Leucine-rich repeat kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinson's disease
and is also linked to its idiopathic form
. LRRK2 has been proposed to function in membrane ...trafficking
and colocalizes with microtubules
. Despite the fundamental importance of LRRK2 for understanding and treating Parkinson's disease, structural information on the enzyme is limited. Here we report the structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported cryo-electron tomography in situ structure
. We propose that the conformation of the LRRK2 kinase domain regulates its interactions with microtubules, with a closed conformation favouring oligomerization on microtubules. We show that the catalytic half of LRRK2 is sufficient for filament formation and blocks the motility of the microtubule-based motors kinesin 1 and cytoplasmic dynein 1 in vitro. Kinase inhibitors that stabilize an open conformation relieve this interference and reduce the formation of LRRK2 filaments in cells, whereas inhibitors that stabilize a closed conformation do not. Our findings suggest that LRRK2 can act as a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.
Cytoplasmic dynein and kinesin-1 are microtubule-based motors with opposite polarity that transport a wide variety of cargo in eukaryotic cells. Many cellular cargos demonstrate bidirectional ...movement due to the presence of ensembles of dynein and kinesin, but are ultimately sorted with spatial and temporal precision. To investigate the mechanisms that coordinate motor ensemble behavior, we built a programmable synthetic cargo using three-dimensional DNA origami to which varying numbers of DNA oligonucleotide-linked motors could be attached, allowing for control of motor type, number, spacing, and orientation in vitro. In ensembles of one to seven identical-polarity motors, motor number had minimal affect on directional velocity, whereas ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one motor species.
Abstract
KM3NeT, a neutrino telescope currently under construction in the Mediterranean Sea, consists of a network of large-volume Cherenkov detectors. Its two different sites, ORCA and ARCA, are ...optimised for few GeV and TeV-PeV neutrino energies, respectively. This allows for studying a wide range of physics topics spanning from the determination of the neutrino mass hierarchy to the detection of neutrinos from astrophysical sources. Deep learning techniques provide promising methods to analyse the signatures induced by charged particles traversing the detector. This document will cover a deep learning based approach using graph convolutional networks to classify and reconstruct events in both the ORCA and ARCA detector. Performance studies on simulations as well as applications to real data will be presented, together with comparisons to classical approaches.
Cytoplasmic dynein is a microtubule-based motor required for intracellular transport and cell division. Its movement involves coupling cycles of track binding and release with cycles of ...force-generating nucleotide hydrolysis. How this is accomplished given the ∼25 nanometers separating dynein's track- and nucleotide-binding sites is not understood. Here, we present a subnanometer-resolution structure of dynein's microtubule-binding domain bound to microtubules by cryo—electron microscopy that was used to generate a pseudo-atomic model of the complex with molecular dynamics. We identified large rearrangements triggered by track binding and specific interactions, confirmed by mutagenesis and single-molecule motility assays, which tune dynein's affinity for microtubules. Our results provide a molecular model for how dynein's binding to microtubules is communicated to the rest of the motor.
Summary Surgical site infections (SSIs) significantly increase post-operative morbidity and mortality. SSI surveillance is an established monitoring tool and reduces SSI rates. The purpose of this ...study was to compare prospective in-hospital SSI surveillance (I) by the surgical staff and (II) additionally by an infection control team (ICT). The reference method (III) was defined by data generated by the surgical team, supplemented by the ICT and completed by post-discharge surveillance with a post-operative follow-up of one year representing the sum of all available resources. During 24 months, all consecutive inpatient procedures ( N = 6283) were prospectively recorded by the surgical staff until patients' discharge (I). SSI rates were compared with the surveillance performed by the ICT (II) and with the reference method (III). The overall SSI rate (reference method) was 4.7% ( N = 293), of which 187 (63.8%) were detected in-hospital and 106 (36.2%) after discharge. (I) The surgical staff detected 91/187 (48.7%) of in-hospital SSIs 91/293 (31.0%) of the reference, (II) the ICT an additional 96/187 (51.3%) during hospitalisation 96/293 (32.8%) of the reference. Further cross-checking as performed in the visceral surgery department increased the surgeons' detection rate (I) to 59/105 (56.2%) of in-hospital SSIs 59/147 (40.1%) of the reference. SSI surveillance by the surgical staff detects almost half of all in-hospital SSIs and has the potential to increase the detection rate by simple interventions such as cross-checking. Such a relatively inexpensive surveillance system is an option for hospitals without an ICT or for low risk surgical procedures. Moreover, trends in SSI rates can easily be detected, allowing early intervention.
The existence and function of actin in the nucleus has been hotly debated
for forty years. Recently, β-actin was found to be a component of
mammalian SWI/SNF-like BAF chromatin remodeling complexes ...and still more
recently other SWI/SNF-related chromatin remodeling complexes in yeast, flies,
and man. Although the function of actin in these chromatin remodeling complexes
is only starting to be explored, the fact that actin is one of the most
regulated proteins in the cell suggests that control of nuclear actin may be a
critical regulatory point in the control of chromatin remodeling. Actin rapidly
shuttles between the nucleus and the cytoplasm offering additional sites and
modes of regulation. In addition, actin-related proteins (Arps) are also
components of these chromatin remodeling complexes and have been implicated in
transcriptional control in yeast. The observation that the BAF chromatin
remodeling complex in which actin was originally identified, is also a human
tumor suppressor complex necessary for the actions of the retinoblastoma
protein indicates that the study of nuclear actin is likely to contribute to
understanding cell growth control.
Celotno besedilo
Dostopno za:
CMK, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
ABSTRACT
Addressing the origin of the astrophysical neutrino flux observed by IceCube is of paramount importance. Gamma-Ray Bursts (GRBs) are among the few astrophysical sources capable of achieving ...the required energy to contribute to such neutrino flux through pγ interactions. In this work, ANTARES data have been used to search for upward going muon neutrinos in spatial and temporal coincidence with 784 GRBs occurred from 2007 to 2017. For each GRB, the expected neutrino flux has been calculated in the framework of the internal shock model and the impact of the lack of knowledge on the majority of source redshifts and on other intrinsic parameters of the emission mechanism has been quantified. It is found that the model parameters that set the radial distance where shock collisions occur have the largest impact on neutrino flux expectations. In particular, the bulk Lorentz factor of the source ejecta and the minimum variability time-scale are found to contribute significantly to the GRB-neutrino flux uncertainty. For the selected sources, ANTARES data have been analysed by maximizing the discovery probability of the stacking sample through an extended maximum-likelihood strategy. Since no neutrino event passed the quality cuts set by the optimization procedure, 90 per cent confidence level upper limits (with their uncertainty) on the total expected diffuse neutrino flux have been derived, according to the model. The GRB contribution to the observed diffuse astrophysical neutrino flux around 100 TeV is constrained to be less than 10 per cent.
The motor protein cytoplasmic dynein is responsible for most of the minus-end-directed microtubule traffic within cells. Dynein contains four evolutionarily conserved AAA (ATPase associated with ...various cellular activities) domains that are thought to bind nucleotide; the role of nucleotide binding and hydrolysis in each of these four AAA domains has constituted an important and unresolved question in understanding dynein's mechanism. Using Saccharomyces cerevisiae cytoplasmic dynein as a model system, we mutagenized residues involved in nucleotide binding or hydrolysis in the four AAA domains and examined the ability of the mutant dyneins to mediate nuclear segregation in vivo and to bind micro-tubules in vitro. Our analysis shows that an AAA1 hydrolysis mutant blocks dynein function, whereas a triple AAA2/3/4 hydrolysis mutant does not, suggesting that nucleotide binding is required at only one site. We also show that nucleotide binding at AAA3, but not hydrolysis, is essential for motor activity in vivo and ATP-induced dissociation of dynein from microtubules, suggesting that this domain acts as a critical allosteric site. In contrast, mutations in AAA2 cause subtle defects in dynein function, whereas mutation in AAA4 produce no obvious defects. These results show that the four conserved dynein AAA domains have distinct functions in dynein's mechanochemical cycle.
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