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Electronic Edition

Volume 44, number 31

November 9, 2018



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Abstracts

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Function of small GTPases in Dictyostelium macropinocytosis



Thomas D. Williams, Peggy I. Paschke, Robert R. Kay



MRC-Laboratory of Molecular Biology, Francis Crick Avenue, 

Cambridge, CB2 0QH, UK.





Phil. Trans. Roy. Soc. B, in press



Macropinocytosis – the large-scale, non-specific uptake of fluid by 

cells – is used by Dictyostelium discoideum amoebae to obtain nutrients. 

These cells form circular ruffles around regions of membrane defined by 

a patch of PI(3,4,5)P3 (PIP3) and the activated forms of the small 

G-proteins Ras and Rac. When this ruffle closes, a vesicle of medium is 

delivered to the cell interior for further processing. It is accepted that 

PIP3 is required for efficient macropinocytosis. Here we assess the roles 

of Ras and Rac in Dictyostelium macropinocytosis. Gain of function 

experiments show that macropinocytosis is stimulated by persistent Ras 

activation and genetic analysis suggests that RasG and RasS are the 

key Ras proteins involved. Among the activating GEFs, GefF is implicated 

in macropinocytosis by an insertional mutant. The individual roles of Rho 

family proteins are little understood but activation of at least some may be 

independent of PIP3.





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

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Structural basis of Gip1 for cytosolic sequestration of G protein in 

wide-range chemotaxis



Takero Miyagawa, Hiroyasu Koteishi, Yoichiro Kamimura, Yukihiro 

Miyanaga, Kohei Takeshita3, Atsushi Nakagawa, Masahiro Ueda





Nature Communicationsvolume 9, Article number: 4635 (2018) 



G protein interacting protein 1 (Gip1) binds and sequesters heterotrimeric 

G proteins in the cytosolic pool, thus regulating G protein-coupled receptor 

(GPCR) signalling for eukaryotic chemotaxis. Here, we report the underlying 

structural basis of Gip1 function. The crystal structure reveals that the region 

of Gip1 that binds to the G protein has a cylinder-like fold with a central 

hydrophobic cavity composed of six alpha-helices. Mutagenesis and 

biochemical analyses indicate that the hydrophobic cavity and the hydrogen 

bond network at the entrance of the cavity are essential for complex formation 

with the geranylgeranyl modification on the G-gamma subunit. Mutations of 

the cavity impair G protein sequestration and translocation to the membrane 

from the cytosol upon receptor stimulation, leading to defects in chemotaxis 

at higher chemoattractant concentrations. These results demonstrate that the 

Gip1-dependent regulation of G protein shuttling ensures wide-range gradient 

sensing in eukaryotic chemotaxis.





submitted by:  Yoichiro Kamimura [[log in to unmask]]

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Localization of all four ZnT zinc transporters in Dictyostelium and impact 

of ZntA and B knockout on bacteria killing



Caroline Barisch1*, Vera Kalinina1,2, Louise H. Lefrançois1, Joddy Appiah1, 

Ana T. López-Jiménez1 and Thierry Soldati1



1Department of Biochemistry, Faculty of Science, University of Geneva, 

30 quai Ernest-Ansermet, Science II, 1211 Geneva-4, Switzerland

2 present address: Institute of Cytology, Russian Academy of Sciences, 

Tikhoretsky ave. 4, 194064 St. Petersburg, Russia

*Corresponding author





Journal of Cell Science, in press

http://jcs.biologists.org/content/early/2018/10/20/jcs.222000)



Professional phagocytes have developed an extensive repertoire of 

autonomous immunity strategies to ensure killing of bacteria. Besides 

phagosome acidification and the generation of reactive oxygen species, 

deprivation of nutrients and the lumenal accumulation of toxic metals are 

essential to kill ingested bacteria or inhibit growth of intracellular pathogens. 

We use the soil amoeba Dictyostelium discoideum, a professional phagocyte 

that digests bacteria for nutritional purposes, to decipher the role of zinc 

poisoning during phagocytosis of non-pathogenic bacteria and visualize the 

temporal and spatial dynamics of compartmentalized, free zinc using 

fluorescent probes. Immediately after particle uptake, zinc is delivered to 

phagosomes by fusion with “zincosomes” of endosomal origin, but also by 

the action of one or more zinc transporters. We localize the four Dictyostelium 

ZnT transporters to endosomes, the contractile vacuole and the Golgi 

apparatus, and study the impact of znt knockouts on zinc homeostasis. Finally, 

we show that zinc is delivered into the lumen of Mycobacterium smegmatis-

containing vacuoles, and that Escherichia coli deficient in the zinc efflux 

P1B-type ATPase ZntA is killed faster than wild type bacteria.





submitted by:  Thierry Soldati [[log in to unmask]]

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[End dictyNews, volume 44, number 31]