Mechanism of Cytoplasmic Localization

9318757个Fucus的Quatrano合子提供了一个模型系统，用于研究合子/细胞不对称的产生和局部细胞质成分向所产生的子细胞的分配所涉及的基本机制。大的单细胞受精卵在培养中同步发育出一个极轴，可以通过施加梯度(如单边光)来实验定位。这些细胞是研究极轴是如何建立的，以及细胞如何定向地将细胞质物质运输到由细胞外载体定义的预定位置的理想系统。为了使光诱导的极轴在Fucus中变得固定或稳定，细胞质中的肌动蛋白微丝网络和细胞外基质必须是完整的。Quatrano博士提出，极轴的固定涉及到在未来的类根部位通过完整的膜蛋白连接肌动蛋白、细胞骨架细丝和细胞壁纤维。这种轴稳定复合体(ASC)在结构上连接细胞质和细胞壁，可以用于固定假定的类根尖端的不对称性，并为囊泡运输到支持类根生长的靶标提供细胞质“轨迹”。ASC与其他系统中的极性结构(即哺乳动物细胞中的局灶性粘连)相似，而Fucus中ASC的形成特征与其他极性细胞(即发芽酵母和极化上皮)非常相似。在这项授权中，Quatrano博士提出了两个主要问题：(1)ASC是否为定位和保留位于新生根状突起中的囊泡和基因产物提供了结构基础，以及在轴固定和顶端生长方面稳定这种复合体的关键联系是什么？(2)酵母中极芽形成的基因产物和机制在Fucus中ASC的形成、囊泡的重新组装和类根生长中是否保守？如果是这样的话，这些产物也能在经历极化、不对称细胞分裂的其他植物细胞中检测到吗？更具体的目标是确定定位在复合体中的蛋白质的特征，并确定不对称变得稳定所需的组分之间的关键联系。为了评估特定蛋白质在固定过程中的作用，并测试该复合体的各种结构模型，Quatrano博士建议通过向受精卵中微量注射抗体来干扰特定蛋白质的功能。他将使用共聚焦显微镜观察复合体在外部梯度下形成的动力学。荧光标记的肌动蛋白和各种染料将被显微注射到受精卵中，以跟随微丝网络和高尔基体小泡在目标位置的组装。此外，定位于发育受精卵中的特定mRNAs将被鉴定和表征，并用作报告程序，以确定ASC在其不对称分布中的作用。最后，Quatrano博士已经证明，酵母中形成极芽所需的颈丝和ras样蛋白的抗体也能识别Fucus中的蛋白质。使用来自这些基因保守区的引物，他已经从褐藻中鉴定出与酵母产物有序列相似性的克隆。他将通过在酵母中进行互补测试来测试这些藻类基因的功能相似性。他将通过用Fucus cDNA文库补充酵母极性突变株来分离Fucus中的其他极性基因。如果在高等植物细胞中发现了与酵母极性基因在结构和功能上具有同源性的任何Fucus基因，他将确定它们是否在经历极性、不对称分裂的植物细胞中特异表达。*##！#“#$#%#&amp；#‘#(#)#*#+#，#-#.#/#0#1#2#3#4#5#6####9318757 Fucus的四分合子为研究合子产生的基本机制提供了一个模型系统7.$！！F(Times New Roman符号&amp；Arial：“h FC e UC e U K+Dana Brigham Dana Brigham

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