The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.
The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.
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DOI：
10.1016/j.ydbio.2014.06.022
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发表时间：
2014-09-15
影响因子：
2.7
通讯作者：
Tootle, Tina L.
中科院分区：
文献类型：
--
作者：
Spracklen, Andrew J.;Fagan, Tiffany N.;Lovander, Kaylee E.;Tootle, Tina L.
肌动蛋白细胞骨架的动态重塑是发育和组织稳态所必需的。虽然固定图像分析提供了显着的洞察这些事件，细胞骨架动力学的完整理解需要实时成像。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，我们比较评估三个这样的工具- Utrophin，Lifeact，和F-tractin -用于表征肌动蛋白重塑事件发生在果蝇中期卵子发生或卵泡发育过程中的生殖源性的护士细胞内。具体来说，我们使用UAS/GAL 4系统在不同水平和不同细胞中表达这些工具，并分析这些工具对生育力的影响，肌动蛋白细胞骨架的改变，以及通过固定和实时成像标记丝状肌动蛋白（F-肌动蛋白）结构的能力。虽然Utrophin和Lifeact都在果蝇种系内强烈标记F-肌动蛋白结构，但当强烈表达时，它们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，导致多核护士细胞，早期F-肌动蛋白丝和第9阶段（S9）期间的聚集体形成，以及第10 B阶段（S10 B）期间的无序平行肌动蛋白丝束。然而，通过使用较弱的种系GAL 4驱动程序与较高的温度组合，Utrophin可以标记具有最小缺陷的F-肌动蛋白。此外，在生殖系内的强Utrophin表达导致在中期卵子发生期间在保育细胞核和生殖囊泡中形成F-肌动蛋白。类似地，Lifeact表达仅在胚泡内产生核F-肌动蛋白。F-tractin的表达水平低于其他两种标记工具，但标记细胞质F-actin结构良好，不会导致不育或引人注目的肌动蛋白缺陷。总之，这些研究揭示了评估组织和细胞类型内每个肌动蛋白标记工具的实用性是多么重要，以便确定代表可接受的标记和观察到的现象的最小破坏之间的最佳折衷的工具。在这种情况下，我们发现，F-tractin，也许Utrophin，当Utrophin的表达水平进行优化，有效地标记，而不会导致肌动蛋白缺陷，可用于研究果蝇的护士细胞内的F-肌动蛋白动力学。
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.
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DOI：
10.1083/jcb.201007095
发表时间：
2011-01-24
期刊：
The Journal of cell biology
影响因子：
--
作者：
Hatan M;Shinder V;Israeli D;Schnorrer F;Volk T
通讯作者：
Volk T
影响因子：
56.9
作者：
Baarlink, Christian;Wang, Haicui;Grosse, Robert
通讯作者：
Grosse, Robert
影响因子：
3.7
作者：
Deibler M;Spatz JP;Kemkemer R
通讯作者：
Kemkemer R
影响因子：
3.3
作者：
Belin BJ;Cimini BA;Blackburn EH;Mullins RD
通讯作者：
Mullins RD
影响因子：
--
作者：
Burkel, Brian M.;von Dassow, George;Bement, William M.
通讯作者：
Bement, William M.