We have previously demonstrated that the myosin essential light chain (ELC) is required for myosin function in a Dictyostelium cell line, 7–11, in which the expression of ELC was inhibited by antisense RNA overexpression. We have now disrupted the gene encoding the ELC (mlcE) in Dictyostelium by gene targeting. The mlcE- mutants provide a clean genetic background for phenotypic analysis and biochemical characterization by removing complications arising from the residual ELC present in 7–11 cells, as well as the possibility of mutations due to insertion of the antisense construct at multiple sites in the genome. The mlcE- mutants, when grown in suspension, exhibited the typical multinucleate phenotype observed in both myosin heavy chain mutants and 7–11 cells. This phenotype was rescued by introducing a construct that expressed the wild-type Dictyostelium ELC cDNA. Myosin purified from the mlcE- cells exhibited significant calcium ATPase activity, but the actin-activated ATPase activity was greatly reduced. The results obtained from the mlcE- mutants strengthen our previous conclusion based on the antisense cell line 7–11 that ELC is critical for myosin function. The proper localization of myosin in mlcE- cells suggests that its phenotypic defects primarily arise from defective contractile function of myosin rather than its mislocalization. The enzymatic defect of myosin in mlcE- cells also suggests a possible mechanism for the observed chemotactic defect of mlcE- cells. We have shown that while mlcE- cells were able to respond to chemoattractant with proper directionality, their rate of movement was reduced. During chemotaxis, proper directionality toward chemoattractant may depend primarily on proper localization of myosin, while efficient motility requires contractile function. In addition, we have analyzed the morphogenetic events during the development of mlcE- cells using lacZ reporter constructs expressed from cell type specific promoters. By analyzing the morphogenetic patterns of the two major cell types arising during Dictyostelium development, prespore and prestalk cells, we have shown that the localization of prespore cells is more susceptible to the loss of ELC than prestalk cells, although localization of both cell types is abnormal when developed in chimeras formed by mixing equal numbers of wild-type and mutant cells. These results suggest that the morphogenetic events during Dictyostelium development have different requirements for myosin.
Targeted disruption of the Dictyostelium myosin essential light chain gene produces cells defective in cytokinesis and morphogenesis
T.L. Chen, P.A. Kowalczyk, G. Ho, R.L. Chisholm; Targeted disruption of the Dictyostelium myosin essential light chain gene produces cells defective in cytokinesis and morphogenesis. J Cell Sci 1 October 1995; 108 (10): 3207–3218. doi: https://doi.org/10.1242/jcs.108.10.3207
Download citation file:
JCS Journal Meeting 2023: Imaging Cell Dynamics
Our 2023 Journal Meeting on ‘Imaging Cell Dynamics’ will be held from 14-17 May 2023 in Lisbon, Portugal. Due to popular demand, we can currently only accept applications for online attendance. Apply now to attend this meeting virtually. Registration deadline: 31 March.
Call for papers: Cell and Tissue Polarity
We are welcoming submissions for our next special issue, which will focus on ‘Cell and tissue polarity’ and will be guest edited by David Bryant. Submission deadline: 15 July.
Editorial: Publishing where it matters
Editor-in-Chief Michael Way outlines Journal of Cell Science’s plans for the upcoming year and introduces Seema Grewal as our new Executive Editor.
preLights 5th Birthday webinar
preLights, our preprint highlighting service, is celebrating its 5th birthday this year. To mark the occasion, join us online on 14 March 2023 at 16:00 GMT for a discussion, led by four preLights alumni, on how to identify and navigate the challenges and opportunities while shaping your career as an early-career researcher.
Cell Scientists to Watch
As a community-focused journal, Journal of Cell Science is keen to support the next generation of cell biologists. Check out Cell Scientists to Watch, our interview series featuring talented researchers who have recently set up their own labs.