Nanoscale control of silica morphology and three-dimensional structure during diatom cell wall formation Mark Hildebrand,a) Evelyn York, Jessica I. Kelz,b) Aubrey K. Davis, and Luciano G. Frigeri Scripps Institution of Oceanography, University of California—San Diego, La Jolla, California 92093-0202
David P. Allison Biological & Nanoscale Systems Group, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6123; Department of Biochemistry & Cellular & Molecular Biology, University of Tennessee, Knoxville, TN, 37996-0840; and Molecular Imaging Inc., Agilent Technologies Tempe, Arizona 85282
Mitchel J. Doktycz Biological & Nanoscale Systems Group, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6123 (Received 25 April 2006; accepted 27 July 2006)
We present a unique approach combining biological manipulation with advanced imaging tools to examine silica cell wall synthesis in the diatom Thalassiosira pseudonana. The innate capabilities of diatoms to form complex 3D silica structures on the nano- to micro-scale exceed current synthetic approaches because they use a fundamentally different formation process. Understanding the molecular details of the process requires identifying structural intermediates and correlating their formation with genes and proteins involved. This will aid in development of approaches to controllably alter structure, facilitating the use of diatoms as a direct source of nanostructured materials. In T. pseudonana, distinct silica morphologies were observed during formation of different cell wall substructures, and three different scales of structural organization were identified. At all levels, structure formation correlated with optimal design properties for the final product. These results provide a benchmark of measurements and new insights into biosilicification processes, potentially also benefiting biomimetic approaches.
I. INTRODUCTION
The development of nanotechnology depends on the generation of nano- and microstructured materials with precise yet diverse structural features suited to a variety of applications, in high abundance and at low cost. A promising route to low-temperature, mild reaction condition syntheses of nanostructured materials is found in biology and in biomimetic approaches1–3 either directly utilizing biological components4–9 or through principles derived from biology.10–15 The unicellular algae known as diatoms produce complex nano- and microscale silica structures in their cell walls, reproduced with fidelity, inexpensively, and in enormous numbers through biological replication. One major determinant of diatom
a)
Address all correspondence to this author. e-mail:
[email protected] b) Present address: U.S. Naval Academy, 121 Blake Road, Annapolis, Maryland 21402-5000. DOI: 10.1557/JMR.2006.0333 J. Mater. Res., Vol. 21, No. 10, Oct 2006
silica polymerization, polypeptides known as silaffins,16–18 has been applied in biomimetic approaches,19–21 and the nearly concomitant discovery of synthetic polyaminecatalyzed silica polymerization22,23 and long-chain polyamines from diatoms with the same ability 24 has spawned detailed investigations into the use of these molecules in silica syntheses.11 Comparison of in vivo and in vitro silicification can be mutually beneficial. Synthetic approaches can provide insight into possible underlying mechanisms for formation of diatom silica5,25 because reaction components and conditions can be controlled, enabling rational design. Because diatoms can make complex three-dimensional nano- and microscale structures exceeding those possible with current synthetic approaches (which are of necessity simpler than the cellular process), understanding the biological process will aid in development of more sophisticated biomimetic approaches. In addition, because diatom silica structure formation is encoded in the form of genes in the genome, developing gene manipulation techniques26 could enable the use of these organisms as a direct source of specifically © 2006 Materials Research Society
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tailored nano- and microstructured materials.27 These approaches are not limited to the chemistry of silica; techniques have been developed to convert diatom silica to other technologically useful metal oxides while maintaining nanostructure.28–31 From our current level of understanding, it is clear that diatom silica structure formation occurs by a fundamentally different mechanism than those used in current synthetic approaches because it does not rely solely on an internal or pre-structured template or repetitive self assembly but rather occurs dynamically inside an expandable and moldable membrane-bound intracellular compartment called the silica deposition vesicle (SDV).32,33 Sophisticated control over altering diatom silica structure is not currently possible because the structure formation process is incompletely understood. Such an understanding requires identification of structural intermediates in the formation of the material itself and elucidating molecular details of the underlying cellular processes responsible. The completion of the entire genome sequence for the diatom Thalassiosira pseudonana34 provides a tremendous opportunity to apply powerful genomic and proteomic approaches to identify cellular components involved in silicification.18,35 Lacking however, is a detailed understanding of structure formation in T. pseudonana, which will be necessary to correlate the involvement of genes and proteins with formation of particular structures. Identification and accurate measurements of intermediates in silica structure formation should elucidate design strategies and mechanisms of control, shed light on the underlying physical/chemical parameters involved, and provide a basis for comparison with genetically or otherwise modified structures. II. EXPERIMENTAL TECHNIQUES
Cultures of T. pseudonana strain CCMP1335 were grown and synchronized as described.35 Acid treatment preparation of cell wall silica for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) was done as described.35 For SEM, samples were sputter-coated (gold/palladium) and observed in an FEI Quanta 600 (FEI Company, Hillsboro, OR) scanning electron microscope at the Scripps Institution of Oceanography Unified Laboratory Facility. Some ultrahigh resolution field-emission SEM (FESEM) images were obtained on a Hitachi S-4800 (Hitachi High Technologies America, Inc., Schaumburg, IL). For TEM, samples were suspended in 100% ethanol, spotted onto Formvarcoated copper grids (Ted Pella, Inc., Redding, CA), and stained with uranyl acetate. Samples were observed in a FEI Technai 12 transmission electron microscope at the San Diego State University Electron Microscopy Facility. For AFM imaging, diatom samples were suspended in water, mounted on gelatin-coated mica disks,36 and 2690
imaged in air. Imaging was accomplished using a Molecular Imaging LE AFM (Molecular Imaging, Agilent Technologies, Tempe, AZ) operating in MacMode, using silicon cantilevers with a manufacturer’s spring constant of 2.8 N/m. Topographic images were first-order flattened and were recorded at a scan rate of 1.0 lines/s with 512 points per line. III. RESULTS
The diatom cell wall is called a frustule, encasing the cellular cytoplasm in a petri dish arrangement, with overlapping upper and lower halves [Fig. 1(a)]. The frustule consists of distinct siliceous structures on the micron scale called “valves” and “girdle bands,” and each half of the frustule consists of a single valve and a series of girdle bands encircling the cell and providing overlap between the halves [Fig. 1(a)]. Valves of T. pseudonana are circular (Fig. 1) with a highly conserved average diameter (under our growth conditions) of 3.8 m (see Table I for a summary and statistics of all measurements), with most between 3.0 and 4.2 m. Length of cells averaged 6.5 m but varied between 4.3 and 9.0 m (occasionally even shorter or longer cells were observed) by incorporation of different numbers of girdle bands. The salinity of the growth medium affected cell length but not valve width. In 200 mM NaCl, cells averaged 7.0 m in length, and in 500 mM NaCl, average length was 5.9 m. The valve of T. pseudonana has silica ribs radiating from the center. The ribs contain cross-connections [Figs. 1(b) and 1(c)], and on the outer valve surface (distal to the cell center) silica ridges are built upon these ribs [Fig. 1(c)]. Valve ribs branched but generally maintained a parallel orientation relative to each other [Fig. 1(b)] instead of increasing in separation with increasing distance from the valve center. Branching was usually positioned to fill in the space with the greatest distance between adjacent ribs, maintaining a consistent average spacing of 145 nm. Between ribs were numerous nanopores [Fig. 1(b)], average diameter of 18 nm, covering about 4% of the valve surface. Generally one larger pore (the fultoportula37) was offset from the center of the valve, and varying numbers (average. 10) of similarly structured pores called rimoportulae37 were around the rim [Fig. 1(b) and 1(c)]. Thickness of the valve rim averaged 80 nm (Table I), but within a given valve this varied, sometimes substantially (up to 6-fold, minimum observed 29 nm, maximum 192 nm). Focused-ion-beam milling across several valve centers (data not shown) also showed variation in thickness in a given valve, with an average of 103 nm, and thinner near the rim. Using a newly developed culture synchrony procedure for T. pseudonana35 we enriched for cells making valves, and identified intermediates in formation (Fig. 2). At the
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TABLE I. Dimensions of components of the T. pseudonana frustule.
FIG. 1. Overall features of the T. pseudonana frustule: (a) diagram of the overall features of a diatom cell (individual parts are labeled; see text for complete description); (b) TEM image of T. pseudonana valve, highlighting the rib and nanopore structure; (c) SEM image of the outer valve surface and first girdle band. The arrow on top indicates the fultoportula, the arrows on the rim indicate two of the rimoportulae, and the bracket at left indicates the first girdle band, called the valvocopula.
Feature
Size
Range
Valve diameter Cell length Rib to rib distance in valve Branch node to rib distance in valve Diameter of valve pores Valve rim thickness Valve thickness (focused ion beam milled) Girdle band width Girdle band thickness Portulae, inside height Portulae, outside height Portulae, inside base Portulae, inside pore Portulae, outside pore Portulae, inside pore rim to rim Portulae, outside pore rim to rim
3.8 ± 0.4 m (n ⳱ 67) 6.5 ± 1.4 m (n ⳱ 74) 145 ± 13 nm (n ⳱ 60)
3.0–4.2 m 4.3–9.0 m
162 ± 16 nm (n ⳱ 59) 18.3 ± 3.1 nm (n ⳱ 16) 80 ± 33 nm (n ⳱ 125) 103 ± 23 nm (n ⳱ 35) 745 ± 120 nm (n ⳱ 30) 41 ± 13 nm (n ⳱ 53) 153 ± 18 nm (n ⳱ 18) 199 ± 43 nm (n ⳱ 7) 334 ± 21 nm (n ⳱ 15) 72 ± 14 nm (n ⳱ 15) 79 ± 13 nm (n ⳱ 11) 176 ± 21 nm (n ⳱ 19) 148 ± 28 nm (n ⳱ 9)
29–192 nm 63–150 nm
earliest recognizable stage, a thin base layer corresponding to an outline of the valve was observed, with the silica ribs radiating from the center [Fig. 2(a)]. In centric diatoms, silicification begins at a central location, and structure is formed radially from there,33 expanding in the x- and y-axis plane [Fig. 2(b)]. Precursors of the rimoportulae and fultoportula were visible at the early stage [Fig. 2(a)]. Subsequently, the rim of the valve became more heavily silicified and the portulae continued to develop, yet the central portion of the valve remained thin, as evidenced by its collapse in Fig. 2(c). During maturation, the rim continued to develop and the central region became more rigid [Figs. 2(d)–2(f)]; eventually the valve assumed a fully rigid mature form [Fig. 2(g)]. During this phase, the SDV expanded in the z-axis direction distal to the cell center [Fig. 2(h)]. Silica forming the ribs was initially deposited as flattened separated strips [Figs. 3(a) and 3(b)], which fuse to form the continuous valve surface. Individual strips consisted of a raised center section flanked by precursors to the nanopores [Fig. 3(b)]. TEM revealed that the initial deposition in the base layer was a branched structure [Fig. 3(c)]. Newly formed nanopores [Fig. 3(c)] were larger and more irregular compared with mature nanopores found toward the center of the valve [Fig. 3(d)]. The ridges on the distal face of mature valves had a distinct silica morphology compared with the base layer of the ribs, consisting of spherical silica particles, with an average cross-section of 50 nm [Fig. 4(a) and 4(b)]. The extent of ridged structure varied from valve to valve and in a given valve could vary in different regions of the surface, but there were no consistent patterns comparing valves. Atomic force microscopy (AFM) on non-ridged portions of the distal valve face (data not shown) identified rib structures similar to those seen in Figs. 3(a) and
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FIG. 2. Intermediates in valve formation (SEM images of successive stages) (a) beginning with deposition of the initial base layer, (c) followed by rim buildup, and (d–g) sequential increase in silicification of the valve surface. (b) Diagram of initial expansion in the x and y plane of the valve formed in (a). (h) Diagram of z-axis expansion toward the distal valve surface in stages (d–g).
3(b), consistent with the ridged structure being a distinct silica layer formed on top the of ribs. In contrast to the distal surface of the valve, the inner (proximal) surface was smooth [Figs. 4(c) and 4(d), note the difference in scale between Figs. 4(b) and 4(d)], though an outline of the ribs
FIG. 3. Formation of the valve base layer and maturation of nanopores: (a) SEM image of initially deposited silica strips, corresponding to ribs in the mature valve, (b) close-up of the strips in which precursors of the nanopores are visible flanking the raised central portion, (c) TEM image of the silica polymerization front during formation of the base layer (note branched structure and irregular shape of the nanopores), and (d) TEM image of a mature portion of the valve, showing the more circular shape of the nanopores. 2692
was visible, and AFM-imaged nanopores were more visible (not being obscured by 50-nm silica particles). The timing of formation of valve and girdle band structures in diatoms has not been directly measured. From synchronized growth experiments, we can measure girdle band formation in T. pseudonana at a maximum rate of one per 30 min and estimate valve formation to take less than 30 min.38 Girdle bands averaged 745 nm in width and had both smooth and textured portions [Figs. 5(a) and 5(b)]. The smooth portion is the underlap between adjacent girdle bands, corresponding to 30% of the entire width. A welldefined row of pores was visible at the junction of the smooth and textured regions [Fig. 5(a)]. Other nanopores were present in varying abundance in individual girdle bands, and most commonly the girdle band immediately adjacent to the valve (the valvocopula) had more pores than subsequent ones [Fig. 5(c)]. The arrangement, density, and size of pores in the valvocopulae were similar to that in the valve. Girdle bands were 41 nm thick (Table I) and had lips on their edges [Figs. 5(a) and 5(b)]. T. pseudonana girdle bands are not complete circles; at the ends they taper to a point and in the next girdle band below, a structure called the ligulum [Fig. 5(b)] projects into this region to fill the gap [Fig. 1(a)]. The ligulae were positioned on opposite sides of the frustule in adjacent girdle bands; on the same side they were staggered by approximately 45° [see Fig. 1(a)], an arrangement that would maximize overall structural integrity. Each girdle band is also made in an SDV, but because of difficulties in imaging these structures, details of their formation are not clear, and little information is available in the literature. It is unclear what distinguishes SDVs involved in valve or girdle band formation.
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FIG. 4. Silica structures on the distal and proximal valve surfaces: (a) SEM image showing spherical particles on the ridges of the distal valve surface, (b) AFM image of spherical particles on the ridges of the distal valve surface (average diameter of particles is 50 nm), (c) SEM image of the proximal valve surface, showing the smooth silica morphology and inner structures of the fulto- and rimo-portulae, and (d) AFM image of the proximal valve surface showing nanopores and the smooth silica structure. Scanned areas in (b) and (d) are approximately 1000 × 1000 nm. Note the scale difference in (b) and (d).
The portulae [Fig. 6(a)] are sites of extrusion of chitin strands and appear to be specifically designed for this purpose.39,40 The inner portion of the rimoportulae consisted of a tube flanked by adjacent horseshoe-shaped pores [Fig. 6(b), arrows], and the outer portion formed a chamber that tapered to the top and was buttressed by ridges on the outer valve surface [Figs. 6(a) and 6(c)]. The portulae openings inside and outside the valve had similar diameters (Table I), but the inner opening was circular whereas the outer could be oval [Fig. 6(c)]. The flanking pores of the rimoportulae were on opposite sides oriented generally along the valve circumference [Fig. 6(b)] but at a slight angle relative to each other, whereas those of the fultoportulae were generally positioned in a radial orientation relative to the valve center, although some variation was seen. In addition to the multiple rimoportulae around the valve circumference, a single pore with a simpler structure was seen [Fig. 6(b), arrowhead]. No protuberance from this pore on the outside of the valve could be identified. Formation of the inner tube of the rimoportulae occurred via circularization and extension of a cylindrical structure at an early stage in deposition [Figs. 6(d) and
FIG. 5. Structure of girdle bands: (a) SEM image of girdle band showing smooth and textured portions and the row of pores at the interface, (b) structure of the girdle band ligula, and (c) different numbers of nanopores in successive girdle bands (valve would be at top).
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FIG. 6. Structure and formation of the portulae. (a) TEM image of cross section of a rimoportula. (b) Ultrahigh resolution SEM image of the inner portion of two rimoportulae, and another type of pore (arrowhead) found once per valve; arrows denote the horseshoe-shaped pores flanking the central tube of one of the rimoportula. (c) Buttressed structure of outer portula. (d) Rimoportulae forming during base layer formation of the valve (view is from the inside of the valve); (inset) enlargement of forming rimoportulae. (e) Outer portion of the rimoportulae during formation; the inner tube is incompletely silicified at the bottom, corresponding to the opening seen in the inset in (d). A distinctive rim structure is visible. (f) TEM image of two forming portulae showing four (left) and two (right) flanking pores, in contrast to the three seen in (e). (g, h) Successive growth of outer rim structure to form the outer chamber of the portulae. (i) Cutaway diagram of portulae structure. The inner tube is on the left, flanking pores are in the center, and the outer chamber is toward the right.
6(e)]. Even though two flanking pores were always observed on the inside of the valve [Fig. 6(b)], there were two, three, or four flanking pores (about 40–45 nm) visible during formation [Figs. 6(e) and 6(f)]. We were unable to determine the relationship between the additional pores and the two visible pores. On the outside of the valve, there was an overgrowth of silica forming a distinctive irregular rim (300–330 nm) separate from the inner tube and the flanking pores [Figs. 6(e) and 6(g)]. This grew in a tapered manner to form the outer chamber [Figs. 6(g) and 6(h)]. The resulting structure had an irregularly shaped chamber on the outside of the valve, and an inner pore resulting from the tube on the inside, flanked by multiple smaller pores [Fig. 6(i)]. The biochemical machinery for chitin synthesis is located precisely beneath the inner tube,39,40 and the overall structure of the portulae suggests a specific function in chitin synthesis, although the particulars are not known. Species of the Thalassiosirales synthesize long, rigid strands of chitin to facilitate buoyancy.41 As such, the portulae represent an adaptation of the silicifying machinery to form a structure used in a biochemical function, synthesizing a product useful to the cell. IV. DISCUSSION
The combination of heavier ribs interspersed with less silicified regions in the valve of T. pseudonana (Figs. 1 and 3) seems optimal in terms of economical use of material while maintaining strength. A previous study42 provides direct experimental support that diatom silica 2694
structures are optimized for strength (crushing resistance) and lightness. In addition to contributing to lightness, the presence of numerous nanopores in T. pseudonana silica would enable uptake and efflux of metabolites across an otherwise impenetrable barrier. The initial deposition of thicker ribs followed by filling-in between them indicates a correlation between the mechanism of formation and optimization of design. We believe that distinct factors, which may act in concert, are involved in generating the different silica morphologies comparing the base layer and the ridges (Fig. 4) and within individual girdle bands [Figs. 5(a) and 5(b)]. As discussed more fully below, one factor is controlled changes in the levels or types of polypeptides known as silaffins, and long chain polyamines, which are involved in silica polymerization and nanoscale structure formation.16–18,24 A second factor involves effects either on, or due to, the SDV membrane, also called the silicalemma.43,44 It has been suggested that smooth silica at the silica/ silicalemma interface is formed by inhibition of expansion of the SDV after it reached its full size.33,45,46 The smooth morphology in the region of underlap between T. pseudonana girdle bands [Figs. 5(a) and 5(b)], which is a region of appression where SDV expansion is inhibited, is consistent with this. Chemical effects could also contribute to formation of smooth silica; in biomimetic experiments, increasing the concentration of hydroxyls changed silica morphology from spherical to a flat sheet structure.6 Perhaps hydroxyls are enriched on specific luminal faces of the silicalemma. Consistencies
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in spherical silica particle sizes and the presence of distinct silica morphologies in evolutionarily diverse diatom species indicate similarities in silica processing strategies (this study and in Refs. 45 and 47). After initial deposition, the valve silicalemma in T. pseudonana apparently expands in the z-axis direction only distally (Fig. 2), as observed in other centric species,48,49 although there are exceptions.44 The mechanism of unidirectional expansion is unclear but could involve spatially specific interactions with membraneassociated proteins, or a tighter association of the silica with the proximal portion of the silicalemma. Another question is how materials are delivered to accomplish distal z-axis expansion. In centric diatoms, the SDV apparently grows by fusion of small vesicles derived from the Golgi.48,50,51 After initial deposition of the base layer (Fig. 2), these vesicles would be physically blocked from the distal portion of the SDV. The initial x and y plane SDV expansion forming a thin structure that outlines the valve [Fig. 2(a) and 2(b)] would be facilitated by the lack of a substantial silica matrix. The buildup of the rim at an early stage [Fig. 2(c)] defines the valve diameter and the space required for subsequent z-axis expansion. This is a “spatial cementing” approach, where the space defining a structure is initially outlined in thin silica and later filled in. The initially deposited branched form of silica [Fig. 3(c)] is an ideal structure for filling in a space, which again shows correlation between a mechanism involved in valve formation and optimal design properties. The valve nanopores are initially irregular [Fig. 3(c)], and their subsequent circularization [Fig. 3(d)] indicates some mechanism that prevents their filling in, possibly involving proteins or phase separation events of longchain polyamines.25 It was proposed that some polysaccharides could play a role in inhibition of silica polymerization in areas of the SDV.33 An attractive feature of protein-mediated pore formation (either intrinsically or by influencing the shape of the silicalemma) is that regular and specific positioning would be possible, as seen in the regular positioning of pores next to the higher central ridge during rib deposition [Fig. 3(b)]. Data in this study combined with other work16–18,33 indicate three scales in formation of diatom silica structure; previously only two were recognized.33 The largest scale is formation of the overall shape of the valve [corresponding to Fig. 2(a)] and girdle bands, involving movements of the SDV directed by the cytoskeleton.33,52 This has been called macromorphogenesis;33,52 however, we suggest that the term microscale structure formation be used to more accurately reflect the micron size range of the structures. The smallest scale is the nanostructured morphology of the initial silica polymerization product, which we suggest should be referred to as nanoscale (1–100 nm) structure formation. The flat and spherical
silica morphologies in the base layer and ridges in the T. pseudonana valve [Figs. 3(c) and 4] are examples of this. The composition of silaffins and polyamines appear to be major determinants of nanoscale structure.16–18,24 The nanoscale silica morphologies in T. pseudonana are consistent with in vitro silica precipitation experiments done with combinations of long-chain polyamines and the three types of silaffins purified from this species.18 In vitro, silaffin 3 formed flat sheet-like structures,18 and we showed that mRNA levels for silaffin 3 were specifically induced during valve synthesis,35 consistent with its contribution to base layer formation (Figs. 2 and 3). Lower concentrations of silaffin 3, or the presence of the light form of silaffin 1 and 2, generated spherical silica structures,18 suggesting mechanisms for formation of spherical silica in the ridges (Fig. 4). These observations represent the first connection between structural, molecular, and biochemical processes in diatom silicification. The details of how silaffins are controlled to produce different silica morphologies in formation of the frustule are currently unclear. One level of control is at the level and timing of expression of the silaffins,35 and it is possible that only the relative ratios of silaffins in the SDV at a given time determine silica morphology. However, it is difficult to imagine that the precise control over silica structure demonstrated by diatoms could be achieved by the SDV simply serving as a “mixing pot” into which ingredients are added, and a detailed structure results. Self assembly and molecular recognition are fundamental design principles in biomineralization.2,3 It seems that more precise control could be achieved via specific interactions between silaffins and/or control over their localization within the SDV. If a silaffin were targeted to a specific substructure, then its ability to form silica of a useful morphology for that substructure would be optimized. This scenario would imply that there are specific proteins in the SDV that interact with the silaffins and are “targets” for their localization. Two approaches could be taken to further clarify the role that particular silaffins play in frustule formation. The first is to determine whether the silaffins are specifically localized. Gene tagging approaches using green fluorescent protein have been developed for diatoms53 and could enable this. If particular silaffins are not localized to specific structures, then perhaps small-scale shaping of the SDV membrane could play a significant role in structure formation, for example, in forming a confined space in which silica polymerization occurs. Of course, combinations of these contributions are possible. The second approach to analyzing the roles of silaffins would be to modify the expression of individual silaffin genes, either by increasing or decreasing their levels or altering their timing, and then determining whether specific changes in structure occurs. In addition to the nano- and microscale aspects of
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diatom silica formation, which are governed by distinct (and at least partially characterized) molecular entities, data in this report and from others33,51,54 indicate a mesoscale, involving formation of organized substructures within the SDV, which was previously called “micromorphogenesis.”33 We suggest that the term mesoscale be used to refer to structures intermediate in size between nano- and microscale and to assembly of initial polymerization structures into higher order structures within the SDV (which still may have sub-100 nm features). Examples of meso-scale structure are individual ribs (Fig. 3), which during formation are not each surrounded by silicalemma; instead, the SDV defines the front of silicification and the ribs are deposited within it, as “fingers in a mitten.”33,51,54 Lack of a spontaneously formed similar rib structure in vitro18 indicates that this arrangement cannot result solely from intrinsic selfassembling properties of the silaffins and polyamines, which do however self-associate (facilitating the polymerization process),17,55 suggesting that nano- and mesoscale processes are interrelated. The complex structure of the portulae (Fig. 6) is another example of mesoscale structure formation, and there are numerous other examples in other diatom species.33 The molecular determinants of mesoscale structure are currently unknown, but it seems likely that these entities can either organize the silaffins, be involved in their specific targeting, and/ or control the shape of the silicalemma. The least characterized components of the SDV are its membraneassociated proteins. Because of the demonstrated importance of expansion and molding of the silicalemma in forming structure,33,52 observations of mesoscale structure associated with SDV expansion (Fig. 3 and Refs. 48 and 49), and the role that proteins can play in shaping membrane structure,56 we believe that silicalemmaassociated proteins play important roles in mesoscale silica structure formation.35 Understanding mechanisms involved in meso- and microscale structure formation in diatoms would provide information for higher-order structural control in biomimetic applications. Although the valve of T. pseudonana is relatively simple compared with many other diatoms, our investigation has identified a succinct design strategy in which structure formation processes correlate with structural requirements. The complete genome sequence of T. pseudonana34 along with characterization of cell wall proteins18,35 allows this species to be used as a model for studying diatom silicification. Identification of cell wall proteins coupled with determining their localization and high resolution imaging analysis will enable elucidation of the interplay between these proteins and membrane systems involved in making structure. Tabulation of feature sizes in this work provides a benchmark for evaluating the dynamic range of structure possible and understanding the effects that control it. The work described 2696
herein is not only essential to correlate structure formation with genes and proteins involved but will enable modeling of the process and provide a basis for comparison in attempts to modify structure by genetic or nongenetic means. ACKNOWLEDGMENTS
The authors thank Tom Croke and Alan Street of Qualcomm Inc., San Diego, CA, for use of their equipment for focused ion beam milling of T. pseudonana valves. We are grateful to Steve Barlow of the San Diego State University Electron Microscopy Facility, San Diego, CA, for training and assistance with TEM, and to Bill Roth of Hitachi High Technologies America (Schaumburg, IL) for ultrahigh resolution FESEM imaging. This work was supported by Air Force Office of Scientific Research Multidisciplinary University Research Initiative Grant No. RF00965521 (M.H., J.I.K., A.K.D. and L.G.F.) and the U.S. Department of Energy Office of Biological and Environmental Sciences Medical Science Program (M.J.D., D.P.A.). A portion of this research was conducted at the Center for Nanophase Materials Sciences, which is sponsored at Oak Ridge National Laboratory by the Division of Scientific User Facilities, U.S. Department of Energy. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under Contract No. DE-AC05-00OR22725. REFERENCES 1. D.E. Morse: Silicon biotechnology: Harnessing biological silica production to construct new materials. Trends Biotechnol. 17, 230 (1999). 2. S. Mann: Biomineralization Principles and Concepts in Bioinorganic Materials Chemistry (Oxford University Press, Oxford, UK, 2001). 3. S.A. Davis, E. Dujardin, and S. Mann: Biomolecular inorganic materials chemistry. Curr. Opin. Solid State Mater. Sci. 7, 273 (2003). 4. J. Cha, G. Stucky, D. Morse, and T. Deming: Biomimetic synthesis of ordered silica structures mediated by block copolypeptides. Nature 403, 289 (2000). 5. R.R. Naik, P.W. Whitlock, F. Rodriguez, L.L. Brott, D.D. Glawe, S.J. Clarson, and M.O. Stone: Controlled formation of biosilica structures in vitro. Chem. Comm. 2, 238 (2003). 6. F. Rodriguez, D.D. Glawe, R.R. Naik, K.P. Hallinan, and M.O. Stone: Study of the chemical and physical influences upon in vitro peptide-mediated silica formation. Biomacromolecules 5, 261 (2004). 7. M.B. Dickerson, R.R. Naik, P.M. Sarosi, G. Agarwal, M.O. Stone, and K.H. Sandhage: Ceramic nanoparticle assemblies with tailored shapes and tailored chemistries via biosculpting and shapepreserving inorganic conversion. J. Nanosci. Nanotechnol. 5, 63 (2005). 8. D. Kisailus, J.H. Choi, J.C. Weaver, W.J. Yang, and D.E. Morse: Enzymatic synthesis and nanostructural control of gallium oxide at low temperature. Adv. Mater. 17, 314 (2005).
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