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dictyNews Volume 32 Number 01
dictyNews
Electronic Edition
Volume 32, number 1
January 9, 2009
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu
or by using the form at
http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit.
Back issues of dictyNews, the Dicty Reference database and other
useful information is available at dictyBase - http://dictybase.org.
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Abstracts
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ReASH- another viable option for in vivo protein labeling in Dictyostelium
Ran-Der Hwang, Chin-Chi Chen and David A. Knecht
Journal of Microscopy, in press
Biarsenical-tetracysteine fluorescent protein tagging has been effectively
used in a variety of cell types. It has the advantage of requiring a
much smaller peptide alteration to existing proteins than fusion to GFP
or RFP. However, there are no reports of the tetracysteine tagging
system being used in Dictyostelium. In order to establish this tagging
system in Dictyostelium, the filamin gene (FLN) was modified to express
a C-terminal tetracysteine sequence and then transfected into cells.
After addition of either FlAsH-EDT2 or ReAsH-EDT2, the fluorescence
intensity of cells increased in a time dependent manner and reached
a plateau after three hours of incubation. ReAsH had a much stronger
and more specifically localized fluorescent signal compared to FlAsH.
After removal of the ReAsH-EDT2 reagent, the fluorescence signal
remained detectable for at least twenty-four hours. The localization
of filamin labeled by ReAsH was similar to that of an mRFP-filamin
fusion protein, but the fluorescence signal from the ReAsH labeled
protein was stronger. Our findings suggest that the ReAsH-tetracysteine
tagging system can be a useful alternative for in vivo protein tagging
in Dictyostelium.
Submitted by: Dave Knecht [david.knecht@uconn.edu]
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Autophagy contributes to degradation of Hirano bodies
Dong-Hwan Kim, Richard C. Davis, Ruth Furukawa and
Marcus Fechheimer
Autophagy, Vol 5;1 P: 44 - 51
Hirano bodies are actin-rich inclusions reported most frequently in the
hippocampus in association with a variety of conditions including
neurodegenerative diseases, and aging. We have developed a model
system for formation of Hirano bodies in Dictyostelium and cultured
mammalian cells to permit detailed studies of the dynamics of these
structures in living cells. Model Hirano bodies are frequently observed in
membrane-enclosed vesicles in mammalian cells consistent with a role
of autophagy in the degradation of these structures. Clearance of Hirano
bodies by an exocytotic process is supported by images from electron
microscopy showing extracellular release of Hirano bodies, and
observation of Hirano bodies in the culture medium of Dictyostelium
and mammalian cells. An autophagosome marker protein Atg8-GFP, was
co-localized with model Hirano bodies in wild type Dictyostelium cells,
but not in atg5- or atg1-1 autophagy mutant strains. Induction of model
Hirano bodies in Dictyostelium with a high level expression of 34 kDa
DeltaEF1 from the inducible discoidin promoter resulted in larger Hirano
bodies and a cessation of cell doubling. The degradation of model Hirano
bodies still occurred rapidly in autophagy mutant (atg5-) Dictyostelium,
suggesting that other mechanisms such as the ubiquitin-mediated
proteasome pathway could contribute to the degradation of Hirano bodies.
Chemical inhibition of the proteasome pathway with lactacystin, significantly
decreased the turnover of Hirano bodies in Dictyostelium providing direct
evidence that autophagy and the proteasome can both contribute to
degradation of Hirano bodies. Short term treatment of mammalian cells with
either lactacystin or 3-methyl adenine results in higher levels of Hirano
bodies and a lower level of viable cells in the cultures, supporting the
conclusion that both autophagy and the proteasome contribute to
degradation of Hirano bodies.
Submitted by: Ruth Furukawa [furukawa@cb.uga.edu]
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Steroids initiate a signaling cascade that triggers rapid sporulation in
Dictyostelium
Christophe Anjard, Yongxuan Su and William F. Loomis*
Center for Molecular Genetics, Division of Biological Sciences,
University of California San Diego, La Jolla, CA 92093-0368
Development, in press
Encapsulation of prespore cells of Dictyostelium discoideum is controlled
by several intercellular signals to ensure appropriate timing during
fruiting body formation. Acyl CoA binding protein, AcbA, is secreted by
prespore cells and processed by the prestalk protease TagC to form the 34
amino acid peptide SDF-2. The SDF-2 receptor is a constitutive histidine
kinase, DhkA, which no longer stimulates the cAMP phosphodiesterase,
RegA, when SDF-2 is bound. The subsequent increase in cAMP and PKA
triggers rapid encapsulation. AcbA is secreted when gamma-aminobutyric
acid (GABA) is released from prespore cells and binds to GrlE, a G protein
coupled receptor (GPCR). Analysis of SDF-2 production in a series of mutant
strains lacking Galpha subunits and GPCRs, either as pure populations or
when mixed with other mutant strains, uncovered the non-cell autonomous
roles of GrlA, a membrane localized GPCR, Galpha4 and Galpha7. We found
that Galpha7 is essential for the response to GABA and is likely to be coupled
to GrlE. GrlA and Galpha4 null cells respond normally to GABA but fail to
secrete it. We found that they are necessary for response to a small,
hydrophobic molecule, SDF-3, which is released late in culmination.
Pharmacological inhibition of steroidogenesis during development blocked
the production of SDF-3. Moreover, the response to SDF-3 can be blocked
by the steroid antagonist mifepristone, while hydrocortisone and other
steroids mimic the effects of SDF-3 when added in the nanomolar range. It
appears that SDF-3 is a steroid that elicits rapid release of GABA by acting
through GrlA coupled to G protein containing the Galpha4 subunit. It may
either stimulate the prespore specific glutamate decarboxylase, GadA, that
synthesizes GABA or inhibit the GABA transaminase, GabT, that degrades
GABA. SDF-3 is at the head of the cascade that amplifies the signal for
encapsulation to ensure rapid, synchronous formation of spores.
Submitted by: Bill Loomis [wloomis@ucsd.edu]
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DNA Passage to Nuclei: Role of Endo/lysosomal circuit in Eukaryotic
Dictyostelium.
Bhavesh Vats#, Harish Padh*
Department of Cell and Molecular Biology, B. V. Patel Pharmaceutical Education
and Research Development (PERD) Centre, Thaltej- Gandhinagar Highway,
Thaltej, Ahmedabad – 380054, INDIA.
Telephone Number: +91-79-27439375, Fax number: +91-79-27450449.
Email: perd@perdcentre.com
*Corresponding author
#Present affiliation: Analytical Development, INTAS Biopharmaceuticals Limited,
Sarkhej- Bavla Highway, Ahmedabad- 382 210, INDIA.
Telephone number: +912717660100, Fax number: +912717251189
Email: bhavesh.vats@intasbiopharma.co.in
Canadian Journal of Microbiology, in press
The understanding of DNA passage in eukaryotic cells is still very ambiguous.
The route to the nucleus is difficult due to the barriers- metabolic as well
as membranous, posed by the eukaryotic cells. Endocytosis appears to be the
most likely process responsible for the transport but is also the major culprit
of low transfection efficiencies. Here, we report a study on a eukaryotic
amoeba, Dictyostelium discoideum, where by disruption of the endocytic
process at the opportune moment, the transformant number increased.
We have observed by disruption of fluid phase uptake of calcium phosphate
DNA nanoparticles, the number of clones increased with probable increase
in number of foreign genes integrating in the host genome. The method
described here leads to the possibility of safe and inexpensive methods
for transfer of genes required for heterologous recombinant protein
production as well as generation therapeutic recombinant cells.
Submitted by: Harish Padh [hpadh@yahoo.com]
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[End dictyNews, volume 32, number 1]
