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Originally published In Press as doi:10.1074/mcp.M500174-MCP200 on October 5, 2005.
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Molecular & Cellular Proteomics 5:182-193, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Identification of Proteins from a Cell Wall Fraction of the Diatom Thalassiosira pseudonana

Insights into Silica Structure Formation*,S

Luciano G. Frigeri{ddagger}, Timothy R. Radabaugh§, Paul A. Haynes§, and Mark Hildebrand{ddagger},||

From the {ddagger} Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093-0202 and the § Department of Biochemistry, University of Arizona, Tucson, Arizona 85721


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

The silicified cell walls of the unicellular algae known as diatoms are fascinating products of biomineralization processes. Each diatom species constructs a uniquely structured cell wall called a frustule, consisting of valves and girdle bands (1, 2) composed of organic material and silica, which is often ornately and intricately shaped with fine features on the nanometer scale. Silica structures are made in the specialized membrane-bound intracellular compartment known as the silica deposition vesicle, or SDV1 (3–6). The SDV is a dynamic organelle that is expanded and molded during frustule formation, and these movements are essential to produce structures characteristic of the valve and girdle bands (1, 7–10). Upon completion, the entire silica structure is exocytosed to form an integral part of the cell wall (1). A first step to understand structure formation at the molecular level would be to isolate a purified SDV fraction and analyze its protein component. Unfortunately in over 3 decades of attempts, the SDV has not been purified. At best, researchers have isolated cell wall fractions enriched in components of the SDV (11, 12) but very likely also containing other constituents. Despite their incomplete purity, examination of cell wall fractions has yielded valuable insights into components involved in cell wall synthesis. Initial attempts relied on complete removal of surrounding cell wall organic material followed by dissolution of the silica with hydrofluoric acid (HF), suggesting that proteins were specifically associated with, and likely occluded within, the silica (12). More refined approaches were developed by Kröger et al. (11, 13), who initially identified calcium-binding cell wall coat proteins called frustulins extracted from the cell wall by EDTA treatment. They then examined proteins tightly associated with the silica by pretreatment with EDTA and SDS followed by dissolution of the silica with HF (and later ammonium fluoride) combined with chromatographic purification (14–17). These studies identified the pleuralins, proteins associated with the region of overlap between the two halves of the cell of the pennate diatom Cylindrotheca fusiformis (17, 18), the silaffins, one of the major components involved in silica polymerization (14, 19), and long chain polyamines, another major silica polymerization component (15). Continuing work highlights the importance of silaffins and polyamines in silica polymerization (20), and it has been proposed that different silaffins, or different mixtures or ratios of silaffins and polyamines, are involved in formation of different silica structures in the diatom cell wall (15, 20–22).

In addition to silica polymerization and the resultant formation of small scale silica structure, larger scale silica structure-forming processes are involved. Electron microscopic studies have provided direct evidence that the overall shape of the valve is controlled by expansion and molding of the SDV and that the cytoskeleton plays an essential role (1, 8, 9, 23–26). It is very likely that there is a direct interaction between the cytoskeleton (microtubules and actin) and the silicalemma (27, 28). If one examines the complexity of structures formed in a given diatom valve and the well defined stages in its development (1, 29, 30), it is apparent that there must be as yet uncharacterized SDV components involved. For example, proteins associated with the silicalemma could be involved in forming smaller scale silica features in the cell wall. Pores in diatom silica could result from inhibition of expansion of the SDV into particular areas (1), but the molecular components involved are unidentified. Even theoretical processes proposed to occur within the SDV, such as phase separations involving polyamines (31), are likely to be strongly influenced by the confined space in the SDV. It is reasonable to suggest that proteins of the SDV membrane can be either directly involved or act as intermediaries in movements or molding of this membrane. Thus, a cell wall fraction worthy of further study would be one enriched in membrane proteins of the SDV.

The recent determination of the complete genome sequence of the diatom Thalassiosira pseudonana (32) offers an unprecedented opportunity to examine the complex cellular processes of cell wall silicification using the tools of genomics and proteomics. Unfortunately because the only components of silicification identified are the silaffins (20) and silicic acid transporters (33, 34), examination of gene sequences in T. pseudonanaat our present level of understanding, will yield little information. To increase this understanding additional genes need to be identified, and genome information will be useful to identify proteins associated with the SDV.

A key experimental tool to study diatom cell wall synthesis is the ability to grow cultures synchronously so that most cells are entrained and then progress through the same stage of the cell cycle at the same time (35–38). This allows one to harvest cells at a time when valve or girdle band synthesis is maximal, which likely will also correspond to a time of high levels of expression of genes (as mRNA) and proteins involved in the process. Synchronized diatom cultures were used previously to compare mRNA or protein levels prior to and during cell wall synthesis, enabling identification of those that were induced (39, 40). Based on previous approaches (37), we have developed a synchrony technique for T. pseudonana2 that involves initial starvation of cells for silicon. After silicon replenishment, girdle band formation begins and continues until about 3 h, and by 4 h valve formation begins, which continues until 7 h. Cells begin to separate at that time, and additional girdle bands are synthesized until 8–9 h when the process begins again. It is unlikely that the actual time of formation of the valve is 2 h, but because of slight differences in cell cycle stage progression of individual cells the process in the entire culture is observed to require that time.

The combination of an available diatom genome sequence (32), the development of cell wall fractionation (11, 13) and synchrony techniques, and the characterization of cell wall-specific proteins and genes to use as markers (20) has set the stage for the work reported herein in which we used a tandem mass spectrometric approach to identify proteins associated with a membrane-associated cell wall fraction from T. pseudonana and their genes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Culture Conditions—
Axenic cultures of T. pseudonana strain CCMP1335 were grown in ASWT medium (37) with Bacto tryptone added to 1 g/liter at 16–18 °C in continuous light at an intensity of 150 µmol/m2/s. Synchronized growth of cultures was achieved as follows. Cells were grown in ASWT to a concentration of 3.5–4.0 x 106/ml (this required addition of silicate to 420 µM when cells were at 0.5–1 x 106/ml) with magnetic stirring and aeration, and then cells were harvested under sterile conditions by centrifugation in a Composite KAD-14.250 rotor (Composite Rotor Inc., Mountain View, CA) at 4200 x g for 12 min, washed once with silicate-free ASWT, and inoculated into silicate-free ASWT at 0.8–1.0 x 106 cells/ml. After 24 h, silicate was added back to a final concentration of 200 µM to initiate progression through the cell cycle and cell wall formation. In a typical synchrony, at least 80% of the cells doubled by 8 h post-silicate addition. New cell wall formation was evaluated by monitoring the fluorescence of rhodamine 123 (added to 2 µg/ml in the culture just prior to silicate addition) incorporated into silica (41) by fluorescence microscopy. To do so, a small aliquot of the culture was harvested every hour; cells were pelleted by centrifugation in a microcentrifuge for 1 min at 14,000 x g, extracted with 1 ml of methanol for 1 min to remove constitutive pigments, and then observed. When 30–40% of the cells analyzed were showing new valve synthesis, the bulk of the culture was harvested by centrifugation at 11,000 x g in a KAD-14.250 rotor for 16 min and resuspended in 3.5% NaCl, and cell number was determined in a hemacytometer using bright field microscopy. Aliquots of cells (5–6 x 108) were pelleted by centrifugation at 4200 x g for 4 min in a Sorvall HB-4 rotor and quickly frozen at –80 °C until used. In the experiments used for these studies, new valve formation was initially observed at 4.5–5.0 h after silicate addition (0 h) and reached an optimum for harvesting at 5.5–6.0 h. From an 8-liter culture we usually recovered 5 x 108 cells/liter.

Preparation of Cell Wall Extracts and Fractions—
The general procedure was based on techniques developed by Kröger et al. (11, 13). All operations were at 4 °C, and an HB-4 rotor was used for all centrifugations. Frozen cells from one tube (equivalent to a 1-liter culture after centrifugation) were washed two to three times with 10 mM Tris-Cl, 1 mM CaCl2, pH 7.4 (TC buffer) by centrifugation at 4200 x g for 4 min until the supernatant was clear. Cells were resuspended in 4 ml of the same buffer, and 1 ml each was aliquoted into 2-ml tubes containing 1 g of acid-washed glass beads, size 212–300 µm (Sigma). Cells were lysed with a Fast Prep cell beater (FP120, Bio 101, Savant Instruments, Holbrook, NY) for 45 s at a speed setting of 5. Tubes were removed quickly and chilled on ice for 5 min before proceeding to the next step. Cell lysates were transferred to 14-ml polypropylene Falcon 2059 tubes, diluted to 3–4 ml with TC buffer, and centrifuged as above to clarify the lysates, and the supernatant was transferred to a new tube, leaving behind the small amount of glass beads carried over from the previous transfer. The unfractionated cell walls were pelleted by centrifugation at 600 x g for 6 min and washed with 5–6 ml of TC buffer several times until the supernatant was clear. The preparation was either used immediately or stored at –80 °C.

Sequential fractionation of cell walls was done as follows. 5–6 x 108 cell equivalents were resuspended in 0.6 ml of 0.1 M EDTA pH 7.8 and extracted overnight (12–16 h) with constant rotation to remove Ca2+-binding cell wall proteins (11). After centrifugation at 9000 x g for 2 min, the supernatant was transferred to a new tube and stored at 4 °C. The pellet was washed twice with MilliQ water and then was resuspended in 0.6 ml 1 M NaCl, extracted with rotation for 30 min at room temperature, and then centrifuged. The supernatant was removed, and the pellet was washed as above. The NaCl extract was stored at 4 °C. The residual pellet was then extracted in 0.6 ml of 8 M urea for 30 min at room temperature and treated as above. From the pellet, lipid-associated proteins were extracted in 0.6 ml 2% SDS by boiling for 15 min, the supernatant was removed, and the pellet was washed twice with water. The pellet from the SDS extract was dried under vacuum, then resuspended in 0.3 ml of concentrated hydrofluoric acid, and incubated at 4 °C for 45 min. The HF extract was centrifuged at 16,000 x g for 2 min at 4 °C to pellet insoluble material, and the supernatant was removed and evaporated to dryness under vacuum. The residual pellet was dissolved in a small volume of water, neutralized with 0.2 M NaOH, and prepared for PAGE by adding an equal amount of 2x SDS sample buffer with 10 mM ß-mercaptoethanol.

SDS-PAGE—
With the exception of the HF extract, all extracts were treated with 7%TCA, 0.014% deoxycholate to precipitate proteins from the extracting agent followed by a cold acetone wash to remove residual TCA. Samples were dissolved in an appropriate amount of 1x SDS sample buffer with 5 mM ß-mercaptoethanol and placed in a boiling water bath for 3 min, and equivalent amounts based on starting cell numbers were analyzed by PAGE using Novex NuPAGE bis-Tris precast gels (4–12%) with MES running buffer (Invitrogen). Depending on the desired application, protein bands were detected either with Coomassie stain (Simply Blue Safestain, Invitrogen), SYPRO Ruby (Bio-Rad), or silver stain (42).

In-gel Digestion of SDS-PAGE Gel Bands—
SDS-PAGE gel slices were destained (43) and digested (44) using a Multiprobe-II liquid handling system (PerkinElmer Life Sciences). Following digestion, tryptic peptides were extracted from the gel pieces with 5% formic acid, 50% CH3CN on the Multiprobe-II liquid handling system. The extracted peptides were transferred to 0.5-ml Eppendorf centrifuge tubes, concentrated to 10 µl within a SpeedVac vacuum centrifuge (Savant, Farmingdale, NY), and replaced in the wells of a 96-well plate for further processing.

Nanoflow High Performance Liquid Chromatography-Tandem Mass Spectrometry—
A microbore HPLC system (Surveyor, ThermoFinnigan, San Jose, CA) was modified to operate at capillary flow rates using a simple T-piece flow splitter. Columns (6 cm x 100-µm inner diameter) were prepared by packing 100-Å, 5-µm Zorbax C18 resin at 500 p.s.i. pressure into columns with integrated electrospray tips made from fused silica pulled to a 5-µm tip using a laser puller (Sutter Instrument Co., Novato, CA). Electrospray voltage of 1.8 kV was applied using a gold electrode via a liquid junction upstream of the column. Samples were introduced onto the analytical column using a Surveyor autosampler (Surveyor, ThermoFinnigan). The HPLC column eluent was eluted directly into the electrospray ionization source of a ThermoFinnigan LCQ-Deca XP Plus ion trap mass spectrometer.

Peptides were eluted in a gradient using buffer A (0.1% formic acid) and buffer B (acetonitrile, 0.1% formic acid) at a flow rate of 400 nl/min. Following an initial wash with buffer A for 10 min, peptides were eluted with a linear gradient from 0 to 50% buffer B over a 60-min interval followed by 50–98% B over 5 min and a 5-min wash at 98% B. Spectra were scanned over the range 400–1500 mass units. Automated peak recognition, dynamic exclusion, and daughter ion scanning of the top three most intense ions were performed using the Xcalibur software as described previously (45).

Database Searching and Data Interpretation—
The following explanation includes all necessary information for complying with guidelines in publication of peptide and protein identification data as outlined by Carr et al. (46). Raw MS/MS data were converted to .dta files for each set of spectra acquired using Xcalibur Bioworks cluster version 3.1 (ThermoFinnigan) with the following parameters: peptide molecular weight range, 500–3500; threshold, 1000; precursor mass tolerance, 1.4; group scan = 1; minimum group count, 1; minimum ion count, 25; fragment ion tolerance = 0; no charge state determination applied.

MS/MS spectral data were analyzed using SEQUEST (Bioworks cluster version 3.1, ThermoFinnigan), a computer program that allows the correlation of experimental data with theoretical spectra generated from known protein sequence (47, 48). No enzyme specificity was used during the searching process, and static modification of cysteine with +57 amu (for iodoacetamide modification) and differential modification of methionine with +16 amu for oxidation were both considered. Parent and fragment masses were both considered as average values, and the maximum number of missed internal cleavage sites allowed was two.

In this work, the criteria for a preliminary positive peptide identification for a doubly charged peptide were a correlation factor (Xcorr) greater than 2.5, a {Delta} cross-correlation factor ({Delta}Cn) greater than 0.1 (indicating a significant difference between the best match reported and the next best match), a minimum of one tryptic peptide terminus, and a high preliminary scoring (49). For triply charged peptides the correlation factor threshold was set at 3.5, and for singly charged peptides the threshold was set at 2.0. At least one spectrum meeting these criteria from each identified protein was visually examined to confirm the presence of a strong Y- or B-ion series that could be matched to the assigned sequence for at least 4 amino acids.

All spectra were searched against release 1.0 of the T. pseudonana predicted protein database downloaded from the Joint Genome Institute (genome.jgi-psf.org/thaps1/thaps1.download.ftp.html). This was supplemented with sequences from trypsin, keratin, and other common laboratory contaminants (50) to produce a total of 11,662 protein sequences. Database search results were filtered and organized, and comparative numerical analysis of datasets was performed using DTAselect and Contrast (51). The same program was used to filter out trypsin and keratin peptides from results. Upon closer examination, many gene models called in the T. pseudonana genome sequence were incomplete, e.g. ORFs extended further upstream or downstream than called. In these cases, full-length ORFs were manually generated and used for subsequent analyses. These included searching for transmembrane domains using TMpred (52) and coiled coil regions using COILS (53) at the ExPASy Proteomics Server (us.expasy.org/), and BLAST searches (54) at the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov/BLAST/).

mRNA Extraction, cDNA Synthesis, and Real Time PCR—
From an 8-liter synchrony culture, prior to and then every hour after silicate addition, 750 ml was harvested for total RNA isolation, which was done using established procedures (55). An aliquot of total RNA was subjected to DNase treatment using an RNeasy (Qiagen Inc., Valencia, CA) kit as recommended. PCR amplification of an RNA sample that was not treated with reverse transcriptase served as a check for DNA contamination, which proved lacking. cDNA was prepared from DNase-treated RNA using a SuperScript II 3' RACE System for Rapid Amplification of cDNA Ends kit from Invitrogen following the manufacturer’s directions. For each protein identified by proteomic analysis, specific primers were designed for its corresponding gene with the aid of the Primer Quest Program (Integrated DNA Technologies, Coralville, IA). Primer sets were designed to specifically amplify an ~200-bp fragment. Real time PCR was done to quantitate mRNA levels using a Light Cycler System and the Fast Start DNA MasterPlus Sybr Green I kit (Roche Applied Science). Standards for real time PCR were dilutions of T. pseudonana genomic DNA. Normalization was done using equivalent amounts of DNase-treated RNA as material for cDNA synthesis. Dilutions of cDNA were done to ensure that amplification was in the linear range.

Growth of T. pseudonana in the Presence of 1,3-Diaminopropane Dihydrochloride (DAPDH)—
DAPDH was added to 10 mM in a 50-ml culture of T. pseudonana inoculated at low cell density (~5 x 104 cells/ml), and the culture was allowed to grow for 3 days after which a portion was harvested for scanning electron microscopy (SEM). The DAPDH culture grew to about 10% of the density of a control. After 3 days of growth, spermidine HCl was added to the culture at 10 mM, and growth rate increased to levels comparable with the control, consistent with an effect on polyamine levels.

Electron Microscopy—
Cells were prepared for SEM by resuspending 1 x 107 cells in 1 ml of concentrated H2SO4, placing the tube in a boiling water bath for 10 min, cooling, adding c.a. 20 mg KNO3, and then boiling an additional 10 min. Cell wall silica was gently pelleted (1000 x g for 3 min using an HB-4 rotor), washed once with 100 mM Tris-HCl, 10 mM EDTA, pH 8.0 and twice with water, and resuspended in absolute ethanol. An aliquot containing 2 x 106 cells was filtered onto a Whatman Cyclopore 1-µm PC10 membrane, which was dried, sputter-coated (gold/palladium), and then observed in an FEI Quanta 600 scanning electron microscope at the Scripps Institution of Oceanography Unified Laboratory Facility.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of Cell Wall Extracts and Electrophoretic Separation—
To isolate a cell fraction enriched in proteins associated with the SDV membrane, we modified the basic procedures developed by Kröger et al. (11, 13) for diatom cell wall preparation, adding additional steps to aid in removal of nonspecifically associated proteins. After preparation of an initial cell wall fraction, material was sequentially extracted (see "Experimental Procedures") with EDTA (to remove calcium-binding cell surface proteins), NaCl (to remove proteins interacting nonspecifically via electrostatic interactions), urea (to remove proteins interacting nonspecifically via hydrogen bonding), SDS (to extract membrane-associated proteins), and HF (to extract silica-associated proteins and polyamines). In the gel shown in Fig. 1, different protein banding patterns were observed in each treatment, indicative of a sequential removal of a specific subset of proteins. Comparison of detergent-extracted protein recovery in a sequentially extracted cell wall fraction compared with whole cells indicated a 45-fold enrichment. To identify proteins that were either induced or repressed during valve synthesis, we compared extracts from a synchronous culture at 0 and 6 h when the cells were inactive and active in making valves. Valve synthesis was monitored by direct observation using rhodamine 123 staining and an increase in abundance in the HF extract of proteins similar in size to two of the silaffins identified in T. pseudonana, SIL1-2 L (19 kDa) and SIL3 (31 kDa), which are known to be directly involved in silica polymerization (20). Samples were loaded on the gel based on identical cell numbers, which did not appreciably change between 0 and 6 h. In each fraction the abundance of specific proteins changed comparing the two time points (Fig. 1). The overall increase in protein in the 6-h SDS fraction may reflect the fact that cells have undergone cytokinesis (increasing their membrane content) but are not yet separated (maintaining a similar amount of cell wall proteins), which would result in an increase in membrane-associated proteins per the same cell number. Also to be considered is that as the SDV forms it becomes the size of an entire valve, therefore its membrane constituents are likely to contribute significantly to the total in the extract.



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  		FIG. 1. PAGE separation of cell wall fractions. Shown is a SYPRO Ruby-stained 4–12% polyacrylamide gel separation of proteins extracted from cell wall fractions of T. pseudonana at 0 and 6 h after silicon replenishment (see "Experimental Procedures" for details). Lanes at the left and right are molecular weight markers (MWM), and molecular masses in kDa are listed at the extreme left. Samples from each type of extract (EDTA, NaCl, urea, SDS, and HF) are grouped so that both time points (0 and 6 h) from each are in adjacent lanes.

 
Our interest in this study was in proteins associated with the SDV membrane. Because many of the protein bands migrated closely to each other, we decided to examine regions in the gel from the 6-h SDS extract that had a general induction of protein at 6 h relative to 0 h. Regions corresponding to approximate sizes of 27, 49, 59, 105, and 180 kDa were excised and subjected to nano-LC-MS/MS peptide sequence determination. Complete details of MS/MS data are available in the supplemental data. Examination of the data identified 31 proteins of interest for further examination. The names of the proteins, corresponding gene models in the T. pseudonana genome, a description of similarity, and E values from BLAST (54) scores are listed in Table I.


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  		TABLE I Proteins identified in the SDS-extracted cell wall fraction

 
Analysis of mRNA Patterns during the Course of the Synchrony by Real Time PCR—
Because the cell wall SDS fraction is not likely to consist only of SDV-associated proteins, we applied an additional level of evaluation to test whether a given protein might be involved in cell wall synthesis. This was done by monitoring mRNA expression patterns during the course of a synchrony. We first identified markers for different stages in cell wall synthesis by examining mRNA levels of two of the silaffin genes identified in T. pseudonana, SIL1 and SIL3 (20), using real time PCR. As seen in Fig. 2, SIL1 mRNA levels peaked at 1 h, decreased, and then were induced to a lesser extent at 5–7 h. For SIL3, they were induced slightly at 1 h, decreased, then induced highly to peak at 6 h, and then decreased afterward, although they remained at preinduction levels until 9 h (Fig. 2). These patterns correlated excellently with girdle band formation for SIL1 and valve formation for SIL3, indicating that these genes are excellent markers for distinct stages in cell wall synthesis.



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  		FIG. 2. mRNA expression patterns of SIL1 and SIL3 during a synchrony. The graph shows relative amounts of mRNA (in ng) for SIL1 and SIL3 at different times prior to (0 h) and after (1–9 h) replenishment of silicate to a silicon-starved culture of T. pseudonana.

 
We next designed specific primers for genes corresponding to each of the proteins we identified and monitored mRNA levels during the course of synchrony by real time PCR. Based on their response, genes were placed into three categories. The first was genes whose mRNA levels varied similarly to the SIL genes (example in Fig. 3, top). One consideration was that a given gene could be specific for either girdle band or valve synthesis or be involved in both. In the example shown in Fig. 3 (top), we see higher induction at 1 h compared with the relationship between 1 and 6 h for SIL3 and higher induction at 6 h compared with the relationship between 1 and 6 h for SIL1, consistent with a possible role in both girdle band and valve synthesis for this gene. mRNA patterns for a total of 10 genes correlated to the silaffins Table I). A second class of response was genes whose mRNA patterns did not correlate with the silaffins, an example of which is shown in Fig. 3 (middle). Thirteen such genes were identified Table I). A third class of response was genes that had mRNA patterns correlating to the silaffins but which had additional features. Seven of these genes were identified Table I). An example is seen in Fig. 3 (bottom) where an induction at 1 and 6 h is visible, but an additional peak at 3 h is also seen. Genes with these responses could encode proteins involved in cell wall synthesis but that also play other roles in the cell (e.g. proteins of the cytoskeleton). In the first and third classes of responses, slight variations in the relative levels and timing of changes in mRNA expression were observed, but in general the overall patterns were remarkably consistent with the silaffins. Data on mRNA patterns for individual genes is available in the supplemental data.



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  		FIG. 3. Three different classes of mRNA patterns from the proteomic analysis. Shown are examples of genes responding similarly to the silaffins (4g, upper panel), dissimilarly to the silaffins (1a, middle panel), and similarly to the silaffins but with additional features (4e, lower panel). mRNA levels in ng are listed at left, and time during synchrony is denoted at the bottom.

 
Characterization of mRNA Responses of Polyamine Metabolism Genes and Effect of Inhibition of Polyamine Synthesis on Valve Structure—
One identified protein Table I) was glutamate acetyltransferase, an enzyme known to be involved in polyamine metabolism. Analysis of mRNA levels for this gene during synchrony revealed that it was down-regulated during the 4–6-h period (Fig. 4A). Examination of the biochemical pathway of polyamine metabolism (Fig. 4B) showed that glutamate acetyltransferase would actually shuttle ornithine away from polyamine synthesis; hence the down-regulation during valve synthesis is consistent with the increased need for polyamines at this time. To further analyze the pathway, we designed specific primers to test mRNA expression patterns of genes in other steps in polyamine biosynthesis. Variation in mRNA level for the arginase gene did not correlate to cell wall synthesis, but a good correlation was observed for the genes encoding ornithine decarboxylase, ornithine carbamoyltransferase, and S-adenosylmethionine decarboxylase (Fig. 4A). These data are consistent with induction of the pathway with citrulline as an initial substrate for polyamine biosynthesis.



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  		FIG. 4. mRNA responses of genes in the polyamine biosynthesis pathway. A, plots of mRNA responses of polyamine metabolism genes during a synchrony. B, polyamine biosynthesis pathway, identifying enzymes (bold), and intermediates (boxed) involved in formation of polyamines. SAM, S-adenosylmethionine.

 
Several specific inhibitors of ornithine decarboxylase have been identified and applied to inhibit polyamine synthesis in a variety of cell types (56–58). We tested the effect of DAPDH on valve synthesis in T. pseudonana. Cells treated with 10 mM DAPDH were inhibited to about 10% of the growth in untreated cells and had a rounded morphology. Examination of valves from the treated culture showed that they were incompletely formed (Fig. 5), having an outline of the structure but not thickened to their full extent. The silica was thin and flexible, consistent with the observed rounded cell morphology.



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  		FIG. 5. Effect of the polyamine synthesis inhibitor DAPDH on valve formation. A, valve from untreated culture. B, valve from culture treated with 10 mM 1,3-diaminopropane dihydrochloride. Arrows denote areas where silicification has not occurred.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
As stated in the introduction, in over 3 decades of attempts, no group has been able to isolate a pure SDV fraction to aid in understanding the involvement of this intracellular compartment in diatom cell wall synthesis. What has been accomplished is removal of organic material from the silica followed by silica dissolution to isolate tightly associated components (12, 16, 17). The approach taken in this study provides a means to identify genes involved in cell wall synthesis even with a less pure fraction enriched in cell wall components and specifically to examine membrane-associated proteins. The addition of NaCl and urea extraction steps have not been applied previously to diatom cell wall preparations and based on the different protein banding patterns observed in Fig. 1 clearly aid in resolving proteins with different characteristics. It is possible that these treatments could have removed proteins involved in structure formation that are peripherally associated with SDV membrane components; thus these fractions are worthy of future study. Because of the additional extraction steps, the membrane-associated protein (SDS) fraction is likely to be more pure than in previous studies, facilitating identification of integral SDV membrane proteins and proteins very tightly associated with these. We also applied other evaluation criteria, including comparison of protein abundance during periods when the valve is and is not being made (6 and 0 h) and monitoring mRNA expression patterns during the course of a synchrony and correlating these patterns to genes known to be involved in the process. Although mRNA levels do not necessarily have to correlate with protein abundance, in general they do (59, 60), and we can clearly relate SIL1 and -3 mRNA patterns to distinct stages in cell wall synthesis. As such, SIL1 and -3 are the first described marker genes for cell wall synthesis. The data in Fig. 2 are also consistent with the hypothesis of Kröger et al. (14, 15), Poulsen and Kröger (20), and Poulsen et al. (22) that different silaffins are involved in formation of different silica structures because SIL1 is induced during girdle band synthesis and SIL3 is induced during valve synthesis.

The validity of the experimental approach was demonstrated in the case of the polyamine biosynthesis pathway. Identifying one gene in this pathway (glutamate acetyltransferase) by nano-LC-MS/MS enabled the testing of other known pathway genes for mRNA response and identified those likely involved in the induction of polyamine synthesis. Application of a specific inhibitor of an enzyme in this pathway (ornithine decarboxylase) resulted in aberrant valve formation (Fig. 5), directly demonstrating the involvement of that enzyme (and its gene) in the process.

Proteins Identified and Their Possible Involvement in Cell Wall Synthesis—
Other proteins identified in this analysis Table I) offer intriguing possibilities in terms of their possible involvement in cell wall synthesis. Two cytoskeletal proteins were identified, a homolog to the myosin type I tail domain and a dynein ß chain homolog. Myosin is an actin motor protein, and the type I tail domain has a membrane association binding site. Specifically myosin I is associated with intracellular membrane vesicles or the cytoplasmic face of the plasma membrane (61, 62). Dynein is a multisubunit microtubule-dependent enzyme whose cytoplasmic isoform acts as a motor for the intracellular retrograde motility of vesicles and organelles along microtubules (63). Both actin and microtubules have been shown to play key roles in the expansion of the SDV during diatom valve formation (27, 28), and identification of motor proteins possibly involved in the process may be beneficial in following the dynamics of valve formation.

Two proteins possibly involved in vesicle trafficking were identified in the SDS-extracted fraction, a homolog to dymeclin and a homolog to CDC48. The mRNA pattern for the dymeclin homolog had excellent correlation to SIL3 (see supplemental data). The function of dymeclin is not clearly established; however, in human cells mutants in this gene have an enlarged endoplasmic reticulum network and a large number of intracytoplasmic vesicles, suggesting its involvement in trafficking (64). CDC48 is an ATPase required for membrane fusion and protein degradation (65–67). It possesses chaperone-like activities and can functionally interact with Hsc70. Yeast CDC48 plays a role in cell division control, whereas other eukaryotic homologs are involved in the budding and transfer of membrane from the transitional endoplasmic reticulum to the Golgi apparatus (65–67). In T. pseudonana, mRNA levels for cdc48 peaked at 1, 3, and 5–6 h, consistent with an involvement in cell wall synthesis but also in other cellular functions. Vesicle trafficking is likely an essential aspect of the growth of the SDV (29), and initial experiments using trafficking inhibitors have generated aberrant forms of the valve (data not shown).

In a complex multicomponent process such as cell wall formation, protein/protein interactions are likely to play important roles. Four proteins identified in the SDS fraction had domains indicative of such interactions. Protein 2b contained a tetratricopeptide repeat, which mediates protein/protein interactions and the assembly of multiprotein complexes in a variety of cellular processes (68, 69). mRNA levels for the corresponding gene correlated strongly with SIL1, consistent with a possible involvement in girdle band synthesis. Protein 4g contained an HSP70 domain and tetratricopeptide repeat, and mRNA levels correlated with SIL3, consistent with a possible role in valve formation. Proteins 3f and 3h also contained protein/protein interaction motifs Table I), but their mRNA patterns did not correlate to cell wall synthesis.

Two protein kinase homologs were identified. Protein 4b had a serine/threonine protein kinase catalytic domain, and its overall mRNA expression pattern was similar to SIL3, but it peaked slightly earlier at 5 h. Further analysis of the sequence identified a possible single membrane-spanning segment and a single region with a coiled-coil motif. Protein 4h was homologous to the {alpha}-kinase protein family, and its mRNA pattern had excellent correlation with SIL3. The {alpha}-kinases are a novel family of eukaryotic protein kinase catalytic domains, which have no detectable similarity to conventional kinases (70). Protein types with the highest BLAST scores were elongation factor 2 and myosin heavy chain kinases. Analysis of the 4h sequence indicated two regions with predicted coiled-coil propensity in exactly the same location as in myosin heavy chain kinases from Dictyostelium discoideum (71), and coiled-coil motifs were absent from elongation factor 2 protein kinase sequences, suggesting that the diatom protein may be a myosin heavy chain kinase. In D. discoideum, phosphorylation induced by these proteins drives the disassembly of myosin II filaments (71). These proteins also have a non-conserved actin binding region (70). Because protein kinases are key regulatory enzymes in cellular processes and regulate the activity of multiple components, further characterization of these genes may shed light on the regulation of silicification as well as aid in identifying other components involved.

Protein processing and degradation are involved in all essential steps of biogenesis of subcellular structure. A total of five proteins in the SDS fraction were homologs of proteases or their inhibitors, and the mRNA patterns of three of these had at least some correlation with cell wall synthesis. Protein 2c was a homolog to a Kazal-type serine protease inhibitor, which can inhibit a variety of serine proteases (72), and its mRNA pattern was similar to SIL3. Protein 2g was a homolog to a trypsin-like serine protease, with highest similarity to the Deg P family, and was predicted to contain a transmembrane domain. mRNA for this gene was induced later during valve formation. Protein 4d was homologous to membrane-associated proteases in the stomatin/prohibitin family. One function of stomatin is in regulating monovalent cation transport through lipid membranes (73), and a second function is to act as a cytoskeletal anchor (74). Some prohibitin homologs have gene repression and antiproliferative activity (75, 76), and others are localized in the mitochondria (77). mRNA levels for this gene were induced during valve formation. Proteins 2f and 3a were also identified as proteases; however, mRNA patterns for neither correlated with cell wall synthesis.

Based on inferential data, it has been suggested that the ancestral form of the SDV could have been the lysosome (78). We decided to consider any proteins in intracellular compartments other than the chloroplast and mitochondria as worthy of further analysis, hence the inclusion of peroxisomal proteins in our analysis (no lysosomal proteins were identified). Protein 1a was a homolog to a 22-kDa peroxisomal membrane protein, and 2e was a homolog to a peroxisomal biogenesis AAA ATPase Table I), but mRNA patterns for neither correlated with cell wall synthesis.

Three transport protein homologs were identified. Protein 2d matched extremely well to P-type ATPases responsible for transporting phospholipids from one leaflet of bilayer membranes to the other (79). Because a substantial amount of lipid would be required for expansion of the SDV, the presence of this protein and induction of its gene during valve formation are consistent. Protein 4c had homology to the Major Facilitator Superfamily transporter family, but its specific function is undefined; mRNA levels for its gene were induced during valve formation and also at 3 h post-silicon replenishment. Protein 3d had homology to a class I carnitine/acylcarnitine translocase, but its mRNA pattern did not correlate to cell wall synthesis.

Two proteins associated with the cell surface were discovered in the SDS fraction. Protein 1b was a homolog to extracellular proteins identified in many organisms and was a member of the SCP/Tpx-1/Ag5/PR-1/Sc7 family of extracellular domains, which include both plant pathogenesis-related and mammalian cell surface proteins (80, 81), representing functional links between plant defense systems and the human immune system. The SCP domain was identified in the genomic gene model; however, we discovered that the ORF was truncated in this model and actually extended an additional 447 amino acids upstream in frame. One membrane-spanning segment was identified in the upstream region as well as a domain homologous to the mucins, the major glycoprotein component of mucilage. Diatom cell walls are covered with mucilage (82), and secretion of mucilage in many organisms is a defense mechanism, consistent with the role of the SCP domain on the protein. The other cell surface protein was 5a, corresponding to a recently described girdle band-associated protein with a chitin binding domain (83). Both cell surface proteins had mRNA patterns similar to SIL3.

Protein 4e was a homolog to a ubiquitin ligase module and more specifically to an anaphase-promoting complex cullin domain (84). The cullins are involved in cell division control in yeasts (85) and probably in various processes in the cell cycle of other organisms (86). Although induced at anaphase, mRNA for this protein was also induced at 1 and 6–7 h, consistent with the pattern of SIL3, suggesting more than one functional role for this protein. Another member of the cullin family is mammalian vasopressin-activated calcium-mobilizing receptor (VACM-1), a kidney-specific protein thought to form a cell surface receptor but which does not have any structural hallmarks of a receptor (87).

Two binding proteins were identified: 1c, which contained a calcium binding domain and predicted transmembrane segments, and 2a, which was a homolog to fibrillarin and related nucleolar RNA-binding proteins, but neither had a mRNA pattern consistent with cell wall synthesis. Five proteins had unknown functions: 3e and 4a had no significant matches in database comparisons, whereas 3c, 3g, and 4f matched to other proteins whose function has not been determined Table I). The mRNA pattern for 4a was consistent with a possible involvement in cell wall synthesis; we were unable to get real time PCR data in repeated attempts for 4f, and mRNA patterns for 3c, 3e, and 3g did not correlate.

The Effect of Polyamine Synthesis Inhibition—
The effect of the polyamine biosynthesis inhibitor DAPDH on valve formation was dramatic (Fig. 5). We have been examining the process of valve formation in T. pseudonana by microscopy2 and have identified two general stages: 1) formation of a thin outline of the valve by expansion and fusion of thin silica strips and 2) thickening of the valve to form the final structure. The image in Fig. 5B is very similar to that seen in initial stages of valve formation. Possible interpretations of these data are that polyamines are more involved in the second stage of valve formation than the first, or if insufficient polyamines are present, the second stage is not initiated. In the initial discovery of polyamines associated with diatom silica, Kröger et al. (15) surveyed a variety of diatom species. In all species other than C. fusiformis, polyamines were abundant, and in C. fusiformis, they were present but at much lower levels (15). Silica in the cell wall of C. fusiformis consists of thin silica strips, and it has been suggested that the lesser amount of polyamines in this species could be due to the lack of need to fill in a voluminous structure (88). Poulsen and Kröger (20) demonstrated that polyamines are required for in vitro silica formation using extracts from T. pseudonana; however, the data in Fig. 5B may suggest that polyamines are more involved in the thickening process than the initial formation of the outline of the valve. Further experiments are required to test this hypothesis more rigorously.

The results presented here are to our knowledge the first proteomic analysis of cell wall fractions from a diatom and outline an approach for further investigations in T. pseudonana and other diatom species. After identifying proteins of interest by nano-LC-MS/MS, monitoring mRNA levels for their genes during a synchrony provided both validation of MS/MS results and another level of functional evaluation. In the case of the polyamines this confirmed the involvement of a biochemical pathway involved in cell wall silicification. As a consequence, inhibition of an enzyme in this pathway resulted in alteration of structure of the valve. We believe that the results presented in this report represent a promising initial study, but it is clear that as more proteins potentially involved in diatom cell wall synthesis are identified, other techniques to evaluate their involvement need to be applied and developed. These include determining intracellular location via gene fusion or immunolocalization techniques (18, 89, 90) and modification of gene expression to determine the effect on cell wall silica structure. The genetic manipulation of diatoms is still at an early stage (91–93), and more progress in this area will be essential to unravel the intricacies of the process of formation of the diatom silica cell wall.


   ACKNOWLEDGMENTS
 
We thank Evelyn York at the Scripps Institution of Oceanography Unified Laboratory Facility for assistance with SEM, Sandra Hazelaar for help with the synchrony, and Nils Kröger for kindly providing the silaffin sequences. P. A. H. thanks Thomas Hyland for continued support.


  FOOTNOTES
 
Received, June 7, 2005, and in revised form, October 3, 2005.

Published, MCP Papers in Press, October 5, 2005, DOI 10.1074/mcp.M500174-MCP200

1 The abbreviations used are: SDV, silica deposition vesicle; ASWT, artificial seawater tryptone; DAPDH, 1,3-diaminoproprane dihydrochloride; HF, hydrofluoric acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; SEM, scanning electron microscopy. Back

2 M. Hildebrand, A. K. Davis, L. G. Frigeri, and J. Kelz, manuscript in preparation. Back

* This work was supported in part by Air Force Office of Scientific Research Multidisciplinary University Research Initiative Grant RF00965521 (to L. G. F. and M. H.). Back

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

Supported by funding from the Bio5 Institute of the University of Arizona. Back

|| To whom correspondence should be addressed: Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0202. E-mail: mhildebrand{at}ucsd.edu


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