Biology subjects

Buczak, K.

Publications and source records attributed to Buczak, K..

8 recordsLinked to original sources

bioRxiv2026

Desmoglein-3 modulates p38MAPK and ERK signaling responses through the mechano-sensitive channel Piezo1

BackgroundSkin is constantly exposed to mechanical forces such as pressure and friction, which need to be sensed and buffered to ensure tissue homeostasis and barrier function. Desmosomes are essential for epidermal integrity, but their role in converting mechanical cues into cellular signaling responses are not well understood. MethodsHere, we combine proteomics and shear-stress assays with live-cell reporters to investigate how desmosomes modulate stress-kinase pathways in keratinocytes. ResultsWe show that the desmosomal adhesion molecule DSG3 is essential not only for cell-cell adhesion but also for modulating p38MAPK and ERK signaling. Loss of DSG3 disrupts mechanotransduction-related protein networks, including the expression of the mechanosensitive channel Piezo1. Under static conditions, DSG3 dampens ERK activity via Piezo1-dependent mechanisms, whereas DSG3 suppresses p38MAPK activity through an independent mechanism. In contrast, DSG3 is required to trigger an activation of both ERK and p38MAPK in response to shear stress in a Piezo1-dependent manner. Experiments with domain-specific DSG3 mutants demonstrate that cell cohesion and signaling responses are partially uncoupled, while maintaining DSG3 tail integrity was crucial for p38MAPK and ERK responses. ConclusionThese findings demonstrate that DSG3 independently coordinates adhesion and mechanotransduction in a domain-specific manner, providing novel insights into how DSG3 contributes to epithelial integrity under dynamic mechanical environments.

cell biology↗

bioRxiv2026

Metabolic-secretory decoupling defines a disease-intrinsic state in rheumatoid arthritis monocytes

ObjectivesCirculating monocytes from rheumatoid arthritis (RA) patients are pre-primed for inflammatory activation, but their disease-intrinsic features have not been systematically characterized. Given the important role of metabolism in shaping immune cell function, we aimed to determine how this pre-primed state is underpinned metabolically and whether these changes persist across different activation states, using an unbiased multi-omics approach. MethodsPeripheral blood CD14 monocytes from RA patients and matched healthy donors were analyzed in an undifferentiated state (M0) and after differentiation into classically activated M(IFN{gamma}+LPS) and alternatively activated M(IL-4) macrophages, followed by acute lipopolysaccharide (LPS) stimulation. Metabolomic (untargeted LC-MS/MS), transcriptomic (RNA-seq), and proteomic (label-free mass LC-MS/MS) profiling were performed. Data was comprehensively analyzed by weighted gene correlation network analysis, differential analysis, gene set enrichment analysis, multi-omics factor analysis and metabolic flux modeling. ResultsRA monocytes exhibited a stable disease-driven signature across activation states. Integration of metabolomic, transcriptomic and proteomic data revealed an unexpected convergence on metabolic-secretory coupling, with depletion of nucleotide and redox metabolites, downregulation of mitochondrial and translational pathways, and remodeling of the secretory apparatus, including loss of cis-Golgi components. Consistently, metabolic modeling predicted reduced glycosylation fluxes, connecting metabolic changes to altered secretory capacity. ConclusionsRA monocytes adopt a stable, disease-intrinsic state that persists across activation conditions. Multi-omics data identify a linked metabolic and secretory defect, with reduced glycosylation capacity as a potential functional consequence. This metabolic-secretory coupling represents a defining feature of RA monocyte dysfunction and a potential therapeutic target.

immunology↗

bioRxiv2026

Cell cycle-dependent protein dynamics in budding yeast resolved by deconvolution of bulk proteomics

The cell division cycle is characterised by oscillatory dynamics in regulatory mechanisms and biosynthesis, coordinated with genome replication and segregation. To understand these dynamics, quantitative cell cycle-dependent protein concentration data is essential. Unfortunately, accurate resolution of cell cycle-dependent protein dynamics is challenging because single-cell proteomics is currently infeasible and bulk proteomics requires inherently imperfect cell synchronisation. Here, we developed a computational method to deconvolve cell cycle-dependent protein concentration dynamics and applied it to new budding yeast bulk proteome data. Key to this method was a yeast population model, parameterised with experimental cell cycle progression and volume growth data, for quantifying the desynchronisation in sampled populations. We performed deconvolution on 3373 proteins, using cross-validation to determine regularisation parameters, and identified 563 proteins with cell cycle-dependent dynamics. Many of these dynamics were consistent with known yeast biology and dynamic proteins were enriched for several metabolic process, extending previous observations and supporting the emerging picture of metabolic activity as varying substantially over cell cycle phases. We consider the generated cell cycle-resolved budding yeast proteome data a key resource.

systems biology↗

bioRxiv2025

Identification and proteomic profiling of CD90+ small EVs using a refined immunocapture separation approach targeting stromal-derived EV subpopulations in synovial fluid of arthritis patients

Extracellular vesicles (EVs) have emerged as essential drivers in disease progression and promising biomarkers across various disease conditions, including autoimmune diseases such as rheumatoid arthritis (RA). RA is characterized by chronic inflammation driven by patient-specific cellular sub-phenotypes within the synovium. Since EVs reflect their cellular origin and are detectable in biofluids like synovial fluid, they offer a promising alternative for novel biomarker discovery or diagnostics in arthritis, potentially reducing the need for invasive tissue biopsies. However, the inherent complexity, viscosity and extracellular matrix (ECM) composition of synovial fluid pose significant challenges for efficient EV isolation. Moreover, most previous studies have focused on heterogenous bulk EV populations, neglecting the diverse EV subsets present in patient biofluids. To address these challenges, we established a novel immunocapture-based separation method, that selectively targets and isolates cell type-specific EV subpopulation from the heterogenous EV pool present in synovial fluid of arthritis patients. As a proof-of-concept approach, we targeted a stromal-derived EV population by leveraging the fibroblast-associated surface marker CD90/THY1, representing a subset of synovial fibroblasts implicated as important drivers in synovitis and chronic inflammation in RA. Western Blot and data independent acquisition (DIA) liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) proteomic analysis of immunocaptured CD90+ EVs isolated from cultured primary synovial fibroblasts and synovial fluid of arthritis patients, confirmed the successful separation of stromal vs a myeloid/lymphoid EV populations. The in-depth proteomic profiling of synovial fluid EVs offers unprecedented insights into EV heterogeneity in arthritis. This analysis enables the classification of cell-type associated EV subsets and the identification of novel EV markers for future immunocapture-based EV separation strategies. Our refined fit-for-purpose EV separation approach, coupled to in depth proteomic profiling, provides a powerful tool to identify disease-relevant EV subpopulations and novel biomarker candidates in synovial fluid, facilitating advances in arthritis diagnostics and personalized medicine

immunology↗

bioRxiv2024

Massive reduction of RyR1 in muscle spindles of mice carrying recessive Ryr1 mutations alters proprioception and causes scoliosis

Muscle spindles are stretch receptors lying deep within the muscle belly involved in detecting changes in muscle length and playing a fundamental role in motor control, posture and synchronized gait. They are made up of an external capsule surrounding 3-5 intrafusal muscle fibers and a nuclear bag complex. Dysfunction of muscle spindles leads to abnormal proprioceptor function, which has been linked to aberrant bone and cartilage development, scoliosis, kyphosis and joint contractures. RYR1, the gene encoding the calcium release channel of the sarcoplasmic reticulum, is the most common target of mutations linked to human congenital myopathies, a condition often accompanied by skeleton alterations and joint contractures. So far, the link between RYR1 mutations, altered muscle spindles and skeletal defects has not been investigated. To this end, we investigated heterozygous mice carrying recessive Ryr1 mutations isogenic to those present in a severely affected child. Here we show that: (i) the RyR1 protein localizes to the polar regions of intrafusal fibers and exhibits a doubled row distribution pattern, typical for junctional sarcoplasmic reticulum proteins; (ii) muscle spindles of compound heterozygous mice show structural defects; (iii) RyR1 content in intrafusal muscle fibers from dHT mice is reduced by 54%. Such a massive reduction of mutant RyR1 in intrafusal muscle fibers leads to altered expression of intrafusal fiber proteins, severe scoliosis, alteration of gait and inter limb coordination. These results support the hypothesis that RYR1 mutations not only affect the function of extrafusal muscles, but might also affect that of intrafusal muscles. The latter may be one of the underlying causes of skeletal abnormalities seen in patients affected by recessive RYR1 mutations.

physiology↗

bioRxiv2022

FKBP35 secures ribosome homeostasis in Plasmodium falciparum

Plasmodium falciparum accounts for the majority of over 600000 malaria-associated deaths annually. Parasites resistant to nearly all antimalarials have emerged and the need for drugs with alternative modes of action is thus undoubted. The FK506-binding protein PfFKBP35 has gained attention as a promising drug target due to its high affinity to the macrolide compound FK506 (tacrolimus). Whilst there is considerable interest in targeting PfFKBP35 with small molecules, a genetic validation of this factor as a drug target is missing and its function in parasite biology remains elusive. Here, we show that limiting PfFKBP35 levels are lethal to P. falciparum and result in a delayed death-like phenotype that is characterized by defective ribosome homeostasis and stalled protein synthesis. Our data furthermore suggest that FK506, unlike the action of this drug in model organisms, exerts its anti-proliferative activity in a PfFKBP35-independent manner and, using cellular thermal shift assays, we identify putative FK506-targets beyond PfFKBP35. In addition to revealing first insights into the function of PfFKBP35, our results show that FKBP-binding drugs can adopt non-canonical modes of action - with major implications for the development of FK506-derived molecules active against Plasmodium parasites and other eukaryotic pathogens.

molecular biology↗

bioRxiv2021

Artificial intelligence reveals nuclear pore complexity

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport. Their intricate 120 MDa architecture remains incompletely understood. Here, we report a near-complete structural model of the human NPC scaffold with explicit membrane and in multiple conformational states. We combined AI-based structure prediction with in situ and in cellulo cryo-electron tomography and integrative modeling. We show that linker Nups spatially organize the scaffold within and across subcomplexes to establish the higher-order structure. Microsecond-long molecular dynamics simulations suggest that the scaffold is not required to stabilize the inner and outer nuclear membrane fusion, but rather widens the central pore. Our work exemplifies how AI-based modeling can be integrated with in situ structural biology to understand subcellular architecture across spatial organization levels. One sentence summaryAn AI-based, dynamic model of the human nuclear pore complex reveals how the protein scaffold and the nuclear envelope are coupled inside cells.

molecular biology↗

bioRxiv2021

Shared and specific functions of Arfs 1-5 at the Golgi revealed by systematic knockouts

The ADP-ribosylation factors (Arfs) are small GTPases regulating membrane traffic in the secretory pathway. They are closely related and appear to have overlapping functions, regulators, and effectors. The functional specificity of individual Arfs and the extent of redundancy in vivo are still largely unknown. We addressed these questions by CRISPR/Cas9-mediated genomic deletion of the human class I (Arfs 1 and 3) and class II (Arfs 4 and 5) Arfs, either individually or in combination. Cells lacking individual Arfs or certain combinations were viable with only a slight growth defect when lacking Arf1 or Arf4. However, Arf1 and 4, and Arf4 and 5 could not be deleted simultaneously. Hence, class I Arfs are not essential and Arf4 alone was found to be sufficient for cell viability. Remarkably, two single knockouts produced specific and distinct phenotypes. Upon deletion of Arf1, the Golgi complex was enlarged and recruitment of vesicle coats decreased, confirming a major role of Arf1 in coat formation at the Golgi. Cell lines deleted for Arf4 exhibited secretion of ER resident proteins, indicating a specific defect in coatomer-dependent ER protein retrieval by the KDEL receptors. The knockout cell lines will be a useful tool to study other Arf-dependent processes.

cell biology↗