In vivo High Angular Resolution Diffusion-Weighted Imaging of Mouse Brain at 16.4 Tesla

Magnetic Resonance Imaging (MRI) of the rodent brain at ultra-high magnetic fields (> 9.4 Tesla) offers a higher signal-to-noise ratio that can be exploited to reduce image acquisition time or provide higher spatial resolution. However, significant challenges are presented due to a combination of longer T ₁ and shorter T ₂/T₂* relaxation times and increased sensitivity to magnetic susceptibility resulting in severe local-field inhomogeneity artefacts from air pockets and bone/brain interfaces. The Stejskal-Tanner spin echo diffusion-weighted imaging (DWI) sequence is often used in high-field rodent brain MRI due to its immunity to these artefacts. To accurately determine diffusion-tensor or fibre-orientation distribution, high angular resolution diffusion imaging (HARDI) with strong diffusion weighting (b >3000 s/mm²) and at least 30 diffusion-encoding directions are required. However, this results in long image acquisition times unsuitable for live animal imaging. In this study, we describe the optimization of HARDI acquisition parameters at 16.4T using a Stejskal-Tanner sequence with echo-planar imaging (EPI) readout. EPI segmentation and partial Fourier encoding acceleration were applied to reduce the echo time (TE), thereby minimizing signal decay and distortion artefacts while maintaining a reasonably short acquisition time. The final HARDI acquisition protocol was achieved with the following parameters: 4 shot EPI, b = 3000 s/mm², 64 diffusion-encoding directions, 125×150 μm² in-plane resolution, 0.6 mm slice thickness, and 2h acquisition time. This protocol was used to image a cohort of adult C57BL/6 male mice, whereby the quality of the acquired data was assessed and diffusion tensor imaging (DTI) derived parameters were measured. High-quality images with high spatial and angular resolution, low distortion and low variability in DTI-derived parameters were obtained, indicating that EPI-DWI is feasible at 16.4T to study animal models of white matter (WM) diseases.     

Multiplexed echo planar imaging for sub-second whole brain FMRI and fast diffusion imaging

Echo planar imaging (EPI) is an MRI technique of particular value to neuroscience, with its use for virtually all functional MRI (fMRI) and diffusion imaging of fiber connections in the human brain. EPI generates a single 2D image in a fraction of a second; however, it requires 2-3 seconds to acquire multi-slice whole brain coverage for fMRI and even longer for diffusion imaging. Here we report on a large reduction in EPI whole brain scan time at 3 and 7 Tesla, without significantly sacrificing spatial resolution, and while gaining functional sensitivity. The multiplexed-EPI (M-EPI) pulse sequence combines two forms of multiplexing: temporal multiplexing (m) utilizing simultaneous echo refocused (SIR) EPI and spatial multiplexing (n) with multibanded RF pulses (MB) to achieve m×n images in an EPI echo train instead of the normal single image. This resulted in an unprecedented reduction in EPI scan time for whole brain fMRI performed at 3 Tesla, permitting TRs of 400 ms and 800 ms compared to a more conventional 2.5 sec TR, and 2-4 times reductions in scan time for HARDI imaging of neuronal fibertracks. The simultaneous SE refocusing of SIR imaging at 7 Tesla advantageously reduced SAR by using fewer RF refocusing pulses and by shifting fat signal out of the image plane so that fat suppression pulses were not required. In preliminary studies of resting state functional networks identified through independent component analysis, the 6-fold higher sampling rate increased the peak functional sensitivity by 60%. The novel M-EPI pulse sequence resulted in a significantly increased temporal resolution for whole brain fMRI, and as such, this new methodology can be used for studying non-stationarity in networks and generally for expanding and enriching the functional information.     

Resting State BOLD Functional Connectivity at 3T: Spin Echo versus Gradient Echo EPI

Previous evidence showed that, due to refocusing of static dephasing effects around large vessels, spin-echo (SE) BOLD signals offer an increased linearity and promptness with respect to gradient-echo (GE) acquisition, even at low field. These characteristics suggest that, despite the reduced sensitivity, SE fMRI might also provide a potential benefit when investigating spontaneous fluctuations of brain activity. However, there are no reports on the application of spin-echo fMRI for connectivity studies at low field. In this study we compared resting state functional connectivity as measured with GE and SE EPI sequences at 3T. Main results showed that, within subject, the GE sensitivity is overall larger with respect to that of SE, but to a less extent than previously reported for activation studies. Noteworthy, the reduced sensitivity of SE was counterbalanced by a reduced inter-subject variability, resulting in comparable group statistical connectivity maps for the two sequences. Furthermore, the SE method performed better in the ventral portion of the default mode network, a region affected by signal dropout in standard GE acquisition. Future studies should clarify if these features of the SE BOLD signal can be beneficial to distinguish subtle variations of functional connectivity across different populations and/or treatments when vascular confounds or regions affected by signal dropout can be a critical issue.     

Cargo surface fluidity can reduce inter-motor mechanical interference,promote load-sharing and enhance processivity in teams of molecular motors

In cells, multiple molecular motors work together as teams to carry cargoes such as vesicles and organelles over long distances to their destinations by stepping along a network of cytoskeletal filaments. How motors that typically mechanically interfere with each other, work together as teams is unclear. Here we explored the possibility that purely physical mechanisms, such as cargo surface fluidity, may potentially enhance teamwork, both at the single motor and cargo level. To explore these mechanisms, we developed a three dimensional simulation of cargo transport along microtubules by teams of kinesin-1 motors. We accounted for cargo membrane fluidity by explicitly simulating the Brownian dynamics of motors on the cargo surface and considered both the load and ATP dependence of single motor functioning. Our simulations show that surface fluidity could lead to the reduction of negative mechanical interference between kinesins and enhanced load sharing thereby increasing the average duration of single motors on the filament. This, along with a cooperative increase in on-rates as more motors bind leads to enhanced collective processivity. At the cargo level, surface fluidity makes more motors available for binding, which can act synergistically with the above effects to further increase transport distances though this effect is significant only at low ATP or high motor density. Additionally, the fluid surface allows for the clustering of motors at a well defined location on the surface relative to the microtubule and the fluid-coupled motors can exert more collective force per motor against loads. Our work on understanding how teamwork arises in cargo-coupled motors allows us to connect single motor properties to overall transport, sheds new light on cellular processes, reconciles existing observations, encourages new experimental validation efforts and can also suggest new ways of improving the transport of artificial cargo powered by motor teams.     

Processive kinesins require loose mechanical coupling for efficient collective motility

Processive motor proteins are stochastic steppers that perform actual mechanical steps for only a minor fraction of the time they are bound to the filament track. Motors usually work in teams and therefore the question arises whether the stochasticity of stepping can cause mutual interference when motors are mechanically coupled. We used biocompatible surfaces to immobilize processive kinesin-1 motors at controlled surface densities in a mechanically well-defined way. This helped us to study quantitatively how mechanical coupling between motors affects the efficiency of collective microtubule transport. We found that kinesin-1 constructs that lack most of the non-motor sequence slow each other down when collectively transporting a microtubule, depending on the number of interacting motors. This negative interference observed for a motor ensemble can be explained quantitatively by a mathematical model using the known physical properties of individual molecules of kinesin-1. The non-motor extension of kinesin-1 reduces this mutual interference, indicating that loose mechanical coupling between motors is required for efficient transport by ensembles of processive motors.     

Kinesin-related proteins in the mammalian testes: candidate motors for meiosis and morphogenesis.

The kinesin superfamily of molecular motors comprises proteins that participate in a wide variety of motile events within the cell. Members of this family share a highly homologous head domain responsible for force generation attached to a divergent tail domain thought to couple the motor domain to its target cargo. Many kinesin-related proteins (KRPs) participate in spindle morphogenesis and chromosome movement in cell division. Genetic analysis of mitotic KRPs in yeast and Drosophila, as well as biochemical experiments in other species, have suggested models for the function of KRPs in cell division, including both mitosis and meiosis. Although many mitotic KRPs have been identified, the relationship between mitotic motors and meiotic function is not clearly understood. We have used sequence similarity between mitotic KRPs to identify candidates for meiotic and/or mitotic motors in a vertebrate. We have identified a group of kinesin-related proteins from rat testes (termed here testes KRP1 through KRP6) that includes new members of the bimC and KIF2 subfamilies as well as proteins that may define new kinesin subfamilies. Five of the six testes KRPs identified are expressed primarily in testes. Three of these are expressed in a region of the seminiferous epithelia (SE) rich in meiotically active cells. Further characterization of one of these KRPs, KRP2, showed it to be a promising candidate for a motor in meiosis: it is localized to a meiotically active region of the SE and is homologous to motor proteins associated with the mitotic apparatus. Testes-specific genes provide the necessary probes to investigate whether the motor proteins that function in mammalian meiosis overlap with those of mitosis and whether motor proteins exist with functions unique to meiosis. Our search for meiotic motors in a vertebrate testes has successfully identified proteins with properties consistent with those of meiotic motors in addition to uncovering proteins that may function in other unique motile events of the SE.     

Stepping, strain gating, and an unexpected force-velocity curve for multiple-motor-based transport

BACKGROUND: Intracellular transport via processive kinesin, dynein, and myosin molecular motors plays an important role in maintaining cell structure and function. In many cases, cargoes move distances longer than expected for single motors; there is significant evidence that this increased travel is in part due to multiple motors working together to move the cargoes. Although we understand single motors experimentally and theoretically, our understanding of multiple motors working together is less developed. RESULTS: We theoretically investigate how multiple kinesin motors function. Our model includes stochastic fluctuations of each motor as it proceeds through its enzymatic cycle. Motors dynamically influence each other and function in the presence of thermal noise and viscosity. We test the theory via comparison with the experimentally observed distribution of step sizes for two motors moving a cargo, and by predicting slightly subadditive stalling force for two motors relative to one. In the presence of load, our predictions for travel distances and mean velocities are different from the steady-state model: with high motor-motor coupling, we predict a form of strain-gating, where-because of the underlying motor's dynamics-the motors share load unevenly, leading to increased mean travel distance of the multiple-motor system under load. Surprisingly, we predict that in the presence of small load, two-motor cargoes move slightly slower than do single-motor cargoes. Unpublished data from G.T. Shubeita, B.C. Carter, and S.P.G. confirm this prediction in vivo. CONCLUSIONS: When only a few motors are active, fluctuations and unequal load sharing between motors can result in significant alterations of ensemble function.     

Properties of the Kinesin-1 motor DdKif3 from Dictyostelium discoideum

The amoeba Dictyostelium discoideum possesses genes for 13 different kinesins. Here we characterize DdKif3, a member of the Kinesin-1 family. Kinesin-1 motors form homodimers that can move micrometer-long distances on microtubules using the energy derived from ATP hydrolysis. We expressed recombinant motors in Escherichia coli and tested them in different in vitro assays. Full-length and truncated Kif3 motors were active in gliding and ATPase assays. They showed a strong dependence on ionic strength. Like the full-length motor, the truncated DdKif3-592 motor (aa 1-592; comprising motor domain, neck, and partial stalk) reached its maximum speed of around 2.0micrcom s(-1) at a potassium acetate concentration of 200mM. The shortened DdKif3-342 motor (aa 1-342; comprising motor domain, partial neck) showed a high ATP turnover, comparable to that of the fungal Kinesin-1, Nkin. Results from the duty cycle calculations and gliding assays indicate that DdKif3 is a processive motor. A GFP-fusion protein revealed a mainly cytoplasmic localization of DdKif3. Immunofluorescence staining makes an association with the endoplasmic reticulum or mitochondria unlikely. Despite a similar phylogenetic distance to both metazoa and fungi, in terms of its biochemical properties DdKif3 revealed a closer similarity to fungal than animal kinesins.     

Iron accumulation in deep cortical layers accounts for MRI signal abnormalities in ALS: correlating 7 Tesla MRI and pathology

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by cortical and spinal motor neuron dysfunction. Routine magnetic resonance imaging (MRI) studies have previously shown hypointense signal in the motor cortex on T(2)-weighted images in some ALS patients, however, the cause of this finding is unknown. To investigate the utility of this MR signal change as a marker of cortical motor neuron degeneration, signal abnormalities on 3T and 7T MR images of the brain were compared, and pathology was obtained in two ALS patients to determine the origin of the motor cortex hypointensity. Nineteen patients with clinically probable or definite ALS by El Escorial criteria and 19 healthy controls underwent 3T MRI. A 7T MRI scan was carried out on five ALS patients who had motor cortex hypointensity on the 3T FLAIR sequence and on three healthy controls. Postmortem 7T MRI of the brain was performed in one ALS patient and histological studies of the brains and spinal cords were obtained post-mortem in two patients. The motor cortex hypointensity on 3T FLAIR images was present in greater frequency in ALS patients. Increased hypointensity correlated with greater severity of upper motor neuron impairment. Analysis of 7T T(2)(*)-weighted gradient echo imaging localized the signal alteration to the deeper layers of the motor cortex in both ALS patients. Pathological studies showed increased iron accumulation in microglial cells in areas corresponding to the location of the signal changes on the 3T and 7T MRI of the motor cortex. These findings indicate that the motor cortex hypointensity on 3T MRI FLAIR images in ALS is due to increased iron accumulation by microglia.     

MRI典型和特殊故障排除方法

介绍了GE公司的磁共振机（Signa Mri 1．OT）的主机显示器、磁体监视器故障排除方法,以及解决伪影干扰的经验。     

Comparative analysis of two C-terminal kinesin motor proteins: KIFC1 and KIFC5A

We have taken advantage of the close structural relationship between two C-terminal motors, KIFC5A and KIFC1, to examine the sequence requirements for targeting of these two motors within the cell. Although KIFC5A and KIFC1 are almost identical in their motor and stalk domains, they differ in well-defined regions of their tail domains. Specific antisera to these motors were used to determine their localization to distinct subcellular compartments, the spindle for KIFC5A or membranous organelles for KIFC1. In addition to defining the intracellular localization of KIFC1, the reactivity of the KIFC1 antibody demonstrates that this motor contains a frame shift with respect to KIFC5A and is likely the product of a separate gene. The divergent tail domains of these motors are predicted to harbor specific information that directs them to their correct intracellular targets. In order to define the sequences responsible for the differential localization of these two motors, GFP was fused to motors with various tail deletions and their localization visualized after transfection. We were able to identify distinct sequences in each motor responsible for its unique cellular localization. The KIFC5A tail contains a 43 amino acid sequence with both nuclear localization and microtubule binding activity while KIFC1 contains a 19 amino acid sequence sufficient to target this motor to membrane-bounded organelles.     

Motor proteins and kinesin-based nanoactuatoric devices

Eukaryotic organisms synthesize diverse motor proteins converting chemical into mechanical energy. Among them, both rotary (e.g., ATP synthase) and linear motors are found. Linear motors comprise highly specialized proteins moving along nucleic acid filaments (in the case of e.g., RNA polymerase) or cytoskeletal filaments. The present paper provides a brief overview on cytoskeleton-associated motors (myosins, dyneins, and kinesins) and summarizes results contributing to elaborate a basic configuration for constructing a kinesin-driven motor device, suitable for e.g. a controlled displacement of objects or specific substances over millimetre distances with nanometre precision.     

Controlled and stochastic retention concentrates dynein at microtubule ends to keep endosomes on track

Bidirectional transport of early endosomes (EEs) involves microtubules (MTs) and associated motors. In fungi, the dynein/dynactin motor complex concentrates in a comet-like accumulation at MT plus-ends to receive kinesin-3-delivered EEs for retrograde transport. Here, we analyse the loading of endosomes onto dynein by combining live imaging of photoactivated endosomes and fluorescent dynein with mathematical modelling. Using nuclear pores as an internal calibration standard, we show that the dynein comet consists of ～55 dynein motors. About half of the motors are slowly turned over (T(1/2): ～98 s) and they are kept at the plus-ends by an active retention mechanism involving an interaction between dynactin and EB1. The other half is more dynamic (T(1/2): ～10 s) and mathematical modelling suggests that they concentrate at MT ends because of stochastic motor behaviour. When the active retention is impaired by inhibitory peptides, dynein numbers in the comet are reduced to half and ～10% of the EEs fall off the MT plus-ends. Thus, a combination of stochastic accumulation and active retention forms the dynein comet to ensure capturing of arriving organelles by retrograde motors.     

Identification of isoforms of a mitotic motor in mammalian spermatogenesis

We have isolated the full-length coding sequence for mouse KIFC5A (kinesin family c-terminal 5A) cDNA, encoding a motor protein found in the testes. The complete sequence of the KIFC5A cDNA is homologous to a group of carboxyl-terminal motors, including hamster CHO2, human HSET, and mouse KIFC1 and KIFC4. The KIFC5A and KIFC1 cDNAs are nearly identical except for the presence of two additional sequence blocks in the 5'-end of KIFC5A and a number of single base-pair differences in their motor domains. Polymerase chain reaction amplification and sequencing of the 5'-end of KIFC5A identified 3 distinct RNA species in testes and other tissues. Sequence comparison and genetic mapping confirmed the existence of a small multi-gene family in the mouse and suggest possible mechanisms of alternative splicing, genetic duplication, and separate genetic loci in the generation of these motors. In order to examine the possible role of these motors in germ cells of the testes, an antibody to a shared epitope was used to localize this group of proteins to different spermatogenic cell types. These experiments suggest that KIFC5-like motor proteins are associated with multiple microtubule complexes in male germ cells, including the meiotic spindle, the manchette, and the flagella.     

Pull or Push? Octopuses Solve a Puzzle Problem

Octopuses have large brains and exhibit complex behaviors, but relatively little is known about their cognitive abilities. Here we present data from a five-level learning and problem-solving experiment. Seven octopuses (Octopus vulgaris) were first trained to open an L shaped container to retrieve food (level 0). After learning the initial task all animals followed the same experimental protocol, first they had to retrieve this L shaped container, presented at the same orientation, through a tight fitting hole in a clear Perspex partition (level 1). This required the octopuses to perform both pull and release or push actions. After reaching criterion the animals advanced to the next stage of the test, which would be a different consistent orientation of the object (level 2) at the start of the trial, an opaque barrier (level 3) or a random orientation of the object (level 4). All octopuses were successful in reaching criterion in all levels of the task. At the onset of each new level the performance of the animals dropped, shown as an increase in working times. However, they adapted quickly so that overall working times were not significantly different between levels. Our findings indicate that octopuses show behavioral flexibility by quickly adapting to a change in a task. This can be compared to tests in other species where subjects had to conduct actions comprised of a set of motor actions that cannot be understood by a simple learning rule alone.     

Diffusion of kinesin motors on cargo can enhance binding and run lengths during intracellular transport

Cellular cargoes, including lipid droplets and mitochondria, are transported along microtubules using molecular motors such as kinesins. Many experimental and computational studies focused on cargoes with rigidly attached motors, in contrast to many biological cargoes that have lipid surfaces that may allow surface mobility of motors. We extend a mechanochemical three-dimensional computational model by adding coupled-viscosity effects to compare different motor arrangements and mobilities. We show that organizational changes can optimize for different objectives: Cargoes with clustered motors are transported efficiently but are slow to bind to microtubules, whereas those with motors dispersed rigidly on their surface bind microtubules quickly but are transported inefficiently. Finally, cargoes with freely diffusing motors have both fast binding and efficient transport, although less efficient than clustered motors. These results suggest that experimentally observed changes in motor organization may be a control point for transport.     

Prospective evaluation of the setup errors and its impact on safety margin for cervical cancer pelvic conformal radiotherapy

AimThe primary objective was to assess set-up errors (SE) and secondary objective was to determine optimal safety margin (SM)BackgroundTo evaluate the SE and its impact on the SM utilizing electronic portal imaging (EPI) for pelvic conformal radiotherapy.Material and methods20 cervical cancer patients were enrolled in this prospective study. Supine position with ankle and knee rest was used during CT simulation. The contouring was done using consensus guideline for intact uterus. 50 Gy in 25 fractions were delivered at the isocenter with ≥95% PTV coverage. Two orthogonal (Anterior and Lateral) digitally reconstructed radiograph (DRR) was constructed as a reference image. The pair of orthogonal [Anterior-Posterior and Right Lateral] single exposure EPIs during radiation was taken. The reference DRR and EPIs were compared for shifts, and SE was calculated in the X-axis, Y-axis, and Z-axis directions.Results320 images (40 DRRs and 280 EPIs) were assessed. The systematic error in the Z-axis (AP EPI), X-axis (AP EPI), and Y-axis (Lat EPI) ranged from -12.0 to 11.8 mm, -10.3 to 7.5 mm, and -8.50 to 9.70 mm, while the random error ranged from 1.60 to 6.15 mm, 0.59 to 4.93 mm, and 1.02 to –4.35 mm. The SM computed were 7.07, 6.36, and 7.79 mm in the Y-axis, X-axis, and Z-axis by Van Herk’s equation, and 6.0, 5.51, and 6.74 mm by Stroom’s equation.ConclusionThe computed SE helps defining SM, and it may differ between institutions. In our study, the calculated SM was approximately 8 mm in the Z-axis, 7 mm in X and Y axis for pelvic conformal radiotherapy.     

Distinct in situ structures of the Borrelia flagellar motor

Bacteria can be propelled in liquids by flagellar filaments that are attached to and moved by flagellar motors. These motors are rotary nanomachines that use the electrochemical potential from ion gradients. The motor can spin in both directions with specific proteins regulating the direction in response to chemotactic stimuli. Here we investigated the structure of flagellar motors of Borrelia spirochetes, the causative agents of Lyme disease in humans. We revealed the structure of the motor complex at 4.6-nm resolution by sub-volume averaging of cryo-electron tomograms and subsequently imposing rotational symmetry. This allowed direct visualisation of individual motor components, the connection between the stator and the peptidoglycan as well as filamentous linkers between the stator and the rod. Two different motor assemblies seem to co-exist at a single bacterial pole. While most motors were completely assembled, a smaller fraction appeared to lack part of the C-ring, which plays a role in protein export and switching the directionality of rotation. Our data suggest a novel mechanism that bacteria may use to control the direction of movement.     

Microscopic analysis of bacterial motility at high pressure

The bacterial flagellar motor is a molecular machine that converts an ion flux to the rotation of a helical flagellar filament. Counterclockwise rotation of the filaments allows them to join in a bundle and propel the cell forward. Loss of motility can be caused by environmental factors such as temperature, pH, and solvation. Hydrostatic pressure is also a physical inhibitor of bacterial motility, but the detailed mechanism of this inhibition is still unknown. Here, we developed a high-pressure microscope that enables us to acquire high-resolution microscopic images, regardless of applied pressures. We also characterized the pressure dependence of the motility of swimming Escherichia coli cells and the rotation of single flagellar motors. The fraction and speed of swimming cells decreased with increased pressure. At 80 MPa, all cells stopped swimming and simply diffused in solution. After the release of pressure, most cells immediately recovered their initial motility. Direct observation of the motility of single flagellar motors revealed that at 80 MPa, the motors generate torque that should be sufficient to join rotating filaments in a bundle. The discrepancy in the behavior of free swimming cells and individual motors could be due to the applied pressure inhibiting the formation of rotating filament bundles that can propel the cell body in an aqueous environment.     

A model of myosin V processivity

Cytoplasmic transport is mediated by a group of molecular motors that typically work in isolation, under conditions where they must move their cargos long distances without dissociating from their tracks. This processive behavior requires specific adaptations of motor enzymology to meet these unique physiologic demands. One of these involves the ability of the two heads of a processive motor to communicate their structural states to each other. In this study, we examine a processive motor from the myosin superfamily myosin V. We have measured the kinetics of nucleotide release, of phosphate release, and of the weak-to-strong transition, as this motor interacts with actin, and we have used these studies to develop a model of how myosin V functions as a transport motor. Surprisingly, both heads release phosphate rapidly upon the initial encounter with an actin filament, suggesting that there is little or no intramolecular strain associated with this step. However, ADP release can be affected by both forward and rearward strain, and under steady-state conditions it is essentially prevented in the lead head until the rear head detaches. Many of these features are remarkably like those underlying the processive movement of kinesin on microtubules, supporting our hypothesis that different molecular motors satisfy the requirement for processive movement in similar ways, regardless of their particular family of origin.