proteins are organized. The Spitzenkörper is intimately involved in the final delivery of vesicles to the growing point of the cell, thus playing a critical role in polarized hyphal extension and regulating the direction of cell growth. The fact that the Zygomycota lack a Spitzenkörper, yet have the ability to undergo polarized growth has long perplexed fungal cell biologists. Ordinary light microscopy methods by themselves cannot aid in the elucidation of the apical cytoplasmic organization and polymerization at the hyphael tip.
Green Fluorescent Protein (GFP) technology may aid in this endeavor via direct binding of GFP to the beta -tubulin subunits of the microtubule cytoskeleton at the DNA level. With a successful GFP transformation, the dynamic polymerization and organization of microtubules at the apex can be directly visualized in vivo, without compromising the integrity of the cell. This work seeks to elucidate the apical cytoplasmic organization in the hyphal apex by means of direct genetic manipulation and transformation of the beta-tubulin::GFP gene in R. Oryzae.