The Cep192-organized aurora A-Plk1 cascade is essential for centrosome cycle and bipolar spindle assembly.

As cells enter mitosis, the two centrosomes separate and grow dramatically, each forming a nascent spindle pole that nucleates a radial array of microtubules. Centrosome growth (and associated microtubule nucleation surge), termed maturation, involves the recruitment of pericentriolar material components via an as-yet unknown mechanism. Here, we show that Cep192 binds Aurora A and Plk1, targets them to centrosomes in a pericentrin-dependent manner, and promotes sequential activation of both kinases via T-loop phosphorylation. The Cep192-bound Plk1 then phosphorylates Cep192 at several residues to generate the attachment sites for the γ-tubulin ring complex and, possibly, other pericentriolar material components, thus promoting their recruitment and subsequent microtubule nucleation. We further found that the Cep192-dependent Aurora A-Plk1 activity is essential for kinesin-5-mediated centrosome separation, bipolar spindle formation, and equal centrosome/centriole segregation into daughter cells. Thus, our study identifies a Cep192-organized signaling cascade that underlies both centrosome maturation and bipolar spindle assembly.

BRCA1 promotes unloading of the CMG helicase from a stalled DNA replication fork.

The tumor suppressor protein BRCA1 promotes homologous recombination (HR), a high-fidelity mechanism to repair DNA double-strand breaks (DSBs) that arise during normal replication and in response to DNA-damaging agents. Recent genetic experiments indicate that BRCA1 also performs an HR-independent function during the repair of DNA interstrand crosslinks (ICLs). Here we show that BRCA1 is required to unload the CMG helicase complex from chromatin after replication forks collide with an ICL. Eviction of the stalled helicase allows leading strands to be extended toward the ICL, followed by endonucleolytic processing of the crosslink, lesion bypass, and DSB repair. Our results identify BRCA1-dependent helicase unloading as a critical, early event in ICL repair.

Centrosomal protein of 192 kDa (Cep192) promotes centrosome-driven spindle assembly by engaging in organelle-specific Aurora A activation.

Centrosomes are primary microtubule (MT)-organizing centers (MTOCs). During mitosis, they dramatically increase their size and MT-nucleating activity and participate in spindle assembly from spindle poles. These events require the serine/threonine kinase, Aurora A (AurA), and the centrosomal protein of 192 kDa (Cep192)/spindle defective 2 (Spd-2), but the underlying mechanism remains unclear. We have found that Cep192, unlike targeting protein for Xklp2 (TPX2), a known MT-localizing AurA activator, is an AurA cofactor in centrosome-driven spindle assembly. Cep192, through a direct interaction, targets AurA to mitotic centrosomes where the locally accumulating AurA forms homodimers or oligomers. The dimerization of endogenous AurA, in the presence of bound Cep192, triggers potent kinase activation that, in turn, drives MT assembly. Depletion of Cep192 or specific interference with AurA-Cep192 binding did not prevent AurA oligomerization on MTs but abrogated AurA recruitment to centrosomes and its activation by either sperm nuclei or anti-AurA antibody (αAurA)-induced dimerization. In these settings, MT assembly by both centrosomes and αAurA-coated beads was also abolished or severely compromised. Hence, Cep192 activates AurA by a mechanism different from that previously described for TPX2. The Cep192-mediated mechanism maximizes AurA activity at centrosomes and appears essential for the function of these organelles as MTOCs.

Are redox changes a critical switch for mitotic progression?

Cell-cycle dependent redox changes result in increased protein oxidation in mitotic cells. We show that oxidative modifications of a conserved cysteine residue within Aurora A kinase (AURKA) can promote its activation during mitosis. Targeting redox-sensitive cysteine residues within AURKA may lead to the development of novel anti-cancer agents with improved clinical efficacy.

Estimation and Observability Analysis of Human Motion on Lie Groups.

This article proposes a framework for human-pose estimation from the wearable sensors that rely on a Lie group representation to model the geometry of the human movement. Human body joints are modeled by matrix Lie groups, using special orthogonal groups SO(2) and SO(3) for joint pose and special Euclidean group SE(3) for base-link pose representation. To estimate the human joint pose, velocity, and acceleration, we develop the equations for employing the extended Kalman filter on Lie groups (LG-EKF) to explicitly account for the non-Euclidean geometry of the state space. We present the observability analysis of an arbitrarily long kinematic chain of SO(3) elements based on a differential geometric approach, representing a generalization of kinematic chains of a human body. The observability is investigated for the system using marker position measurements. The proposed algorithm is compared with two competing approaches: 1) the extended Kalman filter (EKF) and 2) unscented KF (UKF) based on the Euler angle parametrization, in both simulations and extensive real-world experiments. The results show that the proposed approach achieves significant improvements over the Euler angle-based filters. It provides more accurate pose estimates, is not sensitive to gimbal lock, and more consistently estimates the covariances.

Redox priming promotes Aurora A activation during mitosis.

Cell cycle-dependent redox changes can mediate transient covalent modifications of cysteine thiols to modulate the activities of regulatory kinases and phosphatases. Our previously reported finding that protein cysteine oxidation is increased during mitosis relative to other cell cycle phases suggests that redox modifications could play prominent roles in regulating mitotic processes. The Aurora family of kinases and their downstream targets are key components of the cellular machinery that ensures the proper execution of mitosis and the accurate segregation of chromosomes to daughter cells. In this study, x-ray crystal structures of the Aurora A kinase domain delineate redox-sensitive cysteine residues that, upon covalent modification, can allosterically regulate kinase activity and oligomerization state. We showed in both Xenopus laevis egg extracts and mammalian cells that a conserved cysteine residue within the Aurora A activation loop is crucial for Aurora A activation by autophosphorylation. We further showed that covalent disulfide adducts of this residue promote autophosphorylation of the Aurora A kinase domain. These findings reveal a potential mechanistic link between Aurora A activation and changes in the intracellular redox state during mitosis and provide insights into how novel small-molecule inhibitors may be developed to target specific subpopulations of Aurora A.

The Centrosome and the Primary Cilium: The Yin and Yang of a Hybrid Organelle.

Centrosomes and primary cilia are usually considered as distinct organelles, although both are assembled with the same evolutionary conserved, microtubule-based templates, the centrioles. Centrosomes serve as major microtubule- and actin cytoskeleton-organizing centers and are involved in a variety of intracellular processes, whereas primary cilia receive and transduce environmental signals to elicit cellular and organismal responses. Understanding the functional relationship between centrosomes and primary cilia is important because defects in both structures have been implicated in various diseases, including cancer. Here, we discuss evidence that the animal centrosome evolved, with the transition to complex multicellularity, as a hybrid organelle comprised of the two distinct, but intertwined, structural-functional modules: the centriole/primary cilium module and the pericentriolar material/centrosome module. The evolution of the former module may have been caused by the expanding cellular diversification and intercommunication, whereas that of the latter module may have been driven by the increasing complexity of mitosis and the requirement for maintaining cell polarity, individuation, and adhesion. Through its unique ability to serve both as a plasma membrane-associated primary cilium organizer and a juxtanuclear microtubule-organizing center, the animal centrosome has become an ideal integrator of extracellular and intracellular signals with the cytoskeleton and a switch between the non-cell autonomous and the cell-autonomous signaling modes. In light of this hypothesis, we discuss centrosome dynamics during cell proliferation, migration, and differentiation and propose a model of centrosome-driven microtubule assembly in mitotic and interphase cells. In addition, we outline the evolutionary benefits of the animal centrosome and highlight the hierarchy and modularity of the centrosome biogenesis networks.

Inertial measurement unit-based pose estimation: Analyzing and reducing sensitivity to sensor placement and body measures.

INTRODUCTION: Inertial measurement units have been proposed for automated pose estimation and exercise monitoring in clinical settings. However, many existing methods assume an extensive calibration procedure, which may not be realizable in clinical practice. In this study, an inertial measurement unit-based pose estimation method using extended Kalman filter and kinematic chain modeling is adapted for lower body pose estimation during clinical mobility tests such as the single leg squat, and the sensitivity to parameter calibration is investigated. METHODS: The sensitivity of pose estimation accuracy to each of the kinematic model and sensor placement parameters was analyzed. Sensitivity analysis results suggested that accurate extraction of inertial measurement unit orientation on the body is a key factor in improving the accuracy. Hence, a simple calibration protocol was proposed to reach a better approximation for inertial measurement unit orientation. RESULTS: After applying the protocol, the ankle, knee, and hip joint angle errors improved to 4 . 2 ∘ , 6 . 3 ∘ , and 8 . 3 ∘ , without the need for any other calibration. CONCLUSIONS: Only a small subset of kinematic and sensor parameters contribute significantly to pose estimation accuracy when using body worn inertial sensors. A simple calibration procedure identifying the inertial measurement unit orientation on the body can provide good pose estimation performance.

Aurora-PLK1 cascades as key signaling modules in the regulation of mitosis.

Mitosis is controlled by reversible protein phosphorylation involving specific kinases and phosphatases. A handful of major mitotic protein kinases, such as the cyclin B-CDK1 complex, the Aurora kinases, and Polo-like kinase 1 (PLK1), cooperatively regulate distinct mitotic processes. Research has identified proteins and mechanisms that integrate these kinases into signaling cascades that guide essential mitotic events. These findings have important implications for our understanding of the mechanisms of mitotic regulation and may advance the development of novel antimitotic drugs. We review collected evidence that in vertebrates, the Aurora kinases serve as catalytic subunits of distinct complexes formed with the four scaffold proteins Bora, CEP192, INCENP, and TPX2, which we deem "core" Aurora cofactors. These complexes and the Aurora-PLK1 cascades organized by Bora, CEP192, and INCENP control crucial aspects of mitosis and all pathways of spindle assembly. We compare the mechanisms of Aurora activation in relation to the different spindle assembly pathways and draw a functional analogy between the CEP192 complex and the chromosomal passenger complex that may reflect the coevolution of centrosomes, kinetochores, and the actomyosin cleavage apparatus. We also analyze the roles and mechanisms of Aurora-PLK1 signaling in the cell and centrosome cycles and in the DNA damage response.

Classification-based Segmentation for Rehabilitation Exercise Monitoring.

INTRODUCTION: Exercise segmentation, the process of isolating individual repetitions from continuous time series measurement of human motion, is key to providing online feedback to patients during rehabilitation and enables the computation of useful metrics such as joint velocity and range of motion that are otherwise difficult to measure in the clinical setting. METHODS: This paper proposes a classifier-based approach, where the motion segmentation problem is formulated as a two-class classification problem, classifying between segment and non-segment points. The proposed approach does not require domain knowledge of the exercises and generalizes to groups of participants and exercises that were not part of the training set, allowing for more robustness in clinical applications. RESULTS: Using only data from healthy participants for training, the proposed algorithm achieves an average segmentation accuracy of 92% on a 30-participant healthy dataset and 87% on a 44-patient rehabilitation dataset. CONCLUSION: A real-time approach for segmentation of rehabilitation exercises is proposed, based on two-class classification approach. The method is validated on both healthy and rehabilitation motion datasets and generalizes to a variety of demographics and exercises not part of the training set.

Rhythmic Extended Kalman Filter for Gait Rehabilitation Motion Estimation and Segmentation.

This paper proposes a method to enable the use of non-intrusive, small, wearable, and wireless sensors to estimate the pose of the lower body during gait and other periodic motions and to extract objective performance measures useful for physiotherapy. The Rhythmic Extended Kalman Filter (Rhythmic-EKF) algorithm is developed to estimate the pose, learn an individualized model of periodic movement over time, and use the learned model to improve pose estimation. The proposed approach learns a canonical dynamical system model of the movement during online observation, which is used to accurately model the acceleration during pose estimation. The canonical dynamical system models the motion as a periodic signal. The estimated phase and frequency of the motion also allow the proposed approach to segment the motion into repetitions and extract useful features, such as gait symmetry, step length, and mean joint movement and variance. The algorithm is shown to outperform the extended Kalman filter in simulation, on healthy participant data, and stroke patient data. For the healthy participant marching dataset, the Rhythmic-EKF improves joint acceleration and velocity estimates over regular EKF by 40% and 37%, respectively, estimates joint angles with 2.4° root mean squared error, and segments the motion into repetitions with 96% accuracy.

Assays to Study Mitotic Centrosome and Spindle Pole Assembly and Regulation.

Faithful chromosome segregation during cell division requires proper bipolar spindle assembly and critically depends on spindle pole integrity. In most animal cells, spindle poles form as the result of the concerted action of various factors operating in two independent pathways of microtubule assembly mediated by chromatin/RanGTP and by centrosomes. Mutation or deregulation of a number of spindle pole-organizing proteins has been linked to human diseases, including cancer and microcephaly. Our knowledge on how the spindle pole-organizing factors function at the molecular level and cooperate with one another is still quite limited. As the list of these factors expands, so does the need for the development of experimental approaches to study their function. Cell-free extracts from Xenopus laevis eggs have played an instrumental role in the dissection of the mechanisms of bipolar spindle assembly and have recently allowed the reconstitution of the key steps of the centrosome-driven microtubule nucleation pathway (Joukov et al., Mol Cell 55:578-591, 2014). Here we describe assays to study both centrosome-dependent and centrosome-independent spindle pole formation in Xenopus egg extracts. We also provide experimental procedures for the use of artificial centrosomes, such as microbeads coated with an anti-Aurora A antibody or a recombinant fragment of the Cep192 protein, to model and study centrosome maturation in egg extract. In addition, we detail the protocol for a microtubule regrowth assay that allows assessment of the centrosome-driven spindle microtubule assembly in mammalian cells.

Online tracking of the lower body joint angles using IMUs for gait rehabilitation.

An important field in physiotherapy is the rehabilitation of gait. A continuous assessment and progress tracking of a patient's ability to walk is of clinical interest. Unfortunately the tools available to the therapists are very time-consuming and subjective. Non-intrusive, small, wearable, wireless sensors can be worn by the patients and provide inertial measurements to estimate the pose of the lower body during walking. For this purpose, we propose two different kinematic models of the human lower body. We use an Extended Kalman Filter to estimate the joint angles and show that a variety of sensors, such as accelerometers, gyroscopes, and motion capture markers, can be used and fused together to aid the joint angle estimate. The algorithm is validated on gait data collected from healthy participants.

Human motion segmentation by data point classification.

Contemporary physiotherapy and rehabilitation practice uses subjective measures for motion evaluation and requires time-consuming supervision. Algorithms that can accurately segment patient movement would provide valuable data for progress tracking and on-line patient feedback. In this paper, we propose a two-class classifier approach to label each data point in the patient movement data as either a segment point or a non-segment point. The proposed technique was applied to 20 healthy subjects performing lower body rehabilitation exercises, and achieves a segmentation accuracy of 82%.

Aurora kinases and spindle assembly: variations on a common theme?

The Aurora A (AurA) serine/threonine kinase controls multiple aspects of cell division and plays a key role in centrosome maturation and bipolar spindle assembly. The pleiotropic functions of AurA depend on its interaction with several cofactors, the best known of which is TPX2. TPX2 targets AurA to spindle microtubules (MTs) and activates it, both allosterically and by protecting the activation loop (T-loop) of the kinase domain from dephosphorylation. Although several factors have been implicated in the regulation of AurA at centrosomes, the underlying mechanism has remained elusive, and the existence of a distinct centrosome-specific AurA activator has been proposed. Our recent study has identified this activator as Cep192/Spd-2, one of the key factors in centrosome biogenesis. Cep192 targets AurA to centrosomes, where it promotes its activation by a novel, oligomerization-dependent mechanism characterized by extensive T-loop phosphorylation and high kinase activity. This process is key to the function of centrosomes as microtubule-organizing centers. Here, our findings are discussed in the context of other recent studies on the Aurora kinases, with an emphasis on their role in spindle assembly. The collected evidence suggests that the 'hot spots' of MT nucleation, centrosomes and kinetochores, rely on the oligomerization-mediated mechanism of activation of AurA and AurB, respectively.

Mechanism of RAD51-dependent DNA interstrand cross-link repair.

DNA interstrand cross-links (ICLs) are toxic DNA lesions whose repair in S phase of eukaryotic cells is incompletely understood. In Xenopus egg extracts, ICL repair is initiated when two replication forks converge on the lesion. Dual incisions then create a DNA double-strand break (DSB) in one sister chromatid, whereas lesion bypass restores the other sister. We report that the broken sister chromatid is repaired via RAD51-dependent strand invasion into the regenerated sister. Recombination acts downstream of FANCI-FANCD2, yet RAD51 binds ICL-stalled replication forks independently of FANCI-FANCD2 and before DSB formation. Our results elucidate the functional link between the Fanconi anemia pathway and the recombination machinery during ICL repair. In addition, they demonstrate the complete repair of a DSB via homologous recombination in vitro.

The BRCA1/BARD1 heterodimer modulates ran-dependent mitotic spindle assembly.

The heterodimeric tumor-suppressor complex BRCA1/BARD1 exhibits E3 ubiquitin ligase activity and participates in cell proliferation and chromosome stability control by incompletely defined mechanisms. Here we show that, in both mammalian cells and Xenopus egg extracts, BRCA1/BARD1 is required for mitotic spindle-pole assembly and for accumulation of TPX2, a major spindle organizer and Ran target, on spindle poles. This function is centrosome independent, operates downstream of Ran GTPase, and depends upon BRCA1/BARD1 E3 ubiquitin ligase activity. Xenopus BRCA1/BARD1 forms endogenous complexes with three spindle-pole proteins, TPX2, NuMA, and XRHAMM--a known TPX2 partner--and specifically attenuates XRHAMM function. These observations reveal a previously unrecognized function of BRCA1/BARD1 in mitotic spindle assembly that likely contributes to its role in chromosome stability control and tumor suppression.

The nucleosomal surface as a docking station for Kaposi's sarcoma herpesvirus LANA.

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) mediates viral genome attachment to mitotic chromosomes. We find that N-terminal LANA docks onto chromosomes by binding nucleosomes through the folded region of histones H2A-H2B. The same LANA residues were required for both H2A-H2B binding and chromosome association. Further, LANA did not bind Xenopus sperm chromatin, which is deficient in H2A-H2B; chromatin binding was rescued after assembly of nucleosomes containing H2A-H2B. We also describe the 2.9-angstrom crystal structure of a nucleosome complexed with the first 23 LANA amino acids. The LANA peptide forms a hairpin that interacts exclusively with an acidic H2A-H2B region that is implicated in the formation of higher order chromatin structure. Our findings present a paradigm for how nucleosomes may serve as binding platforms for viral and cellular proteins and reveal a previously unknown mechanism for KSHV latency.