Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization.

We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed that when eight gold particles were seen in this iterated array, the signal to noise ratio was greater than 30:1. Furthermore, these gold particles were colinear, often spiral, or circular suggesting detection of a single nucleic acid molecule. Antibodies against actin, vimentin, or tubulin proteins were used after in situ hybridization, allowing simultaneous detection of the protein and its cognate message on the same sample. This revealed that cytoskeletal mRNAs are likely to be extremely close to actin protein (5 nm or less) and unlikely to be within 20 nm of vimentin or tubulin filaments. Actin mRNA was found to be more predominant in lamellipodia of motile cells, confirming previous results. These results indicate that this high resolution in situ hybridization approach is a powerful tool by which to investigate the association of mRNA with the cytoskeleton.

Strategies for ultrastructural visualization of biotinated probes hybridized to messenger RNA in situ.

A progressive development of the application of in situ methodology to ultrastructural procedures has resulted in the ability to detect individual molecules of mRNA with high probability. Beginning with whole-mount cells and then developing myotubes, both in culture and detergent extracted before fixation, we were able to progress to methods which allow detection of mRNA in tissue sections. Initial results confirm that the detection of mRNA in thin-sectioned tissue is very similar to observations on the extracted, cultured cells, and that the same methods of data analysis apply. Current work is devoted to the application of the methodology to other cellular structures, such as the nucleus, and to other tissue-probe systems, such as brain. Acknowledgements. The authors appreciate the skilled help from John McNeil and Shirwin Pockwinse in the laborious and time-consuming preparations of material and photography. FS was on sabbatical leave from the Department of Pathology at Southwestern Medical Center.

Amino acid sequences that determine the nuclear localization of yeast histone 2B.

Histone-beta-galactosidase protein fusions were used to identify the domain of yeast histone 2B, which targets this protein to the nucleus. Amino acids 28 to 33 in H2B were required for nuclear localization of such fusion proteins and thus constitute a nuclear localization sequence. The amino acid sequence in this region (Gly-29 Lys Lys Arg Ser Lys Ala) is similar to the nuclear location signal in simian virus 40 large T antigen (Pro-126 Lys Lys Lys Arg Lys Val) (D. Kalderon, B.L. Roberts, W.D. Richardson, and A.E. Smith, Cell 39:499-509, 1984). A point mutation changing lysine 31 to methionine abolished nuclear localization of an H2B-beta-galactosidase fusion protein containing amino acids 1 to 33 of H2B. However, an H2B-beta-galactosidase fusion protein containing both this point mutation and the H2A interaction domain of H2B was nuclear localized. These results suggest that H2A and H2B may be cotransported to the nucleus as a heterodimer.

Structure and chemical composition of insoluble filamentous components of sperm flagellar microtubules.

By progressive solvent extraction, we have obtained a series of subfragments of flagellar microtubules. Mild treatment gives rise to ribbons that contain longitudinally arranged protofilaments. Further extraction leaves a distinctive residue containing thinner ribbons, of three and eventually two protofilaments. Finally, filaments 2-3 nm in diameter and fibrous ribbons apparently containing 6 or more 2 nm subfibrils are found. This latter solvent-resistant material is consistently enriched in a characteristic set of polypeptides, which are found in flagella of several different species, including echinoderms and a mollusc. These polypeptides appear different from alpha- and beta-tubulin on the basis of their solubilities, isoelectric points and electrophoretic mobilities in sodium dodecyl sulphate/polyacrylamide gels; these conclusions are reinforced by peptide mapping after limited proteolytic digestion, although the latter method reveals certain similarities between these unique flagellar proteins, tubulin, chicken gizzard desmin and rabbit actin. A remarkable feature of the protein in the final fraction is the high alpha-helical content: 71% as measured by circular dichroism. We consider the possible origins of these filaments in the microtubule, in particular the possibility that microtubule protofilaments are heterogeneous in protein composition, and we discuss some of the implications of our findings.

Arrangement of tubulin subunits and microtubule-associated proteins in the central-pair microtubule apparatus of squid (Loligo pealei) sperm flagella.

This study provides a comprehensive, high-resolution structural analysis of the central-pair microtubule apparatus of sperm flagella. It describes the arrangement of several microtubule-associated "sheath" components and suggests, contrary to previous thinking, that microtubules are structurally asymmetric. The two microtubules of the central pair are different in several respects: the C2 tubule bears a single row of 18-nm-long sheath projections with an axial periodicity of 16 nm, whereas the C1 tubule possesses rows of 9-nm globular sheath components with an axial repeat of 32 nm. The lumen of the C2 tubule always appears completely filled with electron-dense material; that of the C1 tubule is frequently hollow. The C2 tubule also possesses a series of beaded chains arranged around the microtubule; the beaded chains are composed of globular subunits 7.5-10 nm in diameter and appear to function in the pairing of the C1 and C2 tubules. These findings indicate: that the beaded chains are not helical, but assume the form of lock washers arranged with a 16-nm axial periodicity on the microtubule; and that the lattice of tubulin dimers in the C2 tubule is not helically symmetric, but that there are seams between certain pairs of protofilaments. Proposed lattice models predict that, because of these seams, central pair and perhaps all singlet microtubules may contain a ribbon of 2-5 protofilaments that are resistant to solubilization; these models are supported by the results of the accompanying paper (R. W. Linck, and G. L. Langevin. 1981. J. Cell Biol. 89: 323-337.

Reassembly of flagellar B (alpha beta) tubulin into singlet microtubules: consequences for cytoplasmic microtubule structure and assembly.

B(alpha beta) tubulin was obtained from a homogeneous class of microtubules, the incomplete B subfiber of sea urchin sperm flagellar doublet microtubules, by thermal fractionation. The thermally derived soluble B tubulin fraction (100, 000 g-h) repolymerizes in vitro, yielding microtubule-like structures. The microtubule-associated protein (MAP) composition and certain assembly parameters of thermally derived B tubulin are different from those reported for sonication-derived flageller tubulin and purified vertebrate tubulin. The "microtubules" reassembled from thermally prepared B tubulin are composed of 12-15 protofilaments (73% possess 14 protofilaments). A certain number possess a single "adlumenal component" applied to their inside walls, regardless of the number of protofilaments. Following the first cycle of polymerization, 81% of the B tubulin and essentially 100% of the MAPs remain cold insoluble. Evidence suggests that B tubulin assembles faithfully into a B lattice, creating a j seam between two protofilaments that are laterally bonded in a A-lattice configuration. The significance of these seams is discussed in relation to the mechanism of microtubule assembly, the stability of observed ribbons of protofilaments, and the three-dimensional organization of microtubule-associated components.