Modulation of the actin cytoskeleton by the nebulin domain protein Lasp

我的NSERC研究计划的总体目标是了解肌动蛋白细胞骨架在发育过程中的调制和调节。丝状肌动蛋白的组装、分解、稳定和成束等特性受多种肌动蛋白结合蛋白的调节，从而在分化的细胞中形成许多不同的结构。在这里，我们建议研究如何Lasp，肌动蛋白结合的星云蛋白结构域蛋白家族的果蝇成员，调节肌动蛋白细胞骨架在精子发生过程中。肌动蛋白锥是睾丸中的重要结构，它沿着轴丝迁移，将合胞体中的精子细胞分离成64个精子。这个过程称为精子细胞个体化。我们可以证明Lasp突变体中肌动蛋白锥迁移不完全，导致精子缺陷。此外，Lasp在睾丸中锚定干细胞龛中发挥作用。因此，我们建议分析Lasp如何调节肌动蛋白丝在果蝇肌动蛋白锥迁移，以及它如何锚定干细胞龛。目的：（1）研究Lasp突变体的肌动蛋白视锥结构。我们将首先分析肌动蛋白锥的大小和迁移速度在Lasp突变体和Lasp过表达孤立的囊肿。我们也将研究其他肌动蛋白调节蛋白，如CapZ，Arp 2/3，profilin，actinin，和绒毛蛋白在Lasp突变体的本地化。然后，我们将测试细胞松弛素D染色和注射荧光G肌动蛋白到睾丸中，如果肌动蛋白丝的倒刺末端在Lasp突变体的影响。我们还将通过透射电子显微镜分析野生型和Lasp突变体的肌动蛋白锥超微结构。(2)通过实时成像分析肌动蛋白锥体中的肌动蛋白周转。我们将采用光漂白后的荧光恢复（FRAP）来评估肌动蛋白丝的周转。我们将漂白GFP-肌动蛋白，并在肌动蛋白锥的不同区域评估Lasp突变体与野生型中丝状肌动蛋白的恢复率，以确定肌动蛋白周转是否受到影响。作为阴性对照，我们将测试加入细胞松弛素后的回收率，细胞松弛素阻断单体的加入，因此应该减缓回收率，以及在活的CapZ突变体中，阳性对照，应该增加回收率。这些实验将提供野生型和Lasp突变体中肌动蛋白视锥的周转和组装的信息。(3)研究Lasp蛋白结构域和相互作用蛋白的功能。我们将使用三重标记的Lasp转基因生物化学分离蛋白质相互作用的Lasp LIM，星云蛋白，和SH 3域在体内。然后通过质谱法鉴定相互作用的蛋白质。与此同时，我们将确定肌动蛋白视锥中结构域特异性转基因的定位及其拯救精子细胞个体化缺陷的能力。这些方法将确定肌动蛋白锥迁移所需的结构域和蛋白质伴侣，但也用于锚定男性干细胞龛，因为这些构建体将在生殖细胞和枢纽细胞中表达。影响：我们对Lasp的研究将有助于更好地理解精子发生和肌动蛋白锥迁移。此外，我们对Lasp的基础细胞生物学研究将有助于阐明nebulin结构域在精子发生中的作用机制。我们相信我们的研究结果将扩展到脊椎动物中的其他nebulin家族成员，揭示nebulin结构域如何在不同的环境中调节肌动蛋白丝。因此，我们的研究结果将提供深入了解肌动蛋白细胞骨架的动态调节，这使得形态发生和延伸发展进行适当的。该研究计划将为研究生和本科生提供细胞生物学，遗传学和生物化学各个方面的优秀培训机会。

The overall objective of my NSERC research program is to understand the modulation and regulation of the actin cytoskeleton during development. The properties of filamentous actin, that is assembly, disassembly, stabilization, and bundling, are regulated by a wide range of actin binding proteins, resulting in many different structures in differentiated cells. Here we propose to investigate how Lasp, the single Drosophila member of the actin-binding nebulin domain protein family, regulates the actin cytoskeleton during spermatogenesis. Actin cones are prominent structures in testes, which migrate along the axoneme to separate spermatids previously in a syncytium into 64 individual sperm. This process is called spermatid individualization. We could show that actin cone migration is incomplete in Lasp mutants, resulting in defective sperm. In addition, Lasp plays a role in anchoring the stem cell niche in testis. Thus, we propose to analyze how Lasp regulates actin filaments during actin cone migration in Drosophila, and how it anchors the stem cell niche. Aims: (1) Characterize actin cone structure in Lasp mutants. We will first analyze actin cone size and migration speed in Lasp mutants and upon Lasp overexpression in isolated cysts. We will likewise study the localization of other actin regulatory proteins such as CapZ, Arp2/3, profilin, actinin, and villin in Lasp mutants. We will then test by cytochalasin D staining and injection of fluorescent G actin into testes if barbed ends of actin filaments are affected in Lasp mutants. We will also analyze actin cone ultrastructure in wild type and Lasp mutants by transmission electron microscopy. (2) Analyze actin turnover in actin cones by live imaging. We will employ fluorescence recovery after photobleaching (FRAP) to assess actin filament turnover. We will bleach GFP-actin and assess the recovery rate of filamentous actin in Lasp mutant versus wild type in different areas of the actin cone to determine if actin turnover is affected. As a negative control, we will test recovery after addition of cytochalasin, which blocks monomer addition and should therefore slow the recovery rate, as well as in a viable CapZ mutant, a positive control, which should increase the recovery rate. These experiments will provide information on turnover and assembly of actin cones in wild type and Lasp mutants. (3) Investigate the function of Lasp protein domains and interacting proteins. We will use a triple-tagged Lasp transgene to biochemically isolate proteins interacting with the Lasp LIM, nebulin, and SH3 domains in vivo. Interacting proteins will then be identified by mass spectrometry. In parallel, we will determine the localization of domain-specific transgenes in actin cones and their ability to rescue defects in spermatid individualization. These approaches will identify the domains and protein partners required for actin cone migration, but also for anchoring the male stem cell niche, as these constructs will be expressed in both germ cells and hub cells. Impact: Our work on Lasp will lead to a better understanding of spermatogenesis and actin cone migration. In addition, our basic cell biological studies on Lasp will shed light on the mechanism of nebulin domain function in spermatogenesis. We believe our results will extend to other nebulin family members in vertebrates, revealing how nebulin domains regulate actin filaments in different contexts. Our results will therefore provide insight into the dynamic regulation of the actin cytoskeleton, which allows morphogenesis and by extension development to proceed properly. This research program will provide outstanding training opportunities for graduate and undergraduate students in all aspects of cell biology, genetics, and biochemistry.