Biology subjects

Zimyanin, V.

Publications and source records attributed to Zimyanin, V..

9 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

RIG-I mediated neuron-specific IFN type 1 signaling in FUS-ALS induces neurodegeneration and offers new biomarker-driven individualized treatment options for (FUS-)ALS

Recent research has demonstrated significant aberrant activation of the innate immune system in ALS model systems due to mutations in SOD1, TARDBP and C9orf72 through stimulation of the TBK1-IRF3 pathway. This pathway can be activated, for example, by cGAS-STING-dependent sensing of cytosolic DNA that accumulates as a result of chronic DNA damage and defective mitochondria, both of which have been identified as early pathology in FUS-ALS spinal motor neurons (sMNs). Therefore, we analysed innate immune pathways in isogenic and non-isogenic FUSmut iPSC-derived sMNs, which revealed upregulation of interferon-stimulated genes (ISGs) and activation of the TBK1-IRF3 pathway in FUSmut sMNs. Notably, we found evidence for accumulation of cytosolic dsRNA and its sensor RIG-I in FUS-ALS. RIG-I, but not MDA5, was found to be significantly upregulated in FUSmut sMNs, and siRNA-mediated knockdown abolished the increased IFN1 activation in FUSmut sMNs. In post-mortem analysis, RIG-I was highly expressed in the remaining -MNs. IFN treatment of FUSwt sMNs phenocopied the axonal degeneration of FUSmut sMNs. Mechanistically, DNA damage induction did not increase ISG expression, but dsRNA was increased in the mitochondria of FUSmut sMNs. Mitochondrial transcription, a known source of dsRNA, was found to be upregulated in compartmental axonal RNAseq analysis and its inhibition reduced ISGs in FUS-ALS sMNs. Furthermore, the JAK-STAT inhibitor ruxolitinib alleviated the upregulated ISG expression and reversed the axonal degeneration of sMNs. Finally, we analysed ISG expression in peripheral blood samples from 18 FUS-ALS patients, eight of whom had a significantly elevated interferon signature. Blood ISGs correlated with disease progression rate and negatively with disease duration. RIG-I-mediated innate immune activation in sMNs may be an interesting novel individualised biomarker-driven therapeutic target in (FUS-) ALS. A one-sentence summary of your paperRIG-I-I-mediated innate immune activation is found in FUS-ALS spinal motor neurons caused by cytosolic dsRNA accumulation due to mitochondrial transcriptional activation and is amenable to JAK-STAT inhibition and might thus be an interesting novel individualized biomarker-driven therapeutic approach in (FUS-) ALS

neuroscience↗

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↗

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

DJ-1 products glycolic acid and D-lactate restore deficient axonal trafficking and DNA damage response in FUS and SOD1-associated amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is the most common motor neuron disease leading to death within 2-5 years. Currently available drugs can only slightly prolong survival. Despite the progress that has been achieved in unravelling the molecular mechanisms of the disease so far, the underlying pathophysiology is not fully understood. We present novel insights into the pathophysiology of Superoxide Dismutase 1 (SOD1)- and in particular Fused In Sarcoma (FUS)-ALS by revealing a putatively central role of the Parkinsons disease (PD) associated glyoxylase DJ-1 and its products glycolic acid (GA) and D-lactic acid (DL). Combined, but not single, treatment with GA and DL restored axonal trafficking deficits of mitochondria and lysosomes in FUS- and SOD1-ALS patient-derived motoneurons (MNs). This was accompanied by restoration of mitochondrial membrane potential as well as mitochondrial fragmentation (FUS-ALS) or elongation (SOD1-ALS). Furthermore, GA and DL restored cytoplasmic mislocalization of FUS and FUS recruitment to DNA damage sites. We further show that despite presenting an early axonal transport deficiency as well, TDP-43 patient-derived MNs did not share this mechanism. While this points towards the necessity of individualized (gene-) specific therapy stratification, it also suggests common therapeutic targets across different gene variants of ALS. Thus, we introduce a putative novel treatment for ALS based on a combination of the two substances GA and DL which might be not only an interesting novel drug candidate in subsets of ALS cases but also in other neurodegenerative diseases characterized by mitochondrial depolarization.

neuroscience↗

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↗

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↗

bioRxiv2021

The Drosophila anterior-posterior axis is polarized by asymmetric myosin activation

The Drosophila anterior-posterior (AP) axis is specified at mid-oogenesis when Par-1 kinase is recruited to the posterior cortex of the oocyte, where it polarises the microtubule cytoskeleton to define where the axis determinants, bicoid and oskar mRNAs localise. This polarity is established in response to an unknown signal from the follicle cells, but how this occurs is unclear. Here we show that the myosin chaperone, Unc-45 and Non-Muscle Myosin II (MyoII) are required in the germ line upstream of Par-1 in polarity establishment. Furthermore, the Myosin regulatory Light Chain (MRLC) is di-phosphorylated at the oocyte posterior in response to the follicle cell signal, inducing longer pulses of myosin contractility at the posterior and increased cortical tension. Over-expression of MRLC-T21A that cannot be di-phosphorylated or acute treatment with the Myosin light chain kinase inhibitor ML-7 abolish Par-1 localisation, indicating that posterior of MRLC di-phosphorylation is essential for polarity. Thus, asymmetric myosin activation polarizes the anterior-posterior axis by recruiting and maintaining Par-1 at the posterior cortex. This raises an intriguing parallel with AP axis formation in C. elegans where MyoII is also required to establish polarity, but functions to localize the anterior PAR proteins rather than PAR-1.

developmental biology↗