9318757 Quatrano Zygotes of Fucus provide a model system to study the basic mechanisms involved in the generation of zygotic/cell asymmetry and partitioning of localized cytoplasmic components to the resulting daughter cells. Large, single-celled zygotes synchronously develop a polar axis in culture that can be experimentally oriented by imposed gradients such as unilateral light. Such cells represent an ideal system to study how a polar axis is established and how a cell directionally transports cytoplasmic material to a predetermined site defined by an extracellular vector. In order for a light-induced polar axis to become fixed or stabilized in Fucus, a network of actin microfilaments in the cytoplasm and an extracellular matrix must be intact. Dr. Quatrano has proposed that the fixation of a polar axis involves linking actin cytoskeletal filaments and cell wall fibrils through integral membrane proteins at the future rhizoid site. This axis stabilizing complex (ASC) that structurally links the cytoplasm with the cell wall could serve to immobilize asymmetries at the presumptive rhizoid tip and to provide cytoplasmic "tracks" for vesicle transport to a targeted patch for support of rhizoid outgrowth. The ASC is similar to polar structures in other systems (i.e., focal adhesions in mammalian cells), and the characteristics of its formation in Fucus is remarkably like other polar cells (i.e., budding yeast and polarized epithelium). In this grant, Dr. Quatrano proposes two major questions: (1) Does the ASC provide the structural basis for the targeting and retention of vesicles and gene products localized in the emerging rhizoid, and what are the critical linkages that stabilize this complex with respect to axis fixation and tip growth? (2) Are gene products and mechanisms of polar bud formation in yeast conserved in the formation of the ASC, vesicle recruitment, and rhizoid growth in Fucus? If so, can these products also be detected in other plant cells undergoing polarized, asymmetric cell divisions? More specific aims are to characterize the proteins localized in the complex and to identify the critical linkages between components that are required for the asymmetries to become stabilized. To assess the role of specific proteins in the fixation process and to test various structural models of the complex, Dr. Quatrano proposes to interfere with the function of specific proteins by microinjecting antibodies into the zygote. He will observe the dynamics of the complex forming in response to external gradients by using confocal microscopy. Fluorescently labeled actin and various dyes will be microinjected into the zygotes to follow the assembly of the microfilament network and Golgi vesicles at the targeted site. Also, specific mRNAs that are localized in the developing zygote will be identified and characterized and used as reporters to determine the role of the ASC in their asymmetric distribution. Finally, Dr. Quatrano has shown that antibodies raised to neck filament and ras-like proteins that are required for polar bud formation in yeast also recognize proteins in Fucus. Using primers from conserved regions in these genes, he has identified clones from brown algae that have sequence similarities with the yeast products. He will test the functional similarities of these algal genes by complementation tests in yeast. He will isolate other polarity genes from Fucus by complementing yeast polarity mutants with Fucus cDNA libraries. If any of the Fucus genes that have structural and functional homologies with the yeast polarity genes are found in higher plant cells, he will determine if they are specifically expressed in plant cells undergoing polar, asymmetric divisions. *** # #!#"###$#%#&#'#(#)#*#+#,#-#.#/#0#1#2#3#4#5#6# # # # # # # # # # # # # # # # # # # # # # # # # # #9318757 Quatrano Zygotes of Fucus provide a model system to study the basic mechanisms involved in the generation of zygotic < 7 < $ ! ! F << ( Times New Roman Symbol & Arial : " h fC e uC e U K + Dana Brigham Dana Brigham
