Identification and localization of proteins immunologically related to intermediate filament proteins in sea urchin eggs and embryos

Abstract

Sea urchin spermatozoa, eggs, and embryos were labeled with the universal antibody against the intermediate filament proteins (anti‐IFA) described by Pruss et al. [Cell 27:419–428, 1981] and with anti‐beta‐tubulin. Localization of these antibodies was by indirect immunofluorescence microscopy. Cytoskeleton of unfertilized eggs, prepared according to a procedure adapted from Kane [Exp. Cell Res. 162:495–506, 1986] or as described by Dufresne et al. [Biochem. Cell Biol. 66:780–791, 1988], and reacted with the anti‐IFA demonstrate a uniformly stained background except for the nuclear areas, which appear as dark rings. During the first cell cycle, the anti‐IFA staining pattern coincides with that of spindle‐associated tubulin but not with the cortical pattern of microtubules. Swimming embryos reacted with the anti‐IFA show a labeling located on the cilia and within the cytoplasm of each individual cell of the larva. In spermatozoa, the labeling occurs all along the flagellae. Immunoblots of proteins from eggs and embryos reveal one major protein of 117 kDa and sometimes a component of 66 kDa, both of which cosediment with tubulin during the isolation procedure of microtubules described by Vallee and Bloom [Proc. Natl. Acad. Sci. USA 80:6259–6263, 1983]. These data show that proteins homologous to the intermediate filament proteins reported in vertebrate cells are present in both gametes of sea urchins. The specific localization ofthese proteins in the spindle, the flagella, and the cilia suggest that they may play a significant role in the organization and function of the microtubular lattice of the spermatozoa and of the embryo.

Sea Urchin Embryo as a Model Organism for the Rapid Functional Screening of Tubulin Modulators

“…These are also involved in cell division and ciliary movement. In the sea urchin embryo, intermediate filaments co-localize with the mitotic spindle and are believed to participate in various microtubule-dependent events (30). Ciliary basal apparatus of the blastula ectoderm cell is connected to cytoplasmic microtubules and cortical actin microfilaments, while intermediate filaments have been detected in cilia (30,31).…”

“…In the sea urchin embryo, intermediate filaments co-localize with the mitotic spindle and are believed to participate in various microtubule-dependent events (30). Ciliary basal apparatus of the blastula ectoderm cell is connected to cytoplasmic microtubules and cortical actin microfilaments, while intermediate filaments have been detected in cilia (30,31). It is likely that all these structures contribute to the stability of the sea urchin embryo cilium, although their specific role in regulating the ciliary beating still remains unclear.…”

Regulation of the pentose phosphate pathway at fertilization in sea urchin eggs

“…One class of proteins that possesses similar low solubility are the intermediate filaments (Klymkowsky et al, 1989;Skalli and Goldberg, 1991). Although immunocytochemical studies have shown that mature sea urchin eggs lack an organized network of intermediate filaments (Boyle and Emst, 1989;St-Pierre and Dufiesne, 1990), one intriguing possibility is that G6PDH is associating with less organized arrays of intermediate filament proteins (e.g., dimers, tetramers or protofilaments). Alternatively, the binding elements could be intermediate filament-like proteins not recognized by antibodies directed against intermediate filaments.…”