PubMed Health⌕ Search

PubMed · 12972346

A quantitative method for assessing co-localization in immunolabeled thin section electron micrographs.

Abstract

A method is introduced for the analysis of nearest neighbor distances between immunogold particles marking proteins on electron micrographs. Deviation from the distribution that is predicted by chance indicates co-localization of the labeled species, and the potential for productive interaction in vivo. Application of this method to the analysis of nearest neighbor distances in experiments with pea leaf thin sections and isozyme-directed antibodies indicates that glyceraldehyde-3-P dehydrogenase is located near P-glycerate kinase and near aldolase in the chloroplast stroma, consistent with the notion that these enzymes are part of a multi-enzyme photosynthetic CO(2)-fixation complex in situ.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

James B Anderson, Andrew A Carol, Vanessa K Brown, Louise E Anderson. 2003. A quantitative method for assessing co-localization in immunolabeled thin section electron micrographs.. https://doi.org/10.1016/s1047-8477(03)00138-2

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

Nuclear and territorial topography of chromosome telomeres in human lymphocytes.

Nuclear and territorial positioning of p- and q-telomeres and centromeres of chromosomes 3, 8, 9, 13, and 19 were studied by repeated fluorescence in situ hybridization, high-resolution cytometry, and three-dimensional image analysis in human blood lymphocytes before and after stimulation. Telomeres were found on the opposite side of the territories as compared with the centromeres for all chromosome territories investigated. Mutual distances between telomeres of submetacentric chromosomes were very short, usually shorter than centromere-to-telomere distances, which means that the chromosome territory is nonrandomly folded. Telomeres are, on average, much nearer to the center of the cell nucleus than centromeres; q-telomeres were found, on average, more centrally localized as compared with p-telomeres. Consequently, we directly showed that chromosome territories in the cell nucleus are (1) polar and (2) partially oriented in cell nuclei. The distributions of genetic elements relative to chromosome territories (territorial distributions) can be either narrower or broader than their nuclear distributions, which reflects the degree of adhesion of an element to the territory or to the nucleus. We found no tethering of heterologous telomeres of chromosomes 8, 9, and 19. In contrast, both pairs of homologous telomeres of chromosome 19 (but not in other chromosomes) are tethered (associated) very frequently.

Cell Compartmentation↗

Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis.

Lysosome-related organelles are cell type-specific intracellular compartments with distinct morphologies and functions. The molecular mechanisms governing the formation of their unique structural features are not known. Melanosomes and their precursors are lysosome-related organelles that are characterized morphologically by intralumenal fibrous striations upon which melanins are polymerized. The integral membrane protein Pmel17 is a component of the fibrils and can nucleate their formation in the absence of other pigment cell-specific proteins. Here, we show that formation of intralumenal fibrils requires cleavage of Pmel17 by a furin-like proprotein convertase (PC). As in the generation of amyloid, proper cleavage of Pmel17 liberates a lumenal domain fragment that becomes incorporated into the fibrils; longer Pmel17 fragments generated in the absence of PC activity are unable to form organized fibrils. Our results demonstrate that PC-dependent cleavage regulates melanosome biogenesis by controlling the fibrillogenic activity of a resident protein. Like the pathologic process of amyloidogenesis, the formation of other tissue-specific organelle structures may be similarly dependent on proteolytic activation of physiological fibrillogenic substrates.

Cell Compartmentation↗

Intracellular localisation of human HIF-1 alpha hydroxylases: implications for oxygen sensing.

Hypoxia-inducible factor1 (HIF-1) is an essential transcription factor for cellular adaptation to decreased oxygen availability. In normoxia the oxygen-sensitive alpha-subunit of HIF-1 is hydroxylated on Pro564 and Pro402 and thus targeted for proteasomal degradation. Three human oxygen-dependent HIF-1 alpha prolyl hydroxylases (PHD1, PHD2, and PHD3) function as oxygen sensors in vivo. Furthermore, the asparagine hydroxylase FIH-1 (factor inhibiting HIF) has been found to hydroxylate Asp803 of the HIF-1 C-terminal transactivation domain, which results in the decreased ability of HIF-1 to bind to the transcriptional coactivator p300/CBP. We have fused these enzymes to the N-terminus of fluorescent proteins and transiently transfected the fusion proteins into human osteosarcoma cells (U2OS). Three-dimensional 2-photon confocal fluorescence microscopy showed that PHD1 was exclusively present in the nucleus, PHD2 and FIH-1 were mainly located in the cytoplasm and PHD3 was homogeneously distributed in cytoplasm and nucleus. Hypoxia did not influence the localisation of any enzyme under investigation. In contrast to FIH-1, each PHD inhibited nuclear HIF-1 alpha accumulation in hypoxia. All hydroxylases suppressed activation of a cotransfected hypoxia-responsive luciferase reporter gene. Endogenous PHD2mRNA and PHD3mRNA were hypoxia-inducible, whereas expression of PHD1mRNA and FIH-1mRNA was oxygen independent. We propose that PHDs and FIH-1 form an oxygen sensor cascade of distinct subcellular localisation.

Cell Compartmentation↗