genetic-engineering-dictyostelium-discoideum-cells-based-on-selection

Genetic Engineering of <em>Dictyostelium discoideum</em> Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

MRC Laboratory of Molecular Biology

Dictyostelium has a mature technology for molecular-genetic manipulation based around transfection using several different selectable markers, marker re-cycling, homologous recombination and insertional mutagenesis, all supported by a well-annotated genome. However this technology is optimized for mutant, axenic cells that, unlike non-axenic wild type, can grow in liquid medium. There is a pressing need for methods to manipulate wild type cells and ones with defects in macropinocytosis, neither of which can grow in liquid media. Here we present a panel of molecular genetic techniques based on the selection of Dictyostelium transfectants by growth on bacteria rather than liquid media. As well as extending the range of strains that can be manipulated, these techniques are faster than conventional methods, often giving usable numbers of transfected cells within a few days. The methods and plasmids described here allow efficient transfection with extrachromosomal vectors, as well as chromosomal integration at a 'safe haven' for relatively uniform cell-to-cell expression, efficient gene knock-in and knock-out and an inducible expression system. We have thus created a complete new system for the genetic manipulation of Dictyostelium cells that no longer requires cell feeding on liquid media.

Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum.

Signaling from chemoattractant receptors activates the cytoskeleton of crawling cells for chemotaxis. We show using phosphoproteomics that different chemoattractants cause phosphorylation of the same core set of around 80 proteins in Dictyostelium cells. Strikingly, the majority of these are phosphorylated at an [S/T]PR motif by the atypical MAP kinase ErkB. Unlike most chemotactic responses, ErkB phosphorylations are persistent and do not adapt to sustained stimulation with chemoattractant. ErkB integrates dynamic autophosphorylation with chemotactic signaling through G-protein-coupled receptors. Downstream, our phosphoproteomics data define a broad panel of regulators of chemotaxis. Surprisingly, targets are almost exclusively other signaling proteins, rather than cytoskeletal components, revealing ErkB as a regulator of regulators rather than acting directly on the motility machinery. ErkB null cells migrate slowly and orientate poorly over broad dynamic ranges of chemoattractant. Our data indicate a central role for ErkB and its substrates in directing chemotaxis.

The Atypical MAP Kinase ErkB Transmits Distinct Chemotactic Signals through a Core Signaling Module.

Macropinocytosis is an actin-driven process of large-scale, non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism  In , macropinocytic cups are organised by 'macropinocytic patches' in the plasma membrane. These contain activated Ras, Rac and PI(3,4,5)P3 and direct actin polymerisation to their periphery. We show that an Akt (PkbA) and an SGK (PkbR1) protein kinase act downstream of PI(3,4,5)P3 and are together nearly essential for fluid uptake. This pathway enables the formation of larger macropinocytic patches and macropinosomes, thereby dramatically increasing fluid uptake. Through phosphoproteomics, we identify a RhoGAP, GacG, as a PkbA/PkbR1 target and show it is required for efficient macropinocytosis and expansion of macropinocytic patches. The function of Akt and SGK in cell feeding through control of macropinosome size has implications for cancer cell biology.

Peggy Paschke

MRC Laboratory of Molecular Biology

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria