Name: visualizing cytoskeleton-dependent trafficking of lipid-containing organelles in drosophila embryos
Uploaded: 2021-12-13T13:00:00
Duration: 8 min 55 s
Description: Watch this Scientific Journal Video about Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos at JoVE.com

We thank Pakinee Phromsiri, Brian Jencik, Jinghong (James) Tang, and Roger White for their comments on the manuscript. We thank Patrick Oakes, Stefano Di Talia, and Victoria Deneke for sharing their expertise on how to perform PIV analysis. This work was supported by National Institutes of Health grants F31 HD100127 (to M. D. K.) and R01 GM102155 (to M. A. W).

     

<ol>
	<li>Chowdhary, S., Tomer, D., Dubal, D., Sambre, D., Rikhy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondrial+organization+and+function+in+the+Drosophila+blastoderm+embryo.">Analysis of mitochondrial organization and function in the Drosophila blastoderm embryo.</a> Scientific Reports. 7 (1), 5502 (2017).</li><li>Mavor, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab8+directs+furrow+ingression+and+membrane+addition+during+epithelial+formation+in+Drosophila+melanogaster.">Rab8 directs furrow ingression and membrane addition during epithelial formation in Drosophila melanogaster.</a> Development. 143 (5), 892-903 (2016).</li><li>Riggs, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+cytoskeleton+remodeling+during+early+Drosophila+furrow+formation+requires+recycling+endosomal+components+Nuclear-fallout+and+Rab11.">Actin cytoskeleton remodeling during early Drosophila furrow formation requires recycling endosomal components Nuclear-fallout and Rab11.</a> Journal of Cell Biology. 163 (1), 143-154 (2003).</li><li>Welte, M. A., Gross, S. P., Postner, M., Block, S. M., Wieschaus, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+vesicle+transport+in+Drosophila+embryos:+forces+and+kinetics.">Developmental regulation of vesicle transport in Drosophila embryos: forces and kinetics.</a> Cell. 92 (4), 547-557 (1998).</li><li>Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=As+the+fat+flies:+The+dynamic+lipid+droplets+of+Drosophila+embryos.">As the fat flies: The dynamic lipid droplets of Drosophila embryos.</a> Biochimica et Biophysica Acta. 1851 (9), 1156-1185 (2015).</li><li>Deneke, V. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organized+nuclear+positioning+synchronizes+the+cell+cycle+in+Drosophila+embryos.">Self-organized nuclear positioning synchronizes the cell cycle in Drosophila embryos.</a> Cell. 177 (4), 925-941 (2019).</li><li>Welte, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+lipid-droplet+transport+by+the+perilipin+homolog+LSD2.">Regulation of lipid-droplet transport by the perilipin homolog LSD2.</a> Current Biology. 15 (14), 1266-1275 (2005).</li><li>Bailey, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant+role+for+lipid+droplets+in+a+stem+cell+niche+of+Drosophila.">Antioxidant role for lipid droplets in a stem cell niche of Drosophila.</a> Cell. 163 (2), 340-353 (2015).</li><li>Johnson, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2Av+buffering+via+dynamic+sequestration+to+lipid+droplets+in+Drosophila+embryos.">H2Av buffering via dynamic sequestration to lipid droplets in Drosophila embryos.</a> Elife. 7, 36021 (2018).</li><li>Lowe, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+expression+patterns,+subcellular+localisations+and+interaction+partners+of+Drosophila+proteins+using+a+pigP+protein+trap+library.">Analysis of the expression patterns, subcellular localisations and interaction partners of Drosophila proteins using a pigP protein trap library.</a> Development. 141 (20), 3994-4005 (2014).</li><li>Rambold, A. S., Cohen, S., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+trafficking+in+starved+cells:+regulation+by+lipid+droplet+lipolysis,+autophagy,+and+mitochondrial+fusion+dynamics.">Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics.</a> Developmental Cell. 32 (6), 678-692 (2015).</li><li>Rand, M. D., Kearney, A. L., Dao, J., Clason, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeabilization+of+Drosophila+embryos+for+introduction+of+small+molecules.">Permeabilization of Drosophila embryos for introduction of small molecules.</a> Insect Biochemistry and Molecular Biology. 40 (11), 792-804 (2010).</li><li>Tran, S. L., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vivo+centrifugation+of+Drosophila+embryos.">In-vivo centrifugation of Drosophila embryos.</a> Journal of Visualized Experiments. (40), (2010).</li><li>Rothwell, W. F., Sullivan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+embryo+collection.">Drosophila embryo collection.</a> CSH Protocols. 2007, (2007).</li><li>Wieschaus, E., N&#252;sslein-Volhard, C., Roberts, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A Practical Approach. , 179-214 (1998).</li><li>. The Interactive Fly: Stages of Development and Mitotic Domains Available from: <a target="_blank" href="https://www.sdbonline.org/sites/fly/aimain/2stages.htm">https://www.sdbonline.org/sites/fly/aimain/2stages.htm</a> (1999)</li><li>Liberzon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> OpenPIV/openpiv-python: OpenPIV - Python (v0.22.2) with a new extended search PIV grid option. , (2020).</li><li>Markow, T. A., Beall, S., Matzkin, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egg+size,+embryonic+development+time+and+ovoviviparity+in+Drosophila+species.">Egg size, embryonic development time and ovoviviparity in Drosophila species.</a> Journal of Evolutionary Biology. 22 (2), 430-434 (2009).</li><li>Gross, S. P., Welte, M. A., Block, S. M., Wieschaus, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynein-mediated+cargo+transport+in+vivo.+A+switch+controls+travel+distance.">Dynein-mediated cargo transport in vivo. A switch controls travel distance.</a> Journal of Cell Biology. 148 (5), 945-956 (2000).</li><li>Debarre, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+lipid+bodies+in+cells+and+tissues+using+third-harmonic+generation+microscopy.">Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy.</a> Nature Methods. 3 (1), 47-53 (2006).</li><li>Dou, W., Zhang, D., Jung, Y., Cheng, J. X., Umulis, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+imaging+of+lipid-droplet+intracellular+motion+in+early+Drosophila+embryos+using+femtosecond-stimulated+Raman+loss+microscopy.">Label-free imaging of lipid-droplet intracellular motion in early Drosophila embryos using femtosecond-stimulated Raman loss microscopy.</a> Biophysical Journal. 102 (7), 1666-1675 (2012).</li><li>Barry, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injecting+Gryllus+bimaculatus+Eggs.">Injecting Gryllus bimaculatus Eggs.</a> Journal of Visualized Experiments. (150), (2019).</li><li>Le Trionnaire, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+protocol+for+targeted+mutagenesis+with+CRISPR-Cas9+system+in+the+pea+aphid.">An integrated protocol for targeted mutagenesis with CRISPR-Cas9 system in the pea aphid.</a> Insect Biochemistry and Molecular Biology. 110, 34-44 (2019).</li></ol>

The authors have no conflicts of interest to disclose.

The Drosophila embryo is a powerful and convenient model to study fundamental questions in cellular and organismal biology. Its relative simplicity, powerful genetics, and small size make it an excellent system for imaging both cellular processes and development. Here, a standard microinjection protocol is adapted to enable FP usage in embryos. This approach allows for fluorescent imaging of specific cellular structures without the need for genetically encoded fluorophores, opening many genetic backgrounds to imaging. Combining multiple dyes plus strategically chosen fluorescently tagged proteins can open multichannel live imaging spanning the whole spectrum of visible light.
Critical steps in the protocol: 
This protocol uses BODIPY 493/503 to label LDs. This approach can easily be adapted to mark other cellular structures. For subsequent image analysis, one of the most important factors is the signal-to-noise ratio, i.e., the brightness of the dye compared to the background signal. Lysosomes have been successfully imaged (LysoTracker Red, 1 mM), as well as mitochondria (Mitoview 633, 200 &#181;M), the ER (ER tracker Green, 10 &#181;M), and microtubules (SiR tubulin, 200 nM in DMSO), as shown in Figure&#160;2, Figure 3, Figure 4, Figure&#160;5. In addition, yolk vesicles are autofluorescent and give off blue light upon UV excitation (image using 405 nm as the excitation wavelength (Figure 6)). For other dyes, aim for the dye concentration to be 100-1,000x of what would be needed for staining cultured cells live; this is similar to the concentration of a stock solution that would be diluted into cell culture media. As this protocol calls for an injection of 100 fL and the Drosophila embryo is roughly 9 nL in volume18, these dye concentrations will average out to an internal embryonic concentration of under 1/100th of what is present in the cell culture media. Temporarily, the local concentration will be higher at the site of injection, which is most relevant for FPs that do not diffuse well (i.e., mitochondrial dyes and SiR-tubulin). For these FPs, start at the recommended high concentrations; if unexpected death is observed, successively dilute two-fold until an acceptable compromise between survival and signal strength is reached.
When co-injecting multiple dyes, both dyes should either be in the same solvent, or both the solvents and dyes should be compatible with the mixture (alcohol concentrations exceeding ~10% are not recommended).
The quality of the needles is essential for the success of this procedure, as the tip needs to be as fine as possible. Otherwise, damage from the injection wound can compromise the subsequent development of the embryo. As commercial needle pullers differ, it is important to follow the suggestions of the manufacturer and try out multiple pulling parameters until the desired shape is achieved. It is critical to perform the quality control step 1.1.3 as working with a cracked, jagged, or large bore needle tip will make the successful injection more difficult or even impossible.
The embryo needs to be partially desiccated so that additional volumes of liquid can be added during the injection. If the embryo is under-desiccated, the needle will not enter easily, and cytoplasm will squirt out as the needle penetrates or as the solution is injected. If the embryo is over-desiccated, it will look deflated and will not develop properly. The exact drying time depends on local conditions, e.g., air humidity, and can change from day to day. It has to be determined empirically for each session.
The ability of the embryo to survive after microinjection depends critically on the quality of the needle, proper desiccation, and limiting injection volume to less than 1 pL (ideally 100 fL). As long as these parameters are optimized, no significant toxicity is apparent when the described dyes are injected at the recommended concentrations. If embryos survive the desiccation and injection steps, they typically develop successfully well into germ-band extension, an exception being the microtubule and ER probes which caused cellularization defects at high levels (100 fL injection of the stock concentration of each). Testing found no obvious developmental defects when DMSO, water, and mixtures of the two were injected at the recommended volumes, with an embryo survival rate through germ-band extension of ~75% or more. Injection volumes more than over 1 pL caused defects and embryos injected with ~4 pL volumes developed for less than 1 h. Therefore, injection volumes need to be kept low, which means that dye concentrations have to be high.
Generally, injections along the lateral edge of the embryo are recommended as those result in the least damage. However, the injection site may need to be adjusted depending on the diffusive properties of the FPs employed. BODIPY 493/503 and LysoTracker diffuse faster across the entire embryo than Lipid Spot 610 (another dye to mark LDs), while SiR-Tubulin and Mitoview 633 never diffuse fully across the embryo (imaging as late as 7 h post-injection). Thus, injection in or near the site of interest may be necessary. When injecting in the anterior or posterior regions, a particularly fine needle is recommended.
Image acquisition relies on confocal microscopy to optically section and resolve small organelles and all cytoskeletal components. Techniques requiring analysis of many images (e.g., STORM or PALM) will not work because the embryonic contents are in motion and the fluorophores are not optimized for photoswitching. Epifluorescence microscopy lacks the lateral and axial resolution to make out most organelles and smaller cellular structures. For these reasons, it is strongly recommended to use a confocal microscope or employ light sheet technology.
The reproducibility of the image analysis relies greatly on consistent imaging data. For the greatest chance of success, optimization of the injection technique and image acquisition is required. Establishing and practicing a technique where the dye(s) of interest, site of injection, age of the embryo, injection volume, and acquisition setup are all consistent will generate the most robust data for image analysis.
Modifications and troubleshooting of the method 
This protocol demonstrates a method for analyzing the bulk flow of LDs in cleavage stage embryos using particle image velocimetry. The same approach can be used for other organelles, other developmental stages, and other analysis methods. For example, Figure 2 shows analysis of LDs and acidic organelles flowing in the syncytial stages of embryogenesis, visualized by co-injecting BODIPY 493/503 and LysoTracker Red. Further successful imaging of LD motion in embryos up to 7 h post-fertilization has been achieved; these embryos do retain the injection wound but are able to develop for several hours.
Data gathered using this protocol has been used for particle image velocimetry, but many other analysis techniques are available. For example, particle tracking programs like those found in ImageJ, Imaris, or manual tracking can be used to obtain velocities and directionalities of moving structures. Note that most such tracking software are built to work with data from planar cell culture systems and do not always adapt well to 3D structures like the Drosophila embryo. Further, for the generation of the best quality particle tracking data, multiple Z planes would need to be imaged; this should be feasible if image stack acquisition times are under ~2 s. This benchmark should be reachable on spinning disc confocal, lattice light sheet, and recent laser scanning confocal systems. However, the feasibility of particle tracking for abundant organelles such as LDs, mitochondria, and lysosomes is low as the amount of positive signal in a field of view is too high for the current tracking methods. Tracking of less abundant structures like nuclei or yolk vesicles may be possible. PIV for flow analysis works well for LDs and acidic organelles in cleavage stages because both organelles move freely. Organelles like nuclei, ER, and mitochondria are tethered to other cellular structures and thus do not move freely and are not suited to software analysis that assumes free motion. The investigator should pick the techniques best suited to the organelle of interest.
During the syncytial and cellular blastoderm stages, LDs (as well as some other organelles) move along radially oriented microtubules5. It is therefore possible to find cross-sectional views (like in Figure 5) where single microtubules are in focus for long distances, allowing particle tracking in 2D. Since these optical planes are deep within the embryo, overall signal strength is diminished, and the signal-to-noise ratio is reduced.
For tracking analysis, imaging as fast as possible can reveal crucial details of the motion and thus of the motile machinery. For example, lipid-droplet motion is a mixture of two motile states, slow-short motion (~200 nm/s; average travel distance ~100 nm) and fast-long motion (~450 nm/s; average travel distance ~1,000 nm)19; thus, if images are taken every second or even less frequently, the slow-short state becomes undetectable. However, frequent imaging also induces fluorophore bleaching and phototoxicity. Imaging conditions, therefore, have to be adjusted depending on the exact question to be addressed.
Limitations of the method 
Depending on the dye desired, the method can be limited by the compatibility between the dye solubility and the toxicity of the injection solution. Alcohols like isopropanol and ethanol are difficult to handle within a needle due to their lower viscosity and appear to damage cellular components and kill the embryo.
The method is also not well suited for visualizing the earliest steps in embryogenesis because it takes 30+ min to prepare the embryos for injection. At room temperature, the initial cell cycles of the embryo are just ~10 min long each; so, even if one were to pick a newly fertilized egg in step 5.2.3, the first few cell cycles would already be completed by the time the embryo is ready for imaging.
Illumination with light in the UV/blue range is considerably more phototoxic than for longer wavelengths. Under such conditions (e.g., to follow autofluorescent yolk vesicles; Figure 6), one has to limit imaging time (leading to shorter time series) or use lower laser power (resulting in a reduced signal-to-noise ratio).
After cellularization, dyes injected in a specific location tend to diffuse poorly, as they must traverse many cell membranes. This limits the region of observation in later developmental stages.
The significance of the method with respect to existing/alternative methods 
The motion of LDs and other lipid-containing organelles in early embryos can be visualized with genetically encoded fluorophores, label-free techniques, and by the introduction of FPs. The latter can be achieved by permeabilization of the vitelline membrane12 or the microinjection approach discussed here.
Genetically encoded fluorophores are versatile markers whose levels are typically highly reproducible from embryo to embryo. However, they have lower quantum yields and bleach more easily than FPs. Typically, they are only available in one or two tagged version(s) (e.g., GFP or mCherry), limiting the choice of which structures can be imaged simultaneously. FPs, on the other hand, often exist in a large variety; for example, various lipid-droplet specific dyes are available with emission spectra from Autodot in the UV/blue spectrum to Lipidtox and LipidSpot 610 in the far-red spectrum. FPs can also be directly applied to any strain of interest, and thus do not require strain construction to, for example, introduce the desired organelle marker into a mutant strain of interest. This advantage is particularly pronounced when multiple structures are to be labeled simultaneously; instead of time-consuming crosses spanning multiple generations, this can be achieved in a single day by mixing the relevant dyes and introducing them at the same time. Finally, if cellular processes are to be probed with pharmacological inhibition, drugs and dyes can be introduced together.
Label-free methods are very powerful approaches for detecting specific cellular structures. For example, LDs can be specifically detected in early embryos by third-harmonic generation microscopy20 or by femtosecond Stimulated Raman Loss microscopy21. Like FPs, these approaches can be applied in any genetic background, and because they do not cause bleaching, they potentially allow for faster image acquisition. However, they are typically limited to specific organelles and thus do not by themselves support multiplex imaging; they also require specialized microscopes.
There are two general strategies for introducing small molecules into embryos. One is the microinjection approach employed here; the other is chemical (terpene) treatment to permeabilize the vitelline membrane. The latter approach12 is less involved than microinjection, but also more variable from embryo to embryo. In addition, after permeabilization, the protection provided by the vitelline membrane is compromised and the embryo proper is accessible to the external medium, making it more challenging to keep it alive. Microinjection is much less likely to derail embryonic development than permeabilization. However, permeabilization is recommended if many embryos need to be monitored simultaneously, e.g., for drug screening purposes. To follow the movement of cellular structures and obtain reproducible image series suitable for image analysis, microinjection is the method of choice.
Importance and potential applications of the method in specific research areas 
The Drosophila embryo is an important model system for studying many cell-biological and developmental processes1,5,6. Tagging organelles with fluorescent proteins has made major contributions to the understanding of how the early embryo develops, how various organelles traffic, and how such trafficking is modulated developmentally and genetically. However, their propensity to bleach and the challenges of generating strains in which multiple organelles are labeled with different colors limit the application of this approach. The use of FPs introduced by microinjection solves many of these challenges and can even be combined with fluorescently tagged proteins. This technique allows for the imaging of multiple organelles, cell structures, and cytoskeletal components in any genetic background. As a result, several genotypes can be compared via live imaging, making it possible to determine the effect of mutations on the trafficking of multiple organelles.
This protocol demonstrates the FP injection approach for embryos of Drosophila melanogaster, but in principle, this approach applies to any insect eggs for which microinjection techniques have been established, including other species of Drosophila, crickets22, and aphids23.

Vital dyes and molecular probes are powerful tools to image specific cellular structures and organelles live. In the Drosophila embryo, many different organelles display cytoskeleton-driven localization1,2,3,4, but the application of these small molecules is challenging because the eggshell is impermeable to many of them. This protocol describes a method to use fluorescent probes (FPs) in live embryos via microinjection in order to detect large-scale trafficking of organelles. The procedure covers preparation of the injection solution, egg collection and preparation of embryos, microinjection, imaging, and image analysis.
Dramatic spatial rearrangements of organelles are common in many animal oocytes, eggs, and embryos, in part because of the large size of these cells. In the Drosophila embryo, for example, lipid droplets (LDs) and yolk vesicles move toward the embryo center just before cellularization5. This motion depends on microtubules and leaves a ~40 &#181;m region all around the periphery of the embryo depleted of the two organelles. During earlier cleavage stages, many organelles are transported by cytoplasmic flows that are driven by actin-myosin-based contractions at the embryo surface6. Although the embryos of many species exhibit similar rearrangements, the Drosophila embryo is particularly suited for following these processes by imaging because it develops externally at standard laboratory &#39;room temperature&#39;, is relatively transparent, small enough to fit on most microscope setups, and can be manipulated using powerful genetic tools.
For some organelles, fluorescently tagged proteins are available that specifically label these structures. For example, LSD-2 (also known as dPLIN2) is a protein that in embryos specifically targets LDs7. Fly lines are available that carry either inducible transgenes encoding a fusion between Green Fluorescent Protein (GFP) and LSD-28 or a gene trap in which yellow fluorescent protein (YFP) is inserted into the coding region of the endogenous LSD-2 gene9,10. However, this approach has limitations, including that these fusion proteins have low quantum yields and tend to bleach easily. In addition, labeling multiple different structures simultaneously can be challenging: for many organelles, only one type of fluorescent tag (often GFP or mCherry) is currently available, so imaging two organelles at the same time may require new transgenes or insertions; also, even if compatible tags are available, introducing them into a single strain can require time-consuming crosses. It also makes using the many powerful genetic resources less convenient, e.g., if two organelle markers, a Gal4 driver, and an inducible RNAi construct all have to be present in the same mother.
In principle, these limitations can be overcome with the use of FPs, including vital dyes (e.g., LysoTracker to mark lysosomes), molecular probes (e.g., SiR-tubulin to label microtubules), and fluorescently labeled biological molecules (e.g., C12 BODIPY to probe fatty acid metabolism11). From use in cultured cells, they are typically well-validated as powerful tools for probing cellular biology. FPs are versatile, have superior photo properties, and are compatible with fluorescent proteins. Multiple dyes can be mixed and applied simultaneously, often with the benefits of separation into the violet/blue and far-red areas of the spectrum and small Stokes shifts, preventing channel bleed through. Small Stokes shifts allow for simultaneous capture of multiple imaging channels, enabling the tracking of several organelles at once. Finally, they can equally be applied to labeling organelles in the embryos of other Drosophila species or even other insects where no fluorescently tagged proteins may be available.
However, most of these FPs cannot traverse the elaborate eggshell of the Drosophila embryo. It consists of five layers: three outer chorionic layers (chorion) that prevent mechanical damage plus a waxy layer surrounding the vitelline membrane that creates a chemical barrier12. For simplicity, the combination of the waxy layer and the vitelline membrane will be referred to as the &#34;vitelline membrane&#34; below. To bypass the eggshell, this protocol adapts an established embryo microinjection approach to introduce FPs into the Drosophila embryo. The protocol describes how to monitor the cytoplasmic flow of LDs in cleavage stage embryos. It includes the preparation of injection needles and egg collection cages, the process of egg collection, and mechanical removal of the chorion. It goes over how to microinject and image the embryos and how to analyze the bulk flow of LDs using particle image velocimetry (PIV, adapted from6). It provides advice on troubleshooting to ensure embryo survival and create the best system for imaging. Also discussed is how the protocol can be modified to simultaneously image LDs and microtubules or to apply it to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the endoplasmic reticulum (ER).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AUTODO</td>
			<td>abcepta</td>
			<td>SM1000a</td>
			<td>Stains lipid droplets in violet/blue</td>
		</tr>
		<tr>
			<td>BODIPY 493/503</td>
			<td>ThermoFisher</td>
			<td>D3922</td>
			<td>Stains lipid droplets in green</td>
		</tr>
		<tr>
			<td>Desiccant Beads &#8211;Desiccant-Anhydrous Indicating Drierite</td>
			<td>W.A. Hammond Drierite Company</td>
			<td>21001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope SteREO DiscoveryV20 with transillumination base</td>
			<td>Zeiss</td>
			<td>4350030000000000</td>
			<td></td>
		</tr>
		<tr>
			<td>Double sided tape-Permanent Double-Sided Tape</td>
			<td>Scotch (3M)</td>
			<td>-</td>
			<td>Sold by many vendors</td>
		</tr>
		<tr>
			<td>ER-Tracker Green (BODIPY FL Glibenclamide)</td>
			<td>ThermoFisher</td>
			<td>E34251</td>
			<td>Stains the ER/nuclear envelope</td>
		</tr>
		<tr>
			<td>Femptotip II</td>
			<td>Eppendorf</td>
			<td>H129354N</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Glass capillaries -Glass Thinw w/fil 1.0mm 4in</td>
			<td>World Precision Instruments</td>
			<td>TW100F-4</td>
			<td>Must be pulled</td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>-</td>
			<td>-</td>
			<td>Buy the appropriate refractive index for your objective lens</td>
		</tr>
		<tr>
			<td>Glass slides -Double Frosted Microscope Slides precleaned</td>
			<td>FisherBrand</td>
			<td>12-550-343</td>
			<td></td>
		</tr>
		<tr>
			<td>Halocarbon oil 27</td>
			<td>Sigma-Aldrich</td>
			<td>H8773-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Halocarbon oil 700</td>
			<td>Sigma-Aldrich</td>
			<td>H8898-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Heptane 
			greener alternative 
			anhydrous, 99%</td>
			<td>Sigma-Aldrich</td>
			<td>246654-1L</td>
			<td>Heptane is considered toxic by the USA&#39;s OSHA</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>Sp5</td>
			<td>Many different types of confocal microscopes will work.</td>
		</tr>
		<tr>
			<td>LipidSpot 610</td>
			<td>Biotium</td>
			<td>#70069</td>
			<td>Stains lipid droplets in far red</td>
		</tr>
		<tr>
			<td>LysoTracker Red</td>
			<td>ThermoFisher</td>
			<td>L7528</td>
			<td>Stains lysosomes in red</td>
		</tr>
		<tr>
			<td>MitoView 633</td>
			<td>Biotium</td>
			<td>#70055</td>
			<td>Stains mitochondria</td>
		</tr>
		<tr>
			<td>Needle loading pipette tips - 20uL microloader tips</td>
			<td>Eppendorf</td>
			<td># 5242956.003</td>
			<td></td>
		</tr>
		<tr>
			<td>P-1000, Next Generation Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000</td>
			<td>The settings used are heat-470, pull-70, velocity-60, delay-90, pressure-200, ramp-479 at 1x1. They refer to the intensity and length of the heating, the timing of pulling force and whether the force is applied linearly. Using these conditions, one capillary generates two usable needles with fine openings at the tip.</td>
		</tr>
		<tr>
			<td>SiR-Tubulin</td>
			<td>Cytosketon Inc</td>
			<td>CY-SC014</td>
			<td>Made by Spirochrome; Cytoskeleton Inc. is the North American distributor. Stains microtubules in far red</td>
		</tr>
		<tr>
			<td>TransferMan</td>
			<td>Eppendorf</td>
			<td>&#160;5178 NK</td>
			<td></td>
		</tr>
		<tr>
			<td>ViaFluor 405</td>
			<td>Biotium</td>
			<td>#70064</td>
			<td>Stains microtubules in violet/blue</td>
		</tr>
	</tbody>
</table>

visualizing cytoskeleton-dependent trafficking of lipid-containing organelles in drosophila embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Following injection, the dye will be localized only at the site where the needle tip was inserted. The dye will then diffuse away from the injection site depending on its diffusive characteristics. Figure 1 shows injection of BODIPY 493/503, soon after injection (panel A) and 24 min later (panel B). After 24 min, the dye has made it to roughly the midpoint of the embryo&#39;s long axis.
Analyzing organelle motility can be achieved through dye injections and time-lapse imaging. In Figure 2, an embryo was co-injected with BODIPY 493/503 (Figure 2A) and LysoTracker Red (Figure 2B) and imaged using laser excitation at 488 and 596 nm, respectively. This embryo was then time-lapse imaged (one frame every 30 s for 30 min, 5 min analyzed). The time series was then run through PIV analysis, the output of which is shown via streamlines in Figure 2A,B. Note that the streamlines do not represent the trajectory of individual particles, but the cytoplasmic flow is inferred from analyzing all the particles in that region of the cytoplasm. Through labeling of two independent cellular structures (LDs and acidic organelles), the PIV analysis finds similar flows, with both labels converging on the central region of the embryo where the cytoplasm is flowing into the embryo&#39;s interior6.
Currently available FPs allow the labeling of many other organelles and cellular structures. Figure 3 shows the labeling of the ER via ER tracker Green. ER tracker provides a nice resolution of the nuclear envelope, allowing visualization of major cell cycle stages. ER tracker Green is imaged using 488 nm excitation.
Labeling of the mitochondria is tricky, as most dyes tested seem to be trapped in the first mitochondrion they enter. On the other hand, no sign of dye toxicity was detected, making it possible to follow labeled mitochondria through cellularization and the ectodermal nuclear cycle 15. Figure 4 shows an ectodermal cell several hours post-injection with Mitoview 633 (excitation wavelength 633 nm).
BODIPY, Lysotracker, and LipidSpot are robust and can be used to acquire ~500+ images at 512 x 512, line average 3, frame rates from 1/s to 0.1/s. ER tracker Green, SiR-tubulin, and the mitochondrial dyes mentioned are less robust and yield ~50-200 images under the same conditions.
Starting at blastoderm stages, LDs move bidirectionally along microtubules, powered by the opposite-polarity motors kinesin-1 and cytoplasmic dynein5. This motion can be visualized by co-labeling LDs and microtubules via injecting both BODIPY 493/503 and SiR-Tubulin (Figure 5). As LDs frequently reverse their direction of movement (as they switch between kinesin-1 and cytoplasmic dynein), higher frame rates during acquisition better capture critical details of LD motility.
Live imaging of autofluorescent yolk vesicles is possible without any form of dye injection (Figure 6). However, the autofluorescence is dim, and the excitation laser is phototoxic. Thus, live imaging of autofluorescent yolk has a poor signal-to-noise ratio relative to dye injection.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63291/63291fig01.jpg" /> 
Figure 1: Diffusion of BODIPY 493/503 through the embryo. The dye was injected along the lateral edge toward the anterior end (top right) and diffuses from this injection site into the embryo, labeling LDs. (A) The dye has diffused through portions of the embryo adjacent to the injection site. (B) Roughly 24 min/2 nuclear cycles later, the dye has diffused past the midpoint of the embryo. Scale bar: 100 &#181;m. A 1024 x 1024 frame (line average 4) was acquired every 30 s. <a href="https://www.jove.com/files/ftp_upload/63291/63291fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63291/63291fig02.jpg" /> 
Figure 2: Particle image velocimetry (PIV) for LDs and acidic organelles. An embryo was injected with both BODIPY 493/503 and LysoTracker Red. (A) BODIPY channel. (B) LysoTracker Red channel. (A&#39;,B&#39;) Streamline diagrams generated by PIV analysis of the two channels generated from 10 sequential frames, including those shown in A and B. A&#39; corresponds to the flow of LDs, and B&#39; corresponds to the flow of acidic organelles. Note that both A&#39; and B&#39; show a left-of-center confluence where embryonic contents are flowing out of the plane of view, into the center of the embryo. Also, note that BODIPY has diffused more than LysoTracker as different dyes have different diffusive properties. Scale bar: 100 &#181;m. A 1024 x 1024 frame (line average 4) was acquired every 30 s. <a href="https://www.jove.com/files/ftp_upload/63291/63291fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63291/63291fig03.jpg" /> 
Figure 3: ER tracker labels syncytial nuclear divisions. A syncytial blastoderm embryo was microinjected with ER tracker and a portion of its surface was imaged over time. (A) Spindle assembly during a nuclear division. (B) Abscission of the nuclear envelope during the same division. (C)&#160;Subsequent interphase. (D) The onset of the next division is indicated by centrosome appearance (occurrence of circular ER-free regions, marked by arrowheads). Note the gradual dye bleaching. Excitation wavelength: 488 nm. Scale bar: 5 &#181;m. A: initial frame, B: 3 min elapsed, C: 10 min elapsed, D: 13 min elapsed. A 1024 x 1024 frame (line average 4) was acquired every 30 s. <a href="https://www.jove.com/files/ftp_upload/63291/63291fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63291/63291fig04.jpg" /> 
Figure 4: Mitoview 633 labeling of mitochondria. An embryo was injected with Mitoview 633 during the syncytial blastoderm stage and imaged 4 h later, after cellularization. The image shows a neuroectodermal cell of an embryo in germ-band extension. Scale bar: 5 &#181;m. A 1024 x 1024 frame (line average 4) was acquired every 30 s. <a href="https://www.jove.com/files/ftp_upload/63291/63291fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63291/63291fig05.jpg" /> 
Figure 5: Co-labeling of LDs and microtubules. A cellularizing embryo was injected with a mixture of BODIPY 493/503 (yellow A,B,C) and SiR Tubulin (magenta A&#39;,B&#39;,C&#39;). A&#39;&#39;, B&#39;&#39;, C&#39;&#39; show the merged channels. Panels A, A&#39;, and A&#39;&#39; show the initial frame, panels B, B&#39; and B&#39;&#39; show the frame after 5 s, and panels C, C&#39; and C&#39;&#39; show the frame after 10 s. Scale bar: 5 &#181;m. A 512 x 512 frame (line average 3) was acquired every 2.5 s. <a href="https://www.jove.com/files/ftp_upload/63291/63291fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63291/63291fig06.jpg" /> 
Figure 6: Imaging yolk vesicle autofluorescence during syncytial cleavage stages. (A) At the start of the acquisition. (B) After 8 min. (C) After 16 min. Excitation wavelength: 405 nm. Low excitation intensity was used to keep the embryo alive. Scale bar: 100 &#181;m. A 1024 x 1024 frame (line average 4) was acquired every 30 s. <a href="https://www.jove.com/files/ftp_upload/63291/63291fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental data. <a href="https://www.jove.com/files/ftp_upload/63291/PIV_analysis_script_Kilwein_Welte_JoVE_2021.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos at JoVE.com

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

In the early Drosophila embryo, many organelles undergo large scale motion. Our protocol describes how to monitor this motion using fluorescent dyes developed for cell culture. Compared to fluorescent proteins, commercially available fluorescent probes are brighter and span a wider range of the spectrum.They allow for multiplex colabeling and can be used in any genetic background. It takes practice and trial and error to know how long to desiccate embryos, so that they either burst during injection or dry up and stop developing. To begin, start preparing the injection needles by placing the capillary in a capillary holder on the needle puller and securing it using wing nuts.Align the center of the capillary with the heating element. According to the manufacturer's instructions, choose appropriate settings on the needle puller. Using a dissecting microscope, perform the quality control test.Press the needles into the putty horizontally with the needle tips suspended in the air. Cover the needles with the lid and store until use. Using needle loading tips attached to a micropipette, draw one microliter of the injection solution into the tip without trapping air bubbles.Using gloves, hold the needle in one hand with its tip pointing away and insert the loading tip into it. Dispense the liquid near the needle tip, remove the pipette and hold the needle vertically until the liquid flows to the tip. Cover the container with aluminum foil without damaging the needle tips to prevent the bleaching of the dyes.Using a transfer pipette, or a micropipetter, place a small drop of heptane glue at the center of a rectangular cover slip, and allow it to evaporate. For removing the chorion mechanically, cover the appropriately aged embryo plate with a thin layer of halocarbon oil 27 to turn the eggshell transparent. View the plate under a dissection microscope with trans illumination to confirm the embryo stage and select the embryos in cleavage stages.Using fine tweezers, pick up an embryo of the desired stage by grabbing the dorsal appendages and transfer it to the prepared glass slide covered with double sided tape. Place the embryo on the tape and minimize the transfer of oil. Roll the embryo gently across the surface of the tape by nudging with the side of the tip of the tweezers without poking it directly.Continue rolling the embryo until the chorion adheres to the tape and cracks. After the chorion cracks, keep rolling the embryo to separate it from the chorion, which remains stuck to the tape. Roll the embryo back onto the chorion, which is less adhesive than the tape.Gently rub the embryo with the tweezers until it attaches to the tweezers for transfer. Then, transfer the embryo to the cover slip and bring it in contact with the glue to hold the embryo in place, separating it from the tweezers. Adjust the orientation of the embryo on the cover slip.Let the embryos desiccate for five to 12 minutes by placing the cover slip with embryos into the desiccation chamber and sealing it. Take off the cover slip from the chamber and put a drop of halocarbon oil 700, covering the embryos to prevent further desiccation. Examine them under the dissecting scope and proceed to microinjection if the embryos were desiccated properly.Place the 4X objective into the light path and using the focus knob on the microscope, adjust the embryos into focus. Move the embryos horizontally into the center of the field of view using the stage controls. Keep the embryos at the edge of the field of view and pan away in preparation for lowering the needle.Staying with the same focal plane, lower the needle tip and ensure it is visible in the field of view along with the embryos. Check that needle is functioning by dispensing some of the injection solution into halocarbon oil 700. If successful, a bubble of solution will appear in the oil near the needle tip.Using the stage controls, move the embryo towards the needle tip until the tip gently punctures the embryo and enters into it. Simultaneously, begin the flow of injection solution until a transient clear spot appears at the site of the needle tip, indicating successful solution transfer into the embryo. Secure the cover slip on the confocal microscope using the slide holder.Use caution as the cover slip is fragile. Inspect the embryos using the epifluorescence function on the confocal microscope with a 40X objective and ensure that the fluoro 4 in the embryo is visible before proceeding. For live imaging, during the syncytial stages, set the image size of 512 by 512 pixels, line average of three, and a frame rate of 0.1 frames per second.After the BODIPY 493 503 dye was injected, it was localized at the site where the needle tip was inserted and then diffused adjacent to the injection site. After 24 minutes, the dye has diffused past the midpoint of the embryo. The organelle motility was monitored by injecting the embryo with BODIPY 493 503 and LysoTracker Red.The particle image velocimetry analysis revealed that the embryonic contents converged on the central region of the embryo, where the cytoplasm is flowing into the embryo's interior. Labeling of the ER, using ER-Tracker Green, provides a vivid resolution of the nuclear envelope, allowing visualization of major cell cycle stages. The embryo was injected with MitoView 633 at the blastoderm stage and imaged four hours later.The image shows a neuroectodermal cell of an embryo in germ band extension. A cellularizing embryo was injected with a mixture of BODIPY 493 503 and SiR Tubulin, which shows that the lipid droplets move bidirectionally along microtubules. The embryo must be partially desiccated so that liquid can be injected.Too little desiccation will cause the embryo to leak when injected, too much desiccation will kill the embryo. Videos of moving organelles can be characterized via diverse image analysis techniques. Particle tracking reveals the behavior of individual organelles, particle imaging velocimetry reveals the bulk flow.

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