Name: analysis of actomyosin dynamics at local cellular and tissue scales using time-lapse movies of cultured drosophila egg chambers
Uploaded: 2019-06-03T12:30:00
Description: Watch this Scientific Journal Video about Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers at JoVE.com

The authors are very thankful to Miriam Osterfield for sharing her advice on the in vitro life imaging of egg chambers using an approach adopted from the Celeste Berg laboratory4.&#160;We also thank to Sebastian Tosi for the Fiji script that enables cell segmentation.

NOTE: The following protocol provides instructions on how to analyze actomyosin at the local cellular and the tissue scale in Drosophila egg chambers. The local-scale approach allows users to analyze detailed actomyosin behavior in up to 15 cells per egg chamber and requires the acquisition of TLMs for a short period of time (5&#8211;10 min) by using high-speed imaging and an inverted confocal microscope. In contrast, the tissue scale provides users with actomyosin information in 50&#8211;100 ce...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bullwinkle+is+required+for+epithelial+morphogenesis+during+Drosophila+oogenesis.">bullwinkle is required for epithelial morphogenesis during Drosophila oogenesis.</a> Developmental Biology. 267, 320-341 (2004).</li><li>Cetera, 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=Epithelial+rotation+promotes+the+global+alignment+of+contractile+actin+bundles+during+Drosophila+egg+chamber+elongation.">Epithelial rotation promotes the global alignment of contractile actin bundles during Drosophila egg chamber elongation.</a> Nature Communications. 5, 5511 (2014).</li><li>Viktorinova, I., Henry, I., Tomancak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+rotation+is+preceded+by+planar+symmetry+breaking+of+actomyosin+and+protects+epithelial+tissue+from+cell+deformations.">Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations.</a> PLOS Genetics. 13, e1007107 (2017).</li><li>Rauzi, M., Lenne, P. F., Lecuit, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+polarized+actomyosin+contractile+flows+control+epithelial+junction+remodelling.">Planar polarized actomyosin contractile flows control epithelial junction remodelling.</a> Nature. 468, 1110-1114 (2010).</li><li>Spracklen, A. J., Fagan, T. N., Lovander, K. E., Tootle, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pros+and+cons+of+common+actin+labeling+tools+for+visualizing+actin+dynamics+during+Drosophila+oogenesis.">The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.</a> Developmental Biology. 393, 209-226 (2014).</li><li>. 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Critical steps and troubleshooting for the dissection and culturing of egg chambers
If too many flies are placed into a small vial, the fly food can turn muddy after 2&#8211;3 days due to extensive amounts of feeding larvae and adult flies getting trapped in the fly food. In such a case, flip the rest of these flies into a new vial with fresh food and downsize their number. In particular, exclude females that were stuck in the food.
The Schneider mi...

The continual development of novel imaging and software technologies with applications in the life sciences has provided an enormous impact on understanding the basic principles of life. One of the main challenges is the reliable visualization of developmental processes in combination with their live imaging in various tissues. Tissues are parts of organs and bodies and, as such, the majority are not easily accessible for imaging. Therefore, protocols that allow their dissection and in vitro culturing have been developed in order to visualize biological events that sufficiently reflect the in vivo situation within a living body.
Over the past decades, the culturing and live imaging of Drosophila egg chambers, acinar-like structures resembling primitive organs, has contributed immensely to the understanding of the basic principles of primitive organ development1,2,3. Currently, there are several culturing protocols available, and their usage depends on acquisition time, cell type to be imaged, and their accessibility (e.g., inner germline vs. outer somatic line)4.
A common feature in all these culturing protocols is the need for the immobilization of analyzed egg chambers that display a high contractile activity in liquid media. The contractile activity of egg chambers is caused mainly by the muscle sheet that covers a long string of connected egg chambers5,6,7. Therefore, to achieve proper immobilization of young egg chambers, various approaches have been developed, involving covering egg chambers with coverslips6,8,9 or flexible blankets4,10 or embedding them in low-melting-point agarose3,11. These approaches are popular as they also allow the imaging of a larger visual plane due to the subtle flattening of the circumferential surface of the egg chambers.
However, recently, it has been shown that young egg chambers (stage 1-8) rotate around their anterior-posterior axis6 and that this tissue motion is powered by a fine actomyosin network close to the circumferential surface of these young egg chambers12. Therefore, artificial alteration of the cellular surface caused by a subtle flattening of this tissue may have a negative impact on the behavior of the force-generating actomyosin machinery. The counterpoint is that if the egg chamber tissue is not flattened, microscopic imaging of proteins at the circumferential surface of egg chambers becomes even more limited by the decreased size of the acquisition plane.
Therefore, we have combined protocols from Prasad et al.9 and the lab of Celeste Berg4,10 and further modified them so that no coverslip/flexible blanket/agarose is used in the developed method. Drosophila egg chambers are freely cultured in media and the protocol presented here applies only inverted microscopy. There are two parts to the protocol. The first part is focused on the analysis of actomyosin signals at the local cellular scale (up to 15 cells) within egg chambers. In the second part, we focus on overcoming the limitations associated with a small acquisition plane caused by the free culturing of egg chambers. In this regard, we have developed a novel Fiji-based computational method with a semi-interactive graphical user interface that selectively extracts and unfolds defined layers of a circumferential tissue surface. This is followed by a protocol that describes how to analyze actomyosin at the tissue scale (i.e., &#62;50 cells). As the selective extraction of a defined thin layer of curved epithelial tissues has not been easily possible using a classical z-stack projection (Figure 1), this easy-to-use method serves as an important prerequisite to comprehensively understand&#160;the behavior of a thin (&#60;1 &#181;m) actomyosin network at the tissue scale in Drosophila egg chambers.
In addition, to facilitate the protocol, we provide example time-lapse movies (TLMs) and sample files of fluorescently tagged nonmuscle conventional Myosin II behavior (see Supplementary File 3). Myosin&#160;II is a motor protein and represents the active contractile part of the actomyosin machinery. In order to image Myosin II, we use Drosophila transgenic lines that contain a modified regulatory light chain of Myosin II called MRLC::GFP (see Table of Materials for details)12,13. In order to visualize cell membranes, the protocol is based on commercial dyes (see Table of Materials). This protocol is suitable not only for the analysis of small subcellular MRLC::GFP signals12 but also for any similar-sized subcellular particles around &#177;300 &#181;M, such as those observed with Life-Act::GFP12,14.
Although both these protocols are presented using in vitro cultured Drosophila egg chambers, the acquisition of actomyosin signals can also be performed using other tissues upon the optimization of the culturing media and depending on the availability of either fluorescently tagged proteins with corresponding commercial dyes or, for example, mRNA microinjections to obtain transient gene expression profiles. Similarly, the Fiji-based protocol for the extraction of a thin layer from a circumferential surface can be applied more generally to ellipsoid and organ-like tissues.

<table>
	<tbody>
		<tr>
			<td>Schneider Medium</td>
			<td>Gibco</td>
			<td>21720-024</td>
			<td>[+]-Glutamine</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>&#160;[+] 10.000 Units/ml Penicillin [+] 10.000 &#181;g/ml Streptomycin</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>&#160;F135</td>
			<td>Heat inactivated</td>
		</tr>
		<tr>
			<td>Insulin Solution Human</td>
			<td>Sigma</td>
			<td>&#160;I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Falcon tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>Millex-GV filter</td>
			<td>Millipore</td>
			<td>SLGV033NS</td>
			<td>33 mm</td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>35 mm Petri dish, 14 mm Microwell, No. 1.5 cover glass</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>A. Dumont &#38; Fils</td>
			<td></td>
			<td>&#160;#55</td>
		</tr>
		<tr>
			<td>CellMask Deep Red (cell membrane dye)</td>
			<td>Invitrogen</td>
			<td>C10046</td>
			<td></td>
		</tr>
		<tr>
			<td>FM4-64 (cell membrane dye)</td>
			<td>ThermoFisher/Invitrogen</td>
			<td>T13320</td>
			<td></td>
		</tr>
		<tr>
			<td>Depression dissection slide</td>
			<td>Fisher Scientific</td>
			<td>12-560B</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopic equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Steromicroscope</td>
			<td>e.g. Zeiss</td>
			<td></td>
			<td>Stemi SV 6</td>
		</tr>
		<tr>
			<td>Inverted confocal microscope</td>
			<td>e.g. Zeiss/Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disc microscope</td>
			<td>e.g. Zeiss/Andor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopic glass/cover glass</td>
			<td>e.g. Thermo Scientific/Menzel Glas</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cactus tool</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wironit needle holder</td>
			<td>Hammacher</td>
			<td>9160020</td>
			<td></td>
		</tr>
		<tr>
			<td>Cactus spine</td>
			<td>Echinocactus grusonii/barrel cactus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila stocks used in the manuscript</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AX3/AX3; sqh-MRLC::GFP/sqh-MRLC::GFP</td>
			<td></td>
			<td></td>
			<td>For detail see Rauzi et al.: Planar polarized actomyosin contractile flows control epithelial junction remodeling. Nature 2010, Vol. 468, pg. 1110-1115.</td>
		</tr>
		<tr>
			<td>AX3/AX3; sqh-MRLC::GFP/sqh-MRLC::GFP; fat258D/fat103C</td>
			<td></td>
			<td></td>
			<td>For detail see Viktorinova et al.: Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations. PLoS Genetics 2017, Vol. 13, Issue 11, pg. e1007107.</td>
		</tr>
	</tbody>
</table>

analysis of actomyosin dynamics at local cellular and tissue scales using time-lapse movies of cultured drosophila egg chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

This protocol enables scientists to investigate the behavior of actomyosin networks in epithelial tissues. This is only possible when a detailed analysis of actomyosin behavior at the local cellular scale (a few cells) is combined with a similar analysis at the tissue scale (many cells). However, epithelial tissues are often curved and the extraction of a thin layer of these tissues was previously not easily possible, as shown in Drosophila egg chambers (Figu...

Watch this Scientific Journal Video about Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers at JoVE.com

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

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