At each cell division, the spindle self-organizes from microtubules and motors. In human spindles, the motors dynein and Eg5 generate contractile and extensile stress, respectively. Inhibiting dynein ...or its targeting factor NuMA leads to unfocused, turbulent spindles, and inhibiting Eg5 leads to monopoles; yet, bipolar spindles form when both are inhibited together. What, then, are the roles of these opposing motors? Here, we generate NuMA/dynein- and Eg5-doubly inhibited spindles that not only attain a typical metaphase shape and size but also undergo anaphase. However, these spindles have reduced microtubule dynamics and are mechanically fragile, fracturing under force. Furthermore, they exhibit lagging chromosomes and a dramatic left-handed twist at anaphase. Thus, although these opposing motors are not required for spindle shape, they are essential to its mechanical and functional robustness. This work suggests a design principle whereby opposing active stresses provide robustness to force-generating cellular structures.
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•Spindles lacking NuMA/dynein and Eg5 motor activities attain normal shape and size•Microtubule organization and flux are disrupted without these opposing motors•Spindles lacking NuMA/dynein and Eg5 are mechanically fragile in metaphase•Opposing forces are required to prevent twisting and segregation errors in anaphase
Successful cell division relies on the robust assembly of the microtubule-based spindle. Neahring et al. show that the opposing mitotic motors NuMA/dynein and Eg5 are not required for human spindle formation but are instead essential for its mechanical, structural, and functional robustness.
This paper reports the design, development, and magnetic resonance imaging (MRI) compatibility evaluation of an actuated transrectal prostate robot for MRI-guided needle intervention in the prostate. ...The robot performs actuated needle MRI guidance with the goals of providing 1) MRI compatibility; 2) MRI-guided needle placement with accuracy sufficient for targeting clinically significant prostate cancer foci; 3) reducing interventional procedure times (thus increasing patient comfort and reducing opportunity for needle targeting error due to patient motion); 4) enabling real-time MRI monitoring of interventional procedures; and 5) reducing the opportunities for error that arise in manually actuated needle placement. The design of the robot, employing piezoceramic-motor actuated needle guide positioning and manual needle insertion, is reported. Results of an MRI compatibility study show no reduction of MRI signal-to-noise ratio (SNR) with the disabled motors. Enabling the motors reduces the SNR by 80% without radio frequency (RF) shielding, but the SNR is only reduced by 40-60% with RF shielding. The addition of RF shielding is shown to significantly reduce image SNR degradation caused by the presence of the robotic device. An accuracy study of MRI-guided biopsy needle placements in a prostate phantom is reported. The study shows an average in-plane targeting error of 2.4 mm with a maximum error of 3.7 mm. These data indicate that the system's needle targeting accuracy is similar to that obtained with a previously reported manually actuated system, and is sufficient to reliably sample clinically significant prostate cancer foci under MRI guidance.
Elucidating the wiring diagram of the human cell is a central goal of the postgenomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry, and data science to ...systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery. We found that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions, and that RNA binding proteins form a specific subgroup defined by unique interaction and localization properties. Paired with a fully interactive website (opencell.czbiohub.org), our work constitutes a resource for the quantitative cartography of human cellular organization.
We report quantitative label-free imaging with phase and polarization (QLIPP) for simultaneous measurement of density, anisotropy, and orientation of structures in unlabeled live cells and tissue ...slices. We combine QLIPP with deep neural networks to predict fluorescence images of diverse cell and tissue structures. QLIPP images reveal anatomical regions and axon tract orientation in prenatal human brain tissue sections that are not visible using brightfield imaging. We report a variant of U-Net architecture, multi-channel 2.5D U-Net, for computationally efficient prediction of fluorescence images in three dimensions and over large fields of view. Further, we develop data normalization methods for accurate prediction of myelin distribution over large brain regions. We show that experimental defects in labeling the human tissue can be rescued with quantitative label-free imaging and neural network model. We anticipate that the proposed method will enable new studies of architectural order at spatial scales ranging from organelles to tissue.
The cell cycle, over which cells grow and divide, is a fundamental process of life. Its dysregulation has devastating consequences, including cancer
. The cell cycle is driven by precise regulation ...of proteins in time and space, which creates variability between individual proliferating cells. To our knowledge, no systematic investigations of such cell-to-cell proteomic variability exist. Here we present a comprehensive, spatiotemporal map of human proteomic heterogeneity by integrating proteomics at subcellular resolution with single-cell transcriptomics and precise temporal measurements of individual cells in the cell cycle. We show that around one-fifth of the human proteome displays cell-to-cell variability, identify hundreds of proteins with previously unknown associations with mitosis and the cell cycle, and provide evidence that several of these proteins have oncogenic functions. Our results show that cell cycle progression explains less than half of all cell-to-cell variability, and that most cycling proteins are regulated post-translationally, rather than by transcriptomic cycling. These proteins are disproportionately phosphorylated by kinases that regulate cell fate, whereas non-cycling proteins that vary between cells are more likely to be modified by kinases that regulate metabolism. This spatially resolved proteomic map of the cell cycle is integrated into the Human Protein Atlas and will serve as a resource for accelerating molecular studies of the human cell cycle and cell proliferation.
We designed an epi-illumination SPIM system that uses a single objective and has a sample interface identical to that of an inverted fluorescence microscope with no additional reflection elements. It ...achieves subcellular resolution and single-molecule sensitivity, and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of 16 cell lines and parallel recording of cellular responses to perturbations.
At each cell division, nanometer-scale motors and microtubules give rise to the micron-scale spindle. Many mitotic motors step helically around microtubules in vitro, and most are predicted to twist ...the spindle in a left-handed direction. However, the human spindle exhibits only slight global twist, raising the question of how these molecular torques are balanced. Here, we find that anaphase spindles in the epithelial cell line MCF10A have a high baseline twist, and we identify factors that both increase and decrease this twist. The midzone motors KIF4A and MKLP1 are together required for left-handed twist at anaphase, and we show that KIF4A generates left-handed torque in vitro. The actin cytoskeleton also contributes to left-handed twist, but dynein and its cortical recruitment factor LGN counteract it. Together, our work demonstrates that force generators regulate twist in opposite directions from both within and outside the spindle, preventing strong spindle twist during chromosome segregation.
At each cell division, nanometer-scale components self-organize to build a micron-scale spindle. In mammalian spindles, microtubule bundles called kinetochore-fibers attach to chromosomes and focus ...into spindle poles. Despite evidence suggesting that poles can set spindle length, their role remains poorly understood. In fact, many species do not have spindle poles. Here, we probe the pole’s contribution to mammalian spindle length, dynamics, and function by inhibiting dynein to generate spindles whose kinetochore-fibers do not focus into poles, yet maintain a metaphase steady-state length. We find that unfocused kinetochore-fibers have a mean length indistinguishable from control, but a broader length distribution, and reduced length coordination between sisters and neighbors. Further, we show that unfocused kinetochore-fibers, like control, can grow back to their steady-state length if acutely shortened by drug treatment or laser ablation: they recover their length by tuning their end dynamics, albeit slower due to their reduced baseline dynamics. Thus, kinetochore-fiber dynamics are regulated by their length, not just pole-focusing forces. Finally, we show that spindles with unfocused kinetochore-fibers can segregate chromosomes but fail to correctly do so. We propose that mammalian spindle length emerges locally from individual k-fibers while spindle poles globally coordinate k-fibers across space and time.
Targeted proteomics is a convenient method determining enzyme expression levels, but a quantitative analysis of these proteomic data has not been fully explored yet. Here, we present and demonstrate ...a computational tool (principal component analysis of proteomics, PCAP) that uses quantitative targeted proteomics data to guide metabolic engineering and achieve higher production of target molecules from heterologous pathways. The method is based on the application of principal component analysis to a collection of proteomics and target molecule production data to pinpoint specific enzymes that need to have their expression level adjusted to maximize production. We illustrated the method on the heterologous mevalonate pathway in Escherichia coli that produces a wide range of isoprenoids and requires balanced pathway gene expression for high yields and titers. PCAP-guided engineering resulted in over a 40% improvement in the production of two valuable terpenes. PCAP could potentially be productively applied to other heterologous pathways as well.
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•We report quantitative analysis method for proteomics data using PCA.•Principal component analysis of proteomics (PCAP) can direct metabolic engineering.•PCAP provides information not apparent by the simple observation of proteomics data.•PCAP was successfully applied to improve the mevalonate pathway (MEV) in E. coli.•Limonene and bisabolene production increased about 40% using PCAP-based strategy.
Mitochondria in cardiac myocytes are critical for generating ATP to meet the high metabolic demands associated with sarcomere shortening. Distinct remodeling of mitochondrial structure and function ...occur in cardiac myocytes in both developmental and pathological settings. However, the factors that underlie these changes are poorly understood. Because remodeling of tissue architecture and extracellular matrix (ECM) elasticity are also hallmarks of ventricular development and disease, we hypothesize that these environmental factors regulate mitochondrial function in cardiac myocytes. To test this, we developed a new procedure to transfer tunable polydimethylsiloxane disks microcontact-printed with fibronectin into cell culture microplates. We cultured Sprague-Dawley neonatal rat ventricular myocytes within the wells, which consistently formed tissues following the printed fibronectin, and measured oxygen consumption rate using a Seahorse extracellular flux analyzer. Our data indicate that parameters associated with baseline metabolism are predominantly regulated by ECM elasticity, whereas the ability of tissues to adapt to metabolic stress is regulated by both ECM elasticity and tissue alignment. Furthermore, bioenergetic health index, which reflects both the positive and negative aspects of oxygen consumption, was highest in aligned tissues on the most rigid substrate, suggesting that overall mitochondrial function is regulated by both ECM elasticity and tissue alignment. Our results demonstrate that mitochondrial function is regulated by both ECM elasticity and myofibril architecture in cardiac myocytes. This provides novel insight into how extracellular cues impact mitochondrial function in the context of cardiac development and disease.
A new methodology has been developed to measure O
consumption rates in engineered cardiac tissues with independent control over tissue alignment and matrix elasticity. This led to the findings that matrix elasticity regulates basal mitochondrial function, whereas both matrix elasticity and tissue alignment regulate mitochondrial stress responses.
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