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

Summary

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.

Abstract

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’ circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (>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.

Introduction

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 pa....

Protocol

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 (5–10 min) by using high-speed imaging and an inverted confocal microscope. In contrast, the tissue scale provides users with actomyosin information in 50–100 ce.......

Discussion

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 2–3 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.......

Materials

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