Biology subjects

Flozak, A. S.

Publications and source records attributed to Flozak, A. S..

12 recordsLinked to original sources

bioRxiv2025

Top-Down Individual Ion Mass Spectrometry Reveals 85-110 kDa Catenin Phospho-Proteoforms Regulated by Actomyosin Contractility

A central challenge in top-down proteomics is the characterization of large proteoforms (>70 kDa) due to their high spectral complexity in mass spectrometers. Here, we advance individual ion mass spectrometry (I2MS) for intact mass and fragmentation analysis of {beta}- and -catenins (85-110 kDa), key components of adherens junctions. Using denatured I2MS, we resolved discrete phosphorylation states of catenins isolated from HEK cells subjected to differential actomyosin tension. Up to 10 phosphorylations were detected on {beta}-catenin and 7 on -catenin, with site-specific changes corresponding to actomyosin contractility. Notably, phosphorylation at -catenin S641 was constitutive, while other sites in the P-linker and actin-binding domains as well as {beta}-catenin S675 and S552 were sensitive to actomyosin perturbation. Application of I2MS for fragment ion detection (I2MS2) also enabled 25-30% sequence coverage for these exceptionally large proteoforms, compared to <1% using conventional methods for top-down mass spectrometry. Our results support a "catenin phospho-code" model, wherein combinatorial phosphorylation patterns encode mechano-transductive signals regulating cell-cell adhesion. This work establishes top-down I2MS as a viable approach for probing complex post-translational modification landscapes in high-mass proteins and highlights proteoforms as functional units in cellular regulation.

biochemistry↗

bioRxiv2025

α-catenin phosphorylation is actomyosin-sensitive and required for epithelial barrier functions through Afadin

Zonula adherens junctions (zAJ) are spatially proximal to tight junctions (TJ), in a superstructure known as the apical junctional complex (AJC). A key component of the AJC is a circumferential ring of filamentous (F)-actin, but how actomyosin contractility drives AJC structure and epithelial barrier function is incompletely understood. Here, we show that a central mechanosensitive component of zAJ, -catenin (-cat), undergoes force-dependent phosphorylation in an unstructured linker region. This modification in turn primes the -cat mechanosensitive Middle-region for effector-binding. We credential Afadin, a multi-domain TJ/AJ scaffold protein, as mechano-chemical binding partner of -cat, identifying residues in -cat required for this interaction. -cat phosphorylation and Afadin-binding are required for their co-enrichment at zAJ and epithelial barrier function. A mouse model that prevents -cat phosphorylation is particularly detrimental to post-natal brain development. These data support a stepwise model where -cat integrates mechanical and chemical signals to progressively promote zAJ enrichment, effector recruitment and epithelial barrier function.

cell biology↗

bioRxiv2025

Profibrotic monocyte-derived alveolar macrophages as a biomarker and therapeutic target in systemic sclerosis-associated interstitial lung disease

Interstitial lung disease (ILD) is present in over 60% of patients with systemic sclerosis (SSc) and is the leading cause of SSc-related deaths. Profibrotic monocyte-derived alveolar macrophages (MoAM) play a causal role in the pathogenesis of pulmonary fibrosis in animal models where their persistence in the niche requires signaling through Colony Stimulating Factor 1 Receptor (CSF1R). We hypothesized that the presence and proportion of MoAM in bronchoalveolar lavage (BAL) fluid from patients with SSc-ILD may be a biomarker of ILD severity. To test this hypothesis, we analyzed BAL fluid from 9 prospectively enrolled patients with SSc-ILD and 13 healthy controls using flow cytometry and single-cell RNA sequencing. Patients with SSc-ILD had more MoAM and interstitial macrophages in BAL fluid than healthy controls, and their abundance was associated with lung fibrosis severity. We identified changes in the MoAM transcriptome as a function of treatment with mycophenolate, an effective therapy for SSc-ILD. In SSc-ILD lung explants, spatial transcriptomics identified an expanded population of interstitial macrophages spilling into the alveolar space. Our findings suggest that the proportion of profibrotic MoAM and interstitial macrophages in BAL fluid may serve as a biomarker of SSc-ILD and credential them as possible targets for therapy.

immunology↗

bioRxiv2024

α-catenin phosphorylation is elevated during mitosis to resist apical rounding and epithelial barrier leak

Epithelial cell cohesion and barrier function critically depend on -catenin, an actin-binding protein and essential constituent of cadherin-catenin-based adherens junctions. -catenin undergoes actomyosin force-dependent unfolding of both actin-binding and middle domains to strongly engage actin filaments and its various effectors, where this mechanosensitivity is critical for adherens junction function. We previously showed that -catenin is highly phosphorylated in an unstructured region that links mechanosensitive middle- and actin-binding domains (known as the P-linker region), but the cellular processes that promote -catenin phosphorylation have remained elusive. Here, we leverage a previously published phospho-proteomic data set to show that the -catenin P-linker region is maximally phosphorylated during mitosis. By reconstituting -catenin Crispr KO MDCK with wild-type, phospho-mutant and mimic forms of -catenin, we show that full phosphorylation restrains mitotic cell rounding in the apical direction, strengthening interactions between dividing and non-dividing neighbors to limit epithelial barrier leak. Since major scaffold components of adherens junctions, tight junctions and desmosomes are also differentially phosphorylated during mitosis, we reason that epithelial cell division may be a tractable system to understand how junction complexes are coordinately regulated to sustain barrier function under tension-generating morphogenetic processes.

cell biology↗

bioRxiv2023

α-catenin mechanosensitivity as a route to cytokinesis failure through sequestration of LZTS2

Epithelial cells can become polyploid upon tissue injury, but mechanosensitive cues that trigger this state are poorly understood. Using -catenin (-cat) knock-out Madin Darby Canine Kidney (MDCK) cells reconstituted with wild-type and mutant forms of -cat as a model system, we find that an established -cat actin-binding domain unfolding mutant designed to reduce force-sensitive binding to F-actin (-cat-H0-FABD+) can promote cytokinesis failure, particularly along epithelial wound-fronts. Enhanced -cat coupling to cortical actin is neither sufficient nor mitotic cell-autonomous for cytokinesis failure, but critically requires the mechanosensitive Middle-domain (M1-M2-M3) and neighboring cells. Disease relevant -cat M-domain missense mutations known to cause a form of retinal pattern dystrophy (-cat E307K or L436P) are associated with elevated binucleation rates via cytokinesis failure. Similar binucleation rates are seen in cells expressing an -cat salt-bridge destabilizing mutant (R551A) designed to promote M2-M3 domain unfurling at lower force thresholds. Since binucleation is strongly enhanced by removal of the M1 as opposed to M2-M3 domains, cytokinetic fidelity is most sensitive to -cat M2-M3 domain opening. To identify -cat conformation-dependent proximity partners that contribute to cytokinesis, we used a biotin-ligase approach to distinguished proximity partners that show enhanced recruitment upon -cat M-domain unfurling (R551A). We identified Leucine Zipper Tumor Suppressor 2 (LZTS2), an abscission factor previously implicated in cytokinesis. We confirm that LZTS2 enriches at the midbody, but discover it also localizes to tight and tricellular junctions. LZTS2 knock-down promotes binucleation in both MDCK and Retinal Pigmented Epithelial (RPE) cells. -cat mutants with persistent M2-M3 domain opening showed elevated junctional enrichment of LZTS2 from the cytosol compared -cat wild-type cells. These data implicate LZTS2 as a mechanosensitive effector of -cat that is critical for cytokinetic fidelity. This model rationalizes how persistent mechano-activation of -cat may drive tension-induced polyploidization of epithelia post-injury and suggests an underlying mechanism for how pathogenic -cat mutations drive macular dystrophy.

cell biology↗

bioRxiv2023

An alpha-catenin actin-binding domain unfolding mutant designed to lower force-dependent actin-binding promotes epithelial strengthbut interferes with cell-cell coordination

-catenin (-cat) displays force-dependent unfolding and binding to actin filaments through direct and indirect means, but features of adherens junction structure and function most vulnerable to loss of these allosteric mechanisms have not been directly compared. By reconstituting an -cat F-actin-binding domain unfolding mutant known to exhibit enhanced binding to actin (-cat-H0-FABD+) into -cat knock-out Madin Darby Canine Kidney (MDCK) cells, we show that partial loss of the -cat catch bond mechanism (via an altered H0 -helix) leads to stronger epithelial sheet integrity with greater co-localization between the -cat-H0-FABD+ mutant and actin. -cat-H0-FABD+-expressing cells are less efficient at closing scratch-wounds, suggesting reduced capacity for more dynamic cell-cell coordination. Evidence that -cat-H0-FABD+ is equally accessible to the conformationally sensitive 18 antibody epitope as WT -cat and shows similar vinculin recruitment suggests this mutant engages lower tension cortical actin networks, as its M-domain is not persistently open. Conversely, -cat-M-domain salt-bridge mutants with persistent recruitment of vinculin and phosphorylated myosin light chain show only intermediate monolayer adhesive strengths, but display less directionally coordinated and thereby slower migration speeds during wound-repair. These data show -cat M- and FABD-unfolding mutants differentially impact cell-cell cohesion and migration properties, and suggest signals favoring -cat-cortical actin interaction without persistent M-domain opening may improve epithelial monolayer strength through enhanced coupling to lower tension actin networks.

cell biology↗

bioRxiv2022

Alpha-T-catenin is expressed in peripheral nerves as a constituent of Schwann cell adherens junctions

The adherens junction component, alpha-T-catenin (Tcat) is an established contributor to cardiomyocyte junction structure and function, but recent genomic studies link CTNNA3 polymorphisms to diseases with no clear cardiac underpinning, including asthma, autism and multiple sclerosis, suggesting causal contributions from a different cell-type. We show Ctnna3 mRNA is highly expressed in peripheral nerves (e.g., vagus and sciatic), where Tcat protein enriches at paranodes and myelin incisure adherens junctions of Schwann cells. We validate Tcat immunodetection specificity using a new Ctnna3-knockout fluorescence reporter mouse line yet find no obvious Schwann cell loss-of-function morphology at the light microscopic level. CTNNA3/Ctnna3 mRNA is also abundantly detected in oligodendrocytes of the central nervous system via public databases, supporting a general role for Tcat in these unique cell-cell junctions. These data suggest that the wide range of diseases linked to CTNNA3 may be through its role in maintaining neuroglial functions of central and peripheral nervous systems.

cell biology↗

bioRxiv2022

Hypercapnia limits β-catenin mediated alveolar type 2 cell progenitor function by altering Wnt production from adjacent fibroblasts

Persistent symptoms and radiographic abnormalities suggestive of failed lung repair are among the most common symptoms in patients with COVID-19 after hospital discharge. In mechanically ventilated patients with ARDS secondary to SARS-CoV-2 pneumonia, low tidal volumes to reduce ventilator-induced lung injury necessarily elevate blood CO2 levels, often leading to hypercapnia. The role of hypercapnia on lung repair after injury is not completely understood. Here, we show that hypercapnia limits {beta}-catenin signaling in alveolar type 2 (AT2) cells, leading to reduced proliferative capacity. Hypercapnia alters expression of major Wnts in PDGFRa+-fibroblasts from those maintaining AT2 progenitor activity and towards those that antagonize {beta}-catenin signaling and limit progenitor function. Activation of {beta}-catenin signaling in AT2 cells, rescues the inhibition AT2 proliferation induced by hypercapnia. Inhibition of AT2 proliferation in hypercapnic patients may contribute to impaired lung repair after injury, preventing sealing of the epithelial barrier, increasing lung flooding, ventilator dependency and mortality.

cell biology↗

bioRxiv2021

Lung injury induces alveolar type 2 cell hypertrophy and polyploidy with implications for repair and regeneration

Epithelial polyploidization post-injury is a conserved phenomenon, recently shown to improve barrier restoration during wound healing. Whether lung injury can induce alveolar epithelial polyploidy is not known. We show that bleomycin injury induces AT2 cell hypertrophy and polyploidy. AT2 polyploidization is also seen in short term ex vivo cultures, where AT2-to-AT1 trans-differentiation is associated with substantial binucleation due to failed cytokinesis. Both hypertrophic and polyploid features of AT2 cells can be attenuated by inhibiting the integrated stress response (ISR) using the small molecule ISRIB. These data suggest that AT2 hypertrophic growth and polyploidization may be a feature of alveolar epithelial injury. As AT2 cells serve as facultative progenitors for the distal lung epithelium, a propensity for injury-induced binucleation has implications for AT2 self-renewal and regenerative potential upon reinjury, which may benefit from targeting the ISR.

cell biology↗

bioRxiv2019

Single-cell RNA-seq reveals spatially restricted multicellular fibrotic niches during lung fibrosis

Ontologically distinct populations of macrophages differentially contribute to organ fibrosis through unknown mechanisms. We applied lineage tracing, spatial methods and single-cell RNA-seq to a spatially-restricted model of asbestos-induced pulmonary fibrosis. We demonstrate that while tissue-resident interstitial macrophages, tissue-resident alveolar macrophages, and monocyte-derived alveolar macrophages are present in the fibrotic niche, only monocyte-derived alveolar macrophages are causally related to fibrosis. Monocyte-derived alveolar macrophages were specifically localized to fibrotic regions in the proximity of fibroblasts where they expressed molecules known to drive fibroblast proliferation, including PDGFA. Moreover, we identified autocrine M-CSF/M-CSFR signaling in monocyte-derived alveolar macrophages as a novel mechanism promoting their self-maintenance and persistence in the fibrotic niche. Pharmacological blockade of M-CSF signaling led to disappearance of the established population of monocyte-derived alveolar macrophages. Thus, our data indicate that monocyte-derived alveolar macrophages are specifically recruited to the fibrotic niche where they are maintained by autocrine signaling and drive fibrosis by stimulating fibroblast proliferation.

immunology↗

bioRxiv2018

Single-Cell Transcriptomic Analysis of Human Lung Reveals Complex Multicellular Changes During Pulmonary Fibrosis

Pulmonary fibrosis is a devastating disorder that results in the progressive replacement of normal lung tissue with fibrotic scar. Available therapies slow disease progression, but most patients go on to die or require lung transplantation. Single-cell RNA-seq is a powerful tool that can reveal cellular identity via analysis of the transcriptome, but its ability to provide biologically or clinically meaningful insights in a disease context is largely unexplored. Accordingly, we performed single-cell RNA-seq on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and one bronchoscopic cryobiospy sample. Integrated single-cell transcriptomic analysis of donors and patients with pulmonary fibrosis identified the emergence of distinct populations of epithelial cells and macrophages that were common to all patients with lung fibrosis. Analysis of transcripts in the Wnt pathway suggested that within the same cell type, Wnt secretion and response are restricted to distinct non-overlapping cells, which was confirmed using in situ RNA hybridization. Single-cell RNA-seq revealed heterogeneity within alveolar macrophages from individual patients, which was confirmed by immunohistochemistry. These results support the feasibility of discovery-based approaches applying next generation sequencing technologies to clinically obtained samples with a goal of developing personalized therapies.\n\nOne Sentence SummarySingle-cell RNA-seq applied to tissue from diseased and donor lungs and a living patient with pulmonary fibrosis identifies cell type-specific disease-associated molecular pathways.

systems biology↗

bioRxiv2017

Arginase 1 Is Negatively Regulated By β-Catenin Signaling In The Lung

We previously demonstrated that mice lacking the Wnt co-receptor, Lrp5, had attenuated pulmonary fibrosis in the bleomycin model. We found that Arginase 1 (Arg1), an enzyme that converts L-arginine to urea and ornithine, was markedly elevated in Lrp5-/- lungs compared with wild-type mice after bleomycin injury. We show that this induction is not apparently due to the expression of Th2 cytokines, IL-4 and IL-13, but instead is due to Wnt/{beta}-catenin signaling, which negatively regulates Arg1 expression in lung macrophages. Although Arg1 expression in macrophages has been used to define an alternatively activated phenotype, flow cytometry analysis of alveolar and interstitial macrophage sub-populations in Lrp5-/- lungs 14 days after bleomycin injury revealed no clear evidence of skewing from a classical to an alternatively activated phenotype. Upregulation of Arg1 expression and arginase activity might diminish lung arginine levels with consequent alterations in collagen or cytokine production. However, dietary supplementation of bleomycin-treated Lrp5-/- mice with the Arg1 substrate, L-arginine, failed to alter lung collagen content or cytokine levels 21 days after bleomycin injury. These findings demonstrate that Arg1 is negatively regulated by {beta}-catenin signaling in macrophages, raising the possibility that Wnt signaling directs alterations in immune cell metabolism that may be relevant to lung repair after injury.

cell biology↗