Single-Cell Transcriptomics Reveals a Conserved Metaplasia Program in Pancreatic Injury.

BACKGROUND & AIMS: Acinar to ductal metaplasia (ADM) occurs in the pancreas in response to tissue injury and is a potential precursor for adenocarcinoma. The goal of these studies was to define the populations arising from ADM, the associated transcriptional changes, and markers of disease progression. METHODS: Acinar cells were lineage-traced with enhanced yellow fluorescent protein (EYFP) to follow their fate post-injury. Transcripts of more than 13,000 EYFP+ cells were determined using single-cell RNA sequencing (scRNA-seq). Developmental trajectories were generated. Data were compared with gastric metaplasia, KrasG12D-induced neoplasia, and human pancreatitis. Results were confirmed by immunostaining and electron microscopy. KrasG12D was expressed in injury-induced ADM using several inducible Cre drivers. Surgical specimens of chronic pancreatitis from 15 patients were evaluated by immunostaining. RESULTS: scRNA-seq of ADM revealed emergence of a mucin/ductal population resembling gastric pyloric metaplasia. Lineage trajectories suggest that some pyloric metaplasia cells can generate tuft and enteroendocrine cells (EECs). Comparison with KrasG12D-induced ADM identifies populations associated with disease progression. Activation of KrasG12D expression in HNF1B+ or POU2F3+ ADM populations leads to neoplastic transformation and formation of MUC5AC+ gastric-pit-like cells. Human pancreatitis samples also harbor pyloric metaplasia with a similar transcriptional phenotype. CONCLUSIONS: Under conditions of chronic injury, acinar cells undergo a pyloric-type metaplasia to mucinous progenitor-like populations, which seed disparate tuft cell and EEC lineages. ADM-derived EEC subtypes are diverse. KrasG12D expression is sufficient to drive neoplasia when targeted to injury-induced ADM populations and offers an alternative origin for tumorigenesis. This program is conserved in human pancreatitis, providing insight into early events in pancreas diseases.

Supermeres are functional extracellular nanoparticles replete with disease biomarkers and therapeutic targets.

Extracellular vesicles and exomere nanoparticles are under intense investigation as sources of clinically relevant cargo. Here we report the discovery of a distinct extracellular nanoparticle, termed supermere. Supermeres are morphologically distinct from exomeres and display a markedly greater uptake in vivo compared with small extracellular vesicles and exomeres. The protein and RNA composition of supermeres differs from small extracellular vesicles and exomeres. Supermeres are highly enriched with cargo involved in multiple cancers (glycolytic enzymes, TGFBI, miR-1246, MET, GPC1 and AGO2), Alzheimer's disease (APP) and cardiovascular disease (ACE2, ACE and PCSK9). The majority of extracellular RNA is associated with supermeres rather than small extracellular vesicles and exomeres. Cancer-derived supermeres increase lactate secretion, transfer cetuximab resistance and decrease hepatic lipids and glycogen in vivo. This study identifies a distinct functional nanoparticle replete with potential circulating biomarkers and therapeutic targets for a host of human diseases.

Mechanical Counterbalance of Kinesin and Dynein Motors in a Microtubular Network Regulates Cell Mechanics, 3D Architecture, and Mechanosensing.

Microtubules (MTs) and MT motor proteins form active 3D networks made of unstretchable cables with rod-like bending mechanics that provide cells with a dynamically changing structural scaffold. In this study, we report an antagonistic mechanical balance within the dynein-kinesin microtubular motor system. Dynein activity drives the microtubular network inward compaction, while isolated activity of kinesins bundles and expands MTs into giant circular bands that deform the cell cortex into discoids. Furthermore, we show that dyneins recruit MTs to sites of cell adhesion, increasing the topographic contact guidance of cells, while kinesins antagonize it via retraction of MTs from sites of cell adhesion. Actin-to-microtubule translocation of septin-9 enhances kinesin-MT interactions, outbalances the activity of kinesins over that of dyneins, and induces the discoid architecture of cells. These orthogonal mechanisms of MT network reorganization highlight the existence of an intricate mechanical balance between motor activities of kinesins and dyneins that controls cell 3D architecture, mechanics, and cell-microenvironment interactions.

The Hippo pathway regulates density-dependent proliferation of iPSC-derived cardiac myocytes.

Inducing cardiac myocytes to proliferate is considered a potential therapy to target heart disease, however, modulating cardiac myocyte proliferation has proven to be a technical challenge. The Hippo pathway is a kinase signaling cascade that regulates cell proliferation during the growth of the heart. Inhibition of the Hippo pathway increases the activation of the transcription factors YAP/TAZ, which translocate to the nucleus and upregulate transcription of pro-proliferative genes. The Hippo pathway regulates the proliferation of cancer cells, pluripotent stem cells, and epithelial cells through a cell-cell contact-dependent manner, however, it is unclear if cell density-dependent cell proliferation is a consistent feature in cardiac myocytes. Here, we used cultured human iPSC-derived cardiac myocytes (hiCMs) as a model system to investigate this concept. hiCMs have a comparable transcriptome to the immature cardiac myocytes that proliferate during heart development in vivo. Our data indicate that a dense syncytium of hiCMs can regain cell cycle activity and YAP expression and activity when plated sparsely or when density is reduced through wounding. We found that combining two small molecules, XMU-MP-1 and S1P, increased YAP activity and further enhanced proliferation of low-density hiCMs. Importantly, these compounds had no effect on hiCMs within a dense syncytium. These data add to a growing body of literature that link Hippo pathway regulation with cardiac myocyte proliferation and demonstrate that regulation is restricted to cells with reduced contact inhibition.

MCL-1 Inhibition by Selective BH3 Mimetics Disrupts Mitochondrial Dynamics Causing Loss of Viability and Functionality of Human Cardiomyocytes.

MCL-1 is a well-characterized inhibitor of cell death that has also been shown to be a regulator of mitochondrial dynamics in human pluripotent stem cells. We used cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) to uncover whether MCL-1 is crucial for cardiac function and survival. Inhibition of MCL-1 by BH3 mimetics resulted in the disruption of mitochondrial morphology and dynamics as well as disorganization of the actin cytoskeleton. Interfering with MCL-1 function affects the homeostatic proximity of DRP-1 and MCL-1 at the outer mitochondrial membrane, resulting in decreased functionality of hiPSC-CMs. Cardiomyocytes display abnormal cardiac performance even after caspase inhibition, supporting a nonapoptotic activity of MCL-1 in hiPSC-CMs. BH3 mimetics targeting MCL-1 are promising anti-tumor therapeutics. Progression toward using BCL-2 family inhibitors, especially targeting MCL-1, depends on understanding its canonical function not only in preventing apoptosis but also in the maintenance of mitochondrial dynamics and function.

Reassessment of Exosome Composition.

The heterogeneity of small extracellular vesicles and presence of non-vesicular extracellular matter have led to debate about contents and functional properties of exosomes. Here, we employ high-resolution density gradient fractionation and direct immunoaffinity capture to precisely characterize the RNA, DNA, and protein constituents of exosomes and other non-vesicle material. Extracellular RNA, RNA-binding proteins, and other cellular proteins are differentially expressed in exosomes and non-vesicle compartments. Argonaute 1-4, glycolytic enzymes, and cytoskeletal proteins were not detected in exosomes. We identify annexin A1 as a specific marker for microvesicles that are shed directly from the plasma membrane. We further show that small extracellular vesicles are not vehicles of active DNA release. Instead, we propose a new model for active secretion of extracellular DNA through an autophagy- and multivesicular-endosome-dependent but exosome-independent mechanism. This study demonstrates the need for a reassessment of exosome composition and offers a framework for a clearer understanding of extracellular vesicle heterogeneity.

Myosin-18B Promotes the Assembly of Myosin II Stacks for Maturation of Contractile Actomyosin Bundles.

Cell adhesion, morphogenesis, mechanosensing, and muscle contraction rely on contractile actomyosin bundles, where the force is produced through sliding of bipolar myosin II filaments along actin filaments. The assembly of contractile actomyosin bundles involves registered alignment of myosin II filaments and their subsequent fusion into large stacks. However, mechanisms underlying the assembly of myosin II stacks and their physiological functions have remained elusive. Here, we identified myosin-18B, an unconventional myosin, as a stable component of contractile stress fibers. Myosin-18B co-localized with myosin II motor domains in stress fibers and was enriched at the ends of myosin II stacks. Importantly, myosin-18B deletion resulted in drastic defects in the concatenation and persistent association of myosin II filaments with each other and thus led to severely impaired assembly of myosin II stacks. Consequently, lack of myosin-18B resulted in defective maturation of actomyosin bundles from their precursors in osteosarcoma cells. Moreover, myosin-18B knockout cells displayed abnormal morphogenesis, migration, and ability to exert forces to the environment. These results reveal a critical role for myosin-18B in myosin II stack assembly and provide evidence that myosin II stacks are important for a variety of vital processes in cells.

Muscle-specific stress fibers give rise to sarcomeres in cardiomyocytes.

The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 µm long filaments that then 'stitch' together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.

UNC-45a promotes myosin folding and stress fiber assembly.

Contractile actomyosin bundles, stress fibers, are crucial for adhesion, morphogenesis, and mechanosensing in nonmuscle cells. However, the mechanisms by which nonmuscle myosin II (NM-II) is recruited to those structures and assembled into functional bipolar filaments have remained elusive. We report that UNC-45a is a dynamic component of actin stress fibers and functions as a myosin chaperone in vivo. UNC-45a knockout cells display severe defects in stress fiber assembly and consequent abnormalities in cell morphogenesis, polarity, and migration. Experiments combining structured-illumination microscopy, gradient centrifugation, and proteasome inhibition approaches revealed that a large fraction of NM-II and myosin-1c molecules fail to fold in the absence of UNC-45a. The remaining properly folded NM-II molecules display defects in forming functional bipolar filaments. The C-terminal UNC-45/Cro1/She4p domain of UNC-45a is critical for NM-II folding, whereas the N-terminal tetratricopeptide repeat domain contributes to the assembly of functional stress fibers. Thus, UNC-45a promotes generation of contractile actomyosin bundles through synchronized NM-II folding and filament-assembly activities.

Cell volume change through water efflux impacts cell stiffness and stem cell fate.

Cells alter their mechanical properties in response to their local microenvironment; this plays a role in determining cell function and can even influence stem cell fate. Here, we identify a robust and unified relationship between cell stiffness and cell volume. As a cell spreads on a substrate, its volume decreases, while its stiffness concomitantly increases. We find that both cortical and cytoplasmic cell stiffness scale with volume for numerous perturbations, including varying substrate stiffness, cell spread area, and external osmotic pressure. The reduction of cell volume is a result of water efflux, which leads to a corresponding increase in intracellular molecular crowding. Furthermore, we find that changes in cell volume, and hence stiffness, alter stem-cell differentiation, regardless of the method by which these are induced. These observations reveal a surprising, previously unidentified relationship between cell stiffness and cell volume that strongly influences cell biology.

Tunable Surface Repellency Maintains Stemness and Redox Capacity of Human Mesenchymal Stem Cells.

Human bone marrow derived mesenchymal stem cells (hMSCs) hold great promise for regenerative medicine due to their multipotent differentiation capacity and immunomodulatory capabilities. Substantial research has elucidated mechanisms by which extracellular cues regulate hMSC fate decisions, but considerably less work has addressed how material properties can be leveraged to maintain undifferentiated stem cells. Here, we show that synthetic culture substrates designed to exhibit moderate cell-repellency promote high stemness and low oxidative stress-two indicators of naïve, healthy stem cells-in commercial and patient-derived hMSCs. Furthermore, the material-mediated effect on cell behavior can be tuned by altering the molar percentage (mol %) and/or chain length of poly(ethylene glycol) (PEG), the repellant block linked to hydrophobic poly(ε-caprolactone) (PCL) in the copolymer backbone. Nano- and angstrom-scale characterization of the cell-material interface reveals that PEG interrupts the adhesive PCL domains in a chain-length-dependent manner; this prevents hMSCs from forming mature focal adhesions and subsequently promotes cell-cell adhesions that require connexin-43. This study is the first to demonstrate that intrinsic properties of synthetic materials can be tuned to regulate the stemness and redox capacity of hMSCs and provides new insight for designing highly scalable, programmable culture platforms for clinical translation.

Focal adhesions control cleavage furrow shape and spindle tilt during mitosis.

The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critical role in stem cell differentiation and the relative positioning of daughter cells during development. Early observations of adhesive cell lines revealed asymmetry in the shape of the cleavage furrow, where the bottom (i.e., substrate attached side) of the cleavage furrow ingressed less than the top (i.e., unattached side). This data suggested substrate attachment could be regulating furrow ingression. Here we report a population of mitotic focal adhesions (FAs) controls the symmetry of the cleavage furrow. In single HeLa cells, stronger adhesion to the substrate directed less ingression from the bottom of the cell through a pathway including paxillin, focal adhesion kinase (FAK) and vinculin. Cell-cell contacts also direct ingression of the cleavage furrow in coordination with FAs in epithelial cells-MDCK-within monolayers and polarized cysts. In addition, mitotic FAs established 3D orientation of the mitotic spindle and the relative positioning of mother and daughter centrosomes. Therefore, our data reveals mitotic FAs as a key link between mitotic cell shape and spindle orientation, and may have important implications in our understanding stem cell homeostasis and tumorigenesis.

A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells.

How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers' attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.

Bayesian localization microscopy reveals nanoscale podosome dynamics.

We describe a localization microscopy analysis method that is able to extract results in live cells using standard fluorescent proteins and xenon arc lamp illumination. Our Bayesian analysis of the blinking and bleaching (3B analysis) method models the entire dataset simultaneously as being generated by a number of fluorophores that may or may not be emitting light at any given time. The resulting technique allows many overlapping fluorophores in each frame and unifies the analysis of the localization from blinking and bleaching events. By modeling the entire dataset, we were able to use each reappearance of a fluorophore to improve the localization accuracy. The high performance of this technique allowed us to reveal the nanoscale dynamics of podosome formation and dissociation throughout an entire cell with a resolution of 50 nm on a 4-s timescale.

A role for actin arcs in the leading-edge advance of migrating cells.

Epithelial cell migration requires coordination of two actin modules at the leading edge: one in the lamellipodium and one in the lamella. How the two modules connect mechanistically to regulate directed edge motion is not understood. Using live-cell imaging and photoactivation approaches, we demonstrate that the actin network of the lamellipodium evolves spatio-temporally into the lamella. This occurs during the retraction phase of edge motion, when myosin II redistributes to the lamellipodial actin and condenses it into an actin arc parallel to the edge. The new actin arc moves rearward, slowing down at focal adhesions in the lamella. We propose that net edge extension occurs by nascent focal adhesions advancing the site at which new actin arcs slow down and form the base of the next protrusion event. The actin arc thereby serves as a structural element underlying the temporal and spatial connection between the lamellipodium and the lamella during directed cell motion.