Identifying valve SDVs in cell lysates
For the rapid identification of valve SDVs in cell lysates, we aimed to establish a fluorescent labeling strategy in combination with correlative fluorescence-electron microscopy imaging, which is outlined in the following. To facilitate following the cell cycle progression in vivo, transgenic cell lines were used that expressed C-terminally tagged silicanin-1 (Sin1-GFPC), which was previously shown to be located in the membrane of T. pseudonana valve and girdle band SDVs (32). By synchronizing the cell culture, the proportion of cells undergoing valve SDV development was enhanced. When the maximum proportion of valve SDV bearing cells was observed, the SDV-specific dye PDMPO was briefly added to the culture to label the silica inside SDVs (48) before it was exocytosed. Cells were gently lysed and the lysate immobilized on a TEM finder grid allowing to rapidly identify the positions of valve SDVs by fluorescence microscopy searching for PDMPO-labeled structures with plate-like morphology. TEM was then employed for imaging the objects at the identified positions.
A Sin1-GFPC expressing T. pseudonana cell line had previously been generated (32), but a corresponding transgenic C. cryptica cell line had to be established here. Previously, gene g20669.t1 had been identified as Sin1 homologue of C. cryptica, because its predicted protein sequence, ccSin1, showed 75.9% global sequence identity to T. pseudonana Sin1 (32). A transgenic C. cryptica cell line was produced that expressed C-terminally GFP-tagged ccSin1 (ccSin1-GFPC) under control of the ccSin1 promoter and terminator sequences. Fluorescence microscopy revealed that ccSin1-GFPC was located in both valve SDVs and girdle band SDVs as well as several other intracellular vesicles (Fig. 1B). These locations match those previously described for Sin1-GFPC in T. pseudonana (Fig. 1A) (32). Based on the sequence and intracellular location, we concluded that ccSin1 indeed represents the functional homologue of T. pseudonana Sin1.
Previously, incubation of diatom cultures in silicon-free medium for extended period of times followed by replenishment of silicic acid (in the following abbreviated Si) has achieved only partial cell cycle synchrony for various diatoms species including T. pseudonana and C. cryptica (38, 49). Here, we have investigated the effect of two consecutive Si starvation-replenishment series on the synchrony of cell division in T. pseudonana and C. cryptica. During the first Si starvation-replenishment series for T. pseudonana, an increase in cell density occurred 2.5 hours after re-addition of Si (Fig. 1C, black dots). The initial cell density had doubled after 5.5 hours, which suggested that each cell underwent one cell division within 3 hours. The percentage of dividing cells at several time points after Si replenishment was estimated based on the proportion of cells that exhibited the characteristic valve-shaped Sin1-GFPC fluorescence in the mid cell region (at each time point 100 cells were examined). The maximum proportion of cells containing valve SDVs was ~30% and was observed 3.5 hours after replenishment of Si. After 9 hours, the cells were subjected again to Si starvation followed by Si replenishment. This time, the cell number doubled between 2.75-5 hours after Si replenishment and thus required only 2.25 hours (Fig. 1C, blue dots). The maximum proportion of dividing cells was 70% and was reached 3 hours after Si replenishment. These data demonstrated that the two consecutive silicon starvation-replenishment series improved the cell division synchrony in T. pseudonana. In contrast, for C. cryptica one Si starvation-replenishment series achieved better cell cycle synchrony than two consecutive ones. In the first series, cell division started 4.75 hours after Si replenishment, and the cell density had doubled after 10 hours (Fig. 1D, black dots). The maximum proportion of dividing cells was observed 5.5 hours after Si replenishment and was only 10%. During the second silicon starvation-replenishment cycle, the cell density increased only 50% within 12 hours after Si replenishment, and the maximum proportion of dividing cells was only 7% (observed after 6 hours; Fig. 1D, blue dots). We assumed, that repeated Si starvation imposes a higher metabolic stress on C. cryptica compared to T. pseudonana, and thus the growth rate of the former slows down after the second Si starvation period.
Fig. 1 Live cell imaging of silicanin-1 GFP fusion proteins in individual cells of (A) T. pseudonana and (B) C. cryptica. The left panels show an individual cell after cytokinesis with one valve SDV (green, plate-like structure) in each sibling cell. The right panel shows a cell during interphase with a girdle band SDV and additional silicanin-1-GFP bearing intracellular vesicles. Green color indicates GFP fluorescence and red color shows chlorophyll autofluorescence. Scale bar: 4 µm. Cell density measurements during the first (black dots) and second (blue dots) series of Si starvation-replenishment with (C) T. pseudonana and (D) C. cryptica. The red arrow indicates the time point at which the highest proportion of dividing cells was observed in the second Si starvation-replenishment cycle. Standard deviations (n=3) for the first Si starvation-replenishment series were ± 0.07∙106 cells∙mL-1 (T. pseudonana) and ± 0.03∙106 cells∙mL-1 (C. cryptica), and for the second series ± 0.1∙106 cells∙mL-1 (T. pseudonana) and ±0.04∙106 cells∙mL-1 (C. cryptica) (see Supporting Information).
To tag valve SDVs with a second fluorescent label, synchronized cells were incubated briefly (10 min) with PDMPO at the time point when the highest proportion of valve SDV bearing cells was present, i.e., 3 and 5.5 hours after the second Si replenishment for T. pseudonana and C. cryptica, respectively (Fig. 1C, D; red arrows). Immediately after PDMPO labelling, the cells were gently lysed, and putative valve SDVs could be readily identified by epifluorescence microscopy as GFP and PDMPO labeled disks (Fig. 2A; yellow arrows). Discs that exhibited much stronger contrast in bright field microscopy than the putative valve SDVs but
Fig. 2 Identification of valve SDVs in the lysates from Sin1-GFPC expressing and PDMPO labeled T. pseudonana cells. (A) Bright field (BF) and epifluorescence images of the same lysate in in the GFP and PDMPO channels. The red color is due to chlorophyll autofluorescence. Yellow arrows point to valve SDVs. The white arrowhead points to a mature valve with neither GFP nor PDMPO fluorescence. Scale bars: 10 µm. (B) PDMPO fluorescence patterns of three individual valve SDVs from a cell lysate. Scale bar: 3 µm (all images have the same magnification).
lacked both GFP and PDMPO fluorescence (Fig. 2B; white arrowhead), were presumed to be mature valves. Inspection of many putative valve SDVs revealed differences in diameters and PDMPO fluorescence patterns (Fig. 2B). Discs with diameters of 2-3 µm showed usually a quite homogeneous distribution of PDMPO fluorescence. In contrast, SDVs with diameters of 4-6 µm showed homogeneous fluorescence only in the central region and exhibited a more strongly fluorescent rim with a dot-like pattern. We hypothesized that the different types of PDMPO labeled disks represent valve SDVs containing biosilica at different developmental stages. To verify this, a correlative fluorescence approach and transmission electron approach was applied, which revealed that PDMPO and GFP labeled disks indeed contained immature valve biosilica (Fig. 3).
Fig. 3 Correlative fluorescence and electron microscopy of a valve SDV (arrow). A PDMPO labeled valve SDV was identified by epifluorescence microscopy (EF) on a finder grid and subsequently analyzed with TEM (middle and right images). Scale bars: 10 µm (left and middle images), 400 nm (right image).
TEM analysis of valve morphogenesis
It is to be expected that silica biogenesis came to a stop when the cells were lysed, because this procedure was performed near 0°C and involved a >25-fold dilution of the cellular content, which should drastically slow down all metabolic activity. Therefore, it is reasonable to assume that each valve that was immobilized on the TEM grid was in the same developmental stage as at the time point of cell lysis. The valve SDVs were then permanently “frozen” in their developmental stages through washing with H2O and drying. As a consequence of this preparation method, it was not possible to follow the development of individual valve SDVs. However, once the cells were not in perfect cell cycle synchrony, the lysate contained valves from many (possibly all) different developmental stages. To obtain a detailed view of valve morphogenesis, more than 150 TEM images of valve SDVs of different developmental stages were obtained from T. pseudonana. This confirmed that SDVs with a homogenous PDMPO pattern corresponded to earlier developmental stages of the biosilica than those exhibiting intense, dot-patterned PDMPO fluorescence at the rim (Fig. S1). An analogous correlated fluorescence microscopy-TEM analysis was performed with lysates from ccSin1-GFPC expressing and PDMPO labeled C. cryptica cells. From these analyses, the sequence of changes in biosilica structure during valve morphogenesis was reconstructed for both T. pseudonana and C. cryptica (Figs. 4, 5). The general criterion to establish the sequence of valve development was the progression of individual morphological features, such as the dendritic branches, the porous layers, or tube-like structures (called fultoportula). EDX mapping of valve SDVs indicated that all developing morphological features visible in TEM images were silica-based (Fig. S2). In the following, valve morphogenesis of T. pseudonana and C. cryptica are comparatively described.
In both species, the smallest discernable biosilica structure is a central silica ring (termed annulus) from which regularly spaced, radial silica ribs (termed costae) emerge (Fig. 4A, 5A). The costae start branching relatively soon after they emerge from the annulus. They have rather rough edges and seem to be composed of an agglomeration of globular particles. As the valve SDV increases in diameter, the costae continue to grow radially in both species (Fig. 4B, C). In C. cryptica thinner costae (termed narrow costae) emanate from the much wider primary costae (Fig. 5B). As the SDV diameter increases, the narrow costae grow radially in parallel to the wide costae (Fig. 5C, D).
Already at early stages during costae growth in T. pseudonana, the formation of pores with ~20 nm diameter (i.e. cribrum pores) occurs in the valve center (Fig. 4C, D). Subsequently, cribrum pore formation propagates radially along the costae and a thin silica layer develops around the pores (Fig. 4D, E) eventually filling the entire space between the costae (Fig. 4F). In C. cryptica the formation of cribrum pores that have similar diameters (~20 nm) as in T. pseudonana also starts in the center relatively early during valve development (Fig. 5C). Cribrum pore formation proceeds radially outward along the wide and narrow costae (Fig. 5C-H). However, as the space between the costae becomes filled with a layer of silica, 1-3 additional rows of cribrum pores are formed that are not adjacent to any costa (Fig. 5H). In contrast, cribrum pores that are not adjacent to costae are quite rare in T. pseudonana.
When the costae have almost reached their full length, the formation of regularly spaced, tube-like structures (each termed fultoportula) is initiated in T. pseudonana. Fultoportula formation is highly synchronized throughout the valve and involves the ends of four (rarely three) costae, which cease normal growth and instead develop into a central tube with three satellite pores (Fig. 4F-H). The inter costae space adjacent to the fultoportula is fully silicified lacking cribrum pores (Fig. 4G). In the regions between the developing fultoportulae, costae continue to extend but cease shortly after fultoportulae formation is completed. Termination of the lateral valve growth is marked by the merging of the costae ends into a patternless, non-porous ring of silica, which represents a continuous margin of the valve. (Fig. 4H). In C. cryptica, each wide costa develops a fultoportula well before the full length of the costae has been reached (Fig. 5E-H). Both wide and narrow costae continue to grow beyond the position of the fultoportulae before they merge into a patternless continuous ring of silica. Both, in T. pseudonana and C. cryptica almost 50% of the valves contain one (rarely two) fultoportula that is positioned slightly off the valve center (Fig. 4G, H and Fig. 5F-H). Formation of these central fultoportulae always precedes the formation of the fultoportulae at the periphery of the valve (Fig. 4E, 5B).
When fultoportulae formation commences in T. pseudonana, neighboring costae in the central part of the valve become connected by silica bridges (Fig. 4F, G). As valve development progresses, the number of silica bridges decreases towards the periphery, however, they become particularly prominent close to the valve margin in the spaces between fultoportulae (Fig. 4H). Two neighboring silica bridges together with the two interjacent costae segments represent so-called areola walls, which together constitute an areola pore. Each areola pore usually encompasses several cribrum pores (Fig. 4F, G).
Fig. 4 Valve development in T. pseudonana. Different valve SDVs were imaged by TEM and ordered based on the stage of silica structure formation. For each valve, an overview image (top row) and a corresponding detail image (bottom row) is shown. Colored arrows highlight characteristic biosilica features: green = annulus, blue = costa, red = cribrum pore, yellow = fultoportula, orange = areola pore. Scale bars: 1 µm (overview images), 400 nm (detail images).
Fig. 5 Valve development in C. cryptica. Different valve SDVs were imaged by TEM and ordered based on the stage of silica structure formation. For each valve an overview image (top row) and a corresponding detail image (bottom row) is shown. Colored arrows highlight characteristic biosilica features: green = annulus, purple = wide costa, blue = narrow costae, red = cribrum pore, yellow = fultoportula, orange = areola pore. Scale bars: 1 µm (overview images), 400 nm (detail images).
The formation of fultoportulae and areolae pores coincides with an apparent increase in thickness of the silica throughout the valve, which has previously been coined “z expansion” (37). In C. cryptica, the silica bridges between neighboring costae start appearing also during fultoportulae formation (Fig. 5F). Whereas in T. pseudonana cribrum pore formation in a given area is always completed before silica bridges arise, both these structural features develop simultaneously in C. cryptica (Fig. 5F, G). In some areas it is evident that an areola pore can be completed even before the cribrum pores are formed (Fig. 5F).
Fig. 6 Schematics of valve morphogenesis in T. pseudonana and C. cryptica. Morphogenesis proceeds from left to right. For simplicity only a quarter of the developing valves are shown.
Valve morphogenesis in T. pseudonana and C. cryptica has been investigated before (37,38). However, the correlative fluorescence and electron microscopy approach that was established in the present work has enabled the imaging of entire immature valves at an unprecedented number of different developmental stages while they are still encased by the SDV membrane. These include the structures of the annulus and costae at early developmental stages, and the differences between T. pseudonana and C. cryptica in the timing of the development of cribrum pores and areola pores. Figure 6 schematically summarizes the main steps in the morphogenesis of the valves of T. pseudonana and C. cryptica to highlight the similarities and differences in structures and development of their valves.
Morphogenesis of cribrum pores in T. pseudonana
The cribrum pores of T. pseudonana are 22 ±2 nm (n = 200) in diameter and positioned in a seemingly irregular pattern within the silica layer between the costae. In the following we refer to the silica layer between the costae as cribrum pore layer. From numerous images of immature valves at various stages of development, the morphogenesis of the cribrum pore layer was reconstructed (Fig. 7). Cribrum pore formation always starts along the costae, which are initially rather smooth (Fig. 7A, A’; white arrowheads). The costae develop wave-like edges as silica grows into the space between the costae (Fig. 7B, B’; yellow arrowheads). The wave peaks are fairly regularly spaced and each develops into an anvil shaped structure with the anvil ends elongating parallel to the long-axis of the costa (Fig. 7B, B’, C; blue arrowheads). The ends of two neighboring anvils that face each other merge, thus establishing a cribrum pore (Fig. 7C, C’; red arrowheads). The rows of cribrum pores associated with neighboring costae usually have very similar periodicities. The space between the two rows of pores becomes filled with silica, when the distance between the costae is <120 nm. When neighboring costae are spaced wider than 120 nm, additional cribrum pores can develop that do not originate from a costa edge (Fig. 7C; green arrowhead).
Fig. 7 Morphogenesis of cribrum pores in T. pseudonana. Images (A-C) show TEM micrographs from valve SDVs in different developmental stages, and images (A’-C’) show schematics of the same developmental stages. The arrows point to characteristic intermediate stages of pore development. The arrowheads point to features that are described in the text. Scale bars: 200 nm.
The elemental composition of biosilica in valve SDVs
In some of the T. pseudonana cell lysate preparations, SDVs were observed that contained many spherical nanoparticles with diameters of 19.4 ± 2.4 nm (n = 100) (Fig. 8A-C). The nanoparticles were also observed in some valve SDVs of C. cryptica (Fig.S3). In T. pseudonana, the nanoparticles were only observed during cribrum pore formation and were primarily associated with the costae. It has previously been hypothesized that silica nanoarticles are the primary building blocks for biosilica morphogenesis inside the SDV (15, 27, 28). The hypothesis was based on rather circumstantial evidence that included the presence of electron dense nanoparticles of unknown chemical composition in the growth zones of SDVs (28) (see also Introduction for further information). To investigate the elemental composition of the nanoparticles that were present in our SDV preparations, energy-dispersive X-ray (EDX) spectroscopy was performed. The EDX spectrum of the nanoparticles (Fig. 8D) showed characteristic signals for carbon (0.27 keV), nitrogen (0.40 keV), oxygen (0.52 keV), and silicon (1.75 keV). The silicon and oxygen signals are indicative of the nanoparticles being silica based. The nitrogen signal inside the nanoparticles might hint towards the presence proteins, long-chain polyamines, and/or amino sugar bearing polysaccharides, all of which are known to be associated with mature diatom biosilica (20–22). However, we cannot rule out that the nitrogen signal originated from nitrogen containing metabolites (e.g., amino acids) or a purely inorganic compound (e.g., nitrate, ammonia). EDX mapping of the valve SDVs confirmed that the costae and the nanoparticles are silica- and nitrogen based (Fig. 8D). The carbon signal in the spectrum originated mainly from the formvar coated grid surface (note that formvar is free of nitrogen and silicon), yet the EDX map clearly indicated the presence of carbon also in the costae (Fig. 8D). This result supports the presence of organic components inside the valve SDV. The silica nanoparticles could not be clearly identified in the carbon map, presumably due to the high carbon signal of the background.
Fig. 8 (A-C) TEM images of early valve SDVs with associated ~20 nm sized spherical nanoparticles. Scale bars: 200 nm. (D) Scanning transmission electron microscopy (STEM) EDX analysis of several nanoparticles within a single valve SDV. The yellow circles in the STEM image highlight the nanoparticles from which the EDX spectrum was obtained. The elemental maps are depicted with the corresponding chemical symbol. Scale bars: 100 nm.
To investigate the elemental composition of the immature valve biosilica independent of the surrounding SDV lipid bilayer and other SDV components that are not or only loosely attached with the developing silica structures, the lysate was extracted with sodium dodecyl sulfate (SDS) before immobilization on a formvar coated grid. Immature valve biosilica could still be easily identified on the grid, because the PDMPO molecules are tightly incorporated into the silica matrix (48). As expected, the EDX spectrum (Fig. 9) of the detergent-treated, immature valve biosilica showed signals for carbon (0.27 keV), oxygen (0.52 keV), nitrogen, (0.4 keV) and silicon (1.75 keV). Additional signals for cobalt (0.77 keV), aluminum (1.48 keV), and phosphorus (2.0 keV) were obtained. The cobalt signal in the EDX spectrum originated from excitations in the pole piece of the microscope rather than the sample. The presence of aluminum in the immature valve biosilica was not unexpected as this element was previously found in biosilica isolated from both naturally grown and cultivated diatoms (50, 51). Carbon, nitrogen and phosphorous are clearly co-located with the silica in the immature valve, which is consistent with the previously proposed presence of long-chain polyamines and highly phosphorylated proteins (i.e., silaffins and silacidins) (20, 22). The phosphorous signal might also be caused by the presence of inorganic phosphate, which has previously been found associated with mature biosilica in the diatom Coscinodiscus granii (52).
Fig. 9 EDX analysis of an SDS-extracted, immature valve from T. pseudonana. The STEM image of the valve is shown on the upper right. The EDX spectrum is derived from the entire valve surface. The elemental maps are depicted with the corresponding chemical symbol. Scale bars: 100 nm.