Western blot analysis using anti-Sin1 antibodies. M total membrane fraction from T. To test whether the predicted transmembrane domain of Sin1 is integrated into the lipid bilayer, isolated total membranes of T. Under these conditions, proteins containing a domain that fully penetrates the lipid bilayer remain bound to the membrane, whereas proteins that are peripherally associated with the membrane become completely solubilized [ 23 ].
Western blot analysis revealed that approximately half of the Sin1 molecules remained associated with the carbonate extracted membranes Additional file 1 : Figure S3a, Additional file 1 : Table S3. Therefore, the fact that approximately half of the Sin1 protein molecules remain associated with the membrane rather than becoming fully extracted by alkaline carbonate buffer is regarded as a proof for transmembrane anchoring of Sin1. Fluorescence microscopy analysis of transformant cells expressing Sin1-GFP N revealed GFP fluorescence in the valve and girdle band regions of live cells and in isolated biosilica Fig.
After complete removal of the silica, the GFP was present in ring-shaped, purely organic structures Fig. This confirmed that Sin1 is a component of the previously described biosilica-associated insoluble organic matrices [ 16 , 17 ] from which it has recently been identified by proteomics analysis [ 17 ].
This result indicated that the Sin1 molecules are largely embedded inside the biosilica, which implies that they are exposed to the lumen of the SDVs during silica deposition in vivo.
The fusion proteins were expressed under control of the endogenous Sin1 promoter and terminator sequences. Green color indicates the GFP fusion proteins and the red color is caused by chlorophyll autofluorescence.
Sin1 becomes cleaved by a protease between the luminal domain lum and the transmembrane helix orange. The luminal domain is incorporated into the biosilica, while the transmembrane helix and the cytosolic domain blue squiggle become degraded. In cells expressing Sin1-GFP C , GFP-fluorescent ring-like structures and plate-like structures appeared transiently during girdle band and valve morphogenesis, respectively Fig.
Strong GFP fluorescence was also associated with intracellular spherical structures that were moving throughout the cytoplasm, whereas GFP fluorescence appeared to be absent from the biosilica cell walls Fig. This seemed to indicate that Sin1 is absent from the biosilica and the organic matrices, which would contradict the result obtained with cells expressing Sin1-GFP N.
To resolve this discrepancy, T. In this fusion protein the cyan fluorescing protein mTurquoise2 mT2 was positioned within the luminal region between the RXL domain and NQ domain and the yellow fluorescing protein Venus was attached to the C-terminus. Live transformant cells exhibited both cyan fluorescence and yellow fluorescence Additional file 1 : Figure S5a , confirming that the full length double fluorescent-tagged Sin1 protein molecules were expressed.
The cyan fluorescence was present in the biosilica of live cells Additional file 1 : Figure S5a , in isolated biosilica, and in the insoluble organic matrices Additional file 1 : Figure S5b. In contrast, the yellow fluorescence was absent from the biosilica and the insoluble organic matrices Additional file 1 : Figure S5a, b.
This result can be explained by assuming a proteolytic cleavage between the luminal region and the C-terminal part of Sin1 during silica biogenesis. Only the luminal domain of Sin1 rather than the transmembrane helix and cytosolic domain would become incorporated into the biosilica Fig.
The heptapeptide motif GGQKFAL, which is right at the transition of the luminal region to the transmembrane domain, is perfectly conserved in all silicanin sequences see Additional file 1 : Figure S1 and might be the recognition site for a silicanin-specific protease.
To investigate the location of Sin1 during the cell cycle, time lapse confocal fluorescence microscopy with individual cells expressing Sin1-GFP C was performed. Biosilica produced during imaging was labeled by pre-loading the cells with the dye 2- 4-pyridyl 4- 2-dimethylaminoethylaminocarbamoyl methoxy phenyl oxazole PDMPO.
PDMPO is known to accumulate in silica deposition vesicles and remains permanently entrapped inside the biosilica also after exocytosis, but it does not stain mature biosilica that is already present on the cell surface [ 26 ]. The particles were quite mobile but most of the time remained close to the region where the cleavage furrow will appear i. During the entire cell cycle, GFP fluorescence is also present throughout the plasma membrane Additional file 2 : Movie S1. In Fig. Additionally, schematic drawings are presented showing the characteristic stages of the cell cycle Fig.
Therefore, all events preceding the completion of valve biogenesis are assigned negative times. For simplicity, intracellular compartments, except for the SDVs, have been omitted. Black and blue colors indicate mature biosilica and newly produced biosilica, respectively. Red and yellow colors depict the plasma and SDV membranes, respectively. All images are projections of nine z-planes.
We assume that, at this time point, cytokinesis has just been completed and thus the GFP-fluorescent plasma membranes of the two sibling cells are adjacent to one another in the mid-cell region. Shortly after cytokinesis, GFP fluorescence strongly increased in the mid-cell region starting from the center Fig.
Fluorescence in the mid-cell region appeared to steadily increase until the end of valve biogenesis Fig. These observations demonstrated 1 the development of a valve biosilica in each sibling cell during the time period from —70 to 0 min, and 2 the co-localization of Sin1 with the valve SDVs during silica biogenesis. During the following 10 minutes, GFP fluorescence intensity in the mid-cell region decreased drastically while GFP fluorescence in the entire plasma membrane region of each sibling cell increased Fig.
This was confirmed by quantitative analysis of the fluorescence intensity, which revealed identical fast kinetics for the GFP loss in the mid-cell region and the increase of GFP fluorescence in the plasma membrane region upon exocytosis of the valves from the two sibling cells Figs.
Consistent with this assumption was the simultaneous sudden drop in fluorescence intensity of the biosilica-bound PDMPO Figs. Upon exocytosis, the pH in the vicinity of the biosilica changes from acidic inside the SDVs [ 27 ] to near neutral on the cell surface.
Furthermore, it is possible that PDMPO molecules, which had accumulated inside the SDVs but were not entrapped inside the newly produced biosilica, rapidly diffused into the surrounding medium upon exocytosis.
From each cell, images were recorded in 3. From each frame, z-projections were generated by combining all nine z-planes. The schematic shows the delineations of the cellular regions analyzed.
The coloring of the cellular regions in the schematic corresponds to the line coloring in the graphs. The black lines represent the sum of the intensities from all regions of the cell. This allowed alignment of the time scale of the four different cells used in our analysis.
The gray-shaded areas represent the standard deviation of the averaged fluorescence intensities obtained from the four cells for each region. No standard deviation is given for time periods for which only a single cell was available for averaging. Shortly after exocytosis of the valves, the distance between the centers of the two sibling cells had increased and several spherical GFP-fluorescent particles and associated GFP-fluorescent strands reappeared in the sibling cells near their contact region Fig.
We assume that the GFP-fluorescent spherical particles and associated strands, which were also observed before the onset of valve biogenesis Fig. During valve biogenesis these structures appeared to have fused with the developing valve SDVs in the mid-cell region.
Between In this location the first girdle band SDV is supposed to develop in each sibling cell. Like with valve exocytosis, GFP fluorescence intensity at the sites of girdle band formation rapidly decreased during exocytosis while GFP fluorescence in the entire plasma membrane simultaneously increased with the exocytosis of each girdle band Fig. This result indicates that the development of the valve SDVs precedes the deposition of silica, which is in agreement with the results from a previous ion-abrasion electron microscopy study on T.
The sudden decrease of GFP fluorescence in the mid-cell region of the cell and the simultaneous increase in GFP fluorescence in the plasma membrane during exocytosis of the valve Fig. This enabled the SDV-derived Sin1-GFP C molecules to diffuse across the entire plasma membrane, thus substantially decreasing in abundance at the site of biosilica exocytosis. However, the decrease of GFP fluorescence in the mid-cell region during valve exocytosis was only partially compensated by the increase of GFP fluorescence in the plasma membrane.
During this time, GFP fluorescence in the cytoplasm increased only moderately blue trace in Fig. As GFP fluorescence outside the cell did not increase, the result suggests that a fraction of the Sin1-GFP C molecules was proteolytically degraded during valve exocytosis.
From the normalized GFP fluorescence data Fig. This degradation-prone fraction may be formed by Sin1-GFP C molecules from which the luminal region was proteolytically cleaved off and incorporated into the biosilica.
The remaining GFP-tagged C-terminal part containing the transmembrane and cytosolic domains of Sin1 may have then become rapidly degraded, thus eliminating the GFP fluorescence. In contrast, Sin1-GFP C molecules that retain the luminal region during silica biogenesis may be resistant to such fast proteolysis, and after valve exocytosis would become components of the plasma membrane. To further investigate the function of Sin1 in silica biogenesis, we studied the properties of the recombinantly expressed luminal region of Sin1 rSin1 lum , aa 25— , which contains most of the RXL domain, the NQ domain, and a C-terminal hexahistidine tag Additional file 1 : Figures S2b, S9a.
Dynamic light scattering DLS revealed that rSin1 lum has a hydrodynamic diameter of 6. When the solution was acidified to pH 5. A further moderate increase of acidity to pH 5. After decreasing the acidity by adjusting the pH to 6. This result indicated that pH-induced assembly of rSin1 lum clusters is a reversible process. Cryo-electron microscopy revealed that the rSin1 lum clusters had spherical shapes with a relatively wide size distribution that increased with decreasing pH, and was within the size range determined by DLS Fig.
Growth of the clusters appeared to occur through fusion Fig. We assume that the pH-triggered, reversible formation of aggregates is a physiologically relevant property of Sin1, because diatom SDVs are acidic compartments [ 27 ].
Dynamic light scattering analysis of rSin1 lum at different pH. A solution of rSin1 lum was adjusted to increasingly acidic pH values black traces , and then titrated back to near neutral pH blue trac e. The black traces show the particle distribution by mass. The dotted lines show the particle distribution by intensity to highlight the presence of small amounts of rSin1 lum clusters. Cryo-electron microscopy analysis of rSin1 lum clusters. The black arrows point to small clusters that consist of only 10—20 protein monomers.
The arrowhead s point to neck regions between two clusters that are indicative of fusion events. As the luminal domain of Sin1 is embedded inside the silica Additional file 1 : Table S4 , we investigated whether this part may be directly involved in the deposition of silica inside the SDVs. Therefore, we analyzed the silica formation activity of rSin1 lum in vitro at pH 5.
Previously, it has been shown that strongly negatively charged diatom phosphoproteins e. The luminal region of Sin1 is predicted to be highly negatively charged at pH 5.
These data demonstrated that rSin1 lum interacts with LCPA, resulting in a high silica formation activity at near physiological pH conditions.
In the present work, we have identified Sin1 as the first SDV transmembrane protein. Bioinformatics analysis revealed that Sin1 is highly conserved throughout the diatom realm, and homologous proteins were also identified in two non-diatom organisms.
One of them, the amoeboid alga Rhizochromulina marina , is not reported to produce biosilica, yet it belongs to the taxon Dictyochophyceae , which also includes silicoflagellates that form siliceous skeletons [ 34 , 35 , 36 ]. The other non-diatom homologue of Sin1 is present in the colepid ciliate Tiarina fusa , which is a protozoan that forms a shell made of calcium carbonate [ 37 ]. This suggests an evolutionary relationship between the mechanisms for the biomineralization of silica and calcium carbonate, which has recently been also demonstrated for three coccolithophore species [ 19 ].
These coccolithophore species encode silicic acid transporter-like proteins and the biomineralization of their calcium carbonate scales was perturbed by germanic acid i. The absence of Sin1 genes in other non-diatom organisms that produce biosilica e.
Given the high degree of sequence similarity among Sin1 homologues in centric and pennate diatoms, we assume that Sin1 may have a fundamental role in the biogenesis of diatom biosilica, which will be discussed below. Based on the data presented in this study, we hypothesize that there are two populations of Sin1 in the cell.
The Sin1 molecules of one population in the following referred to as Sin1 cross become covalently cross-linked via their luminal regions to organic components in the SDV lumen e. This event is part of the self-assembly process of organic components in the SDV lumen that results in a silica-forming insoluble organic matrix. Being part of the silica-forming organic matrix, the luminal regions of the Sin1 cross population become encapsulated by silica during silica biogenesis in the SDV lumen.
In contrast, the molecules of the other Sin1 subpopulation only loosely interact with the components of the silica-forming organic matrix and do not become encapsulated by silica. We assume that, during exocytosis, both the cytosolic domain and the transmembrane helix of the Sin1 cross molecules are cleaved off and are then rapidly proteolytically degraded, whereas the luminal region becomes an integral component of the extracellular biosilica.
The other population of Sin1 molecules retain their membrane anchors, and can diffuse throughout the plasma membrane—SDV membrane continuum during exocytosis. The fate of the SDV membrane after biosilica exocytosis has thus far remained a conundrum.
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Try again? Cited by. Download options Please wait Supplementary information PDF K. Article type Paper. Submitted 29 Dec Accepted 05 Mar First published 05 Mar Download Citation. Author version available. Download author version PDF. Nonpolar molecules are hydrophobic, meaning they do not interact or mix with water. Students will investigate the physical properties of lipids through a laboratory activity.
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