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dictyNews Volume 21 Number 11
Dicty News
Electronic Edition
Volume 21, number 11
October 10, 2003
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu.
Back issues of Dicty-News, the Dicty Reference database and other
useful information is available at dictyBase - http://dictybase.org.
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Abstracts
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Macroautophagy is dispensable for intracellular replication of
Legionella pneumophila in Dictyostelium discoideum
Grant P. Otto, Mary Y. Wu, Margaret Clarke, Hao Lu, O. Roger
Anderson, Hubert Hilbi, Howard A. Shuman, and Richard H. Kessin.
Accepted, Molecular Microbiology
SUMMARY
The gram-negative bacterium Legionella pneumophila is a facultative intracellular
pathogen of free-living amoebae and mammalian phagocytes. L. pneumophila is
engulfed in phagosomes that initially avoid fusion with lysosomes. The phagosome
associates with endoplasmic reticulum and mitochondria, and eventually resembles
ER. The morphological similarity of the replication vacuole to autophagosomes,
and enhanced bacterial replication in response to macroautophagy-inducing
starvation, led to the hypothesis that L. pneumophila infection requires
macroautophagy. Since L. pneumophila replicates in D. discoideum, and
macroautophagy genes have been identified and mutated in D. discoideum, we have
taken a genetic and cell biological approach to evaluate the relationship between
host macroautophagy and intracellular replication of L. pneumophila. Mutation of
the apg1 , apg5, apg6, apg7 and apg8 genes produced typical macroautophagy
defects, including reduced bulk protein degradation and cell viability during
starvation. We show that L. pneumophila replicates normally in D. discoideum
macroautophagy mutants and produces replication vacuoles that are
morphologically indistinguishable from those in wild-type D. discoideum.
Furthermore, a GFP-tagged marker of autophagosomes, Apg8, does not
systematically co-localise with DsRed-labelled L. pneumophila. We conclude that
macroautophagy is dispensable for L. pneumophila intracellular replication in
D. discoideum.
Submitted by: Grant Otto [go25@columbia.edu]
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A mechanical unfolding intermediate in an actin-crosslinking protein.
Ingo Schwaiger, Angelika Kardinal, Michael Schleicher, Angelika Noegel and
Matthias Rief
Accepted, Nature Structural Biology
A large number of F-actin cross-linking proteins consists of two
actin-binding domains separated by a rod domain that can vary considerably
in length and structure. We used single-molecule force-experiments to
investigate the mechanics of the immunoglobulin (Ig) rod domains of filamin
from Dictyostelium discoideum (ddFLN). We find that one of the 6 Ig-domains
unfolds at lower forces than all other domains and exhibits a stable
unfolding intermediate on its mechanical unfolding pathway. Amino acid
inserts into various loops of this domain lead to length changes in the
single molecule unfolding pattern which allowed us to map the stable core
of ~60 amino acids that constitute the unfolding intermediate. Fast
refolding in combination with low unfolding forces suggest a potential in
vivo role for this domain as a mechanically extensible element within the
ddFLN rod.
Submitted by: Michael Schleicher [schleicher@lrz.uni-muenchen.de]
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Disruption of aldehyde reductase increases group size in Dictyostelium
Karen Ehrenman1, Gong Yang1, Wan-Pyo Hong1, Tong Gao1, Wonhee Jang2,
Debra A. Brock1, R. Diane Hatton1, James D. Shoemaker3, and Richard H. Gomer1,2
1Howard Hughes Medical Institute and 2Department of Biochemistry and Cell
Biology, MS-140, Rice University, Houston, TX and 3Metabolic Screening Laboratory,
Department of Biochemistry and Molecular Biology, St. Louis University, St. Louis, MO
Journal of Biological Chemistry, in press
ABSTRACT
Developing Dictyostelium cells form structures containing ~20,000 cells.
The size regulation mechanism involves a secreted counting factor (CF)
repressing cytosolic glucose levels. Glucose or a glucose metabolite affect
cell-cell adhesion and motility; these in turn affect whether a group stays
together, loses cells, or even breaks up. NADPH-coupled aldehyde reductase
reduces a wide variety of aldehydes to the corresponding alcohols, including
converting glucose to sorbitol. The levels of this enzyme previously appeared
to be regulated by CF. We find that disrupting alrA, the gene encoding aldehyde
reductase, results in the loss of alrA mRNA and AlrA protein and a decrease in
the ability of cell lysates to reduce both glyceraldehyde and glucose in a
NADPH-coupled reaction. Counterintuitively, alrA¯ cells grow normally and
have decreased glucose levels compared to parental cells. The alrA¯ cells
form long unbroken streams and huge groups. Expression of AlrA in alrA¯ cells
causes cells to form normal fruiting bodies, indicating that AlrA affects group
size. alrA¯ cells have normal adhesion but a reduced motility, and computer
simulations suggest that this could indeed result in the formation of large groups.
alrA¯ cells secrete low levels of countin and CF50, two components of CF, and
this could partially account for why alrA¯ cells form large groups. alrA¯
cells are responsive to CF and are partially responsive to recombinant
countin and CF50, suggesting that disrupting alrA inhibits but does not
completely block the CF signal transduction pathway. Gas chromatography/
mass spectroscopy indicates that the concentrations of several metabolites are
altered in alrA¯ cells, suggesting that the Dictyostelium aldehyde reductase
affects several metabolic pathways in addition to converting glucose to sorbitol.
Together, our data suggest that disrupting alrA affects CF secretion, causes
many effects on cellular metabolism, and has a major effect on group size.
Submitted by: Richard Gomer [richard@rice.edu]
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Uniform cAMP stimulation of Dictyostelium cells induces localized patches of
signal transduction and pseudopodia
Marten Postma, Jeroen Roelofs, Joachim Goedhart, Theodorus W.J. Gadella,
Antonie J.W.G. Visser, and Peter J.M. Van Haastert
Department of Biochemistry, University of Groningen, Nijenborgh 4,
9747 AG Groningen, the Netherlands
Molecular Biology of the Cell, in press
ABSTRACT
The chemoattractant cAMP induces the translocation of cytosolic PHCrac-GFP to
the plasma membrane. PHCrac-GFP is a green fluorescent protein fused to a PH domain
that presumably binds to phosphatydylinositol polyphosphates in the membrane.
We determined the relative concentration of PHCrac-GFP in the cytosol and at
different places along the cell boundary. In cells stimulated homogeneously
with 1 microM cAMP we observed two distinct phases of PHCrac-GFP translocation.
The first translocation is transient and occurs to nearly the entire boundary of
the cell; the response is maximal at 6-8 seconds after stimulation and disappears
after about 20 seconds. A second translocation of PHCrac-GFP starts after about
30 seconds and persists as long as cAMP remains present. Translocation during
this second response occurs to small patches with radius of about 4-5 µm,
each covering about 10 % of the cell surface. Membrane patches of PHCrac-GFP are
both temporally and spatially closely associated with pseudopodia, which are
extended at about 10 s from the area with a PHCrac-GFP patch. These signaling
patches in pseudopodia of homogeneously stimulated cells resemble the single
patch of PHCrac-GFP at the leading edge of a cell in a gradient of cAMP, suggesting
that PHCrac-GFP is a spatial cue for pseudopod formation also in uniform cAMP.
Submitted by: Peter J.M. Van Haastert [P.J.M.van.Haastert@chem.rug.nl]
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[End Dicty News, volume 21, number 11]
