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Lysosomes are important sites for macromolecular degradation, defined by an acidic lumenal pH of ∼4.5. To better understand lysosomal pH, we designed a novel, genetically encoded, fluorescent protein (FP)-based pH biosensor called Fluorescence Indicator REporting pH in Lysosomes (FIRE-pHLy). This biosensor was targeted to lysosomes with lysosomal-associated membrane protein 1 (LAMP1) and reported lumenal pH between 3.5 and 6.0 with monomeric teal fluorescent protein 1 (mTFP1), a bright cyan pH-sensitive FP variant with a pKa of 4.3. Ratiometric quantification was enabled with cytosolically oriented mCherry using high-content quantitative imaging. We expressed FIRE-pHLy in several cellular models and quantified the alkalinizing response to bafilomycin A1, a specific V-ATPase inhibitor. In summary, we have engineered FIRE-pHLy, a specific, robust, and versatile lysosomal pH biosensor, that has broad applications for investigating pH dynamics in aging- and lysosome-related diseases, as well as in lysosome-based drug discovery.

Microglia, the innate immune cells of the brain, are exquisitely sensitive to dynamic changes in the neural environment. Using single cell RNA sequencing of the postnatal somatosensory cortex during topographic remapping, we identified a type I interferon (IFN-I) responsive microglia population that expanded with this developmental stressor. Using the marker gene IFITM3 we found that IFN-I responsive microglia were engulfing whole neurons. Loss of IFN-I signaling (

Cancer cells often co-opt post-transcriptional regulatory mechanisms to achieve pathologic expression of gene networks that drive metastasis. Translational control is a major regulatory hub in oncogenesis, however its effects on cancer progression remain poorly understood. To address this, we used ribosome profiling to compare genome-wide translation efficiencies of poorly and highly metastatic breast cancer cells and patient-derived xenografts. We developed novel regression-based methods to analyze ribosome profiling and alternative polyadenylation data, and identified HNRNPC as a translational controller of a specific mRNA regulon. Mechanistically, HNRNPC, in concert with PABPC4, binds near to poly(A) signals, thereby governing the alternative polyadenylation of a set of mRNAs. We found that HNRNPC and PABPC4 are downregulated in highly metastatic cells, which causes HNRNPC-bound mRNAs to undergo 3’ UTR lengthening and subsequently, translational repression. We showed that modulating HNRNPC expression impacts the metastatic capacity of breast cancer cells in xenograft mouse models. We also found that a small molecule, previously shown to induce a distal-to-proximal poly(A) site switching, counteracts the HNRNPC-PABPC4 driven deregulation of alternative polyadenylation and decreases the metastatic lung colonization by breast cancer cells in vivo.

SummaryMicroglia are brain resident phagocytes that can engulf synaptic components and extracellular matrix as well as whole neurons. However, whether there are unique molecular mechanisms that regulate these distinct phagocytic states is unknown. Here we define a molecularly distinct microglial subset whose function is to engulf neurons in the developing brain. We transcriptomically identified a cluster of Type I interferon (IFN-I) responsive microglia that expanded 20-fold in the postnatal day 5 somatosensory cortex after partial whisker deprivation, a stressor that accelerates neural circuit remodeling.In situ, IFN-I responsive microglia were highly phagocytic and actively engulfed whole neurons. Conditional deletion of IFN-I signaling (Ifnar1fl/fl) in microglia but not neurons resulted in dysmorphic microglia with stalled phagocytosis and an accumulation of neurons with double strand DNA breaks, a marker of cell stress. Conversely, exogenous IFN-I was sufficient to drive neuronal engulfment by microglia and restrict the accumulation of damaged neurons. IFN-I deficient mice had excess excitatory neurons in the developing somatosensory cortex as well as tactile hypersensitivity to whisker stimulation. These data define a molecular mechanism through which microglia engulf neurons during a critical window of brain development. More broadly, they reveal key homeostatic roles of a canonical antiviral signaling pathway in brain development.

Synapse remodeling is essential to encode experiences into neuronal circuits. Here, we define a molecular interaction between neurons and microglia that drives experience-dependent synapse remodeling in the hippocampus. We find that the cytokine interleukin-33 (IL-33) is expressed by adult hippocampal neurons in an experience-dependent manner and defines a neuronal subset primed for synaptic plasticity. Loss of neuronal IL-33 or the microglial IL-33 receptor leads to impaired spine plasticity, reduced newborn neuron integration, and diminished precision of remote fear memories. Memory precision and neuronal IL-33 are decreased in aged mice, and IL-33 gain of function mitigates age-related decreases in spine plasticity. We find that neuronal IL-33 instructs microglial engulfment of the extracellular matrix (ECM) and that its loss leads to impaired ECM engulfment and a concomitant accumulation of ECM proteins in contact with synapses. These data define a cellular mechanism through which microglia regulate experience-dependent synapse remodeling and promote memory consolidation.

Protein phosphorylation is a central mechanism of signal transduction that both positively and negatively regulates protein function. Large-scale studies of the dynamic phosphorylation states of cell signaling systems have been applied extensively in cell lines and whole tissues to reveal critical regulatory networks, and candidate-based evaluations of phosphorylation in rare cell populations have also been informative. However, application of comprehensive profiling technologies to adult stem cell and progenitor populations has been challenging, due in large part to the scarcity of such cells in adult tissues. Here, we combine multicolor flow cytometry with highly efficient 3-dimensional high performance liquid chromatography/mass spectrometry to enable quantitative phosphoproteomic analysis from 200 000 highly purified primary mouse hematopoietic stem and progenitor cells (HSPCs). Using this platform, we identify ARHGAP25 as a novel regulator of HSPC mobilization and demonstrate that ARHGAP25 phosphorylation at serine 363 is an important modulator of its function. Our approach provides a robust platform for large-scale phosphoproteomic analyses performed with limited numbers of rare progenitor cells. Data from our study comprises a new resource for understanding the molecular signaling networks that underlie hematopoietic stem cell mobilization.

Daley and collaborators show that endogenous Lin28b drives erythroid-dominant fetal hematopoiesis and that decreases in Lin28b activate adult granulocyte-predominant hematopoiesis., For appropriate development, tissue and organ system morphogenesis and maturation must occur in synchrony with the overall developmental requirements of the host. Mistiming of such developmental events often results in disease. The hematopoietic system matures from the fetal state, characterized by robust erythrocytic output that supports prenatal growth in the hypoxic intrauterine environment, to the postnatal state wherein granulocytes predominate to provide innate immunity. Regulation of the developmental timing of these myeloerythroid states is not well understood. In this study, we find that expression of the heterochronic factor Lin28b decreases in common myeloid progenitors during hematopoietic maturation to adulthood in mice. This decrease in Lin28b coincides with accumulation of mature let-7 microRNAs, whose biogenesis is regulated by Lin28 proteins. We find that inhibition of let-7 in the adult hematopoietic system recapitulates fetal erythroid-dominant hematopoiesis. Conversely, deletion of Lin28b or ectopic activation of let-7 microRNAs in the fetal state induces a shift toward adult-like myeloid-dominant output. Furthermore, we identify Hmga2 as an effector of this genetic switch. These studies provide the first detailed analysis of the roles of endogenous Lin28b and let-7 in the timing of hematopoietic states during development.

Call to action The developing brain initially makes more synapses than it needs. With further development, excess synapses are pruned away, leaving mature circuits. Synapses can be eliminated by microglia, which engulf and destroy them. Vainchtein et al. found that the microglia are called into action by astrocytes, supportive cells on which neurons rely. Astrocytes near a redundant synapse release the cytokine interleukin-33 (IL-33), which recruits microglia to the site. In mice, disruptions in this process, as caused by deficiency in IL-33, led to too many excitatory synapses and overactive brain circuitry. Science , this issue p. 1269

We propose a new numerical model to describe thrombus formation in cerebral aneurysms. This model combines CFD simulations with a set of bio-mechanical processes identified as being the most important to describe the phenomena at a large space and time scales. The hypotheses of the model are based on in vitro experiments and clinical observations. We document that we can reproduce very well the shape and volume of patient specific thrombus segmented in giant aneurysms.

Mast cells are critical components of the innate immune system and important for host defense, allergy, autoimmunity, tissue regeneration, and tumor progression. Dysregulated mast cell development leads to systemic mastocytosis, a clinically variable but often devastating family of hematologic disorders. Here we report that induced expression of Lin28, a heterochronic gene and pluripotency factor implicated in driving a fetal hematopoietic program, caused mast cell accumulation in adult mice in target organs such as the skin and peritoneal cavity. In vitro assays revealed a skewing of myeloid commitment in LIN28B-expressing hematopoietic progenitors, with increased levels of LIN28B in common myeloid and basophil-mast cell progenitors altering gene expression patterns to favor cell fate choices that enhanced mast cell specification. In addition, LIN28B-induced mast cells appeared phenotypically and functionally immature, and in vitro assays suggested a slowing of mast cell terminal differentiation in the context of LIN28B upregulation. Finally, interrogation of human mast cell leukemia samples revealed upregulation of LIN28B in abnormal mast cells from patients with systemic mastocytosis (SM). This work identifies Lin28 as a novel regulator of innate immune function and a new protein of interest in mast cell disease.

We propose a Proton beam writing (PBW) system with an auto scanning program and stage movement in order to entirely solve the narrow scanning and low design problems of PBW technique. We developed the auto scanning program to transform from 3D CAD (Computer Aided Design) data to scanning pattern data which enables to pattern the complex structures more accurate and much faster. On the other hand, we combined the XY-stage control and electrostatic scanning to expand the fabrication area of our previous system from micrometers range to centimeters range which able to fabricate the whole microstructures network by one exposing time. Our PBW system provides a versatile tool with the unique advantages of proton beam for 3D machining, particularly useful for high-aspect ratio and complicated microstructures.

Cellular functions are largely effected by proteins, but protein-level analysis of hematopoietic stem and progenitor cell (HSPC) functions has historically been challenged by the difficulty of performing comprehensive and robust proteomic studies of rare cell populations. To confront this challenge, we developed a novel nanoscale multidimensional mass spectrometry-based phosphoproteomic platform that allows, for the first time, comprehensive and unbiased analysis of the activated protein circuits in blood stem cells, as assessed by protein phosphorylation status. We used this platform to interrogate the proteomic features responsible for the growth and maintenance of hematopoietic progenitors. Our analysis pipeline is capable of returning 12,000 unique phosphopeptide sequences (corresponding to several thousand proteins) from an input of 400,000 FACS-sorted primary mouse HSPCs. Among these phosphorylated proteins, the novel Rac-GAP Arhgap25 emerged as an important regulator of mobilization in HSPCs. Arhgap25 is phosphorylated upon treatment of HSPCs with a standard cyclophosphamide-GCSF mobilization protocol. Germline deletion of Arhgap25 in mice impairs HSPC egress from the bone marrow, both at rest and after mobilizing stimuli. These findings validate the use of this platform in the discovery of new therapeutic targets in hematopoiesis, and present a clear pathway for identifying novel targets in other rare subsets of human progenitor cells, including leukemia stem cells. Disclosures No relevant conflicts of interest to declare.

Dysregulated mast cell development leads to systemic mastocytosis, a clinically variable but often devastating family of hematologic disorders. Lin28 is a heterochronic gene and pluripotency factor implicated in many types of malignancy, and prior studies suggest that Lin28 expression can restore a fetal hematopoietic program in adult mice. However, the role of Lin28 in hematologic malignancy remains controversial. In our study, we induced expression of Lin28 in adult mice using a doxycycline-responsive transgenic system. Lin28 induction caused marked mast cell accumulation in target organs such as the skin and peritoneal cavity. In vitro assays revealed a skewing of myeloid commitment in Lin28-expressing hematopoietic progenitors, with increased levels of Lin28 in common myeloid progenitors and basophil-mast cell progenitors altering gene expression patterns to favor cell fate choices that enhance mast cell specification. In addition, Lin28-induced mast cells appeared phenotypically and functionally immature, and in vitro assays suggested a slowing of mast cell terminal differentiation in the context of Lin28 upregulation. Finally, interrogation of human mast cell leukemia samples revealed upregulation of LIN28 in abnormal mast cells from patients with aggressive systemic mastocytosis (ASM). This work identifies Lin28 as a novel regulator of innate immune function and a new protein of interest in mast cell disease. Disclosures No relevant conflicts of interest to declare.

Within the mammalian fetal liver (FL) or adult bone marrow (ABM), terminally differentiated neutrophils, monocytes, and red blood cells emerge from a pool of multipotent common myeloid progenitors (CMPs). At steady state, the output of these cells is finally balanced but highly adaptable to the pathophysiologic requirements of the host. In the FL, erythroid output predominates to generate adequate oxygen transport for the metabolic demands of rapid organism growth in a hypoxic intrauterine environment; while granulocytic production comprises a larger balance of myelopoietic output in the ABM to provide innate immunity. The genetic regulators that specify timing of developmental stage-specific granulocytic/erythrocytic output from the myeloid progenitor pool have not been described. Initially defined as regulators of developmental timing in C. elegans, the mammalian Lin28a and Lin28b genes encode RNA binding-proteins capable of contributing to pluripotency induction in fully differentiated cells. Lin28 proteins exert their effects in part through inhibition of the maturation of the let-7 family of microRNAs - of which there are eight forms in mammals - that collectively drive cell terminal differentiation. Therefore, Lin28a/b expression is largely confined to embryonic tissues and is associated with the undifferentiated pluripotent or multipotent state in vivo. Given this role in regulation of multipotent cell function, we assessed the relative roles of theLin28/let-7 axis in developmental timing of myeloid progenitor phenotypes. We first analyzed patterns of let-7 expression during FL and ABM myelopoiesis. We found that several mature let-7 forms accumulated in CMPs during the transition from FL to ABM hematopoiesis (up to 30-fold induction as observed in let-7b in ABM compared to FL), coincident with 5-fold downregulation of the let-7 target and fetal-specific hematopoietic regulator Hmga2, indicating that the presence of mature let-7species may specify ABM-like myelopoiesis. To investigate this concept, we utilized a model of ectopic expression of LIN28B to block maturation of all let-7 species in ABM. Mice with activated LIN28B expression had a FL-like erythroid-dominant hematopoietic phenotype compared to controls (megakaryocyte-erythroid progenitor [MEP]:granulocyte-macrophage progenitor [GMP] ratio 3.9 ± 0.9 vs 0.8 ± 0.1, P = 0.005), along with blunted output of mature neutrophils in the ABM (P = 0.0001). Expression of LIN28B decreased formation of mature let-7 species in CMPs. Taken together, these results suggest that LIN28B expression reprograms ABM CMPs to a FL-like phenotype. To further define the Lin28-let-7 interaction in myelopoiesis, we analyzed the expression patterns of Lin28a and Lin28b as well as precursor let-7 microRNAs and their host genes during myeloid development. We observed up to 30-fold activation of precursor let-7 microRNAs and 4-fold activation of the polycistron encoding let-7b and let-7c2 coincident with 5-fold downregulation of Lin28b in CMPs during the transition from FL to ABM myelopoiesis. These results are consistent with mechanisms regulating levels of mature let-7 microRNAs at both the transcriptional and post-transcriptional level. We next characterized the effects of dysregulation of the Lin28b-let-7 interaction in the FL. Enforced expression of let-7g in mouse embryos drove increased granulocytic output in FL myeloid progenitors in culture (P = 0.001). While ectopic expression of Lin28 has been shown to confer fetal hematopoietic programs in other systems, the impact of loss of Lin28 genes on fetal hematopoiesis has not been reported. Fetal loss of Lin28b skewed the myeloid progenitor pool toward an ABM-like phenotype compared to wild-type animals (MEP:GMP 2.8 ± 0.5 vs. 4± 0.4, P = 0.1), though this phenotype is blunted due to the relative absence of let-7 precursors in the FL compared to the ABM, resulting in deregulated maturation of only a subset of mature let-7 forms. However, Lin28b-null FLs contained increased numbers of mature neutrophils (P = 0.005). Together, these data identify a Lin28/let-7 genetic switch with multiple levels of regulation that controls timing of the developmental myelopoietic phenotype. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow is critical for successful maintenance of hematologic homeostasis, and is important clinically in hematopoietic stem cell transplant and for recovery after chemotherapy. Pharmacologic mobilization of HSPCs induces proliferation and migration of progenitor cells into the peripheral circulation, hastening hematologic recovery. However, there are very few agents that can achieve robust mobilization of HSPCs in vivo, partly because the mechanisms by which HSPCs emigrate from the bone marrow are diverse and complex. Understanding the central pathways responsible for HSPC mobilization promises to facilitate the development of highly effective mobilizing agents. We compared resting bone marrow HSPCs (bmHSPCs) with mobilized HSPCs (mobHSPCs) to identify unique pathways responsible for blood cell mobilization. This type of comparison has been performed at a transcriptional level, but cellular functions are ultimately effected by proteins. Additionally, mRNA abundance correlates poorly with levels of corresponding proteins and is insensitive to changes in functional activity of those proteins. Therefore, we developed a mass spectrometry-based platform to allow for the detection of comprehensive phosphoproteomes from small numbers of cells, enabling for the first time analysis of rare cell populations like HSPCs, which constitute only 0.01-0.1% of all bone marrow hematopoietic cells. Using flow cytometry, stable-isotope labeling, and a novel multidimensional nanoscale phosphoproteomic platform, we have successfully compared the phosphoproteomes of rigorously defined bmHSPCs and mobHSPCs. Analysis of as few as 2x105 flow-sorted cells by 3D RP-SAX-RP-MS/MS coupled to an Orbitrap Velos mass spectrometer resulted in detection of more than 3,600 unique phosphopeptide sequences, corresponding to about 1,000 proteins. Hierarchical clustering and pathway analysis generated priority lists of candidate proteins that are more phosphorylated either in the resting state or in the mobilized state. Of these, we focused on the novel Rac-GAP Arhgap25 as a protein potentially central to the process of HSPC mobilization. Arhgap25 is highly phosphorylated in mobilized HSPCs, but not in resting HSPCs. Although the function of Arhgap25 phosphorylation is unknown, Rac is well-described as necessary for HSPC engraftment and retention in the bone marrow. Thus, absence of Arhgap25 would be anticipated to increase Rac activation and promote bone marrow retention of HSPCs; this suggests that the phosphorylation of Arhgap25 may be inhibitory. Arhgap25 knockout mice were generated; as predicted, Arhgap25-/- mice have significantly higher numbers of Lin-Sca-1+c-Kit+(LSK) cells in the bone marrow, and lower numbers of LSKs in the spleen and peripheral blood, as compared to control mice. The hematopoietic compartment of these mice was further analyzed in both the mobilized state and in a transplant setting. These studies have delineated the role of Arhgap25 in HSC and HPC mobilization and engraftment. We have developed a nanoscale phosphoproteomics platform able to analyze at high resolution small numbers of rare but biologically important cells. This technology has identified many proteins that are activated in HSPC mobilization, and has identified Arhgap25 as a key moderator of HSPC mobility. Additional candidates are undergoing validation. Further studies are underway to take therapeutic advantage of Arhgap25’s role in mobilization. Disclosures: Wagers: BD: Consultancy; iPierian: Consultancy.