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The different components of a multisubunit cell number-counting factor have both unique and overlapping functions

¹ Howard Hughes Medical Institute and
² Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 South Main Street, Houston, TX 77005-1892, USA

View larger version (37K): [in a new window]   Fig. 1. Sequence of the cf50 gene. The sequence starts with genomic DNA; the 5' end of known cDNA sequence starts at nt 28 and continues to 83 nt past the stop codon, and is an exact match to the genomic sequence. The first ATG is at nucleotide 25. The N-terminal amino acid sequence obtained from the purified 50 kDa protein is indicated with a heavy underline. Two potential N-linked glycosylation sites (Alexander, 1997) are indicated by shaded boxes. Vertical arrows mark the beginning and end of the sequence expressed in bacteria and then used to immunize rabbits for antibody production. An arrow pointing to the left over nucleotide 260 marks the 3' end of the region that was replaced with a blasticidin resistance cassette to make a gene disruption transformant; the 5' end of the region that was replaced was 681 bp upstream of the first ATG. A double underline indicates a potential AAUAAA poly(A) addition signal. The GenBank accession number is AF405695.

View larger version (45K): [in a new window]   Fig. 2. cf50– cells do not have cf50 mRNA or CF50 protein. (A) A northern blot of RNA from vegetative cells was probed with a fragment of the cf50 cDNA. A 0.9 kB band (arrow) is present in Ax2 wild-type parental cells but not in the cf50– cells. Aliquots of the samples were electrophoresed on separate gels and stained with ethidium bromide to verify equal loading and that the ribosomal bands were not degraded. (B) A western blot of vegetative cells was stained with anti-CF50 antibodies. A 50 kDa band is present in Ax2, countin– and smlA– cells, but is absent from cf50– cells. (C) A western blot of conditioned starvation media from the above cell lines was stained with anti-CF50 antibodies. A 50 kDa band is present in Ax2, countin– and smlA– CMs, but is absent from cf50– CM. (D) A similar blot stained with anti-countin antibodies, showing countin present in the wild-type and smlA– CMs, absent from the countin– CM and present at low levels with what appears to be a lower molecular mass band in the cf50– CM. (E) A blot of conditioned HL5 growth media showing that CF50 is secreted from growing Ax2, countin– and smlA– cells, but not from cf50– cells. (F) A similar blot of conditioned HL5 growth media stained with anti-countin antibodies. For B-F, SDS-polyacrylamide gels of aliquots of the samples used for the western blots were stained with Coomassie Blue to verify that equal amounts of protein were loaded for each blot.

View larger version (29K): [in a new window]   Fig. 3. Developing cf50– cells form unusually large structures. Cells were grown on bacteria on agar plates, and as the cells consumed the bacteria they starved and formed fruiting bodies. Slices of agar with fruiting bodies or aggregates were tilted and photographed. The left panel shows the parental Ax2 cells; the center and right panels show examples of the structures formed by cf50– cells. Bar is 1 mm.

View larger version (54K): [in a new window]   Fig. 4. Starvation of cf50– cells in the presence of smlA– conditioned starvation medium (CM) causes a decrease in group size. Cells were starved on filters sitting on pads soaked with either buffer or CM from smlA– cells. Bar is 1 mm.

View larger version (18K): [in a new window]   Fig. 5. Addition of recombinant CF50 causes an increase in group number. Cells were starved on filters sitting on pads soaked with the indicated concentration of recombinant CF50. The number of groups the streams broke up into was then counted 18 hours later. Values are means ± s.e.m. from at least 3 separate experiments.

View larger version (61K): [in a new window]   Fig. 6. cf50 mRNA and CF50 protein are present in growing and developing cells. Ax2 wild-type cells were starved on filters, and samples were harvested at the indicated times (in hours) after starvation; V indicates vegetative cells. (A) A northern blot of mRNA extracted from cells; (B) a western blot of whole cells stained with anti-CF50 antibodies.

View larger version (67K): [in a new window]   Fig. 7. Distribution of CF50 in cells. (A) Cells of the indicated type were starved on filters for 6 hours, harvested, allowed to settle on glass slides, fixed, and stained for CF50 by immunofluorescence and observed by conventional immunofluorescence microscopy. (B) Wild-type cells were further examined by deconvolution fluorescence microscopy. Bar in A is 20 µm and bar in B is 5 µm.

View larger version (52K): [in a new window]   Fig. 8. CF50 is present in an 450 kDa complex in CM. CM was prepared from Ax2 and smlA– cells, and concentrated 10-fold. (A) The material was size-fractionated on a sieving gel column, and the fractions were assayed for CF50 by western blotting. The 50 kDa CF50 band from both wild-type and smlA– CM elutes at approximately 450 kDa; a 30 kDa band eluting below 66 kDa is also present in the smlA– CM. The elution position of size markers is shown at the bottom. (B) CM from Ax2 and cf50– cells was similarly size fractionated and the fractions were assayed for countin by western blotting. As previously observed, countin in Ax2 CM elutes at 450 kDa (Brock and Gomer, 1999), while the countin in cf50– CM elutes primarily at a lower molecular weight. The position of size markers is the same as in A.

View larger version (11K): [in a new window]   Fig. 9. Developing cf50– cells move more slowly than parental Ax2 cells. Cells were starved in submerged culture for 6 hours, and then fields of cells were observed by time-lapse videomicroscopy. For each cell, the approximate center of the cell was tracked for 5 minutes, and the cell speeds were then binned in 1 µm/minute increments. The average speeds were 3.3±0.2 µm/minute for Ax2 and 2.3±0.1 µm/minute for cf50– (means ± s.e.m. for 60 cells each, combining the observations from 20 cells in three separate experiments).