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Targeted disruption of the ABP-120 gene leads to cells with altered motility
D Cox1, J Condeelis, D Wessels
1Albert Einstein College of Medicine, Yeshiva University, Bronx, New York.
Abstract:
The actin-binding protein ABP-120 has been proposed to play a role in cross-linking F-actin filaments during pseudopod formation in motile Dictyostelium amebas. We have tested this hypothesis by analyzing the phenotype of mutant cell lines which do not produce ABP-120. Two different transformation vectors capable of targeted disruption of the ABP-120 gene locus have been constructed using a portion of an ABP-120 cDNA clone. Three independent cell lines with different disruption events have been obtained after transformation of amebas with these vectors. The disruption of the ABP-120 gene by vector sequences results in either the production of a small amount of truncated ABP-120 or no detectable protein at all. The phenotypes of two different clones lacking ABP-120, generated in strains AX3 and AX4, have been characterized and show identical results. ABP-120- cells tend to remain rounder before and after cAMP stimulation, and do not reextend pseudopods normally after rapid addition of cAMP. In addition, ABP-120- cells translocating in buffer exhibit defects in both the rate and extent of pseudopod formation. The amount of F-actin cross-linked into the cytoskeleton after cAMP stimulation of ABP-120- cells is reduced at times when ABP-120 has been shown to be incorporated into the cytoskeleton, and this correlates temporally with the absence of reextension of pseudopods after cAMP stimulation. The instantaneous velocity is significantly reduced both before and after cAMP stimulation in the ABP-120- cells, and the cells show decreased chemotactic efficiency compared to ABP-120+ controls. This phenotype is consistent with a role for ABP-120 in pseudopod extension by cross-linking actin filaments as proposed by the "cortical expansion model" (Condeelis, J., A. Bresnick, M. Demma, C. Dharmawardhane, R. Eddy, A. L. Hall, R. Sauterer, and V. Warren. 1990. Dev. Genet. 11:333-340).
Insights
Mutant Dictyostelium amebas lacking actin-binding protein ABP-120 show impaired pseudopod formation and reduced cell motility. This supports ABP-120's role in cross-linking F-actin for cell movement.
Area of Science:
- Cell Biology
- Biochemistry
- Genetics
Background:
- Actin-binding protein ABP-120 is hypothesized to cross-link F-actin filaments during pseudopod formation in Dictyostelium amebas.
- Understanding ABP-120's function is crucial for elucidating mechanisms of cell motility.
Purpose of the Study:
- To investigate the role of ABP-120 in pseudopod formation and cell motility in Dictyostelium.
- To analyze the phenotype of mutant cell lines lacking ABP-120.
Main Methods:
- Gene disruption using transformation vectors to create ABP-120- cell lines.
- Phenotypic characterization of ABP-120- cells, including pseudopod extension, cell shape, and F-actin cross-linking.
- Assessment of cell velocity and chemotactic efficiency.
Main Results:
- ABP-120- cells exhibited defects in pseudopod reextension after cAMP stimulation and reduced rate/extent of pseudopod formation during translocation.
- Reduced F-actin cross-linking was observed in ABP-120- cells, correlating with impaired pseudopod extension.
- Instantaneous velocity and chemotactic efficiency were significantly reduced in ABP-120- cells compared to controls.
Conclusions:
- The findings support the hypothesis that ABP-120 is essential for pseudopod extension by cross-linking actin filaments.
- ABP-120 plays a critical role in Dictyostelium cell motility and chemotaxis, consistent with the "cortical expansion model".