Biology subjects

Schwartz, M. A.

Publications and source records attributed to Schwartz, M. A..

33 records · Page 2Linked to original sources

bioRxiv2023

cSTAR analysis identifies endothelial cell cycle as a key regulator of flow-dependent artery remodeling

Fluid shear stress (FSS) from blood flow is sensed by vascular endothelial cells (ECs) to determine vessel stability, remodeling and susceptibility to atherosclerosis and other inflammatory diseases but the regulatory networks that govern these behaviors are only partially understood. We used cSTAR, a powerful new computational method, to define EC transcriptomic states under low shear stress (LSS) that triggers vessel inward remodeling, physiological shear stress (PSS) that stabilizes vessels, high shear stress (HSS) that triggers outward remodeling, and oscillatory shear stress (OSS) that confers disease susceptibility, all in comparison to cells under static conditions (STAT). We combined these results with the LINCS database where EC transcriptomic responses to drug treatments to define a preliminary regulatory network in which the cyclin-dependent kinases CDK1/2 play a central role in promoting vessel stability. Experimental analysis showed that PSS induced a strong late G1 cell cycle arrest in which CDK2 was activated. EC deletion of CDK2 in mice resulted in inward artery remodeling and both pulmonary and systemic hypertension. These results validate use of cSTAR to determine EC state and in vivo vessel behavior, reveal unexpected features of EC phenotype under different FSS conditions, and identify CDK2 as a key element within the EC regulatory network that governs artery remodeling.

cell biology↗

bioRxiv2023

Mechanosensing through talin 1 contributes to tissue mechanical homeostasis

It is widely believed that tissue mechanical properties, determined mainly by the extracellular matrix (ECM), are actively maintained. However, despite its broad importance to biology and medicine, tissue mechanical homeostasis is poorly understood. To explore this hypothesis, we developed mutations in the mechanosensitive protein talin1 that alter cellular sensing of ECM stiffness. Mutation of a novel mechanosensitive site between talin1 rod domain helix bundles 1 and 2 (R1 and R2) shifted cellular stiffness sensing curves, enabling cells to spread and exert tension on compliant substrates. Opening of the R1-R2 interface promotes binding of the ARP2/3 complex subunit ARPC5L, which mediates the altered stiffness sensing. Ascending aortas from mice bearing these mutations show increased compliance, less fibrillar collagen, and rupture at lower pressure. Together, these results demonstrate that cellular stiffness sensing regulates ECM mechanical properties. These data thus directly support the mechanical homeostasis hypothesis and identify a novel mechanosensitive interaction within talin that contributes to this mechanism.

cell biology↗

bioRxiv2022

Calcium flux through ER-TGN contact sites facilitates cargo export

Ca2+ influx into the trans-Golgi Network (TGN) promotes secretory cargo sorting by the Ca2+-ATPase SPCA1 and the luminal Ca2+ binding protein Cab45. Cab45 oligomerizes upon a local Ca2+ influx, and Cab45 oligomers sequester and separate soluble secretory cargo from the bulk flow of proteins in the TGN. However, how this Ca2+ flux into the lumen of the TGN is achieved remains elusive, as the cytosol has a very low steady-state Ca2+ concentration. The TGN forms membrane contact sites (MCS) with the Endoplasmic Reticulum (ER), whereby the close apposition of the two organelles allows the protein-mediated exchange of molecular species such as lipids. Here we show that the TGN export of Cab45 clients requires the integrity of ER-TGN MCS and IP3R-dependent Ca2+ fluxes in the MCS, suggesting Ca2+ transfer between these organelles. Using an MCS-targeted Ca2+ FRET sensor module, we measure the Ca2+ flow in these sites in real-time. These data show for the first time that ER-TGN MCS facilitates Ca2+ transfer required for SPCA1-dependent cargo sorting and export from the TGN, thus solving a fundamental question in cell biology. SummaryThe current study demonstrates that the trafficking of COMP and LyzC relies on Ca2+ flux between the endoplasmic reticulum (ER) and trans-Golgi Network (TGN). This process requires the activity of IP3 receptors, present in ER membranes, and depends on the integrity of the membrane contact site between these two organelles.

cell biology↗

bioRxiv2022

ESCRT-dependent STING degradation curtails steady-state and cGAMP-induced signaling

STING is an intracellular sensor of cyclic di-nucleotides involved in response to pathogen- or self-derived DNA that induces protective immunity, or if dysregulated, autoimmunity. STING trafficking is tightly linked to its activity. We aimed to systematically characterize genes regulating STING trafficking and to define their impact on STING responses. Based on proximity-ligation proteomics and genetic screens, an ESCRT complex containing HGS, VPS37A and UBAP1 was found to be required for STING degradation and signaling shutdown. Analogous to phosphorylated STING creating a platform for IRF3 recruitment, oligomerization-driven STING ubiquitination by UBE2N formed a platform for ESCRT recruitment at the endosome, responsible for STING signaling shutdown. A UBAP1 mutant that underlies human spastic paraplegia and disrupts ESCRT function led to STING-dependent type I IFN responses at the steady-state, defining ESCRT as a homeostatic regulator of STING signaling.

immunology↗

bioRxiv2022

A novel cancer-associated cassette exon in TLN1 alters Talin 1 mechanosensitivity

Talin 1 is the core mechanosensitive adapter protein linking integrins to the cytoskeleton. The TLN1 gene is comprised of 57 exons that encode the 2541 amino acid TLN1 protein. TLN1 was previously considered to be expressed as a single isoform. However, through differential pre-mRNA splicing analysis, we discovered a cancer-enriched, non-annotated 51-nucleotide exon in TLN1 between exons 17 and 18, which we refer to as exon 17b. TLN1 is comprised of an N-terminal FERM domain, linked to 13 force-dependent switch domains, R1-R13. Inclusion of exon 17b results in an in-frame insertion of 17 amino acids immediately after Gln665 in the region between R1-R2 which lowers the force required to open the R1-R2 switches potentially altering downstream mechanotransduction. Biochemical analysis of this isoform revealed enhanced vinculin binding, and cells expressing this variant show altered adhesion dynamics. Finally, we show that the TGF-{beta}/SMAD3 signaling pathway regulates this isoform switch. Future studies will need to consider the balance of these two TLN1 isoforms.

cancer biology↗

bioRxiv2022

Shear stress induced KLF4 is a key determinant in AVM pathogenesis

BackgroundVascular networks form, remodel and mature under the influence of both fluid shear stress (FSS) and soluble factors. For example, FSS synergizes with Bone Morphogenic Protein 9 (BMP9) and BMP10 to promote and maintain vascular stability. Mutation of the BMP receptors ALK1, Endoglin or the downstream effector SMAD4 leads to Hereditary Hemorrhagic Telangiectasia (HHT), characterized by fragile and leaky arterial-venous malformations (AVMs). But how endothelial cells (EC) integrate FSS and BMP signals in normal vascular development and homeostasis, and how mutations give rise to malformations is not well understood. ResultsHere we show that loss of Smad4 in murine ECs increases cells sensitivity to flow and the resulting AVMs are characterized by excessive elongation and polarity against the flow. Smad4 deletion also blocks the anti-proliferative response to high FSS, leading to loss of arterial identity. Our data show that loss of cell cycle arrest leads to loss of arterial identity, which is essential in AVM formation upon Smad4 depletion in ECs. Excessive flow-induced activation of KLF4-PI3K/AKT due to Cyclin dependent Kinase (CDK) activation mediates the aberrant morphological responses to flow triggering AVM formation. ConclusionsOur results show that loss of polarization against the flow is not required for AVM formation upon EC Smad4 depletion. Instead, increased EC proliferation-mediated loss of arterial identity due to flow-induced PI3K/Akt/Cdks hyperactivation and Klf4 over-expression are the main events associated with AVM formation.

molecular biology↗

bioRxiv2021

The IPF fibroblastic focus is an active collagen biosynthesis factory embedded in a distinct extracellular matrix

BackgroundThe Fibroblastic Focus (FF) is the signature lesion of Idiopathic Pulmonary Fibrosis (IPF) where myofibroblasts accumulate and extracellular matrix (ECM) is produced. However, the molecular composition and function of the FF and surrounding tissue remain undefined. MethodsUtilizing laser capture microdissection coupled mass spectrometry (LCM-MS), we interrogated the FF, adjacent mature scar, and adjacent alveoli in 6 IPF specimens plus 6 non-fibrotic alveolar specimens as controls. The data were subject to qualitative and quantitative analysis, and validation by immunohistochemistry. ResultsWe found that the protein signature of IPF alveoli is defined by immune deregulation as the strongest category. The IPF mature scar was classified as end-stage fibrosis whereas the FF contained an overabundance of a distinctive ECM compared to non-fibrotic control. ConclusionSpatial proteomics demonstrated distinct protein compositions in the histologically defined regions of IPF tissue. These data revealed that the FF is the main site of collagen biosynthesis and that the alveoli adjacent to the FF are abnormal. This new and essential information will inform future mechanistic studies on mechanisms of IPF progression.

pathology↗

bioRxiv2021

High-throughput RNA isoform sequencing using programmable cDNA concatenation

Alternative splicing is a core biological process that enables profound and essential diversification of gene function. Short-read RNA sequencing approaches fail to resolve RNA isoforms and therefore primarily enable gene expression measurements - an isoform unaware representation of the transcriptome. Conversely, full-length RNA sequencing using long-read technologies are able to capture complete transcript isoforms, but their utility is deeply constrained due to throughput limitations. Here, we introduce MAS-ISO-seq, a technique for programmably concatenating cDNAs into single molecules optimal for long-read sequencing, boosting the throughput >15 fold to nearly 40 million cDNA reads per run on the Sequel IIe sequencer. We validated unambiguous isoform assignment with MAS-ISO-seq using a synthetic RNA isoform library and applied this approach to single-cell RNA sequencing of tumor-infiltrating T cells. Results demonstrated a >30 fold boosted discovery of differentially spliced genes and robust cell clustering, as well as canonical PTPRC splicing patterns across T cell subpopulations and the concerted expression of the associated hnRNPLL splicing factor. Methods such as MAS-ISO-seq will drive discovery of novel isoforms and the transition from gene expression to transcript isoform expression analyses.

genetics↗

bioRxiv2021

MEKK3-TGFβ crosstalk regulates inward arterial remodeling

Arterial remodeling is an important adaptive mechanism that maintains normal fluid shear stress in a variety of physiologic and pathologic conditions. Inward remodeling, a process that leads to reduction in arterial diameter, plays a critical role in progression of such common diseases as hypertension and atherosclerosis. Yet despite its pathogenic importance, molecular mechanisms controlling inward remodeling remain undefined. Mitogen-activated protein kinases (MAPKs) perform a number of functions ranging from control of proliferation to migration and cell fate transitions. While the MAPK ERK1/2 signaling pathway has been extensively examined in the endothelium, less is known about the role of the MEKK3/ERK5 pathway in vascular remodeling. To better define the role played by this signaling cascade, we studied the effect of endothelial-specific deletion of its key upstream MAP3K, MEKK3, in adult mice. The genes deletion resulted in a gradual inward remodeling of both pulmonary and systematic arteries leading to spontaneous hypertension in both vascular circuits and accelerated progression of atherosclerosis in hyperlipidemic mice. Molecular analysis revealed activation of TGF{beta} signaling both in vitro and in vivo. Endothelial-specific TGF{beta}R1 knockout prevented inward arterial remodeling in MEKK3 endothelial knockout mice. These data point to the unexpected participation of endothelial MEKK3 in regulation of TGF{beta}R1-Smad2/3 signaling and inward arterial remodeling in artery diseases. SignificanceInward remodeling of arteries to reduce lumen diameter is a major factor in disease progression and morbidity in multiple vascular diseases, including hypertension and atherosclerosis. However, molecular mechanisms controlling inward arterial remodeling remain largely undefined. In this study, we identify endothelial MEKK3 as an unexpected regulator of inward remodeling via inhibition of TGF{beta}-Smad2/3 signaling. Genetic deletion of MEKK3 in adult endothelium results in induction of TGF{beta}-Smad2/3 signaling, endothelial-to-mesenchymal transition and inward remodeling in both pulmonary and arterial circuits. The latter process results in pulmonary and systemic hypertension and accelerates atherosclerosis. These results provide a new basis for understanding the inward artery remodeling that leads to reduced blood flow to affected tissues and exacerbates hypertension in vascular disease.

pathology↗

bioRxiv2021

Defective flow-migration coupling causes arteriovenous malformations in hereditary hemorrhagic telangiectasia

BackgroundActivin receptor-like kinase 1 (ACVRL1, hereafter ALK1) is an endothelial transmembrane serine threonine kinase receptor for BMP family ligands that plays a critical role in cardiovascular development and pathology. Loss-of-function mutations in the ALK1 gene cause type 2 hereditary hemorrhagic telangiectasia (HHT), a devastating disorder that leads to arteriovenous malformations (AVMs). Here we show that ALK1 controls endothelial cell polarization against the direction of blood flow and flow-induced endothelial migration from veins through capillaries into arterioles. MethodsUsing Cre lines that recombine in different subsets of arterial, capillary-venous or endothelial tip cells, we showed that capillary-venous Alk1 deletion was sufficient to induce AVM formation in the postnatal retina. ResultsALK1 deletion impaired capillary-venous endothelial cell polarization against the direction of blood flow in vivo and in vitro. Mechanistically, ALK1 deficient cells exhibited increased integrin signaling interaction with VEGFR2, which enhanced downstream YAP/TAZ nuclear translocation. Pharmacological inhibition of integrin or YAP/TAZ signaling rescued flow migration coupling and prevented vascular malformations in Alk1 deficient mice. ConclusionsOur study reveals ALK1 as an essential driver of flow-induced endothelial cell migration and identifies loss of flow-migration coupling as a driver of AVM formation in HHT disease. Integrin-YAP/TAZ signaling blockers are new potential targets to prevent vascular malformations in HHT patients.

cell biology↗

bioRxiv2021

Fibronectin-mediated inflammatory signaling through integrin α5 in vascular remodeling

Adhesion of vascular endothelial cells (ECs) to the underlying basement membrane potently modulates EC inflammatory activation. The normal basement membrane proteins laminin and collagen IV attenuate inflammatory signaling in part through integrin 2{beta}1. In contrast, fibronectin, the provisional matrix protein found in injured, remodeling or inflamed vessels, sensitizes ECs to inflammatory stimuli through integrins 5{beta}1and and v{beta}3. A chimeric integrin in which the cytoplasmic domain of 5 is replaced with that of 2 pairs with {beta}1 and binds fibronectin but signals like 2{beta}1. Here, we examined mice in which integrin 5 is replaced with the 5/2 chimera, using the transverse aortic constriction (TAC) and partial carotid ligation (PCL) models of vessel remodeling. Following TAC and PCL surgery, WT mice showed increased fibronectin deposition and expression of inflammatory markers, which were strongly attenuated in a5/2 mice. 5/2 mice also showed reduced artery wall hypertrophy in the TAC model and diminished inward remodeling in the PCL model. Acute atherosclerosis after PCL in hyperlipidemic ApoE-/- mice on a high fat diet was dramatically decreased in 5/2 mice. These results underscore the key role for integrin 5 signaling in pathological vascular remodeling and support its potential as a therapeutic target.

physiology↗

bioRxiv2020

Latrophilins are essential for endothelial junctional fluid shear stress mechanotransduction

Endothelial cell (EC) responses to fluid shear stress (FSS) are crucial for vascular development, adult physiology and disease. PECAM1 is an important transducer but earlier events remain poorly understood. We therefore investigated heterotrimeric G proteins in FSS sensing. Knockdown (KD) in ECs of single G proteins had little effect but combined depletion of Gi and Gq/11 blocked all known PECAM1-dependent responses. Re-expression of Gi2 and Gq but not Gi1 and Gi3 rescued these effects. Sequence alignment and mutational studies identified that K307 in Gi2 and Gq/11 (Q306 in Gi1/3), determines participation in flow signaling. We developed pull-down assays for measuring G activation and found that this residue, localized to the GPCR interface, determines activation by FSS. We developed a protocol for affinity purification of GPCRs on activated Gs, which identified latrophilins (ADGRLs) as specific upstream interactors for Gi2 and Gq/11. Depletion of latrophilin-2 blocked EC activation of Gi2 and Gq, downstream events in vitro, and flow-dependent vascular morphogenesis in zebrafish embryos. Surprisingly, latrophilin-2 depletion also blocked flow activation of two additional pathways activated at cell-cell junctions, Smad1/5 and Notch1, independently of G proteins. Latrophilins are thus central mediators of junctional shear stress mechanotransduction via G protein-dependent and -independent mechanisms.

molecular biology↗

bioRxiv2019

Laser capture microdissection coupled mass spectrometry (LCM-MS) for spatially resolved analysis of formalin-fixed and stained human lung tissues

Haematoxylin and eosin (H&E) - which respectively stain nuclei blue and other cellular and stromal material pink - are routinely used for clinical diagnosis based on the identification of morphological features. A richer characterization can be achieved by laser capture microdissection coupled to mass spectrometry (LCM-MS), giving an unbiased assay of the proteins that make up the tissue. However, the process of fixing, and H&E staining of tissues is poorly compatible with standard sample preparation methods for mass spectrometry, resulting in low protein yield. Here we describe a microproteomics technique optimized to analyze H&E-stained, formalin-fixed paraffin-embedded (FFPE) tissues. We advance our methodology by combining 3 techniques shown to individually enhance protein yields (heat extraction, physical disruption, and in column digestion) into one optimized pipeline for the analysis of H&E stained FFPE tissues. Micro-dissected morphologically normal human lung alveoli (0.082 mm3) and human lung blood vessels (0.094 mm3) from FFPE fixed section from Idiopathic Pulmonary Fibrosis (IPF) specimens were then subject to comparative proteomics using this methodology. This approach yielded 1252 differentially expressed proteins including 137 extracellular matrix (ECM) proteins. In addition, we offer proof of principal that MS can identify distinct, characteristic proteomic compositions of anatomical features within complex tissues.

biochemistry↗

bioRxiv2019

Fibronectin receptor integrin α5β1 regulates assembly of PP2A complexes through PDE4D: modulation of vascular inflammation and atherosclerosis

Fibronectin in the vascular wall promotes inflammatory activation of the endothelium during vascular remodeling and atherosclerosis. These effects are mediated in part by fibronectin binding to integrin 5, which recruits and activates phosphodiesterase 4D5 (PDE4D5) by inducing its dephosphorylation on an inhibitory site Ser651. Active PDE then hydrolyzes anti-inflammatory cAMP to facilitate inflammatory signaling. To test this model in vivo, we mutated the integrin binding site in PDE4D5 in mice. This mutation reduced endothelial inflammatory activation in athero-prone regions of arteries, and, in a hyperlipidemia model, reduced atherosclerotic plaque size while increasing markers of plaque stability. We then investigated the mechanism of PDE4D5 activation. Proteomics identified the PP2A regulatory subunit B55 as the factor recruiting PP2A to PDE4D5. The B55-PP2A complex localized to adhesions and directly dephosphorylated PDE4D5. This interaction also unexpectedly stabilized the PP2A-B55 complex. The integrin-regulated, pro-atherosclerotic transcription factor Yap is also dephosphorylated and activated through this pathway. PDE4D5 therefore mediates matrix-specific regulation of EC phenotype via an unconventional adapter role, assembling and anchoring a multifunctional PP2A complex with other targets. These results are likely to have widespread consequences for control of cell function by integrins.

cell biology↗

bioRxiv2017

New Insights into the Conformational Activation of Full-Length Integrin

Integrin binding to extracellular matrix proteins is regulated by conformational transitions from closed, low affinity states to open, high affinity states. However, the pathways of integrin conformational activation remain incompletely understood. Here, by combining all-atom molecular dynamics simulation, coarse-graining, heterogeneous elastic network modeling, and experimental ligand binding measurements, we test the effect of integrin {beta} mutations that destabilize the closed conformation. Our results support a \"deadbolt\" model of integrin activation, where extension of the headpiece is not coupled to leg separation, consistent with recent cryo-EM reconstructions of integrin intermediates. Moreover, our results are inconsistent with a \"switchblade-like\" mechanism. The data show that locally correlated atomistic motions are likely responsible for extension of integrin headpiece before separation of transmembrane legs, without persistence of these correlations across the entire protein. By combining modeling and simulation with experiment, this study provides new insight into the structural basis of full-length integrin activation.

biophysics↗