Spracklen Andrew J, Fagan Tiffany N, Lovander Kaylee E, Tootle Tina L
Anatomy and Cell Biology Department, Carver College of Medicine, University of Iowa, 51 Newton Rd, Iowa City, IA 52242, USA.
Anatomy and Cell Biology Department, Carver College of Medicine, University of Iowa, 51 Newton Rd, Iowa City, IA 52242, USA.
Dev Biol. 2014 Sep 15;393(2):209-226. doi: 10.1016/j.ydbio.2014.06.022. Epub 2014 Jul 1.
Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools--Utrophin, Lifeact, and F-tractin--for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool within the tissue and cell type of interest in order to identify the tool that represents the best compromise between acceptable labeling and minimal disruption of the phenomenon being observed. In this case, we find that F-tractin, and perhaps Utrophin, when Utrophin expression levels are optimized to label efficiently without causing actin defects, can be used to study F-actin dynamics within the Drosophila nurse cells.
肌动蛋白细胞骨架的动态重塑对于发育和组织稳态都是必需的。虽然固定图像分析为这类事件提供了重要见解，但要全面理解细胞骨架动力学则需要实时成像。通过将肌动蛋白相互作用蛋白的肌动蛋白结合结构域与荧光蛋白融合，已经产生了许多用于肌动蛋白实时成像的工具。在这里，我们比较评估了三种这样的工具——肌萎缩蛋白相关蛋白（Utrophin）、生命肌动蛋白（Lifeact）和F - 肌动蛋白（F-tractin）——在果蝇卵子发生中期或卵泡发育过程中，用于表征生殖系来源的滋养细胞内发生的肌动蛋白重塑事件的效用。具体而言，我们使用UAS/GAL4系统在不同水平和不同细胞中表达这些工具，并通过固定和实时成像分析这些工具对生育力的影响、肌动蛋白细胞骨架的改变以及标记丝状肌动蛋白（F-肌动蛋白）结构的能力。虽然肌萎缩蛋白相关蛋白和生命肌动蛋白都能有力地标记果蝇生殖系中的F-肌动蛋白结构，但当它们强烈表达时会导致不育和严重的肌动蛋白缺陷，包括皮质肌动蛋白分解导致多核滋养细胞、在第9阶段（S9）早期形成F-肌动蛋白丝和聚集体，以及在第10B阶段（S10B）时平行肌动蛋白丝束紊乱。然而，通过使用较弱的生殖系GAL4驱动子并结合较高温度，肌萎缩蛋白相关蛋白可以以最小的缺陷标记F-肌动蛋白。此外，生殖系中强烈的肌萎缩蛋白相关蛋白表达会在卵子发生中期导致滋养细胞核和生发泡中形成F-肌动蛋白。同样，生命肌动蛋白的表达仅在生发泡内导致核F-肌动蛋白形成。F - 肌动蛋白的表达水平低于其他两种标记工具，但能很好地标记细胞质F-肌动蛋白结构，且不会导致不育或明显的肌动蛋白缺陷。这些研究共同揭示了在感兴趣的组织和细胞类型中评估每种肌动蛋白标记工具的效用是多么关键，以便确定在可接受的标记和对所观察现象的最小干扰之间达到最佳平衡的工具。在这种情况下，我们发现F - 肌动蛋白，也许还有肌萎缩蛋白相关蛋白（当肌萎缩蛋白相关蛋白的表达水平经过优化以有效标记而不引起肌动蛋白缺陷时），可用于研究果蝇滋养细胞内的F-肌动蛋白动力学。
