Robust microtubule dynamics facilitate low-tension kinetochore detachment in metaphase.

During mitosis, sister chromatids are stretched apart at their centromeres via their attachment to oppositely oriented kinetochore microtubules. This stretching generates inwardly directed tension across the separated sister centromeres. The cell leverages this tension signal to detect and then correct potential errors in chromosome segregation, via a mechanical tension signaling pathway that detaches improperly attached kinetochores from their microtubules. However, the sequence of events leading up to these detachment events remains unknown. In this study, we used microfluidics to sustain and observe low-tension budding yeast metaphase spindles over multiple hours, allowing us to elucidate the tension history prior to a detachment event. We found that, under conditions in which kinetochore phosphorylation weakens low-tension kinetochore-microtubule connections, the mechanical forces produced via the dynamic growth and shortening of microtubules is required to efficiently facilitate detachment events. Our findings underscore the critical role of robust kinetochore microtubule dynamics in ensuring the fidelity of chromosome segregation during mitosis.

Centromere mechanical maturation during mammalian cell mitosis.

During mitosis, tension develops across the centromere as a result of spindle-based forces. Metaphase tension may be critical in preventing mitotic chromosome segregation errors, however, the nature of force transmission at the centromere and the role of centromere mechanics in controlling metaphase tension remains unknown. We combined quantitative, biophysical microscopy with computational analysis to elucidate the mechanics of the centromere in unperturbed, mitotic human cells. We discovered that the mechanical stiffness of the human centromere matures during mitotic progression, which leads to amplified centromere tension specifically at metaphase. Centromere mechanical maturation is disrupted across multiple aneuploid cell lines, leading to a weak metaphase tension signal. Further, increasing deficiencies in centromere mechanical maturation are correlated with rising frequencies of lagging, merotelic chromosomes in anaphase, leading to segregation defects at telophase. Thus, we reveal a centromere maturation process that may be critical to the fidelity of chromosome segregation during mitosis.

A Gradient in Metaphase Tension Leads to a Scaled Cellular Response in Mitosis.

During mitosis, motor proteins associate with microtubules to exert pushing forces that establish a mitotic spindle. These pushing forces generate opposing tension in the chromatin that connects oppositely attached sister chromatids, which may then act as a mechanical signal to ensure the fidelity of chromosome segregation during mitosis. However, the role of tension in mitotic cellular signaling remains controversial. In this study, we generated a gradient in tension over multiple isogenic budding yeast cell lines by genetically altering the magnitude of motor-based spindle forces. We found that a decreasing gradient in tension led to an increasing gradient in the rates of kinetochore detachment and anaphase chromosome mis-segregration, and in metaphase time. Simulations and experiments indicated that these tension responses originate from a tension-dependent kinetochore phosphorylation gradient. We conclude that the cell is exquisitely tuned to the magnitude of tension as a signal to detect potential chromosome segregation errors during mitosis.

Physical limits on kinesin-5-mediated chromosome congression in the smallest mitotic spindles.

A characteristic feature of mitotic spindles is the congression of chromosomes near the spindle equator, a process mediated by dynamic kinetochore microtubules. A major challenge is to understand how precise, submicrometer-scale control of kinetochore micro-tubule dynamics is achieved in the smallest mitotic spindles, where the noisiness of microtubule assembly/disassembly will potentially act to overwhelm the spatial information that controls microtubule plus end-tip positioning to mediate congression. To better understand this fundamental limit, we conducted an integrated live fluorescence, electron microscopy, and modeling analysis of the polymorphic fungal pathogen Candida albicans, which contains one of the smallest known mitotic spindles (<1 µm). Previously, ScCin8p (kinesin-5 in Saccharomyces cerevisiae) was shown to mediate chromosome congression by promoting catastrophe of long kinetochore microtubules (kMTs). Using C. albicans yeast and hyphal kinesin-5 (Kip1p) heterozygotes (KIP1/kip1∆), we found that mutant spindles have longer kMTs than wild-type spindles, consistent with a less-organized spindle. By contrast, kinesin-8 heterozygous mutant (KIP3/kip3∆) spindles exhibited the same spindle organization as wild type. Of interest, spindle organization in the yeast and hyphal states was indistinguishable, even though yeast and hyphal cell lengths differ by two- to fivefold, demonstrating that spindle length regulation and chromosome congression are intrinsic to the spindle and largely independent of cell size. Together these results are consistent with a kinesin-5-mediated, length-dependent depolymerase activity that organizes chromosomes at the spindle equator in C. albicans to overcome fundamental noisiness in microtubule self-assembly. More generally, we define a dimensionless number that sets a fundamental physical limit for maintaining congression in small spindles in the face of assembly noise and find that C. albicans operates very close to this limit, which may explain why it has the smallest known mitotic spindle that still manifests the classic congression architecture.

Identification of a cell-of-origin for fibroblasts comprising the fibrotic reticulum in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the middle aged and elderly with a prevalence of one million persons worldwide. The fibrosis spreads from affected alveoli into contiguous alveoli, creating a reticular network that leads to death by asphyxiation. Lung fibroblasts from patients with IPF have phenotypic hallmarks, distinguishing them from their normal counterparts: pathologically activated Akt signaling axis, increased collagen and α-smooth muscle actin expression, distinct gene expression profile, and ability to form fibrotic lesions in model organisms. Despite the centrality of these fibroblasts in disease pathogenesis, their origin remains uncertain. Here, we report the identification of cells in the lungs of patients with IPF with the properties of mesenchymal progenitors. In contrast to progenitors isolated from nonfibrotic lungs, IPF mesenchymal progenitor cells produce daughter cells manifesting the full spectrum of IPF hallmarks, including the ability to form fibrotic lesions in zebrafish embryos and mouse lungs, and a transcriptional profile reflecting these properties. Morphological analysis of IPF lung tissue revealed that mesenchymal progenitor cells and cells with the characteristics of their progeny comprised the fibrotic reticulum. These data establish that the lungs of patients with IPF contain pathological mesenchymal progenitor cells that are cells of origin for fibrosis-mediating fibroblasts. These fibrogenic mesenchymal progenitors and their progeny represent an unexplored target for novel therapies to interdict fibrosis.