Ultrastructure and Membrane Traffic During Cell Division in the Marine Pennate Diatom Phaeodactylum tricornutum

Abstract

The marine pennate diatom Phaeodactylum tricornutum has become a model for diatom biology, due to its ease of culture and accessibility to reverse genetics approaches. While several features underlying the molecular mechanisms of cell division have been described, morphological analyses are less advanced than they are in other diatoms. We therefore examined cell ultrastructure changes prior to and during cytokinesis. Following chloroplast division, cleavage furrows are formed at both longitudinal ends of the cell and are accompanied by significant vesicle transport. Although neither spindle nor microtubules were observed, the nucleus appeared to be split by the furrow after duplication of the Golgi apparatus. Finally, centripetal cytokinesis was completed by fusion of the furrows. Additionally, F-actin formed a ring structure and its diameter became smaller, accompanying the ingrowing furrows. To further analyse vesicular transport during cytokinesis, we generated transgenic cells expressing yellow fluorescent protein (YFP) fusions with putative diatom orthologs of small GTPase Sec4 and t-SNARE protein SyntaxinA. Time-lapse observations revealed that SyntaxinA-YFP localization expands from both cell tips toward the center, whereas Sec4-YFP was found in the Golgi and subsequently relocalizes to the future division plane. This work provides fundamental new information about cell replication processes in P. tricornutum.

Keywords: Diatom; Sec4; Syntaxin; actin; contractile ring.; cytokinesis.

Figure 1

Electron micrographs of cytokinesis in P. tricornutum. A and B. Whole cell images during cytokinesis. C-F. A part of cleavage furrows. G-J. Duplication of Golgi apparatus. K-N. Serial sections of nucleus. A. Completion of chloroplast duplication before initiation of cytokinesis. A single nucleus and Golgi apparatus are observed. B. Initial phase of cytokinesis. Arrows indicate the both edges of the cleavage furrows. Interestingly, nucleus is invaginated and seemly bisected by the cleavage furrow. C. Vesicles in the cleavage furrow. Front portion of the cleavage furrow is indicated by arrow. D. Magnified image of C. Vesicular membrane is similar to the new plasma membrane. E. Cleavage furrow in cryo-fixed cell. Front portion is indicated by arrow. F. Magnified image of the front portion of the cleavage furrow in E. Electron-dense materials are localized under the membrane of cleavage furrow (double arrowheads). G. Magnified Golgi apparatus in A. H. Curved Golgi apparatus covering the protrusion of nucleus (arrowhead). I. Duplicated Golgi apparatuses located with both sides of nuclear protrusion. J. Cleavage furrow (arrow) separating each set of nucleus and Golgi apparatus. K. A large vesicle in mid part of nucleus (white arrowhead). It seemed that the two parts of the nucleus were connected only with a narrow portion between the furrow and the large vesicle. L. The large vesicle (white arrowhead) became unclear and the connecting portion extended. M. The large vesicle completely disappeared and furrow only passes through nucleus unilaterally (arrow). N. Nucleus maintaining both protruded and penetrated portions. Spindle apparatus and microtubules were not observed in any sections. c: chloroplast, G: Golgi apparatus, n: nucleus. Scale bar: 0.5 μm (A and B), 0.2 μm (C and E-N) and 20 nm (D).

Figure 2

Mitochondrial dynamics during cell division. A-D. Doubly transgenic lines expressing unCPS-YFP and CFP-H4. Green, blue and red fluorescence correspond to mitochondrion, nucleus and chloroplast, respectively. E and G. Mitochondrion and nucleus stained with MitoTracker Orange and DAPI. F and H. DIC images of same cells in E and G respectively. A. Branched mitochondrion is stretched to the tip of the cell along the chloroplast. B. Two spots of mitochondrial signals beside the chloroplast constrictions (arrowheads). C. Extended mitochondria located in the future dividing plane prior to karyokinesis. D. Two identical cells with elongated mitochondria just after cell division. E. Branched mitochondrion is extended throughout the cell. DIC image of the cell is shown in F. G. Signals from mitochondria accumulate between two daughter nuclei (arrowheads). DIC image is shown in H. Scale bar: 1 μm.

Figure 3

Actin dynamics throughout the cell cycle. A-G. F-actin, nucleus and chloroplast were visualized with FITC-phalloidin (green), DAPI (blue) and chlorophyll autofluorescence (red), respectively. A. Interphase cell surrounded by actin bundle under the plasma membrane. B. Actin ring appeared after the duplication of chloroplast. C. Contracted actin ring with fibrous actin in the cell. D. Smaller contractile ring after karyokinesis. The ring positioned between two daughter nuclei. E. Oblique view of the cell at a similar stage as in D. Actin fibers connected with the contractile ring. F. Final stage of cytokinesis. Actin accumulated in the cytokinetic face. G. Completion of cell division. Two daughter cells were surrounded by actin bundles. H. Bright field image in same frame with G. Cell walls for both daughter cells had been completely generated. Scale bar: 1 μm.

Figure 4

Neighbor-joining (NJ) trees of Rab family (A) and other small GTPases (B). The 19 small GTPases from P. tricornutum were aligned with selected small GTPases from the centric diatom Thalassiosira pseudonana (Tp), the oomycete Phytophthora sojae (Ps) and, as a reference, yeast Saccharomyces cerevisiae (Sc). Detailed information about sequences used here is listed in Supplementary TableS1. Numbers at branch points indicate bootstrap percentages from NJ analyses with 1000 replicates. Only values greater than 60% are shown. ScSec4 was clustered with Pt_30139 and this monophyletic clade was supported with significant bootstrap value. Diatom T. pseudonana and oomycete P. sojae were almost always clustered together with P. tricornutum as they all belonged to Heterokonta. It is noteworthy that there are three unknown monophyletic clades, which are completely independent from the others. These could be Heterokont-specific Rab family small GTPases.

Figure 5

Neighbor-joining (NJ) tree of Syntaxins. Each phylum forms a monophyletic clade. The 7 Syntaxins from P. tricornutum were aligned with selected Syntaxins from the centric diatom Thalassiosira pseudonana (Tp), the oomycete Phytophthora sojae (Ps) and, as references, yeast Saccharomyces cerevisiae (Sc), the plants Arabidopsis thaliana (At) and Oryza sativa (Os), and Homo sapiens (Hs). Pt_19932 was clustered with KNOLLE, Sso, Syt1 and Syt2 with significant bootstrap support. There was also one monophyletic clade consisting of only diatom sequences and this might correspond to a diatom-specific Syntaxin.

Figure 6

Dynamics of Sec4 distribution. A-L. Images from cells transformed with SEC4-YFP/CFP-H4 constructs. Sec4 (green) and H4 (blue) are displayed together with red chlorophyll autofluorescence. M and N. Micrographs of wild type cell. A: Interphase cell. The Sec4 core signals are presumably in the Golgi apparatus (based on similarity of morphology compared with TEM images). B-L. Time-lapse images for 6 hours and 48 minutes. Numbers in each image indicate the passage of time. Nuclear division occurred in G-I. Two daughter nuclei then started to move separately (J-L) and cell division was completed at the end. M. Ultrastructures just after the duplication of chloroplast and Golgi apparatus. Two Golgi apparatuses secreted a lot of vesicles to the future dividing plane (arrows). N. Magnified image of arrowed part of M. Scale bar: 2 μm (A-L) and 0.2 μm (M and N).

Figure 7

Localization of SyntaxinA during cell cycle. SytA signals (green) and chlorophyll autofluorescence (red) are shown here. A. SytA signals localized to cytoplasmic periphery of the cell. B. The signals began to accumulate in both tips of the cell following chloroplast duplication. C. The accumulation of SytA-YFP fluorescence extended to the center of the cell. D. The extended signals from both tips ultimately combined to make a contiguous line in the future dividing plane. Scale bar: 1 μm.