589 research outputs found
Using in vivo-biotinylated ubiquitin to describe a mitotic exit ubiquitome from human cells
Mitotic division requires highly regulated morphological and biochemical changes to the cell. Upon commitment to exit mitosis, cells begin to remove mitotic regulators in a temporally and spatially controlled manner to bring about the changes that re-establish interphase. Ubiquitin-dependent pathways target these regulators to generate polyubiquitin-tagged substrates for degradation by the 26S proteasome. However, the lack of cell-based assays to investigate in vivo ubiquitination limits our knowledge of the identity of substrates of ubiquitin-mediated regulation in mitosis. Here we report an in vivo ubiquitin tagging system in human cells that allows efficient purification of ubiquitin conjugates from synchronised cell populations. Coupling purification with mass spectrometry, we identify a series of mitotic regulators that are targeted for polyubiquitination in mitotic exit. We show that some are new substrates of the Anaphase Promoting Complex/Cyclosome (APC/C), and validate KIFC1 and RacGAP1/Cyk4 as two such targets involved respectively in timely mitotic spindle disassembly and cell spreading. We conclude that in vivo biotin-tagging of ubiquitin can provide valuable information about the role of ubiquitin-mediated regulation in processes required for rebuilding interphase cells
Long-pulse laser launch and ionization of tailored large neutral silver nanoparticles with atomic mass assignment
We explore the synthesis, characterization, neutral launch and vacuum ultraviolet ionization of massive perfluorinated-alkyl-capped nanoparticles. The presence of the ligand coating in solution is corroborated by Fourier transform infrared spectroscopy (FT-IR) and the particle size distribution is analyzed by transmission electron microscopy (TEM). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identifies perfluoralkyl coated silver nanoparticles as the most stable species among the materials studied here. They can be launched in high vacuum using long-pulse low-power laser heating-orders of magnitude below typical thresholds for laser desorption. Energy-dispersive X-ray spectroscopy (EDX) of the recaptured silver clusters confirms the expected elemental distribution. Volatilization with subsequent ionization of the neutral nanoparticle beam in high vacuum by 157 nm light allows analyzing their mass with atomic resolution
Quantitative proteomic analysis of Parkin substrates in Drosophila neurons.
Parkin (PARK2) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson's Disease (PD). In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1. PINK1 activates Parkin activity leading to ubiquitination of multiple proteins, which in turn promotes clearance of mitochondria by mitophagy. Many substrates have been identified using cell culture models in combination with depolarising drugs or proteasome inhibitors, but not in more physiological settings.Here we utilized the recently introduced BioUb strategy to isolate ubiquitinated proteins in flies. Following Parkin Wild-Type (WT) and Parkin Ligase dead (LD) expression we analysed by mass spectrometry and stringent bioinformatics analysis those proteins differentially ubiquitinated to provide the first survey of steady state Parkin substrates using an in vivo model. We further used an in vivo ubiquitination assay to validate one of those substrates in SH-SY5Y cells.We identified 35 proteins that are more prominently ubiquitinated following Parkin over-expression. These include several mitochondrial proteins and a number of endosomal trafficking regulators such as v-ATPase sub-units, Syx5/STX5, ALiX/PDCD6IP and Vps4. We also identified the retromer component, Vps35, another PD-associated gene that has recently been shown to interact genetically with parkin. Importantly, we validated Parkin-dependent ubiquitination of VPS35 in human neuroblastoma cells.Collectively our results provide new leads to the possible physiological functions of Parkin activity that are not overtly biased by acute mitochondrial depolarisation
Vibrational spectrum of topologically disordered systems
The topological nature of the disorder of glasses and supercooled liquids
strongly affects their high-frequency dynamics. In order to understand its main
features, we analytically studied a simple topologically disordered model,
where the particles oscillate around randomly distributed centers, interacting
through a generic pair potential. We present results of a resummation of the
perturbative expansion in the inverse particle density for the dynamic
structure factor and density of states. This gives accurate results for the
range of densities found in real systems.Comment: Completely rewritten version, accepted in Physical Review Letter
Nonequilibrium functional RG with frequency dependent vertex function: A study of the single impurity Anderson model
We investigate nonequilibrium properties of the single impurity Anderson
model by means of the functional renormalization group (fRG) within Keldysh
formalism. We present how the level broadening Gamma/2 can be used as flow
parameter for the fRG. This choice preserves important aspects of the Fermi
liquid behaviour that the model exhibits in case of particle-hole symmetry. An
approximation scheme for the Keldysh fRG is developed which accounts for the
frequency dependence of the two-particle vertex in a way similar but not
equivalent to a recently published approximation to the equilibrium Matsubara
fRG. Our method turns out to be a flexible tool for the study of weak to
intermediate on-site interactions U <= 3 Gamma. In equilibrium we find
excellent agreement with NRG results for the linear conductance at finite gate
voltage, magnetic field, and temperature. In nonequilibrium, our results for
the current agree well with TD-DMRG. For the nonlinear conductance as function
of the bias voltage, we propose reliable results at finite magnetic field and
finite temperature. Furthermore, we demonstrate the exponentially small scale
of the Kondo temperature to appear in the second order derivative of the
self-energy. We show that the approximation is, however, not able to reproduce
the scaling of the effective mass at large interactions.Comment: [v2] - minor changes throughout the text; added new Fig. 3; corrected
pert.-theory data in Figs. 10, 11; published versio
Dynamic structure factor of the Ising model with purely relaxational dynamics
We compute the dynamic structure factor for the Ising model with a purely
relaxational dynamics (model A). We perform a perturbative calculation in the
expansion, at two loops in the high-temperature phase and at one
loop in the temperature magnetic-field plane, and a Monte Carlo simulation in
the high-temperature phase. We find that the dynamic structure factor is very
well approximated by its mean-field Gaussian form up to moderately large values
of the frequency and momentum . In the region we can investigate,
, , where is the correlation
length and the zero-momentum autocorrelation time, deviations are at
most of a few percent.Comment: 21 pages, 3 figure
Pushing the mass limit for intact launch and photoionization of large neutral biopolymers
Since their first discovery by Louis Dunoyer and Otto Stern, molecular beams have conquered research and technology. However, it has remained an outstanding challenge to isolate and photoionize beams of massive neutral polypeptides. Here we show that femtosecond desorption from a matrix-free sample in high vacuum can produce biomolecular beams at least 25 times more efficiently than nanosecond techniques. While it has also been difficult to photoionize large biomolecules, we find that tailored structures with an abundant exposure of tryptophan residues at their surface can be ionized by vacuum ultraviolet light. The combination of these desorption and ionization techniques allows us to observe molecular beams of neutral polypeptides with a mass exceeding 20,000 amu. They are composed of 50 amino acids – 25 tryptophan and 25 lysine residues – and 26 fluorinated alkyl chains. The tools presented here offer a basis for the preparation, control and detection of polypeptide beams
A new test for random number generators: Schwinger-Dyson equations for the Ising model
We use a set of Schwinger-Dyson equations for the Ising Model to check
several random number generators. For the model in two and three dimensions, it
is shown that the equations are sensitive tests of bias originated by the
random numbers. The method is almost costless in computer time when added to
any simulation.Comment: 6 pages, 3 figure
The search for magnetic fields in mercury-manganese stars
We performed a highly sensitive search for magnetic fields on a large set of
HgMn stars. With the aid of a new polarimeter attached to the HARPS
spectrometer at the ESO 3.6m-telescope, we obtained high-quality circular
polarization spectra of 41 single and double HgMn stars. Using a multi-line
analysis technique on each star, we co-added information from hundreds of
spectral lines resulting in significantly greater sensitivity to the presence
of magnetic fields, including very weak fields. For the 47 individual objects
studied, including 6 components of SB2 systems, we do not detect any magnetic
fields at greater than the 3 sigma level. The lack of detection in the circular
polarization profiles indicates that if strong fields are present on these
stars, they must have complex surface topologies. For simple global fields, our
detection limits imply upper limits to the fields present of 2-10 Gauss in the
best cases. We conclude that HgMn stars lack large-scale magnetic fields,
typical for spotted magnetic Ap stars, sufficient to form and sustain the
chemical spots observed on HgMn stars. Our study confirms that in addition to
magnetically altered atomic diffusion, there exists another differentiation
mechanism operating in the atmospheres of late-B main sequence stars which can
compositional inhomogeneities on their surfaces.Comment: 12 pages, 8 figures, 2 table
- …