Microscopic Investigation of Protein Function in C. elegans Using Fluorescent Imaging

Cliff J. Luke1, Linda P. O'Reilly1

1 Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.41
DOI:  10.1002/0471142956.cy1241s74
Online Posting Date:  October, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Caenorhabditis elegans is a powerful model organism for studying human biology and disease due to its surprisingly high genetic homology to Homo sapiens. Its genetic amenability, small size, short generation time, and transparent body make it an ideal organism for multiple scientific disciplines. Fluorescent microscopy is essential for studying protein biological function. However, C. elegans, mainly due to its high motility, has been more difficult to adapt to fluorescence imaging, especially live‐imaging. We present here several protocols for the study of protein location, function and dynamics in context of a whole animal. These protocols, especially when combined with existing genetic procedures, can yield a great deal of insight in the physiological roles of proteins in C. elegans, which can be directly translated into mammalian systems. © 2015 by John Wiley & Sons, Inc.

Keywords: Caenorhabditis elegans; fluorescent protein; immobilization; antibody; time‐lapse imaging; fluorescence microscopy

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Fixation of C. elegans for Immunofluorescence
  • Immobilization of C. elegans for Live Fluorescence Imaging
  • Basic Protocol 2: Chemical Immobilization using Sodium Azide or Levamisole
  • Mechanical Immobilization
  • Basic Protocol 3: Immobilization by Weight of Coverglass
  • Basic Protocol 4: Immobilization by Cyanoacrylate Glue
  • Basic Protocol 5: Immobilization by Agarose and Polystyrene Beads
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Fixation of C. elegans for Immunofluorescence

  Materials
  • Phosphate‐buffered saline (PBS; see recipe)
  • Caenorhabditis elegans (C. elegans)
  • 4% paraformaldehyde (PFA) in PBS (see recipe)
  • 2‐mercaptoethanol solution (see recipe)
  • Immunowash solution (see recipe)
  • Collagenase solution
  • Blocking buffer (see recipe)
  • Primary antibody
  • Fluorescent secondary antibody
  • Fluoromount‐G (Southern Biotech)
  • Nail polish
  • 6‐cm NGM petri dishes seeded with OP50
  • Microcentrifuge
  • Rotator at 4°C
  • Glass microscope slide
  • 200‐μl pipets
  • 22 × 50–mm coverglass no. 1.5
  • Upright or inverted widefield or confocal microscope
CAUTION: Paraformaldehyde is corrosive and an irritant. 2‐mercaptothanol is a toxin and irritant. Always perform steps using paraformaldehyde and 2‐mercaptoethanol in the fume hood with personal protective equipment, such as gloves and eyeglasses.

Basic Protocol 2: Chemical Immobilization using Sodium Azide or Levamisole

  Materials
  • 2% agarose in water, melted and kept at 65°C (see recipe)
  • 25 mM NaN 3or 1 mM levamisole in PBS or M9 (see recipe)
  • Caenorhabditis elegans (C. elegans)
  • Glass microscope slide
  • Laboratory tape
  • Transfer or Pasteur pipets
  • 22 × 50–mm coverglass no. 1.5
  • C. elegans platinum wire pick
  • Scalpel or razor blade
  • Stereomicroscope
  • Upright or inverted widefield or confocal microscope
CAUTION: Hot agarose can cause burns and should be handled appropriately. Use gloves and personal protective wear when making agarose pads. NaN 3 and levamisole are toxins and should be handled with care. Always make these solutions in a fume hood and wear personal protective clothing. Care must also be taken when using sharps such as a scalpel or razor blade to prevent personal injury.

Alternate Protocol 1:

  Additional Materials (also see protocol 2)
  • Coverglass‐bottomed 35‐mm petri dish (e.g., MatTek glass‐bottom dish, cat. no. P35G‐1.5‐14‐C; http://glass‐bottom‐dishes.com/)
  • Platinum wire pick
  • Coverslip forceps
  • 12‐mm diameter round coverslip
  • 22 × 22–mm square coverslip

Basic Protocol 3: Immobilization by Weight of Coverglass

  Materials
  • Phosphate‐buffered saline (PBS; see recipe) or M9 containing OP50 (see recipe)
  • Glass microscope slide
  • Platinum wire pick
  • 22 × 50–mm coverglass no. 1.5
  • Inverted widefield or confocal microscope

Basic Protocol 4: Immobilization by Cyanoacrylate Glue

  Materials
  • 4% agarose in water, melted and kept at 65°C
  • Caenorhabditis elegans (C. elegans)
  • Cyanoacrylate glue
  • Phosphate‐buffered saline (PBS; see recipe) or M9 containing OP50 (see recipe)
  • Glass microscope slide
  • Laboratory tape
  • Transfer or Pasteur pipet
  • Thin glass capillary
  • Bunsen burner
  • Platinum wire pick
  • 22 × 50–mm coverglass no. 1.5
  • Inverted widefield or confocal microscope

Basic Protocol 5: Immobilization by Agarose and Polystyrene Beads

  Materials
  • 6% agarose in water, melted and kept at ∼95°C on a stirring hotplate
  • 0.05‐μm polystyrene microsphere, 2.5% (w/v)
  • Microspheres
  • 40× phosphate‐buffered saline (PBS)
  • OP50 (see recipe) in phosphate‐buffered saline (PBS)
  • Caenorhabditis elegans (C. elegans)
  • 1:1 mix of paraffins:petroleum jelly
  • Deionized water
  • Coverglass‐bottomed 35 mm petri dish
  • Spatula
  • 22 × 22–mm square coverslip
  • 1.5‐ml microcentrifuge tubes
  • Coverglass forceps
  • Low‐lint tissue
  • Parafilm
  • Inverted widefield or confocal microscope
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Alavez, S., Vantipalli, M.C., Zucker, D.J., Klang, I.M., and Lithgow, G.J. 2011. Amyloid‐binding compounds maintain protein homeostasis during ageing and extend lifespan. Nature 472:226‐229.doi: 10.1038/nature09873.
  Allison, A.C. and Young, M.R. 1964. Uptake of dyes and drugs by living cells in culture. Life Sci. 3:1407‐1414. doi: 10.1016/0024‐3205(64)90082‐7.
  Anderson, R.G. and Orci, L. 1988. A view of acidic intracellular compartments. J. Cell Biol. 106:539‐543.doi: 10.1083/jcb.106.3.539.
  Berezikov, E., Bargmann, C.I., and Plasterk, R.H. 2004. Homologous gene targeting in Caenorhabditis elegans by biolistic transformation. Nucleic Acids Res. 32:e40‐e46. doi: 10.1093/nar/gnh033.
  Brenner, S. 1974. The genetics of Caenorhabditis elegans. Genetics 77:71‐94.
  Broverman, S., MacMorris, M., and Blumenthal, T. 1993. Alteration of Caenorhabditis elegans gene expression by targeted transformation. Proc. Natl. Acad. Sci. U.S.A. 90:4359‐4363.doi: 10.1073/pnas.90.10.4359.
  Clokey, G.V. and Jacobson, L.A. 1986. The autofluorescent "lipofuscin granules" in the intestinal cells of Caenorhabditis elegans are secondary lysosomes. Mech. Ageing Dev. 35:79‐94. doi: 10.1016/0047‐6374(86)90068‐0.
  Coburn, C., Allman, E., Mahanti, P., Benedetto, A., Cabreiro, F., Pincus, Z., Matthijssens, F., Araiz, C., Mandel, A., Vlachos, M., Edwards, S.A., Fischer, G., Davidson, A., Pryor, R.E., Stevens, A., Slack, F.J., Tavernarakis, N., Braeckman, B.P., Schroeder, F.C., Nehrke, K., and Gems, D. 2013. Anthranilate fluorescence marks a calcium‐propagated necrotic wave that promotes organismal death in C. elegans. PLoS Biol. 11:e1001613.
  Consortium, T.C.e.S. 1998. Genome sequence of the nematode C. elegans: A platform for investigating biology. Science 282:2012‐2018.doi: 10.1126/science.282.5396.2012.
  Dickinson, D.J., Ward, J.D., Reiner, D.J., and Goldstein, B. 2013. Engineering the Caenorhabditis elegans genome using Cas9‐triggered homologous recombination. Nat. Methods 10:1028‐1034. 10.1038/nmeth.2641.
  Duerr, J.S. 2006. Immunohistochemistry. WormBook 1‐61.
  Friedland, A.E., Tzur, Y.B., Esvelt, K.M., Colaiacovo, M.P., Church, G.M., and Calarco, J.A. 2013. Heritable genome editing in C. elegans via a CRISPR‐Cas9 system. Nat. Methods 10:741‐743.doi: 10.1038/nmeth.2532.
  Frokjaer‐Jensen, C., Davis, M.W., Hopkins, C.E., Newman, B.J., Thummel, J.M., Olesen, S.P., Grunnet, M., and Jorgensen, E.M. 2008. Single‐copy insertion of transgenes in Caenorhabditis elegans. Nat. Genet. 40:1375‐1383. doi: 10.1038/ng.248.
  Goodman, M.B., Hall, D.H., Avery, L., and Lockery, S.R. 1998. Active currents regulate sensitivity and dynamic range in C. elegans neurons. Neuron 20:763‐772.doi: 10.1016/S0896‐6273(00)81014‐4.
  Hadwiger, G., Dour, S., Arur, S., Fox, P., and Nonet, M.L. 2010. A monoclonal antibody toolkit for C. elegans. PLoS One 5:e10161. doi: 10.1371/journal.pone.0010161.
  Kerr, R., Lev‐Ram, V., Baird, G., Vincent, P., Tsien, R.Y., and Schafer, W.R. 2000. Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron 26:583‐594.doi: 10.1016/S0896‐6273(00)81196‐4.
  Kim, E., Sun, L., Gabel, C.V., and Fang‐Yen, C. 2013. Long‐term imaging of Caenorhabditis elegans using nanoparticle‐mediated immobilization. PLoS One 8:e53419. doi: 10.1371/journal.pone.0053419.
  Kostich, M., Fire, A., and Fambrough, D.M. 2000. Identification and molecular‐genetic characterization of a LAMP/CD68‐like protein from Caenorhabditis elegans. J. Cell Sci. 113:2595‐2606.
  Kramer, J.M. 1994. Structures and functions of collagens in Caenorhabditis elegans. FASEB J. 8:329‐336.
  Long, O.S., Benson, J.A., Kwak, J.H., Luke, C.J., Gosai, S.J., O'Reilly, L.P., Wang, Y., Li, J., Vetica, A.C., Miedel, M.T., Stolz, D.B., Watkins, S.C., Zuchner, S., Perlmutter, D.H., Silverman, G.A., and Pak, S.C. 2014. A C. elegans model of human alpha1‐antitrypsin deficiency links components of the RNAi pathway to misfolded protein turnover. Hum. Mol. Genet. 23:5109‐5122.doi: 10.1093/hmg/ddu235.
  Luke, C.J., Niehaus, J.Z., O'Reilly, L.P., and Watkins, S.C. 2014. Non‐microfluidic methods for imaging live C. elegans. Methods 68:542‐547.
  Luke, C.J., Pak, S.C., Askew, Y.S., Naviglia, T.L., Askew, D.J., Nobar, S.M., Vetica, A.C., Long, O.S., Watkins, S.C., Stolz, D.B., Barstead, R.J., Moulder, G.L., Bromme, D., and Silverman, G.A. 2007. An intracellular serpin regulates necrosis by inhibiting the induction and sequelae of lysosomal injury. Cell 130:1108‐1119. doi: 10.1016/j.cell.2007.07.013.
  Mello, C. and Fire, A. 1995. DNA transformation. Methods Cell Biol. 48:451‐482.
  Mello, C.C., Kramer, J.M., Stinchcomb, D., and Ambros, V. 1991. Efficient gene transfer in C. elegans: Extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10:3959‐3970.
  Miedel, M.T., Graf, N.J., Stephen, K.E., Long, O.S., Pak, S.C., Perlmutter, D.H., Silverman, G.A., and Luke, C.J. 2012. A pro‐cathepsin L mutant is a luminal substrate for endoplasmic reticulum‐associated degradation in C. elegans. PLoS One 7:e40145. doi: 10.1371/journal.pone.0040145.
  San‐Miguel, A. and Lu, H. 2013. Microfluidics as a tool for C. elegans research. WormBook 1‐19. doi: 10.1895/wormbook.1.162.1.
  Shaye, D.D. and Greenwald, I. 2011. OrthoList: A compendium of C. elegans genes with human orthologs. PLoS One 6:e20085.doi: 10.1371/journal.pone.0020085.
  Wadsworth, W.G. and Riddle, D.L. 1988. Acidic intracellular pH shift during Caenorhabditis elegans larval development. Proc. Natl. Acad. Sci. U.S.A. 85:8435‐8438. doi: 10.1073/pnas.85.22.8435.
  Yuan, J., Shaham, S., Ledoux, S., Ellis, H.M., and Horvitz, H.R. 1993. The C. elegans cell death gene ced‐3 encodes a protein similar to mammalian interleukin‐1 beta‐converting enzyme. Cell 75:641‐652.doi: 10.1016/0092‐8674(93)90485‐9.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library