Centrin Scaffold in Chlamydomonas reinhardtii Revealed by Immunoelectron Microscopy

ABSTRACT In the flagellate green alga Chlamydomonas reinhardtii the Ca2+-binding EF-hand protein centrin is encoded by a single-copy gene. Previous studies have localized the protein to four distinct structures in the flagellar apparatus: the nucleus-basal body connector, the distal connecting fiber, the flagellar transitional region, and the axoneme. To explain the disjunctive distribution of centrin, the interaction of centrin with as yet unknown specific centrin-binding proteins has been implied. Here, we demonstrate using serial section postembedding immunoelectron microscopy of isolated cytoskeletons that centrin is located in additional structures (transitional fibers and basal body lumen) and that the centrin-containing structures of the basal apparatus are likely part of a continuous filamentous scaffold that extends from the nucleus to the flagellar bases. In addition, we show that centrin is located in the distal lumen of the basal body in a rotationally asymmetric structure, the V-shaped filament system. This novel centrin-containing structure has also been detected near the distal end of the probasal bodies. Taken together, these results suggest a role for a rotationally asymmetric centrin “seed” in the growth and development of the centrin scaffold following replication of the basal apparatus.

Dictyostelium gamma-tubulin: molecular characterization and ultrastructural localization

The centrosome of Dictyostelium discoideum is a nucleus-associated body consisting of an electron-dense, three-layered core surrounded by an amorphous matrix, the corona. To elucidate the molecular and supramolecular architecture of this unique microtubule-organizing center, we have isolated and sequenced the gene encoding gamma-tubulin and have studied its localization in the Dictyostelium centrosome using immunofluorescence and postembedding immunoelectron microscopy. D. discoideum possesses a single copy of a gamma-tubulin gene that is related to, but more divergent from, other gamma-tubulins. The low-abundance gene product is localized to the centrosome in an intriguing pattern: it is highly concentrated in the corona in regularly spaced clusters whose distribution correlates with the patterning of dense nodules that are a prominent feature of the corona. These observations lend support to the notion that the corona is the functional homologue of the pericentriolar matrix of ‘higher’ eukaryotic centrosomes, and that nodules are the functional equivalent of gamma-tubulin ring complexes that serve as nucleation sites for microtubules in animal centrosomes.

Immunocytochemical Localization of Chymase to Cytoplasmic Vesicles After Rat Peritoneal Mast Cell Stimulation by Compound 48/80

The subcellular events responsible for release of mediators by mast cells may help to clarify roles for mast cells in health and disease. In this study we show that the granule-associated protease chymase is also within cytoplasmic vesicles in appropriately stimulated rat peritoneal mast cells. Rat peritoneal mast cells were recovered before or 1–10 sec after exposure to the secretogogue compound 48/80 (10 μg/ml) and then were examined by radioimmunoassay to quantify histamine release or were processed, using routine methods for postembedding immunoelectron microscopy, to identify the subcellular localization of chymase. In comparison to unstimulated cells, compound 48/80 stimulated cells in two independent experiments showed an increase (15%, 28%) in the surface area of the cell and a decrease (12%, 6%) in the surface area of the total granule compartment before degranulation channel formation. These global cellular changes occurred in a background of transient but significant ( p<0.01) increases in the area and number of chymase-immunoreactive vesicles per μ2 cytoplasm. These changes were detectable at 5 or 7 sec after stimulation with compound 48/80 but returned to near prestimulation levels by 9 or 10 sec after addition of compound 48/80 (total cumulative histamine release was 28% by 8 sec and 47% by 14 sec). These observations suggest that vesicles participate in the early stages of regulated secretion of chymase from rat peritoneal mast cells.

Microfibril-associated glycoprotein-1 (MAGP-1) is specifically located on the beads of the beaded-filament structure for fibrillin-containing microfibrils as visualized by the rotary shadowing technique.

This study used immunoelectron microscopic techniques to define the ultrastructural location of MAGP-1 on the fibrillin-containing microfibrils of the ocular zonule. A specific anti-MAGP-1 monoclonal antibody (MAb), 11B, was produced that did not crossreact with fibrillin-1 or other microfibrillar proteins. MAb 11B was shown by immunofluorescence to localize intensely to zonular tissue. Postembedding immunoelectron microscopy showed that MAGP-1 was associated with microfibrils throughout the zonule, with the exception of a narrow band of microfibrils at the junction with the lens capsule. With preembedding labeling, the anti-MAGP-1 MAb was found to localize in a crossbanding pattern, at intervals of about 50 nm, to microfibrils throughout the zonule and along bundles of microfibrils in surrounding vitreous tissue. Rotary shadowing of isolated microfibrils showed a "beads on a string" morphology with a periodicity of about 50 nm. With immunogold labeling, the anti-MAGP-1 antibody specifically localized on the beads in a symmetrical manner. Occasionally two gold partides were attached to the same bead, suggesting that multiple MAGP-1 molecules were present in the structure. The results indicate that MAGP-1 is intimately and regularly associated with the bead regions of fibrillin-containing microfibrils. The findings are consistent with a major structural role for MAGP-1 in microfibril biology.