Characterization and Localization of Insoluble Organic Matrices Associated with Diatom Cell Walls: Insight into Their Roles during Cell Wall Formation

Organic components associated with diatom cell wall silica are important for the formation, integrity, and function of the cell wall. Polysaccharides are associated with the silica, however their localization, structure, and function remain poorly understood. We used imaging and biochemical approaches to describe in detail characteristics of insoluble organic components associated with the cell wall in 5 different diatom species. Results show that an insoluble organic matrix enriched in mannose, likely the diatotepum, is localized on the proximal surface of the silica cell wall. We did not identify any organic matrix embedded within the silica. We also identified a distinct material consisting of glucose polymer with variable localization depending on the species. In some species this component was directly involved in the morphogenesis of silica structure while in others it appeared to be only a structural component of the cell wall. A novel glucose-rich structure located between daughter cells during division was also identified. This work for the first time correlates the structure, composition, and localization of insoluble organic matrices associated with diatom cell walls. Additionally we identified a novel glucose polymer and characterized its role during silica structure formation.     

A low-latitude bloom of the surf-zone diatom, Anaulus australis (Centrales, Bacillariophyta) on the coast of Southern Queensland (Australia)

A novel bloom of the surf diatom Anaulus australis Drebes etSchultz was observed in subtropical waters off Surfers' Paradise,Queensland, Australia (27°55'S; 153°23'E) in early May2000. This is the lowest latitude in which an Anaulus australissurf diatom bloom has been reported. Nitrogen stable isotopeanalysis of surf diatoms may indicate anthropogenic nutrientinputs in this environment.     

Subcellular location and composition of the wall and secreted extracellular sulphated polysaccharides/proteoglycans of the diatomStauroneis amphioxys Gregory

Summary Extracellular polysaccharide/proteoglycan (EPS) mucilages play a crucial role in maintaining the structure of the extensive algal sheets that appear along the undersurface of nearshore Antarctic sea ice during the austral spring. In this study we have determined the composition and ultrastructural location of a family of novel sulphated polysaccharides/proteoglycans from the pennate ice diatomStauroneis amphioxys Gregory. They occur as soluble EPS in the culture supernatant, as an intercellular mucilage sheet, and as components of a distinct organic layer (diatotepum) underlying the silicious cell wall. The ultrastructural location and quantitative extraction of the mucilage EPS and the major diatotepum polysaccharides with hot water and alkali, respectively, was monitored by light and electron microscopy. The EPS and wall components were purified by Ultrafiltration, anion exchange and gel filtration chromatographies, and their monosaccharide composition was determined by gas-chro-matography mass spectrometry. The soluble and mucilage EPS, and major diatotepum polysaccharides/proteoglycans had an apparent molecular mass greater than 2 × 10⁶ Da on gel. They contained a similar complex monosaccharide composition that includes glucuronic acid and galactose as the major sugars and significant levels of rhamnose, fucose, arabinose, xylose, mannose, glucose and the mono-O-methylated monosaccharides 3-O-methylrhamnose, 3-O-methylfucose, 3-O- and 4-O-methylxylose. The ratios of Gal to GlcA, which together account for 45% of the monosaccharides, varied from 0.8 (in the soluble EPS) to 2.3 (in diatotepum polysaccharides). The level of sulphation also varied from 5–15% (w/w), with the mucilage EPS being the most highly sulphated. The soluble EPS also contains a small amount of protein (ca. 5%, v/w) which cochromatographs with the polysaccharide during gel filtration and anion exchange chromatographies suggesting that it may be a sulphated proteoglycan. They are clearly distinct from a sulphated glucuronomannan that remained in the alkali-insoluble fraction and may be tightly associated with the silica wall components. The amount of mucilage EPS increased during logarithmic growth but decreased during stationary phase, when most of the EPS was found in the soluble pool. These changes correlate with the breakdown of the mucilage sheet and dispersal of diatom colonies during stationary growth. Interestingly, the soluble EPS from stationary-growth cultures was indistinguishable from the mucilage EPS of logarithmic- or stationary-phase cells, suggesting that the dissolution of the intercellular mucilage was not due to a change in EPS composition. The possibility that cell motility may be required for mucilage formation and the significance of these polysaccharides in the under-ice community is discussed.     

CULTURES OF CHYTRIDS - PARASITES OF ALGAE IN BIOLOGICAL INSTITUTE OF PETERSBURG UNIVERSITY

Although diatom extracellular matricies are usually thought of exclusively in terms of the beautiful, architecturally complex silicious frustule, polymers exuded through the frustule are critical mediators of interactions with the external environment. In several species, complex proteoglycans appear to be the primary components involved in adhesion and motility. When viewed with high-resolution cryo-scanning electron microscopy methods, the ubiquity and pervasiveness of these polymers was revealed in both freshwater and marine taxa. Monoclonal antibody mapping of carbohydrate epitopes characterized by NMR, methylation and monosaccharide analysis and correlated with structural observations by EM revealed an organizational pattern far more complex than previously proposed. Modeling assembly of extracellular "stalks" in the marine biofouling diatom Achnanthes longipes involves intracellular sequestering of multiple components, deposition at the protoplasmic membrane/diatotepum interface, transport through the multilayered diatotepum and holes in the silica, extrusion from the frustule, and assembly into a very complex multi-laminate biocomposite structure. The mechanism of extracellular polymer participation in motility is complex in a different way, as some current models of raphe associated motility involve cytoskeletal interactions and molecular motors.     

EXTRACELLULAR MATRIX PROTEO‐GLYCANS INVOLVED IN DIATOM ADHESION AND MOTILITY

Although diatom extracellular matricies are usually thought of exclusively in terms of the beautiful, architecturally complex silicious frustule, polymers exuded through the frustule are critical mediators of interactions with the external environment. In several species, complex proteoglycans appear to be the primary components involved in adhesion and motility. When viewed with high‐resolution cryo‐scanning electron microscopy methods, the ubiquity and pervasiveness of these polymers was revealed in both freshwater and marine taxa. Monoclonal antibody mapping of carbohydrate epitopes characterized by NMR, methylation and monosaccharide analysis and correlated with structural observations by EM revealed an organizational pattern far more complex than previously proposed. Modeling assembly of extracellular “stalks” in the marine biofouling diatom Achnanthes longipes involves intracellular sequestering of multiple components, deposition at the protoplasmic membrane/diatotepum interface, transport through the multilayered diatotepum and holes in the silica, extrusion from the frustule, and assembly into a very complex multi‐laminate biocomposite structure. The mechanism of extracellular polymer participation in motility is complex in a different way, as some current models of raphe associated motility involve cytoskeletal interactions and molecular motors.     

Analytical studies of silica biomineralization: towards an understanding of silica processing by diatoms

Diatoms have continued to attract research interest over a long time. One important reason for this research interest is the amazingly beautiful microstructured and nanostructured patterning of the silica-based diatom cell walls. These materials become increasingly important from the materials science point of view. However, many aspects of diatom cell wall formation and patterning are still not fully understood. The present minireview article summarizes our recent knowledge especially with respect to two major topics related to diatom cell wall formation and patterning: (1) uptake and metabolism of silicon by living diatom cells and (2) understanding of the genetic control of cell wall formation. Analytical techniques as well as recent results concerning these two topics are highlighted in this review.     

Identification of proteins from a cell wall fraction of the diatom Thalassiosira pseudonana: insights into silica structure formation

Diatoms are unicellular eucaryotic algae with cell walls containing silica, intricately and ornately structured on the nanometer scale. Overall silica structure is formed by expansion and molding of the membrane-bound silica deposition vesicle. Although molecular details of silica polymerization are being clarified, we have limited insight into molecular components of the silica deposition vesicle, particularly of membrane-associated proteins that may be involved in structure formation. To identify such proteins, we refined existing procedures to isolate an enriched cell wall fraction from the diatom Thalassiosira pseudonana, the first diatom with a sequenced genome. We applied tandem mass spectrometric analysis to this fraction, identifying 31 proteins for further evaluation. mRNA levels for genes encoding these proteins were monitored during synchronized progression through the cell cycle and compared with two previously identified silaffin genes (involved in silica polymerization) having distinct mRNA patterns that served as markers for cell wall formation. Of the 31 proteins identified, 10 had mRNA patterns that correlated with the silaffins, 13 had patterns that did not, and seven had patterns that correlated but also showed additional features. The possible involvements of these proteins in cell wall synthesis are discussed. In particular, glutamate acetyltransferase was identified, prompting an analysis of mRNA patterns for other genes in the polyamine biosynthesis pathway and identification of those induced during cell wall synthesis. Application of a specific enzymatic inhibitor for ornithine decarboxylase resulted in dramatic alteration of silica structure, confirming the involvement of polyamines and demonstrating that manipulation of proteins involved in cell wall synthesis can alter structure. To our knowledge, this is the first proteomic analysis of a diatom, and furthermore we identified new candidate genes involved in structure formation and directly demonstrated the involvement of one enzyme (and its gene) in the structure formation process.     

QUANTITATIVE NANOMECHANICAL MAPPING OF MARINE DIATOM IN SEAWATER USING PEAK FORCE TAPPING ATOMIC FORCE MICROSCOPY1

It is generally accepted that a diatom cell wall is characterized by a siliceous skeleton covered by an organic envelope essentially composed of polysaccharides and proteins. Understanding of how the organic component is associated with the silica structure provides an important insight into the biomineralization process and patterning on the cellular level. Using a novel atomic force microscopy (AFM) imaging technique (Peak Force Tapping), we characterized nanomechanical properties (elasticity and deformation) of a weakly silicified marine diatom Cylindrotheca closterium (Ehrenb.) Reimann et J. C. Lewin (strain CCNA1). The nanomechanical properties were measured over the entire cell surface in seawater at a resolution that was not achieved previously. The fibulae were the stiffest (200 MPa) and the least deformable (only 1 nm). Girdle band region appeared as a series of parallel stripes characterized by two sets of values of Young’s modulus and deformation: one for silica stripes (43.7 Mpa, 3.7 nm) and the other between the stripes (21.3 MPa, 13.4 nm). The valve region was complex with average values of Young’s modulus (29.8 MPa) and deformation (10.2 nm) with high standard deviations. After acid treatment, we identified 15 nm sized silica spheres in the valve region connecting raphe with the girdle bands. The silica spheres were neither fused together nor forming a nanopattern. A cell wall model is proposed with individual silica nanoparticles incorporated in an organic matrix. Such organization of girdle band and valve regions enables the high flexibility needed for movement and adaptation to different environments while maintaining the integrity of the cell.     

TARGETING AND COVALENT MODIFICATION OF CELL WALL AND MEMBRANE PROTEINS HETEROLOGOUSLY EXPRESSED IN THE DIATOM CYLINDROTHECA FUSIFORMIS (BACILLARIOPHYCEAE)

Diatoms are unicellular organisms encased by silica-based cell walls that display species-specific structures. Morphogenesis of diatom cell walls is believed to be controlled by a polysaccharide/protein-matrix that remains associated with mature cell walls. Recently, a family of calcium-binding glycoproteins, the frustulins, has been identified as major diatom cell wall component. Here we describe a transformation-based approach to investigate intracellular targeting and function of frustulins. When ε-frustulin from the diatom Navicula pelliculosa is expressed in Cylindrotheca fusiformis, it is correctly targeted into the cell wall. Furthermore, the unique N-terminus of ε-frustulin was properly modified, indicating that C. fusiformis and N. pelliculosa contain homologous frustulin-processing proteases. In a different transformation experiment, a modified version of the Chlorella kessleri hexose/H⁺ symporter bearing a bacterial biotinyl-acceptor domain was expressed in C. fusiformis. The transporter became biotinylated in vivo and was functionally incorporated into the plasma membrane, allowing C. fusiformis to take up ¹⁴C-glucose and ¹⁴C-glucosamine. Stage-specific radioactive labeling with this transformant revealed that secretion of frustulins is strongly enhanced during cell wall development. The data presented in this study demonstrate for the first time functional expression of a membrane protein and correct targeting of a cell wall protein heterologously expressed in a diatom cell.     

STUDIES ON THE BIOCHEMISTRY AND FINE STRUCTURE OF SILICA SHELL FORMATION IN DIATOMS : I. The Structure of the Cell Wall of Cylindrotheca fusiformis Reimann and Lewin

An electron microscope study on the cell wall of the diatom Cylindrotheca fusiformis was carried out using stereoscopic and sectioning techniques. Material prepared by an enzyme treatment or by a mechanical method showed that the wall consists of two major components: a silica shell and organic material. Vapor of hydrofluoric acid was employed to remove the silica and thereby reveal the arrangement of the organic material. An attempt was made to increase the contrast of the organic component by "staining." Uranylacetate not only increased the electron opacity of the organic material but also apparently decreased the electron opacity of the silica shell. In ultrathin sections of complete cells, the structure as revealed by stereoscopy could be confirmed and extended. Every part of the silica shell is tightly enclosed by organic material. In the valve region the silica enclosed in this way is located between other layers of organic material. The whole cell wall is surrounded by a mucilaginous substance which stains with ruthenium red.     

Grazing-induced changes in cell wall silicification in a marine diatom

In aquatic environments, diatoms (Bacillariophyceae) constitute a central group of microalgae which contribute to about 40% of the oceanic primary production. Diatoms have an absolute requirement for silicon to build-up their silicified cell wall in the form of two shells (the frustule). To date, changes in diatom cell wall silicification have been only studied in response to changes in the growth environment, with consistent increase in diatom silica content when specific growth rates decrease under nutrient or light limitations. Here, we report the first evidence for grazing-induced changes in cell wall silicification in a marine diatom. Cells grown in preconditioned media that had contained both diatoms and herbivores are significantly more silicified than diatoms grown in media that have contained diatoms alone or starved herbivores. These observations suggest that grazing-induced increase in cell wall silicification can be viewed as an adaptive reaction in habitats with variable grazing pressure, and demonstrate that silicification in diatoms is not only a constitutive mechanical protection for the cell, but also a phenotypically plastic trait modulated by grazing. In turn, our results corroborate the idea that plant-herbivore interactions, beyond grazing sensu stricto, contribute to drive ecosystem structure and biogeochemical cycles in the ocean.     

ULTRASTRUCTURE OF A CHAIN-FORMING DIATOM PHAEODACTYLUM TRICORNUTUM1

The ultrastructure of a chain-forming clone of the polymorphic diatom Phaeodactylum tricornutum Bohlin has been studied by scanning and transmission electron microscopy. Both fusiform and tri-radiate cells are capable of forming chains. The cells, lacking any silica shell, are attached to each other at the central region of the theca, leaving the arms free. Neither homogenization nor sonication completely disrupts the chains. The attachment is due to fusion of the cell wall in the central region of the cell during cell wall deposition. This fusion results from failure of the cytoplasmic cleavage furrow to separate the plasma membranes of the two daughter cells sufficiently so that a single wall is deposited instead of two separate walls. Possible explanations for this are discussed.     

Extensive and intimate association of the cytoskeleton with forming silica in diatoms: control over patterning on the meso- and micro-scale

BACKGROUND: The diatom cell wall, called the frustule, is predominantly made out of silica, in many cases with highly ordered nano- and micro-scale features. Frustules are built intracellularly inside a special compartment, the silica deposition vesicle, or SDV. Molecules such as proteins (silaffins and silacidins) and long chain polyamines have been isolated from the silica and shown to be involved in the control of the silica polymerization. However, we are still unable to explain or reproduce in vitro the complexity of structures formed by diatoms. METHODS/PRINCIPAL FINDING: In this study, using fluorescence microscopy, scanning electron microscopy, and atomic force microscopy, we were able to compare and correlate microtubules and microfilaments with silica structure formed in diversely structured diatom species. The high degree of correlation between silica structure and actin indicates that actin is a major element in the control of the silica morphogenesis at the meso and microscale. Microtubules appear to be involved in the spatial positioning on the mesoscale and strengthening of the SDV. CONCLUSIONS/SIGNIFICANCE: These results reveal the importance of top down control over positioning of and within the SDV during diatom wall formation and open a new perspective for the study of the mechanism of frustule patterning as well as for the understanding of the control of membrane dynamics by the cytoskeleton.     

A new calcium binding glycoprotein family constitutes a major diatom cell wall component.

Diatoms possess silica-based cell walls with species-specific structures and ornamentations. Silica deposition in diatoms offers a model to study the processes involved in biomineralization. A new wall is produced in a specialized vesicle (silica deposition vesicle, SDV) and secreted. Thus proteins involved in wall biogenesis may remain associated with the mature cell wall. Here it is demonstrated that EDTA treatment removes most of the proteins present in mature cell walls of the marine diatom Cylindrotheca fusiformis. A main fraction consists of four related glycoproteins with a molecular mass of approximately 75 kDa. These glycoproteins were purified to homogeneity. They consist of repeats of Ca2+ binding domains separated by polypeptide stretches containing hydroxyproline. The proteins in the EDTA extract aggregate and precipitate in the presence of Ca2+. Immunological studies detected related proteins in the cell wall of the freshwater diatom Navicula pelliculosa, indicating that these proteins represent a new family of proteins that are involved in the biogenesis of diatom cell walls.     

Dynamics of silica cell wall morphogenesis in the diatom Cyclotella cryptica: Substructure formation and the role of microfilaments

Diatoms are unicellular algae that make cell walls out of silica with highly ornate features on the nano- to microscale. The complexity and variety of diatom cell wall structures exceeds those possible with synthetic materials chemistry approaches. Understanding the design and assembly processes involved in diatom silicification should provide insight into patterning on the unicellular level, and information for biomimetic approaches for materials synthesis. In this report we examine the formation of distinct cell wall structures (valves and girdle bands) in the diatom Cyclotella cryptica by high resolution imaging using SEM, AFM, and fluorescence microscopy. Special attention was paid to imaging structural intermediates, which provided insight into the underlying design and assembly principles involved. Distinct stages in valve formation were identified, indicating a transition from a fractally organized structure to a dynamic pathway-dependent process. Substructures in the valves appeared to be pre-positioned prior to complete silicification, suggesting that organics responsible for these structures were pre-assembled and put in place. Microtubules and microfilamentous actin play significant roles in the positioning process, and actin is also important in the pathway-dependent expansion of the front of silicification. Our results indicate that even though all silica structures in C. cryptica are made of assemblies of nanoparticulate silica, control of meso- and microscale structure occurs on a higher order. It is apparent that diatoms integrate bottom up and top down control and synthesis mechanisms to form the diversity of structures possible.     

Possible role of ubiquitin in silica biomineralization in diatoms: identification of a homologue with high silica affinity

In diatom silicon biomineralization peptides are believed to play a role in silica precipitation and the consequent structure direction of the cell wall. Characterization of such peptides should reveal the nature of this organic-inorganic interaction, knowledge that may eventually well be used to expand the existing range of artificial silicas ("biomimicking"). Biochemical studies on Navicula pelliculosa revealed a set of proteins, which have a high affinity for a solid silica matrix; some were only eluted from the matrix when SDS-denaturation was applied. One of the proteins with an affinity for silica, about 8.5 kDa, is shown to be a homologue of ubiquitin on the basis of its N-terminal amino acid sequence; ubiquitin itself is a highly conserved 8.6 kDa protein that is involved in protein degradation. This finding is in line with a model of silica biomineralization in diatoms that implies the removal of templating polypeptides when pores in the growing cell wall develop. Western blotting with specific anti-ubiquitin antibodies confirmed cross-reactivity. Immunocytochemical localization of ubiquitin indicates that it is present along the diatom cell wall and inside pores during different stages of valve formation.     

SILICON METABOLISM IN DIATOMS: IMPLICATIONS FOR GROWTH

Diatoms are the world's largest contributors to biosilicification and are one of the predominant contributors to global carbon fixation. Silicon is a major limiting nutrient for diatom growth and hence is a controlling factor in primary productivity. Because our understanding of the cellular metabolism of silicon is limited, we are not fully knowledgeable about intracellular factors that may affect diatom productivity in the oceans. The goal of this review is to present an overview of silicon metabolism in diatoms and to identify areas for future research. Numerous studies have characterized parameters of silicic acid uptake by diatoms, and molecular characterization of transport has begun with the isolation of genes encoding the transporter proteins. Multiple types of silicic acid transporter gene have been identified in a single diatom species, and multiple types appear to be present in all diatom species. The controlled expression and perhaps localization of the transporters in the cell may be factors in the overall regulation of silicic acid uptake. Transport can also be regulated by the rate of silica incorporation into the cell wall, suggesting that an intracellular sensing and control mechanism couples transport with incorporation. Sizable intracellular pools of soluble silicon have been identified in diatoms, at levels well above saturation for silica solubility, yet the mechanism for maintenance of supersaturated levels has not been determined. The mechanism of intracellular transport of silicon is also unknown, but this must be an important part of the silicification process because of the close coupling between silica incorporation and uptake. Although detailed ultrastructural analyses of silica deposition have been reported, we know little about the molecular details of this process. However, proteins occluded within silica that promote silicification in vitro have recently been characterized, and the application of molecular techniques holds the promise of great advances in this area. Cellular energy for silicification and transport comes from aerobic respiration without any direct involvement of photosynthetic energy. As such, diatom silicon metabolism differs from that of other major limiting nutrients such as nitrogen and phosphorous, which are closely linked to photosynthetic metabolism. Cell wall silicification and silicic acid transport are tightly coupled to the cell cycle, which results in a dependency in the extent of silicification on growth rate. Silica dissolution is an important part of diatom cellular silicon metabolism, because dissolution must be prevented in the living cell, and because much of the raw material for mineralization in natural assemblages is supplied by dissolution of dead cells. Perhaps part of the reason for the ecological success of diatoms is due to their use of a silicified cell wall, which has been calculated to impart a substantial energy savings to organisms that have them. However, the growth of diatoms and other siliceous organisms has depleted the oceans of silicon, such that silicon availability is now a major factor in the control of primary productivity. Much new progress in understanding silicon metabolism in diatoms is expected because of the application of molecular approaches and sophisticated analytical techniques. Such insight is likely to lead to a greater understanding of the role of silicon in controlling diatom growth, and hence primary productivity, and of the mechanisms involved in the formation of the intricate silicified structures of the diatom cell wall.     

A STRESS‐INDUCED PROTEIN ASSOCIATED WITH THE GIRDLE BAND REGION OF THE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYTA)1

We report the characterization of a cell‐surface protein isolated from the centric diatom Thalassiosira pseudonana Hasle and Heimdal. This protein has an apparent molecular weight of 150 kDa, is highly acidic, and is intimately associated with the cell wall. Although originally identified in cells experiencing copper toxicity, it is also induced by silicon and iron limitation but not by phosphate or nitrate limitation. Using immunofluorescence techniques, the 150‐kDa protein was localized to the girdle band region and covered the elongated girdle band region of morphologically aberrant cells suffering from copper toxicity. Although having biochemical similarities to girdle band associated proteins identified in pennate diatoms known as pleuralins, the 150‐kDa protein is not a sequence homolog and is predicted to have a number of unique features, such as chitin binding domains and a possible RGD cell attachment motif. Results presented here suggest that this protein is normally cell cycle regulated and may be involved in stabilizing cells during the division process.     

Silicification of auxospores in the araphid diatom Tabularia fasciculata (Bacillariophyta)

We report on auxospore wall structure and development in the araphid pennate diatom Tabularia fasciculata. Similar to most other pennates, these auxospores showed a typical bidirectional elongation, but unexpectedly bore no transverse perizonium, and with no detectable silicon during much of their expansion. Energy dispersive X-ray spectroscopy (EDS) analyses segregated auxospores into two types: (1) those containing no detectable silicon and (2) those with measureable amounts. Both types were of similar size. Silica precipitation began throughout the auxospore at or near maximal length, but initially was detectable in isolated regions throughout the structure. Following this initial condition, silicon was consistently detectable throughout auxospores of comparable size and corresponded to deposition of longitudinal perizonium (visible through the thin organic outer layer of the wall in some auxospores), followed by the deposition of the initial valves. Our results raise the question as to how the tubular shape of bidirectionally expanding auxospores up to ∼90 μm long is maintained in the absence of transverse siliceous elements restricting isodiametric expansion of the cell, which are present in all other known pennate auxospores and all but one other diatom. Our study is the first to systematically examine mineral elements of the auxospore wall analytically.     

Pleuralins are involved in theca differentiation in the diatom Cylindrotheca fusiformis

Diatom cells are encased within a silica-based cell wall (frustule) that serves as armour-like protection for the enclosed protoplast. Maintaining the integrity of the frustule requires a precise coupling between the biogenesis of new frustule components and the cell cycle. Thus far, the molecular mechanisms by which this coupling is achieved are unknown. This study demonstrates that pleuralins (formerly HEPs), a previously characterized family of diatom cell wall proteins, are involved in cell cycle-dependent frustule development. The frustule is made up of two, overlapping half-shells termed the epitheca and hypotheca. Both thecae are morphologically identical, yet immunolocalisation with anti-pleuralin antibodies demonstrates that their protein composition is clearly different. During interphase, pleuralins are associated only with the epitheca, where they are confined to the inner surface of the terminal elements (pleural bands) in the region of overlap with the hypotheca. At cell division, pleuralins also become associated with the newly formed pleural bands of the hypotheca. Remarkably, this process is concomitant with the functional conversion of the parental hypotheca into the epitheca of one of the progeny cells. These results indicate that developmentally controlled association of pleuralins with the frustule is involved in hypotheca-epitheca differentiation, which is a crucial process to ensure proper frustule development.