Mechanism of Cytoplasmic Localization

小行星9318757 墨角藻的合子提供了一个模型系统，以研究在产生合子/细胞的不对称性和分区的本地化的细胞质成分所产生的子细胞的基本机制。 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（如单侧光）进行实验定向。 这样的细胞代表了研究如何建立极轴以及细胞如何将细胞质物质定向转运到由细胞外载体限定的预定位点的理想系统。 为了使光诱导的极轴在墨角藻中变得固定或稳定，细胞质和细胞外基质中的肌动蛋白微丝网络必须是完整的。 Quatrano博士提出，极轴的固定涉及通过未来假根部位的完整膜蛋白连接肌动蛋白细胞骨架丝和细胞壁原纤维。 这种轴稳定复合物（ASC），在结构上连接细胞质与细胞壁，可以用于在假定的假根尖端的不对称性，并提供细胞质的“轨道”的小泡运输到一个有针对性的补丁支持假根的生长。 ASC类似于其他系统中的极性结构（即，在哺乳动物细胞中的粘着斑），并且其在墨角藻中形成的特征显著地类似于其它极性细胞（即，芽殖酵母和极化上皮）。 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兴假根中的囊泡和基因产物提供了结构基础，以及在轴固定和尖端生长方面稳定这种复合物的关键联系是什么？ (2)在墨角藻属中形成ASC、囊泡募集和假根生长的过程中，酵母中极芽形成的基因产物和机制是否保守？ 如果是这样的话，这些产物是否也可以在其他进行极化、不对称细胞分裂的植物细胞中检测到？ 更具体的目标是表征定位在复合物中的蛋白质，并确定不对称性稳定所需的组件之间的关键联系。 为了评估特定蛋白质在固定过程中的作用，并测试复合物的各种结构模型，Quatrano博士提出通过将抗体显微注射到受精卵中来干扰特定蛋白质的功能。 他将通过使用共聚焦显微镜观察复合物形成对外部梯度的响应的动力学。 将标记的肌动蛋白和各种染料显微注射到受精卵中，以跟踪微丝网络和高尔基体囊泡在靶位点的组装。 此外，将鉴定和表征位于发育中的合子中的特定mRNA，并将其用作报告基因以确定ASC在其不对称分布中的作用。 最后，Quatrano博士已经证明，针对酵母中极芽形成所需的颈丝和ras样蛋白质的抗体也识别墨角藻中的蛋白质。 利用这些基因保守区域的引物，他已经从褐藻中鉴定出与酵母产物具有序列相似性的克隆。 他将通过酵母中的互补试验来测试这些藻类基因的功能相似性。 他将通过用墨角藻cDNA文库补充酵母极性突变体，从墨角藻中分离出其他极性基因。 如果在高等植物细胞中发现任何与酵母极性基因具有结构和功能同源性的墨角藻基因，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性表达。 *** # #!# "###$#%##'#(#)#*#+#,#-#.#/# 0#1#2#3#4#5#6# # # ### 墨角藻的合子为研究合子产生的基本机制提供了一个模式系统 7 $ ! ! F ( Times New Roman 符号Arial : " H fC e uC e U K + 丹娜·布里格姆

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