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. Author manuscript; available in PMC: 2021 May 1.
Published in final edited form as: Cytoskeleton (Hoboken). 2020 Feb 8;77(5-6):229–237. doi: 10.1002/cm.21599

Comparative analysis of taxol-derived fluorescent probes to assess microtubule networks in a complex live three-dimensional tissue

Gregory Logan 1, Brooke McCartney 1
PMCID: PMC7354890  NIHMSID: NIHMS1578564  PMID: 32012482

Abstract

Drosophila oogenesis is an excellent in vivo model for investigating cytoskeletal dynamics because of the rapid cytoskeletal remodeling that occurs at the end of stage 10; however, there are few robust tools for detecting microtubules in live complex tissues. The recent development of membrane permeable taxol-based fluorescent probes to label microtubules is significant technical progress, but the effectiveness of these probes and the potential stabilizing effects of the taxol derivative have not been well characterized in vivo. Here, we compared three commercially available taxol-derived microtubule labels to determine their efficacy and potential artifacts. We found that all three probes labeled microtubules with differences in permeability, brightness, and signal to noise ratio. Like taxol, however, all of the probes disrupted the F-actin cytoskeleton at higher concentrations. We also found that the efflux pump inhibitor, verapamil, increased the intensity of the label and modestly increased the severity of the F-actin defects. Of the three probes, Tubulin Tracker (ThermoScientific) was the most permeable and was brightest, with the highest signal to noise ratio. Furthermore, washing out the probe after a 30-min incubation significantly reduced the F-actin artifacts without compromising signal brightness.

Keywords: cytoskeleton, drosophila, F-actin, microtubules, oogenesis, taxol

1 ∣. INTRODUCTION

A dynamic cytoskeleton is required to maintain cell structure and to carry out vital cellular functions including cell movement, growth, and division (Fletcher & Mullins, 2010). Many vital components of the cytoskeleton—including F-actin, microtubules, and their regulatory proteins—have been identified and characterized (Goodson & Jonasson, 2018; Pollard, 2016); however, it remains unclear how these proteins interact together to create higher order cytoskeletal structures at specific times and locations in the cell. To answer questions about these specific spatiotemporal events, we require reliable and tractable models for studying F-actin, microtubules, and the proteins that regulate them, live and in vivo. Studies in cultured cells have been invaluable to our understanding of fundamental principles of cytoskeletal organization and dynamics, but they do not adequately represent the diversity of cytoskeletal systems that exist in complex, developing tissues. Developing three-dimensional systems, including whole embryos and adult tissues, fill those gaps (Huelsmann & Brown, 2014; Viktorinová & Dahmann, 2013), but present additional technical challenges for live imaging of the cytoskeleton because the tissues are thicker which can reduce permeability, resolution, and signal to noise ratio (Review, Legent, Tissot, & Guichet, 2015).

Drosophila oogenesis has been an excellent model for discovering many proteins and molecular mechanisms for controlling cytoskeletal assembly, organization, and dynamics (Hudson & Cooley, 2002; Huelsmann & Brown, 2014; Xue & Cooley, 1993). Each Drosophila ovary is composed of 16–20 strings of egg chambers (ovarioles); each egg chamber produces a single oocyte. By late oogenesis (stages 9–11), each egg chamber consists of a syncytium of germ cells including the single oocyte and 15 nurse cells interconnected by F-actin-based ring canals. These are surrounded by a layer of either squamous or columnar somatic follicle cells (Figure 1a, McLaughlin & Bratu, 2015). The nurse cells drive the development of the oocyte by providing it with mRNAs, proteins, and organelles transported into the oocyte through the ring canals. In stage 11, the nurse cell cortex rapidly contracts, expelling the remaining cytoplasmic contents into the oocyte—a process called “dumping” (Figure 1a”). To prevent the large nurse cell nuclei from blocking ring canals while dumping, during stage 10B the nurse cells assemble enormous arrays of F-actin cables that position nurse cell nuclei away from ring canals (Figure 1a’, Hudson & Cooley, 2002; Huelsmann, Ylänne, & Brown, 2013; Tilney, Tilney, & Guild, 1996). We have observed that the cytoplasmic microtubule network at this stage is dense (Figure 1c-e), and some microtubules coalign with individual F-actin cables (Molinar and McCartney, unpublished observations).

FIGURE 1.

FIGURE 1

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Relative fluorescent intensity, signal to noise ratio, and permeability of taxol-derived fluorescent probes in Drosophila nurse cells. (a–a”) Schematic of egg chambers during stages 10A through 11 shows that egg chambers are composed of an oocyte and nurse cells connected by ring canals; F-actin cable arrays develop during stage 10B; and nurse cells contract during stage 11 in a process called “dumping.”

(b) Microtubules can be visualized, but with very poor resolution, by expressing UAS-α-tubulin-GFP using a germline Gal4 driver (MTD-Gal4). (c–e) Brightness corrected images of maximum projections show labeling of nurse cell microtubules after 90-min incubations with Tubulin Tracker, Viafluor, and SiR-Tubulin, respectively. Yellow boxes represent the high magnification insets (c’–e’). (c”–e”) Raw images of c–e show relative signal intensity of the three probes. (f) The maximum pixel intensity along the midline of the raw images like c”–e” (yellow dashed line in c”, normalized to intensity at t0) increased over time, with Tubulin Tracker being significantly brighter. (g) The signal to noise ratio is highest with Tubulin Tracker. (h) Early timepoints during live imaging show microtubules (arrowheads) only at the peripheral edge of the egg chamber.

(i) Microtubules at the peripheral edge of the egg chambers appear significantly earlier in Tubulin Tracker treated egg chambers compared to Viafluor and SiR-Tubulin. (j) Late timepoints during live imaging show microtubules (arrowheads) throughout the egg chamber. (k) Microtubules at the center of the egg chambers appear significantly earlier in Tubulin Tracker treated egg chambers compared to Viafluor and SiR-Tubulin. Scale Bar = 25 μm. *One-way ANOVA, Tukey's Multiple Comparison, N = 6 egg chambers/treatment

Observing F-actin cables live during stage 10B has been possible using several tools to visualize F-actin in egg chambers, including genetically expressing fluorescent F-actin and F-actin-binding proteins (Spracklen, Fagan, Lovander, & Tootle, 2014), and F-actin labeling probes like SiR-actin (our unpublished work), that have been used extensively in cultured cells (Lukinavičius et al., 2014). While these tools have been helpful for understanding the dynamic spatiotemporal events that regulate F-actin cable assembly (Huelsmann & Brown, 2014), thorough analysis of these tools during oogenesis has revealed significant developmental and cytoskeletal artifacts in some cases (Spracklen et al., 2014). Probes for studying microtubules have, until recently, been unreliable or unavailable for systems beyond cultured cells. Fixing and staining microtubules is challenging (Hudson & Cooley, 2014), and while genetically expressed GFP-tagged tubulin labels microtubules in cultured cells with only mild effects on microtubule dynamics (Rusan, Fagerstrom, Yvon, & Wadsworth, 2001), it does not effectively label microtubules in egg chambers at stage 10B (Figure 1b). This is likely due to a high level of free cytosolic tubulin, and reduced resolution due to the thickness of the egg chamber (Legent et al., 2015). Recently, taxol-derived fluorescent probes have been created to bind and label microtubules in live tissue including SiR-Tubulin (Spirochrome, 2016), Viafluor (Biotium, 2017), and Tubulin Tracker (ThermoScientific Inc., 2018). While these probes are potentially useful, they have not been rigorously tested or compared in vivo. Furthermore, some of these probes exhibit microtubule stabilizing effects in vitro, though not as strong as taxol itself (Lukinavičius et al., 2014). Here, we report an analysis of Viafluor, Tubulin Tracker, and SiR-Tubulin in Drosophila nurse cells, comparing fluorescence intensity, signal to noise ratio, permeability, and their potential side effects on the cytoskeleton.

2 ∣. RESULTS AND DISCUSSION

2.1 ∣. Taxol-derived dyes effectively label microtubules in Drosophila nurse cells

We compared three commercially available taxol-derived fluorescent probes coupled to dyes with similar excitation/emission spectra, Tubulin Tracker Deep Red (Ex/Em = 652/669, ThermoScientific), Viafluor-647 (650/675, Biotium), and SiR-Tubulin-652 (652/674, Spirochrome). We imaged egg chambers by incubating them in the recommended dilution of each probe (1:1,000:1 μM for SiR-Tubulin and Tubulin Tracker, unknown concentration for Viafluor) in imaging media (see Section 4) and imaging every 5 min for 90 min. By the end of the imaging period, all three probes labeled microtubules throughout the nurse cells (Figure 1c-e). The signal intensity for all three probes increased steadily through the imaging period, and by the end, the intensity of Tubulin Tracker was significantly higher than the intensity of Viafluor and SiR-Tubulin (Figure 1c”-e”, f). Tubulin Tracker also had the highest signal to noise ratio (Figure 1g). To assess how quickly the probes penetrate the tissue, we determined when microtubules became visible at the periphery of the cells, and when they became visible in the central cytoplasm (Figure 1h-k). Tubulin Tracker labeled peripheral (Figure 1h,i, arrowheads) microtubules faster than Viafluor and labeled central microtubules (Figure 1j, k, arrowheads) faster than either ViaFluor or SiR-Tubulin. As with fluorescence intensity and signal to noise ratio, there were no significant differences between ViaFluor and SiRTubulin. (Figure 1i-k). Thus, Tubulin Tracker was significantly better than the other two probes, demonstrating the highest fluorescence intensity and signal to noise ratio, and the fastest penetration in this tissue. Because the probes tested here are coupled to dyes with very similar excitation/emission spectra, it is unlikely that changing the imaging conditions (e.g., filter optimization) would have a significant effect on the results.

2.2 ∣. Reducing the concentration of Tubulin Tracker significantly decreased brightness, signal to noise ratio, and speed of penetration

Although the recommended dilution for Tubulin Tracker is 1:1000, the manufacturer also recommends optimizing the dilution for the specific tissue (ThermoScientific Inc., 2018). To determine the effective concentration range for Tubulin Tracker, we tested the ability for Tubulin Tracker to label microtubules at lower concentrations (1:5,000 and 1:10,000). While microtubules were labeled at the lower concentrations (Figure 2a-c), the brightness of the signal decreased significantly with each dilution, (Figure 2d). In addition, at the end of the imaging period, the signal to noise ratio was significantly lower with 1:5,000 and 1:10,000 Tubulin Tracker compared to 1:1,000 (Figure 2e). Microtubules also become visible at peripheral and central parts of the egg chamber at significantly earlier times with 1:1,000 than 1:5,000 and 1:10,000 (Figure 2f-g). These results suggest that while diluting Tubulin Tracker below the recommended 1:1,000 allows for microtubule visualization, 1:1,000 is optimal for visualizing microtubules in egg chambers.

FIGURE 2.

FIGURE 2

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Reducing the concentration of Tubulin Tracker significantly decreased brightness, signal to noise ratio, and permeability. (a–c) Brightness corrected images show that microtubules are visible in egg chambers treated with Tubulin Tracker at three dilutions 1:1,000 (a), 1:5,000 (b), and 1:10,000 (c), although microtubules become less visible with each dilution. Yellow boxes represent the high magnification insets (a’–c’). (a”–c”) Raw images of (a)–(c) show relative signal intensity of the three dilutions. (d) The maximum pixel intensity along the midline of egg chambers increased over time, and has lower intensity with each dilution. (e) The signal to noise ratio is significantly lower in egg chambers treated with 1:5,000 and 1:10,000 Tubulin Tracker. (f,g) Microtubules at the peripheral edge and at the center of the egg chambers appear later with lower concentrations of Tubulin Tracker. Scale bar = 25 μm. *One-way ANOVA, Tukey's Multiple Comparison, N = 6 egg chambers/concentration

2.3 ∣. Taxol and taxol-derived probes can cause F-actin cable abnormalities in Drosophila egg chambers

All three fluorescent probes bind microtubules through a taxol-derived domain (See datasheets for SiR-Tubulin, Tubulin Tracker, and Viafluor). Cytochrome has shown that SiR-Tubulin has similar microtubule-stabilizing effects as taxol in tubulin polymerization assays (Lukinavičius et al., 2014), which is possible for Tubulin Tracker and Viafluor as well. Ideally, we would examine microtubule dynamic instability to assess the effects of taxol and these taxol-derived probes on microtubule stability. However, we have been unable to identify microtubule ends, perhaps due to the high density of microtubules in this tissue. Instead, we examined the array of F-actin cables that develop at stage 10B, as their organization and morphology is easily assayed. We first asked whether taxol itself disrupts the F-actin cable array by treating dissected Drosophila ovaries with three different concentrations of taxol: 10 μM (known to stabilize microtubules, Shannon, Canman, Moree, Tirnauer, & Salmon, 2005), 2 μM (showing minimal stabilizing effects, Yang, Inaki, Cliffe, & Rørth, 2012), and 0.2 μM (expected to have minimal effects) or DMSO alone (vehicle). Ovaries were treated for 90 min before fixing and staining with phalloidin to measure the effect of microtubule stabilization on the F-actin cytoskeleton (Figure 3a). In the presence of taxol, but not DMSO alone, we observed concentration dependent defects in the F-actin cable array. The highest concentration of taxol (10 μM) completely blocked F-actin cable assembly in the majority of egg chambers (10/13; Figure 3b,e), and instead we observed clumps of phalloidin positive cytoplasmic F-actin near the cortex (Figure 3b, arrowheads). At 2 μM taxol we observed significantly more F-actin cable assembly (17/22), but found that F-actin cables detached from the cortex (Figure 3c, arrowheads) in approximately one third of the egg chambers with F-actin cables (8/22 cases; Figure 3e). At the lowest concentration of taxol (0.2 μM), all egg chambers (16/16) had F-actin cable assembly, and only 4/16 exhibited F-actin cable detachment (Figure 3d and e). In this way, we established a baseline for the effect of microtubule stabilization on the F-actin cable array.

FIGURE 3.

FIGURE 3

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The effects of taxol and taxol-derived probes on F-actin cables. (a) The paradigm for determining the effect of taxol and taxol-derived probes was to collect whole ovaries, incubate them in differing doses of taxol or taxol-drived probes for 90 min, followed by fixing and staining with phalloidin to observe the effect of the probes on the F-actin cytoskeleton. (b–d) Treating egg chambers with taxol resulted in two concentration dependent F-actin cable defects including a complete loss of cables (b). This was often accompanied by the presence of clumps of phalloidin positive material near the membrane (arrowheads). (c) We also observed F-actin cable detachment from the cortex (arrowheads) in a fraction of taxol-treated egg chambers. (d) Normal F-actin cables were also observed. (e) Quantification of taxol dose-dependent F-actin cable defects. DMSO is the vehicle control. (f and f”) Quantification of dose dependent F-actin cable defects by taxol-derived probes. (g) For the analysis of dose-dependent effects of the fluorescent microtubule probes on F-actin cables, we combined the egg chambers without cables and those with detached cables into a single category of “abnormal cables.” The effects of the three probes were indistinguishable and dose dependent. (ANOVA Tukey's Multiple Comparison, N = 3 replicates, over five egg chambers/replicate). Scale bar = 25 μm

Next we asked whether treatment with the taxol-derived fluorescent probes produced similar defects. Because absolute concentration information is only available for SiR-Tubulin, we could not make direct comparisons between the dilutions of the probes and the concentrations of taxol we used. We treated egg chambers with three dilutions of each probe (1:1,000; 1:5,000; and 1:10,000) for 90 min before fixing and staining with phalloidin, and assessing the egg chambers for loss of F-actin cables or detached F-actin cables. Like taxol, treatment with any of the probes resulted in both types of defects. Complete loss of F-actin cables was rare, and only occurred when the probes were used at 1:1000 (Figure 3f-f”). For statistical analysis, egg chambers that had either F-actin cable loss or detached F-actin cables were grouped into a single category of “abnormal F-actin cables” (Figure 3g). The effect of the three probes on F-actin cables was indistinguishable at all concentrations (Figure 3g). At the recommended dilution of 1:1,000, 55–65% of the egg chambers had abnormal F-actin cables, with the frequency decreasing with decreasing concentration (Figure 3g). These results suggest that using the taxol-derived fluorescent probes at the recommended concentrations results in microtubule stabilization that can have secondary effects on the F-actin cytoskeleton, and presumably other cellular processes that rely on a dynamic microtubule network. Our analysis demonstrates that reducing the working concentration of the probes does significantly reduced the defects (Figure 3g), but our initial characterization of the three probes (Figures 1 and 2) suggests that only Tubulin Tracker would still be effective for microtubule visualization at lower concentrations.

2.4 ∣. The effect of verapamil on microtubule labeling and stabilization by SiR-Tubulin

Although the fluorescence intensity, signal to noise ratio, and permeability of SiR-Tubulin was less than that of Tubulin Tracker (Figure 1f,g), the manufacturer of SiR-Tubulin recommends increasing the signal intensity by using verapamil to inhibit the efflux of the dye (Spirochrome, 2016). We found that adding verapamil (10 μM) to the live imaging media caused the signal intensity of SiR-Tubulin (1:1,000) to increase five-fold compared to treatment with DMSO (Figure 4a”, b”, and c), without affecting the resolution of the microtubules (Figure 4a,a’ and b,b’). Verapamil also slightly increased the signal to noise ratio (Figure 4d), and dramatically decreased the amount of time to visualize microtubules at the peripheral edge, and at the center of the egg chamber (Figure 4e,f). The addition of verapamil also modestly increased the frequency of abnormal F-actin cables compared to vehicle control (Figure 4g). These data suggest that while verapamil may be effective at increasing the brightness, signal to noise ratio, and permeability of SiR-Tubulin, it does so by increasing the internal concentration of the probe, which in turn increases the stabilizing effect of the probe on the MT network.

FIGURE 4.

FIGURE 4

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Effect of verapamil on the labeling efficacy of SiR-Tubulin and associated F-actin cable defects. (a and b) Brightness corrected images show microtubule labeling of nurse cells after 90-min incubations with SiR-Tubulin and verapamil (a) or SiR-Tubulin and DMSO (b). Yellow boxes represent the high magnification insets (a’ and b’). (a” and b”) Raw images of (a) and (b) show relative signal intensity of SiR-Tubulin with verapamil versus DMSO. (c) The maximum pixel intensity along the midline of egg chambers is five-fold higher in egg chambers treated with SiR-Tubulin and verapamil compared to SiR-Tubulin and DMSO. (d) Egg chambers treated with SiR-Tubulin and verapamil have a slightly higher signal to noise ratio than egg chambers treated with SiR-Tubulin and DMSO. (e and f) Microtubules at the peripheral edge of the egg chambers (e) and at the center of the egg chambers (f) appeared earlier when treated with verapamil and SiR-Tubulin. (g) Treating egg chambers with SiR-Tubulin and verapamil significantly increased the percent of abnormal egg chambers compared to SiR-Tubulin and DMSO (Two-way ANOVA, N = 3 replicates, over five egg chambers/replicate). Scale Bar = 25 μm. *Two-way t test, N = 6 egg chambers/treatment

2.5 ∣. Tubulin Tracker incubation and washout decreased the side effects on the F-actin cytoskeleton while maintaining effective microtubule labeling

The protocols for each probe suggest that the tissues should be incubated for 30 min in the probe followed by a washout before imaging (ThermoScientific Inc., 2018). We tested whether shortening exposure to the probe through a washout could decrease the artifacts associated with Tubulin Tracker while maintaining robust microtubule labeling. We treated egg chambers with Tubulin Tracker at 1:1,000 for 30 min, washed three times with live imaging buffer, and then imaged the egg chambers for 90 min (Figure 5a). We compared this to a control, where we treated the egg chambers with DMSO alone for 30 min, washed three times with live imaging buffer, and then imaged the egg chambers for 90 min while incubating the tissue in live imaging buffer containing Tubulin Tracker at 1:1,000. Tubulin Tracker labeled microtubules in both the washout (Figure 5b-b”) and the control (Figure 5c-c”). At the end of the imaging period, the signal intensity of the washout group was significantly lower than the control (Figure 5d), but there was a small but significant increase in signal to noise ratio (Figure 5e). Measuring the signal intensity over time demonstrated that the washout was effective at preventing the gradual increase in signal intensity that was observed in the control (Figure 5d). Peripheral and central microtubules were immediately visible at the beginning of the imaging period after washout (Figure 5f-g). Furthermore, the washout treatment reduced the frequency of artifacts at 1:1,000 (Figure 5h). Although the percent of egg chambers with abnormal F-actin cables in the control group (Figure 5h, control) is less than we observed in our previous measures of Tubulin Tracker (Figure 3g, Tubulin Tracker), these measurements are not significantly different (p = .7 Two-way ANOVA). These results suggest that washout may be an effective way of decreasing the stabilizing effects and consequent artifacts caused by this taxol-derived fluorescent probe.

FIGURE 5.

FIGURE 5

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The effect of Tubulin Tracker washout on microtubule labeling and F-actin cytoskeleton disruption. (a) For the washout experiments, we treated egg chambers for 30 min with Tubulin Tracker (Washout) or DMSO (Control) followed by three rinses in live imaging media and 90 min of imaging in live imaging media with DMSO (Washout) or Tubulin Tracker (Control). (b and c) Brightness corrected images show labeling efficiency of nurse cells in the washout group treated with 1:1,000 Tubulin Tracker (b) or the control (c) at the end of the 90-min imaging period. Yellow boxes represent the high magnification insets (b’ and c’). (b” and c”) Raw images of (b) and (c) show relative signal intensity of the washout versus control. (d) The maximum pixel intensity along the midline of egg chambers remains relatively constant over the imaging period for the washout and increases in the control. (e) Washout treatment increased the signal to noise ratio compared to control. (f and g) Microtubules are immediately visible at the peripheral edge and in the center of the egg chambers after washout. (h) Washout of Tubulin Tracker significantly decreased the frequency of F-actin cable defects on the F-actin cytoskeleton at the highest concentration of Tubulin Tracker (Two-way ANOVA, Bonferroni's Multiple Comparison; N = 3 replicates, over five egg cambers per replicate). Scale bar = 25 μm. *Two-way t test, N = 6 egg chambers/treatment

3 ∣. CONCLUSION

Here we tested the efficacy and side effects of three taxol-derived microtubule-binding fluorescent probes in Drosophila egg chambers. While all three probes labeled microtubules, Tubulin Tracker exhibited the highest fluorescence intensity and signal to noise ratio, and was the fastest to penetrate the tissue. Like taxol alone, SiR-Tubulin, ViaFluor, and Tubulin Tracker caused defects in the F-actin cytoskeleton that we attribute to taxol-dependent microtubule stabilization. In all cases, reducing the concentration of the probe significantly reduced the frequency of F-actin defects, but our data suggest that only Tubulin Tracker retains strong labeling at those lower concentrations. While the recommended efflux inhibitor verapamil used with SiR-Tubulin did increase fluorescence intensity and signal to noise ratio, and reduce penetration time to levels similar to Tubulin Tracker, verapamil with SiR-Tubulin also modestly increased the frequency of F-actin defects. Finally, we showed that labeling with the most effective probe, Tubulin Tracker, could be improved by washing out the probe before imaging. Taken together, these results demonstrate that with careful optimization these probes are an important addition to the cell biologist's toolkit. While Tubulin Tracker outperformed the others in the challenging cellular environment of the Drosophila ovary, it is possible that in simpler cell culture systems SiR-Tubulin and Viafluor may have improved performance. Because all of the probes have stabilizing properties, their utility for measuring microtubule dynamics may be limited. However, our results suggest that they will be useful for assessing microtubule organization, assembly, and inter-actions with other proteins and organelles. Furthermore, their ease of use will greatly enhance the efficiency of screening mutant tissues for microtubule-associated changes without the significant labor of introducing a genetically encoded microtubule label into each mutant background.

4 ∣. MATERIALS AND METHODS

4.1 ∣. Flies

The following stocks were used: w1118, MTD-Gal4 (Bloomington #31777), UAS-α-TubulinGFP (Bloomington #7373).

4.2 ∣. Live imaging of microtubules and quantitative assays

24 hr before live imaging, w1118 flies were fed wet yeast paste to promote egg production. Stage 10B egg chambers were isolated for live imaging as described by Spracklen and Tootle (2013). Egg chambers were imaged in live imaging medium consisting of Schneider's media plus 20% FBS, 5 μg/ml insulin, 2 mg/mL trehalose, 5 μM methoprene, 1 μg/ml 20-hydroxyecdysone, and 50 ng/ml adenosine deaminase. For each treatment, 1 mL of imaging medium was spiked with 1 μL of Tubulin Tracker Deep Red (ThermoScientific #T34077, Waltham, MA, USA), Viafluor 647 (Biotium, 70063, Hayward, CA, USA), SiR-tubulin (Spirochrome, CY-SC002, Stein am Rhein, Switzerland), or DMSO (Sigma, 67-68-5) and imaged in glass bottom culture (MatTek #P35G-0.170-14-C). For each egg chamber, 10 z-sections were taken every 0.5 μm between 22.5 and 27.5 μm from the coverslip. Each image was acquired with a 100 ms exposure every 5 min for 90 min on a spinning disc microscope with a X-Light V2 scan head (Crest Optics) and a Prime95B CMOS camera (Photometrics) on a Zeiss Axiovert 200 M using Metamorph software (Molecular Devices). We excited the fluorophores using a Celesta laser (Lumencore) at a wavelength of 635 nm and used a 697/60 emissions filter (Chroma). All intensity measurements and brightness adjustments were done on maximum projections using ImageJ. To measure fluorescence intensity of microtubules, five lines were drawn across the center axis of the egg chamber (Figure 1c”), and the values were reported as the average maximum intensity of the three lines. Signal to noise ratio was measured as the maximum intensity along a microtubule divided by the average intensity along a line directly adjacent to the microtubule. Peripheral and central microtubule labeling was measured as the time point when microtubules first became visible at the peripheral or central regions of each egg chamber (Figure 1h-k). All measurements were plotted using Prism 8 software (GraphPad).

4.3 ∣. Assessing the F-actin cytoskeleton in fixed tissue

w1118 flies were fed with yeast paste 24 hr before ovaries were collected, and incubated in live imaging media with taxol, taxol-derived dyes, or DMSO on an orbital shaker (80 RPM) for 90 min at room temperature. Ovaries were fixed and stained as described previously (McCartney, Price, & Webb, 2006) using phalloidin-488 (ThermoFisher, A12379). Egg chambers were imaged using either a Zeiss Axiovert 200M (described above) or an Andor Revolution XD spinning disk confocal microscope. All stage 10B egg chambers were imaged and categorized as having normal, detached, or absent F-actin cables. Each treatment was replicated three times with at least five egg chambers classified per replicate. The percent of abnormal cables was determined as the combined percent of detached and absent F-actin cables.

ACKNOWLEDGEMENTS

Thank you to Haibing Teng and the Molecular Biosensor and Imaging Center (MBIC) at Carnegie Mellon University for providing us with imaging equipment. This work was funded by a grant from the National Institutes of Health (R01-GM120378).

Funding information

National Institutes of Health, Grant/Award Number: R01-GM120378

Footnotes

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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