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dictyNews Volume 42 Number 22
dictyNews
Electronic Edition
Volume 42, number 22
September 23, 2016
Please submit abstracts of your papers as soon as they have been
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=========
Abstracts
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Autophagy in Dictyostelium: mechanisms, regulation and disease
in a simple biomedical model
Ana Mesquita,1,7 Elena Cardenal-Muñoz,2 Eunice Dominguez,1,5
Sandra Muñoz-Braceras,1 Beatriz Nuñez-Corcuera,1 Ben A. Phillips,3
Luis C. Tábara,1 Qiuhong Xiong,4 Roberto Coria,5 Ludwig Eichinger,4
Pierre Golstein,6 Jason S. King,3 Thierry Soldati,2 Olivier Vincent1
and Ricardo Escalante1
Autophagy, in press
Autophagy is a fast-moving field with an enormous impact on human
health and disease. Understanding the complexity of the mechanism
and regulation of this process often benefits from the use of simple
experimental models such as the social amoeba Dictyostelium discoideum.
Since the publication of the first review describing the potential of
D. discoideum in autophagy, significant advances have been made that
demonstrate both the experimental advantages and interest in using
this model. Since our previous review, research in D. discoideum has
shed light on the mechanisms that regulate autophagosome formation
and contributed significantly to the study of autophagy-related pathologies.
Here, we review these advances, as well as the current techniques to
monitor autophagy in D. discoideum. The comprehensive bioinformatics
search of autophagic proteins that was a substantial part of the previous
review has not been revisited here except for those aspects that challenged
previous predictions such as the composition of the Atg1 complex. In recent
years our understanding of, and ability to investigate autophagy in
D. discoideum has evolved significantly and will surely enable and
accelerate future research using this model.
submitted by: Ricardo Escalante [rescalante@iib.uam.es]
———————————————————————————————————————
Analysis of Relevant Parameters for Autophagic Flux Using HeLa Cells
Expressing EGFP-LC3.
Sandra Muñoz-Braceras and Ricardo Escalante
Methods Mol Biol. 2016;1449:313-29
Macroautophagy (called just autophagy hereafter) is an intracellular
degradation machinery essential for cell survival under stress
conditions and for the maintenance of cellular homeostasis. The
hallmark of autophagy is the formation of double membrane vesicles
that engulf cytoplasmic material. These vesicles, called autophagosomes,
mature by fusion with endosomes and lysosomes that allows the
degradation of the cargo. Autophagy is a dynamic process regulated at
multiple steps. Assessment of autophagy is not trivial because the number
autophagosomes might not necessarily reflect the real level of autophagic
degradation, the so-called autophagic flux. Here, we describe an optimised
protocol for the analysis of relevant parameters of autophagic flux using
HeLa cells stably expressing EGFP-LC3. These cells are a convenient tool
to determine the influence of the downregulation or overexpression of
specific proteins in the autophagic flux as well as the analysis of
autophagy-modulating compounds. Western blot analysis of relevant
parameters, such as the levels of EGFP-LC3, free EGFP generated by
autophagic degradation and endogenous LC3·I-II are analyzed in the
presence and absence of the autophagic inhibitor chloroquine.
submitted by: Ricardo Escalante [rescalante@iib.uam.es]
———————————————————————————————————————
Glutathione S-transferase 4 is a putative DIF-binding protein that regulates
the size of fruiting bodies in Dictyostelium discoideum
Hidekazu Kuwayama*, Haruhisa Kikuchi**, Yoshiteru Oshima**, and
Yuzuru Kubohara***
*Faculty of Life and Environmental Sciences, University of Tsukuba,
Tsukuba 305-8572, Japan
**Laboratory of Natural Product Chemistry, Graduate School of
Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan
***Department of Molecular and Cellular Biology, Institute for Molecular
and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan
***Laboratory of Health Life Science, Graduate School of Health and S
ports Science, Juntendo University, Inzai, Chiba 270-1695, Japan
Biochem. Biophys. Rep., in press
In the development of the cellular slime mold Dictyostelium discoideum,
two chlorinated compounds, the differentiation-inducing factors DIF-1
and DIF-2, play important roles in the regulation of both cell
differentiation and chemotactic cell movement. However, the receptors
of DIFs and the components of DIF signaling systems have not previously
been elucidated. To identify the receptors for DIF-1 and DIF-2, we here
performed DIF-conjugated affinity gel chromatography and liquid
chromatography–tandem mass spectrometry and identified the glutathione
S-transferase GST4 as a major DIF-binding protein. Knockout and
overexpression mutants of gst4 (gst4– and gst4OE, respectively) formed
fruiting bodies, but the fruiting bodies of gst4– cells were smaller than
those of wild-type Ax2 cells, and those of gst4OE cells were larger than
those of Ax2 cells. Both chemotaxis regulation and in vitro stalk cell
formation by DIFs in the gst4 mutants were similar to those of Ax2 cells.
These results suggest that GST4 is a DIF-binding protein that regulates
the sizes of cell aggregates and fruiting bodies in D. discoideum.
submitted by: Yuzuru Kubohara [ykuboha@juntendo.ac.jp]
———————————————————————————————————————
Acanthamoeba and Dictyostelium use different foraging strategies
Nick A. Kuburich#, Nirakar Adhikari#, and Jeffrey A. Hadwiger*
Department of Microbiology and Molecular Genetics, Oklahoma
State University, Stillwater, OK 74078-3020
# These authors contributed equally to this work
Protist, in press
Amoeba often use cell movement as a mechanism to find food, such as
bacteria, in their environment. The chemotactic movement of the soil
amoeba Dictyostelium to folate or other pterin compounds released by
bacteria is a well-documented foraging mechanism. Acanthamoeba can
also feed on bacteria but relatively little is known about the mechanism(s)
by which this amoeba locates bacteria. Acanthamoeba movement in the
presence of folate or bacteria was analyzed in above agar assays and
compared to that observed for Dictyostelium. The overall mobility of
Acanthamoeba was robust like that of Dictyostelium but Acanthamoeba
did not display a chemotactic response to folate. In the presence of
bacteria, Acanthamoeba only showed a marginal bias in directed
movement whereas Dictyostelium displayed a strong chemotactic response.
A comparison of genomes revealed that Acanthamoeba and Dictyostelium
share some similarities in G protein signaling components but that specific
G proteins used in Dictyostelium chemotactic responses were not present in
current Acanthamoeba genome sequence data. The results of this study
suggest that Acanthamoeba does not use chemotaxis as the primary
mechanism to find bacterial food sources and that the chemotactic
responses of Dictyostelium to bacteria may have co-evolved with
chemotactic responses that facilitate multicellular development.
submitted by: Jeff Hadwiger [jeff.hadwiger@okstate.edu]
———————————————————————————————————————
WASH drives early recycling from macropinosomes and phagosomes to maintain
surface phagocytic receptors
Catherine M. Buckley1,2*, Navin Gopaldass3,4*, Cristina Bosmani3, Simon A.
Johnston2,5, Thierry Soldati3 and Robert H. Insall6# and Jason S. King1,2#
Proc. Nat. Acad. Sciences USA, in press
http://www.pnas.org/content/early/2016/09/15/1524532113.full
Macropinocytosis is an ancient mechanism that allows cells to harvest
nutrients from extracellular media, which also allows immune cells to sample
antigens from their surroundings. During macropinosome formation, bulk
plasma membrane is internalized with all its integral proteins. It is vital
for cells to salvage these proteins before degradation, but the mechanisms
for sorting them are not known. Here we describe the evolutionarily conserved
recruitment of the WASH (WASP and SCAR homolog) complex to both
macropinosomes and phagosomes within a minute of internalization. Using
Dictyostelium, we demonstrate that WASH drives protein sorting and recycling
from macropinosomes and is thus essential to maintain surface receptor levels
and sustain phagocytosis. WASH functionally interacts with the retorter
complex at both early and late phases of macropinosome maturation, but
mediates recycling via retromer-dependent and -independent pathways.
WASH mutants consequently have decreased membrane levels of integrins
and other surface proteins. This study reveals an important pathway enabling
cells to sustain macropinocytosis without bulk degradation of plasma
membrane components.
submitted by: Jason King [jason.king@sheffield.ac.uk]
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[End dictyNews, volume 42, number 22]
