DYSTROPHIN PROTEIN COMPLEX ASSEMBLY IN LIVING CELLS

The Duchenne and Limb Girdle Muscular Dystrophies (DMD, LGMD) are a heterogeneous group of genetic disorders. Primary mutations in the dystrophin gene result in the absence of the protein in DMD, and mutations in any one of four sarcoglycan (á, â, ä, ã) genes results in a loss of the entire sarcoglycan complex in LGMD. Mutations of the á-sarcoglycan gene are clinically the most frequently observed, and of these cases, one-third have a missense substitution of a cysteine for an arginine at residue 77 (R77C) of the á-sarcoglycan protein. The function of á-sarcoglycan and the implications of the R77C mutation on protein traffic are currently unknown. Here a model system has been developed to study dystrophin protein complex (DPC) assembly in living cells. We report that a minidystrophin gene construct, currently the most promising avenue for adeno-associated virus mediated gene therapy, properly assembles and integrates into the DPC in vivo, utilizing similar mechanisms as wild type dystrophin. We also demonstrate by a variety of assays that in the absence of sarcoglycan complex assembly, á-sarcoglycan is recycled from the plasma membrane. Furthermore, I provide evidence that R77C, the most commonly occurring LGMD mutation, causes a fundamental defect in protein biosynthesis, trapping the mutant protein in the endoplasmic reticulum in vitro and in vivo. Additionally, I show through re-introduction of selected sarcoglycans that the sarcoglycans are able to associate intracellularly to form specific sub-complexes. Central to sarcoglycan complex assembly is the formation of a â-ä-core complex which promotes the deposition of both the core complex and á-sarcoglycan at the plasma membrane, as seen clinically in the microscopic pathology of some cases of LGMD-2C (ã-sarcoglycan deficiency). Taken together these data show the DPC follows a systematic and sequential assembly process, where proper integration, delivery and deposition of each protein into the complex is dependent on several protein-protein associations that in turn allow appropriate trafficking and assembly at the plasma membrane. The multi-factorial reconstruction of the DPC must therefore be carefully evaluated when treating the muscular dystrophies in humans

Opportunities and challenges for deep learning in cell dynamics research

With the growth of artificial intelligence (AI), there has been an increase in the adoption of computer vision and deep learning (DL) techniques for the evaluation of microscopy images and movies. This adoption has not only addressed hurdles in quantitative analysis of dynamic cell biological processes, but it has also started supporting advances in drug development, precision medicine and genome-phenome mapping. Here we survey existing AI-based techniques and tools, and open-source datasets, with a specific focus on the computational tasks of segmentation, classification, and tracking of cellular and subcellular structures and dynamics. We summarise long-standing challenges in microscopy video analysis from the computational perspective and review emerging research frontiers and innovative applications for deep learning-guided automation for cell dynamics research

Deep learning techniques and mathematical modeling allow 3D analysis of mitotic spindle dynamics

Time-lapse microscopy movies have transformed the study of subcellular dynamics. However, manual analysis of movies can introduce bias and variability, obscuring important insights. While automation can overcome such limitations, spatial and temporal discontinuities in time-lapse movies render methods such as 3D object segmentation and tracking difficult. Here, we present SpinX, a framework for reconstructing gaps between successive image frames by combining deep learning and mathematical object modeling. By incorporating expert feedback through selective annotations, SpinX identifies subcellular structures, despite confounding neighbor-cell information, non-uniform illumination, and variable fluorophore marker intensities. The automation and continuity introduced here allows the precise 3D tracking and analysis of spindle movements with respect to the cell cortex for the first time. We demonstrate the utility of SpinX using distinct spindle markers, cell lines, microscopes, and drug treatments. In summary, SpinX provides an exciting opportunity to study spindle dynamics in a sophisticated way, creating a framework for step changes in studies using time-lapse microscopy

Roles for the Conserved Spc105p/Kre28p Complex in Kinetochore-Microtubule Binding and the Spindle Assembly Checkpoint

Background: Kinetochores attach sister chromatids to microtubules of the mitotic spindle and orchestrate chromosome disjunction at anaphase. Although S. cerevisiae has the simplest known kinetochores, they nonetheless contain ∼70 subunits that assemble on centromeric DNA in a hierarchical manner. Developing an accurate picture of the DNA-binding, linker and microtubule-binding layers of kinetochores, including the functions of individual proteins in these layers, is a key challenge in the field of yeast chromosome segregation. Moreover, comparison of orthologous proteins in yeast and humans promises to extend insight obtained from the study of simple fungal kinetochores to complex animal cell kinetochores. Principal Findings: We show that S. cerevisiae Spc105p forms a heterotrimeric complex with Kre28p, the likely orthologue of the metazoan kinetochore protein Zwint-1. Through systematic analysis of interdependencies among kinetochore complexes, focused on Spc105p/Kre28p, we develop a comprehensive picture of the assembly hierarchy of budding yeast kinetochores. We find Spc105p/Kre28p to comprise the third linker complex that, along with the Ndc80 and MIND linker complexes, is responsible for bridging between centromeric heterochromatin and kinetochore MAPs and motors. Like the Ndc80 complex, Spc105p/Kre28p is also essential for kinetochore binding by components of the spindle assembly checkpoint. Moreover, these functions are conserved in human cells. Conclusions/Significance: Spc105p/Kre28p is the last of the core linker complexes to be analyzed in yeast and we show it to be required for kinetochore binding by a discrete subset of kMAPs (Bim1p, Bik1p, Slk19p) and motors (Cin8p, Kar3p), all of which are nonessential. Strikingly, dissociation of these proteins from kinetochores prevents bipolar attachment, even though the Ndc80 and DASH complexes, the two best-studied kMAPs, are still present. The failure of Spc105 deficient kinetochores to bind correctly to spindle microtubules and to recruit checkpoint proteins in yeast and human cells explains the observed severity of missegregation phenotypes

Dynamic modelling reveals the separable contributions to achieving correct spindle orientation in a noisy system

The mechanisms by which the mammalian mitotic spindle is guided to a predefined orientation through microtubule-cortex interactions have recently received considerable interest, but there has been no dynamic model that describes spindle movements toward the preferred axis in human cells. Here, we develop a dynamic model based on stochastic activity of cues anisotropically positioned around the cortex of the mitotic cell and we show that the mitotic spindle does not reach equilibrium before chromosome segregation. Our model successfully captures the characteristic experimental behavior of noisy spindle rotation dynamics in human epithelial cells, including a weak underlying bias in the direction of rotation, suppression of motion close to the alignment axis, and the effect of the aspect ratio of the interphase cell shape in defining the final alignment axis. We predict that the force exerted per cue has a value that minimizes the deviation of the spindle from the predefined axis. The model has allowed us to systematically explore the parameter space around experimentally relevant configurations, and predict the mechanistic function of a number of established regulators of spindle orientation, highlighting how physical modeling of a noisy system can lead to functional biological understanding. We provide key insights into measurable parameters in live cells that can help distinguish between mechanisms of microtubule and cortical-cue interactions that jointly control the final orientation of the spindle.This work was supported by Cancer Research UKThis is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.bpj.2015.08.01

How are Dynamic Microtubules Stably Tethered to Human Chromosomes?

During cell division, microtubules capture and pull chromosomes apart into two equal sets. Without the establishment of proper chromosome-microtubule attachment, microtubules cannot impart the pulling forces needed to separate sister chromatid pairs. How are chromosomes captured along microtubule walls? How is the attachment of chromosomes to dynamic microtubule-ends achieved and monitored? We discuss these key questions by considering the roles of kinetochore-bound microtubule regulating proteins and also the complex regulatory loops of kinases and phosphatases that control chromosome-microtubule attachment and ensure the accurate segregation of chromosomes

DNA repair. PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair.

XRCC4 and XLF are two structurally related proteins that function in DNA double-strand break (DSB) repair. Here, we identify human PAXX (PAralog of XRCC4 and XLF, also called C9orf142) as a new XRCC4 superfamily member and show that its crystal structure resembles that of XRCC4. PAXX interacts directly with the DSB-repair protein Ku and is recruited to DNA-damage sites in cells. Using RNA interference and CRISPR-Cas9 to generate PAXX(-/-) cells, we demonstrate that PAXX functions with XRCC4 and XLF to mediate DSB repair and cell survival in response to DSB-inducing agents. Finally, we reveal that PAXX promotes Ku-dependent DNA ligation in vitro and assembly of core nonhomologous end-joining (NHEJ) factors on damaged chromatin in cells. These findings identify PAXX as a new component of the NHEJ machinery.T.O. and T.L.B. are supported by the Wellcome Trust. The Jackson lab is funded by Cancer Research UK (CRUK) program grant C6/A11224, the European Research Council and the European Community Seventh Framework Programme grant agreement no. HEALTH-F2-2010- 259893 (DDResponse). Core infrastructure funding to the Jackson lab is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, supplemented by CRUK. V.M.D. is a CRUK Career Development Fellow. The Draviam lab is funded by a CRUK CDA (C28598/A9787).This is the accepted manuscript version. The final version is available from AAAS at http://www.sciencemag.org/content/347/6218/185.full

Spindle rotation in human cells is reliant on a MARK2-mediated equatorial spindle-centering mechanism

This work was supported by a Cancer Research UK Career Development Award (C28598/A9787), Biotechnology and Biological Sciences Research Council Project grant (BB/R01003X/1), and a Queen Mary University of London Laboratory startup grant to V.M. Draviam, a Universiti Brunei Darussalam PhD studentship to I. Zulkipli, a Queen Mary University of London PhD studentship to M. Hart, a London Interdisciplinary Biosciences Consortium Biotechnology and Biological Sciences Research Council–Doctoral Training Partnerships PhD studentship to D. Dang (cosupervised by V.M. Draviam and N. Sastry; BB/M009513/1), and an Islamic Development Bank PhD studentship to P. Gul