Biology subjects

Redemann, S.

Publications and source records attributed to Redemann, S..

16 recordsLinked to original sources

bioRxiv2026

Eg5 activity and density-driven bundling organize the human metaphase mitotic spindle independently of spindle bipolarity

The mitotic spindle segregates chromosomes through the coordinated actions of microtubules and molecular motors. Classic models propose that microtubules nucleate at spindle poles and grow inward to capture chromosomes; however, recent structural studies show that spindles contain short microtubules that do not span the distance between poles and chromosomes. It is unclear how these short microtubules assemble a bipolar spindle. Using cryo-electron tomography to map microtubule polarity in human metaphase spindles, we find that microtubules form locally antiparallel bundles with consistent 8 nm wall-to-wall spacing. We utilized motor perturbations and centriole depletion, which generated motor-active monopolar spindles, to reveal that the kinesin-5 motor Eg5 organizes local antiparallel overlap independently of spindle bipolarity. We found that bundles are organized by density-driven steric interactions rather than motor-mediated crosslinking. These findings support a self-organized, bottom-up model in which local microtubule-motor interactions within dense bundles generate forces that build the bipolar spindle, challenging pole-centric models. KEY FINDINGSO_LIMicrotubules in metaphase spindles organize in locally antiparallel bundles with consistent 8 nm wall-to-wall spacing. C_LIO_LIThe Eg5 motor generates antiparallel microtubule overlap independently of spindle bipolarity. C_LIO_LIMicrotubule spacing scales with density through steric interactions, not direct motor crosslinking. C_LIO_LIDynein regulates microtubule density to control bundle architecture; balanced Eg5-dynein density regulation maintains spindle bipolarity. C_LI

biophysics↗

bioRxiv2024

Accurate and fast segmentation of filaments and membranes in micrographs and tomograms with TARDIS

Segmentation of macromolecular structures is the primary bottleneck for studying biomolecules and their organization with electron microscopy in 2D/3D - requiring months of manual effort. Transformer-based Rapid Dimensionless Instance Segmentation (TARDIS) is a deep learning framework that automatically and accurately annotates membranes and filaments. Pre-trained TARDIS models can segment electron tomography (ET) reconstructions from both 3D and 2D electron micrographs of cryo and plastic-embedded samples. Furthermore, by implementing a novel geometric transformer architecture, TARDIS is the only method to provide accurate instance segmentations of these structures. Reducing the annotation time for ET data from months to minutes, we demonstrate segmentation of membranes and filaments in over 13,000 tomograms in the CZII Data Portal. TARDIS thus enables quantitative biophysical analysis at scale for the first time. We show this in application to kinetochore-microtubule attachment and viral-membrane interactions. TARDIS can be extended to new biomolecules and applications and open-source at https://github.com/SMLC-NYSBC/TARDIS.

bioinformatics↗

bioRxiv2024

Axonal transcriptome reveals upregulation of PLK1 as a protective mechanism in response to increased DNA damage in FUSP525L spinal motor neurons

Mutations in the gene FUSED IN SARCOMA (FUS) are among the most frequently occurring genetic forms of amyotrophic lateral sclerosis (ALS). Early pathogenesis of FUS-ALS involves impaired DNA damage response and axonal degeneration. However, it is still poorly understood how these gene mutations lead to selective spinal motor neuron (MN) degeneration and how nuclear and axonal phenotypes are linked. To specifically address this, we applied a compartment specific RNA-sequencing approach using microfluidic chambers to generate axonal as well as somatodendritic compartment-specific profiles from isogenic induced pluripotent stem cells (iPSCs)-derived MNs. We demonstrate high purity of axonal and soma fractions and show that the axonal transcriptome is unique and distinct from that of somas including significantly fewer number of transcripts. Functional enrichment analysis revealed that differentially expressed genes (DEGs) in axons were mainly enriched in key pathways like RNA metabolism and DNA damage, complementing our knowledge of early phenotypes in ALS pathogenesis and known functions of FUS. In addition, we demonstrate a strong enrichment for cell cycle associated genes including significant upregulation of polo-like kinase 1 (PLK1) in FUSP525L mutant MNs. PLK1 was increased upon DNA damage induction and PLK1 inhibition further increased the number of DNA damage foci in etoposide-treated cells, an effect that was diminished in case of FUS mutant MNs. In contrast, inhibition of PLK1 increased late apoptotic or necrosis-induced neuronal cell death in mutant neurons. Taken together, our findings provide insights into compartment-specific transcriptomics in human FUS-ALS MNs and we propose that specific upregulation of PLK1 might represent an early event in the pathogenesis of ALS, possibly modulating DNA damage response and other associated pathways.

neuroscience↗

bioRxiv2024

Mechanisms of Meiotic Spindle Initiation in Caenorhabditis elegans Oocytes

In many animal species, the oocyte meiotic spindle, which is required for chromosome segregation, forms without centrosomes. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in C. elegans oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly occurred after auxin-induced degradation of Ran-GEF but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown. We found that the concentration of soluble tubulin in the metaphase spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules. Measurement of the volume occupied by yolk granules and mitochondria indicated that volume exclusion would be sufficient to explain the concentration of tubulin in the spindle volume. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes.

cell biology↗

bioRxiv2023

Microtubule length correlates with spindle length in C. elegans meiosis

The accurate segregation of chromosomes during female meiosis relies on the precise assembly and function of the meiotic spindle, a dynamic structure primarily composed of microtubules. Despite the crucial role of microtubule dynamics in this process, the relationship between microtubule length and spindle size remains elusive. Leveraging C. elegans as a model system, we combined electron tomography and live imaging to investigate this correlation. Our analysis revealed significant changes in spindle length throughout meiosis, coupled with alterations in MT length. Surprisingly, while spindle size decreases during the initial stages of anaphase, the size of antiparallel microtubule overlap decreased as well. Detailed electron tomography shows a positive correlation between microtubule length and spindle size, indicating a role of microtubule length in determining spindle dimensions. Notably, microtubule numbers displayed no significant association with spindle length, highlighting the dominance of microtubule length regulation in spindle size determination. Depletion of the microtubule depolymerase KLP-7 led to elongated metaphase spindles with increased microtubule length, supporting the link between microtubule length and spindle size. These findings underscore the pivotal role of regulating microtubule dynamics, and thus microtubule length, in governing spindle rearrangements during meiotic division, shedding light on fundamental mechanisms dictating spindle architecture.

cell biology↗

bioRxiv2023

Lack of chromokinesin Klp-19 creates a more rigid midzone and affects force transmission during anaphase in C. elegans

Recent studies have highlighted the significance of the spindle midzone, the region between the segregating chromosomes, in ensuring proper chromosome segregation. By combining 3D electron tomography, cutting-edge light microscopy and a novel single cell in vitro essay allowing single molecule tracking, we have discovered a previously unknown role of the regulation of microtubule dynamics within the spindle midzone of C. elegans by the chromokinesin KLP-19, and its relevance for proper spindle function. Using Fluorescence recovery after photobleaching and a combination of second harmonic generation and two-photon fluorescence microscopy, we found that the length of the antiparallel microtubule overlap zone in the spindle midzone is constant throughout anaphase, and independent of cortical pulling forces as well as the presence of the microtubule bundling protein SPD-1. Further investigations of SPD-1 and KLP-19 in C. elegans, the homologs of PRC1 and KIF4a, suggest that KLP-19 regulates the overlap length and functions independently of SPD-1. Our data shows that KLP-19 plays an active role in regulating the length of microtubules within the midzone as well as the size of the antiparallel overlap region throughout mitosis. Depletion of KLP-19 in mitosis leads to an increase in microtubule length and thus microtubule-based interactions in the spindle midzone, which affects spindle dynamics and force transmission. Our data shows that by localizing KLP-19 to the spindle midzone in anaphase microtubule dynamics can be locally controlled allowing the formation of a functional midzone. SummaryKLP-19 controls microtubule length in the spindle midzone of C. elegans, affecting spindle dynamics and force transmission during mitosis.

cell biology↗

bioRxiv2023

Collapse of late endosomal pH elicits a rapid Rab7 response via V-ATPase and RILP.

Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of proper pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). We used the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyper-activation of Rab7 and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR) recycling on pH-neutralized LEs. Either pH neutralization (NH4Cl) or Rab7 hyper-active mutants alone can phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds or in disease states. Summary StatementMulligan et al. show that V-ATPase assembly and RILP mediate hyperactivation of the small GTPase Rab7 when the late endosomal pH gradient collapses disrupting endosome tubulation and M6PR trafficking.

cell biology↗

bioRxiv2023

Mechanisms of cilia regeneration in Xenopus multiciliated epithelium in vivo.

Cilia regeneration is a physiological event, and while studied extensively in unicellular organisms, it remains poorly understood in vertebrates. In this study, using Xenopus multiciliated cells (MCCs) as a model, we demonstrate that, unlike unicellular organisms, deciliation removes the transition zone (TZ) and the ciliary axoneme. While MCCs immediately begin the regeneration of the ciliary axoneme, surprisingly, the assembly of TZ is delayed. However, ciliary tip proteins, Sentan and Clamp, localize to regenerating cilia without delay. Using cycloheximide (CHX) to block new protein synthesis, we show that the TZ protein B9d1 is not a component of the cilia precursor pool and requires new transcription/translation, providing insights into the delayed repair of TZ. Moreover, MCCs in CHX treatment assemble fewer ([~] 10 vs. [~]150 in controls) but near wild-type length (ranging between 60 to 90%) cilia by gradually concentrating ciliogenesis proteins like IFTs at a select few basal bodies. Using mathematical modeling, we show that cilia length compared to cilia number influences the force generated by MCCs more. In summary, our results question the requirement of TZ in motile cilia assembly and provide insights into how cells determine organelle size and number.

cell biology↗

bioRxiv2023

Live cell imaging of ATP levels reveals metabolic compartmentalization within motoneurons and early metabolic changes in FUS ALS motoneurons.

Motoneurons are one of the highest energy demanding cell types and a primary target in Amyotrophic lateral sclerosis (ALS), a debilitating and lethal neurodegenerative disorder without currently available effective treatments. Disruption of mitochondrial ultra-structure, transport and metabolism is a commonly reported phenotype in ALS models and can critically affect survival and proper function of motor neurons. However, how changes in metabolic rates contribute to ALS progression are not fully understood yet. Here we utilize hiPCS derived motoneuron cultures and live imaging quantitative techniques to evaluate metabolic rates in Fused in Sarcoma (FUS)-ALS model cells. We show that differentiation and maturation of motoneurons is accompanied by an overall upregulation of mitochondrial components and significant increase in metabolic rates that corresponds to their high energy-demanding state. Detailed compartment-specific live measurements using a fluorescent ATP sensor and FLIM imaging show significantly lower levels of ATP in the somas of cells carrying FUS-ALS mutations. These changes lead to the increased vulnerability of disease motoneurons to further metabolic challenges with mitochondrial inhibitors and could be due to the disruption of mitochondrial inner membrane integrity and an increase in its proton leakage. Furthermore, our measurements demonstrate heterogeneity between axonal and somatic compartments with lower relative levels of ATP in axons. Our observations strongly support the hypothesis that mutated FUS impacts metabolic states of motoneurons and makes them more susceptible to further neurodegenerative mechanisms.

neuroscience↗

bioRxiv2023

Mitotic events depend on regulation of PLK-1 levels by the mitochondrial protein SPD-3

In metazoans, Polo Kinase (Plk1) controls several mitotic events including nuclear envelope breakdown, centrosome maturation and kinetochore assembly. Here we show that mitotic events regulated by Polo Like Kinase (PLK-1) in early C. elegans embryos depend on the mitochondrial-localized protein SPD-3. spd-3 mutant one-cell embryos contain abnormally positioned mitotic chromosomes and prematurely and asymmetrically disassemble the nuclear lamina. Nuclear envelope breakdown (NEBD) in C. elegans requires direct dephosphorylation of lamin by PLK-1. In spd-3 mutants PLK-1 levels are ~6X higher in comparison to control embryos and PLK-1::GFP was highly accumulated at centrosomes, the nuclear envelope, nucleoplasm, and chromosomes prior to NEBD. Partial depletion of plk-1 in spd-3 mutant embryos rescued mitotic chromosome and spindle positioning defects indicating that these phenotypes result from higher PLK-1 levels and thus activity. Our data suggests that the mitochondrial SPD-3 protein controls NEBD and chromosome positioning by regulating the endogenous levels of PLK-1 during early embryogenesis in C. elegans. This finding suggests a novel link between mitochondria and mitotic events by controlling the amount of a key mitotic regulator, PLK-1 and thus may have further implications in the context of cancers or age-related diseases and infertility as it provides a novel link between mitochondria and mitosis.

cell biology↗

bioRxiv2022

Liquid-liquid phase separation of the chromosomal passenger complex drives parallel bundling of midzone microtubules

The spindle midzone is a dynamic structure that forms during anaphase, mediates chromosome segregation, and provides a signaling platform to position the cleavage furrow. The spindle midzone comprises two antiparallel bundles of microtubules (MTs) but the process of their formation is poorly understood. Here, we show that the Chromosomal Passenger Complex (CPC) undergoes liquid-liquid phase separation (LLPS) to generate parallel MT bundles in vitro when incubated with free tubulin and GTP. MT bundles emerge from CPC droplets with protruding minus-ends that then grow into long, tapered MT structures. During this growth, the CPC in condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for LLPS or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data uncovers a kinase-independent function of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle.

cell biology↗

bioRxiv2021

NDC1 is necessary for stable assembly of the nuclear pore scaffold to establish nuclear transport in early C. elegans embryos

Nuclear pore complexes (NPCs) are large protein assemblies that facilitate transport of macromolecules across the nuclear envelope (NE) [1, 2]. How thousands of NPCs rapidly assemble after open mitosis to form a functional NE is not known. Recruitment of the Nup107-160 outer ring scaffold to chromatin initiates NPC assembly. The Nup53/93 complex bridges the outer ring to the central channel to form a functional pore [3-6]. Nup53 interacts with the conserved transmembrane nucleoporin Ndc1; however, how Ndc1 contributes to post-mitotic NPC assembly is unclear [7-9]. Here, we use C. elegans embryos to show that the timely formation of a functional NE after mitosis depends on Ndc1. Endogenously tagged Ndc1 is recruited early to the reforming NE and is highly mobile in the nuclear rim. 3D analysis of post-mitotic NE formation revealed a decreased NPC density in NEs of ndc1 deleted embryos - continuous nuclear membranes contained few holes where assembling NPCs are normally located. Nup160 is highly mobile in NEs depleted of Ndc1 and outer ring scaffold components are less enriched at the rim. When both ndc1 and nup53 are absent, nuclear assembly fails. Together, these data show that Ndc1 dynamically associates with the NE and promotes stable association of the outer ring scaffold in the NE to facilitate NPC assembly after open mitosis. Furthermore, Ndc1 and Nup53 function in parallel to drive nuclear assembly. We propose that Ndc1 is a dynamic membrane adaptor that helps recruit and promote the self-assembly of the nuclear pore scaffold to drive post-mitotic NPC assembly.

cell biology↗

bioRxiv2020

Semi-automatic stitching of filamentous structures in image stacks from serial-section electron tomography

We present a software-assisted workflow for the alignment and matching of filamentous structures across a 3D stack of serial images. This is achieved by combining automatic methods, visual validation, and interactive correction. After an initial alignment, the user can continuously improve the result by interactively correcting landmarks or matches of filaments. Supported by a visual quality assessment of regions that have been already inspected, this allows a trade-off between quality and manual labor. The software tool was developed to investigate cell division by quantitative 3D analysis of microtubules (MTs) in both mitotic and meiotic spindles. For this, each spindle is cut into a series of semi-thick physical sections, of which electron tomograms are acquired. The serial tomograms are then stitched and non-rigidly aligned to allow tracing and connecting of MTs across tomogram boundaries. In practice, automatic stitching alone provides only an incomplete solution, because large physical distortions and a low signal-to-noise ratio often cause experimental difficulties. To derive 3D models of spindles despite the problems related to sample preparation and subsequent data collection, semi-automatic validation and correction is required to remove stitching mistakes. However, due to the large number of MTs in spindles (up to 30k) and their resulting dense spatial arrangement, a naive inspection of each MT is too time consuming. Furthermore, an interactive visualization of the full image stack is hampered by the size of the data (up to 100 GB). Here, we present a specialized, interactive, semi-automatic solution that considers all requirements for large-scale stitching of filamentous structures in serial-section image stacks. The key to our solution is a careful design of the visualization and interaction tools for each processing step to guarantee real-time response, and an optimized workflow that efficiently guides the user through datasets. Author summaryElectron tomography of biological samples is used for a 3D reconstruction of filamentous structures, such as microtubules (MTs) in mitotic and meiotic spindles. Large-scale electron tomography can be applied to increase the reconstructed volume for the visualization of full spindles. For this, each spindle is cut into a series of semi-thick physical sections, of which electron tomograms are acquired. The serial tomograms are then stitched and non-rigidly aligned to allow tracing and connecting of MTs across tomogram boundaries. Previously, we presented fully automatic approaches for this 3D reconstruction pipeline. However, large volumes often suffer from imperfections (i.e. physical distortions) caused during sectioning and imaging, making it difficult to apply fully automatic approaches for matching and stitching of numerous tomograms. Therefore, we developed an interactive, semi-automatic solution that considers all requirements for large-scale stitching of microtubules in serial-section image stacks. We achieved this by combining automatic methods, visual validation and interactive error correction, thus allowing the user to continuously improve the result by interactively correcting landmarks or matches of filaments. We present large-scale reconstructions of spindles in which the automatic workflow failed and where different steps of manual corrections were needed. Our approach is also applicable to other biological samples showing 3D distributions of MTs in a number of different cellular contexts.

cell biology↗

bioRxiv2020

Microtubule re-organization during female meiosis in C. elegans

The female meiotic spindles of most animals are acentrosomal and undergo striking morphological changes while transitioning from metaphase to anaphase. The ultra-structure of acentrosomal spindles, and how changes to this structure correlate with such dramatic spindle rearrangements remains largely unknown. To address this, we applied light microscopy, large-scale electron tomography and mathematical modeling of female meiotic C. elegans spindles undergoing the transition from metaphase to anaphase. Combining these approaches, we find that meiotic spindles are dynamic arrays of short microtubules that turn over on second time scales. The results show that the transition from metaphase to anaphase correlates with an increase in the number of microtubules and a decrease in their average length. Detailed analysis of the tomographic data revealed that the length of microtubules changes significantly during the metaphase-to-anaphase transition. This effect is most pronounced for those microtubules located within 150 nm of the chromosome surface. To understand the mechanisms that drive this transition, we developed a mathematical model for the microtubule length distribution that considers microtubule growth, catastrophe, and severing. Using Bayesian inference to compare model predictions and data, we find that microtubule turn-over is the major driver of the observed large-scale reorganizations. Our data suggest that in metaphase only a minor fraction of microtubules, those that are closest to the chromosomes, are severed. The large majority of microtubules, which are not in close contact with chromosomes, do not undergo severing. Instead, their length distribution is fully explained by growth and catastrophe alone. In anaphase, even microtubules close to the chromosomes show no signs of cutting. This suggests that the most prominent drivers of spindle rearrangements from metaphase to anaphase are changes in nucleation and catastrophe rate. In addition, we provide evidence that microtubule severing is dependent on the presence of katanin.

cell biology↗

bioRxiv2019

Soluble tubulin is locally enriched at mitotic centrosomes in C. elegans

During mitosis, the centrosome expands its capacity to nucleate microtubules. Understanding the mechanisms of centrosomal microtubule nucleation is, however, constrained by a lack of knowledge of the amount of soluble and polymer tubulin at mitotic centrosomes. Here we combined light microscopy and serial-section electron tomography to measure the amount of dimer and polymer at mitotic centrosomes in early C. elegans embryos. We show that a C. elegans one-cell stage centrosome at metaphase contains more than ten thousand microtubules with a total polymer concentration of 230 M. Centrosomes concentrate soluble /{beta} tubulin by about tenfold over the cytoplasm, reaching peak values of 470 M, giving a combined total monomer and polymer tubulin concentration at centrosomes of up to 660 M. These findings support in vitro data suggesting that microtubule nucleation in C. elegans centrosomes is driven in part by concentrating soluble tubulin.

cell biology↗

bioRxiv2019

Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

Spindle microtubules, whose dynamics vary over time and at different locations, cooperatively drive chromosome segregation. Measurements of microtubule dynamics and spindle ultrastructure can provide insight into the behaviors of microtubules, helping elucidate the mechanism of chromosome segregation. Much work has focused on the dynamics and organization of kinetochore microtubules, i.e. on the region between chromosomes and poles. In comparison, microtubules in the central spindle region, between segregating chromosomes, have been less thoroughly characterized. Here, we report measurements of the movement of central spindle microtubules during chromosome segregation in human mitotic spindles, and Caenorhabditis elegans mitotic and female meiotic spindles. We found that these central spindle microtubules slide apart at the same speed as chromosomes, even as chromosomes move towards spindle poles. In these systems, damaging central spindle microtubules by laser ablation caused an immediate and complete cessation of chromosome motion, suggesting a strong coupling between central spindle microtubules and chromosomes. Electron tomographic reconstruction revealed that the analyzed anaphase spindles all contain microtubules with both ends between segregating chromosomes. Our results provide new dynamical, functional, and ultrastructural characterizations of central spindle microtubules during chromosome segregation in diverse spindles, and suggest that central spindle microtubules and chromosomes are strongly coupled in anaphase.

cell biology↗