dictyNews
Electronic Edition
Volume 36, number 3
Jan 28, 2011
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Abstracts
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Bio-electrospraying and aerodynamically assisted bio-jetting the model
eukaryotic Dictyostelium discoideum: assessing stress and developmental
competency post-treatment
Nicholl K. Pakes, Suwan N. Jayasinghe and Robin S. B. Williams
Journal of the Royal Society Interface, in press
Bio-electrospraying (BES) and aerodynamically assisted bio-jetting (AABJ)
have recently been established as important novel biospray technologies
for directly manipulating living cells. To elucidate their potential in medical
and clinical sciences, these bio-aerosol techniques have been subjected
to increasingly rigorous investigations. In parallel to these studies, we wish
to introduce these unique biotechnologies for use in the basic biological
sciences, for handling a wide range of cell types and systems, thus
increasing the range and the scope of these techniques for modern
research. Here, the authors present the analysis of the new use of these
biospray techniques for the direct handling of the simple eukaryotic
biomedical model organism Dictyostelium discoideum. These cells are
widely used as a model for immune cell chemotaxis and as a simple
model for development. We demonstrate that AABJ of these cells did not
cause cell stress, as defined by the stress-gene induction, nor affect cell
development. Furthermore, although BES induced the increased
expression of one stress-related gene (gapA), this was not a generalized
stress response nor did it affect cell development. These data suggest
that these biospray techniques can be used to directly manipulate single
cells of this biomedical model without inducing a generalized stress
response or perturbing later development.
Submitted by Robin Williams [[log in to unmask]]
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eIF2alpha Kinases Control Chalone Production In Dictyostelium
discoideum
Robert L. Bowman*, Yanhua Xiong, Janet H. Kirsten and
Charles K. Singleton
Department of Biological Sciences, Vanderbilt University,
VU Station B 351634, Nashville, TN 37235
Eukaryotic Cell, in press
Growing Dictyostelium cells secrete CfaD and AprA, two proteins that
have been characterized as chalones. They exist within a high molecular
weight complex that reversibly inhibits cell proliferation but not growth via
cell surface receptors and a signaling pathway that includes G proteins.
How the production of these two proteins is regulated is unknown.
Dictyostelium cells possess three GCN2 type eIF2alpha kinases, proteins
that phosphorylate the translational initiation factor eIF2alpha and possess
a tRNA binding domain involved in their regulation. The Dictyostelium
kinases have been shown to function during development in regulating
several processes. We show here that expression of an unregulated,
activated kinase domain greatly inhibits cell proliferation. The inhibitory
effect on proliferation is not due to a general inhibition of translation.
Instead it is due to an enhanced production of a secreted factor(s).
Indeed, extracellular CfaD and AprA proteins, but not their mRNAs, are
overproduced in cells expressing the activated kinase domain. The
inhibition of proliferation is not seen when the activated kinase domain
is expressed in cells lacking CfaD or AprA or in cells that contain a non-
phosphorylatable eIF2alpha. We conclude that production of the chalones
CfaD and AprA is translationally regulated by eIF2alpha phosphorylation.
Both proteins are upregulated during culmination of development, and
this enhanced production is lacking in a strain that possesses a non-
phosphorylatable eIF2alpha.
Submitted by Charles Singleton [[log in to unmask]]
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Atg1 allows second-signalled autophagic cell death in Dictyostelium
Marie-Françoise Luciani,1,2,3 Corinne Giusti,1,2,3 Birthe Harms,1,2,3
Yoshiteru Oshima,4 Haruhisa Kikuchi,4 Yuzuru Kubohara,5 and
Pierre Golstein1,2,3,*
1Centre d’Immunologie de Marseille-Luminy (CIML), Faculté des Sciences
de Luminy, Aix-Marseille Université, Marseille F-13288, France;
2INSERM U631, Marseille F-13288, France;
3CNRS UMR6102, Marseille F-13288, France;
4Laboratory of Natural Product Chemistry, Graduate School of
Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan;
5Institute for Molecular & Cellular Regulation, Gunma University,
Maebashi 371-8512, Japan
Autophagy 2011, in press
We investigated the role of atg1 in autophagic cell death (ACD) in a
Dictyostelium monolayer model. The model is especially propitious, not
only because of genetic tractability and absence of apoptosis machinery,
but also because induction of ACD requires two successive exogenous
signals, first the combination of starvation and cAMP, second the
differentiation factor DIF-1. This enables one to analyze separately
first-signal-induced autophagy and subsequent second-signal-induced
ACD. We used mutants of atg1, a gene that plays an essential role in
the initiation of autophagy. Upon starvation/cAMP, in contrast to parental
cells, atg1 mutant cells showed irreversible lesions, clearly establishing
a protective role for atg1. Upon subsequent exposure to DIF-1 or to
more ACD-specific second signals, starved parental cells progressed
to ACD, but starved atg1 mutant cells did not, showing that atg1 was
required for ACD. Thus, in the same cells atg1 was required in two
apparently opposite ways, upon first-signalling for cell survival and upon
second-signalling for ACD. Our findings strongly suggest that atg1, thus
presumably autophagy, protects the cells from starvation-induced cell
death, allowing subsequent induction of ACD by the second signal.
ACD is therefore not only "with" autophagy (since it showed signs of
autophagy throughout), but is also "allowed by" autophagy. This does not
exclude a role for autophagy also after second signalling. These results
may account for discrepancies reported in the literature, encourage
searches for second signals in different developmental models of
ACD, and incite caution in autophagy-related therapeutic attempts.
Submitted by Pierre Golstein [[log in to unmask]]
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Origin and function of the stalk-cell vacuole in Dictyostelium
Toru Uchikawa(a), Akitsugu Yamamoto(b), Kei Inouye(a)
(a) Department of Botany, Graduate School of Science, Kyoto University
(b) Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology
Dev. Biol., in press
Large vacuoles are characteristic of plant and fungal cells, and their origin
has long attracted interest. The cellular slime mould provides a unique
opportunity to study the de novo formation of vacuoles because, in its life
cycle, a subset of the highly motile animal-like cells (prestalk cells) rapidly
develop a single large vacuole and cellulosic cell wall to become plant-like
cells (stalk cells). Here we describe the origin and process of vacuole
formation using live-imaging of Dictyostelium cells expressing GFP-tagged
ammonium transporter A (AmtA-GFP), which was found to reside on the
membrane of stalk-cell vacuoles. We show that stalk-cell vacuoles originate
from acidic vesicles and autophagosomes, which fuse to form autolysosomes.
Their repeated fusion and expansion accompanied by concomitant cell wall
formation enables the stalk cells to rapidly develop turgor pressure necessary
to make the rigid stalk to hold the spores aloft. Contractile vacuoles, which
are rich in H+-ATPase as in plant vacuoles, remained separate from these
vacuoles. We further argue that AmtA may play an important role in the
control of stalk-cell differentiation by modulating the pH of autolysosomes.
Submitted by Kei Inouye [[log in to unmask]]
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Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase
and alpha-N-acetylglucosaminyltransferase enzymes involved in
O2-signaling in Dictyostelium
Hanke van der Wel‡, Jennifer M. Johnson‡, Yuechi Xu‡, Chamini V.
Karunaratne§, Kyle D. Wilson‡, Yusuf Vohra¶, Geert-Jan Boons¶,
Carol M. Taylor§, Brad Bendiak#, and Christopher M. West‡
‡Department of Biochemistry and Molecular Biology, Oklahoma Center
for Medical Glycobiology, University of Oklahoma Health Sciences Center,
Oklahoma City, OK 73104 USA;
§Department of Chemistry, 742 Choppin Hall, Louisiana State University,
Baton Rouge, LA 70803 USA;
¶Dept. of Chemistry and Complex Carbohydrate Research Center,
315 Riverbend Road, University of Georgia, Athens, GA 30602 USA;
#Department of Cell and Developmental Biology and Structural Biology
and Biophysics Program, University of Colorado Denver, Anschutz
Medical Campus, Mail Stop 8108, RC-1 South Bldg., L18-12120, 12801
East 17th Avenue, Aurora, CO 80045 USA
Biochemistry, in press.
The social amoeba Dictyostelium expresses a hypoxia inducible factor-
alpha (HIFalpha)-type prolyl 4-hydroxylase (P4H1) and an
alpha-N-acetylglucosaminyltransferase (Gnt1) that sequentially modify
proline-143 of Skp1, a subunit of the SCF (Skp1/Cullin/F-box protein)-class
of E3 ubiquitin-ligases. Prior genetic studies have implicated Skp1 and its
modification by these enzymes in O2-regulation of development, suggesting
the existence of an ancient O2-sensing mechanism related to modification of
the transcription factor HIFalpha by animal prolyl 4-hydroxylases (PHDs). To
better understand the role of Skp1 in P4H1-dependent O2-signaling,
biochemical and biophysical studies were conducted to characterize the
reaction product and the basis of Skp1 substrate selection by P4H1 and
Gnt1. 1H-NMR demonstrated formation of 4(trans)-hydroxyproline as
previously found for HIFalpha, and highly purified P4H1 was inhibited by
Krebs cycleintermediates and other compounds that affect animal P4Hs.
However, in contrast to hydroxylation of HIFalpha by PHDs, P4H1
depended on features of full-length Skp1, based on truncation, mutagenesis,
and competitive inhibition studies. These features are conserved during
animal evolution, as even mammalian Skp1, which lacks the target proline,
became a good substrate upon its restoration. P4H1 recognition may
depend on features conserved for SCF complex formation as
heterodimerization with an F-box protein blocked Skp1 hydroxylation.
The hydroxyproline-capping enzyme Gnt1 exhibited similar requirements
for Skp1 as a substrate. These and other findings support a model in
which the protist P4H1 conditionally hydroxylates Skp1 of
E3SCFubiquitin-ligases to control half-lives of multiple targets, rather
than the mechanism of animal PHDs where individual proteins are
hydroxylated leading to ubiquitination by the evolutionarily-related
E3VBCubiquitin-ligases.
Submitted by: Chris West [[log in to unmask]]
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[End dictyNews, volume 36, number 3]
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