dictyNews

Electronic Edition

Volume 44, number 13

April 27, 2018



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Abstracts

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The cAMP-induced G protein subunits dissociation monitored in live 

Dictyostelium cells by BRET reveals two activation rates, a negative

 effect of caffeine and potential role of microtubules.



AFM T. Islam,a, Haicen Yue,b, Margarethakay Scavello,a, Pearce 

Haldemana,c, Wouter-Jan Rappel,b, and Pascale G. Charest,a*.





Cellular Signaling, in press



To study the dynamics and mechanisms controlling activation of the 

heterotrimeric G protein Galpha/beta/gamma in Dictyostelium in 

response to stimulation by the chemoattractant cyclic AMP (cAMP), 

we monitored the G protein subunit interaction in live cells using 

bioluminescence resonance energy transfer (BRET). We found that 

cAMP induces the cAR1-mediated dissociation of the G protein 

subunits to a similar extent in both undifferentiated and differentiated 

cells, suggesting that only a small number of cAR1 (as expressed in 

undifferentiated cells) is necessary to induce the full activation of 

Galpha/beta/gamma. In addition, we found that treating cells with 

caffeine increases the potency of cAMP-induced Galpha/beta/gamma 

activation; and that disrupting the microtubule network but not F-actin 

inhibits the cAMP-induced dissociation of Galpha/beta/gamma. Thus, 

microtubules are necessary for efficient cAR1-mediated activation of 

the heterotrimeric G protein. Finally, kinetics analyses of 

Galpha/beta/gamma subunit dissociation induced by different cAMP 

concentrations indicate that there are two distinct rates at which the 

heterotrimeric G protein subunits dissociate when cells are stimulated 

with cAMP concentrations above 500 nM versus only one rate at lower 

cAMP concentrations. Quantitative modeling suggests that the kinetics 

profile of Galpha/beta/gamma subunit dissociation results from the 

presence of both uncoupled and G protein pre-coupled cAR1 that 

have differential affinities for cAMP and, consequently, induce G 

protein subunit dissociation through different rates. We suggest that 

these different signaling kinetic profiles may play an important role 

in initial chemoattractant gradient sensing.





submitted by:  Pascale Charest [[log in to unmask]]

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Rapid and efficient genetic engineering of both wild type and axenic 

strains of Dictyostelium discoideum



Peggy Paschke1, David A. Knecht2, Augustinas Silale1, David Traynor1, 

Thomas D. Williams1,Peter A. Thomason3, Robert H. Insall3, 

Jonathan R. Chubb4, Robert R. Kay1, Douwe M.Veltman1



1 MRC Laboratory of Molecular Biology, Cambridge, UK

2 Department of Molecular and Cell Biology, University of Connecticut, USA.

3 Cancer Research UK Beatson Institute Glasgow, UK.

3 Laboratory for Molecular Cell Biology and Department of Cell and 

Developmental Biology, University College London, UK.





PLoS One, in press



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.





submitted by:  Rob Kay  [[log in to unmask]]

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[End dictyNews, volume 44, number 13]