Biology subjects

Witt, H.

Publications and source records attributed to Witt, H..

10 recordsLinked to original sources

bioRxiv2025

Condensin I confers Stiffness and Centromeric Cohesion to Mitotic Chromosomes

When cells divide, the newly replicated sister chromatids must be segregated evenly to the daughter cells. During mitosis, mechanical force is applied by spindle microtubules in 2 ways: first by pushing on chromosome arms to promote chromosome congression to the cell equator in metaphase, and then by pulling on kinetochores to promote sister chromatid disjunction during anaphase. For segregation to proceed faithfully, the pliable interphase chromatin must be transformed into stiff mitotic chromosomes able to withstand these forces. However, it is unclear how the cell establishes chromosome stiffness and what the consequences are for dividing cells if this stiffness is disrupted. Many of the structural changes imposed on chromosomes in mitosis are driven by Condensin complexes, in conjunction with Topoisomerase II. Here, we have combined rapid protein depletion and live cell imaging with in-depth mechanical characterization of purified mitotic chromosomes to probe the roles of Condensins I and II in the establishment and maintenance of the mechanical strength of mitotic chromosomes. We show that Condensin I, but not Condensin II, is required to establish chromosome stiffness and chromatin elasticity, and yet ceases to be required for the maintenance of these properties once chromosome formation has been completed. Nevertheless, depletion of Condensin I from already formed chromosomes still impacts centromeric chromatin and leads to a loss of sister centromere cohesion. We propose that the extensive chromatin loop network established by Condensin I is locked in place by Topoisomerase II mediated DNA catenation.

cell biology↗

bioRxiv2024

Resolving interface structure and local internal mechanics of mitotic chromosomes

The interface of chromosomes enables them to interact with the cell environment and is crucial for their mechanical stability during mitosis. Here, we use Atomic Force Microscopy (AFM) to probe the interface and local micromechanics of the highly condensed and complex chromatin network of native human mitotic chromosomes. Our AFM images provide detailed snapshots of chromatin loops and Sister-Chromatids Intertwines. A scaling analysis of these images reveals that the chromatin surface has fractal nature. AFM-based Force Spectroscopy and microrheology further show that chromosomes can resist severe deformations, elastically recovering their initial shape following two characteristic timescales. Localized indentations over the chromatids reveal that the spatially varying micromechanics of the chromatin network is largely governed by chromatin density. Together, our AFM investigation provides new insights into the structure and local mechanics of mitotic chromosomes, offering a toolbox for further characterization of complex biological structures, such as chromosomes, down to the nanoscale.

biophysics↗

bioRxiv2024

Generative modeling of biological shapes and images using a probabilistic α-shape sampler

Understanding morphological variation is an important task in many areas of computational biology. Recent studies have focused on developing computational tools for the task of sub-image selection which aims at identifying structural features that best describe the variation between classes of shapes. A major part in assessing the utility of these approaches is to demonstrate their performance on both simulated and real datasets. However, when creating a model for shape statistics, real data can be difficult to access and the sample sizes for these data are often small due to them being expensive to collect. Meanwhile, the current landscape of generative models for shapes has been mostly limited to approaches that use black-box inference--making it difficult to systematically assess the power and calibration of sub-image models. In this paper, we introduce the -shape sampler: a probabilistic framework for generating realistic 2D and 3D shapes based on probability distributions which can be learned from real data. We demonstrate our framework using proof-of-concept examples and in two real applications in biology where we generate (i) 2D images of healthy and septic neutrophils and (ii) 3D computed tomography (CT) scans of primate mandibular molars. The -shape sampler R package is open-source and can be downloaded at https://github.com/lcrawlab/ashapesampler. Author SummaryUsing shapes and images to understand genotypic and phenotypic variation has proven to be an effective strategy in many biological applications. Unfortunately, shape data can be expensive to collect and, as a result, sample sizes for analyses are often small. Despite methodological advancements in shape statistics and machine learning, benchmarking standards for evaluating new computational tools via data simulation is still underdeveloped. In this paper, we present a probability-based pipeline called the -shape sampler which has the flexibility to generate new and unobserved shapes based on an input set of data. We extensively evaluate the generative capabilities of our pipeline using 2D cellular images of neutrophils and 3D mandibular molars from two different suborders of primates.

bioinformatics↗

bioRxiv2023

Ion-mediated condensation controls the mechanics of mitotic chromosomes

During mitosis in eukaryotic cells, mechanical forces generated by the mitotic spindle pull the sister chromatids into the nascent daughter cells. How do mitotic chromosomes achieve the necessary mechanical stiffness and stability to maintain their integrity under these forces? Here, we use optical tweezers to show that ions involved in physiological chromosome condensation are crucial for chromosomal stability, stiffness and viscous dissipation. We combine these experiments with high-salt histone-depletion and theory to show that chromosomal elasticity originates from the chromatin fiber behaving as a flexible polymer, whereas energy dissipation can be explained by interactions between chromatin loops. Taken together, we show how collective properties of mitotic chromosomes, a biomaterial of incredible complexity, emerge from molecular properties, and how they are controlled by the physico-chemical environment.

biophysics↗

bioRxiv2019

Context-dependent role of vinculin in neutrophil adhesion, motility and trafficking

Neutrophils are innate immune effector cells that traffic from the peripheral blood to extravascular sites of inflammation. {beta}2 integrins are involved during multiple phases of neutrophil recruitment, including the transition from rolling to arrest, firm attachment and motility within the vasculature. Following neutrophil arrest, adhesion stabilization occurs as the neutrophil interacts with the endothelial surface and crawls into a favorable position for extravasation. The cytoskeletal protein vinculin has been implicated in other cell types as a regulator of adhesion strength by promoting focal adhesion maturation and as a sensor of the mechanical properties of the microenvironment. Neutrophils express vinculin but do not form mature focal adhesions. Here, we characterize the role of vinculin in {beta}2 integrin-dependent neutrophil adhesion, motility, mechanosensing, and recruitment. We observe that knockout of vinculin attenuates, but does not completely abrogate, neutrophil adhesion, spreading, and crawling under static conditions. In the presence of forces from fluid flow, vinculin was not required for neutrophil adhesion or migration. Vinculin deficiency only mildly attenuated neutrophil traction stresses and spreading on stiff, but not soft, polyacrylamide gels indicating a minor role for vinculin in the mechanosensing of the neutrophil as compared to slower moving mesenchymal cells that form mature focal adhesions. Consistent with these findings, we observe in vivo neutrophil recruitment into the inflamed peritoneum of mice remains intact in the absence of vinculin. Together, these data suggest that while vinculin regulates some aspects of neutrophil adhesion and spreading, it may be dispensable for neutrophil recruitment and motility in vivo.

immunology↗

bioRxiv2019

Pre-stress and cortical stiffness affect fluidity of living cells

Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on pre-stress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical pre-stress, area compressibility modulus, and the power law (fluidity) exponent. Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic pre-stress and area compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicted power law exponent for transiently cross-linked polymer networks. In contrast to the widely used Hertzian mechanics, our model provides viscoelastic parameters independent of indenter geometry and compression velocity.

biophysics↗

bioRxiv2019

Genetic analysis of the STIM1 gene in chronic pancreatitis

Chronic pancreatitis is a complex disease that involves many factors, both genetic and environmental. Over the past two decades, molecular genetic analysis of five genes that are highly expressed in human pancreatic acinar cells, namely PRSS1, PRSS2, SPINK1, CTRC and CTRB1/CTRB2, has established that a trypsin-dependent pathway plays a key role in the etiology of chronic pancreatitis. Since Ca2+ deregulation can lead to intracellular trypsin activation in experimental acute pancreatitis, we analyzed STIM1 (encoding stromal interaction molecule-1, the main regulator of Ca2+ homeostasis in pancreatic acinar cells) as a candidate modifier gene in French, German and Chinese patients with chronic pancreatitis. The French and German subjects were analyzed by Sanger sequencing whereas the Chinese subjects were analyzed by targeted next-generation sequencing confirmed by Sanger sequencing. A total of 37 rare coding variants (35 missense and 2 nonsense) were identified, which were enriched in patients as compared with controls [2.28% (47/2,057) vs. 0.99% (33/3,322); odds ratio = 2.33, P = 0.0001]. This is the first large case-control study to demonstrate a putative association of rare STIM1 coding variants with chronic pancreatitis. Functional analysis will be required to clarify whether or not the rare STIM1 variants detected predispose to pancreatitis.

genetics↗

bioRxiv2019

Lateral Subunit Coupling Determines Intermediate Filament Mechanics

The cytoskeleton is a composite network of three types of protein filaments, among which in-termediate filaments (IFs) are the most extensible ones. Two very important IFs are keratin and vimentin, which have similar molecular architectures, but different mechanical behaviors. Here we compare the mechanical response of single keratin and vimentin filaments using optical tweezers. We show that the mechanics of vimentin strongly depends on the ionic strength of the buffer and that its force-strain curve suggests a high degree of cooperativity between subunits. Indeed, a computational model indicates that in contrast to keratin, vimentin is characterized by strong lateral subunit coupling of its charged monomers during unfolding of -helices. We conclude that cells can tune their mechanics by differential use of keratin versus vimentin.

biophysics↗

bioRxiv2019

Vimentin intermediate filaments undergo irreversible conformational changes during cyclic loading

Intermediate filaments (IFs) are part of the cytoskeleton of eukaryotic cells and are thus largely responsible for the cells mechanical properties. IFs are characterized by a pronounced extensibility and remarkable resilience that enable them to support cells in extreme situations. Previous experiments showed that under strain, -helices in vimentin IFs might unfold to {beta}-sheets. Upon repeated stretching, the filaments soften, however, the remaining plastic strain is negligible. Here we observe that vimentin IFs do not recover their original stiffness on reasonable time scales, and we explain these seemingly contradicting results by introducing a third, less well-defined conformational state. Reversibility on the nanoscale can be fully rescued by introducing crosslinkers that prevent transition to the {beta}-sheet. Our results classify IFs as a nano-material with intriguing mechanical properties, which is likely to play a major role for the cells local adaption to external stimuli.

biophysics↗

bioRxiv2019

Restored alveolar epithelial differentiation and reversed human lung fibrosis upon Notch inhibition

Alveolar epithelial cell type II (AEC2) injury underlies idiopathic pulmonary fibrosis (IPF). Here we show increased Notch1 signaling in AEC2s in human IPF and IPF models, causing enhanced proliferation and de-differentiation of AEC2s. As a result, we observed defective surfactant protein (SP)-B/C processing, elevated alveolar surface tension, repetitive alveolar collapse and development of lung fibrosis. Similar changes were encountered upon pharmacological inhibition of SP-B/C processing in vivo by pepstatin A. Inhibition of Notch signaling in cultured human IPF precision cut lung slices improved surfactant processing capacity of AEC2s and reversed fibrosis. Notch1 therefore offers as novel therapeutic target.\n\nOne sentence summaryNotch1 inhibition restores alveolar epithelial differentiation and surface tension and reverses matrix deposition in lung fibrosis

cell biology↗