秀丽隐杆线虫体壁肌线粒体结构分析

Shaarika  Sarasija; Kenneth R. Norman

doi:10.21769/BioProtoc.2801

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Peer-reviewed

Analysis of Mitochondrial Structure in the Body Wall Muscle of Caenorhabditis elegans

秀丽隐杆线虫体壁肌线粒体结构分析

Shaarika Sarasija email

KN Kenneth R. Norman email

发布: 2018年04月05日第8卷第7期 DOI: 10.21769/BioProtoc.2801 浏览次数: 8669

评审: Sanjib GuhaAnonymous reviewer(s)

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Abstract

Mitochondrial function is altered in various pathologies, highlighting the crucial role mitochondria plays in maintaining cellular homeostasis. Mitochondrial structure undergoes constant fission and fusion in response to changing cellular environment. Due to this, analyzing mitochondrial structure could provide insight into the physiological state of the cell. In this protocol, we describe a method to analyze mitochondrial structure in body wall muscles in the nematode Caenorhabditis elegans, using both transgenic and dye-based approaches.

Keywords: C. elegans (秀丽隐杆线虫)

Mitochondria (线粒体)

Calcium (钙)

Mitochondrial membrane potential (线粒体膜电位)

TMRE (TMRE)

Mitotracker (Mitotracker)

Background

itochondria are involved in ATP production, cellular respiration, calcium buffering and reactive oxidative species (ROS) metabolism (Brookes et al., 2004). Mitochondrial structure and function are dynamic and closely linked, therefore analyzing mitochondrial structure can provide clues to the status of mitochondrial health (Sarasija and Norman, 2015). We developed two sets of protocols to assess mitochondrial structure in the body wall muscle of Caenorhabditis elegans. In the first protocol, we used transgenic ccIs4251 strain in which GFP is targeted to the matrix of the body wall muscle mitochondria to visualize the mitochondria (Fire et al., 1998). In the second protocol, we used mitochondrially targeted dyes, MitoTracker^TM Red CMXRos and tetramethylrhodamine ethyl ester (TMRE) to determine the structural integrity of the body wall muscle mitochondria. Normally, animals used in in-vivo imaging are anesthetized, however anesthetizing the animals could lead to mitochondrial morphological changes (Han et al., 2012), complicating data analysis. Our protocols allow for the in-vivo imaging of mitochondrial structure in live, un-anaesthetized nematodes.

Materials and Reagents

100 mm, 60 mm Petri dishes (Kord-Valmark Labware Products, catalog numbers: 2900 , 2901 )
Glass Pasteur pipettes (Krackeler Scientific, catalog number: 6-72050-900 )
15-ml centrifuge tubes (Globe Scientific, catalog number: 6285 )
22 x 22 mm coverslip (Globe Scientific, catalog number: 1404-10 )
1.5 ml Micro Centrifuge tube (CELLTREAT Scientific, catalog number: 229443 )
50 ml conical tubes (Corning, catalog number: 430829 )
15 ml conical tubes (Corning Centristar, catalog number: 430791 )
C. elegans strains including strain SD1347, ccIs4251 [(pSAK2) myo-3p::GFP::LacZ::NLS + (pSAK4) myo-3p::mitochondrial GFP + dpy-20(+)] (Liu et al., 2009) and OP50 (Caenorhabditis Genetics Center (CGC), University of Minnesota)
Deionized water (dH₂O)
Polybead polystyrene 0.10 μm microspheres (Polysciences, catalog number: 00876-15 )
Agarose (RPI, catalog number: A20090-500.0 )
Clear nail polish (generic)
Carl Zeiss^TM Immersol^TM Immersion Oil (ZEISS, catalog number: 444960-0000-000 )
Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358-10 )
Agar (Fisher Scientific, catalog number: BP1423-2 )
Bacto peptone (BD, Bacto^TM, catalog number: 211677 )
Calcium chloride dihydrate (CaCl₂·2H₂O) (Fisher Scientific, catalog number: C79-500 )
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Fisher Scientific, catalog number: BP213-1 )
Cholesterol (Fisher Scientific, catalog number: C314-500 )
Potassium phosphate dibasic (K₂HPO₄) (Fisher Scientific, catalog number: BP363-1 )
Potassium phosphate monobasic (KH₂PO₄) (Fisher Scientific, catalog number: P285-500 )
Sodium phosphate dibasic anhydrous (Na₂HPO₄) (Fisher Scientific, catalog number: BP332-1 )
Bleach (generic, plain)
Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: BP359-500 )
Bacto tryptone (BD, Bacto^TM, catalog number: 211705 )
Bacto yeast extract (BD, Bacto^TM, catalog number: 212750 )
MitoTracker Red CMXRos (Thermo Fisher Scientific, Invitrogen^TM, catalog number: M7512 )
Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: T669 )
Standard worm (NGM) plates (see Recipes)
Sterile solutions (see Recipes)
Sterile stocks for NGM (see Recipes)
1. 1 M CaCl₂
2. 1 M MgSO₄
3. 1 M K₂HPO₄
4. 1 M KH₂PO₄
5. 1 M KPO₄ pH 6.0
M9 buffer (1 L) (see Recipes)
Bleach solution (see Recipes)
10 N NaOH (see Recipes)
Mitotracker^TM Red CMXRos stock (see Recipes)
TMRE stock (see Recipes)

Equipment

Single channel pipettes (Rainin, models: PR-10 , PR-20 , PR-200 , PR-1000 )
Finnpipette II Multichannel pipettes (Fisher Scientific, model: Fisherbrand^TM Finnpipette^TM II, catalog number: 21377830 )
20 °C Incubator (Percival Scientific, model: I-41NL )
Centrifuges (Eppendorf, models: 5415 D , 5415 R ; Thermo Fischer Scientific, Thermo Scientific^TM, model: IEC Centra CL2 )
Zeiss SteREO Discovery.V8 microscope with SCHOTT Ace^® I light source for maintaining (ZEISS, model: SteREO Discovery.V8 )
Zeiss SteREO Discovery.V12 microscope with SCHOTT Ace^® I light source and X-Cite^® Series 120 Fluorescence Illuminator for transgenic selection (ZEISS, model: SteREO Discovery.V12 )
Zeiss AxioObserver microscope with Andor Clara CCD camera and X-Cite^® Series 120 Fluorescence Illuminator for imaging (ZEISS, model: Axio Observer )
PYREX^® Griffin beakers (Corning, catalog number: 1000-PACK )
PYREX^® Reusable Media Storage Bottles (Fisher Scientific)

Software

MetaMorph^® Microscopy Automation & Image Analysis Software (Molecular Devices)
ImageJ (https://imagej.nih.gov/ij/)
Microsoft Office 2011 Excel (Microsoft Corporation, Redmond, USA)
GraphPad Prism software package (GraphPad Software Inc., San Diego, USA)

Part I: Determining mitochondrial structure using transgenic lines

Procedure

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如何引用

Sarasija, S. and NORMAN, K. R. (2018). Analysis of Mitochondrial Structure in the Body Wall Muscle of Caenorhabditis elegans. Bio-protocol 8(7): e2801. DOI: 10.21769/BioProtoc.2801.