Calcium, pH and the Cytoskeleton in Pollen Tube Growth

9601087 Hepler花粉管生长是将雄性配子输送到卵器的过程，因此在高等植物有性生殖中是必不可少的。理解这些机制可以解决经济和基本问题。最近的研究表明，生长中的花粉管具有一个陡峭的、尖端聚焦的细胞内游离钙（Ca）梯度，以及一个明显的尖端导向的细胞外钙流入，这两者对生长都是必不可少的。此外，梯度在大小上波动，与增长率的变化相关。因此，Ca似乎在花粉管生长的控制中占有关键地位。具体来说，Ca可能刺激细胞壁形成和细胞伸长所需的囊泡胞吐的发生并确定其位置。钙也可能控制细胞骨架的形成，细胞骨架负责将囊泡运送到管的顶端区域。我们对氢离子(H)的作用知之甚少，但似乎存在局部pH梯度，并且它们对生长和发育至关重要。阐明钙梯度、推定氢梯度和细胞骨架元素的组织、活性和相互作用，并了解这些因素如何影响花粉管的极性和生长，是本研究的目标。将特别强调在活花粉管中发生的事件，使用细胞内显微注射作为引入特定荧光类似物、探针和指示染料的手段。例如，细胞内Ca可以通过装载指示剂fura-2葡聚糖的花粉管上的荧光比例离子成像来分析，而H可以用bccf -葡聚糖或snafl -葡聚糖来检测。通过快速、连续的图像采集，Ca或H水平将与生长直接相关。同样，在注射了少量的荧光phalloidin （F-actin特异性）的细胞中，可以观察到生长、细胞质流动和微丝组织之间的关系。活细胞研究将通过使用免疫荧光和免疫金来空间定位关键蛋白，如肌球蛋白，以进一步阐明其功能。提出的研究解决花粉管生长的基本方面，特别是在活细胞的背景下。虽然我们知道某些基本过程的存在，例如尖端聚焦的钙离子进入，以及结构复合体，例如肌动蛋白微丝，但我们对这些过程如何在细胞内整合，以及它们如何相互作用以产生高度极化和快速伸长的花粉管知之甚少。提出的工作试图弥合这一差距，从而提供一个新的层次的理解，不仅可以应用于尖端生长细胞，也可以应用于一般的植物细胞。＊＊＊

9601087 Hepler Pollen tube growth is the process that delivers the male gametes to the egg apparatus and thus is essential for sexual reproduction in higher plants. Understanding the mechanisms addresses issues of both economic and fundamental importance. Recent work reveals that growing pollen tubes posses a steep, tip-focused gradient of intracellular free calcium (Ca), and a pronounced tip-directed influx of extracellular Ca, of which both are essential for growth. In addition the gradient fluctuates in magnitude, being correlated with changes in the growth rate. Ca thus appears to occupy a pivotal position in the control of pollen tube growth. Specifically, Ca may stimulate the occurrence and define the location of vesicle exocytosis required for cell wall formation and cell elongation. Ca might also control the formation of the cytoskeleton, which is responsible for transporting the vesicles to the apical region of the tube. We know much less about the role of hydrogen ions (H), but it is plausible that local gradients in pH exist and that they are essential for growth and development. It is a goal of the proposed work to elucidate the organization, activity and interaction of the Ca gradient, the putative H gradient, and cytoskeletal elements, and to understand how these factors contribute to the polarity and growth of the pollen tube. Particular emphasis will be given to events as they occur in living pollen tubes using intracellular microinjection as a means for introducing specific fluorescent analogs, probes and indicator dyes. Intracellular Ca, for example, will be analyzed by fluorescence ratiometric ion imaging on pollen tubes loaded with the indicator dye, fura-2 dextran, while H can be detected with BCECF-dextran or SNAFL-dextran. Through rapid, sequential image acquisition, Ca or H levels will be directly correlated with growth. Similarly, in cells injected with low amounts of fluorescent phalloidin, specific for F-actin, it will be possible to observe the relati onship between growth, cytoplasmic streaming and the organization of the microfilaments. The live cell studies will be augmented by those using immunofluorescence and immunogold to spatially localize key proteins, e.g., myosin, in an attempt to further elucidate function. The proposed studies address basic aspects of pollen tube growth, particularly within the context of the live cell. Whereas we know the existence of certain fundamental processes, e.g., tip-focused Ca entry, and structural complexes, e.g., the actin microfilaments, we understand much less about how these are integrated within the cell, and how they interact to produce the highly polarized and rapidly elongating pollen tube. The proposed work attempts to bridge this gap and thus to provide a new level of understanding that can apply not only to tip growing cells, but also to plant cells in general. ***