APC couples neuronal mRNAs to multiple kinesins,EB1, and shrinking microtubule ends for bidirectional mRNA motility

Understanding where in the cytoplasm mRNAs are translated is increasingly recognized as being as important as knowing the timing and level of protein expression. mRNAs are localized via active motor-driven transport along microtubules (MTs) but the underlying essential factors and dynamic interactions are largely unknown. Using biochemical in vitro reconstitutions with purified mammalian proteins, multicolor TIRF-microscopy, and interaction kinetics measurements, we show that adenomatous polyposis coli (APC) enables kinesin-1- and kinesin-2-based mRNA transport, and that APC is an ideal adaptor for long-range mRNA transport as it forms highly stable complexes with 3′UTR fragments of several neuronal mRNAs (APC–RNPs). The kinesin-1 KIF5A binds and transports several neuronal mRNP components such as FMRP, PURα and mRNA fragments weakly, whereas the transport frequency of the mRNA fragments is significantly increased by APC. APC–RNP-motor complexes can assemble on MTs, generating highly processive mRNA transport events. We further find that end-binding protein 1 (EB1) recruits APC–RNPs to dynamically growing MT ends and APC–RNPs track shrinking MTs, producing MT minus-end-directed RNA motility due to the high dwell times of APC on MTs. Our findings establish APC as a versatile mRNA-kinesin adaptor and a key factor for the assembly and bidirectional movement of neuronal transport mRNPs.

The spatial control of gene expression via local translation of mRNAs is an essential prerequisite for the maintenance of cell polarity (1, 2), cell development, cell motility (3, 4), and the cotranslational assembly of protein complexes (5). In mammalian neurons, hundreds of mRNAs need to be distributed via microtubule (MT) -dependent, motor protein-driven transport in the form of small packages (6, 7) into a complex network of dendritic branches and the axon until the finest filopodia tips (8–10). As cells control intracellular cargo distribution through a combination of MT-associated proteins (MAPs) and MT post-translational modifications (PTMs) which enable or block the activity of distinct motors (11–13), it is expected that the complete localization process of an mRNA requires either several different adaptors for different motors or a versatile adaptor that can bind several motors (14). For instance in Xenopus laevis, a combination of a kinesin-1 and a kinesin-2 motor is required to localize Vg1 mRNA (15).Arriving at a destination such as filopodia (16) or axonal branch points (17), mRNAs further need to be deposited or “anchored” at cellular structures of the cell cortex at locations where the encoded protein is needed. To date, a major mechanism known to deposit polarity factors at cell cortical elements is microtubule end-binding protein (EB)-dependent MT plus-end-tracking (18–20). MT plus-end-tracking creates transient density waves of plus-end-tracking proteins around polymerizing MT ends (21), and these high local concentrations are thought to drive the formation of polarity factor complexes at the cortex (22, 23). Hence, the question arises whether a mechanism exists that enables mRNA deposition at the cortex, in a similar way as for protein deposition, through MT plus-end–tracking-dependent, concentration-driven complex assembly. While a trailing of mRNPs behind polymerizing MT ends has been observed in axonal growth cones (16), a mechanism that could enable mRNP MT plus-end-tracking or trailing remains unknown.Identifying a versatile adaptor which on the one hand specifically recognizes localization elements of mRNAs and on the other hand can also bind to different kinesins and eventually EB proteins would close a major gap in our understanding of how mRNPs could be distributed in mammalian cells. While we identified adenomatous polyposis coli (APC) as an mRNA adaptor that directly binds G-rich 3′UTR elements and the kinesin-2 KIF3AB-KAP3 (“KIF3ABK”) with high affinities (24), more evidence exists from methods not proving direct interactions which hints toward an alternative, kinesin-1-based transport route for APC (25). This is of high interest, as the kinesin-1 KIF5 was long thought to be a major motor for mRNP transport in different mammalian cell types (26–28) but a minimal adaptor sufficient to couple mRNAs to KIF5 is not known. Further, APC interacts with EB proteins (SI Appendix, Fig. S1A, (29)) and tracks polymerizing MT plus ends (30), which predicts that APC–mRNPs potentially localize to polymerizing MT ends. mRNP plus-end-tracking would be a previously unconsidered mechanism to concentrate mRNPs locally, which could promote concentration-driven mRNP remodeling or anchoring of mRNPs at cortical cell structures for subsequent local protein production.Using a combination of biochemical in vitro reconstitutions with pure proteins and RNA fragments (31), in vitro motility assays, in vitro microtubule dynamics assays (32), and surface plasmon resonance (SPR) interaction measurements, we show that APC couples neuronal mRNAs to the kinesin-1 KIF5A and the MT plus-end-tracking protein EB1. This results in kinesin-1-driven, fast, and highly processive plus-end-directed APC–RNP motility and EB1-mediated concentration of APC–RNPs at MT plus ends. In addition, we found that lattice-diffusing APC–RNPs can track shrinking MT ends resulting in minus end-directed RNA motility. Thus, APC is a versatile adaptor that links multiple G-rich fragments from at least four different neuronal mRNA-3′UTRs to KIF3ABK, KIF5A, and EB1, enabling bidirectional mRNP movements and potentially permitting APC–RNPs to access a wide range of dendritic and axonal segments.     

West Nile Virus Antibody Prevalence in Horses in Ukraine

West Nile virus (WNV) is a mosquito-borne virus of global importance. Over the last two decades, it has been responsible for significant numbers of cases of illness in humans and animals in many parts of the world. In Ukraine, WNV infections in humans and birds were first reported more than 25 years ago, yet the current epidemiological status is quite unclear. In this study, serum samples from over 300 equines were collected and screened in order to detect current WNV activity in Ukraine with the goal to estimate the risk of infection for humans and horses. Sera were tested by enzyme-linked immunosorbent assay (ELISA) and virus neutralization assay (NT) to detect WNV-specific antibodies. The results clearly revealed that WNV circulates in most of the regions from which samples were obtained, shown by a WNV seroprevalence rate of 13.5% of examined horses. This is the first topical report indicating the presence of WNV infections in horses in Ukraine, and the results of this study provide evidence of a widespread WNV circulation in this country.     

A comparison of the grip force distribution in natural hands and in prosthetic hands

Purpose:?The aim of this study is to analyse the grip force distribution for different prosthetic hand designs and the human hand fulfilling a functional task.

Method:?A cylindrical object is held with a power grasp and the contact forces are measured at 20 defined positions. The distributions of contact forces in standard electric prostheses, in a experimental prosthesis with an adaptive grasp, and in human hands as a reference are analysed and compared. Additionally, the joint torques are calculated and compared.

Results:?Contact forces of up to 24.7?N are applied by the middle and distal phalanges of the index finger, middle finger, and thumb of standard prosthetic hands, whereas forces of up to 3.8?N are measured for human hands. The maximum contact forces measured in a prosthetic hand with an adaptive grasp are 4.7?N. The joint torques of human hands and the adaptive prosthesis are comparable.

Conclusions:?The analysis of grip force distribution is proposed as an additional parameter to rate the performance of different prosthetic hand designs.     

Chromatin loops are responsible for higher counts of small DNA fragments induced by high-LET radiation,while chromosomal domains do not affect the fragment sizes

Purpose: To apply a polymer model of DNA damage induced by high-LET (linear energy transfer) radiation and determine the influence of chromosomal domains and loops on fragment length distribution.

Materials and methods: The yields of DSB (double-strand breaks) induced by high-LET radiation were calculated using a track structure model along with a polymer model of DNA packed in the cell nucleus. The cell nucleus was constructed to include the chromosomal domains and chromatin loops. The latter were generated by the random walk method.

Results and conclusions: We present data for DSB yields per track per cell, DNA fragment sizes, the radial distribution of DSB with respect to the track center, and the distribution of 0, 1, 2, and more DSB from a single particle. Calculations were carried out for a range of particles including He (40 keV/μm), N (225 keV/μm), and Fe ions (150 keV/μm). Situations relevant to PFGE (pulsed-field gel electrophoresis) and microbeam experiments with direct irradiation of the cell nucleus were simulated to demonstrate the applicability of the model. Data show that chromosomal domains do not have a significant influence on fragment-size distribution, while the presence of DNA loops increases the frequencies of smaller fragments by nearly 30% for fragment sizes in the range from 2 kbp (bp = base pair) to 20 kbp.     

Modified Girdlestone arthroplasty and hip arthrodesis using the Ilizarov external fixator as a salvage method in the management of severely infected total hip replacement

Background:

Resection arthroplasty or hip arthrodesis after total hip replacement (THR) can be used to salvage the limb in case with deep infection and severe bone loss. The Ilizarov fixator provides stability, axial correction, weight-bearing and good fusion rates.

Materials and Methods:

We retrospectively assessed the outcomes of 37 patients with severe periprosthetic infection after THR treated between 1999 and 2011. The treatment included implant removal, debridement and a modified Girdestone arthroplasty (29 cases) or hip arthrodesis (seven cases) using the Ilizarov fixator. The Ilizarov fixation continued from 45 to 50 days in the modified arthroplasty group and 90 days in the arthrodesis group. One case was treated using the conventional resection arthroplasty bilaterally.

Results:

Eighteen months after treatment, infection control was seen in 97.3% cases. Six hips were fused as one patient died in this group. Limb length discrepancy (LLD) averaged 5.5 cm. The Harris hip score ranged from 35 to 92 points. Hip joint motion ranged from 10° to 30° in the modified arthroplasty group. All subjects could walk independently or using support aids. No subluxation or LLD progression was observed.

Conclusion:

The modified Girdlestone arthroplasty and hip arthrodesis using the Ilizarov apparatus results in sufficient ability for ambulation and good infection control in cases of failed THR associated with severe infection.     

A B1‐insensitive high resolution 2D T1 mapping pulse sequence for dGEMRIC of the HIP at 3 Tesla

Early detection of cartilage degeneration in the hip may help prevent onset and progression of osteoarthritis in young patients with femoroacetabular impingement. Delayed gadolinium‐enhanced MRI of cartilage is sensitive to cartilage glycosaminoglycan loss and could serve as a diagnostic tool for early cartilage degeneration. We propose a new high resolution 2D T₁ mapping saturation–recovery pulse sequence with fast spin echo readout for delayed gadolinium‐enhanced magnetic resonance imaging of cartilage of the hip at 3 T. The proposed sequence was validated in a phantom and in 10 hips, using radial imaging planes, against a rigorous multipoint saturation–recovery pulse sequence with fast spin echo readout. T₁ measurements by the two pulse sequences were strongly correlated (R² > 0.95) and in excellent agreement (mean difference = ?8.7 ms; upper and lower 95% limits of agreement = 64.5 and ?81.9 ms, respectively). T₁ measurements were insensitive to B₁₊ variation as large as 20%, making the proposed T₁ mapping technique suitable for 3 T. Magn Reson Med, 2011. © 2011 Wiley‐Liss, Inc.     

NMDA receptors at the endplate of rat skeletal muscles: precise postsynaptic localization

In this study we demonstrate expression of the N-methyl-D-aspartate receptor NR1 subunit in the rat neuromuscular junction of skeletal muscles of different functional types (extensor digitorum longus, soleus, and diaphragm muscles) using fluorescence immunocytochemistry. Electron microscopic immunocytochemistry has shown that the NR1 subunit is localized solely on the sarcolemma in the depths of the postsynaptic folds. These findings suggest participation of the glutamatergic signaling system in functioning of the adult mammalian neuromuscular junction.