Biology subjects

Rux, C. J.

Publications and source records attributed to Rux, C. J..

6 recordsLinked to original sources

bioRxiv2026

A Computational Pipeline for Quantifying Kinetochore Morphological Changes in Live Cells

To segregate chromosomes kinetochores must resist yet deform under spindle forces. Measuring changes in kinetochore morphology can provide insight into kinetochore structure and function. This remains challenging in live cells because kinetochores are diffraction-limited with irregular, changing shapes. Here, we present a computational pipeline for quantifying kinetochore morphology in live cells, using mammalian cells with fluorescently tagged kinetochore proteins. First, the pipeline tracks, pairs and rotates kinetochores to align with their load-bearing axis. Second, it segments kinetochore signal from background, removing frames with overlapping neighboring kinetochore signals. Third, it provides metrics to define complex, non-Gaussian shape changes: (i) a non-parametric size metric that is more robust than the commonly used full-width-at-half-maximum (FWHM); (ii) analysis to classify common morphological patterns such as asymmetry, low intensity "tails" and multimodality; (iii) a 2D protein1-to-protein2 kinetochore vector as a reporter of structural rearrangements, if two kinetochore proteins were imaged. Finally, we validate the method using simulations, convolving ground-truth objects with the measured point spread function. Although kinetochore shape diversity makes assigning kinetochore size challenging, we show that our metrics better capture kinetochore size and shape changes than FWHM. Together, this pipeline provides a framework for analyzing complex kinetochore shape changes, with potential applications to other small and dynamic cellular structures.

cell biology↗

bioRxiv2025

Mammalian metaphase kinetochores are elastic and require condensin for robust structure and function

SummaryTran et al. show that mammalian metaphase kinetochores undergo large-scale, complex, and elastic deformations under forces of different timescales, magnitudes and directions. They demonstrate that condensin is required for kinetochore local structural organization and microtubule attachment under force. AbstractThe mammalian kinetochore connects chromosomes to dynamic spindle microtubules. To remain attached, it must maintain its structural integrity under force, but to what extent and how it does so remain unclear. Under spindle forces, we find using super resolution microscopy that inner (CENP-A) and outer (Hec1) metaphase kinetochores undergo correlated, large-scale (1 {micro}m) deformations along the force axis, suggesting dynamic, relative sliding of parallel protein linkages. Kinetochore shape changes can be asymmetric, with centromere-facing "tails" correlating with erroneous attachment geometries. Applying microneedle pulling forces, we demonstrate that kinetochores are elastic, stretching under force and relaxing in seconds afterwards. Finally, we show that SMC2 depletion results in more variable kinetochore deformations, despite maintained elasticity, and in reduced microtubule attachment stability. Thus, the kinetochore is structurally highly dynamic and requires a stable centromere base to maintain its structure and function under force. We propose a model whereby individual protein linkages are stiff yet global kinetochore structure flexible to accommodate different attachment geometries and forces while maintaining function.

cell biology↗

bioRxiv2025

Mechanical force locally damages, remodels and stabilizes the lattice of spindle microtubules

To segregate chromosomes at cell division, the spindle must maintain its structure under force. How it does so remains poorly understood. To address this question, we use microneedle manipulation to apply local force to spindle microtubule bundles, kinetochore-fibers (k-fibers), inside mammalian cells. We show that local load directly fractures k-fibers, and that newly created plus-ends often have arrested dynamics, resisting depolymerization. Force alone, without fracture, is sufficient for spindle microtubule stabilization, as revealed by laser ablating k-fibers under local needle force. Doublecortin, which binds a compacted microtubule lattice, is lost around the force application site, suggesting local force-induced structural remodeling. In turn, EB1, which recognizes GTP-tubulin, is locally enriched at stabilization sites, both before and after force-induced fracture. Together, our findings support a model where force-induced damage leads to local spindle microtubule lattice remodeling and stabilization, which we propose reinforces the spindle where it experiences critical loads.

cell biology↗

bioRxiv2024

The biophysical mechanism of mitochondrial pearling

Mitochondrial networks exhibit remarkable dynamics that are driven in part by fission and fusion events. However, there are other reorganizations of the network that do not involve fission and fusion. One such exception is the elusive, "beads-on-a-string" morphological transition of mitochondria. During such transitions, the cylindrical tubes of the mitochondrial membrane transiently undergo shape changes to a string of "pearls" connected along thin tubes. These dynamics have been anecdotally observed in many contexts and given disparate explanations. Here we unify these observations by proposing a common underlying mechanism based on the biophysical properties of tubular fluid membranes for which it is known that, under particular regimes of tension and pressure, membranes reach an instability and undergo a shape transition to a string of connected pearls. First, we use high-speed light-sheet microscopy to show that transient, short-lived pearling events occur spontaneously in the mitochondrial network in every cell type we have examined, including primary fibroblasts, T-cells, neurons, and budding yeast. We present evidence that transient mitochondrial pearling occurs during important biological events, particularly during T cell activation, neuronal firing, and replicative senescence. Using our high-temporal resolution data, we identify two distinct categories of spontaneous pearling, i) internal pressure-driven pearling generated by ionic flux, and ii) external tension-driven pearling generated by the cytoskeleton. By applying live-cell STED and FIB-SEM imaging we document the structural reorganization of inner cristae membranes during mitochondrial pearling and the role of the MICOS complex in regulating the frequency of pearling events. We then establish numerous methods for inducing pearling, including the ability to induce these dynamics with single mitochondrion precision. These methods include ionophores, channel activators, osmotic shock, detergents, laser stimulation, membrane intercalating molecules, chemical fixation, and micro-needle force. These disparate inducers establish three main physical causes of pearling, i) ionic flux producing internal osmotic pressure, ii) membrane packing lowering bending elasticity, and iii) external mechanical force increasing membrane tension. Pearling dynamics thereby reveal a fundamental biophysical facet of mitochondrial biology. We suggest that pearling should take its place beside fission and fusion as a key process of mitochondrial dynamics, with implications for physiology, disease, and aging.

biophysics↗

bioRxiv2023

Torques within and outside the human spindle balance twist at anaphase

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, using lattice light sheet microscopy, 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 redundantly 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.

cell biology↗

bioRxiv2021

Perilacunar bone tissue exhibits sub-micrometer modulus gradation which depends on the recency of osteocyte bone formation

Osteocytes are capable of resorbing and replacing bone local to the lacunar-canalicular system (LCS remodeling). However, the impacts of these processes on perilacunar bone quality are not understood. It is well established that aging is associated with reduced whole-bone fracture resistance, reduced osteocyte viability, and truncated LCS geometries, but it remains unclear if aging changes perilacunar bone quality. In this study, we employed atomic force microscopy (AFM) to quantify sub-micrometer gradations from 2D maps surrounding osteocyte lacunae in young (5 mo) and aged (22 mo) female mice. AFM-mapped lacunae were also imaged with confocal laser scanning microscopy to determine which osteocytes had recently deposited bone as determined by the presence of fluorochrome labels. These assays allowed us to quantify gradations in nanoscale mechanical properties of bone-forming/non-bone-forming osteocytes in young and aged mice. This study reports for the first time that there are sub-micrometer gradations in modulus surrounding lacunae and that these gradations are dependent upon recent osteocyte bone formation. Perilacunar bone adjacent to bone-forming osteocytes demonstrated lower peak and bulk modulus values when compared to bone near non-bone-forming osteocytes from the same mouse. Bone-forming osteocytes also showed increased perilacunar modulus variability. Age reduced lacunar size but did not significant effect modulus gradation or variability. In general, lacunar morphology was not a strong predictor of modulus gradation patterns. These findings support the idea that lacunar-canalicular remodeling activity changes the material properties of surrounding bone tissue on a sub-micrometer scale. Therefore, conditions that affect osteocyte health have the potential to impact bone quality. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/461298v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1fe5a68org.highwire.dtl.DTLVardef@1e17e90org.highwire.dtl.DTLVardef@13c2e3corg.highwire.dtl.DTLVardef@1ff9f38_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗