Immunofluorescence signal amplification by the enzyme-catalyzed deposition of a fluorescent reporter substrate (CARD)

Chao, Jean; DeBiasio, Richard; Zhu, Zhengrong; Giuliano, Kenneth A.; Schmidt, Brigitte F.

doi:10.1002/(sici)1097-0320(19960101)23:1<48::aid-cyto7>3.0.co;2-i

Cited by 54 publications

(31 citation statements)

References 18 publications

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“…A horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Molecular Probes) was used at a final dilution of 50 mg/ml. Visualization was by 10 min of tyramine signal amplification (Bobrow et al 1989;Chao et al 1996) with Alexa Fluor 488 conjugated to tyramine (Molecular Probes). Mouse monoclonal antibody 1CB4 (Okamoto and Thomson 1985;Arduengo et al 1998) was used at 1:2000 to detect the fibrous body-membranous organelle (FB-MO) complexes and was visualized using Texas Red-conjugated, affinity purified goat anti-mouse IgG secondary antibody (Fab 1 ) 2 fragment at a final concentration of 0.75 mg/ml ( Jackson ImmunoResearch Laboratories, West Grove, PA).…”

Section: Methodsmentioning

confidence: 99%

spe-10 Encodes a DHHC–CRD Zinc-Finger Membrane Protein Required for Endoplasmic Reticulum/Golgi Membrane Morphogenesis During Caenorhabditis elegans Spermatogenesis

et al. 2006

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C. elegans spermatogenesis employs lysosome-related fibrous body-membranous organelles (FB-MOs) for transport of many cellular components. Previous work showed that spe-10 mutants contain FB-MOs that prematurely disassemble, resulting in defective transport of FB components into developing spermatids. Consequently, spe-10 spermatids are smaller than wild type and contain defective FB-MO derivatives. In this article, we show that spe-10 encodes a four-pass integral membrane protein that has a DHHC-CRD zinc-finger motif. The DHHC-CRD motif is found in a large, diverse family of proteins that have been implicated in palmitoyl transfer during protein lipidation. Seven spe-10 mutants were analyzed, including missense, nonsense, and deletion mutants. An antiserum to SPE-10 showed significant colocalization with a known marker for the FB-MOs during wild-type spermatogenesis. In contrast, the spe-10(ok1149) deletion mutant lacked detectable SPE-10 staining; this mutant lacks a spe-10 promoter and most coding sequence. The spe-10(eb64) missense mutation, which changes a conserved residue within the DHHC-CRD domain in all homologues, behaves as a null mutant. These results suggest that wild-type SPE-10 is required for the MO to properly deliver the FB to the C. elegans spermatid and the DHHC-CRD domain is essential for this function.

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Section: Methodsmentioning

confidence: 99%

spe-10 Encodes a DHHC–CRD Zinc-Finger Membrane Protein Required for Endoplasmic Reticulum/Golgi Membrane Morphogenesis During Caenorhabditis elegans Spermatogenesis

et al. 2006

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“…The hybridized probe is visualized by the enzyme-mediated deposition of a fluorochrome-conjugated reporter molecule (Chao et al 1996;Adams 1992;Bobrow 1989).…”

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confidence: 99%

“…Amplification and visualization are accomplished by enzy-matic deposition of the novel fluorescent HRP substrate Cy3.29-tyramide. Cy3.29-tyramide is a bright fluorescent substrate of high photostability and water solubility (Chao et al 1996).…”

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confidence: 99%

Signal Amplification in the Detection of Single-copy DNA and RNA by Enzyme-catalyzed Deposition (CARD) of the Novel Fluorescent Reporter Substrate Cy3.29-Tyramide

Schmidt

Chao

Zhu

et al. 1997

J Histochem Cytochem.

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“…The SPE-5 Mab was incubated with samples overnight at a 1:1000 dilution for all immunofluorescent experiments described in this article. The secondary antibody was a goat anti-mouse IgG conjugated to horseradish peroxidase (Molecular Probes), and visualization was by 5 min of tyramine signal amplification (TSA) (Bobrow et al 1989;Chao et al 1996) with Alexa Fluor 488 conjugated to tyramine (Invitrogen). Mouse monoclonal antibody 1CB4 (Okamoto and Thomson 1985;Arduengo et al 1998) was used after completion of the SPE-5 Mab incubation/TSA enhancement step at 1:2000 to detect the fibrous body-membranous organelle (FB-MO) complexes.…”

Section: Immunocytochemistrymentioning

confidence: 99%

Developmental Genetics of Secretory Vesicle Acidification DuringCaenorhabditis elegansSpermatogenesis

et al. 2012

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Secretory vesicles are used during spermatogenesis to deliver proteins to the cell surface. In Caenorhabditis elegans, secretory membranous organelles (MO) fuse with the plasma membrane to transform spermatids into fertilization-competent spermatozoa. We show that, like the acrosomal vesicle of mammalian sperm, MOs undergo acidification during development. Treatment of spermatids with the V-ATPase inhibitor bafilomycin blocks both MO acidification and formation of functional spermatozoa. There are several spermatogenesis-defective mutants that cause defects in MO morphogenesis, including spe-5. We determined that spe-5, which is on chromosome I, encodes one of two V-ATPase B paralogous subunits. The spe-5 null mutant is viable but sterile because it forms arrested, multi-nucleate spermatocytes. Immunofluorescence with a SPE-5-specific monoclonal antibody shows that SPE-5 expression begins in spermatocytes and is found in all subsequent stages of spermatogenesis. Most SPE-5 is discarded into the residual body during spermatid budding, but a small amount remains in budded spermatids where it localizes to MOs as a discrete dot. The other V-ATPase B subunit is encoded by vha-12, which is located on the X chromosome. Usually, spe-5 mutants are self-sterile in a wild-type vha-12 background. However, an extrachromosomal transgene containing wild-type vha-12 driven by its own promoter allows spe-5 mutant hermaphrodites to produce progeny, indicating that VHA-12 can at least partially substitute for SPE-5. Others have shown that the X chromosome is transcriptionally silent in the male germline, so expression of the autosomally located spe-5 gene ensures that a V-ATPase B subunit is present during spermatogenesis. V ESICULAR organelles in eukaryotic cells frequently maintain an acidic pH (reviewed by Paroutis et al. 2004) that is created by the vacuolar H+-ATPase (V-ATPase). The V-ATPase is a large (910-kDa) molecular machine that couples ATP hydrolysis to the movement of protons across biological membranes. The V-ATPase has a V 0 -sector that creates the pore-for-proton translocation through the lipid bilayer and a V 1 -sector, located in the cytoplasm, that is the site of ATP hydrolysis. Each V-ATPase holoenzyme is composed of 14 different subunits, some of which are present in multiple copies (reviewed by Toei et al. 2010). In yeast, there is one gene for each V-ATPase subunit, except for the "a" subunit, which is encoded by two genes (reviewed by Kane 2006). The physiological properties of the V-ATPase are in part determined by which of these two "a" subunits it contains (Kawasaki-Nishi et al. 2001). In humans and other animals, the "a" and other V-ATPase subunits are encoded by more than one gene (reviewed by Toei et al. 2010). Subunit diversity presumably allows the V-ATPase to be either customized for a specific function or utilized in a tissuespecific fashion.In this article, we use pH-sensitive vital dyes and specific inhibitors to show that sperm-specific MOs use the V-ATPase to acidify their interior a...

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Immunofluorescence signal amplification by the enzyme-catalyzed deposition of a fluorescent reporter substrate (CARD)

Cited by 54 publications

References 18 publications

spe-10 Encodes a DHHC–CRD Zinc-Finger Membrane Protein Required for Endoplasmic Reticulum/Golgi Membrane Morphogenesis During Caenorhabditis elegans Spermatogenesis

spe-10 Encodes a DHHC–CRD Zinc-Finger Membrane Protein Required for Endoplasmic Reticulum/Golgi Membrane Morphogenesis During Caenorhabditis elegans Spermatogenesis

Signal Amplification in the Detection of Single-copy DNA and RNA by Enzyme-catalyzed Deposition (CARD) of the Novel Fluorescent Reporter Substrate Cy3.29-Tyramide

Developmental Genetics of Secretory Vesicle Acidification DuringCaenorhabditis elegansSpermatogenesis

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