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Analysis of Chlamydomonas SF-assemblin by GFP tagging and expression of antisense constructs

Botanisches Institut, Universität zu Köln, Germany

View larger version (88K): [in a new window]   Fig. 1. Analysis of Chlamydomonas cells expressing GFP-tagged SFA. (A) Gene structure of sfa (accession number AJ344366). The size and positions of the introns are indicated. The positions of the codons of the skip residues are marked by dots. Note the regular spacing of the introns. (B) Gene construct used for the expression of SFA-GFP. The arrow indicates the position of the start of translation. (C) Comparison of the SMAFs in control cells (control; anti-SFA staining) with the GFP signals observed in GFP3 and GFP8. Cells were fixed with methanol (-20°C) for 20 seconds. GFP3 expressed moderated amounts of SFA-GFP, forming cross-like structures similar to that observed in control cells by indirect immunofluorescence, whereas GFP8 cells overexpressed SFA-GFP, causing oversized fibers, when cultivated with a light/dark-cycle of 14/10 hours (GFP3-L and GFP8-L). Small fibers were present in GFP8 when cultivated in the dark for 2 days (GFP8-D). Bar, 10 µm. (D) Western blot of control (C) and GFP8 cells. The membrane strip on the left was stained with amidoblack and the positions of the standard proteins (Mr) are indicated on the left. Anti-SFA reacted with a band of 34 kDa in control and GFP8 cells, and a band of approximately 60 kDa in GFP8. (E) Details of strain GFP8. (a) Frequently one (indicated by arrowheads) of the four SFA-GFP fibers was much longer than the other three. (b,c) GFP signal (b) or indirect immunofluorescence with anti-SFA (c) of a GFP8 cell with a gap in one of the SFA-GFP fibers. Bars, 5 µm.

View larger version (62K): [in a new window]   Fig. 2. Analysis of GFP8 cells with antibodies for acetylated tubulin. Top: isolated cytoskeleton of a control cell stained with polyclonal anti--tubulin and 6-11B-1 (acetylated tubulin), revealing the numerous microtubules of the cytoskeleton and the four acetylated flagellar roots. Bottom: GFP8 cytoskeletons stained with 6-11B-1 and corresponding GFP images, revealing that prominent SFA-GFP fibers were mostly associated with microtubular bundles characterized by short acetylated regions. The arrowheads mark microtubular roots acetylated over most of their length. Bar, 10 µm.

View larger version (96K): [in a new window]   Fig. 5. Observation of SFA-GFP in dividing GFP8 cells. Living cells: the recording time of each picture is indicated (MESZ hours in large numbers; minutes — and in c-e, also seconds — in small numbers). (a,b) Two examples of cells prior to the first division. (c-e) Cells during the first division. (e) Phase contrast images of the cell documented in d showing the four pulsing contractile vacuoles (arrowheads). Arrowhead in c: moving dot of SFA-GFP. Arrowheads in d: #1, rapidly moving dot; #2, slowly moving dot; #3, dot appearing from the back of the cell. Bars, 5 µm (c,d), 4 µm (a,b). Methanol-fixed cells: (a,c) SFA-GFP and corresponding anti-SFA signals (b,d) showing that SFA-GFP is underrepresented in two of the fibers (arrowhead in b) and in dividing cells (arrowheads in c). The differences documented in a/b are characteristic for cells before division and indicate a rapid removal of SFA-GFP from the axisforming fibers (arrowheads in b) along the two-stranded roots. Bars, 2 µm (a,b), 10 µm (c,d).

View larger version (151K): [in a new window]   Fig. 3. Analysis of GFP8 cells by standard and immunogold electron microscopy. (a-c) Postembedding immunogold electron microscopy. Sections were stained with anti-rabbit-IgG 10 nm gold and either anti-SFA (a) or anti-GFP (b and c, which are consecutive sections). Arrowheads in a and c show SMAFs, and arrowheads in b indicate the immunodecorated region between the two basal bodies at the origin of the SMAFs. (d-f) Three consecutive sections showing a basal apparatus with prominent SMAFs. Open arrows: microtubules originating on or near the SMAFs; arrows: two-stranded microtubular roots alongside the SMAFs; double arrowheads: new basal bodies. (g) Control cell showing new basal bodies (double arrowhead) and thin SMAFs (arrows), visible as electron-opaque bands along the microtubular roots. For better images of wild-type SMAFs see Goodenough and Weiss (Goodenough and Weiss, 1978). (h) Section through a GFP8 cell with prominent SMAF, secondary cytoskeletal microtubules (open arrow) and new basal body (double arrowhead). Bars, 1 µm.

View larger version (93K): [in a new window]   Fig. 4. The effect of heat shock on GFP8 and control cells. (A) A heat shock of 40°C was applied for 60 minutes. Cells were immobilized on slides and fixed by rinsing in -20°C methanol for 20 seconds. T0, cells before heat shock; T60, cells after heat shock. SFA-GFP fibers were largely depolymerized with the exception of a dot-like region near the basal bodies. T120, thin fibers extend from the brighter dot; T240, globular aggregates have formed, in addition to the cross-like structure. These aggregates were also visible in phase contrast (arrows) and were often located at the end of the developing SFA-GFP fibers (insert). T360, SFA-GFP fibers were reassembled almost completely. Bar, 10 µm. (B) In vivo observation of cells embedded in agarose during heat shock and recovery. Each picture represents an average of several images taken at six to seven different focus levels over a period of 5-6 seconds. For heat treatment the slide with the embedded cells was incubated on a temperature-controlled metal block. T0, embedded cells were still able to rotate, causing the SMAFs to appear several times on the averaged image. T70, cells have resorbed the flagella and the fibers were depolymerized. T130, T210, T290, repolymerization of SMAFs first visible as thread-like extensions from the stable core region. Bar, 10 µm. (C) Immunofluorescence analysis of heat-shocked control (1) and GFP8 cells (2, 3) using anti-SFA. (1) In control cells, SMAFs seem to be unaffected by heat shock. (2) GFP fluorescence and corresponding antibody staining (3) of a GFP8 cell after heat shock. The resulting signals show differences in shape and size, indicating an unequal distribution of wild-type and GFP-tagged protein. It should be noted that anti-SFA binds both SFA and SFA-GFP (compare also Fig. 5, bottom). Bar, 1 µm. (D) Northern blot of control and GFP8 cells using a partial cDNA of SFA as a probe. In GFP8 cells, several transcripts at and below 3.2 kb were detected 45 minutes after heat shock (T105), which were not present before (T0) or three hours after heat shock (T240), or in control cells. 24hD, RNA from cells cultivated in the dark for 24 hours. 30 µg of total RNA were loaded in each lane. The positions of RNA standard molecules are indicated. (E) Western blot analysis of GFP8 and control cells during heat shock using anti-SFA. Time points and sample type (whole cell, pellet or supernatant) are indicated above the bars and from each sample type similar amounts of protein were loaded. A heat shock of 60 minutes duration was applied from T0 to T60. Upper rows: in whole cells only minor variations in the amount of SFA and SFA-GFP were observed during heat shock. Middle rows: analysis of pellets and supernatants of GFP8 cells during heat shock. SFA-GFP was largely removed from the insoluble fraction after the heat shock. Note the difference in apparent molecular weight between soluble and insoluble SFA-GFP. Anti-centrin staining (BAS6.8) of the same samples is shown as a loading control. Lower rows: supernatant and pellet of control cells. Note the shift in molecular weight between soluble and insoluble forms of SFA. wc-c, whole cell control.

View larger version (52K): [in a new window]   Fig. 8. Effect of cycloheximide on temperature-shifted GFP8 cells. (A) Cells, cultivated at 15°C, were either left untreated (lane 1), heat shocked and incubated at the conditions indicated (lane 2-4), or shifted to 32°C without heat shock (lane 5). After 6 hours, cells were extracted with detergent and the insoluble fractions analyzed by western blotting with anti-SFA. The strong decrease in the amount of SFA-GFP observed after a shift from 15 to 32°C (compare lanes 1 and 5) was partially inhibited by cycloheximide (lane 4). Furthermore, cells treated with cycloheximide (lane 4) contained less wild-type SFA in comparison with controls (lane 3 or 5). (B) Cells, cultivated at 15°C (lane 1), were shifted to 32°C for 6 hours either with (+, lane 3) or without (-, lane 2) cycloheximide. After removal of the cycloheximide (+ in brackets, lane 4), we observed a strong increase in the amount of SFA-GFP, and especially SFA. Temperature and time points are indicated (temp./hours). Anti-SFA was used for detection of SFA and SFA-GFP and similar amounts of cytoskeletons were loaded in lanes 3 and 4. (C) Fluorescence images of GFP8 cells corresponding to the experiment documented in the western blot shown in B. (1) GFP8 cells from a 15°C culture after 6 hours at 32°C; (2) as 1 but two hours later and centrifuged as the sample with cyloheximide; (3) GFP8 cells from a 15°C culture after 6 hours at 32°C in the presence of cycloheximide; (4) as 3, but two hours after cycloheximide has been removed. The cells had assembled long SFA-GFP fibers and some cells contained additional globular aggregates (insert). Bars, 10 µm (overviews) or 5 µm (insert).

View larger version (42K): [in a new window]   Fig. 6. Analysis of cells expressing GFP-tagged modified SFAs. (A) Sequence of the C-terminal domain of SFA. Underlined residues are truncated in C18; the deletion in C6 is double underlined. Lower rows show the point mutations used to modify the penultimate serine. (B) Phase contrast and fluorescent image of strain C18-11 cells fixed with -20°C methanol. The GFP-tagged protein was concentrated near the basal bodies. Bar, 10 µm. (C) Western blot analysis of strains expressing truncated or mutated SFA molecules using anti-SFA. Upper panel: distribution of SFA, and GFP-tagged SFA or SFAC18 in control (C), GFP8 and C18-11 cells. Note the reduced amounts of SFA in GFP8 and 18-11. In the latter, most of the GFP-tagged protein remained in the supernatant, whereas full-length SFA-GFP was mostly insoluble. The amounts loaded in each lane corresponded to 3.6x105 cells (whole cell), 1.8x106 cells (pellets) and 3.3x105 cells (supernatants). Lower panel: comparison of pellets and supernatants from GFP8, SE12 and SA11. Note that most of the SFA-GFP-SE was soluble, whereas SFA-GFP-SA was highly insoluble. Similar amounts of pellets and supernatants were loaded. (D) Fluorescence images of strain SA11 cultivated at 15°C before and immediately after a heat shock of 60 minutes. Note the star-like arranged SFA fibers and the dotted signals after heat shock. Bar, 10 µm.

View larger version (41K): [in a new window]   Fig. 7. Effect of environmental temperature on the size and composition of the SMAFs in GFP8 cells. Cells, previously maintained at 15°C, were grown for 48 hours at the temperatures indicated. (A) GFP fluorescence images of cells grown at 15, 25 or 32°C. Bar, 5 µm. (B) Western blot analysis of control and GFP8 cells cultivated at 15, 25 or 32°C with anti-SFA. The amount of SFA-GFP decreased with rising temperatures, whereas slightly more SFA was detected in cells cultivated at 32°C. The amount of SFA in control cells remained almost constant under the conditions chosen. (C) Comparison of insoluble and soluble fractions from GFP8 cells cultivated at 25 or 32°C, revealing higher levels of SFA in the pellet at elevated temperature. Note the constant amount of SFA-GFP in the supernatant of GFP8 cells cultivated at 25 or 32°C. Anti-SFA was used to detect wild-type and GFP-tagged SFA. Similar amounts of whole cells, pellets and supernatants were loaded.

View larger version (51K): [in a new window]   Fig. 9. Characterization of Chlamydomonas cells transformed with pCB-AS1. (A) The construct used to reduce the expression of SFA. The solid bars represent introns of 71 bp at +4 and of 90 bp at +91; genomic and complementary DNA of SFA were joined 35 bp after the second intron. (B) Nothern blot documenting the expression of the antisense construct in AS5 and AS8. A transcript of 900 bp was detected in cells 45 minutes after a heat shock of 60 minutes (T105). The transcript was not detected before (T0) or 3 hours after heat shock (T240), or in a sample isolated from cells maintained in the dark for 24 hours (24hD). Equal amounts of total RNA (30 µg) were loaded as documented by the ethidium bromide-stained gel. (C) Western blots of control (C) cells and various AS-strains. Equal amounts of cytoskeletons isolated from AS8 and control cells were loaded and stained either with amidoblack (left membrane strip) or probed with anti-SFA (middle membrane strip). Right: membrane strip comparing SFA in insoluble fractions from AS3 and AS5 with that of control cells. Prolonged development of membrane strips visualized some residual SFA in the antisense strains (arrow). (D) AS8 cells permeabilized with -20°C methanol and stained with anti-tubulin (6-11B-1) and anti-SFA. Open arrowheads: uniflagellate cells; arrow: stronger SFA signal. Bar, 5 µm.

View larger version (115K): [in a new window]   Fig. 10. Expression of SFA-GFP in Chlamydomonas AS5 cells. (A) Phase contrast and fluorescent images of AS5-GFP25 and AS5-GFP34 cultivated at 25°C. The cells contained globular aggregates, in addition to a slightly elongated signal near the cell apex (arrows). Both signals were comparably weak. (B) Western blots stained with anti-SFA (top, bottom) or anti-GFP (middle) of whole cells, cytoskeletons, and supernatant of AS5-GFP34 cells. SFA-GFP was detected almost exclusively in the soluble fraction of AS5-GFP34 cells. Note the reduced amounts of wild-type SFA in GFP8 and especially AS5-GFP34 when compared with controls (bottom).