Gap junctions in Turing-type periodic feather pattern formation.

Periodic patterning requires coordinated cell-cell interactions at the tissue level. Turing showed, using mathematical modeling, how spatial patterns could arise from the reactions of a diffusive activator-inhibitor pair in an initially homogeneous 2D field. Most activators and inhibitors studied in biological systems are proteins, and the roles of cell-cell interaction, ions, bioelectricity, etc. are only now being identified. Gap junctions (GJs) mediate direct exchanges of ions or small molecules between cells, enabling rapid long-distance communications in a cell collective. They are therefore good candidates for propagating nonprotein-based patterning signals that may act according to the Turing principles. Here, we explore the possible roles of GJs in Turing-type patterning using feather pattern formation as a model. We found 7 of the 12 investigated GJ isoforms are highly dynamically expressed in the developing chicken skin. In ovo functional perturbations of the GJ isoform, connexin 30, by siRNA and the dominant-negative mutant applied before placode development led to disrupted primary feather bud formation. Interestingly, inhibition of gap junctional intercellular communication (GJIC) in the ex vivo skin explant culture allowed the sequential emergence of new feather buds at specific spatial locations relative to the existing primary buds. The results suggest that GJIC may facilitate the propagation of long-distance inhibitory signals. Thus, inhibition of GJs may stimulate Turing-type periodic feather pattern formation during chick skin development, and the removal of GJ activity would enable the emergence of new feather buds if the local environment were competent and the threshold to form buds was reached. We further propose Turing-based computational simulations that can predict the sequential appearance of these ectopic buds. Our models demonstrate how a Turing activator-inhibitor system can continue to generate patterns in the competent morphogenetic field when the level of intercellular communication at the tissue scale is modulated.

Gate-Tunable Proximity Effects in Graphene on Layered Magnetic Insulators.

The extreme versatility of van der Waals materials originates from their ability to exhibit new electronic properties when assembled in close proximity to dissimilar crystals. For example, although graphene is inherently nonmagnetic, recent work has reported a magnetic proximity effect in graphene interfaced with magnetic substrates, potentially enabling a pathway toward achieving a high-temperature quantum anomalous Hall effect. Here, we investigate heterostructures of graphene and chromium trihalide magnetic insulators (CrI3, CrBr3, and CrCl3). Surprisingly, we are unable to detect a magnetic exchange field in the graphene but instead discover proximity effects featuring unprecedented gate tunability. The graphene becomes highly hole-doped due to charge transfer from the neighboring magnetic insulator and further exhibits a variety of atypical gate-dependent transport features. The charge transfer can additionally be altered upon switching the magnetic states of the nearest CrI3 layers. Our results provide a roadmap for exploiting proximity effects arising in graphene coupled to magnetic insulators.

The chaperone GRP78 is a host auxiliary factor for SARS-CoV-2 and GRP78 depleting antibody blocks viral entry and infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 global pandemic, utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) for viral entry. However, other host factors might also play important roles in SARS-CoV-2 infection, providing new directions for antiviral treatments. GRP78 is a stress-inducible chaperone important for entry and infectivity for many viruses. Recent molecular docking analyses revealed putative interaction between GRP78 and the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (SARS-2-S). Here we report that GRP78 can form a complex with SARS-2-S and ACE2 on the surface and at the perinuclear region typical of the endoplasmic reticulum in VeroE6-ACE2 cells and that the substrate-binding domain of GRP78 is critical for this interaction. In vitro binding studies further confirmed that GRP78 can directly bind to the RBD of SARS-2-S and ACE2. To investigate the role of GRP78 in this complex, we knocked down GRP78 in VeroE6-ACE2 cells. Loss of GRP78 markedly reduced cell surface ACE2 expression and led to activation of markers of the unfolded protein response. Treatment of lung epithelial cells with a humanized monoclonal antibody (hMAb159) selected for its safe clinical profile in preclinical models depleted cell surface GRP78 and reduced cell surface ACE2 expression, as well as SARS-2-S-driven viral entry and SARS-CoV-2 infection in vitro. Our data suggest that GRP78 is an important host auxiliary factor for SARS-CoV-2 entry and infection and a potential target to combat this novel pathogen and other viruses that utilize GRP78 in combination therapy.

Characterization and mechanism of stress-induced translocation of 78-kilodalton glucose-regulated protein (GRP78) to the cell surface.

Glucose-regulated protein (GRP78)/BiP, a major chaperone in the endoplasmic reticulum, is recently discovered to be preferably expressed on the surface of stressed cancer cells, where it regulates critical oncogenic signaling pathways and is emerging as a target for anti-cancer therapy while sparing normal organs. However, because GRP78 does not contain classical transmembrane domains, its mechanism of transport and its anchoring at the cell surface are poorly understood. Using a combination of biochemical, mutational, FACS, and single molecule super-resolution imaging approaches, we discovered that GRP78 majorly exists as a peripheral protein on plasma membrane via interaction with other cell surface proteins including glycosylphosphatidylinositol-anchored proteins. Moreover, cell surface GRP78 expression requires its substrate binding activity but is independent of ATP binding or a membrane insertion motif conserved with HSP70. Unexpectedly, different cancer cell lines rely on different mechanisms for GRP78 cell surface translocation, implying that the process is cell context-dependent.

Liver-specific knockout of GRP94 in mice disrupts cell adhesion, activates liver progenitor cells, and accelerates liver tumorigenesis.

UNLABELLED: Liver cancer is one of the most common solid tumors, with poor prognosis and high mortality. Mutation or deletion of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is strongly correlated with human liver cancer. Glucose-regulated protein 94 (GRP94) is a major endoplasmic reticulum (ER) chaperone protein, but its in vivo function is still emerging. To study the role of GRP94 in maintaining liver homeostasis and tumor development, we created two liver-specific knockout mouse models with the deletion of Grp94 alone, or in combination with Pten, using the albumin-cre system. We demonstrated that while deletion of GRP94 in the liver led to hyperproliferation of liver progenitor cells, deletion of both GRP94 and PTEN accelerated development of liver tumors, including both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC), suggestive of progenitor cell origin. Furthermore, at the premalignant stage we observed disturbance of cell adhesion proteins and minor liver injury. When GRP94 was deleted in PTEN-null livers, ERK was selectively activated. CONCLUSION: GRP94 is a novel regulator of cell adhesion, liver homeostasis, and tumorigenesis.

Inducible knockout of GRP78/BiP in the hematopoietic system suppresses Pten-null leukemogenesis and AKT oncogenic signaling.

Traditionally, GRP78 is regarded as protective against hypoxia and nutrient starvation prevalent in the microenvironment of solid tumors; thus, its role in the development of hematologic malignancies remains to be determined. To directly elucidate the requirement of GRP78 in leukemogenesis, we created a biallelic conditional knockout mouse model of GRP78 and PTEN in the hematopoietic system. Strikingly, heterozygous knockdown of GRP78 in PTEN null mice is sufficient to restore the hematopoietic stem cell population back to the normal percentage and suppress leukemic blast cell expansion. AKT/mTOR activation in PTEN null BM cells is potently inhibited by Grp78 heterozygosity, corresponding with suppression of the PI3K/AKT pathway by GRP78 knockdown in leukemia cell lines. This is the first demonstration that GRP78 is a critical effector of leukemia progression, at least in part through regulation of oncogenic PI3K/AKT signaling. In agreement with PI3K/AKT as an effector for cytosine arabinoside resistance in acute myeloid leukemia, overexpression of GRP78 renders human leukemic cells more resistant to cytosine arabinoside-induced apoptosis, whereas knockdown of GRP78 sensitizes them. These, coupled with the emerging association of elevated GRP78 expression in leukemic blasts of adult patients and early relapse in childhood leukemia, suggest that GRP78 is a novel therapeutic target for leukemia.

Gap junctions in Turing-type periodic feather pattern formation.

Periodic patterning requires coordinated cell-cell interactions at the tissue level. Turing showed, using mathematical modeling, how spatial patterns could arise from the reactions of a diffusive activator-inhibitor pair in an initially homogenous two-dimensional field. Most activators and inhibitors studied in biological systems are proteins, and the roles of cell-cell interaction, ions, bioelectricity, etc. are only now being identified. Gap junctions (GJs) mediate direct exchanges of ions or small molecules between cells, enabling rapid long-distance communications in a cell collective. They are therefore good candidates for propagating non-protein-based patterning signals that may act according to the Turing principles. Here, we explore the possible roles of GJs in Turing-type patterning using feather pattern formation as a model. We found seven of the twelve investigated GJ isoforms are highly dynamically expressed in the developing chicken skin. In ovo functional perturbations of the GJ isoform, connexin 30, by siRNA and the dominant-negative mutant applied before placode development led to disrupted primary feather bud formation, including patches of smooth skin and buds of irregular sizes. Later, after the primary feather arrays were laid out, inhibition of gap junctional intercellular communication in the ex vivo skin explant culture allowed the emergence of new feather buds in temporal waves at specific spatial locations relative to the existing primary buds. The results suggest that gap junctional communication may facilitate the propagation of long-distance inhibitory signals. Thus, the removal of GJ activity would enable the emergence of new feather buds if the local environment is competent and the threshold to form buds is reached. We propose Turing-based computational simulations that can predict the appearance of these ectopic bud waves. Our models demonstrate how a Turing activator-inhibitor system can continue to generate patterns in the competent morphogenetic field when the level of intercellular communication at the tissue scale is modulated.

Hierarchical nanosheets built from superatomic clusters: properties, exfoliation and single-crystal-to-single-crystal intercalation.

Tuning the properties of atomic crystals in the two-dimensional (2D) limit is synthetically challenging, but critical to unlock their potential in fundamental research and nanotechnology alike. 2D crystals assembled using superatomic blocks could provide a route to encrypt desirable functionality, yet strategies to link the inorganic blocks together in predetermined dimensionality or symmetry are scarce. Here, we describe the synthesis of anisotropic van der Waals crystalline frameworks using the designer superatomic nanocluster Co3(py)3Co6Se8L6 (py = pyridine, L = Ph2PN(Tol)), and ditopic linkers. Post-synthetically, the 3D crystals can be mechanically exfoliated into ultrathin flakes (8 to 60 nm), or intercalated with the redox-active guest tetracyanoethylene in a single-crystal-to-single-crystal transformation. Extensive characterization, including by single crystal X-ray diffraction, reveals how intrinsic features of the nanocluster, such as its structure, chirality, redox-activity and magnetic profile, predetermine key properties of the emerging 2D structures. Within the nanosheets, the strict and unusual stereoselectivity of the nanocluster's Co edges for the low symmetry (α,α,ß) isomer gives rise to in-plane structural anisotropy, while the helically chiral nanoclusters self-organize into alternating Δ- and Λ-homochiral rows. The nanocluster's high-spin Co edges, and its rich redox profile make the nanosheets both magnetically and electrochemically active, as revealed by solid state magnetic and cyclic voltammetry studies. The length and flexibility of the ditopic linker was varied, and found to have a secondary effect on the structure and stacking of the nanosheets within the 3D crystals. With these results we introduce a deterministic and versatile synthetic entry to programmable functionality and symmetry in 2D superatomic crystals.

The COOH-Terminal Proline-Rich Region of GRP78 Is a Key Regulator of Its Cell Surface Expression and Viability of Tamoxifen-Resistant Breast Cancer Cells.

Translocation of 78-kDa glucose-regulated protein (GRP78) from endoplasmic reticulum (ER) to plasma membrane represents a paradigm shift beyond its traditional function as an ER chaperone protein. Cell surface GRP78 (csGRP78) exerts novel signaling functions, and mechanisms underlying its cell surface expression are just emerging. Acquired tamoxifen resistance of breast cancer cells is accompanied with elevated level of csGRP78. Therefore, the tamoxifen-resistant MCF7 breast cancer cells (MCF7-LR) represents a clinically relevant model to study mechanisms of csGRP78 expression. We discovered that a proline-rich region (PRR) containing three consecutive prolines close to the COOH-terminus of GRP78 is important for its ability to form a complex with the partner protein, CD44v, as demonstrated by in vitro glutathione S-transferase pull-down assay. Proline to alanine mutations at the PRR compromised GRP78 expression level on the cell surface as evidenced by purification of biotinylated cell surface proteins. Reconstitution of MCF7-LR cells with the PRR mutant after knockdown of endogenous GRP78 diminished the capacity of GRP78 to stimulate STAT3 activation. The enforced expression of a short peptide bearing the PRR region of GRP78 led to reduction of CD44v and Cyclin D1 protein levels as well as cell viability, accompanied with increase in apoptotic signaling including cleaved Caspase-3 and PARP. These findings suggest that the COOH-terminal PRR of GRP78 is critical for its interaction with CD44v as well as its cell surface expression, and enforced expression of the short peptide bearing the PRR region may provide a new approach to lower the viability of tamoxifen-resistant breast cancer cells.

GRP78 regulates CD44v membrane homeostasis and cell spreading in tamoxifen-resistant breast cancer.

GRP78 conducts protein folding and quality control in the ER and shows elevated expression and cell surface translocation in advanced tumors. However, the underlying mechanisms enabling GRP78 to exert novel signaling functions at cell surface are just emerging. CD44 is a transmembrane protein and an important regulator of cancer metastasis, and isoform switch of CD44 through incorporating additional variable exons to the extracellular juxtamembrane region is frequently observed during cancer progression. Using super-resolution dual-color single-particle tracking, we report that GRP78 interacts with CD44v in plasma membrane nanodomains of breast cancer cells. We further show that targeting cell surface GRP78 by the antibodies can effectively reduce cell surface expression of CD44v and cell spreading of tamoxifen-resistant breast cancer cells. Our results uncover new functions of GRP78 as an interacting partner of CD44v and as a regulator of CD44v membrane homeostasis and cell spreading. This study also provides new insights into anti-CD44 therapy in tamoxifen-resistant breast cancer.

Calcium oscillations coordinate feather mesenchymal cell movement by SHH dependent modulation of gap junction networks.

Collective cell migration mediates multiple tissue morphogenesis processes. Yet how multi-dimensional mesenchymal cell movements are coordinated remains mostly unknown. Here we report that coordinated mesenchymal cell migration during chicken feather elongation is accompanied by dynamic changes of bioelectric currents. Transcriptome profiling and functional assays implicate contributions from functional voltage-gated Ca2+ channels (VGCCs), Connexin-43 based gap junctions, and Ca2+ release activated Ca2+ (CRAC) channels. 4-Dimensional Ca2+ imaging reveals that the Sonic hedgehog-responsive mesenchymal cells display synchronized Ca2+ oscillations, which expand progressively in area during feather elongation. Inhibiting VGCCs, gap junctions, or Sonic hedgehog signaling alters the mesenchymal Ca2+ landscape, cell movement patterns and feather bud elongation. Ca2+ oscillations induced by cyclic activation of opto-cCRAC channels enhance feather bud elongation. Functional disruption experiments and promoter analysis implicate synergistic Hedgehog and WNT/ß-Catenin signaling in activating Connexin-43 expression, establishing gap junction networks synchronizing the Ca2+ profile among cells, thereby coordinating cell movement patterns.

Deficiency of GRP94 in the hematopoietic system alters proliferation regulators in hematopoietic stem cells.

We have previously reported that acute inducible knockout of the endoplasmic reticulum chaperone GRP94 led to an expansion of the hematopoietic stem and progenitor cell pool. Here, we investigated the effectors and mechanisms for this phenomenon. We observed an increase in AKT activation in freshly isolated GRP94-null HSC-enriched Lin(-) Sca-1(+) c-Kit(+) (LSK) cells, corresponding with higher production of PI(3,4,5)P3, indicative of PI3K activation. Treatment of GRP94-null LSK cells with the AKT inhibitor MK2206 compromised cell expansion, suggesting a causal relationship between elevated AKT activation and increased proliferation in GRP94-null HSCs. Microarray analysis demonstrated a 97% reduction in the expression of the hematopoietic cell cycle regulator Ms4a3 in the GRP94-null LSK cells, and real-time quantitative PCR confirmed this down-regulation in the LSK cells but not in the total bone marrow (BM). A further examination comparing freshly isolated BM LSK cells with spleen LSK cells, as well as BM LSK cells cultured in vitro, revealed specific down-regulation of Ms4a3 in freshly isolated BM GRP94-null LSK cells. On examining cell surface proteins that are known to regulate stem cell proliferation, we observed a reduced expression of cell surface connexin 32 (Cx32) plaques in GRP94-null LSK cells. However, suppression of Cx32 hemichannel activity in wild-type LSK cells through mimetic peptides did not lead to increased LSK cell proliferation in vitro. Two other important cell surface proteins that mediate HSC-niche interactions, specifically Tie2 and CXCR4, were not impaired by Grp94 deletion. Collectively, our study uncovers novel and unique roles of GRP94 in regulating HSC proliferation.

Cancer cells resistant to therapy promote cell surface relocalization of GRP78 which complexes with PI3K and enhances PI(3,4,5)P3 production.

Traditionally, GRP78 has been regarded as an endoplasmic reticulum (ER) lumenal protein due to its carboxyl KDEL retention motif. Recently, a subfraction of GRP78 is found to localize to the surface of specific cell types, serving as co-receptors and regulating signaling. However, the physiological relevance of cell surface GRP78 (sGRP78) expression in cancer and its functional interactions at the cell surface are just emerging. In this report, we combined biochemical, imaging and mutational approaches to address these issues. For detection of sGRP78, we utilized a mouse monoclonal antibody highly potent and specific for GRP78 or epitope-tagged GRP78, coupled with imaging and biochemical techniques that allowed detection of sGRP78 but not intracellular GRP78. Our studies revealed that breast and prostate cancer cells resistant to hormonal therapy actively promote GRP78 to the cell surface, which can be further elevated by a variety of ER stress-inducing conditions. We showed that sGRP78 forms complex with PI3K, and overexpression of sGRP78 promotes PIP3 formation, indicative of PI3K activation. We further discovered that an insertion mutant of GRP78 at its N-terminus domain, while retaining stable expression and the ability to translocate to the cell surface as the wild-type protein, exhibited reduced complex formation with p85 and production of PIP3. Thus, our studies provide a mechanistic explanation for the regulation of the PI3K/AKT signaling by sGRP78. Our findings suggest that targeting sGRP78 may suppress therapeutic resistance in cancer cells and offer a novel strategy to suppress PI3K activity.