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First published online 6 January 2009
doi: 10.1242/jcs.036822

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Regulation of Rap1 activity is required for differential adhesion, cell-type patterning and morphogenesis in Dictyostelium

¹ Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK
² Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada
³ MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

View larger version (49K): [in this window] [in a new window]   Fig. 1. Identification of a Dictyostelium RapGAP. (A) Alignment of the potential RapGAP domain of Dictyostelium RapGAPB (Dicty) with RapGAPs from human (human), Drosophila (Drome) and C. elegans (Cele). Identical residues are highlighted in dark grey and conserved residues in light grey. Dictyostelium RapGAPB shows approximately 20% conservation with the human RapGAP1 sequence. (B) Structure of the Dictyostelium rapGAPB gene to illustrate the position of the insertion cassette. Numbers indicate base pairs, grey area encodes the predicted rapGAP domain.

View larger version (46K): [in this window] [in a new window]   Fig. 2. RapGAPB is required to regulate Rap1 activity. (A) Western blot showing levels of activated Rap1 (top panel) and total Rap1 (bottom panel). In wild-type cells, maximal increase in levels of active Rap1 was observed after 10 seconds of cAMP stimulation, followed by a steady decrease over 60 seconds of stimulation. In rapGAPB– cells, the maximal increase after 10 seconds of stimulation is slightly higher than that of wild-type cells. In addition, the level of active Rap1 does not decrease over 60 seconds, instead the maximal level is maintained. The level of total Rap1 is equal in all cell lysates. The graph shows fold change in amount of GTP-Rap1 relative to AX4 cells at 0 seconds. (B) Cell-substrate adhesion was measured by counting the number of vegetative cells that detached from a plate after shaking for 20 minutes. Approximately 60% of wild-type cells (AX4 and DH1) detached. Significantly fewer rapGAPB– and gbpDOE cells detached. By contrast, more rapGAPBOE and gbpD– cells detached. (C) Cells were stained with FITC conjugated phalloidin in order to observe F-actin distribution and viewed by deconvolution microscopy. rapGAPB– and gbpDOE cells exhibit a clearly different F-actin distribution and cell morphology compared with wild-type AX4 cells, whereas the rapGAPBOE cells appeared more like the wild type. Scale bar: 10 µm.

View larger version (62K): [in this window] [in a new window]   Fig. 3. RapGAPB expression is required for developmentally regulated Rap1 activity and for normal morphogenesis. (A) Developmental regulation of activated Rap1 levels. In wild-type cells, levels of GTP-Rap1 peak after 12 hours of development before decreasing. In rapGAPB– cells, GTP-Rap1 levels are higher at all time points and do not decrease during later developmental stages. The graph shows relative amounts of GTP-Rap1 compared with levels in AX4 cells at time 0. (B) Development of wild-type AX4, rapGAPB– and gbpDOE cells. Developing rapGAPB– and gbpDOE cells both formed streams that broke up, resulting in many small mounds. rapGAPB– and gbpDOE cells formed abnormal tip mound structures. The development of rapGAPB– cells temporarily halted at the mound stage before eventually forming small culminants. The small mounds formed by the gbpDOE cells made small slugs that culminated normally to form culminants. Scale bar: 2 mm. (C) Cell-cell adhesion of streaming cells was measured by disaggregating cells and counting the number of cells that reaggregated at different time points. rapGAPB– and gbpDOE cells are less adhesive because fewer cells reaggregated at all time points. (D) cAMP-induced activation of Rap1 in 12 hour developed cells. Western blot shows levels of activated Rap1 (top panel) and total Rap1 (bottom panel). In wild-type cells, levels of active Rap1 peaked after 10 seconds of cAMP stimulation, followed by a steady decrease. In rapGAPB– cells, the level in unstimulated cells was similar to the maximal level in wild-type cells at all time points. Graph shows relative levels of GTP-Rap1 compared with that in AX4 cells at 0 seconds. (E) To determine the expression pattern of rapGAPB, the rapGAPB promoter was placed upstream of lacZ and transformed into wild-type cells. When stained for a short time, expression is enriched in the collar region of slugs (i) and in the upper and lower cups of culminants (iii). After longer periods of staining, stained cells were predominantly found in the collar region, but also found scattered throughout the entire slug (ii) and in the apical tip, upper and lower cups and spores of culminants (iv). Scale bar: 1 mm.

View larger version (115K): [in this window] [in a new window]   Fig. 4. PrestalkA and prestalkO markers are misexpressed in the rapGAPB– mutant. AX4 or rapGAPB– cells were transformed with cell-specific reporter genes. Expression was examined at both slug and early culminant stages. Clear differences were found in the expression of both ecmA-lacZ and ecmO-lacZ. In developing AX4 cells, ecmA-lacZ expression was found mainly in the front of the prestalk region at the slug stage and in the apical tip and stalk of culminants. By contrast, in developing rapGAPB– cells, ecmA-lacZ expression was found in the tip and collar (filled arrowhead) of the equivalent slug stage and mainly in the upper cups (filled arrow) of culminants, with less staining in the apical tip. Dotted line shows stalk tube. In developing AX4 cells, ecmO-lacZ expression was found mainly in the collar region at the slug stage and in the upper cup of culminants. By contrast, in developing rapGAPB– cells, ecmO-lacZ expression was strongly expressed in the tip (open arrowhead) of the equivalent slug stage and mainly in the apical tip (open arrow) and stalk (open arrow) of culminants. Scale bars: 1 mm.

View larger version (68K): [in this window] [in a new window]   Fig. 5. Prestalk A and prestalk O cells are intermingled in the rapGAPB– mutant. AX4 and rapGAPB– cells expressing both ecmO-GFP and ecmAO-RFP were developed to the slug and early culminant stages. In developing AX4 cells, ecmO-GFP expression (green) was found mainly in the collar region of slugs and was mostly absent from the tip (arrowed). ecmAO-RFP expression (red) was found throughout the prestalk region of slugs. As a result, the merged image shows orange-yellow cells only in the collar (arrowed in the magnified image) and only red cells in tips of slugs. By contrast, in developing rapGAPB– cells, ecmO-GFP-expressing cells were found in both the collar and tip regions of the slug (arrowed) and ecmAO-RFP expression was found throughout the entire prestalk region. The resulting merged image shows orange-yellow cells in both the collar and tip of the slug (arrowed on the magnified image). A similar defect was observed at the early culminant stage. In AX4 culminants, ecmO-GFP expression was highest in the upper cup and largely absent from the apical tip (arrowed), whereas ecmAO-RFP expression was found in both the collar and apical tip. The merged image shows orange-yellow cells in only the upper cup (arrowed on the magnified image) and only red cells in the apical tip. However, in rapGAPB– culminants, ecmO-GFP and ecmAO-RFP expression were both found in the upper cup and apical tip (arrowed). The merged image shows orange-yellow cells in both the upper cup and apical tip (arrowed in the magnified image). Scale bars: 1 mm and 0.3 mm (zoom panels).

View larger version (78K): [in this window] [in a new window]   Fig. 6. RapGAPB is required for prestalk cell patterning in chimeric development. (A) 10% GFP-labeled cells were mixed in chimera with 90% unlabeled cells and allowed to develop. In control homotypic mixes, GFP-labeled cells were evenly distributed throughout the slug and culminant. However, when GFP-labeled rapGAPB– cells were mixed with AX4 cells, the labeled cells were absent from the tip of slugs and from the upper and lower cups and stalk of culminants. The opposite pattern was observed when GFP labeled AX4 cells were mixed with rapGAPB– cells. (B) When GFP-labeled ecmAO-gbpDOE or ecmAO-rapAG12V cells were mixed with AX4 cells, the labeled cells were absent from the prestalk region of slugs and from the upper and lower cups and stalk of culminants. Scale bars: 1 mm.

View larger version (115K): [in this window] [in a new window]   Fig. 7. rapGAPB– mutant cells show defects in ecmAO-lacZ, ecmA-lacZ and ecmO-lacZ expression in chimeras. AX4 or rapGAPB– cells were transformed with cell-specific markers. 10% of cells expressing these markers were mixed in chimeras with 90% unlabeled cells and developed. Expression of the markers was observed at both the slug and early culminant stages of development. Clear defects were observed in the expression of ecmAO-lacZ, ecmA-lacZ and ecmO-lacZ in chimeras. When AX4 cells expressing ecmAO-lacZ were mixed with AX4 cells, ecmAO expression was found in the entire prestalk region of slugs and in the apical tip, upper cup and lower cup of culminants. However, when rapGAPB– cells expressing ecmAO-lacZ were mixed with AX4 cells, ecmAO expression was absent from the tip of slugs and only present in the collar region. In addition, in the culminant expression was absent from the apical tip. When AX4 cells expressing ecmA-lacZ were mixed with AX4 cells, ecmA expression was found in tip of slugs and in the apical tip and stalk of culminants. However, when rapGAPB– cells expressing ecmA-lacZ were mixed with AX4 cells, ecmA expression was absent from the tip of slugs and instead present in the collar region. In addition, expression was absent from the apical tip and found instead in the upper and lower cups of culminants. When AX4 cells expressing ecmO-lacZ were mixed with AX4 cells, ecmO expression was found in the collar region of slugs and in the upper cup and lower cups of culminants. However, when rapGAPB– cells expressing ecmO-lacZ were mixed with AX4 cells, ecmO expression was largely found at the rear of the slug and in the slime trail (open arrow). In addition, expressing cells were found mainly in the basal disk of the culminant and scattered on the agar (filled arrows). Scale bar: 1 mm.

View larger version (14K): [in this window] [in a new window]   Fig. 8. rapGAPB– mutant cells show defects in prestalk and prespore cell-cell adhesion at the finger stage of development. Structures were disaggregated and the percentage of cells that aggregated after different times counted. (A) Cell-cell adhesion of wild-type (dotted line) and rapGAPB– cells (solid line). (B) Measurement of overall cell-cell adhesion. rapGAPB– cells are more adhesive than the wild type. Measurement of prespore and prestalk cell adhesion (psA-RFP or ecmAO-RFP expressing cells, respectively). rapGAPB– prespore cells are more adhesive than wild-type prespore cells and rapGAPB– prestalk cells are less adhesive than wild-type prestalk cells. As a consequence rapGAPB– prestalk and prespore cells show similar adhesion, whereas wild-type prestalk cells are more adhesive than wild-type prespore cells.

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