A monoclonal antibody toolkit for C. elegans

Abstract

Background: Antibodies are critical tools in many avenues of biological research. Though antibodies can be produced in the research laboratory setting, most research labs working with vertebrates avail themselves of the wide array of commercially available reagents. By contrast, few such reagents are available for work with model organisms.

Methodology/principal findings: We report the production of monoclonal antibodies directed against a wide range of proteins that label specific subcellular and cellular components, and macromolecular complexes. Antibodies were made to synaptobrevin (SNB-1), a component of synaptic vesicles; to Rim (UNC-10), a protein localized to synaptic active zones; to transforming acidic coiled-coil protein (TAC-1), a component of centrosomes; to CENP-C (HCP-4), which in worms labels the entire length of their holocentric chromosomes; to ORC2 (ORC-2), a subunit of the DNA origin replication complex; to the nucleolar phosphoprotein NOPP140 (DAO-5); to the nuclear envelope protein lamin (LMN-1); to EHD1 (RME-1) a marker for recycling endosomes; to caveolin (CAV-1), a marker for caveolae; to the cytochrome P450 (CYP-33E1), a resident of the endoplasmic reticulum; to beta-1,3-glucuronyltransferase (SQV-8) that labels the Golgi; to a chaperonin (HSP-60) targeted to mitochondria; to LAMP (LMP-1), a resident protein of lysosomes; to the alpha subunit of the 20S subcomplex (PAS-7) of the 26S proteasome; to dynamin (DYN-1) and to the alpha-subunit of the adaptor complex 2 (APA-2) as markers for sites of clathrin-mediated endocytosis; to the MAGUK, protein disks large (DLG-1) and cadherin (HMR-1), both of which label adherens junctions; to a cytoskeletal linker of the ezrin-radixin-moesin family (ERM-1), which localized to apical membranes; to an ERBIN family protein (LET-413) which localizes to the basolateral membrane of epithelial cells and to an adhesion molecule (SAX-7) which localizes to the plasma membrane at cell-cell contacts. In addition to working in whole mount immunocytochemistry, most of these antibodies work on western blots and thus should be of use for biochemical fractionation studies.

Conclusions/significance: We have produced a set of monoclonal antibodies to subcellular components of the nematode C. elegans for the research community. These reagents are being made available through the Developmental Studies Hybridoma Bank (DSHB).

Figure 1. Whole mount immunocytochemistry of C. elegans larvae using monoclonal antibodies.

A) The head of an adult animal shows the synaptic rich regions of the nervous system detected by the anti-synaptobrevin SB1 antibody. B1) Head of an L4 animal showing punctate synaptic staining of the nervous system detected by the RIM antibody. Note that some pharyngeal staining is also observed. B2) Head of an L4 animal showing loss of the punctate nervous system staining, but not the pharyngeal staining, in unc-10(md1117). C1) Head of an L4 animal showing punctate synaptic staining in the nervous system detected by the RIM2 antibody. C2) Head of an L4 animal showing loss of the punctate nervous system staining in unc-10(md1117). D) Head of an adult animal showing anti-SAX-7 staining of the neurons in the nerve ring ganglia and pharynx. E) An L1 animal showing staining of the nervous system with the anti-LMN-1 antibody. F) L4 animal showing punctate cytosolic anti-PAS-7 staining. G) An L2/L3 animal showing staining of the nervous system with the anti DYN-1 antibody. H) L1 (top) and L2 animals showing the staining of the intestine with the anti-LMP-1 antibody. I) an egg and L1 animal showing the APA-2 staining. APA-2 is particularly strong in the seam cells in L1. Scale bar = 20 µm.

Figure 2. Western analysis using monoclonal antibodies.

Western blots of whole wild type C. elegans extracts divided into three panels based upon acrylamide concentration used for SDS-polyacrylamide gel electrophoresis. The antibody used is labeled above each blot fragment. Approximately 20 to 30 µg of extract mixed staged extract was loaded in each lane. Molecular weight markers (BioRad Precision Plus Protein All Blue Standards) from top to bottom 250,150, 100, 75, 50, 37, 25, 20, 15, 10 kDa. the contrast was lowered on the images of the CAV1 and DAO5 westerns to permit visualization of multiple bands in a strong exposure.

Figure 3. Whole mount immunocytochemistry of embryos using monoclonal antibodies.

A) Pre-comma staged embryo showing anti-DLG-1 staining in developing hypodermal cells. B) A three-fold embryo showing anti-HMR-1 staining of adherens junctions. C) A three-fold embryo showing anti-ERM-1 staining in the apical junctions in the developing pharynx and intestine. D) A pre-comma embryo showing anti-LET-413 staining. E) A one and half-staged embryo showing anti-HSP-60 staining. F) A ∼40 cell embryo showing anti-CAV-1 staining. G) A pre-comma staged animal showing anti-SQV-8 staining. H) A pre-comma staged embryo showing anti-RME-1 staining. I) An ∼60 staged embryo showing anti-PAS-7 staining. Scale Bar = 10 µm.

Figure 4. Whole mount immunocytochemistry of 2, 4 and 6 cell embryos using monoclonal antibodies.

A) A two cell embryo showing anti HCP-4 staining of chromosomes. B) A two cell embryo showing anti-LMN-1 staining of the nuclear envelope. C) A six cell embryo showing ORC-2 staining of chromosomes. D) A two cell embryo showing staining of anti-TAC-1 of centrosomes. E) Double labeling using the anti-TAC-1 monoclonal and an anti-LMN-1 polyclonal antibody. F) A six cell embryo showing anti-DYN-1 staining. Scale Bar 10 µm.

Figure 5. Immunocytochemistry of dissected gonad and intestines using monoclonal antibodies.

A) Dissected gonad stained with DAPI (A1) to visualize nuclei and immunostained with anti-DAO-5 antibodies (A2). B) Dissected intestine stained with DAPI (B1) to visualize nuclei (arrows) and immunostained with anti CYP-33E1 antibodies (B2) and the merged view (B3). C) Dissected intestine stained with DAPI (C1) to visualize nuclei (arrows) and immunostained with anti LMP-1 antibodies (C2) and the merged view (C3).