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dictyNews Volume 23 Number 10
Dicty News
Electronic Edition
Volume 23, number 10
September 24, 2004
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 Dicty-News, the Dicty Reference database and other
useful information is available at dictyBase - http://dictybase.org.
=============
Abstracts
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GFP-golvesin constructs to study Golgi tubulation and post-Golgi vesicle
dynamics in phagocytosis
Guenther Gerisch , Aleksander Benjak 1a, Jana Koehler, Igor Weberb, and
Natalie Schneiderc
Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany
a Present address: EMBL Heidelberg, Meyerhofstr. 1, D-69117 Heidelberg, Germany
b Present address: Rudjer Boskovic Institute, Bijenicka cesta 54,
10000 Zagreb, Croatia
c Present address: Kyoto University Biosimulation Center, Kyoto Research Park,
Bldg. #4, 8F, Chudoji Awata-cho 93, Shimogyoku, Kyoto, Japan 600-8815
European Journal of Cell Biology, in press
Dictyostelium cells are professional phagocytes that are optimally suited for
the imaging of phagosome processing from particle uptake to exocytosis. In
order to design fluorescent probes for monitoring membrane trafficking in the
endocytic pathway, we have dissected a membrane protein, golvesin, and have
linked fragments of its sequence to GFP. Endogenous golvesin is partitioned
between the ER, the Golgi apparatus, endosomes, and the contractile vacuole
complex. We have localized signals that are required for exit from the Golgi
to post-Golgi compartments to the C-terminal region of the golvesin sequence.
One GFP-tagged fragment turned out to be a highly specific Golgi marker and
was used to demonstrate the interaction of Golgi tubules with phagosomes.
Signals essential for the retrieval of golvesin at the end of phagosome
processing were localized to the N-terminal region. A truncated golvesin
construct escaping retrieval was employed in recording the delivery of a
phagosomal protein to the plasma membrane. Applying this construct to a
phagosome filled with multiple particles, we observed that the phagosome is
segmented during exocytosis, meaning that sequential release of particles
alternates with membrane fusion.
Submitted by: Guenther Gerisch [gerisch@biochem.mpg.de]
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Mobile actin clusters and traveling waves in cells recovering from actin
depolymerization
Guenther Gerisch 1, Till Bretschneider 1, Annette Mueller-Taubenberger 1,
Evelyn Simmeth 1, Mary Ecke 1, Stefan Diez 2, and Kurt Anderson 2
1 Max-Planck-Institut fuer Biochemie, D-82152 Martinsried;
2 Max-Planck-Institut fuerÊmolekulare Zellbiologie und Genetik,
Pfotenhauer Str. 108, D-01307 Dresden;
Biophysical Journal, in press
At the leading edge of a motile cell, actin polymerizes in close apposition
to the plasma membrane. Here we ask how the machinery for force generation at
a leading edge is established de-novo after the global depolymerization of
actin. The depolymerization is accomplished by latrunculin A, and the
re-organization of actin upon removal of the drug is visualized in
Dictyostelium cells by total internal reflection fluorescence (TIRF)
microscopy. The actin filament system is reorganized in three steps. First,
F-actin assembles into globular complexes that move along the bottom surface
of the cells at velocities up to 10 µm per minute. These clusters are
transient structures that eventually disassemble, fuse or divide. In a second
step, clusters merge into a contiguous zone at the cell border that spreads
and gives rise to actin waves traveling on a planar membrane. Finally, normal
cell shape and motility are resumed. These data show that the initiation of
actin polymerization is separated in Dictyostelium from front protrusion, and
that the coupling of polymerization to protrusion is a later step in the
reconstitution of a leading edge.
Submitted by: Guenther Gerisch [gerisch@biochem.mpg.de]
-----------------------------------------------------------------------------
Chemotaxis of aggregating Dictyostelium cells
Guenther Gerisch, and Mary Ecke
Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany
In: Key Experiments in Practical Developmental Biology
(Eds. Manuel Mar-Beffa and Jennifer Knight)
Cambridge University Press, in press
Objective of the Experiment
In the course of Dictyostelium development, a multicellular organism is
established by the aggregation of single cells. In the following experiment,
the chemoattractant that guides cell movement in an aggregation field is
replaced by treating cells with cyclic AMP diffusing out of a micropipette.
By the use of a micropipette that is easily moved by a micromanipulator, the
direction of diffusion gradients can be changed fast enough to study the
response of the cells within the first few seconds of reorientation.
Submitted by: Guenther Gerisch [gerisch@biochem.mpg.de]
-----------------------------------------------------------------------------
GUANYLYL CYCLASES ACROSS THE TREE OF LIFE
Pauline Schaap
School of Life Sciences, University of Dundee, UK
Frontiers in Bioscience, in press
TABLE OF CONTENTS
1. Abstract
2. Introduction
3. Animals
3.1. The cyclase catalytic domain
3.2. Vertebrate cGMP signaling, a brief survey
3.3. Invertebrate guanylyl cyclases
4. Fungi
5. Dictyostelids
6. Plants
6.1. Flowering plants
6.2. Chlorophyte green algae
7. Alveolates
8. Discicristates
9. Prokaryotes
10. Summary and perspective
11. Acknowledgement
12. References
1. Abstract
This review explores the origins, diversity and functions of guanylyl cyclases
in cellular organisms. In eukaryotes both cGMP and cAMP are produced by the
conserved class III cyclase domains, while prokaryotes use five more unrelated
catalysts for cyclic nucleotide synthesis. The class III domain is found
embedded in proteins with a large variety of membrane topologies and other
functional domains, but the vertebrate guanylyl cyclases take only two forms,
the receptor guanylyl cyclases with single transmembrane domain and the
soluble enzymes with heme binding domain. The invertebrates additionally show
a soluble guanylyl cyclase that cannot bind heme, while the more basal
metazoans may lack the heme binding enzymes altogether. Fungi, the closest
relatives of the metazoans, completely lack guanylyl cyclases, but they appear
again in the Dictyostelids, the next relative in line. Remarkably, the two
Dictyostelid guanylyl cyclases have little in common with the vertebrate
enzymes. There is a soluble guanylyl cyclase, which shows greatest sequence
and structural similarity to the vertebrate soluble adenylyl cyclase, and a
membrane-bound form with the same configuration as the dodecahelical adenylyl
cyclases of vertebrates. There is a difference, the pseudosymmetric C1 and C2
catalytic domains have swapped position in the Dictyostelium enzyme. Unlike
the vertebrate guanylyl cyclases, the Dictyostelium enzymes are activated by
heterotrimeric G-proteins. Swapped C1 and C2 domains are also found in the
structurally similar guanylyl cyclases of ciliates and apicomplexans, but
these enzymes additionally harbour an amino-terminal ATPase module with ten
transmembrane domains. G-protein regulation could not be demonstrated for
these enzymes. Higher plants lack class III cyclase domains, but an
unexplored wealth of guanylyl cyclases is present in the green alga
Chlamydomonas. Progenitors of all structural variants of the eukaryote
guanylyl cyclases are found among the prokaryote adenylyl cyclases. This
and the close similarity of many guanylyl cyclases to adenylyl cyclases
suggests a paraphyletic origin for the eukaryote enzymes with multiple
events of conversion of substrate specificity.
Submitted by: Pauline Schaap [p.schaap@dundee.ac.uk]
-----------------------------------------------------------------------------
A Rapid and Efficient Method to Generate Multiple Gene Disruptions in
Dictyostelium Using a Single Selectable Marker and the Cre-loxP System
Jan Faix ^ ", Lisa Kreppel * ` ", Gad Shaulsky +,
Michael Schleicher ^, and Alan R. Kimmel *
* Laboratory of Cellular and Developmental Biology, NIDDK
National Institutes of Health, Bethesda, MD 20892-8028, USA
^ A. Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-Universitt
80336 Mnchen, Germany
+ Department of Molecular and Human Genetics
Baylor College of Medicine, Houston, TX 77030, USA
Nucelic Acids Research, in press
Dictyostelium has proven an exceptionally powerful system for studying
numerous aspects of cellular and developmental functions. The relatively
small (~34 Mb) chromosomal genome of Dictyostelium and high efficiency of
targeted gene disruption have enabled researchers to characterize many
specific gene functions. However, the number of selectable markers in
Dictyostelium is restricted, as is the ability to perform effective
genetic crosses between strains. Thus, it has been difficult to create
multiple mutations within an individual cell to study epistatic
relationships among genes or potential redundancies between various
pathways. We now describe a robust system for the production of multiple
gene mutations in Dictyostelium by recycling a single selectable marker,
Blasticidin S-resistance, using the Cre-loxP system. We confirm the
effectiveness of the system by generating a single cell carrying 4 separate
gene disruptions. Furthermore, the cells remain sensitive to transformation
for additional targeted or random mutagenesis requiring Blasticidin
selection and for functional expression studies of mutated or tagged
proteins using other selectable markers.
Submitted by: Alan Kimmel [ark1@helix.nih.gov]
Please Note: all vectors essential for the procedure will be made available
thru the Dicty Stock Center - Alan
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[End Dicty News, volume 23, number 10] 
