Reference: Biol Bull 179: 358-365. (December, 1990) Biochemical and Functional Effects of Sulfate Restriction in the Marine Sponge, Microciona prolifera WILLIAM J. KUHNS,* GRADIMIR MISEVIC, AND MAX M. BURGER Hospital tor Sick Children. Toronto, Ontario, Canada, Marine Biological Laboratory, Woods Hole, Massachusetts, ami the Frieilrich Miescher Institute. L'nivcrsity Hospital of Basel. Basel, Switzerland Abstract. The functional and biochemical consequences of sulfate restriction were studied in chemically dissociated Microciona sponge cells maintained in artificial seawater with or without SO 4 2 ~. In cells pre-treated to reduce pre-formed secretions, SO 4 2 deprivation reduced cell motility judged by the lack of aggregates in rotating or stationary cultures in comparison with controls. Microscopic ex-amination showed that cells that customarily demonstrate cytoplasmic processes, such as filopodia and pseudopodia, exhibited marked decreases in these cellular processes when maintained in SO 4 2 -deprived artificial seawater. Uptake and incorporation of 35 SO 4 2 ~ by disaggregated and pre-treated cells was higher under SO 4 2 ~-free conditions relative to controls; this effect was time dependent, rising to a maximum at 12 h, when a three-to seven-fold dif-ference could be demonstrated. 3 H-leucine incorporation indicated that protein synthesis was similar in test and control populations. Comparative high voltage electro-phoresis of supernatants containing 35 SO 4 macromole-cules from chemically dissociated cells indicated deficien-cies of such 35 SO 4 macromolecules if the rotated cells that released these secretions had been pre-treated in SO 4 2 ~ free artificial seawater. The results of SO 4 2 restriction suggest that secretion of macromolecules or Microciona aggregation factor (MAP), and aggregation and locomotion of Microciona cells depend upon an adequate extracellular source of Introduction Both vertebrate and in vertebrate cells require sulfated macromolecules on cell surface receptors and in intra-Received 25 April 1990; accepted 25 September 1990. * On leave, University of North Carolina School of Medicine. De-partment of Pathology. Chapel Hill, NC. SO 4 2 . sulfate transport, and sulfation of macromolecules such as polysaccharides. cellular fluid (Cassaro and Dietrich, 1977; Hogsett and Quantrano, 1978; Mulder. 1981; Klebe et a/.. 1986; Mulder et ai. 1987). For example, mesenchymal migra-tion of sea urchin embryos is blocked in situ in sulfate-deprived medium (Katow and Solursh, 1981), and cell motility and morphology in cell cultures have been influ-enced by sulfated glycosaminoglycans (Venkatasubra-manian and Solursh, 1984). Blebbing has been observed on cell surfaces of sea urchin embryos maintained in sul-fate-free seawater. but not the prolonged processes that accompany mesenchymal cell migration. In this instance it appeared that sulfate deprivation was capable of causing an inhibition of the formation of stable cell attachments to the basal lamina (Venkatasubramanian and Solursh. 1984; Akasaka et ai. 1980). The defect could be reversed by a 6-h pre-treatment in normal seawater. Sulfate availability appears to be particularly important during early embryogenesis and differentiation in several species (Cassaro and Dietrich, 1977; Katow and Solursh, 1981; Lindahl. 1942: Immers and Runnstrom. 1965; Wenzl and Sumper, 198 1 ). Particularly vital in this regard are the sulfated mucopolysaccharides: their presence cor-relates well with tissue-level organization and normal de-velopment (Wenzl and Sumper. 1981; Kinoshita and Saiga, 1979; Yamaguchi and Kinoshita, 1985). This interesting background prompted us to address sulfation, using as a model Microciona, a relatively well studied marine sponge (Humphreys. 1963, 1967; Henkart et ai. 1973; Burger et ai. 1975; Jumblatt et ai, 1980; Misevic and Burger, 1986: Misevic et ai, 1987). These sponges are multicellular. but the relatively loose orga-nization of embryonic and differentiated cells is easily disaggregated. If divalent cations are deleted from the 358
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