Dictyostelium as model system for studies of the actin cytoskeleton by molecular genetics

Abstract

The actin cytoskeleton is an essential structure for most movements at the cellular and intracellular level. Whereas for contraction a muscle cell requires a rather static organisation of cytoskeletal proteins, cell motility of amoeboid cells relies on a tremendously dynamic turnover of filamentous networks in a matter of seconds and at distinct regions inside the cell. The best model system for studying cell motility is Dictyostelium discoideum. The cells live as single amoebae but can also start a developmental program that leads to multicellular stages and differentiation into simple types of tissues. Thus, cell motility can be studied on single cells and on cells in a tissue‐like aggregate. The ability to combine protein purification and biochemistry with fairly easy molecular genetics is a unique feature for investigation of the cytoskeleton and cell motility. The actin cytoskeleton in Dictyostelium harbours essentially all classes of actin‐binding proteins that have been found throughout eukaryotes. By conventional mutagenesis, gene disruption, antisense approaches, or gene replacements many genes that code for cytoskeletal proteins have been disrupted, and altered phenotypes in transformants that lacked one or more of those cytoskeletal proteins allowed solid conclusions about their in vivo function. In addition, tagging the proteins or selected domains with green fluorescent protein allows the monitoring of protein redistribution during cell movement. Gene tagging by restriction enzyme mediated integration of vectors and the ongoing international genome and cDNA sequencing projects offer the chance to understand the dynamics of the cytoskeleton by identification and functional characterisation of all proteins involved. Microsc. Res. Tech. 47:124–134, 1999. © 1999 Wiley‐Liss, Inc.

RNAi Silenced Dd-grp94 (Dictyostelium discoideum Glucose-Regulated Protein 94 kDa) Cell Lines in Dictyostelium Exhibit Marked Reduction in Growth Rate and Delay in Development

“…The actin microfilaments (F-actin) are formed by reversible polymerization of mono- (9,43). Both the chemoattractants are responsible for directed migration and bind to the cell surface recep-meric actin molecules called G-actin (11). The actin proteins constitute about 8% of total cellular proteins tors, which lead to several biochemical changes inside the cell such as increased level of cGMP and in Dictyostelium and in resting Dictyostelium cell Gactin (monomer) and F-actin (polymer) molecules oc-calcium, which then leads to dramatic reorganization of actin cytoskeleton, resulting in the extension of cur in equal ratio as certain cellular factors maintain a balance between actin monomers and polymers pseudopodia in new direction and consequently change in the direction of migration (39).…”

“…These cytosolic proteins, which are large and elongated, are regulated by calcium, phos-aggregation of Dictyostelium amoebae (18). For measuring the cell speed, starving amoebae pholipids, phosphorylated proteins, pH (11,13), and other changes in extracellular and intracellular envi-(approximately 1 × 10 6 ) were evenly spread on a 100mm petri dish and the movement of cells was video ronment of the cell. The proteins make the cytoplasm very dynamic by controlling polymerization of G-recorded for 30 min but the direction of the cell movement was not controlled in this measurement.…”

Crystal Structure of an Archaeal Actin Homolog

“…the concentration is much lower than that of actin in unicellular eukaryotes (e.g. w8% (w100 mM) in Dictyostelium discoideum 16 ).…”

Replacement of the essential Dictyostelium Arp2 gene by its Entamoeba homologue using parasexual genetics

“…That it is one of the easiest organisms to manipulate experimentally, is reflected in the ease with which genes can be disrupted, silenced, overexpressed and/or tagged [12]. Dictyostelium genes can be further manipulated using the technique of parasexual genetics [13,14].…”