Biology subjects

Suresh, P.

Publications and source records attributed to Suresh, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Beyond Segmentation Accuracy: Evaluating Biological Fidelity in Spatial Pathology

Multiplex immunofluorescence (MxIF) resolves cellular identity, state, and spatial organization within tumor tissue, enabling higher-order representations of cancer ecosystems including immune infiltration, multicellular neighborhoods, and tissue niches. Yet these analyses depend on a deceptively simple first step: accurately identifying individual cells. Segmentation errors can alter marker quantification, cellular phenotypes, cell-cell relationships, and ultimately the spatial organization inferred from an image. Consequently, improvements in conventional segmentation benchmarks matter biologically only if they propagate to more faithful representations of the tumor microenvironment. We test this relationship directly using an expert-curated benchmark of 31,924 expert-reviewed cells on three slides under a leave-slide-out protocol. We compare a domain-specific self-supervised encoder, adapted to multiplex tissue imaging, with supervised, microscopy-specific, prompted generalist, and general-purpose vision models, and pass every method's predicted cells through an identical downstream pipeline. We evaluate fidelity across four levels: cell segmentation, phenotype assignment, spatial neighborhood structure, and tissue organization, comprising niches and tumor-immune interactions. The resulting hierarchy reveals that these levels do not improve in lockstep. Cellular phenotype assignment is comparatively robust to segmentation differences, whereas missed cells increasingly disrupt neighborhood structure and tumor-immune interactions. As a stringent spatial-interaction test, tumor-CD8+ contact recovery is poorly predicted by conventional segmentation quality (Panoptic Quality: {rho} = 0.18), while errors specifically at tumor-immune interfaces are strongly associated with contact fidelity ({rho} = -0.80). Methods with nearly identical segmentation accuracy can therefore produce substantially different biological representations. These results argue that cell segmentation for spatial pathology should be evaluated not only by boundary and instance accuracy, but also by preservation of the biological relationships that downstream analyses are intended to measure.

bioinformatics↗

bioRxiv2026

2,6-diaminopurine enhances Aqp4 stop codon readthrough and AQP4 perivascular localization in the brain

In the brain, the water channel Aquaporin 4 (AQP4) is largely restricted to astrocytes, enriched at perivascular astrocytic processes, and involved in fluid balance and neurological disease. The perivascular pool is functionally unique as it is depolarized in diseases, targeted by neuromyelitis optica autoantibodies, and implicated in the clearance of brain metabolites such as amyloid beta. The perivascular AQP4 was recently shown to be AQP4X, an extended isoform arising from readthrough, where approximately 20% of translating ribosomes continue through the Aqp4 stop codon. Because Aqp4 terminates with a UGA, here we tested whether Aqp4 readthrough can be enhanced by 2,6-diaminopurine, a purine analog known to promote UGA decoding. Using dual luciferase and immunoblotting for AQP4X in cultured cells, we show that the drug promotes Aqp4 readthrough and increases AQP4X levels. For in vivo validation, we use the wild-type mouse as well as a genetically engineered mouse with a stop-to-sense mutation allowing Aqp4 readthrough at 100%. 2,6-diaminopurine elevates perivascular AQP4X levels by approximately 10% after 20 hours of single intracranial injection. It fails to enhance AQP4X levels in animals with stop-to-sense mutation, suggesting its action involves Aqp4 readthrough. Aqp4 readthrough can be a pharmacological target for modulating perivascular AQP4X in neurological disease models.

neuroscience↗

bioRxiv2026

Alveolar Epithelial Cell Loss of the Mitochondrial Regulator TFAM Drives Progressive Lung Fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by failed alveolar epithelial repair and progressive fibrotic remodeling. Although aberrant reprogramming of alveolar type 2 (AT2) cells and accumulation of transitional AT2 states are increasing recognized as central features of IPF, the epithelial-intrinsic mechanisms that initiate these pathogenic states remain incompletely understood. Here, we identify mitochondrial transcription factor A (TFAM), a regulator of mitochondrial DNA maintenance, as a critical regulator of AT2 cell homeostasis. TFAM expression was reduced in AT2 cells from human IPF lungs. Inducible AT2 cell-specific Tfam deletion in mice caused spontaneous fibrotic remodeling and increased susceptibility to bleomycin-induced lung injury. TFAM-deficient AT2 cells acquired KRT8+ transitional and p21+ senescence-associated features before the onset of fibrotic transformation, accompanied by impaired oxidative phosphorylation, redox imbalance, mitochondrial superoxide accumulation, repression of mtDNA-encoded respiratory genes, and disrupted mitochondrial ultrastructure. TFAM-deficient AT2 cells developed a profibrotic secretory program that promoted extracellular matrix deposition and fibroblast activation. We further identified insulin-like growth factor-binding protein 2 (IGFBP2) as a secreted mediator induced in TFAM-deficient AT2 cells. IGFBP2 was elevated in AT2 cells in human IPF lung tissue and bronchoalveolar lavage fluid (BALF) from patients with IPF. IGFBP2 was detected in supernatants from fibrotic human precision-cut lung slices (hPCLS). IGFBP2 neutralization attenuated profibrotic remodeling in fibrotic hPCLS. Collectively, our findings identify TFAM-dependent mitochondrial homeostasis as an epithelial checkpoint linking AT2 cell-state stability to impaired epithelial-mesenchymal crosstalk driving pulmonary fibrosis.

pathology↗

bioRxiv2024

A pair of congenic mice for imaging of transplants by positron emission tomography using anti-transferrin receptor nanobodies

Two anti-transferrin receptor (TfR) nanobodies, VHH123 specific for mouse TfR and VHH188 specific for human TfR (huTfR) were used to track transplants non-invasively by PET/CT in mouse models, without the need for genetic modification of the transferred cells. We provide a comparison of the specificity and kinetics of the PET signals acquired when using nanobodies radiolabeled with 89Zr, 64Cu and 18F, and find that the chelation of the 89Zr and 64Cu radioisotopes to anti-TfR nanobodies results in radioisotope release upon endocytosis of the radiolabeled nanobodies. We used a knock-in mouse that expresses a TfR with a human ectodomain (huTfR +/+) as a source of bone marrow for transplants into C57BL/6 recipients and show that VHH188 detects such transplants by PET/CT. Conversely, C57BL/6 bone marrow and B16.F10 melanoma cell-line transplanted into huTfR +/+ recipients can be imaged with VHH123. In C57BL/6 mice impregnated by huTfR+/+ males we saw an intense VHH188 signal in the placenta, showing that TfR-specific VHHs accumulate at the placental barrier but do not enter the fetal tissue. We were unable to observe accumulation of the anti-TfR radiotracers in the central nervous system (CNS) by PET/CT but show evidence of CNS accumulation by radiospectrometry. The model presented here can be used to track many transplanted cell types by PET/CT, provided cells express TfR, as is typically the case for proliferating cells such as tumor lines.

immunology↗

bioRxiv2024

Imaging the interactions of chimeric antigen receptor-expressing T-cells with colorectal cancer cells

Chimeric antigen receptor (CAR) T-cell therapy has shown unprecedented success in haematological cancers but faces challenges in solid tumours. Although carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is differentially expressed in many solid tumours, anti-CEACAM5 CAR T-cells are ineffective. Here, we have studied the interaction of CEACAM5 targeting primary CAR T-cells with colorectal cancer (CRC) cells using fluorescence microscopy. We found that CRC cells glycocalyx is much deeper than that of the CAR T cell causing delayed activation. Oscillating calcium fluxes, indicative of non-sustained CAR T cell activation and reduced cytotoxicity, were observed when CAR T cells interacted with CRC cells, which increased with increasing cell-seeding time. Imaging revealed that this effect correlated with a progressive loss of accessible CEACAM5 antigen on the CRC cell surface, possibly due to their sequestration in the intercellular junction, rendering CAR T cell engagement less effective. Local proteolytic treatment with trypsin to disrupt the CRC cell monolayer, using a micropipette, increased CEACAM5 availability, decreased glycocalyx thickness, and restored sustained CAR T cell calcium fluxes, increasing the killing of CRC cells. Similar enhanced interaction was observed after treatment of CRC cell monolayer with hyaluronidase, approved for use in humans. We observed limited availability of CEACAM5 on human colorectal cancer tissues, whereas treatment with trypsin or hyaluronidase increased accessibility. Our results reveal why CAR T cells targeting CEACAM5 are ineffective and suggest possible routes to improved therapy for CRC.

immunology↗

bioRxiv2023

The delayed kinetics of Myddosome formation explains why Aβ aggregates trigger TLR4 less efficiently than LPS

The Myddosome is a key innate immune signalling platform. It forms at the cell surface and contains MyD88 and IRAK proteins which ultimately coordinate the production of pro-inflammatory cytokines. Toll-like receptor 4 signals via the Myddosome when triggered by Lipopolysaccharide (LPS) or Amyloid-Beta (A{beta}) aggregates but the magnitude and time duration of the response are very different for reasons that are unclear. Here we followed the formation of Myddosomes in live macrophages using local delivery of TLR4 agonist to the cell surface and visualisation with 3D rapid light sheet imaging. This was complemented by super-resolution imaging of Myddosomes in fixed macrophages to determine the size of the signalling complex at different times after triggering. Myddosomes formed more rapidly after LPS than in response to sonicated A{beta} 1-42 fibrils (80 seconds vs 372 seconds). The mean lifetimes of the Myddosomes was also shorter when triggered by LPS compared to sonicated A{beta} fibrils (170 and 220 s) respectively. In both cases a range of Myddosome of different sizes (50-500 nm) were formed. In particular, small round Myddosomes around 100 nm in size formed at early time points, then reduced in proportion over time. Collectively our data suggests that compared to LPS the multivalency of A{beta} fibrils leads to the formation of larger Myddosomes which form more slowly and, due to their size, take longer to disassemble. This explains why sonicated A{beta} fibrils results in less efficient triggering of TLR4 signalling and may be a general property of protein aggregates.

biophysics↗

bioRxiv2023

Replication and Refinement of Brain Age Model for adolescent development.

The discrepancy between chronological age and estimated brain age, known as the brain age gap, may serve as a biomarker to reveal brain development and neuropsychiatric problems. This has motivated many studies focusing on the accurate estimation of brain age using different features and models, of which the generalizability is yet to be tested. Our recent study has demonstrated that conventional machine learning models can achieve high accuracy on brain age prediction during development using only a small set of selected features from multimodal brain imaging data. In the current study, we tested the replicability of various brain age models on the Adolescent Brain Cognitive Development (ABCD) cohort. We proposed a new refined model to improve the robustness of brain age prediction. The direct replication test for existing brain age models derived from the age range of 8-22 years onto the ABCD participants at baseline (9 to 10 years old) and year-two follow-up (11 to 12 years old) indicate that pre-trained models could capture the overall mean age failed precisely estimating brain age variation within a narrow range. The refined model, which combined broad prediction of the pre-trained model and granular information with the narrow age range, achieved the best performance with a mean absolute error of 0.49 and 0.48 years on the baseline and year-two data, respectively. The brain age gap yielded by the refined model showed significant associations with the participants information processing speed and verbal comprehension ability on baseline data.

neuroscience↗

bioRxiv2023

A semiconductor 96-microplate platform for real-time impedance-based high-throughput screening

Profiling compounds and genetic perturbations via high-content imaging has become increasingly popular for drug discovery, but the technique is limited to endpoint images of fixed cells. In contrast, electronic-based devices offer label-free, functional information of live cells, yet current approaches suffer from low-spatial resolution or single-well throughput. Here, we report a semiconductor 96-microplate platform designed for high-resolution real-time impedance "imaging" at scale. Each well features 4,096 electrodes at 25 {micro}m spatial resolution while a miniaturized data interface allows 8x parallel plate operation (768 total wells) within each incubator for enhanced throughputs. New electric field-based, multi-frequency measurement techniques capture >20 parameter images including tissue barrier, cell-surface attachment, cell flatness, and motility every 15 min throughout experiments. Using these real-time readouts, we characterized 16 cell types, ranging from primary epithelial to suspension, and quantified heterogeneity in mixed epithelial and mesenchymal co-cultures. A proof-of-concept screen of 904 diverse compounds using 13 semiconductor microplates demonstrates the platforms capability for mechanism of action (MOA) profiling with 25 distinct responses identified. The scalability of the semiconductor platform combined with the translatability of the high dimensional live-cell functional parameters expands high-throughput MOA profiling and phenotypic drug discovery applications.

bioengineering↗

bioRxiv2022

Lipid Driven Inter-leaflet Coupling of Plasma Membrane Order Regulates FcεRI Signaling in Mast Cells

Engagement of high affinity immunoglobulin E (IgE) receptor Fc{varepsilon}RI with extracellular, multivalent antigen (Ag) stabilizes co-existing ordered and disordered phases in the inner leaflet of the plasma membrane. This optimally controls biochemical interactions between signaling components required for transmembrane (TM) signaling in mast cells. The biophysical organization of the resting inner leaflet is poised to respond appropriately to this extracellular stimulation. The resting inner leaflet is generally less ordered than the outer leaflet, with a lipid composition that does not spontaneously phase separate in model membranes. We proposed that coupling between the two leaflets mediates separation into different phase-like domains in the inner leaflet. To test this hypothesis in live cells, we first established a straightforward approach to evaluate changes in membrane order due to inter-leaflet coupling by measuring inner leaflet diffusion of phase-specific lipid probes with Imaging Fluorescence Correlation Spectroscopy (ImFCS) before and after methyl--cyclodextrin (mCD)-catalyzed exchange of outer leaflet lipids (LEX) with exogenous order- or disorder-promoting phospholipids. We examined the functional impact of LEX by monitoring two Ag-stimulated cellular responses, namely early-stage recruitment of Syk kinase to the inner leaflet and late-stage exocytosis of secretory granules (degranulation). Based on changes in probe diffusion, we observed global increase or decrease of inner leaflet order when outer leaflet is exchanged with order or disorder promoting lipids, respectively, in unstimulated cells. Furthermore, the degree of stimulated Syk recruitment and degranulation correlates with the inner leaflet order of the resting cells, which was varied using LEX. Overall, combined LEX and ImFCS platform provides strong evidence of lipid-based control of stimulated TM signaling in live mast cells. In addition, our functional results imply that resting-state lipid composition and ordering of the outer leaflet sets the ordering of the inner leaflet, likely via interleaflet coupling, and correspondingly modulates TM signaling initiated by antigen-activated IgE-Fc{varepsilon}RI. STATEMENT OF SIGNIFICANCECoupling between plasma membrane leaflets, which are biochemically and biophysically asymmetric, results in a steady-state membrane organization that is thought to play fundamental roles in cellular functions. Here, we present a straightforward assay built around mCD-catalyzed lipid exchange (LEX) and Imaging Fluorescence Correlation Spectroscopy (ImFCS) to quantitatively characterize a novel, lipid-driven, interleaflet coupling mechanism and its functional impact in live mast cells. We showed that elevation of outer leaflet lipid order induces ordering throughout the inner leaflet in resting cells. This ordering enhances protein-based reactions during Ag-stimulated Fc{varepsilon}RI signaling and consequent cellular response. Overall, we provide a compelling evidence of functional relevance of plasma membrane organizational heterogeneity driven by lipid-based interleaflet coupling.

biophysics↗

bioRxiv2022

Recombinant Human ACE2-Fc : A promising therapy for SARS-CoV2 infection

SARS-CoV2 entry is mediated by binding of viral spike-protein(S) to the transmembrane Angiotensin-Converting Enzyme-2 (ACE2) of the host cell. Thus, to prevent transmission of disease, strategies to abrogate the interaction are important. However, ACE2 cannot be blocked since its normal function is to convert the Angiotensin II peptide to Angiotensin(1-7) to reduce hypertension. This work reports a recombinant cell line secreting soluble ACE2-ectopic domain (MFcS2), modified to increase binding and production efficacy and fused to human immunoglobulin-Fc. While maintaining its enzymatic activity, the molecule trapped and neutralized SARS CoV2 virus in vitro with an IC50 of 64 nM. In vivo, with no pathology in the vital organs, it inhibited the viral load in lungs in SARS-CoV2 infected Golden-Syrian-hamster. The Intravenous pharmacokinetic profiling of MFcS2 in hamster at a dose of 5 mg/Kg presented a maximum serum concentration of 23.45 {micro}g/mL with a half-life of 29.5 hrs. These results suggest that MFcS2 could be used as an effective decoy based therapeutic strategy to treat COVID19. This work also reports usage of a novel oral-cancer cell line as in vitro model of SARS-Cov2 infection, validated by over expressing viral-defence pathways upon RNA-seq analysis and over-expression of ACE2 and TMPRSS upon growth in hyperglycaemic condition.

molecular biology↗

bioRxiv2022

Modeling and mechanical perturbations reveal how spatially regulated anchorage gives rise to spatially distinct mechanics across the mammalian spindle

During cell division, the spindle generates force to move chromosomes. In mammals, microtubule bundles called kinetochore-fibers (k-fibers) attach to and segregate chromosomes. To do so, k-fibers must be robustly anchored to the dynamic spindle. We previously developed microneedle manipulation to mechanically challenge k-fiber anchorage, and observed spatially distinct response features revealing the presence of heterogeneous anchorage (Suresh et al. 2020). How anchorage is precisely spatially regulated, and what forces are necessary and sufficient to recapitulate the k-fibers response to force remain unclear. Here, we develop a coarse-grained k-fiber model and combine with manipulation experiments to infer underlying anchorage using shape analysis. By systematically testing different anchorage schemes, we find that forces solely at k-fiber ends are sufficient to recapitulate unmanipulated k-fiber shapes, but not manipulated ones for which lateral anchorage over a 3 m length scale near chromosomes is also essential. Such anchorage robustly preserves k-fiber orientation near chromosomes while allowing pivoting around poles. Anchorage over a shorter length scale cannot robustly restrict pivoting near chromosomes, while anchorage throughout the spindle obstructs pivoting at poles. Together, this work reveals how spatially regulated anchorage gives rise to spatially distinct mechanics in the mammalian spindle, which we propose are key for function.

biophysics↗

bioRxiv2021

The Astrin/SKAP Complex Reduces Friction at the Kinetochore-Microtubule Interface

The kinetochore links chromosomes to spindle microtubules to drive chromosome segregation at cell division. While we know nearly all mammalian kinetochore proteins, how these give rise to the strong yet dynamic microtubule attachments required for function remains poorly understood. Here, we focus on the Astrin-SKAP complex, which localizes to bioriented kinetochores and is essential for chromosome segregation, but whose mechanical role is unclear. Live imaging reveals that SKAP depletion dampens movement and decreases coordination of metaphase sister kinetochores, and increases tension between them. Using laser ablation to isolate kinetochores bound to polymerizing vs depolymerizing microtubules, we show that without SKAP kinetochores move slower on both polymerizing and depolymerizing microtubules, and that more force is needed to rescue microtubules to polymerize. Thus, in contrast to previously described kinetochore proteins that increase grip on microtubules under force, Astrin-SKAP reduces grip, increasing attachment dynamics and force responsiveness and reducing friction. Together, our findings suggest a model where the Astrin-SKAP complex effectively "lubricates" correct, bioriented attachments to help preserve them.

cell biology↗

bioRxiv2021

Phospholipid exchange shows insulin receptor activity is supported by both the propensity to form wide bilayers and ordered domains (rafts)

Using efficient methyl-alpha-cyclodextrin mediated lipid exchange, we studied the effect of altering plasma membrane outer leaflet phospholipid composition upon the activity of insulin receptor (IR) in mammalian cells. After substitution of endogenous lipids with lipids having an ability to form liquid ordered (Lo) domains (sphingomyelins) or liquid disordered (Ld) domains (unsaturated phosphatidylcholines (PCs)), we found that the propensity of lipids to form ordered domains is required for high IR activity. Additional substitution experiments using a series of saturated PCs showed that IR activity increased substantially with increasing acyl chain length. Increasing acyl chain length increases both bilayer width and the propensity to form ordered domains. To distinguish the effects of membrane width and domain formation, we incorporated purified IR into alkyl maltoside micelles with increasing hydrocarbon lengths. IR activity increases with increased chain length, but more modestly than by increasing lipid acyl chain length in cells. This suggests that the ability to form Lo domains as well as wide bilayer width contributes to increased IR activity. Inhibition of phosphatases with sodium orthovanadate showed that some of the lipid dependence of IR activity upon lipid structure reflected protection from phosphatases by lipids that support Lo domain formation. The results are consistent with a model in which a combination of bilayer width and ordered domain formation modulate IR activity via effects upon IR conformation and accessibility to phosphatases. SignificanceThis study shows how methyl--cyclodextrin mediated lipid exchange can be used to probe the influence of lipid structure upon the functioning of a transmembrane receptor. Plasma membranes having a propensity to form Lo domains are required to support a high level of IR activity. The studies indicate this may reflect an effect of lipid environment upon IR domain localization, which in turn alters its conformation and vulnerability to phosphatases. Alterations in lipid composition could conceivably regulate IR activity in vivo.

cell biology↗

bioRxiv2021

H3K4 Methylation in β-cells Prevents Transcriptional Downregulation and Variance Associated with Type 2 Diabetes

Pancreatic {beta}-cells control glucose homeostasis via regulated production and secretion of insulin. This function arises from a highly specialized gene expression program which is established during development and then sustained, with limited flexibility, in terminally differentiated {beta}-cells. Dysregulation of this program is seen in type 2 diabetes (T2D) but mechanisms that preserve gene expression or underlie its dysregulation in mature {beta}-cells are not well resolved. Here we show that trithorax group-dependent histone H3 lysine 4 trimethylation (H3K4me3) maintains expression of genes important for insulin biosynthesis and glucose-responsiveness in {beta}-cells. Transcriptional changes in H3K4me3-deficient {beta}-cells lead to severe hyperglycemia in adult mice. We show that H3K4me3 deficiency leads to a less active and more repressed epigenome profile, which locally correlates with gene expression deficits but does not globally reduce gene expression. Instead, developmentally regulated genes and genes in weakly active or suppressed states particularly rely on H3K4 methylation. We then show that H3K4me3 is re-organized in diabetic Leprdb/db mouse islets in favour of weakly active and disallowed genes at the expense of terminal {beta}-cell markers with broad H3K4me3 peaks. Our results point to key roles of H3K4me3 in maintaining mature {beta}-cell function and establishing a dysfunctional transcriptome in diabetic islets.

cell biology↗

bioRxiv2021

Insights from Genomes and Genetic Epidemiology of SARS-CoV-2 isolates from the state of Andhra Pradesh

Coronavirus disease (COVID-19) emerged from a city in China and has now spread as a global pandemic affecting millions of individuals. The causative agent, SARS-CoV-2 is being extensively studied in terms of its genetic epidemiology using genomic approaches. Andhra Pradesh is one of the major states of India with the third-largest number of COVID-19 cases with limited understanding of its genetic epidemiology. In this study, we have sequenced 293 SARS-CoV-2 genome isolates from Andhra Pradesh with a mean coverage of 13,324X. We identified 564 high-quality SARS-CoV-2 variants, out of which 15 are novel. A total of 18 variants mapped to RT-PCR primer/probe sites, and 4 variants are known to be associated with an increase in infectivity. Phylogenetic analysis of the genomes revealed the circulating SARS-CoV-2 in Andhra Pradesh majorly clustered under the clade A2a (94%), while 6% fall under the I/A3i clade, a clade previously defined to be present in large numbers in India. To the best of our knowledge, this is the most comprehensive genetic epidemiological analysis performed for the state of Andhra Pradesh.

genomics↗

bioRxiv2020

Accurate brain age prediction using recurrent slice-based networks

BrainAge (a subjects apparent age predicted from neuroimaging data) is an important biomarker of brain aging. The deviation of BrainAge from true age has been associated with psychiatric and neurological disease, and has proven effective in predicting conversion from mild cognitive impairment (MCI) to dementia. Conventionally, 3D convolutional neural networks and their variants are used for brain age prediction. However, these networks have a larger number of parameters and take longer to train than their 2D counterparts. Here we propose a 2D slice-based recurrent neural network model, which takes in an ordered sequence of sagittal slices as input to predict the brain age. The model consists of two components: a 2D convolutional neural network (CNN), which encodes the relevant features from the slices, and a recurrent neural network (RNN) that learns the relationship between slices. We compare our method to other recently proposed methods, including 3D deep convolutional regression networks, information theoretic models, and bag-of-features (BoF) models (such as BagNet) - where the classification is based on the occurrences of local features, without taking into consideration their global spatial ordering. In our experiments, our proposed model performs comparably to, or better than, the current state of the art models, with nearly half the number of parameters and a lower convergence time.

neuroscience↗

bioRxiv2019

Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure

At cell division, the mammalian kinetochore binds many spindle microtubules that make up the kinetochore-fiber. To segregate chromosomes, the kinetochore-fiber must be dynamic and generate and respond to force. Yet, how it remodels under force remains poorly understood. Kinetochore-fibers cannot be reconstituted in vitro, and exerting controlled forces in vivo remains challenging. Here, we use microneedles to pull on mammalian kinetochore-fibers and probe how sustained force regulates their dynamics and structure. We show that force lengthens kinetochore-fibers by persistently favoring plus-end polymerization, not by increasing polymerization rate. We demonstrate that force suppresses depolymerization at both plus- and minus-ends, rather than sliding microtubules within the kinetochore-fiber. Finally, we observe that kinetochore-fibers break but do not detach from kinetochores or poles. Together, this work suggests an engineering principle for spindle structural homeostasis: different physical mechanisms of local force dissipation by the k-fiber limit force transmission to preserve robust spindle structure. These findings may inform how other dynamic, force-generating cellular machines achieve mechanical robustness.

cell biology↗

bioRxiv2019

Microneedle manipulation of the mammalian spindle reveals specialized, short-lived reinforcement near chromosomes

The spindle generates force to segregate chromosomes at cell division. In mammalian cells, kinetochore-fibers connect chromosomes to the spindle. The dynamic spindle anchors kinetochore-fibers in space and time to coordinate chromosome movement. Yet, how it does so remains poorly understood as we lack tools to directly challenge this anchorage. Here, we adapt microneedle manipulation to exert local forces on the spindle with spatiotemporal control. Pulling on kinetochore-fibers reveals that the spindle retains local architecture in its center on the seconds timescale. Upon pulling, sister, but not neighbor, kinetochore-fibers remain tightly coupled, restricting chromosome stretching. Further, pulled kinetochore-fibers freely pivot around poles but not around chromosomes, retaining their orientation within 3 {micro}m of chromosomes. This local reinforcement has a 20 s lifetime, and requires the microtubule crosslinker PRC1. Together, these observations indicate short-lived, specialized reinforcement of the kinetochore-fiber in the spindle center. This could help the spindle protect local structure near chromosomes from transient forces while allowing its remodeling over longer timescales, thereby supporting robust chromosome attachments and movements.

cell biology↗