Breaking Caenorhabditis elegans the easy way using the Balch homogenizer: an old tool for a new application

Abstract

The nematode Caenorhabditis elegans is a model organism best known for its powerful genetics. There is an increasing need in the worm community to couple genetics with biochemistry. Isolation of functionally active proteins or nucleic acids without the use of strong oxidizing denaturants or of subcellular compartments from C. elegans has, however, been challenging because of the worms' thick surrounding cuticle. The Balch homogenizer is a tool that has found much use in mammalian cell culture biology. The interchangeable single ball-bearing design of this instrument permits rapid permeabilization, or homogenization, of cells. Here we demonstrate the utility of the Balch homogenizer for studies with C. elegans. We describe procedures for the efficient breakage and homogenization of every larval stage, including dauers, and show that the Balch homogenizer can be used to extract functionally active proteins. Enzymatic assays for catalase and dihydrolipoamide dehydrogenase show that sample preparation using the Balch homogenizer equals or outperforms conventional methods employing boiling, sonication, or Dounce homogenization. We also describe phenol-free techniques for isolation of genomic DNA and RNA. Finally, we used the tool to isolate coupled mitochondria and polysomes. The reusable Balch homogenizer represents a quick and convenient solution for undertaking biochemical studies on C. elegans.

Figure 1. Balch Homogenizer

(A) Instrument construction - stainless steel chamber (1) with a hollow barrel into which a single tungsten carbide ball bearing (2) of defined tolerance (+/− 0.0001 mm) is inserted. The barrel is sealed with bolted steel caps (3). Glass syringes (4) force worm samples around the ball bearing. (B) Assembled instrument - placement of the Balch homogenizer on an ice-cold metal platform is ideal for maintaining the metal chamber at 4°C.

Figure 2. Fragmentation of staged, wild type C. elegans using the Balch homogenizer

Left panels: before homogenization; right panels, after homogenization. (A, B) L2 larvae – 10 μm clearance, 25 passes. (C, D) L4 larvae – 14 μm clearance, 25 passes. (E, F) Gravid adults - 18 μm clearance, 25 passes. (G, H) daf-2(e1370) dauer larvae - 2 μm clearance, 25 passes. In all cases >90% of animals were disrupted. Scale bar: 500 μm.

Figure 3. Quality of C. elegans protein after various homogenization techniques

Adult worms (wild type) were separated into four equal volumes then total protein extracted using one of four techniques – Balch homogenization, boiling, Dounce homogenization or sonication. (A) Extracted protein was separated into supernatant (S) and pellet (P) fractions as described under Materials and Methods, then protein concentration in each fraction determined. (B) Equal quantities of protein (25μg) were analyzed by 10% SDS-PAGE. Simply Blue Stain (Invitrogen) was used to assess relative protein abundance. (C, D) Western analysis was used to assess relative protein stability during each of the four extraction techniques. The E1α subunit of pyruvate dehydrogenase (PDH) was used as a marker protein (arrowhead). (D) is a longer exposure of (C).

Figure 4. RNA integrity following Balch Homogenization

Two methods were employed to extract RNA from C. elegans following sample disruption with the Balch Homogenizer. (A) Guanididium isothiocyanate method employing a Nucleospin RNA Isolation Kit. (B) Trizol (phenol-based) method. In both instances, worm samples were homogenized before molecular disruptants were added. No obvious compromise in RNA integrity was observed in either method. Listed in each panel are - DNA marker sizes (base pairs, bp), and RNA species (28S, 18S, and 5S rRNA, transfer RNA).

Figure 5. Isolation of high quality gDNA using the Balch Homogenizer

(A-C) SD1241 worms contain a sur-5∷GFP transgene that is genomic localized. Different stages of egg development are shown (A). A single round of freeze-thawing (-80°C) is sufficient to disrupt lipid membrane integrity (B). Balch Homogenization results in almost complete dissolution of eggs. A rare piece of debris is shown (C). For panes A-C: left panels, DIC images; right panels, GFP fluorescence. (D) Agarose gel showing phenol-free gDNA purified from either freeze-thawed nuclei or fresh eggs. For both extractions, eggs were homogenized with the Balch homogenizer. Size marker: HindIII-restricted λ DNA (left lane). (E) Purified gDNA isolated from egg nuclei is free of restriction enzyme-inhibiting factors. gDNA smear following EcoR1 digestion (1.5μg gDNA in 30μL, 40U EcoR1, 6hrs, 37°C), indicates sample is highly enriched in gDNA (compare right two lanes). Size markers: Gibco 1kb ladder and HindIII-restricted λ DNA (left two lanes). (F) Q-PCR using primers specific for gDNA (daf-12), mtDNA, or E. coli DNA confirms gDNA purified from egg nuclei is highly pure. Q-PCR amplicon abundance (Sytox stain) is plotted against PCR cycle number. Line marks cycle threshold cutoff (C_t). Genome sizes are C. elegans mtDNA: 13,794 bp, C. elegans genomic DNA: 1×10⁹ bp, and E. coli genomic DNA: 4.6 ×10⁶ bp.

Figure 6. Isolation of coupled mitochondria from C. elegans using the Balch Homogenizer

(A) Adult, wild type worms were gently sheared using the Balch homogenizer (18 μm clearance, 3 passes) and then a mitochondria-enriched fraction isolated. Mitochondrial bioenergetics were assessed using a Hansatech oxygen electrode in conjunction with a tetraphenylphosphonium (TPP⁺)-selective electrode. (B) Functional mitochondria were also isolated from mouse liver following established procedures (Frezza et al [21]). Data in both panels have been normalized to 500 μg protein (1μg/μl). Arrows mark points of substrate or inhibitor addition: mitochondria (Mito.); malate/glutamate (M/G); adenosine diphosphate (ADP); rotenone (Rot.); antimycin A (Ant A); tetramethyl phenylene diamine in conjunction with ascorbate (TMPD + Asc); oligomycin (Oligo) and p-trifluoromethoxy carbonyl cyanide phenyl hydrozone (FCCP).

Figure 7

Polysomal Profiling using the Balch Homogenizer. A whole-cell extract from adult worms was prepared using the Balch Homogenizer (16 μm clearance, 25 passes). Samples were fractionated into polysomes using continuous density gradient centrifugation (7% - 47% sucrose). Total RNA was extracted from each fraction then separated on a 1% agarose gel. Fraction number is shown under lanes (labeled from least to most dense). Identity of various RNA species is marked on right; corresponding ribosomal species are marked at the top of each panel [34]. Size marker (M): HindIII-restricted λ DNA.