Biology subjects

Stolz, D. B.

Publications and source records attributed to Stolz, D. B..

14 recordsLinked to original sources

bioRxiv2026

Sequential- vs. density gradient- centrifugation for the isolation of mitochondria-containing extracellular vesicles

A subset of extracellular vehicles (EVs) with particle diameters >200 nm, large vesicles (lEVs) contain mitochondria that increase recipient cell bioenergetics. To date, sequential centrifugation (SC) is the most reported protocol to separate lEVs from the smaller EVs (<200 nm)/exosomes. We have previously demonstrated that lEVs derived from brain endothelial cells (BECs) using the standard SC method transferred their innate mitochondria to recipient BECs, increased recipient BEC bioenergetics, reduced brain infarct volume, and improved behavioral outcomes in a mouse model of transient ischemic stroke. Despite their promising therapeutic activity, SC-isolated lEVs are likely a mixture of mitochondria-containing lEVs and non-mitochondria-containing lEVs. We hypothesized that subsequent purification of SC-isolated lEVs using density-gradient centrifugation (DGC) may yield a purer sample of mitochondria-containing lEVs. We established a DGC protocol to purify lEVs. In this pilot study, lEVs isolated using SC and DGC protocols were compared to determine their physicochemical characteristics and their effects on recipient BEC bioenergetics. SC-lEVs and DGC-lEVs both significantly restored ATP levels in OGD-injured BECs with no difference between groups. However, a Seahorse mitochondrial function assay revealed distinct functional effects: SC-lEVs did not significantly alter respiration, whereas DGC-lEVs induced a dose-dependent increase in oxygen consumption rate, indicating enhanced oxidative phosphorylation. These findings demonstrate that DGC purification yields a more mitochondria-enriched and functionally potent lEV preparation with an enhanced capacity to restore oxidative phosphorylation in ischemic BECs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/732469v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@102b68borg.highwire.dtl.DTLVardef@11e7df1org.highwire.dtl.DTLVardef@de7161org.highwire.dtl.DTLVardef@1e5d64c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Mitochondria-containing large extracellular vesicles target mouse motor neurons upon intramuscular injection

Amyotrophic Lateral Sclerosis (ALS) is a neurological disorder that causes progressive degeneration of motor neurons. Mitochondrial dysfunction accelerates neurodegeneration aggravating the severity of ALS. We hypothesized that increasing the mitochondrial function of motor neurons may promote neuronal survival. Therefore, we investigated the potential of neuron-derived mitochondria containing extracellular vehicles (EVs) as a novel therapeutic approach for ALS using differentiated NSC-34 cells as a surrogate for neurons. Neuron derived-large EVs (lEVs) but not small EVs (sEVs) contained mitochondria. However, we observed increased cell viability and oxygen consumption rates in heat-stressed neurons treated with both sEVs and lEVs suggesting improved mitochondrial function in recipient neurons. The increased oxygen consumption rates in sEV-treated heat-stressed neurons was accompanied by a greater proton leak compared to lEV treatment. The greater proton leak observed with sEVs likely suggests a lower efficiency of oxidative phosphorylation compared to that achieved by cells treated with mitochondria-containing lEVs. These findings suggest that mitochondrial components present in sEVs, such as proteins and mitochondrial DNA, may too contribute to improving cellular respiration. Furthermore, we have demonstrated that lEV mitochondria are transported into the lumbar spinal cord motor neurons following intramuscular injection in C57BL/6 mice in a EV dose-dependent manner. Collectively, for the first time, we have demonstrated the therapeutic effects of neuronal EVs in recipient heat-stressed neurons and the delivery of lEV mitochondria to spinal cord motor neurons in vivo without any EV surface modifications for neuronal targeting. Further studies will determine the therapeutic efficacy of mitochondria-containing EVs in the SOD1G93A transgenic mouse model of ALS. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=166 HEIGHT=200 SRC="FIGDIR/small/675842v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@af590aorg.highwire.dtl.DTLVardef@1fed11dorg.highwire.dtl.DTLVardef@52d339org.highwire.dtl.DTLVardef@1a97182_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Genetic regulation and targeted reversal of lysosomal dysfunction and inflammatory sterol metabolism in pulmonary arterial hypertension

Vascular inflammation critically regulates endothelial cell (EC) pathophenotypes, particularly in pulmonary arterial hypertension (PAH). Dysregulation of lysosomal activity and cholesterol metabolism have known inflammatory roles in disease, but their relevance to PAH is unclear. In human pulmonary arterial ECs and in PAH, we found that inflammatory cytokine induction of the nuclear receptor coactivator 7 (NCOA7) both preserved lysosomal acidification and served as a homeostatic brake to constrain EC immunoactivation. Conversely, NCOA7 deficiency promoted lysosomal dysfunction and proinflammatory oxysterol/bile acid generation that, in turn, contributed to EC pathophenotypes. In vivo, mice deficient for Ncoa7 or exposed to the inflammatory bile acid 7-hydroxy-3-oxo-4-cholestenoic acid (7HOCA) displayed worsened PAH. Emphasizing this mechanism in human PAH, an unbiased, metabolome-wide association study (N=2,756) identified a plasma signature of the same NCOA7-dependent oxysterols/bile acids associated with PAH mortality (P<1.1x10-6). Supporting a genetic predisposition to NCOA7 deficiency, in genome-edited, stem cell-derived ECs, the common variant intronic SNP rs11154337 in NCOA7 regulated NCOA7 expression, lysosomal activity, oxysterol/bile acid production, and EC immunoactivation. Correspondingly, SNP rs11154337 was associated with PAH severity via six-minute walk distance and mortality in discovery (N=93, P=0.0250; HR=0.44, 95% CI [0.21-0.90]) and validation (N=630, P=2x10-4; HR=0.49, 95% CI [0.34-0.71]) cohorts. Finally, utilizing computational modeling of small molecule binding to NCOA7, we predicted and synthesized a novel activator of NCOA7 that prevented EC immunoactivation and reversed indices of rodent PAH. In summary, we have established a genetic and metabolic paradigm and a novel therapeutic agent that links lysosomal biology as well as oxysterol and bile acid processes to EC inflammation and PAH pathobiology. This paradigm carries broad implications for diagnostic and therapeutic development in PAH and in other conditions dependent upon acquired and innate immune regulation of vascular disease. One Sentence SummaryPulmonary arterial hypertension pathophenotypes arise from allele-specific NCOA7 regulation of lysosome function and inflammatory oxysterol generation, as demonstrated by genomic and metabolomic association studies coupled with genetic and pharmacologic mechanistic evidence. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/582142v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@896f6forg.highwire.dtl.DTLVardef@f31c2eorg.highwire.dtl.DTLVardef@bf46dcorg.highwire.dtl.DTLVardef@1c55686_HPS_FORMAT_FIGEXP M_FIG Graphical abstract. C_FIG

physiology↗

bioRxiv2024

Mitochondria-containing extracellular vesicles from mouse vs. human brain endothelial cells for ischemic stroke therapy

Ischemic stroke-induced mitochondrial dysfunction in the blood-brain barrier-forming brain endothelial cells (BECs) results in long-term neurological dysfunction post-stroke. We previously data from a pilot study where intravenous administration of human BEC (hBEC)-derived mitochondria-containing extracellular vesicles (EVs) showed a potential efficacy signal in a mouse middle cerebral artery occlusion (MCAo) model of stroke. We hypothesized that EVs harvested from donor species homologous to the recipient species (e.g., mouse) may improve therapeutic efficacy, and therefore, use of mouse BEC (mBEC)-derived EVs may improve post-stroke outcomes in MCAo mice. We investigated potential differences in the mitochondria transfer of EVs derived from the same species as the recipient cell (mBEC-EVs and recipient mBECs or hBECs-EVs and recipient hBECs) vs. cross-species EVs and recipient cells (mBEC-EVs and recipient hBECs or vice versa). Our results showed that while both hBEC- and mBEC-EVs transferred EV mitochondria, mBEC-EVs outperformed hBEC-EVs in increasing ATP levels and improved recipient mBEC mitochondrial function via increasing oxygen consumption rates. mBEC-EVs significantly reduced brain infarct volume and neurological deficit scores compared to vehicle-injected MCAo mice. The superior therapeutic efficacy of mBEC-EVs in a mouse MCAo stroke support the continued use of mBEC-EVs to optimize the therapeutic potential of mitochondria-containing EVs in preclinical mouse models. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/575903v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d1deb4org.highwire.dtl.DTLVardef@74157corg.highwire.dtl.DTLVardef@12eb991org.highwire.dtl.DTLVardef@990a47_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Modelling Human Post-Implantation Development via Extra-Embryonic Niche Engineering

Implantation of the human embryo commences a critical developmental stage that comprises profound morphogenetic alteration of embryonic and extra-embryonic tissues, axis formation, and gastrulation events. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons. Additionally, human stem cell models of early post-implantation development with both embryonic and extra-embryonic tissue morphogenesis are lacking. Here, we present iDiscoid, produced from human induced pluripotent stem cells via an engineered a synthetic gene circuit. iDiscoids exhibit reciprocal co-development of human embryonic tissue and engineered extra-embryonic niche in a model of human post-implantation. They exhibit unanticipated self-organization and tissue boundary formation that recapitulates yolk sac-like tissue specification with extra-embryonic mesoderm and hematopoietic characteristics, the formation of bilaminar disc-like embryonic morphology, the development of an amniotic-like cavity, and acquisition of an anterior-like hypoblast pole and posterior-like axis. iDiscoids offer an easy-to-use, high-throughput, reproducible, and scalable platform to probe multifaceted aspects of human early post-implantation development. Thus, they have the potential to provide a tractable human model for drug testing, developmental toxicology, and disease modeling.

developmental biology↗

bioRxiv2023

In vivo evolution of a Klebsiella pneumoniae capsule defect promotes complement-mediated opsono-phagocytosis and persistence during recurrent infection

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) bloodstream infections rarely overwhelm the host but are associated with high mortality. The complement system is a key host defense against bloodstream infection. However, there are varying reports of serum resistance among KPC-Kp isolates. We assessed growth of 59 KPC-Kp clinical isolates in human serum and found increased resistance in 16/59 (27%). We identified five genetically-related bloodstream isolates with varying serum resistance profiles collected from a single patient during an extended hospitalization marked by recurrent KPC-Kp bloodstream infections. We noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ, that emerged during infection was associated with decreased polysaccharide capsule content, and resistance to complement-mediated killing. Surprisingly, disruption of wcaJ increased deposition of complement proteins on the microbial surface compared to the wild-type strain and led to increased complement-mediated opsono-phagocytosis in human whole blood. Disabling opsono-phagocytosis in the airspaces of mice impaired in vivo control of the wcaJ loss-of-function mutant in an acute lung infection model. These findings describe the rise of a capsular mutation that promotes KPC-Kp persistence within the host by enabling co-existence of increased bloodstream fitness and reduced tissue virulence. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/542722v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@155736eorg.highwire.dtl.DTLVardef@10d47c0org.highwire.dtl.DTLVardef@e1ab83org.highwire.dtl.DTLVardef@1c33131_HPS_FORMAT_FIGEXP M_FIG Graphical abstract Created with BioRender.com C_FIG

microbiology↗

bioRxiv2023

Genetically engineering endothelial niche in human kidney organoids enables multilineage maturation, vascularization, and de novo cell types

Vascularization plays a critical role in organ maturation and cell type development. Drug discovery, organ mimicry, and ultimately transplantation in a clinical setting thereby hinges on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcome this hurdle by combining an inducible ETS translocation variant 2 (ETV2) human induced pluripotent stem cell (iPSC) line, which directs endothelial fate, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive vascularization by endothelial cells with an identity most closely related to endogenous kidney endothelia. Vascularized organoids also show increased maturation of nephron structures including more mature podocytes with improved marker expression, foot process interdigitation, an associated fenestrated endothelium, and the presence of renin+ cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Furthermore, this approach is orthogonal to native tissue differentiation paths, hence readily adaptable to other organoid systems and thus has the potential for a broad impact on basic and translational organoid studies. Translational StatementDeveloping therapies for patients with kidney diseases relies on a morphologically and physiologically representative in vitro model. Human kidney organoids are an attractive model to recapitulate kidney physiology, however, they are limited by the absence of a vascular network and mature cell populations. In this work, we have generated a genetically inducible endothelial niche that, when combined with an established kidney organoid protocol, induces the maturation of a robust endothelial cell network, induces a more mature podocyte population, and induces the emergence a functional renin population. This advance significantly increases the clinical relevance of human kidney organoids for etiological studies of kidney disease and future regenerative medicine strategies. Graphical AbstractGenetically engineered endothelial niche induces mature cell populations in human kidney organoids O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/542848v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1b129d4org.highwire.dtl.DTLVardef@313ae3org.highwire.dtl.DTLVardef@111c91org.highwire.dtl.DTLVardef@16a8d45_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

A genome-edited β-cell model of Prader-Willi syndrome reveals chronic deficits in endoplasmic reticulum chaperones and insulin secretion

Prader-Willi syndrome (PWS) is a multisystem disorder with neurobehavioral, metabolic, and hormonal phenotypes, caused by loss of expression of a paternally-expressed imprinted gene cluster. Prior evidence from a PWS mouse model identified abnormal pancreatic islet development with retention of aged insulin and deficient insulin secretion. To determine the collective roles of PWS genes in {beta}-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal (control) or active, paternal PWS-alleles. PWS {beta}-cells demonstrated a significant cell-autonomous reduction in basal and glucose-stimulated insulin secretion. Further, proteomic analyses revealed reduced levels of cellular and secreted hormones, including all insulin peptides and amylin, concomitant with reduction of at least ten endoplasmic reticulum (ER) chaperones, including GRP78 and GRP94. Critically, transcriptomic studies demonstrated that the broad reduction of ER chaperones originated from transcriptional downregulation without corresponding changes for Ins1 and Ins2. In contrast to the dosage compensation previously seen for ER chaperones in Grp78 or Grp94 gene knockouts or knockdown, compensation is precluded by the stress-independent deficiency of ER chaperones in PWS {beta}-cells. Consistent with reduced ER chaperones levels, PWS INS-1 {beta}-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. These findings suggest that a chronic shortage of ER chaperones in PWS {beta}-cells leads to a deficiency of protein folding and/or delay in ER transit of insulin and other cargo. In summary, our results illuminate the pathophysiological basis of pancreatic {beta}-cell hormone deficits in PWS, with evolutionary implications for the multigenic PWS-domain, and indicate that PWS-imprinted genes coordinate concerted regulation of ER chaperone biosynthesis and {beta}-cell secretory pathway function.

genetics↗

bioRxiv2021

Extracellular Vesicles Deliver Mitochondria and HSP27 Protein to Protect the Blood-Brain Barrier

Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial load of endothelial cell-derived extracellular vesicles (EVs) and utilized mixtures of EV/exogenous heat shock protein 27 (HSP27) as a one-two punch strategy to increase BEC survival (via EV mitochondria) and preserve their tight junction integrity (via HSP27 effects). We demonstrated that the medium-to-large (m/lEV) but not small EVs (sEV) transferred their mitochondrial load, which subsequently colocalized with the mitochondrial network of the recipient primary human BECs. BECs treated with m/lEVs increased relative ATP levels and displayed superior mitochondrial function. Importantly, m/lEVs isolated from oligomycin (mitochondrial complex V inhibitor) or rotenone (mitochondrial complex I inhibitor)-exposed BECs (RTN-m/lEVs or OGM-m/lEVs) did not increase BECs ATP levels compared to naive m/lEVs. In contrast, RTN-sEV and OGM-sEV functionality in increasing cellular ATP levels was minimally impacted in comparison to naive sEVs. Intravenously administered m/lEVs showed a reduction in brain infarct sizes compared to vehicle-injected mice in a mouse middle cerebral artery occlusion model of ischemic stroke. We formulated binary mixtures of human recombinant HSP27 protein with EVs: EV/HSP27 and ternary mixtures of HSP27 and EV with cationic polymer poly (ethylene glycol)-b-poly (diethyltriamine): (PEG-DET/HSP27)/EV. (PEG-DET/HSP27)/EV and EV/HSP27 mixtures decreased the paracellular permeability of small and large molecular mass fluorescent tracers in oxygen glucose-deprived primary human BECs. This one-two-punch approach to increase BEC metabolic function and tight junction integrity is a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/466491v5_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@dc5493org.highwire.dtl.DTLVardef@134ad7forg.highwire.dtl.DTLVardef@16a8929org.highwire.dtl.DTLVardef@15315bc_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMedium-to-large extracellular vesicles (m/lEVs), not small EVs contain mitochondria C_LIO_LIm/lEVs increased ATP and mitochondrial function in brain endothelial cells (BECs) C_LIO_LIm/lEVs from oligomycin-exposed BECs did not increase recipient BEC ATP levels C_LIO_LIIntravenously injected m/lEVs reduced brain infarct sizes in a mouse stroke model C_LIO_LIEV/HSP27 mixtures reduced small and large dextran molecule permeability across BECs C_LI

bioengineering↗

bioRxiv2021

ANALYSIS OF CHD-7 DEFECTIVE DAUER NEMATODES IMPLICATES COLLAGEN MISREGULATION IN CHARGE SYNDROME FEATURES.

CHARGE syndrome is a complex developmental disorder caused by mutations in the chromodomain helicase DNA-binding protein7 (CHD7) and characterized by retarded growth and malformations in the heart and nervous system. Despite the public health relevance of this disorder, relevant targets of CHD7 that relate to disease pathology are still poorly understood. Here we report that chd-7, the nematode ortholog of Chd7, is required for dauer morphogenesis, lifespan determination, and stress response. Consistent with our discoveries, we found chd-7 to be allelic to scd-3, a previously identified dauer suppressor from the TGF-{beta} pathway. Notably, DAF-12 promoted chd-7 expression, which is necessary to repress daf-9 for execution of the dauer program. Transcriptomic analysis comparing chd-7-defective and normal dauers showed enrichment of collagen genes, consistent with a conserved role for the TGF-{beta} pathway in formation of the extracellular matrix. To validate a conserved function for chd-7 in vertebrates, we used Xenopus laevis embryos, an established model to study craniofacial development. Morpholino mediated knockdown of Chd7 led to a reduction in col2a1 mRNA levels. Both embryonic lethality and craniofacial defects in Chd7-depleted tadpoles were partially rescued by over-expression of col2a1 mRNA. We suggest that pathogenic features of CHARGE syndrome caused by Chd7 mutations, such as craniofacial malformations, result from the reduction of collagen levels, implying that the extracellular matrix might represent a critical target of Chd7 in CHARGE development. SIGNIFICANCE STATEMENTCHARGE Syndrome is a complex developmental disorder caused by mutations in the chromodomain helicase DNA-binding protein-7 (CHD7). Unfortunately, the cellular events that lead to CHARGE syndrome are still poorly understood. In C. elegans, we identified chd-7 in a screen for suppressors of dauer formation, an alternative larval stage that develops in response to sensory signals of a harsh environment. We found that chd-7 regulates expression of collagens, which constitute the worms cuticle, a specialized extracellular matrix. In frogs embryos, we show that Chd7 inhibition leads to poor Col2a1, which is necessary and sufficient to exhibit CHARGE features. These studies establish C. elegans as an amenable animal model to study the etiology of the developmental defects associated with pathogenic Chd7.

genetics↗

bioRxiv2021

Recapitulate Human Cardio-pulmonary Co-development Using Simultaneous Multilineage Differentiation of Pluripotent Stem Cells

The extensive crosstalk between the developing heart and lung is pivotal for their proper morphogenesis and maturation. However, there remains a lack of model systems for investigating the critical cardio-pulmonary mutual interaction during human embryogenesis. Here, we reported a novel stepwise strategy for directing simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages within a single differentiation of human pluripotent stem cells (hPSCs) via temporal specific tuning of WNT and TGF-{beta} signaling in the absence of exogenous growth factors. Using 3D suspension culture, we established concentric cardio-pulmonary micro-Tissues (Ts), and observed expedited alveolar maturation in the presence of cardiac accompany. Upon withdrawal of WNT agonist, the cardiac and pulmonary components within each dual-lineage T effectively segregated from each other with concurrent initiation of cardiac contraction. We expect our multilineage differentiation model to offer an experimentally tractable system for investigating human cardio-pulmonary interplay and tissue boundary formation during embryogenesis.

developmental biology↗

bioRxiv2019

Vinculin anchors contractile actin to the cardiomyocyte adherens junction

The adherens junction (AJ) couples the actin cytoskeletons of neighboring cells to allow mechanical integration and tissue organization. The physiological demands of intercellular adhesion require that the AJ be responsive to dynamic changes in force while maintaining mechanical load. These demands are tested in the heart, where cardiomyocyte AJs must withstand repeated cycles of actomyosin-mediated contractile force. Here we show that force-responsive cardiomyocyte AJs recruit actin-binding ligands to selectively couple actin networks and promote contact maturation. We employed a panel of N-cadherin-E-catenin fusion proteins to rebuild AJs with specific actin linkages in N-cadherin-null cardiomyocytes. In this system, vinculin recruitment was required to rescue myofibril integration and desmosome assembly at nascent contacts. In contrast, loss of vinculin disrupted junction morphology and blocked myofibril integration. Our results identify vinculin as a critical link to contractile actomyosin and offer insight to how actin integration at the AJ is regulated to provide mechanical stability and cellular organization.

cell biology↗

bioRxiv2018

The N-cadherin interactome in primary cardiomyocytes as defined by quantitative proximity proteomics.

The junctional complexes that couple cardiomyocytes must transmit the mechanical forces of contraction while maintaining adhesive homeostasis. The adherens junction (AJ) connects the actomyosin networks of neighboring cardiomyocytes and is required for proper heart function. Yet little is known about the molecular composition of the cardiomyocyte AJ or how it is organized to function under mechanical load. Here we define the architecture, dynamics and proteome of the cardiomyocyte AJ. Mouse neonatal cardiomyocytes assemble stable AJs along intercellular contacts with organizational and structural hallmarks similar to mature contacts. We combine quantitative mass spectrometry with proximity labeling to identify the N-cadherin (CDH2) interactome. We define over 350 proteins in this interactome, nearly 200 of which are unique to CDH2 and not part of the E-cadherin (CDH1) interactome. CDH2-specific interactors are comprised primarily of adaptor and adhesion proteins that promote junction specialization. Finally, we find evidence of dynamic interplay between AJ and Z-disc proteins. Together, our results provide novel insight into the cardiomyocyte AJ and provide a proteomic atlas for defining the molecular complexes that regulate cardiomyocyte intercellular adhesion.\n\nSummary StatementProximity proteomics reveals a specific and specialized N-cadherin (CDH2) interactome along the cell-cell contacts of primary cardiomyocytes.

cell biology↗

bioRxiv2018

An unconventional myosin, myosin 1d regulates Kupffer’s vesicle morphogenesis and laterality

INTRODUCTION INTRODUCTION RESULTS MATERIALS AND METHODS AUTHOR CONTRIBUTIONS REFERENCES Establishing left-right (LR) asymmetry is a fundamental process essential for arrangement of visceral organs during development. In vertebrates, motile cilia driven fluid flow in the left-right organizer (LRO) is essential for initiating symmetry breaking event1-3. Without a definite LRO structure in invertebrates, LR asymmetry is initiated at a cellular level by actin-myosin driven chirality4, 5. In Drosophila, myosin1D drives tissue-specific chirality in hind-gut looping6, 7. Here, we show that myosin 1d (myo1d) is essential for establishing LR asymmetry in zebrafish. Using super-r ...

developmental biology↗