Re-expression of ABP-120 rescues cytoskeletal, motility, and phagocytosis defects of ABP-120- Dictyostelium mutants.
The actin binding protein ABP-120 has been proposed to cross-link actin filaments in nascent pseudopods, in a step required for normal pseudopod extension in motile Dictyostelium amoebae. To test this hypothesis, cell lines that lack ABP-120 were created independently either by chemical mutagenesis or homologous recombination. Different phenotypes were reported in these two studies. The chemical mutant shows only a subtle defect in actin cross-linking, while the homologous recombinant mutants show profound defects in actin cross-linking, cytoskeletal structure, pseudopod number and size, cell motility and chemotaxis and, as shown here, phagocytosis. To resolve the controversy as to what the ABP-120- phenotype is, ABP-120 was re-expressed in an ABP-120- cell line created by homologous recombination. Two independently "rescued" cell lines that express wild-type levels of ABP-120 were analyzed. In both rescued cell lines, actin incorporation into the cytoskeleton, pseudopod formation, cell morphology, instantaneous velocity, phagocytosis, and chemotaxis were restored to wild-type levels. There is no alteration in the expression levels of several related actin binding proteins in either the original ABP-120- cell line or in the rescued cell lines, leading to the conclusion that neither the aberrant phenotype observed in ABP-120- cells nor the normal phenotype reasserted in rescued cells can be attributed to alterations in the levels of other abundant and related actin binding proteins. Re-expression of ABP-120 in ABP-120- cells reestablishes normal structural and behavioral parameters, demonstrating that the severity and properties of the structural and behavioral defects of ABP-120- cell lines produced by homologous recombination are the direct result of the absence of ABP-120.
Cox
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Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Wessels
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D
Soll
D R
DR
Hartwig
J
J
Condeelis
J
J
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D048429:Cell Size / Q000235:genetics / Q000502:physiology; D002633:Chemotaxis / Q000187:drug effects; D000242:Cyclic AMP / Q000494:pharmacology; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D004023:Dictyostelium / Q000235:genetics* / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D008840:Microfilament Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D010587:Phagocytosis / Q000235:genetics / Q000502:physiology; D010873:Pinocytosis / Q000235:genetics / Q000502:physiology; D011554:Pseudopodia / Q000235:genetics / Q000502:physiology*; D014170:Transformation, Genetic
D000199:Actins; D002352:Carrier Proteins; D008840:Microfilament Proteins; C060680:abpC protein, Dictyostelium; D000242:Cyclic AMP