Figure 1.
Cell cycle synchrony and flagella dynamics.
(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 Fig. S1.
Figure 2.
Cell cycle regulation of IFT mRNAs.
(A–F) Quantitative RT-PCR from samples taken at different time points during the cell cycle. Samples were normalized first to an internal control (GBLP/CBLP, Genbank ID X53574.1) and then relative to the maximum expression value that was set to 1. The curves for panels B and C are superimposed to show the phase shift in IFT27 expression compared with a message for a representative cell cycle regulatory gene, CYCB1.
Figure 3.
Cell cycle regulation of IFT proteins.
(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 Fig. 1B.
Figure 4.
Summary of IFT27 protein and mRNA synthesis during the cell cycle.
The relative concentrations of IFT27 protein (solid dark line) and IFT27 mRNA (thin dashed line) are plotted along with flagella length (solid gray line) and progression through S phase and mitosis (thick dashed line). IFT27 protein concentration drops continuously during G1 and is at its lowest just before division. IFT27 mRNA and protein are normally synthesized during S/M which resets its levels for the next cell cycle.
Figure 5.
Location of IFT27 during interphase.
(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.
Figure 6.
Location of three IFT27, IFT46 and IFT72 during cleavage furrow formation.
(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.
Figure 7.
The subcellular location of IFT27 and other IFT complex B proteins are summarized in a cartoon diagram. The left image depicts an interphase cell where IFT27 (green) is found concentrated principally at the basal bodies and on IFT trains in the flagella (microtubules are drawn in red). In addition, IFT27 is found on a structure located between the basal bodies and the nucleus (blue) and on diffuse puncta in the cytoplasm during interphase. The right image depicts a dividing cell at the time of cleavage furrow formation. Here, IFT27 and other IFT complex B proteins are no longer associated with the basal bodies (purple), and instead cluster along the nascent furrow.
Figure 8.
TEM immunogold labeling of IFT27 during cleavage furrow formation.
(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.