Title

Mutant Rac1B expression in Dictyostelium: effects on morphology, growth, endocytosis, development, and the actin cytoskeleton

UMMS Affiliation

Department of Cell Biology

Date

8-30-2000

Document Type

Article

Subjects

Actins; Animals; Cell Adhesion; Cell Division; Cell Membrane; Cell Movement; Cells, Cultured; Chemotaxis; Cytoskeleton; Dictyostelium; *Endocytosis; Humans; Mutagenesis, Site-Directed; Neuropeptides; Recombinant Fusion Proteins; Sequence Homology; Signal Transduction; rac GTP-Binding Proteins; rac1 GTP-Binding Protein

Disciplines

Cell Biology | Life Sciences | Medicine and Health Sciences

Abstract

Rac1 is a small G-protein in the Ras superfamily that has been implicated in the control of cell growth, adhesion, and the actin-based cytoskeleton. To investigate the role of Rac1 during motile processes, we have established Dictyostelium cell lines that conditionally overexpress epitope-tagged Dictyostelium discoideum wild-type Rac1B (DdRac1B) or a mutant DdRac1B protein. Expression of endogenous levels of myc- or GFP-tagged wild-type DdRac1B had minimal effect on cellular morphologies and behaviors. By contrast, expression of a constitutively active mutant (G12-->V or Q61-->L) or a dominant negative mutant (T17-->N) generated amoebae with characteristic cellular defects. The morphological appearance of actin-containing structures, intracellular levels of F-actin, and cellular responses to chemoattractant closely paralleled the amount of active DdRac1B, indicating a role in upregulating actin cytoskeletal activities. Expression of any of the three mutants inhibited cell growth and cytokinesis, and delayed multicellular development, suggesting that DdRac1B plays important regulatory role(s) during these processes. No significant effects were observed on binding or internalization of latex beads in suspension or on intracellular membrane trafficking. Cells expressing DdRac1B-G12V exhibited defects in fluid-phase endocytosis and the longest developmental delays; DdRac1B-Q61L produced the strongest cytokinesis defect; and DdRac1B-T17N generated intermediate phenotypes. These conditionally expressed DdRac1B proteins should facilitate the identification and characterization of the Rac1 signaling pathway in an organism that is amenable to both biochemical and molecular genetic manipulations.

Rights and Permissions

Citation: Cell Motil Cytoskeleton. 2000 Aug;46(4):285-304.

Related Resources

Link to article in PubMed

PubMed ID

10962483