How to analyze mitochondrial morphology in healthy cells and apoptotic cells in Caenorhabditis elegans.

CHAPTER FOUR

How to Analyze Mitochondrial Morphology in Healthy Cells and Apoptotic Cells in Caenorhabditis elegans Stéphane G. Rolland1 LMU Biocenter, Department Biology II, Ludwig-Maximilians-University, Munich, Germany 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Why Address These Questions in C. elegans 3. How to Inactivate or Overexpress a Particular Candidate Gene in C. elegans 3.1 Inactivation of a particular candidate gene 3.2 Overexpression of a particular candidate gene 4. How to Visualize Mitochondria in C. elegans 4.1 General considerations for imaging 4.2 Mitochondrial membrane potential-sensitive dyes 4.3 Mitochondrial-targeted fluorescent proteins 4.4 Visualizing mitochondria in apoptotic cells 4.5 Quantification of mitochondrial morphology 5. Potential Outcomes and Future Experiments 6. Concluding Remarks and Future Challenges Acknowledgments References

76 79 79 79 81 82 82 82 87 92 93 94 95 95 96

Abstract Mitochondria constantly undergo fusion and fission events. A proper balance of fusion and fission is essential in healthy cells, as disrupting this balance is associated with several neurodegenerative diseases. Mitochondrial fission has also been shown to play an important role during apoptosis. Hence, the machineries that control mitochondrial morphology have both nonapoptotic and apoptotic functions. Seminal work in yeast has identified some of the key components of these machineries. However, the list is certainly not complete and new factors that are specific to metazoans are being identified every year. In this review, we describe methodologies to test whether a particular candidate gene plays a role in the control of mitochondrial morphology in healthy cells and apoptotic cells using Caenorhabditis elegans. Methods in Enzymology, Volume 544 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-417158-9.00004-2

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2014 Elsevier Inc. All rights reserved.

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Stéphane G. Rolland

1. INTRODUCTION Mitochondria are highly dynamic organelles that constantly fuse and divide (Chan, 2012; Nunnari & Suomalainen, 2012; Okamoto & Shaw, 2005; Westermann, 2010). The processes of mitochondrial fusion and fission are controlled by a conserved family of dynamin-related GTPases. DRP-1, the homolog of mammalian Drp1, is responsible for mitochondrial fission in Caenorhabditis elegans (Labrousse, Zappaterra, Rube, & van der Bliek, 1999). Conversely, C. elegans FZO-1 and EAT-3, the homologs of mammalian Mfn1,2 and Opa1, are responsible for the fusion of the outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM), respectively (Ichishita et al., 2008; Kanazawa et al., 2008). Mitochondrial morphology is defined by a balance of mitochondrial fusion and fission. This morphology has been shown to change in response to several cellular signals, such as metabolic, cell cycle, or apoptotic signals (Antico Arciuch, Elguero, Poderoso, & Carreras, 2012; Liesa & Shirihai, 2013; Wang & Youle, 2009). During apoptosis of mammalian cells, mitochondria undergo fission, hereafter referred to as “fragmentation” (Wang & Youle, 2009). In C. elegans, mitochondria also fragment during apoptosis and this fragmentation is required for efficient execution of the apoptotic process ( Jagasia, Grote, Westermann, & Conradt, 2005). Whereas mitochondrial fragmentation during apoptosis in mammalian cells is associated with the release of proapoptotic factors such as cytochrome-c (Landes & Martinou, 2011), the role of mitochondrial fragmentation during C. elegans apoptosis remains elusive. Understanding the role of mitochondrial morphology changes during apoptosis requires a better understanding of the machineries that control mitochondrial fusion and fission as well as how these machineries are regulated. In addition, these machineries play roles in both apoptotic and nonapoptotic cells. For example, mitochondrial fission is important during apoptosis as well as cell division (Antico Arciuch et al., 2012; Labrousse et al., 1999). The function and regulation of the mitochondrial fusion and fission machineries must therefore be understood in both apoptotic and nonapoptotic contexts. Although the general components of these machineries (DRP-1, FZO-1, and EAT-3) are known in C. elegans, several additional factors have been recently identified and probably more will be identified in the future (Table 4.1) (Head et al., 2011; Ichishita et al., 2008; Lee et al., 2009). This

Table 4.1 Factors controlling mitochondrial morphology in C. elegans Loss-offunction Gene in Human Loss-of-function phenotype C. elegans homologs allele

Overexpression phenotype

Overall function

drp-1

Drp-1

Mitochondrial fragmentation; ectopic Mitochondrial fission tm1108 Mitochondrial elongation; mitochondrial distribution defect apoptosis ( Jagasia et al., 2005) (Labrousse et al., 1999)

fzo-1

Mitofusin 1 and 2

tm1133 Mitochondrial fragmentation; no Mitochondrial clustering (Rolland, ectopic apoptosis (Breckenridge Lu, David, & Conradt, 2009) et al., 2008)

OMM fusion

eat-3

Opa1

Mitochondrial fragmentation tm1107 Mitochondrial fragmentation; (Rolland et al., 2009) abnormal cristae; no ectopic apoptosis (Kanazawa et al., 2008)

Inner mitochondrial membrane fusion

tm1912 Swollen mitochondria; abnormal ND cristae (Head, Zulaika, Ryazantsev, & van der Bliek, 2011) tm1730 ND

Cristae maintenance

moma-1

my025 or APOOL

immt-1

Mitofilins

chch-3

CHCHD3 tm2336 Swollen mitochondria (Head et al., 2011)

immt-2

Mitofilins

dic-1

DICE1

Cristae maintenance

Cristae maintenance

ND

Cristae maintenance

tm2366 Interconnected mitochondria (Head et al., 2011)

ND

Cristae maintenance

tm1615 Reduced cristae number (Lee et al., 2009)

Increased cristae number (Lee et al., 2009)

Cristae maintenance Continued

Table 4.1 Factors controlling mitochondrial morphology in C. elegans—cont'd Loss-ofGene in Human function C. elegans homologs allele Loss-of-function phenotype Overexpression phenotype

n2812

Overall function

ced-9

BCL-2 like proteins

egl-1

BH3-only n3082 proteins

Mitochondrial elongation (Lu, Rolland, & Conradt, 2011)

Mitochondrial fragmentation (Lu et al., 2011)

Regulation of mitochondrial morphology

mma-1

LRPPRC RNAi

Mitochondrial hyperfusion (Rolland et al., 2013)

ND

Regulation of mitochondrial translation

fis-1

hFis1

tm1867 No obvious phenotype (Breckenridge et al., 2008) gk363

ND

Unknown

Mff

tm2955 ND

ND

Unknown

tm3041 ND

ND

Unknown

fis-2 mff-1 mff-2 ND, not determined.

No obvious phenotype (Rolland Mitochondrial hyperfusion (Rolland Regulation of et al., 2009; Tan et al., 2008) et al., 2009; Tan et al., 2008) mitochondrial morphology

Analysis of Mitochondrial Morphology in C. elegans

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review describes methodologies to test whether the inactivation and/or overexpression of a particular gene in C. elegans affects mitochondrial morphology in apoptotic and/or nonapoptotic cells.

2. WHY ADDRESS THESE QUESTIONS IN C. ELEGANS Despite being a relatively simple metazoan with only 959 somatic cells, C. elegans is great model organism for cell biologists as it contains all major cell types (germ cells, muscle cells, neurons, and intestinal cells). Due to its essentially invariant cell lineage (Sulston, Schierenberg, White, & Thomson, 1983), it is also well suited for the study of developmentally regulated processes. Furthermore, with its several powerful genetic tools that have been developed over almost five decades, C. elegans has proven to be an excellent model for gene discovery (Brenner, 1974). More specifically concerning the study of the regulation of mitochondrial morphology, all the genes that control mitochondrial morphology in C. elegans are conserved in humans (Table 4.1). Hence, the new factors discovered in C. elegans are likely to be conserved and the understanding of the regulation of mitochondrial morphology in C. elegans will likely increase our understanding of these processes in humans as well.

3. HOW TO INACTIVATE OR OVEREXPRESS A PARTICULAR CANDIDATE GENE IN C. ELEGANS In this section, an overview of how to inactivate or overexpress a candidate gene is described, with an emphasis on genes that regulate mitochondrial morphology.

3.1. Inactivation of a particular candidate gene 3.1.1 Loss-of-function mutants After the genome of C. elegans was published in 1998, efforts have been made to generate mutations in more than 20,000 genes of C. elegans (C. elegans Consortium, 1998). By 2012, with the effort of individual laboratories and the C. elegans Deletion Mutant Consortium, almost 30% of the C. elegans genes have been targeted (C. elegans Consortium, 2012). More recently, with the development of next-generation sequencing, a new library of mutants has been generated by the “million mutations project.” For each C. elegans gene, on average eight different mutant alleles are

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available in this library (Thompson et al., 2013). The list of all these mutants or mutations can be found on Wormbase (http://www.wormbase.org/) and all these mutants are available from the Caenorhabditis Genetics Center (CGC) (http://www.cbs.umn.edu/cgc). Table 4.1 lists all the mutations (loss-of-function alleles) of genes controlling mitochondrial morphology in C. elegans. To test the effect of a new candidate gene on mitochondrial morphology in embryos, animals carrying a mutation in this gene can be analyzed by TMRE staining (Section 4.2.2). To analyze the effect of such a mutation on mitochondrial morphology in other cell types, the mutant strain can be transformed with fluorescent reporters that label mitochondria (Section 4.3). (For detailed protocols to generate C. elegans transgenic animals; see Evans, 2006.)

3.1.2 Inactivation by RNA interference RNA interference (RNAi) is a powerful tool for reverse genetics in C. elegans. Specifically, feeding C. elegans with an Escherichia coli strain expressing double-strand RNA (dsRNA) of a particular gene is often sufficient to knock-down its function (Timmons, Court, & Fire, 2001). Ahringer and coworkers have generated a RNAi feeding library that contains 87% of the C. elegans genes (Kamath et al., 2003). In addition, Marc Vidal and coworkers have generated a RNAi-ORF feeding library (Rual et al., 2004). Whereas the Ahringer’s library targets 500 bp conserved exon sequences, the RNAi-ORF library targets the entire ORF of a particular gene but only covers 55% of C. elegans genes (http://www.geneservice. co.uk and http://www.lifesciences.sourcebioscience.com/). Even though RNAi-mediated inactivation of drp-1, fzo-1, and eat-3 recapitulates the phenotypes observed in the loss-of-function mutants, their penetrances vary. For example, RNAi by feeding of drp-1 induces mitochondrial elongation in 100% of treated animals (Rolland et al., 2009). In contrast, only 66% of animals have fragmented mitochondria upon fzo-1 RNAi (Rolland et al., 2009). Injection of in vitro synthesized dsRNA into animals often results in a more penetrant phenotype compared to the feeding protocol. Hence, depending on the gene, injection of dsRNA may be more appropriate (Ahringer, 2006). A protocol to analyze mitochondrial morphology in embryos or muscle cells in response to the inactivation of a candidate gene using RNAi by feeding is described below (Sections 4.2.2 and 4.3.4).


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3.2. Overexpression of a particular candidate gene 3.2.1 In embryos, using heat-shock promoters hsp-16.2 and hsp-16.41 promoters are heat-inducible promoters that have been successfully used in embryos to overexpress genes that regulates mitochondrial morphology, such as ced-9, egl-1, fzo-1, eat-3, and drp-1 (Table 4.1) ( Jagasia et al., 2005; Lu et al., 2011; Rolland et al., 2013). hsp-16.41 drives the expression of a transgene in the intestine and pharyngeal tissue, whereas hsp-16.2 drives the expression of a transgene in neurons and hypodermal cells (Fire, Harrison, & Dixon, 1990; Stringham, Dixon, Jones, & Candido, 1992). Since these two promoters have a complementary pattern of expression, placing the candidate gene under the control of both promoters will ensure the expression of the transgene in the majority of the cells. (Subcloning can be performed into pPD49.78 and pPD49.83, which contain the hsp-16.2 and hsp-16.41 promoters, respectively; generated by A. Fire, Stanford School of Medicine and available at http://www.addgene. org/; plasmids 1447 and 1448.) Another important consideration is the presence of introns in the gene of interest, since introns are necessary to maximize its expression. Synthetic introns have been shown to increase expression of cDNA-based transgenes in C. elegans (Okkema, Harrison, Plunger, Aryana, & Fire, 1993). Such synthetic introns are present in pPD49.78 and pPD49.83 and were shown to be sufficient to properly drive the expression of ced-9, egl-1, fzo-1, and eat-3 cDNA ( Jagasia et al., 2005; Lu et al., 2011; Rolland et al., 2009). To test the effect of the overexpression of a new candidate gene on mitochondrial morphology, transgenic animals expressing the cDNA of this gene and a mitochondrial matrix-targeted GFP (mitoGFP) under the control of the heat-shock promoters can be analyzed using the protocols described below (Section 4.3.3).

3.2.2 In specific cell types, using smg-inducible system Different promoters can be used to drive the expression of a particular candidate gene in specific cell types (Section 4.3.2). However, constitutive expression of a particular gene may be toxic. In that case, a cell-type specific inducible expression system, such as the smg-inducible system, can be used. The smg-inducible system takes advantage of a temperature-sensitive allele of the Smg RNA surveillance machinery (smg-1(cc546ts)) (Pulak & Anderson, 1993). The candidate gene is cloned under the control of a cell-type specific promoter and upstream of an abnormal 30 UTR (e.g., in

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pPD118.60; provided by A. Fire, Stanford School of Medicine, and available at http://www.addgene.org/; plasmid 1598). At permissive temperature (15 C), the mRNA of the transgene is degraded due to its abnormal 30 UTR. At restrictive temperature (25 C), the Smg RNA surveillance machinery is not functional, which results in the stabilization of the transgene mRNA and its translation. This system has been successfully used to drive the inducible expression of ced-9 and fzo-1 in body wall muscle cells (Rolland et al., 2009). To analyze the role of a candidate gene on mitochondrial morphology in a specific cell-type, the smg-inducible system can be used in conjunction with the corresponding cell-type specific mitochondrial fluorescent reporter (Section 4.3).

4. HOW TO VISUALIZE MITOCHONDRIA IN C. ELEGANS 4.1. General considerations for imaging This review describes methodologies to visualize mitochondria using epifluorescence microscopy. Indeed, deconvolution of a Z-stack acquired with an epifluorescence microscope results in a quality that is sufficient to visualize and quantify mitochondrial morphology. Nonetheless, all the fluorescent imaging described below can also be performed using confocal microscopy.

4.2. Mitochondrial membrane potential-sensitive dyes 4.2.1 TMRE, Mitotracker, and others dyes Several mitochondrial membrane potential-sensitive dyes have been used to visualize mitochondria in C. elegans (Table 4.2). Some of these dyes also allow one to measure the dynamics of mitochondrial membrane potential as they are rapidly and reversibly taken up by mitochondria (Loew, Tuft, Carrington, & Fay, 1993). For example, protocols using TMRE or DiSC3(3) have been established in C. elegans to measure mitochondrial membrane potential at the organismal level (Gaskova et al., 2007; Yoneda et al., 2004). The dyes described in this section have been used to stain mitochondria in several tissues (germline, embryonic cells, body wall muscle cells, hypodermis, neurons). However, the choice of the dye will depend on the tissue to be analyzed. For example, TMRE specifically stains mitochondria in embryos, whereas Mitotracker Red CMXRos does not (Rolland et al., 2009). In contrast, Mitotracker Red CMXRos stains specifically mitochondria in muscle cells (Kimura et al., 2007).

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Table 4.2 C. elegans mitochondrial membrane potential-sensitive dyes Dye Concentration Tissue analyzed References

Rhodamine B hexyl ester

30 mM

Embryonic cells

Jagasia et al. (2005)

TMRE

30 mM

Embryonic cells

Breckenridge et al. (2008), Jagasia et al. (2005), and Rolland et al. (2009)

MitoTracker 500 nM–5 mM Muscle, sperm Red CMXRos

Al Rawi et al. (2011) and Kimura, Tanaka, Nakamura, Takano, and Ohkuma (2007)

Rhodamine 6G 5 mM

Germline, embryonic cells

Badrinath & White (2003) and Labrousse et al. (1999) Gaskova, DeCorby, and Lemire (2007)

DiS-C3(3)

4 mM

NAa

HRB and HR101

100 pM

Mottram, Forbes, Ackley, Hypodermis, muscle, neurons, and Peterson (2012) germline

a

Lemire and coworkers used DiS-C3(3) to measure mitochondrial membrane potential (Gaskova et al., 2007).

In most protocols, mitochondrial dyes are simply incorporated in nematode growth medium (NGM) agar plates. C. elegans animals are fed with E. coli bacteria which are stained with the dye. An important issue with this approach is the background staining present in the animals’ intestines. While it is not a problem to analyze mitochondrial morphology in embryos, the background staining considerably affects the mitochondria visualization in other tissues and requires several washing steps. The concentration of the mitochondrial dye and the duration of exposure are important parameters to consider since extended period of staining or a high concentration of the dyes are toxic (Mottram et al., 2012). Recently, the development of new rhodamine derivatives (HRB and HR101) has improved both these parameters as these dyes are rapidly taken up (within 2 h) at low concentrations (100 pM) and exhibit low toxicity (Mottram et al., 2012). 4.2.2 Protocol to visualize mitochondria in embryos using TMRE 4.2.2.1 Media and solutions

– Solutions for NGM agar plates (1 M CaCl2, 1 M MgSO4, 1 M potassium phosphate buffer, pH 6.0, and cholesterol solution (5 mg/ml in 100% ethanol)) (Stiernagle, 2006).

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– NGM agar medium: prepare 50 ml aliquots using the following recipe: • 0.15 g NaCl • 0.37 g peptone • 0.85 g agar • qsp 50 ml with dH2O After autoclaving, the aliquots can be stored for several weeks at 4 C. – 0.1 M TMRE: dissolve 51.4 mg/ml of TMRE (molecular probes) in DMSO and store 50 ml aliquots at 20 C in the dark. (Aliquots can be thawed and frozen three to four times.) – E. coli OP50 culture: grow a E. coli OP50 culture as described (Stiernagle, 2006). (To ensure reproducibility, use a fresh culture inoculated the day before.) 4.2.2.2 Materials

– – – –

32 mm Petri dishes Microscope slides and cover glasses 25G needles Epifluorescence microscope equipped with Nomarski optics and TRITC filter (e.g., Zeiss Axioskop 2 microscope with Zeiss Filter F41-032)

4.2.2.3 Protocol

Day 1: 1. Melt one aliquot of NGM agar medium using a microwave. When the medium has cooled down to 55 C, add the following (in that order): – 50 ml 1 M CaCl2 – 50 ml 1 M MgSO4 – 1.25 ml 1 M potassium phosphate buffer pH 6.0 – 100 ml cholesterol (5 mg/ml) – 15 ml 0.1 M TMRE 2. Pour 5 ml of medium per 32-mm Petri dish. Let the plates dry overnight in the dark. (Protect TMRE plates from light, since TMRE is light sensitive.) Day 2: 1. Transfer 10 L4 larvae of the strain of interest and control strain on 60 mm NGM plates (without TMRE) and incubate at 20 C for 24 h. 2. Seed TMRE plates with 50 ml of OP50 culture and incubate at 20 C for 24 h in the dark.


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Day 3: Transfer 10 adults of each strain to the TMRE plates and incubate at 20 C for 15 h (i.e., overnight). Day 4: 1. Transfer 2–3 adults from the TMRE plate to a regular NGM plate (without TMRE). Let the animals crawl on the plate for 5 min. (This step is required to remove the excess of TMRE positive bacteria, which creates background signal.) (see Fig. 4.1). 2. Prepare a 2% agarose pad on a microscope slide using the setup described in Fig. 4.1. 3. Transfer the adults in 5 ml of M9 on a cover glass. (Alternatively, M9 containing 1 mM of levamisole can be used to paralyze the animals.) 4. Bisect adult animals using 25G needles to extract the embryos. 5. Transfer the cover glass up-side-down on the agarose pad. 6. Image mitochondria using an epifluorescence microscope. The microscope slides can be analyzed for 10–15 min. To analyze the slides for an extended period of time, the cover glass can be sealed by brushing melted white vaseline along the edge of the cover glass. To further process the images with a deconvolution software, it is necessary to acquire a Z-stack of the embryo. To define the top and bottom sections of the Z-stack, it is preferable to use Nomarski optics, in order to minimize the bleaching of the TMRE signal. For the same reason, during the acquisition of the TMRE signal, it is recommended to increase the imaging exposure time while using a neutral density filter to decrease the intensity of the excitation light. These values need to be adjusted depending on the microscope, excitation light, and camera setup. As an indication, using a Zeiss Axioskop 2 equipped with a 100 1.3 NA oil lens, a TRITC filter (F41-032; Zeiss), a HAL-100 mercury lamp housing, a CCD camera (1300; Micromax), and a 50% neutral density filter, 100 ms exposure acquisition time results in sufficient quality for further processing. Examples of TMRE stained embryos acquired with this setup are provided in Fig. 4.2. 4.2.2.4 Specific protocol for TMRE staining of animals treated with RNAi

RNAi is performed essentially as previously described (Ahringer, 2006). On Day 1, 32 mm RNAi plate supplemented with Carbenicillin (25 mg/ml) and 1 mM IPTG are inoculated with a fresh culture of the

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Figure 4.1 Flowchart of the protocol to visualize mitochondria in C. elegans embryos using TMRE staining. (1) 1-day post-L4 C. elegans adults grown for 15 h on TMRE medium are transferred for 5 min onto normal NGM medium. (2) 2% agarose thin pad is prepared between two microscope slides. The laboratory tape present on the two “spacer” microscope slides defines the thickness of the pad. (3) 2–3 adults are then transferred from the normal NGM medium to a microscope cover glass in 5 ml of M9 medium (that can be supplemented with 1 mM Levamisole). (4) Using two 25G needles, adults are bisected to extract the embryos. (5) The cover glass is then transferred up-side-down on the agarose pad. (6) The microscope slide is analyzed by Nomarski and fluorescent microcopy.

RNAi clone. On Day 2, 10 L4 larvae of the strain of interest are transferred onto the RNAi plate. On Day 3, adults exposed to RNAi for 24 h are transferred onto the TMRE plate. The rest of the protocol is performed as described above.


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Figure 4.2 Representative images of TMRE stained embryos of C. elegans. 2 cells and 30 cells embryos were stained with TMRE and analyzed by Nomarski and fluorescent microscopy. The fluorescent Z-stacks were deconvolved using Autodeblur. A single plane image for each embryo is shown.

4.3. Mitochondrial-targeted fluorescent proteins 4.3.1 Which fluorescent proteins to use As shown in Table 4.3, several fluorescent proteins have been used to label mitochondria in C. elegans: GFP, YFP, CFP, and the dsRedT3 variant. These proteins have been targeted to the mitochondrial matrix (Labrousse et al., 1999; Rolland et al., 2013). In addition, an OMM marker has been generated by fusing the N-terminal 30 amino acids of yeast TOM70 to YFP (TOM70::YFP) (Labrousse et al., 1999). Furthermore, a photoactivable mitoGFP (PAmitoGFP) has been recently generated (Rolland et al., 2013). Unlike mitoGFP, mitoCFP, PAmitoGFP, and TOM70::YFP, the expression of mitodsRed seems to affect viability and growth of the transgenic animals. For this reason, mitodsRed reporters (such as pBC914) have to be used at lower concentrations (0.2 ng/ml) compared to other mitochondrial reporters such as mitoGFP (pVDB#1; 5 ng/ml) (Rolland et al., 2013).

Table 4.3 Plasmids encoding the C. elegans mitochondria fluorescent reporters Fluorescent proteins targeted to Matrix Tissue

Promoter

Embryonic cells

Phsp-16.2 and pBC307 and pBC308 Phsp-16.41 ( Jagasia et al., 2005)

Body wall muscle cells

Pmyo-3

pVDB#1 (Labrousse et al., 1999)

Apoptotic cells

Pegl-1

pBC306 ( Jagasia et al., 2005)

mitoGFP

OMM mitoCFP

mitodsRed

PAmitoGFP

TOM70::YFP


pBC914 (Rolland et al., 2013)

pBC900 (Rolland et al., 2013)



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4.3.2 Which promoter to drive the expression of the mitochondrial marker Since the heat-shock promoters hsp-16.2 and hsp-16.41 have a complementary pattern of expression, the two heat-shock plasmids expressing mitoGFP (Table 4.3) are usually cotransformed to maximize the number of mitoGFPpositive cells. These reporters become inducible at mid-gastrulation (150 min after the first cell division at 20 C) (Stringham et al., 1992). They are therefore suitable to study mitochondrial morphology in apoptotic cells (since the first cell death occurs at 220 min at 20 C) (Section 4.4). An example of an embryo carrying the PHS mitoGFP transgene is shown in Fig. 4.3A and A0 . Van der Bliek and coworkers have pioneered the visualization of mitochondria in body wall muscle cells (muscles which are required for C. elegans locomotion) using the myo-3 promoter to drive the expression of mitoGFP, mitoCFP, and TOM70::YFP (Table 4.3; Labrousse et al., 1999). More

Figure 4.3 Representative images of different mitochondrial fluorescent reporters. MD2416 embryo analyzed by Nomarski (A) and fluorescent microscopy (A0 ). MD2416 animals are mosaic for the presence of the transgene and thus its expression. Only some cells (arrowhead) express the mitoGFP reporter whereas others do not (arrow) (n: nucleus). (B) MD2517 L4 larvae body wall muscle cells mitochondria labeled with mitoGFP and analyzed by fluorescent microscopy. Both fluorescent images are single plane images out of a Z-stack deconvolved using Autodeblur.

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recently, mitodsRed and PAmitoGFP have been placed under the control of the myo-3 promoter (Table 4.3). All these plasmids are often cotransformed in C. elegans with the pRF4 plasmid. pRF4 contains the rol-6(su1006dm) allele, which causes the transgenic animals to exhibit a dominant roller phenotype (Kramer, French, Park, & Johnson, 1990). This phenotype is accompanied by a helical twisting of the four muscle quadrants, which facilitates the visualization of the muscle cells and their mitochondria. An example of a transgenic animal carrying the Pmyo-3 mitoGFP transgene is provided in Fig. 4.3B. 4.3.3 Visualizing embryonic mitochondria using the PHS mitoGFP strain 4.3.3.1 Strain, media, and solutions

– MD2416 (PHS mitoGFP strain; Table 4.4) – 60 mm NGM plate seeded with E. coli OP50 strain (Stiernagle, 2006) – M9 liquid media (Stiernagle, 2006) 4.3.3.2 Materials

– 20 and 32 C incubators – Microscope slides and cover glasses – Epifluorescence microscope equipped with Nomarski optics and FITC filter (e.g., Zeiss Axioskop 2 microscope with Zeiss Filter F41-012) Table 4.4 Transgenic lines expressing the C. elegans mitochondria fluorescent reporters Strain Genotype References

Jagasia et al. MD2416 lin-15(n765ts); bcEx555 [pL15EK (50 ng/ml) + pBC307 (1 ng/ml) + pBC308 (2005) (1 ng/ml)] MD2517 bcEx620 [pRF4 (80 ng/ml) + pVDB#1 (5 ng/ml)]

Rolland et al. (2009)

MD2922 bcEx856 [pRF4 (80 ng/ml) + pBC900 (2 ng/ml) + pBC914 (0.2 ng/ml)]

Rolland et al. (2013)

MD1948 unc-76(e911); bcIs50 [pBC306 (5 ng/ml) + p76-16B (75 ng/ml)]


MD1386 unc-76(e911); bcEx383 [pBC307 (5 ng/ml) + pBC27 (5 ng/ml) + p76-16B (75 ng/ml)]



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4.3.3.3 Protocol

Day 1: Transfer 20 MD2416 L4 larvae on 60 mm NGM plates seeded with OP50 and incubate at 20 C for 24 h. MD2416 carries n765ts, a temperature-sensitive mutation in the lin-15AB gene and bcEx555, an extra-chromosomal array composed of PHS mitoGFP and pL15EK. At restrictive temperature (20 C), nontransgenic animals exhibit a multivulva (Muv) phenotype, whereas transgenic animals are non-Muv or “wild-type” (due to the rescue of the lin-15AB(n765ts) mutation by the plasmid pL15EK) (Sternberg, 2005). At permissive temperature (15 C), both transgenic and nontransgenic animals appear “wild-type.” The MD2416 strain must be therefore kept at 20 C. In addition, the Muv phenotype is only visible with a dissecting microscope at the adult stage. Therefore, it is essential to transfer 20 L4 larvae on Day 1, in order to have at least 10 transgenic adult animals on Day 2. Day 2: 1. Transfer 10 transgenic (non-Muv) MD2416 adults animals onto a new 60 mm NGM plate seeded with OP50. 2. Incubate the plate at 20 C for 2 h. 3. Incubate the plate at 32 C for 45 min. 4. Incubate the plate at 20 C for 1 h and 15 min. 5. Prepare a 2% agarose pad as described earlier (Fig. 4.1). 6. Transfer embryos that were laid on the plate in 5 ml of M9 on the agarose pad. 7. Cover the pad with a cover glass. 8. Image mitochondria using an epifluorescence microscope. In order to further process the images with a deconvolution software, it is necessary to acquire a Z-stack of the embryo. Unlike for the TMRE staining, the top and bottom section of the Z-stack can be defined using fluorescence microscopy, since all the mitochondrial fluorescent reporters are stable. In addition, as mentioned earlier, MD2416 transgenic animals carry extra-chromosomal arrays. They are therefore mosaic for the presence of the transgene and consequently its expression. Hence, not all the cells of a transgenic embryo will express the mitoGFP reporter (Fig. 4.3A and A0 ). 4.3.4 Visualizing muscle cell mitochondria using the Pmyo-3 mitoGFP strain 4.3.4.1 Strain, media, and solutions

– MD2517 (Pmyo-3 mitoGFP strain; Table 4.4)

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– 60 mm NGM plate seeded with E. coli OP50 strain (Stiernagle, 2006) – M9 liquid media (Stiernagle, 2006) supplemented with 1 mM levamisole 4.3.4.2 Materials

– Microscope slides and cover glasses – Epifluorescence microscope equipped with FITC filter (e.g., Zeiss Axioskop 2 microscope equipped with Zeiss Filter F41-012) 4.3.4.3 Protocol

1. Prepare a 2% agarose pad as described earlier (Fig. 4.1). 2. Transfer 10 transgenic (roller) L4 larvae animals in 5 ml of M9 supplemented with 1 mM levamisole on the agarose pad. 3. Cover the agarose pad with a cover glass. 4. Image mitochondria using an epifluorescence microscope. It is critical to image mitochondria within 10–15 min. Longer incubations lead to the death of the animal and an artificial mitochondrial fragmentation. Recently, microfluidic devices have been developed to image C. elegans animals for an extended period of time (Mondal, Ahlawat, Rau, Venkataraman, & Koushika, 2011). An example of muscle cells mitochondria after deconvolution of a Z-stack is provided in Fig. 4.3C. 4.3.4.4 Specific protocol for RNAi treatment of Pmyo-3 mitoGFP strain

RNAi is performed essentially as previously described (Ahringer, 2006). On Day 1, 32 mm RNAi plate supplemented with Carbenicillin (25 mg/ml) and 1 mM IPTG are inoculated with a fresh culture of the RNAi clone. On Day 2, two transgenic (roller) MD2517 L4 larvae are inoculated on the RNAi plate. At 20 C, the next generation of L4 larvae can be analyzed using fluorescence microscopy as described above, 4–5 days later.

4.4. Visualizing mitochondria in apoptotic cells One hundred and thirteen cells undergo programmed cell death during C. elegans embryogenesis (Sulston et al., 1983). The identity of each dying cell, the time at which it dies and its position in the embryo, is highly reproducible from individual to individual. This unique characteristic makes C. elegans an excellent model to study changes in mitochondrial morphology in response to apoptotic signals during development. Apoptotic cells can be identified by Nomarski optics because they exhibit a characteristic “button-like” structure, appearing as flat, round disks. However, since mitochondrial fragmentation occurs before the


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apoptotic cell exhibits this characteristic structure ( Jagasia et al., 2005), other tools have been developed to investigate mitochondrial morphology changes during apoptosis. Specifically, a reporter that expresses mitoGFP under the control of the promoter of the C. elegans BH3-only gene egl-1 has been used to specifically label mitochondria in apoptotic cells ( Jagasia et al., 2005) (Pegl-1:mitoGFP; Table 4.3). Since egl-1 transcription is an early apoptotic event, this reporter allows visualizing mitochondrial morphology during the entire apoptotic process. Analysis by confocal time-lapse microscopy of a strain carrying this reporter revealed that mitochondrial fragmentation is an early feature of apoptosis in C. elegans ( Jagasia et al., 2005). As an alternative, overexpression of egl-1, using the heat-shock promoters described earlier, has been shown to induce ectopic apoptosis (Conradt & Horvitz, 1998). A C. elegans strain carrying the PHS egl-1 (pBC27) and PHS mitoGFP (pBC307) transgenes (Table 4.4) can therefore be used to analyze changes in mitochondrial morphology during ectopic cell death ( Jagasia et al., 2005).

4.5. Quantification of mitochondrial morphology 4.5.1 Qualitative analysis of mitochondrial morphology in embryos Quantitative measurement of mitochondrial morphology in C. elegans embryos represents a technical challenge because of the three-dimensional structure of the mitochondrial network. In addition, during later stages of embryonic development, the small size of the cells makes the quantification even more challenging. For instance, at the stage when the first cell undergoes apoptosis, cells have a diameter of 2 mm. In comparison, HeLa cells have a diameter of 20 mm and C. elegans body wall muscle cells in L4 larvae have a size of 60 mm 8 mm. To my knowledge, automated system for the quantification and classification of mitochondrial morphology has not yet been developed for C. elegans. As a consequence, the data are analyzed qualitatively using different mitochondrial morphology categories (such as fragmented, tubular, and highly elongated). To ensure unbiased analysis, the data are often analyzed blind by two independent investigators. 4.5.2 Quantitative morphometric analysis of mitochondrial morphology in larval muscle cells Body wall muscle cells are well suited for quantitative measurement of mitochondrial morphology, because of the two-dimensional structure of their

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mitochondrial network. In order to increase the signal-to-noise ratio, it is preferable to acquire a Z-stack and to further process it using deconvolution. Mitochondria morphology can then be analyzed using a morphometric analysis software such as MetaMorph (MDS Analytical Technologies) or ImageJ (NIH). Among the parameters that can be measured, mitochondrial length is a good indicator of mitochondrial morphology (Lu et al., 2011; Rolland et al., 2009; Tan et al., 2008). Circularity or shape factor (whose values vary between 0 and 1; 0 indicating a perfect line and 1 indicating a perfect circle) is a good indicator of mitochondrial fragmentation (Bess, Crocker, Ryde, & Meyer, 2012). Finally, the mean area to perimeter ratio has been used as a measure of interconnectivity (Bess et al., 2012). 4.5.3 Quantification of mitochondrial morphology and mitochondrial fusion/fission rates using a photoactivable mitoGFP Fluorescence loss in photobleaching with mitoGFP and more recently photoactivation of PAmitoGFP have been used to measure mitochondrial interconnectivity in body wall muscle cells (Labrousse et al., 1999; Rolland et al., 2013). The recent development of PAmitoGFP in C. elegans opens up the possibility to also measure mitochondrial fusion and fission rates, but this technique need to be further developed.

5. POTENTIAL OUTCOMES AND FUTURE EXPERIMENTS Inactivation of a particular candidate gene or its overexpression may not lead to any obvious mitochondrial morphology changes. In order to conclude that this gene is not involved in the regulation of mitochondrial morphology, it is necessary to confirm that its inactivation or overexpression was successful. In the case of loss-of-function mutation, analysis of the DNA sequence can often predict if the mutation will affect protein function (e.g., deletion of an important domain of the protein or frame shift generating an early stop codon). However, if antibodies against the protein of interest are available, it is good to verify these predictions using western analysis. In the case of RNAi, before concluding on the absence of phenotype, it is important to confirm the efficiency of the inactivation (using RT-PCR or western analysis). Concerning the overexpression, no phenotype can simply mean that the transgene was not expressed or localized properly. This can be tested using antibodies against the protein of interest or alternatively by tagging this protein with GFP.


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When the inactivation of a gene by RNAi leads to a change in mitochondrial morphology, it is recommended to confirm the phenotype using a strain carrying a loss-of-function mutation in this gene. In addition, lossof-function mutations should always be outcrossed, as other mutations in the background may be responsible for the observed phenotype. Finally, if a different loss-of-function allele of the gene of interest is available, it should also be tested. Once the phenotype has been confirmed, further experiments can be performed to gain a better understanding of how the newly discovered factor regulates mitochondrial morphology. For example, epistasis analysis can be performed to determine whether this new factor acts upstream or downstream of the known fusion and fission machineries or alternatively in a new pathway. The expression pattern and localization of this new factor can also be determined by using a GFP fusion protein. This can be performed with the help of new resources such as the TransgeneOme project, which has already generated a library of fosmid-based GFP transgene covering 70% of the C. elegans proteome (Sarov et al., 2012). Finally, if this factor is shown to regulate mitochondrial morphology in apoptotic cells, its role in the execution of the apoptotic process can be tested (Schwartz, 2007).

6. CONCLUDING REMARKS AND FUTURE CHALLENGES This review describes tools for a candidate gene approach to test whether a particular factor control mitochondrial morphology in healthy and/or apoptotic cells. C. elegans being an excellent model for gene discovery, one could imagine a more unbiased approach such as a genetic screen for new factors regulating mitochondrial morphology. Some attempts have been made using RNAi (Ichishita et al., 2008) but with the development of next-generation sequencing, forward genetic screen can now be envisioned. The tools described in this review can be used to test the effect of new identified mutations on mitochondrial morphology in healthy and apoptotic cells.

ACKNOWLEDGMENTS I thank Barbara Conradt, Saroj Regmi, and Nadin Memar for their comments on the manuscript. This work was supported by funding from the National Institutes of Health Grant GM076651 and the Center for Integrated Protein Science Munich to Barbara Conradt.

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Exposure to mitochondrial genotoxins and dopaminergic neurodegeneration in Caenorhabditis elegans.

Kinetics and specificity of paternal mitochondrial elimination in Caenorhabditis elegans.

Mitochondrial Morphology and Fundamental Parameters of the Mitochondrial Respiratory Chain Are Altered in Caenorhabditis elegans Strains Deficient in Mitochondrial Dynamics and Homeostasis Processes.

Caenorhabditis elegans EXO-3 contributes to longevity and reproduction: differential roles in somatic cells and germ cells.

Changes in apoptotic microRNA and mRNA expression profiling in Caenorhabditis elegans during the Shenzhou-8 mission.

Duplications in Caenorhabditis elegans.

In vivo Mitophagy Monitoring in Caenorhabditis elegans to Determine Mitochondrial Homeostasis.

Tools and methods to analyze autophagy in C. elegans.

A fluorogenic aryl fluorosulfate for intraorganellar transthyretin imaging in living cells and in Caenorhabditis elegans.

Automatic quantitative analysis of morphology of apoptotic HL-60 cells.

Loss of hif-1 promotes resistance to the exogenous mitochondrial stressor ethidium bromide in Caenorhabditis elegans.

Epigallocatechin-3-gallate promotes healthy lifespan through mitohormesis during early-to-mid adulthood in Caenorhabditis elegans.

hParkin negatively regulates the phagocytosis of apoptotic cell corpses in Caenorhabditis elegans.

Myosin activity drives actomyosin bundle formation and organization in contractile cells of the Caenorhabditis elegans spermatheca.

Communication between oocytes and somatic cells regulates volatile pheromone production in Caenorhabditis elegans.

Embryonic development in Caenorhabditis elegans.

Indirect suppression in Caenorhabditis elegans.

From cell fates to morphology: developmental genetics of the Caenorhabditis elegans male tail.

Chromosome rearrangements in Caenorhabditis elegans.

Apoptotic transition of senescent cells accompanied with mitochondrial hyper-function.

Polyploids and sex determination in Caenorhabditis elegans.

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay.

Spatial control of active CDC-42 during collective migration of hypodermal cells in Caenorhabditis elegans.

The BED finger domain protein MIG-39 halts migration of distal tip cells in Caenorhabditis elegans.