Developmental Biology 393 (2014) 209–226
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Developmental Biology
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Resource The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis
Andrew J. Spracklen, Tiffany N. Fagan, Kaylee E. Lovander 1, Tina L. Tootle n
Anatomy and Cell Biology Department, Carver College of Medicine, University of Iowa, 51 Newton Rd, Iowa City, IA 52242, USA article info abstract
Article history: Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. Received 12 December 2013 While fixed image analysis has provided significant insight into such events, a complete understanding Received in revised form of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been 4 April 2014 generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Accepted 17 June 2014 Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, and F-tractin – for Available online 1 July 2014 characterizing the actin remodeling events occurring within the germline-derived nurse cells during Keywords: Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express Actin these tools at different levels and in different cells, and analyzed these tools for effects on fertility, Live Imaging alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both Oogenesis 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. & 2014 Elsevier Inc. All rights reserved.
Introduction the functions of actin binding proteins and regulatory factors (reviewed in (Hudson and Cooley, 2002)). Here we focus on events Drosophila oogenesis or follicle development consists of 14 mor- occurring within the germline-derived nurse cells. The main purpose phologically defined stages (reviewed in (Spradling, 1993)). Each egg of the nurse cells is to supply organelles, mRNA, and proteins to the chamber or follicle is comprised of 16 interconnected germline cells oocyte, thereby providing the oocyte with everything needed to – 15 nurse cells and one oocyte – and approximately 1000 somatic complete embryogenesis. cells termed follicle cells. Production of a viable egg requires dynamic Spatial and temporal regulation of actin remodeling is required remodelingoftheactincytoskeletoninboththesomaticandgermline for this multi-stage transport process that is essential for the cells. Thus, Drosophila oogenesis is an ideal system for studying the production of a viable egg. Transport occurs through the ring canals, actin cytoskeleton and has been widely used to identify and elucidate remnants of incomplete cytokinesis that connect the nurse cells to each other and the oocyte. In the earlier stages of follicle develop- ment (prior to stage 10B (S10B)) the transport of materials from the n Corresponding author. Fax: þ1 319 335 7198. nurse cells into the oocyte is slow (reviewed in (Hudson and Cooley, E-mail address: [email protected] (T.L. Tootle). 1 Present Address: Biochemistry Department, Carver College of Medicine, 2002)). Little actin remodeling is seen within the nurse cells University of Iowa, Iowa City, IA 52242, USA during these stages. Intriguingly, we have observed that some actin http://dx.doi.org/10.1016/j.ydbio.2014.06.022 0012-1606/& 2014 Elsevier Inc. All rights reserved. 210 A.J. Spracklen et al. / Developmental Biology 393 (2014) 209–226 remodeling is occurring at the ring canals connected to the oocyte extensive actin filament and/or aggregate formation (see Fig. 1B in the posterior nurse cells during stage 9 (S9) (Spracklen et al., and B0). Such remodeling is regulated as we have found that genetic 2014). This normally results in minimal actin filaments near the ring loss of the Drosophila COX-like enzyme, Pxt, results in a significant canals (see Fig. 1AandA0), but occasionally results in more increase in the prevalence of these early actin structures (Spracklen et al., 2014). During S10B, the nurse cells undergo dynamic actin remodeling S9 DAPI to facilitate the second, rapid phase of transport that occurs during Phalloidin stage 11 (S11), termed nurse cell dumping. Nurse cell dumping requires two distinct actin remodeling events during S10B: 1) the A’ strengthening of nurse cell cortical actin, which ultimately under- goes an actomyosin based contraction to squeeze the nurse cell cytoplasm into the oocyte (Wheatley et al., 1995) and 2) the generation of parallel actin filament bundles that traverse from S9 the nurse cell membrane, inward towards the nucleus, to form a cage that prevents the nucleus from plugging the ring canals during contraction (Guild et al., 1997; Huelsmann et al., 2013). The B’ system of nurse cell dumping has been widely used to both identify actin regulators and define their functions in cytoskeletal remodeling (Cooley et al., 1992; Gates et al., 2009; Mahajan-Miklos and Cooley, 1994a, b). We have shown that Pxt, and thus S10A prostaglandins (PGs) are required to regulate these dynamic remodeling events (Groen et al., 2012; Tootle and Spradling, 2008). Such studies have relied on analyses of actin structures using fixed samples labeled with phalloidin to visualize filamen- tous actin (F-actin). While fixed imaging has provided important insights into the actin cytoskeletal remodeling events occurring within the Droso- phila germline, many questions remain regarding the spatial and temporal regulation of this remodeling. For example, it has Early S10B previously been shown that the remodeling during S10B begins along the nurse cell oocyte boundary and then progresses ante- riorly (see Fig. 1D F) (Guild et al., 1997). However, this spatial and temporal progression is not widely appreciated and the mechan- isms underlying this regulation remain poorly understood. Speci- fically, the roles of particular actin regulators in establishing spatial and temporal regulation over these cytoplasmic actin remodeling events remain unknown. Additionally, fixed analyses cannot be used to assess nurse cell actin filament bundle dynamics, or how pharmacologic or genetic perturbations alter those dynamics. One Mid S10B means of addressing these knowledge gaps is to utilize live imaging. One actin cytoskeleton live imaging reagent routinely used in Drosophila is the GFP-tagged actin binding domain of Moesin, GFP- Moe (Edwards et al., 1997). GFP-Moe has successfully been used to examine actin dynamics in the Drosophila embryo during dorsal closure (Peralta et al., 2007; Toyama et al., 2008), tracheal morphogenesis (Kato et al., 2004), and hemocyte migration (Zanet et al., 2009). It has also been used to label actin dynamics Late S10B
Fig. 1. Actin remodeling is temporally and spatially regulated during mid- oogenesis. (A G) Maximum projections of 3 5 confocal slices of fixed and stained wild-type (yw) follicles, staged as indicated, taken at 20 magnification. Anterior is to the left. F-actin (phalloidin)¼white, DNA (DAPI)¼cyan. (A B0) S9. (C) S10A. (D F) S10B. (G) S11. During S9, the border cells and main-body follicle cells are undergoing migration and the nurse cell cytoplasm is largely devoid of actin filament structures (A A0), but occasionally there are actin filament and aggregate structures emanating from the ring canals in the posterior nurse cells 0 S11 adjacent to the oocyte (B B ). Both the border cell and main-body follicle cell migrations are completed by S10A and the nurse cells lack cytoplasmic F-actin structures (C). During S10B, dynamic actin remodeling is occurring within the nurse cells. Actin filament bundles first form in the posterior nurse cells, at the nurse cell-oocyte boundary (D). These bundles continue to elongate and bundle formation initiates on all of the nurse cell membranes that are directly attached to their neighboring nurse cell by a ring canal (E). At the completion of S10B, the bundles are uniformly distributed along the nurse cell membranes and extend all the way to the nucleus (F). During S11, the cortical actin contracts to squeeze the cytoplasmic contents of the nurse cells into the growing oocyte (G). Scale bars¼50 μm, except in A0 and B0, where scale bars¼10 μm. A.J. Spracklen et al. / Developmental Biology 393 (2014) 209–226 211
Table 1 Summary of the actin binding domain fusions to fluorescent proteins that were used to generate transgenic Drosophila lines.
Reagent Actin binding domain Promoter Tag Reagent reference Fly reference
Utrophin Human ubiquitous dystrophin UASp and sqh EGFP Burkel et al. 2007. Rauzi et al. 2010. Lifeact Yeast ABP140 UASp mEGFP Riedl et al. 2008 This work F-tractin Rat inositol triphosphate 3-kinase A (ITPKA) UASp mEGFP tdTomato Johnson and Schell. 2009 This work
within the somatic cells of developing Drosophila follicles (Boyle et (reviewed in (Hudson and Cooley, 2002)). During S9, besides the al., 2010; Edwards et al., 1997), and was recently used to label cortical actin, there is little F-actin observed within the nurse cells cortical actin structures within the germline (Weil et al., 2010). We (Fig. 1A and A0). Fixation conditions designed to preserve actin have found that GFP-Moe (sqh promoter driven GFP-Moe) strongly structures reveal that there are small actin structures at the labels both germline and follicle cell cortical actin during oogen- posterior ring canals leading into the oocyte. Occasionally, more esis, but fails to label the initial stages of actin bundle formation extensive actin structures, including elongated actin filament during S10B and only weakly labels the fully formed actin bundles bundles and aggregates, are observed (Fig. 1B and B0, yellow (Spracklen and Tootle, unpublished observations), reducing the arrows) (Spracklen et al., 2014). At S10A, these “early” actin utility of this tool for assessing germline actin dynamics during structures are no longer apparent (Fig. 1C). During S10B, rapid Drosophila mid-oogenesis. cytoplasmic actin remodeling occurs (Fig. 1D F) (Cooley et al., Numerous additional tools for live imaging of actin dynamics 1992; Gates et al., 2009; Mahajan-Miklos and Cooley, 1994a, b). In have been developed and utilized in other systems and, in some early S10B, parallel actin filament bundles (subsequently referred cases, in Drosophila. These include directly tagging the N-terminus to as actin bundles) begin to form along the nurse cell/oocyte of actin with a fluorescent protein, i.e. GFP-Actin. While GFP-Actin boundary (Fig. 1D) (Guild et al., 1997; Huelsmann et al., 2013); the does label actin filaments, such labeling has been associated with barbed ends of the filaments are positioned at the nurse cell alterations in actin structure and dynamics (Aizawa et al., 1997; membrane with the pointed ends proximal to the nucleus. By mid- Hird, 1996; Kovar et al., 2006; Roper et al., 2005; Wu and Pollard, S10B, these posterior actin bundles have elongated toward the 2005). An alternative means of labeling actin is by fusing a nucleus and actin bundle formation has initiated along all nurse fluorescent protein to the actin-binding domain from a known cell membranes sharing a ring canal with an adjacent nurse cell actin binding protein. It is thought that this “indirect” method of (Fig. 1E). These actin bundles continue to elongate throughout labeling actin filaments is less likely to disrupt the actin cytoske- S10B, such that the actin bundles extend all the way to the nucleus leton. Examples of this type of tool include Utrophin, Lifeact, and by late S10B (Fig. 1F) (Guild et al., 1997; Huelsmann et al., 2013). F-Tractin (see Table 1). Utrophin is composed of the calponin During S10B, the cortical actin is also strengthened, and, during homology domains of human ubiquitous dystrophin (Utrophin) S11, the cortical actin contracts in an actomyosin dependent (Burkel et al., 2007), Lifeact is the actin-binding domain from yeast manner to squeeze all the nurse cell cytoplasmic contents into actin binding protein 140 (ABP140) (Riedl et al., 2008), and F- the oocyte in a process termed nurse cell dumping (Fig. 1G) tractin is the actin-binding domain from rat inositol triphosphate (Wheatley et al., 1995). It is important to note that we assume 3-kinase (Johnson and Schell, 2009). that the actin cytoskeletal structures observed in fixed sample While numerous studies have shown the utility of these tools for analyses accurately recapitulate what is occurring. In all fixed assessing actin dynamics in other systems (Brock et al., 2012; Hatan et experimental analyses presented here, the actin structures are al., 2011; Rauzi et al., 2010; Zanet et al., 2009) and some of these tools being compared to controls (both GAL4 and UASp only) raised have been used to label actin within the Drosophila germline under the same conditions and processed in the same manner. (Huelsmannetal.,2013;Zanetetal.,2012), the tools have not been To gain further insight into not only the temporal and spatial comparatively assessed for their utility in monitoring the extensive regulation of actin remodeling, but the dynamic nature of these cytoplasmic F-actin remodeling events occurring within the Drosophila remodeling events within the nurse cells, we have obtained or germline. We have generated transgenic lines that express Lifeact- generated transgenic lines to indirectly label F-actin live, includ- mEGFP, F-tractin mEGFP and F-tractin tdTomato (tdTom) under the ing: GFP-Utrophin (Burkel et al., 2007; Rauzi et al., 2010); Lifeact- control of the UASp promoter, which can be expressed in the germline. mEGFP (Riedl et al., 2008); and F-tractin mEGFP and td-Tomato Additionally, we have obtained a similar GFP-Utrophin line (Rauzi et (Johnson and Schell, 2009)(Table 1). These transgenic lines al., 2010)(Table 1). Here we compare the utility of these three labeling express the actin labeling tool under the control of the UASp tools for examining the actin remodeling occurring within the nurse promoter, allowing us to use a variety of GAL4 lines to express the cells. Specifically, we assess whether actin remodeling occurs normally, tools in either the soma or germline (Brand and Perrimon, 1993; using both fertility and F-actin morphology as readouts. Additionally, Rorth, 1998). In our initial experiments, we tested a number of we assess the extent to which each marker colocalizes with F-actin by GAL4 lines to determine their expression patterns during follicle phalloidin staining, and whether these tools are effective for live development (STable 1). Based on those experiments, we chose to imaging of actin dynamics within the Drosophila germline. utilize: c355 GAL4 to drive expression in the somatic follicle cells (Skora and Spradling, 2010); nanos-VP16 GAL4 to drive weak germline expression during mid-oogenesis; and either maternal α-tubulin (3rd chromosome insert or 2nd chromosome insert Results [MK]; referred to as mat3 and mat2MK, respectively) or oskar GAL4 (3rd chromosome insert, referred to as osk3)(Telley et al., Actin cytoskeletal remodeling events occurring within the Drosophila 2012), interchangeably, to drive strong germline expression during nurse cells during mid-oogenesis mid-oogenesis. Importantly, heterozygosity for these GAL4 drivers does not result in any significant actin remodeling abnormalities Fixed image analysis has revealed that tight temporal and (SFig. 1), with the exception that two of the strong germline spatial actin remodeling within the germline-derived nurse cells drivers, mat3 and osk3 GAL4, exhibit an increased frequency of is required for Drosophila follicle development and female fertility early F-actin structures during S9 (SFig. 1D and E, arrows). 212 A.J. Spracklen et al. / Developmental Biology 393 (2014) 209–226
Strong germline expression of GFP-Utrophin results in female sterility utilized at each temperature to define what represented the and severe actin remodeling defects “normal” actin phenotype. Fixed image analysis (phalloidin staining and anti-GFP immu- Actin remodeling, both within the soma and the germline of nofluorescence) reveals that expression of GFP-Utrophin results in developing Drosophila follicles, is required for female fertility. F-actin defects during S9. While somatic expression of GFP- Transgenic lines expressing GFP-Utrophin (the first 261 aa resi- Utrophin does not result in any F-actin abnormalities within the dues, containing the calponin homology domains, of Utrophin nurse cells, when GFP-Utrophin is expressed in the border cells (UtrCH); also known as UTR261 or UtrNT) (Burkel et al., 2007) (c355 GAL4 driving GFP-Utrophin, 25 1C) a delay in migration is under the control of the UASp promoter were previously gener- often observed (Fig. 2B and B″, yellow arrow). Weak germline ated by Rauzi et al. (2010). We assessed fertility of GAL4 driven expression of GFP-Utrophin during S9 (nanos-VP16 GAL4) results GFP-Utrophin expressing females by mating them to wild-type in no obvious F-actin defects in the nurse cells and border cell males (yw) at room temperature ( 21 1C) and quantifying the migration appears normal (Fig. 2C and C″). However, the expres- resulting number of progeny produced per female from a 24 h egg sion is patchy and often weak. When GFP-Utrophin is expressed lay. We find that while somatic (c355) or weak germline (nanos- strongly in the germline of S9 follicles, the F-actin structures are VP16) expression of GFP-Utrophin has little to no effect on female labeled well with GFP-Utrophin; however, severe actin defects are fertility, strong germline expression results in almost complete observed. In particular, clumps of ring canals and multi-nucleate sterility (Table 2, SFig. 2A). This finding suggested that high levels nurse cells (Fig. 2 D F″, red arrowheads and red circles, respec- of Utrophin within the germline might cause actin abnormalities tively) are observed. These defects are not due to incomplete that preclude normal follicle development. cytokinesis but are the result of nurse cell cortical actin break To determine the cause of the fertility defects, we used fixed down, as the cortical actin is intact in the earlier stages of image analysis to assess if Utrophin expression resulted in any oogenesis (data not shown). Early actin filaments and aggregates actin abnormalities and the extent to which Utrophin labels F- are also seen in the posterior nurse cells adjacent to the oocyte actin structures during follicle development. Specifically we when GFP-Utrophin is strongly expressed in the germline at a focused on the actin remodeling events occurring during S9 and much higher frequency and severity than observed in control S10B. As the UAS/GAL4 system is known to exhibit a temperature follicles (Fig. 2D F″, orange boxed regions); this suggests that dependence, with higher temperatures resulting in higher levels of GFP-Utrophin expression may stabilize and/or promote such expression, the crosses and the resulting progeny were reared at cytoskeletal remodeling. Interestingly, similar, but less severe early three temperatures: 18, 21, and 25 1C. Western blot analysis actin structures are observed when PG signaling is lost (Spracklen reveals that GFP-Utrophin expression is indeed highly tempera- et al., 2014). ture dependent with c355 and nanos-VP16 GAL4 drivers, but The most surprising and striking F-actin defect observed by exhibits a smaller change in expression level at the different fixed image analysis, when GFP-Utrophin is strongly expressed in temperatures with the strong germline GAL4 drivers (mat3, the germline line, is the presence of GFP-Utrophin and phalloidin mat2MK, and oskar GAL4) (SFig. 2B). Similarly, the actin defects positive F-actin in the nurse cell nuclei and germinal vesicle observed by phalloidin staining are more severe and occur at a (Fig. 2D F″, green circles). To verify that the F-actin is within higher frequency in flies raised at 25 1C compared to either 18 or and not on the outside of the nucleus, we used wheat germ 21 1C (data not shown). Both GAL4 and UASp only controls were agglutinin (WGA) to label the nuclear envelope. We find that the F- actin is indeed within the nurse cell nuclei (SFig. 3A A‴). Given the coiled appearance of the nuclear F-actin and its unknown Table 2 structure, we have termed these nuclear F-actin structures The relative effects of the different actin labeling tools on female fertility. “threads.” Utrophin has been previously observed on nuclear F-
n actin structures and nuclear-targeted Utrophin may cause F-actin GAL4 Actin labeling tool % Relative fertility polymerization or stabilization (reviewed in (Grosse and none Utrophin-EGFP 78.0 Vartiainen, 2013)). Interestingly, the nuclear actin threads we Lifeact-mEGFP 13a 151 observe are only seen in particular stages of follicle development. Lifeact-mEGFP 14a 85.8 Specifically, S6 S9 follicles exhibit the highest frequency of these F-tractin tdTomato 10C 158 F-tractin tdTomato 15A 171 structures, with a few nurse cell nuclei retaining them into S10A-B c355 Utrophin-EGFP 70.9 (fixed image data not shown and Fig. 4E, live imaging). The Lifeact-mEGFP 13a 72.3 temporal presence of these nuclear actin threads suggests that Lifeact-mEGFP 14a 80.9 there may be a different concentration or composition of nuclear F-tractin tdTomato 10C 118 fi F-tractin tdTomato 15A 104 actin during these stages. Further supporting this idea, we nd nanos VP16 Utrophin-EGFP 93.8 that strong germline expression of particular Drosophila GFP- Lifeact-mEGFP 13a 109 Actins (Roper et al., 2005), including 42A and 57B, results in Lifeact-mEGFP 14a 96.6 shorter nuclear F-actin structures, termed “rods”, during S6-9 F-tractin tdTomato 10C 70.1 (Fagan and Tootle, unpublished observation) that are distinct from F-tractin tdTomato 15A 71.0 osk3 Utrophin-EGFP 5.94 what we observe when GFP-Utrophin is strongly expressed in the Lifeact-mEGFP 13a 0.78 germline. Lifeact-mEGFP 14a 0 We next wanted to assess, by fixed image analysis (phalloidin F-tractin tdTomato 10C 137.5 staining and anti-GFP immunofluorescence), whether GFP- F-tractin tdTomato 15A 72.2 mat3 Utrophin-EGFP 1.77 Utrophin labels the actin bundles that form during S10B and Lifeact-mEGFP 13a 0 whether expression of GFP-Utrophin alters these F-actin struc- Lifeact-mEGFP 14a 0 tures. As expected, somatic expression of GFP-Utrophin has no F-tractin tdTomato 10C 81.2 effect on actin bundle formation during S10B (Fig. 3B B″). Weak F-tractin tdTomato 15A 102 germline expression of GFP-Utrophin results in poor labeling of Fertility was assessed by scoring the number of adult progeny per female from a the actin bundles by Utrophin, and the actin bundles are relatively 24 h egg collection. normal in appearance (Fig. 3C C″), although some disorganiza- n % Relative fertility is compared to GAL4 only controls. tion is observed. However, while strong germline expression of olcetkna 63 at taken follicle rjcin f3 of projections i.2. Fig. uliadgria eil hnGPUrpi ssrnl xrse ntegrln (E germline the in expressed strongly is GFP-Utrophin when vesicle germinal and nuclei aasi h otro us el (D cells nurse posterior the in canals aa -ci aeswl hnGPUrpi ssrnl xrse ntegrln,hg eeso F-tohnrsl nnmru eet including defects numerous in result GFP-Utrophin of levels (D high germline, canals the ring in of expressed strongly aggregates is with GFP-Utrophin when well labels F-actin canal aibei h ee fepeso n blt olblcria n igcnlFatn n osntatrFatnmrhlg (C morphology F-actin alter not does and F-actin, canal ring and cortical label to ability and expression of level the in variable (either (B (A toggrln xrsino F-tohnrslsi otclatnbekonadteacmlto fFatni h us elnce uigS.(A S9. during nuclei cell nurse the in F-actin of accumulation the and breakdown actin cortical in results GFP-Utrophin of expression germline Strong ″