dictyNews
Electronic Edition
Volume 37, number 14
December 2, 2011
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Abstracts
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Actin Crosslinking Proteins, Cortexillin I and II, Are Required for
cAMP-signaling during Dictyostelium Chemotaxis and Development
Shi Shu,* Xiong Liu,* Paul W. Kriebel,† Mathew P. Daniels,‡ and
Edward D. Korn*
*Laboratory of Cell Biology, NHLBI, National Institutes of Health,
Bethesda, Maryland 20892, USA.
†Laboratory of Cellular and Molecular Biology, Center for Cancer
Research, NCI, National Institutes of Health, Bethesda, Maryland 20892,
USA.
‡Electron Microscopy Core Facility, NHLBI, National Institutes of Health,
Bethesda, Maryland 20892, USA.
Molecular Biology of the Cell, in press
Starvation induces Dictyostelium amoebae to secrete cAMP towards which
other amoebae stream forming multi-cellular mounds that differentiate and
develop into fruiting bodies containing spores. We find that the double
deletion of cortexillin (ctx) I and II alters the actin cytoskeleton and
substantially inhibits all molecular responses to extracellular cAMP.
Synthesis of cAMP-receptor and adenylyl cyclase A (ACA) is inhibited and
activation of ACA, RasC and RasG, phosphorylation of ERK2, activation of
TORC2 and stimulation of actin polymerization and myosin assembly are
greatly reduced. As a consequence, cell streaming and development are
completely blocked. Expression of ACA-YFP in the ctxI/ctxII-null cells
significantly rescues the wild-type phenotype indicating that the primary
chemotaxis and development defect is the inhibition of ACA synthesis and
cAMP production. These results demonstrate the critical importance of a
properly organized actin cytoskeleton for cAMP-signaling pathways,
chemotaxis and development in Dictyostelium.
Submitted by Edward Korn [[log in to unmask]]
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Functional characterization of CP148, a novel key component for
centrosome integrity in Dictyostelium
Oliver Kuhnert, Otto Baumann, Irene Meyer and Ralph Gräf*
University of Potsdam, Institute for Biochemistry and Biology, Dept.
of Cell Biology, D-14476 Potsdam-Golm, Germany
Cell. Mol. Life Sci., in press
The Dictyostelium centrosome consists of a layered core structure
surrounded by a microtubule-nucleating corona. A tight linkage through
the nuclear envelope connects the cytosolic centrosome with the
clustered centromeres within the nuclear matrix. At G2/M the corona
dissociates, and the core structure duplicates yielding two spindle
poles. CP148 is a novel coiled coil protein of the centrosomal corona.
GFP-CP148 exhibited cell cycle-dependent presence and absence at
the centrosome, which correlates with dissociation of the corona in
prophase and its reformation in late telophase. During telophase,
GFP-CP148 formed cytosolic foci, which coalesced and joined the
centrosome. This explains the hypertrophic appearance of the corona
upon strong overexpression of GFP-CP148. Depletion of CP148 by
RNAi caused virtual loss of the corona and disorganization of interphase
microtubules. Surprisingly, formation of the mitotic spindle and astral
microtubules was unaffected. Thus, microtubule nucleation complexes
associate with centrosomal core components through different means
during interphase and mitosis. Furthermore, CP148 RNAi caused
dispersal of centromeres and altered Sun1 distribution at the nuclear
envelope, suggesting a role of CP148 in the linkage between
centrosomes and centromeres. Taken together, CP148 is an essential
factor for the formation of the centrosomal corona, which in turn is
required for centrosome/centromere linkage.
Submitted by Ralph Gräf [[log in to unmask]]
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The Skp1 Prolyl 4-hydroxylase of Dictyostelium Mediates
Glycosylation-Independent and -Dependent Responses to O2
without Affecting Skp1 Stability
Dongmei Zhang (1,2), Hanke van der Wel (1), Jennifer M. Johnson (3),
Christopher M. West (1,3)
(1) Dept. of Biochemistry & Molecular Biology, University of Oklahoma
Health Sciences Center, 975 NE 10th St., Oklahoma City, OK, 73104,
(2) Key Laboratory of Biopesticide and Chemical Biology, Ministry of
Education, Fujian Agriculture and Forestry University, Fuzhou 350002,
PR China,
(3) Oklahoma Center for Medical Glycobiology, University of Oklahoma
Health Sciences Center, 975 NE 10th St., Oklahoma City, OK, 73104
J. Biol. Chem., in press
Cytoplasmic prolyl 4-hydroxylases (PHDs) have a primary role in O2-sensing
in animals via modification of the transcriptional factor subunit HIFalpha, resulting
in its poly-ubiquitination by the E3VHLubiquitin (Ub)-ligase and degradation in the
26S-proteasome. Previously thought to be restricted to animals, a homolog (P4H1)
of HIFalpha-type PHDs is expressed in the social amoeba Dictyostelium where it
also exhibits characteristics of an O2-sensor: for development. Dictyostelium lacks
HIFalpha and P4H1 modifies a different protein, Skp1, an adaptor of the SCF-class
of E3-Ub ligases related to the E3VHLUb-ligase that targets animal HIFalpha.
Normally, the HO-Skp1 product of the P4H1 reaction is capped by a GlcNAc sugar
that can be subsequently extended to a pentasaccharide by novel
glycosyltransferases. To analyze the role of glycosylation, the Skp1 GlcNAc-
transferase locus gnt1 was modified with a missense mutation to block catalysis or
a stop codon to truncate the protein. Despite the accumulation of the hydroxylated
form of Skp1, Skp1 was not destabilized based on metabolic labeling. However,
hydroxylation alone allowed for partial correction of the high O2-requirement of
P4H1-null cells, therefore revealing both glycosylation-independent and -dependent
roles for hydroxylation. Genetic complementation of the latter function required an
enzymatically active form of Gnt1. Since the effect of the gnt1-deficiency depended
on P4H1, and Skp1 was the only protein labeled when the GlcNAc-transferase was
restored to mutant extracts, Skp1 apparently mediates the cellular functions of both
P4H1 and Gnt1. Though Skp1 stability itself is not affected by hydroxylation, its
modification may affect the stability of targets of Skp1-dependent Ub-ligases.
Submitted by Chris West [[log in to unmask]]
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[End dictyNews, volume 37, number 14]
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