IFT Proteins Accumulate during Cell Division and Localize to the Cleavage Furrow in Chlamydomonas

Intraflagellar transport (IFT) proteins are well established as conserved mediators of flagellum/cilium assembly and disassembly. However, data has begun to accumulate in support of IFT protein involvement in other processes elsewhere in the cell. Here, we used synchronous cultures of Chlamydomonas to investigate the temporal patterns of accumulation and localization of IFT proteins during the cell cycle. Their mRNAs showed periodic expression that peaked during S and M phase (S/M). Unlike most proteins that are synthesized continuously during G1 phase, IFT27 and IFT46 levels were found to increase only during S/M phase. During cell division, IFT27, IFT46, IFT72, and IFT139 re-localized from the flagella and basal bodies to the cleavage furrow. IFT27 was further shown to be associated with membrane vesicles in this region. This localization pattern suggests a role for IFT in cell division

Cell cycle regulation of IFT proteins.

<p>(A) Top panel is Coomassie Blue (CB) stained gel of total protein from indicated times. Equal protein was loaded in each lane. The last lane contains molecular weight markers. Bottom panels show Western blots from the same set of samples using indicated antisera. (B) Top panel is Coomassie Blue stained gel loaded with equal cell number per lane. The amount loaded was not recalibrated after mitosis and represents equal mother cell equivalents. Bottom panels show Western blots from the same set of samples using indicated antisera. (C,D) Quantitation of Western signals from (A,B) respectively. CB staining was used to normalize in (C), while HSP70B signal was used to normalize in (D). Normalized data were plotted relative to the signal at 0 hrs that was set to a value of 1. (E) Fraction of flagellated cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030729#pone-0030729-g001" target="_blank">Fig. 1B</a>.</p

Cell cycle synchrony and flagella dynamics.

<p>(A) Top, schematic of cell size and cell cycle progression during G1, S phase and mitosis (S/M), and post-mitosis G0, in phototrophic cultures grown in an alternating light-dark cycle whose phases are shown by the shaded bars. Cells grow during the light phase, resorb flagella, and then divide up to three times in succession to produce daughters that regrow flagella. Bottom, graph of progression through successive cell divisions as assessed by mother cells that completed their first, second and third rounds of division. S/M is marked by light blue shading in this graph and all subsequent graphs. Total cell mass increased by about six-fold during G1 and total cell number increased by about six-fold during S/M. (B) Graph showing fraction of cells with flagella (n = 200). (C) Graph of average flagella length (n = 200) with standard deviation versus average cell size with standard deviation (n≥5000). Cell growth ceases in the dark and was not determined after 12 hours. See also Supporting Text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030729#pone.0030729.s001" target="_blank">Fig. S1</a>.</p

TEM immunogold labeling of IFT27 during cleavage furrow formation.

<p>(A). A representative TEM micrograph of a forming cleavage furrow (CF) is shown at a magnification of 12,000×. A nucleus is marked (N) near the top, and numerous membrane vesicles are observed surrounding the furrow. The dark stained structures are starch granules. A scale bar representing 1 µm is placed at the lower left. (B) A region from the same section as A is shown at higher magnification (80,000×) so that IFT27-specific, 12 nm gold particles are visible. Five particles appear in this image and are marked by black arrows (the arrow at upper right points to two gold particles). Each 12 nm particle is directly adjacent to a membrane surface. Smaller 6 nm gold particles seen here are specific for α-tubulin. A scale bar representing 100 nm is placed at the lower left. (C) The total number of gold particles found and photographed in seven individual cell sections, labelled with IFT27-specific antibodies, were counted and categorized by their subcellular location within the cytoplasmic region around the furrow, nuclei, chloroplasts, or cell walls. Shown in the upper table is the total area of each of these regions and the number of gold particles found there per square micron. The lower table shows the results of a control experiment in which the process was repeated for an additional seven cell sections that were subjected to labeling in the absence of primary antibody. (D) The distance between each of the gold particles found in the cytoplasmic region, as quantified in the upper table in C, and the nearest cytoplasmic vesicle or plasma membrane surface was measured and displayed here as a distribution.</p

Location of three IFT27, IFT46 and IFT72 during cleavage furrow formation.

<p>(A,E,I) DIC micrographs of three different dividing Chlamydomonas cells at the time of first cleavage furrow formation. (B,F,J) The immunofluorescence locations of α-tubulin (red), DAPI stained DNA (blue), and the indicated IFT protein (green) observed by widefield microscopy. (B,C) IFT27. (F,G) IFT72. (J,K) IFT46. (D,H,L) 3D reconstructions of optical sections obtained by laser scanning confocal microscopy of three additional dividing Chlamydomonas cells that were subjected to indirect immunofluorescence labeling at the time of cleavage furrow formation. Appearing in red is a reconstruction of each cell's microtubule cytoskeleton shown in side-view at the upper left and right of each panel. The locations of the cleavage furrows (cf) are marked by dotted lines. The 3D reconstructions are rotated about the y-axis giving a view directly into the cleavage furrows in the images shown at the bottom of each panel. The immunofluorescence signals corresponding to IFT27, IFT46 and IFT72 are included and shown as green to the right in panels D, H and L respectively. All three IFT proteins are clustered at the cleavage furrow.</p

Location of IFT27 during interphase.

<p>(A) Representative interphase cell imaged with DIC microscropy. (B) The cell in panel A was subjected to widefield indirect immunofluorescence microscopy with antibodies specific for IFT27 (green) and α-tubulin (red). The nuclear and chloroplast DNA are stained with DAPI (blue). (C) Merged image from panels A and B. Inset panels 1–3 display overlays of immunofluorescence and DAPI signals at the basal body regions of three different cells at three different angles of rotation with respect to the longitudinal axis of the cell body. The location of basal bodies (bb), an IFT train, and the upper edges of DAPI stained nuclei are marked by dotted lines. A prominent structure extending between the basal bodies and nuclei is revealed by IFT27 immunofluorescence in each cell.</p