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Rap1 overexpression reveals that activated RasD induces separable defects during Dictyostelium development
S A Louis1, G Weeks, G B Spiegelman
1Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
Developmental Biology
|November 5, 1997
Summary
Overexpression of Dictyostelium rasD (RasD[G12T]) causes developmental defects, but expressing rap1 or its activated mutant Rap1[G12V] partially rescues these issues, specifically the differentiation block. This suggests RasD[G12T] has two distinct effects, with rap1 only reversing the later one.
Area of Science:
- Cellular and developmental biology
- Molecular genetics
- Biochemistry
Background:
- The Dictyostelium rasD gene is crucial for cellular development, with specific mutations causing aberrant phenotypes.
- RasD[G12T] overexpression leads to multitipped aggregates and developmental arrest.
- The Dictyostelium rap1 gene's role in suppressing rasD-induced developmental defects is unknown.
Purpose of the Study:
- To investigate the suppressive ability of the Dictyostelium rap1 gene on the RasD[G12T]-induced developmental phenotype.
- To determine if activated or dominant-negative rap1 mutants can alter the aberrant development caused by RasD[G12T].
- To elucidate the specific effects of RasD[G12T] and the role of rap1 in Dictyostelium cellular differentiation.
Main Methods:
- Constructing rap1 and mutant rap1 genes (Rap1[G12V], Rap1[G10V]) linked to the rasD promoter.
- Transforming Dictyostelium M1 cells (expressing RasD[G12T]) with these constructs.
- Analyzing developmental phenotypes, including aggregate formation and fruiting body development.
- Assessing cell-type-specific gene expression (ecmA, tagB, cotC, car1) in transformed cell lines.
Main Results:
- Rap1 or Rap1[G12V] expression in M1 cells partially rescued the developmental block, forming 'multitipped escape' (ME) cells that completed development.
- Rap1[G10V] did not alter the M1 phenotype.
- ME cells still exhibited defects in low-density monolayer differentiation, indicating a cell-autonomous defect was not corrected.
- Rap1/Rap1[G12V] expression restored ecmA mRNA levels and reduced tagB mRNA levels in ME cells, suggesting reversal of the later differentiation defect.
Conclusions:
- RasD[G12T] overexpression induces two temporally separated defects: aggregate formation and a subsequent differentiation block.
- Rap1 and Rap1[G12V] expression specifically reverse the differentiation block, not the initial aggregate defect.
- The cell-autonomous defect in monolayer differentiation remains uncorrected in ME cells.
- These findings highlight distinct roles for RasD and Rap1 in regulating Dictyostelium development.