Phospho‐regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore

Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the Saccharomyces cerevisiae outer kinetochore, assembling into 3 MDa‐sized microtubule‐embracing rings, but how ring assembly is specifically initiated in vivo remains to be understood. Here, we describe a molecular pathway that provides local control of ring assembly during the establishment of sister kinetochore bi‐orientation. We show that Dam1c and the general microtubule plus end‐associated protein (+TIP) Bim1/EB1 form a stable complex depending on a conserved motif in the Duo1 subunit of Dam1c. EM analyses reveal that Bim1 crosslinks protrusion domains of adjacent Dam1c heterodecamers and promotes the formation of oligomers with defined curvature. Disruption of the Dam1c‐Bim1 interaction impairs kinetochore localization of Dam1c in metaphase and delays mitosis. Phosphorylation promotes Dam1c‐Bim1 binding by relieving an intramolecular inhibition of the Dam1 C‐terminus. In addition, Bim1 recruits Bik1/CLIP‐170 to Dam1c and induces formation of full rings even in the absence of microtubules. Our data help to explain how new kinetochore end‐on attachments are formed during the process of attachment error correction.     

Bayesian inference in populations of cortical neurons: a model of motion integration and segmentation in area MT

A major issue in cortical physiology and computational neuroscience is understanding the interaction between extrinsic signals from feedforward connections and intracortical signals from lateral connections. We propose here a computational model for motion perception based on the assumption that the local cortical circuits in the medio-temporal area (area MT) implement a Bayesian inference principle. This approach establishes a functional balance between feedforward and lateral, excitatory and inhibitory, inputs. The model reproduces most of the known properties of the neurons in area MT in response to moving stimuli. It accounts for important motion perception phenomena including motion transparency, spatial and temporal integration/segmentation. While integrating several properties of previously proposed models, it makes specific testable predictions concerning, in particular, temporal properties of neurons and the architecture of lateral connections in area MT. In addition, the proposed mechanism is consistent with the known properties of local cortical circuits in area V1. This suggests that Bayesian inference may be a general feature of information processing in cortical neuron populations. Received: 3 December 1997 / Accepted in revised form: 21 July 1998     

Molecular architecture of the Dam1 complex–microtubule interaction

Mitosis is a highly regulated process that allows the equal distribution of the genetic material to the daughter cells. Chromosome segregation requires the formation of a bipolar mitotic spindle and assembly of a multi-protein structure termed the kinetochore to mediate attachments between condensed chromosomes and spindle microtubules. In budding yeast, a single microtubule attaches to each kinetochore, necessitating robustness and processivity of this kinetochore–microtubule attachment. The yeast kinetochore-localized Dam1 complex forms a direct interaction with the spindle microtubule. In vitro, the Dam1 complex assembles as a ring around microtubules and couples microtubule depolymerization with cargo movement. However, the subunit organization within the Dam1 complex, its higher-order oligomerization and how it interacts with microtubules remain under debate. Here, we used chemical cross-linking and mass spectrometry to define the architecture and subunit organization of the Dam1 complex. This work reveals that both the C termini of Duo1 and Dam1 subunits interact with the microtubule and are critical for microtubule binding of the Dam1 complex, placing Duo1 and Dam1 on the inside of the ring structure. Integrating this information with available structural data, we provide a coherent model for how the Dam1 complex self-assembles around microtubules.     

Molecular requirements for the formation of a kinetochore–microtubule interface by Dam1 and Ndc80 complexes

Kinetochores are large protein complexes that link sister chromatids to the spindle and transduce microtubule dynamics into chromosome movement. In budding yeast, the kinetochore–microtubule interface is formed by the plus end–associated Dam1 complex and the kinetochore-resident Ndc80 complex, but how they work in combination and whether a physical association between them is critical for chromosome segregation is poorly understood. Here, we define structural elements required for the Ndc80–Dam1 interaction and probe their function in vivo. A novel ndc80 allele, selectively impaired in Dam1 binding, displayed growth and chromosome segregation defects. Its combination with an N-terminal truncation resulted in lethality, demonstrating essential but partially redundant roles for the Ndc80 N-tail and Ndc80–Dam1 interface. In contrast, mutations in the calponin homology domain of Ndc80 abrogated kinetochore function and were not compensated by the presence of Dam1. Our experiments shed light on how microtubule couplers cooperate and impose important constraints on structural models for outer kinetochore assembly.     

CENP-A exceeds microtubule attachment sites in centromere clusters of both budding and fission yeast

The stoichiometries of kinetochores and their constituent proteins in yeast and vertebrate cells were determined using the histone H3 variant CENP-A, known as Cse4 in budding yeast, as a counting standard. One Cse4-containing nucleosome exists in the centromere (CEN) of each chromosome, so it has been assumed that each anaphase CEN/kinetochore cluster contains 32 Cse4 molecules. We report that anaphase CEN clusters instead contained approximately fourfold more Cse4 in Saccharomyces cerevisiae and ~40-fold more CENP-A (Cnp1) in Schizosaccharomyces pombe than predicted. These results suggest that the number of CENP-A molecules exceeds the number of kinetochore-microtubule (MT) attachment sites on each chromosome and that CENP-A is not the sole determinant of kinetochore assembly sites in either yeast. In addition, we show that fission yeast has enough Dam1-DASH complex for ring formation around attached MTs. The results of this study suggest the need for significant revision of existing CEN/kinetochore architectural models.     

Mps1 phosphorylation of Dam1 couples kinetochores to microtubule plus ends at metaphase

BACKGROUND: Duplicated chromosomes are equally segregated to daughter cells by a bipolar mitotic spindle during cell division. By metaphase, sister chromatids are coupled to microtubule (MT) plus ends from opposite poles of the bipolar spindle via kinetochores. Here we describe a phosphorylation event that promotes the coupling of kinetochores to microtubule plus ends. RESULTS: Dam1 is a kinetochore component that directly binds to microtubules. We identified DAM1-765, a dominant allele of DAM1, in a genetic screen for mutations that increase stress on the spindle pole body (SPB) in Saccharomyces cerevisiae. DAM1-765 contains the single mutation S221F. We show that S221 is one of six Dam1 serines (S13, S49, S217, S218, S221, and S232) phosphorylated by Mps1 in vitro. In cells with single mutations S221F, S218A, or S221A, kinetochores in the metaphase spindle form tight clusters that are closer to the SPBs than in a wild-type cell. Five lines of experimental evidence, including localization of spindle components by fluorescence microscopy, measurement of microtubule dynamics by fluorescence redistribution after photobleaching, and reconstructions of three-dimensional structure by electron tomography, combined with computational modeling of microtubule behavior strongly indicate that, unlike wild-type kinetochores, Dam1-765 kinetochores do not colocalize with an equal number of plus ends. Despite the uncoupling of the kinetochores from the plus ends of MTs, the DAM1-765 cells are viable, complete the cell cycle with the same kinetics as wild-type cells, and biorient their chromosomes as efficiently as wild-type cells. CONCLUSIONS: We conclude that phosphorylation of Dam1 residues S218 and S221 by Mps1 is required for efficient coupling of kinetochores to MT plus ends. We find that efficient plus-end coupling is not required for (1) maintenance of chromosome biorientation, (2) maintenance of tension between sister kinetochores, or (3) chromosome segregation.     

Kinesin velocity increases with the number of motors pulling against viscoelastic drag

Although the properties of single kinesin molecular motors are well understood, it is not clear whether multiple motors pulling a single vesicle in a cell cooperate or interfere with one another. To learn how small numbers of motors interact, microtubule gliding assays were carried out with full-length Drosophila kinesin in a novel motility medium containing xanthan, a stiff, water-soluble polysaccharide. At 2 mg/ml xanthan, the zero-shear viscosity of this medium is 1,000 times the viscosity of water, similar to cellular viscosity. To mimic the rheological drag force on the motors when attached to a vesicle in a cell, we attached a 2 μm bead to one end of the microtubule (MT). During gliding assays in our novel medium, the moving bead exerted a drag force of 4–15 pN on the kinesins pulling the MT. The velocity of MTs with an attached bead increased with MT length and with kinesin concentration. The increase with MT length arose because the number of motors is directly proportional to MT length. Our results show that small numbers of kinesins cooperate constructively when pulling against a viscoelastic drag. In the absence of a bead but still in the viscous medium, MT velocity was independent of MT length and kinesin concentration because the thin MT, like a snake moving through grass, was able to move between xanthan molecules with little resistance. A minimal shared-load model in which the number of motors is proportional to MT length fits the observed dependence of gliding velocity on MT length and kinesin concentration.     

Subunit organization in the Dam1 kinetochore complex and its ring around microtubules

All eukaryotic cells must segregate their chromosomes equally between two daughter cells at each division. This process needs to be robust, as errors in the form of loss or gain of genetic material have catastrophic effects on viability. Chromosomes are captured, aligned, and segregated to daughter cells via interaction with spindle microtubules mediated by the kinetochore. In Saccharomyces cerevisiae one microtubule attaches to each kinetochore, requiring extreme processivity from this single connection. The yeast Dam1 complex, an essential component of the outer kinetochore, forms rings around microtubules and in vitro recapitulates much of the functionality of a kinetochore-microtubule attachment. To understand the mechanism of the Dam1 complex at the kinetochore, we must know how it binds to microtubules, how it assembles into rings, and how assembly is regulated. We used electron microscopy to map several subunits within the structure of the Dam1 complex and identify the organization of Dam1 complexes within the ring. Of importance, new data strongly support a more passive role for the microtubule in Dam1 ring formation. Integrating this information with previously published data, we generated a structural model for the Dam1 complex assembly that advances our understanding of its function and will direct future experiments.     

Evidence for a novel affinity mechanism of motor-assisted transport along microtubules

In microtubule (MT) translocation assays, using colloidal gold particles coupled to monoclonal tubulin antibodies to mark positions along MTs, we found that relative motion is possible between the gold particle and an MT, gliding on dynein or kinesin. Such motion evidently occurred by an affinity release and rebinding mechanism that did not require motor activity on the particle. As the MTs moved, particles drifted to the trailing edge of the MT and then were released. Sometimes the particles transferred from one MT to another, moving orthogonally. Although motion of the particles was uniformly rearward, movement was toward the (-) or (+) end of the MT, depending on whether dynein or kinesin, respectively, was used in the assay. These results open possibilities for physiological mechanisms of organelle and other movement that, although dependent on motor-driven microtubule transport, do not require direct motor attachment between the organelle and the microtubule. Our observations on the direction of particle drift and time of release may also provide confirmation in a dynamic system for the conclusion that beta tubulin is exposed at the (+) end of the MT.     

Two-dimensional substructure of stereo and motion interactions in macaque visual cortex

The analysis of object motion and stereoscopic depth are important tasks that are begun at early stages of the primate visual system. Using sparse white noise, we mapped the receptive field substructure of motion and disparity interactions in neurons in V1 and MT of alert monkeys. Interactions in both regions revealed subunits similar in structure to V1 simple cells. For both motion and stereo, the scale and shape of the receptive field substructure could be predicted from conventional tuning for bars or dot-field stimuli, indicating that the small-scale interactions were repeated across the receptive fields. We also found neurons in V1 and in MT that were tuned to combinations of spatial and temporal binocular disparities, suggesting a possible neural substrate for the perceptual Pulfrich phenomenon. Our observations constrain computational and developmental models of motion-stereo integration.     

Neural network models of bilateral coordination

Two mechanisms are described for controlling the movement of a pair of arms. The first is an engineered motion planner that finds solutions to the ill-posed problem of making noncolliding, goal-directed movements. The second uses neural networks that learn to emulate the coordinated behaviors of the motion planner using considerably less computational resources. Analysis of the networks shows in general terms how they work, and allows us to make testable predictions about some of the response properties that might be observed in the brain systems serving bilateral coordination. Received: 12 March 1998 / Accepted in revised form: 10 November 1998     

Object Segmentation from Motion Discontinuities and Temporal Occlusions–A Biologically Inspired Model

Background

Optic flow is an important cue for object detection. Humans are able to perceive objects in a scene using only kinetic boundaries, and can perform the task even when other shape cues are not provided. These kinetic boundaries are characterized by the presence of motion discontinuities in a local neighbourhood. In addition, temporal occlusions appear along the boundaries as the object in front covers the background and the objects that are spatially behind it.

Methodology/Principal Findings

From a technical point of view, the detection of motion boundaries for segmentation based on optic flow is a difficult task. This is due to the problem that flow detected along such boundaries is generally not reliable. We propose a model derived from mechanisms found in visual areas V1, MT, and MSTl of human and primate cortex that achieves robust detection along motion boundaries. It includes two separate mechanisms for both the detection of motion discontinuities and of occlusion regions based on how neurons respond to spatial and temporal contrast, respectively. The mechanisms are embedded in a biologically inspired architecture that integrates information of different model components of the visual processing due to feedback connections. In particular, mutual interactions between the detection of motion discontinuities and temporal occlusions allow a considerable improvement of the kinetic boundary detection.

Conclusions/Significance

A new model is proposed that uses optic flow cues to detect motion discontinuities and object occlusion. We suggest that by combining these results for motion discontinuities and object occlusion, object segmentation within the model can be improved. This idea could also be applied in other models for object segmentation. In addition, we discuss how this model is related to neurophysiological findings. The model was successfully tested both with artificial and real sequences including self and object motion.     

Theoretical modeling of aging effects in microtubule dynamics

The microtubule (MT) network, an important part of the cytoskeleton, is constantly remodeled by alternating phases of growth and shrinkage of individual filaments. Plus-end tracking proteins (+TIPs) interact with the MT and in many cases alter its dynamics. Although it is established that some +TIPs modify MT dynamics by increasing rescues, the plus-end tracking mechanism is still under debate. We present a model for MT dynamics in which a rescue factor is dynamically added to the filament during growth. As a consequence, the filament shows aging behavior that should be experimentally accessible and thus allow one to exclude some hypothesized models regarding the inclusion of rescue factors at the MT plus end. This result is not limited to +TIPs and can be extended to any kind of mechanism shifting the parameters of dynamic instability. Additionally, we show that the cell geometry has a strong influence on the quantitative results.     

Mathematical analysis and modeling of motion direction selectivity in the retina

Motion detection is one of the most important and primitive computations performed by our visual system. Specifically in the retina, ganglion cells producing motion direction-selective responses have been addressed by different disciplines, such as mathematics, neurophysiology and computational modeling, since the beginnings of vision science. Although a number of studies have analyzed theoretical and mathematical considerations for such responses, a clear picture of the underlying cellular mechanisms is only recently emerging. In general, motion direction selectivity is based on a non-linear asymmetric computation inside a receptive field differentiating cell responses between preferred and null direction stimuli. To what extent can biological findings match these considerations? In this review, we outline theoretical and mathematical studies of motion direction selectivity, aiming to map the properties of the models onto the neural circuitry and synaptic connectivity found in the retina. Additionally, we review several compartmental models that have tried to fill this gap. Finally, we discuss the remaining challenges that computational models will have to tackle in order to fully understand the retinal motion direction-selective circuitry.     

Differences in binding sites of two melatonin receptors help to explain their selectivity to some melatonin analogs: a molecular modeling study

Numerous diseases have been linked to the malfunction of G-protein coupled receptors (GPCRs). Their adequate treatment requires rational design of new high-affinity and high-selectivity drugs targeting these receptors. In this work, we report three-dimensional models of the human MT(1) and MT(2) melatonin receptors, members of the GPCR family. The models are based on the X-ray structure of bovine rhodopsin. The computational approach employs an original procedure for optimization of receptor-ligand structures. It includes rotation of one of the transmembrane alpha-helices around its axis with simultaneous assessment of quality of the resulting complexes according to a number of criteria we have developed for this purpose. The optimal geometry of the receptor-ligand binding is selected based on the analysis of complementarity of hydrophobic/hydrophilic properties between the ligand and its protein environment in the binding site. The elaborated "optimized" models are employed to explore the details of protein-ligand interactions for melatonin and a number of its analogs with known affinity to MT(1) and MT(2) receptors. The models permit rationalization of experimental data, including those that were not used in model building. The perspectives opened by the constructed models and by the optimization procedure in the design of new drugs are discussed.     

Neural Correlates of Illusory Line Motion

Illusory line motion (ILM) refers to a motion illusion in which a flash at one end of a bar prior to the bar''s instantaneous presentation or removal results in the percept of motion. While some theories attribute the origin of ILM to attention or early perceptual mechanisms, others have proposed that ILM results from impletion mechanisms that reinterpret the static bar as one in motion. The current functional magnetic resonance imaging study examined participants while they made decisions about the direction of motion in which a bar appeared to be removed. Preceding the instantaneous removal of the bar with a flash at one end resulted in a motion percept away from the flash. If this flash and the bar''s removal overlapped in time, it appeared that the bar was removed towards the flash (reverse ILM). Independent of the motion type, brain responses indicated activations in areas associated with motion (MT+), endogenous and exogenous attention (intraparietal sulcus, frontal eye fields, and ventral frontal cortex), and response selection (ACC). ILM was associated with lower percept scores and higher activations in ACC relative to real motion, but no differences in shape-selective areas emerged. This pattern of brain activation is consistent with the attentional gradient model or bottom-up accounts of ILM in preference to impletion.     

MetaMQAP: A meta-server for the quality assessment of protein models

Background  

Computational models of protein structure are usually inaccurate and exhibit significant deviations from the true structure. The utility of models depends on the degree of these deviations. A number of predictive methods have been developed to discriminate between the globally incorrect and approximately correct models. However, only a few methods predict correctness of different parts of computational models. Several Model Quality Assessment Programs (MQAPs) have been developed to detect local inaccuracies in unrefined crystallographic models, but it is not known if they are useful for computational models, which usually exhibit different and much more severe errors.     

采用Reichardt运动检测器和Boltzmann Machine神经网络的运动计算

建立了一个探讨灵长类视皮层从Ｖ１区到ＭＴ区的运动信息加工原理的计算模型,这个过程的突出特征是视觉运动信息经过了从局部检测进步到整体感知。模型的第一层由用于抽提运动模式的局部速度以及结构性质的Ｒｅｉｃｈａｒｄｔ运动检测器组成,进一步的加工是通过Ｂｏｌｔｚｍａｎｎ Ｍａｃｈｉｎｅ神经网络来实现的。这种网络的学习算法具有局部更新的显著性质,在学习阶段,网络不断地修改联结权重以形成对于记录在网络的显单元上     

Mutational and structural analyses of the hinge region of membrane type 1-matrix metalloproteinase and enzyme processing

Membrane type 1 (MT1)-matrix metalloproteinase (MMP) is a major mediator of collagen degradation in the pericellular space in both physiological and pathological conditions. Previous evidence has shown that on the cell surface, active MT1-MMP undergoes autocatalytic processing to a major membrane-tethered 44-kDa product lacking the catalytic domain and displaying Gly285 at its N terminus, which is at the beginning of the hinge domain. However, the importance of this site and the hinge region in MT1-MMP processing is unknown. In the current study, we generated mutations and deletions in the hinge of MT1-MMP and followed their effect on processing. These studies established Gly284-Gly285 as the main cleavage site involved in the formation of the 44-kDa species. However, alterations at this site did not prevent processing. Instead, they forced downstream cleavages within the stretch of residues flanked by Gln296 and Ser304 in the hinge region, as determined by the processing profile of various hinge deletion mutants. Also, replacement of the hinge of MT1-MMP with the longer MT3-MMP hinge did not prevent processing of MT1-MMP. Molecular dynamic studies using a computational model of MT1-MMP revealed that the hinge region is a highly motile element that undergoes significant motion in the highly exposed loop formed by Pro295-Arg302 consistent with being a prime target for proteolysis, in agreement with the mutational data. These studies suggest that the hinge of MT1-MMP evolved to facilitate processing, a promiscuous but compulsory event in the destiny of MT1-MMP, which may play a key role in the control of pericellular proteolysis.