Faculty Bibliography

Phagocytosis is an intensely physical process that depends on the mechanical properties of both the phagocytic cell and its chosen target. Here, we employed differentially deformable hydrogel microparticles to examine the role of cargo rigidity in the regulation of phagocytosis by macrophages. Whereas stiff cargos elicited canonical phagocytic cup formation and rapid engulfment, soft cargos induced an architecturally distinct response, characterized by filamentous actin protrusions at the center of the contact site, slower cup advancement, and frequent phagocytic stalling. Using phosphoproteomics, we identified β2 integrins as critical mediators of this mechanically regulated phagocytic switch. Macrophages lacking β2 integrins or their downstream effectors, Talin1 and Vinculin, exhibited specific defects in phagocytic cup architecture and selective suppression of stiff cargo uptake. We conclude that integrin signaling serves as a mechanical checkpoint during phagocytosis to pair cargo rigidity to the appropriate mode of engulfment.

Professional phagocytes like neutrophils and macrophages tightly control what they consume, how much they consume, and when they move after cargo uptake. We show that plasma membrane abundance is a key arbiter of these cellular behaviors. Neutrophils and macrophages lacking the G protein subunit Gβ₄ exhibited profound plasma membrane expansion, accompanied by marked reduction in plasma membrane tension. These biophysical changes promoted the phagocytosis of bacteria, fungus, apoptotic corpses, and cancer cells. We also found that Gβ₄-deficient neutrophils are defective in the normal inhibition of migration following cargo uptake. Sphingolipid synthesis played a central role in these phenotypes by driving plasma membrane accumulation in cells lacking Gβ₄. In Gβ₄ knockout mice, neutrophils not only exhibited enhanced phagocytosis of inhaled fungal conidia in the lung but also increased trafficking of engulfed pathogens to other organs. Together, these results reveal an unexpected, biophysical control mechanism central to myeloid functional decision-making.

Upstream frameshift 1 (UPF1) is an RNA helicase involved in a number of mRNA regulatory processes including nonsense-mediated decay. Mutations in the UPF1 locus that reduce its expression have been associated with adenosquamous carcinoma of the pancreas, a particularly aggressive form of the disease. To determine the effect of Upf1 suppression in a murine model of pancreatic adenocarcinoma, we silenced with shRNA Upf1 in cells derived from an autochthonous tumor in an LSL-Kras^G12D/+; Trp53^R172H/+; Pdx-1^Cre/+ mouse (KPC) and orthotopically implanted these cells in the pancreas of C57BL/6 mice. Tumors derived from Upf1-deficient cells were markedly larger than those derived from control cells, a difference observed only in immunocompetent mice. The immune infiltrate of Upf1-deficient tumors was enriched in myeloid-derived suppressor cells (MDSCs) and depleted of CD8⁺ cells compared to control KPC tumors. Upf1-deficient KPC cells secreted inflammatory cytokines including G-CSF and CXCL2, known to recruit MDSCs. Cytokine secretion from Upf1-deficient KPC cells was induced by increased levels of mitochondrial reactive oxygen species (ROS), which in turn were due to an increase in complex I activity in the electron transport chain. Thus, Upf1 helicase deficiency leads to increased mitochondrial complex I activity which produces ROS that signals for cytokine release that drives immune suppression and enhanced tumor growth.

A key aspect of nutrient absorption is the exquisite division of labour across the length of the small intestine, with individual nutrients taken up at different proximal:distal positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum and ileum. By examining the fine-scale longitudinal transcriptional patterns that span the mouse and human small intestine, we instead identified five domains of nutrient absorption that mount distinct responses to dietary changes, and three regional stem cell populations. Molecular domain identity can be detected with machine learning, which provides a systematic method to computationally identify intestinal domains in mice. We generated a predictive model of transcriptional control of domain identity and validated the roles of Ppar-δ and Cdx1 in patterning lipid metabolism-associated genes. These findings represent a foundational framework for the zonation of absorption across the mammalian small intestine.

Ectodermal appendages, such as the mammary gland (MG), are thought to have evolved from hair-associated apocrine glands to serve the function of milk secretion. Through the directed differentiation of mouse embryonic stem cells (mESCs), here, we report the generation of multilineage ESC-derived mammary organoids (MEMOs). We adapted the skin organoid model, inducing the dermal mesenchyme to transform into mammary-specific mesenchyme via the sequential activation of Bone Morphogenetic Protein 4 (BMP4) and Parathyroid Hormone-related Protein (PTHrP) and inhibition of hedgehog (HH) signaling. Using single-cell RNA sequencing, we identified gene expression profiles that demonstrate the presence of mammary-specific epithelial cells, fibroblasts, and adipocytes. MEMOs undergo ductal morphogenesis in Matrigel and can reconstitute the MG in vivo. Further, we demonstrate that the loss of function in placode regulators LEF1 and TBX3 in mESCs results in impaired skin and MEMO generation. In summary, our MEMO model is a robust tool for studying the development of ectodermal appendages, and it provides a foundation for regenerative medicine and disease modeling.

We describe a modular bond-centric approach to protein nanomaterial design inspired by the rich diversity of chemical structures that can be generated from the small number of atomic valencies and bonding interactions. We design protein building blocks with regular coordination geometries and bonding interactions that enable the assembly of a wide variety of closed and opened nanomaterials using simple geometrical principles. Experimental characterization confirms successful formation of more than twenty multi-component polyhedral protein cages, 2D arrays, and 3D protein lattices, with a high (10-50 %) success rate and electron microscopy data closely matching the corresponding design models. Because of the modularity, individual building blocks can assemble with different partners to generate distinct regular assemblies, resulting in an economy of parts and enabling the construction of reconfigurable systems.

The morphology of cells in vivo can arise from a variety of mechanisms. In the Caenorhabditis elegans hermaphrodite gonad, the distal tip cell (DTC) elaborates into a complex plexus over a relatively short developmental time period, but the mechanisms underlying this change in cell morphology are not well defined. We correlated the time of DTC elaboration with the L4-to-adult molt, but ruled out a relevant heterochronic pathway as a cue for DTC elaboration. Instead, we found that the timing of gonad elongation and aspects of underlying germline flux influence DTC elaboration. We propose a 'hitch and tow' aspect of organ-level dynamics that contributes to cellular morphogenesis, whereby germline flux drags the flexible DTC cell cortex away from its stationary cell body. More broadly, we speculate that this mechanism may contribute to cell shape changes in other contexts with implications for development and disease.

BACKGROUND/UNASSIGNED:A deeper understanding of acute rejection in vascularized composite allotransplantation is paramount for expanding its utility and longevity. There remains a need to develop more precise and accurate tools for diagnosis and prognosis of these allografts, as well as alternatives to traditional immunosuppressive regimens. METHODS/UNASSIGNED:Twenty-seven skin biopsies collected from 3 vascularized composite allotransplantation recipients, consisting of face and hand transplants, were evaluated by histology, immunohistochemistry staining, and gene expression profiling. RESULTS/UNASSIGNED:significantly predicted inflammation specific to vascularized composite allografts that required therapeutic intervention. CONCLUSIONS/UNASSIGNED:The mechanism of vascularized composite allograft-specific inflammation and rejection appears to be conserved across different patients and skin on different anatomical sites. A concise gene signature can be utilized to ascertain graft status along with a continuous scale, providing valuable diagnostic and prognostic information to supplement current gold standards of graft evaluation.

The severity of aortic stenosis (AS) is associated with acquired von Willebrand syndrome (AVWS) and gastrointestinal bleeding, leading to anemia (Heyde's syndrome). We investigated how anemia is linked with AS and AVWS using the LA100 mouse model and patients with AS. Induction of anemia in LA100 mice increased transforming growth factor (TGF)-β1 activation, AVWS, and AS progression. Patients age >75 years with severe AS had higher plasma TGF-β1 levels and more severe anemia than AS patients age <75 years, and there was a correlation between TGF-β1 and anemia. These data are compatible with the hypothesis that the blood loss anemia of Heyde's syndrome contributes to AS progression via WSS-induced activation of platelet TGF-β1 and additional gastrointestinal bleeding via WSS-induced AVWS.