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First published online 22 February 2006
doi: 10.1242/dev.02287

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A retinoblastoma ortholog controls stalk/spore preference in Dictyostelium

¹ Biozentrum der Ludwig-Maximilians-Universität, Grosshadernerstrasse 2, 82152 Planegg-Martinsried, Germany.
² Centre for Structural and Functional Genomics, Concordia University Montreal, Quebec H4B 1R6, Canada.
³ Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Ospedale S.Luigi, 10043 Orbassano, Torino, Italy.

View larger version (74K): [in a new window]   Fig. 1. Structure of the Dictyostelium retinoblastoma ortholog rblA with Rb family members from other species. (A) Overview showing blocks of similarity. The gray rectangle N corresponds to the conserved N-terminal region, while blocks A and B are the RbA and RbB domains, respectively. The regions that interact with SP1 and Rb kinase in the human gene are indicated by thin lines. (B,C) Alignment of rblA with Rb proteins from the indicated organisms. Conserved residues are boxed, and identical shaded in gray. (B) The N-terminal region; (C) the RbA and RbB boxes, and the intervening spacer. Black circles indicate positions relevant to the folding of RbA and RbB domains; asterisks indicate amino acids interacting with the LXCXE peptides. (D) Comparison between rblA and human members of the pocket protein family. The scale is the same as in A. The sequences have been arbitrarily aligned at the N-terminal side of the B box.

View larger version (56K): [in a new window]   Fig. 2. Expression of rblA. (A) Northern blot showing expression beginning at 12 hours. (B) ß-Galactosidase activity in extracts of developing cells carrying the rblA::pgal reporter, detected using chemoluminescence. Two independent clones were measured. The gray curves (left scale) show vegetative activity and an increase beginning at 8-10 hours; the black curves (right scale) show an ultimate 200-fold upregulation. (C) Colony lift of growing clone of rblA::pgal cells, stained with X-gal and counterstained with Ponceau Red. The heavy red band (left) is the clone edge, made up of proliferating cells. Multicellular stages (slugs and fruiting bodies) are on the right. Expression is visible only at multicellular stages.

View larger version (113K): [in a new window]   Fig. 3. Expression pattern of the rblA::pgal during development. (A) In a tight mound (14 hours), the central core, which is occupied by stalk precursors, remains unstained. (B) In the slug stage (17 hours), staining is confined to the rear two-thirds; the anterior prestalk region is negative. (C) In a fruiting body (22 hours), staining is almost exclusively found in the future spore mass. For clarity, the prestalk region and the stalk have been outlined in B and C.

View larger version (27K): [in a new window]   Fig. 4. Nuclear DNA contents as determined using Dapi and image quantification. (A) Comparison of vegetative cells and spores of the strain Ax2. The mean values of 11.5 ±0.3 (spores) and 11.7 ±0.3 (cells) do not differ significantly. As vegetative cells are largely G2 (Weijer et al., 1984), it appears that the spores are also G2 under our conditions. (B) Nuclear DNA content of wild-type and A15::rblA cells, normalized to wild-type spores. A minor G2 population is present but the cells appear to be largely G1.

View larger version (122K): [in a new window]   Fig. 5. Pathway preference in rblA-null cells. (A-C) rblA-null cells were stained with Cell Tracker CMFDA and mixed with excess wild-type cells (proportions 20:80) and allowed to develop. The stained cells predominate in the stalk (A) and the basal disk (B). In the spore head (C), they are found in the upper cup (top); a few form spores (bottom). (D) RblA-null cells transformed with actin6::-ß-galactosidase reporter were mixed with wild-type cells in the same proportion; at the slug stage, the marked cells predominantly occupy the prestalk zone (left) and the rearguard zone (right).

View larger version (26K): [in a new window]   Fig. 6. Cell-cycle regulation of the rblA promoter. Wild-type cells bearing the rblA::pgal reporter were synchronized by cold shock. Over an 8-hour period, reporter activity was followed using chemoluminescence, nuclear DNA synthesis was monitored by BrdU pulse-labeling and cell number measured using a Coulter counter. Gal activities in RLU/µg protein and BrdU incorporation in percent of total nuclei were normalized to the respective average over the entire experiment.

View larger version (23K): [in a new window]   Fig. 7. Regulation of the rblA promoter by glucose and the growth curve. Stationary-phase wild-type cells bearing the rblA::pgal reporter were diluted into fresh normal or glucose-free medium; reporter activity and the cell concentration were followed until the cells again reached stationary phase. The promoter activity is higher in the presence of glucose; however, regardless of glucose, the activity is highest during the exponential phase.

View larger version (114K): [in a new window]   Fig. 8. Absence of pathway preference in rblA-null cells grown with and without glucose. The figures show chimeric slugs containing 95% unmarked, glucose-grown rblA-null cells and 5% gfp-marked rblA-null cells grown without glucose. The gfp-marked cells are distributed over the length of the slug, rather than being concentrated at the anterior ends (*), as would occur in the same experiment conducted with wild-type cells.

View larger version (19K): [in a new window]   Fig. 9. DIF sensitivity, measured by ecmB induction in wild-type and rblA-null cells. The curves show the averaged responses (mean+s.e.m.) in four experiments with wild-type cells and six experiments with rblA-null lines. rblA-null cells are approximately threefold more sensitive to DIF.

View larger version (22K): [in a new window]   Fig. 10. Distribution of cell sizes in wild-type and rblA-null cells. The curves are the averages of three independent experiments with the wild-type and six with rblA-null cells, three with each of the two rblA-null clones. Cells were grown adherently in bacteriological plates, and harvested at about 10% confluency after a minimum of 48 hours exponential growth.

View larger version (163K): [in a new window]   Fig. 11. Acceleration of development in rblA-null cells. (A) Aggregation streams formed by cells in growth medium. (B) At higher magnification, the cells show the elongate form typical of aggregating cells. (C) Development of wild-type cells, plus cells of the independent rblA-null clones 3a612 and 3d5 at 6, 11 and 15 hours. At 6 hours, the rblA-null cells appear similar to the wild-type cells at 11 hours.