Biology subjects

Linder, M.

Publications and source records attributed to Linder, M..

9 recordsLinked to original sources

bioRxiv2026

Cardiomyocyte mechanical activity counteracts intraluminal calcium depletion in the transverse-axial tubular system during fast electrical stimulation

The transverse-axial tubular system (TATS) enables close structural and functional coupling between plasma membrane and sarcoplasmic reticulum of cardiomyocytes. It supports fast and efficient Ca2+-induced Ca2+ release upon cell depolarisation, crucial for excitation-contraction coupling in the heart. Due to the small diameter and tortuosity of individual tubules, the TATS forms a domain of restricted diffusive transport. It has previously been suggested that, as a consequence of an uneven distribution of Ca2+ influx and efflux pathways in TATS compared to outer surface plasma membrane domains of cardiomyocytes, cyclic electrical activity may lead to a gradual depletion of Ca2+ in the TATS. Here, we show experimentally that in mechanically uncoupled rabbit ventricular cardiomyocytes, electrical stimulation does indeed lead to an L-type Ca2+ channel-dependent gradual depletion of Ca2+ inside TATS, an effect that scales with pacing frequency. Ca2+ depletion was absent in freely contracting cardiomyocytes, presumably as a result of cyclic TATS deformation during cell shortening. This squeezes transverse TATS tubules and adds an advective contribution to, and thereby accelerates the, intra-TATS content exchange with bulk extracellular fluid. Our results reveal a novel mechanism of cardiac mechano-dependent auto-regulation, where the increased propensity for development of intra-TATS Ca2+ gradients at high electrical stimulation rates is mitigated by the coinciding mechanically induced TATS deformation, twice on each cycle in the heart (during diastolic stretch and systolic shortening), which accelerates luminal content exchange. Our study provides first insight into a novel facet of cardiac mechano-biology, whose auto-regulatory benefit may be reduced by TATS remodelling in disease.

cell biology↗

bioRxiv2025

Sample-level modeling of single-cell data at scale with tinydenseR

Single-cell studies now routinely encompass hundreds of samples and millions of cells, offering unprecedented opportunities to link sample-level phenotypes with cellular and molecular states. However, current workflows often depend on cell-level inference and rigid clustering, which can distort significance and obscure subtle, continuous variation, in particular for complex experimental designs. Here, we present tinydenseR, a clustering-independent framework that enables robust, scalable, and statistically sensitive detection of differential cell state density, outperforming existing workflows in speed and memory usage. Technology-agnostic at its core, tinydenseR works seamlessly on scRNA-seq, flow, mass and spectral cytometry. Across synthetic benchmarks, a preclinical xenograft model, a publicly available COVID-19 study, two immuno-oncology trials and a multi-study atlas, tinydenseR uncovers disease and treatment history-associated effects, including subtle cell state heterogeneity, while embedding samples in a quantitative manner. Designed to accelerate discovery in clinical, preclinical, and translational research, the open-source package is available at GitHub.com/Novartis/tinydenseR.

bioinformatics↗

bioRxiv2024

Sympathetic Stimulation Can Compensate for Hypocalcaemia-Induced Bradycardia in Human and Rabbit Sinoatrial Node Cells

Regular activation of the heart originates from cyclic spontaneous depolarisations of sinoatrial node cells (SANC). Variations in electrolyte levels, commonly observed in haemodialysis (HD) patients, and the autonomic nervous system (ANS) profoundly affect the SANC function. Thus, we investigated the effects of hypocalcaemia and sympathetic stimulation on SANC beating rate (BR). The {beta}-adrenergic ({beta}-AR) signalling cascade, as described by Behar et al., was incorporated into the SANC models of Severi et al. (rabbit) and Fabbri et al. (human). Simulations were conducted across various extracellular calcium ([Ca2+]o) (0.6 to 1.8 mM) and isoprenaline concentrations [ISO] (0 to 1000 nM) for a sufficient time to allow transient oscillations to equilibrate and reach a limit cycle. The {beta}-AR cell response of the extended models was validated against new Langendorff-perfused rabbit heart experiments and literature data. The extended models revealed that decreased [Ca2+]o necessitated an exponential-like increase in [ISO] to restore the basal BR. Specifically, at 1.2 mM [Ca2+]o, the Severi and Fabbri model required 15.5 and 7.3 nM [ISO], respectively, to restore the initial BR. Further reduction of [Ca2+]o to 0.6 mM required 60.0 and 41.7 nM [ISO] to compensate for hypocalcaemia. A sudden loss of sympathetic tone at low [Ca2+]o resulted in extreme bradycardia or even loss of automaticity within seconds. These findings suggest that hypocalcaemic bradycardia can be compensated for by an elevated sympathetic tone. The integration of the {beta}-AR pathways led to a logarithmic BR increase and offers insights into potential pathomechanisms underlying sudden cardiac death (SCD) in HD patients. Key Points- We extended the sinoatrial node cell (SANC) models of Severi et al. (rabbit) and Fabbri et al. (human) with the {beta}-adrenergic ({beta}-AR) signalling cascade Behar et al. described. - Simulations were conducted across various extracellular calcium ([Ca2+]o) (0.6 to 1.8 mM) and isoprenaline concentrations [ISO] (0 to 1000 nM) to mimic conditions in haemodialysis patients. - An exponential-like increase in [ISO] compensated for hypocalcaemia-induced bradycardia in both models, while inter-species differences lead to more sensitivity of the extended Fabbri model towards hypocalcaemia and increased sympathetic tone. - The extended models may help to further understand the pathomechanisms of several cardiovascular diseases affecting pacemaking, such as the high occurrence of sudden cardiac death (SCD) in chronic kidney disease (CKD) patients.

cell biology↗

bioRxiv2023

Deletion of SMC renders FtsK essential in Corynebacterium glutamicum

Structural maintenance of chromosomes (SMC) are ubiquitously distributed proteins involved in chromosome organization. Deletion of smc causes severe growth phenotypes in many organisms. Surprisingly, smc can be deleted in Corynebacterium glutamicum, a member of the Actinomycetota phylum, without any apparent growth phenotype. Earlier work has shown that SMC in C. glutamicum is loaded in a ParB-dependent fashion to the chromosome and functions in replichore cohesion. The unexpected absence of a growth phenotype in the smc mutant prompted us to screen for unknown synthetic interactions within C. glutamicum. Therefore, we generated a high-density Tn-5 library based on wild-type and smc-deleted C. glutamicum strains. The transposon sequencing (Tn-seq) data revealed that the DNA-translocase FtsK is essential in a smc deletion strain. FtsK localized to the septa and cell poles in wild type cells, however deletion of smc resulted in a decreased polar FtsK localization. Single-particle tracking analysis further suggests that prolonged FtsK complex activity is both required and sufficient to make up for the absence of SMC, thus achieving efficient chromosome segregation in C. glutamicum. Further, single molecule dynamics of FtsK is influenced, albeit indirectly, by DNA-loaded SMC. Deletion of ParB results in an increased of both SMC and FtsK mobility. While the first change agrees with previous data that show how ParB is essential for SMC loading on DNA, the latter suggests that FtsK mobility is affected in cells with defects in chromosome organization. Based on our data we propose a simple, yet efficient mechanism for efficient DNA segregation in C. glutamicum, even in absence of SMC proteins. ImportanceFaithful DNA segregation is of fundamental importance for life. Bacteria have efficient systems to coordinate chromosome compaction, DNA segregation and cell division. A key factor in DNA compaction is the SMC-complex that is found to be essential in many bacteria. In members of the Actinomycetota smc is dispensable, but the reason for this was unclear. We show here that the divisome associated DNA-pump FtsK can compensate SMC loss and the subsequent loss in correct chromosome organization. In cells with distorted chromosomes, FtsK functions for an extended period of time at the septum, until chromosomes are segregated.

microbiology↗

bioRxiv2021

The proteasome regulator PSME4 drives immune evasion and abrogates anti-tumor immunity in NSCLC

Protein degradation by proteasomes is important for the immune response against tumors. Antigens generated by the proteasome promote immune cell infiltration into tumors and improve tumors responses to immunotherapy. For example, immunoproteasomes - a subset of proteasomes induced by inflammatory signals - may improve the response of melanomas to immune checkpoint inhibitors (ICI) by eliciting tumor inflammation. Yet, it is unclear whether and how protein degradation by proteasomes impacts cancer progression and contributes to immune evasion and resistance. Here, we profile the proteasome-cleaved peptides in lung cancers and find that PSME4 serves as a novel inhibitory regulator of the immunoproteasome, playing an anti-inflammatory role in cancer. Biochemical assays combined with scRNA-seq, immunopeptidomics and in vivo analyses demonstrate that PSME4 promotes an immunosuppressive environment around the tumor and abrogates anti-tumor immunity by inhibiting antigen presentation and attenuating tumor inflammation. Furthermore, we find that PSME4 expression is correlated with responsiveness to ICI across several cancer types. Our findings suggest that PSME4-mediated regulation of proteasome activity is a novel mechanism of immune evasion in non-small-cell lung carcinoma and may be targeted therapeutically for restoring anti-tumor immunity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/464690v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@e87910org.highwire.dtl.DTLVardef@641843org.highwire.dtl.DTLVardef@175092corg.highwire.dtl.DTLVardef@ad34c2_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMapping the degradation landscape in Non-Small Cell Lung Cancer (NSCLC) uncovers altered proteasome activity and composition C_LIO_LIProteasome regulator PSME4 plays an anti-inflammatory role in NSCLC by attenuating immunoproteasome activity C_LIO_LIPSME4 restricts tumor antigen presentation and cytokine secretion, defining a cold tumor environment C_LIO_LIPSME4 drives tumor immune evasion and is associated with resistance to immunotherapy C_LI

cancer biology↗

bioRxiv2021

Liquid-Liquid Phase Separation Protects Amyloidogenic and Aggregation-Prone Peptides in Heterologous Expression Systems

Studying pathogenic effects of amyloids requires homogeneous amyloidogenic peptide samples. Recombinant production of these peptides is challenging due to their susceptibility to aggregation and chemical modifications. Thus, chemical synthesis is primarily used to produce amyloidogenic peptides suitable for high resolution structural studies. Here, we exploited the shielded environment of protein condensates formed via liquid-liquid phase separation (LLPS) as a protective mechanism against premature aggregation. We designed a fusion protein tag undergoing LLPS in E. coli and linked it to highly amyloidogenic peptides, including A{beta} amyloid. We find that the fusion proteins form membraneless organelles during overexpression and remain soluble. We also developed a facile purification method of functional A{beta} peptides free of chromatography steps. The strategy exploiting LLPS can be applied to other amyloidogenic, hydrophobic, and repetitive peptides that are otherwise difficult to produce.

molecular biology↗

bioRxiv2020

Colitis is associated with loss of LHPP and up-regulation of histidine phosphorylation in intestinal epithelial cells

Protein histidine phosphorylation (pHis) is a posttranslational modification involved in cell cycle regulation, ion channel activity and phagocytosis (1). Using novel monoclonal antibodies to detect pHis (2), we recently reported that loss of the histidine phosphatase LHPP results in elevated pHis levels in hepatocellular carcinoma (3). Here, we show that intestinal inflammation correlates with loss of LHPP, in DSS-treated mice and in inflammatory bowel disease (IBD) patients. Increased histidine phosphorylation was observed in intestinal epithelial cells (IECs), as determined by pHis immunofluorescence staining of colon samples from a colitis mouse model. However, ablation of Lhpp did not cause increased pHis or promote intestinal inflammation in physiological conditions or after DSS treatment. Our observations suggest that increased histidine phosphorylation plays a role in colitis, but loss of LHPP is not sufficient to increase pHis or to cause inflammation in the intestine.

biochemistry↗

bioRxiv2020

Cortical actin properties controlled by Drosophila Fos aid macrophage infiltration against surrounding tissue resistance

The infiltration of immune cells into tissues underlies the establishment of tissue resident macrophages, and responses to infections and tumors. Yet the mechanisms immune cells utilize to negotiate tissue barriers in living organisms are not well understood, and a role for cortical actin has not been examined. Here we find that the tissue invasion of Drosophila macrophages, also known as plasmatocytes or hemocytes, utilizes enhanced cortical F-actin levels stimulated by the Drosophila member of the fos proto oncogene transcription factor family (Dfos, Kayak). RNA sequencing analysis and live imaging show that Dfos enhances F-actin levels around the entire macrophage surface by increasing mRNA levels of the membrane spanning molecular scaffold tetraspanin TM4SF, and the actin cross-linking filamin Cheerio which are themselves required for invasion. Both the filamin and the tetraspanin enhance the cortical activity of Rho1 and the formin Diaphanous and thus the assembly of cortical actin, which is a critical function since expressing a dominant active form of Diaphanous can rescue the Dfos macrophage invasion defect. In vivo imaging shows that Dfos enhances the efficiency of the initial phases of macrophage tissue entry. Genetic evidence argues that this Dfos-induced program in macrophages counteracts the constraint produced by the tension of surrounding tissues and buffers the properties of the macrophage nucleus from affecting tissue entry. We thus identify strengthening the cortical actin cytoskeleton through Dfos as a key process allowing efficient forward movement of an immune cell into surrounding tissues.

cell biology↗

bioRxiv2018

SUMO-based expression and purification of dermcidin-derived DCD-1L, a human antimicrobial peptide, in Escherichia coli

The dermcidin-derived peptide DCD-1L has a broad spectrum of antimicrobial activity over a wide pH range and in high salt concentrations. Thus, it offers a promising alternative to conventional antibiotics. Furthermore, it plays a role in wound healing, atopic dermatitis and acne vulgaris, indicating applications in cosmetic industries. Recently, dermcidin has been identified as a tumor marker improving cancer prognosis. Hence, large quantities of purified DCD-1L peptide are required to meet the needs of basic research and clinical trials. In the current study, we demonstrate SUMO-based heterologous DCD-1L production in Escherichia coli, followed by affinity chromatography purification. The SUMO tag is cleaved with SUMO-specific protease following purification, leaving free DCD-1L peptide without any additional amino acids. The mass of the peptide was further confirmed by MALDI-TOF-TOF analysis. Furthermore, the cleaved DCD-1L showed antimicrobial activity against the E. coli DH5 alpha test strain. The production and purification of DCD-1L using SUMO tag compare advantageously to other protocols previously described. Thus, the SUMO tag system enables large scale recombinant production of the antimicrobial peptide DCD-1L, which constitutes pharmaceutical and therapeutic potential as an alternative antibiotic.

molecular biology↗