Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

Summary

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

Abstract

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actin^Red) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actin^Red polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actin^Red into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

Introduction

This protocol describes how to inject G-actin^Red to visualize the assembly of intranuclear actin rods in heat-stressed embryos that are undergoing an inducible Actin Stress Response (ASR)¹. We developed this protocol to aid studies of the ASR, which in embryos leads to disrupted morphogenesis and reduced viability, and in adult human cell types is associated with pathologies including renal failure², muscle myopathies³, and Alzheimer’s and Huntington’s Disease^4,5,6,

Protocol

1. Prepare embryo collection cups and apple juice agar plates

1. Five days prior to the injection experiment, construct²⁸ or procure at least two small embryo collection cups. Make fresh 60 mm apple juice agar plates to be used with small collection cups²⁸. Store plates in plastic boxes covered with damp paper towels at 4 °C.
   NOTE: Small embryo collection cups, populated with fly numbers as described in step 1.3, will provide sufficient embryo numbers per experiment, while also ensuring that embryo handling and injection can be done in a short enough time to allow imaging of early developmental ....

Results

A schematic workflow of embryo handling is depicted in Figure 1, and a timetable for a typical experiment is presented in Table 1. An estimate for a good experimental outcome is that for every 10 embryos injected, at least half of the embryos viewed will be at the correct developmental stage, undamaged, and exhibit a robust ASR with heat stress at 32 °C. This ASR will be evidenced by the assembly of intranuclear actin rods as shown in the representative surface view image of.......

Discussion

The significance of this method is that it utilizes the well-established protocol of microinjection in Drosophila embryos^{21,22,23,24,25,26,27} to enable new research regarding the ASR and accompanying actin rod assembly. A major advantage of injecting G-actin^Red into live .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adenosine triphosphate (ATP)	Millipore-Sigma	A23835G	Component of G buffer
Apple juice, Mott's, 64 fl oz	Mott's	014800000344	Component of apple juice plates
Bacto Agar	BD	214010	Component of apple juice plates
Bleach, PureBright Germicidal, 6.0% sodium hypochlorite	KIK International	059647210020	For dechorionating embryos
Calcium chloride	Millipore-Sigma	C1016500G	Component of G buffer
Cell strainer, 70 μm	Falcon	352350	For collecting dechorionated embryos
Confocal microscope, LSM 880 34-channel with Airyscan	Zeiss	0000001994956	For imaging intranuclear actin rods
Desiccant	Drierite	24001	For desiccating embryos
Dissecting microscope, Stemi 508 Stereoscope with 8:1 zoom	Zeiss	4350649000000	For arranging embryos on agar wedge
Dissecting needle, 5 in	Fisher Scientific	08965A	For arranging embryos on agar wedge
Dithiothreitol (DTT)	Fisher Scientific	BP1725	Component of G buffer
Double-sided Tape, Scotch Permanent, 0.5 in x 250 in	3M	021200010323	For making embryo glue
Embryo collection cage	Genessee Scientific	59100	For housing adult flies and collecting embryos
Fine tip tweezers, Dumont Tweezer, Style 5	Electron Microscopy Sciences	72701D	For arranging embryos on agar wedge
Glass capillaries, Borosillicate glass, thin 1 mm x 0.75 mm	World Precision Instruments, Inc.	TW1004	For microneedles
Halocarbon oil 27	Millipore-Sigma	H8773100ML	For hydration of embryos
Heated stage incubator	Zeiss	4118579020000, 4118609020000, 4118609010000	For confocal imaging
Lab Tissue Wipers, KimWipes	Kimberly-Clark	34155	Lab tissue wipers
Light microscope, Invertoskop 40C Inverted Phase contrast microscope, refurbished	Zeiss	Discontinued	Injection microscope
Methyl-4-hydroxybenzoate	Millipore-Sigma	H36471KG	Component of apple juice plates
Microinjector, FemtoJet4x	Eppendorf	5253000025	Microinjector
Micro loader tips, epT.I.P.S. 20 μL	Eppendorf	5242956003	For loading microneedles
Micromanipulator and injection stage with x,y,z dials for needle adjustment	Bernard Instruments, Inc (Houston, TX)	Custom	For performing microinjections
Micropipette puller, Model P-97, Flaming/Brown	Sutter Instruments	P97	For pulling capillary tubes to make microneedles
Microscope cover glass 24x50-1.5	Fisher Scientific	12544E	For mounting embryos
Microscope slides, Lilac Colorfrost, Precleaned, 25 x 75 x 1mm	Fisher Scientific	22037081	For mounting embryos for injection
n-Heptane	Fisher Scientific	H3601	Component of embryo glue
Objective, 10x	Zeiss	Discontinued	10x objective for injection microscope
Objective, C-Apochromat 40x/1,2 W Korr. FCS	Zeiss	4217679971711	40x water objective for confocal
Objective, LD LCI Plan-Apochromat 25x/0.8 Imm Cor DIC M27 for oil, water, silicone oil or glycerine immersion (D=0-0.17mm) (WD=0.57mm at D=0.17mm)	Zeiss	4208529871000	25x mixed immersion objective for confocal
Objective, Plan-Apocrhomat 63x/1.40 Oil DIC f/ELYRA	Zeiss	4207829900799	63x oil objective for confocal
Paintbrush, Robert Simmons Expression E85 Pointed Round size 2	Daler-Rowney	038372016954	For transferring embryos
Paper towels, Kleenex C-fold paper towels, white	Kimberly-Clark	884266344845	For blotting cell strainer
Pasteur pipette, 5 3/4 in	Fisher Scientific	1367820A	For covering embryos with oil
Petri dish, glass, 100 x 20 mm	Corning	3160102	For humid incubation chamber
Petri dish, plastic, 60 x 15 mm	VWR	25384092	For apple juice plates
Pipette, Eppendorf Reference 0.5-10 μL	Eppendorf	2231000604	For loading the microneedle
Pipette tip, xTIP4 250 μL	Biotix	63300006	For adding embryo glue to coverslip
Razor blade	VWR	55411050	For cutting agar wedge, tape, pipette tips
Rhodamine-conjugated globular actin, human platelet (non-muscle; 4x10 μg)	Cytoskeleton, Inc.	APHR-A	G-actin^Red
Scintillation vial, 20 mL Glass borosillicate with polyethylene liner and urea caps	Fisher Scientific	033377	For making embryo glue
Screw top jar, 16 oz	Nalgene	000194414195	For desiccating embryos
Stage micrometer	Electron Microscopy Sciences	602104PG	For calibrating volume of G-actin injection
Sucrose	Millipore-Sigma	840971KG	Component of apple juice plates
Trizma base	Millipore-Sigma	T15031KG	Component of G buffer
Yeast, Lesaffre Yeast Corporation Yeast, Red Star Active Dry, 32 oz	Lesaffre Yeast Corporation	117929157002	Component of yeast paste