dictyNews
Electronic Edition
Volume 38, number 25
September 28, 2012
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Abstracts
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Heteromeric p97/p97R155C complexes induce dominant negative changes
in wild-type and autophagy 9-deficient Dictyostelium strains
Khalid Arhzaouy1, Karl-Heinz Strucksberg1,2, Sze Man Tung1, Karthikeyan
Tangavelou1, Maria Stumpf1, Jan Faix3, Rolf Schröder2, Christoph S.
Clemen1, and Ludwig Eichinger1
1Intitute for Biochemistry I, Medical Faculty, University of Cologne,
Cologne, Germany
2Institute of Neuropathology, University Hospital Erlangen, Erlangen, Germany
3Institute for Biophysical Chemistry, Hannover Medical School, Hannover,
Germany
PLos ONE, in press
Heterozygous mutations in the human VCP (p97) gene cause autosomal-dominant
IBMPFD (inclusion body myopathy with early onset Paget's disease of bone
and frontotemporal dementia), ALS14 (amyotrophic lateral sclerosis with or
without frontotemporal dementia) and HSP (hereditary spastic paraplegia).
Most prevalent is the R155C point mutation. We studied the function of p97
in the social amoeba Dictyostelium discoideum and have generated strains
that ectopically express wild-type (p97) or mutant p97 (p97R155C) fused to
RFP in AX2 wild-type and autophagy 9 knock-out (ATG9KO) cells. Native gel
electrophoresis showed that both p97 and p97R155C assemble into hexamers.
Co-immunoprecipitation studies revealed that endogenous p97 and
p97R155C-RFP form heteromers. The mutant strains displayed changes in cell
growth, phototaxis, development, proteasomal activity, ubiquitinylated
proteins, and ATG8(LC3) indicating mis-regulation of multiple essential
cellular processes. Additionally, immunofluorescence analysis revealed an
increase of protein aggregates in ATG9KO/p97R155C-RFP and ATG9KO cells.
They were positive for ubiquitin in both strains, however, solely
immunoreactive for p97 in the ATG9KO mutant. A major finding is that the
expression of p97R155C-RFP in the ATG9KO strain partially or fully rescued
the pleiotropic phenotype. We also observed dose-dependent effects of p97
on several cellular processes. Based on findings in the single versus the
double mutants we propose a novel mode of p97 interaction with the core
autophagy protein ATG9 which is based on mutual inhibition.
Submitted by Ludwig Eichinger [[log in to unmask]]
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Fast Characterization of Cell-Derived Extracellular Vesicles by Nanoparticles
Tracking Analysis, Cryo-Electron Microscopy and Raman Tweezers
Microspectroscopy
Irène Tatischeff1*, Eric Larquet2, Juan M. Falcón-Pérez3, Pierre-Yves Turpin1
and Sergei G. Kruglik1
Journal of Extracellular Vesicles, in press
The joint use of three complementary techniques, i.e. Nanoparticles Tracking
Analysis, Cryo-Electron Microscopy and Raman Tweezers Microspectroscopy,
is proposed for a rapid characterization of extracellular vesicles of various
origins. Nanoparticles Tracking Analysis is valuable for studying the size
distribution and concentration, Cryo-Electron Microscopy is outstanding for the
morphological characterization, including observation of vesicles heterogeneity,
while Raman Tweezers Microspectroscopy provides the global chemical
composition without using any exogenous label. The capabilities of this
approach are evaluated on the example of cell-derived vesicles of Dictyostelium
discoideum, a convenient general model for eukaryotic extracellular vesicles. At
least two separate species differing in chemical composition (relative amounts
of DNA, lipids and proteins, presence of carotenoids) were found for each of the
two physiological states of this non-pathogenic microorganism, i.e. cell growth
and starvation-induced aggregation. These findings demonstrate the specific
potency of Raman Tweezers Microspectroscopy. In addition, the first Raman
spectra of human urinary exosomes are reported, presumably constituting the
primary step towards Raman characterization of extracellular vesicles for the
purpose of human diseases diagnosis.
Submitted by Irène Tatischeff [[log in to unmask]]
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Rules of engagement: centrosome-nuclear connections in a closed mitotic system
Meredith Leo, Diana Santino, Irina Tikhonenko, Valentin Magidson,
Alexey Khodjakov, and Michael P. Koonce.
Biology Open, in press
The assembly of a functional mitotic spindle is essential for cell reproduction
and requires a precise coordination between the nuclear cycle and the
centrosome. This coordination is particularly prominent in organisms that
undergo closed mitosis where centrosomes must not only respond to temporal
signals, but also to spatial considerations, e.g. switching from the production
of cytoplasmic microtubule arrays to the generation of dynamic intra-nuclear
microtubules required for spindle assembly. We utilize a geneknockout of Kif9,
a Dictyostelium discoideum Kin-I kinesin, to destabilize the physical association
between centrosomes and the nuclear envelope. This approach presents a
unique opportunity to reveal temporal and spatial components in the regulation
of centrosomal activities in a closed-mitosis organism. Here we report that
centrosome-nuclear engagement is not required for the entry into mitosis.
Although detached centrosomes can duplicate in the cytoplasm, neither they
nor nuclei alone can produce spindle-like microtubule arrays. However, the
physical association of centrosomes and the nuclear envelope is required to
progressthrough mitosis beyond prometaphase.
Submitted by Michael Koonce [[log in to unmask]]
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[End dictyNews, volume 38, number 25]
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