Evagination of Cells Controls Bio-Silica Formation and Maturation during Spicule Formation in Sponges

et al. (2011) Evagination of Cells Controls Bio-Silica Formation and Maturation during Spicule Formation in Sponges. PLoS ONE 6(6): e20523. doi:10.1371/journal.pone.0020523 Evagination of Cells Controls Bio-Silica Formation and Maturation during Spicule Formation in Sponges Xiaohong Wang 0 Matthias Wiens 0 Heinz C. Schro der 0 Ute Schlomacher 0 Dario Pisignano 0 Klaus Peter Jochum 0 Werner E. G. Mu ller 0 Jian R. Lu, The University of Manchester, United Kingdom 0 1 National Research Center for Geoanalysis , Beijing , China , 2 European Research Council Advanced Grant Research Group, Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz , Mainz, Germany, 3 Dipartimento di Ingegneria dell'Innovazione , Universita` del Salento and National Nanotechnology Laboratory of CNR-Istituto Nanoscienze , Lecce , Italy , 4 Max Planck Institute for Chemistry , Mainz , Germany The enzymatic-silicatein mediated formation of the skeletal elements, the spicules of siliceous sponges starts intracellularly and is completed extracellularly. With Suberites domuncula we show that the axial growth of the spicules proceeds in three phases: (I) formation of an axial canal; (II) evagination of a cell process into the axial canal, and (III) assembly of the axial filament composed of silicatein. During these phases the core part of the spicule is synthesized. Silicatein and its substrate silicate are stored in silicasomes, found both inside and outside of the cellular extension within the axial canal, as well as all around the spicule. The membranes of the silicasomes are interspersed by pores of <2 nm that are likely associated with aquaporin channels which are implicated in the hardening of the initial bio-silica products formed by silicatein. We can summarize the sequence of events that govern spicule formation as follows: differential GENETIC READOUT (of silicatein) R FRACTAL ASSOCIATION of the silicateins R EVAGINATION of cells by hydro-mechanical forces into the axial canal R and finally PROCESSIVE BIO-SILICA POLYCONDENSATION around the axial canal. We termed this process, occurring sequentially or in parallel, BIOINORGANIC SELF-ORGANIZATION. - Funding: W.E.G.M. is a holder of a European Research Council Advanced Investigator Grant (no 268476 BIOSILICA). This work was supported by grants from the German Bundesministerium f ur Bildung und Forschung (project Center of Excellence BIOTECmarin), the Deutsche Forschungsgemeinschaft (Schr 277/10-1), the European Commission/EUREKA (EUROSTARS, no. 4289 - SILIBACTS), the International Human Frontier Science Program, the European Commission (project no. 244967 - Mem-S Project), the Public Welfare Project of Ministry of Land and Resources of the Peoples Republic of China (Grant No. 201011005-06) and the International S & T Cooperation Program of China (Grant No. 2008DFA00980). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The siliceous skeletal elements of the sponges [phylum: Porifera], termed spicules, possess several unique features which distinguish them from the skeletal elements found in other Metazoa. They are made of silica [(SiO2)n] instead of Ca-based minerals [1] with an unparalleled precision, giving rise to speciesspecific complex structures [2]. These genetically controlled and biologically produced structures are formed at ambient, mild physiological conditions, without high temperatures, pressures, or caustic chemicals [3]. The spicules are the critical structural determinant that controls the morphology of the sponges [4,5]. In the center of the spicules lies a 0.54.0 mm wide axial canal which harbors the organic axial filament [6,7]. Since its discovery the axial filament has been considered to be a template that controls the morphology of the spicules [8]. A major step towards an understanding of the genetically controlled morphogenesis of sponges was the identification of the structural protein of the spicules, termed silicatein which is located in the axial filament [9] as well as on the surface of the spicules [10]. Silicatein is an enzyme which forms the bio-silica required for the construction of the sponge spicules [1114]. The formation of spicules is a rapid process, which lasts for a spicule with a length of 190 mm and a diameter of 6 to 8 mm at 21uC only 40 hrs [15]. Because of this high growth rate it remained unclear for a long time if spicule formation starts intra- or extracellularly [16,17]. Detailed cell biological and biochemical studies on the intracellular spicule formation have been performed with the sponge Suberites domuncula [18,19]. These studies became possible since the establishment of a suitable cell culture system (the primmorphs) from S. domuncula, which allowed time-lapse developmental studies of spicule formation under controlled conditions [20]. The 3D-cell culture is composed of proliferating and differentiating stem cells, and of sclerocytes that initially form the spicules [21]. In these studies we described that silicatein-mediated spicule growth proceeds in two directions. Firstly, in axial, longitudinal direction in which the growth of the spicule is driven by the 23 kDa processed form of silicatein. Secondly, the radial thickening of the spicules, their appositional growth, occurs after extrusion of the spicules into the extracellular space. Accumulation of silica on the surface of the growing spicule in centripetal direction is mediated by the 34.7 kDa silicatein [10,18]. This form of silicatein is distinguished from the 23 kDa enzyme by the presence of the N-terminal pro-peptide sequence that is presumably cleaved off autocatalytically immediately before the onset of bio-silica synthesis [18]. In this study no conclusive evidence has been obtained for the existence of collagen either in the axial filament or on the surface of the spicules that would be causatively involved in bio-silica formation, as has been speculated [22]. Earlier studies on silicatein-driven spicule synthesis did not answer the question of how elongation of the spicule in axial direction occurs [18,19]. Two observations have been published which showed that even after the release of the spicules into the extracellular space the axial filament undergoes maturation steps. These data revealed that thereby an alteration from a less compact organization of the organic components within the axial canal, which also includes membraneous structures, to a compact axial filament occurs [18,19]. In support it was found that during maturation of the spicules the diameter of the axial canal decreases from approximately 4 mm to 0.5 mm. The release of the intracellularly formed spicules, their extrusion into the extracellular space, was assumed to be facilitated by spicule associated filaments [18,23]. The final shapi (...truncated)