Species that propagate by sexual reproduction actively guard against the fertilization of an egg by multiple sperm (polyspermy). Flowering plants rely on pollen tubes to transport their immotile sperm to fertilize the female gametophytes inside ovules. In Arabidopsis , pollen tubes are guided by cysteine-rich chemoattractants to target the female gametophyte 1 , 2 . The FERONIA receptor kinase has a dual role in ensuring sperm delivery and blocking polyspermy 3 . It has previously been reported that FERONIA generates a female gametophyte environment that is required for sperm release 4 . Here we show that FERONIA controls several functionally linked conditions to prevent the penetration of female gametophytes by multiple pollen tubes in Arabidopsis . We demonstrate that FERONIA is crucial for maintaining de-esterified pectin at the filiform apparatus, a region of the cell wall at the entrance to the female gametophyte. Pollen tube arrival at the ovule triggers the accumulation of nitric oxide at the filiform apparatus in a process that is dependent on FERONIA and mediated by de-esterified pectin. Nitric oxide nitrosates both precursor and mature forms of the chemoattractant LURE1 1 , respectively blocking its secretion and interaction with its receptor, to suppress pollen tube attraction. Our results elucidate a mechanism controlled by FERONIA in which the arrival of the first pollen tube alters ovular conditions to disengage pollen tube attraction and prevent the approach and penetration of the female gametophyte by late-arriving pollen tubes, thus averting polyspermy.

Cells maintain integrity despite changes in their mechanical properties elicited during growth and environmental stress. How cells sense their physical state and compensate for cell-wall damage is poorly understood, particularly in plants. Here we report that FERONIA (FER), a plasma-membrane-localized receptor kinase from Arabidopsis, is necessary for the recovery of root growth after exposure to high salinity, a widespread soil stress. The extracellular domain of FER displays tandem regions of homology with malectin, an animal protein known to bind di-glucose in vitro and important for protein quality control in the endoplasmic reticulum. The presence of malectin-like domains in FER and related receptor kinases has led to widespread speculation that they interact with cell-wall polysaccharides and can potentially serve a wall-sensing function. Results reported here show that salinity causes softening of the cell wall and that FER is necessary to sense these defects. When this function is disrupted in the fer mutant, root cells explode dramatically during growth recovery. Similar defects are observed in the mur1 mutant, which disrupts pectin cross-linking. Furthermore, fer cell-wall integrity defects can be rescued by treatment with calcium and borate, which also facilitate pectin cross-linking. Sensing of these salinity-induced wall defects might therefore be a direct consequence of physical interaction between the extracellular domain of FER and pectin. FER-dependent signaling elicits cell-specific calcium transients that maintain cell-wall integrity during salt stress. These results reveal a novel extracellular toxicity of salinity, and identify FER as a sensor of damage to the pectin-associated wall.

Hepcidin is a peptide hormone that negatively regulates iron efflux and plays an important role in controlling the growth of breast tumors. In patients with breast cancer, the combined expression of hepcidin and its membrane target, ferroportin, predict disease outcome. However, mechanisms that control hepcidin expression in breast cancer cells remain largely unknown. Here, we use three-dimensional breast cancer spheroids derived from cell lines and breast cancer patients to probe mechanisms of hepcidin regulation in breast cancer. We observe that the extent of hepcidin induction and pathways of its regulation are markedly changed in breast cancer cells grown in three dimensions. In monolayer culture, BMPs, particularly BMP6, regulate hepcidin transcription. When breast cancer cells are grown as spheroids, there is a > 10-fold induction in hepcidin transcripts. Microarray analysis combined with knockdown experiments reveal that GDF-15 is the primary mediator of this change. The increase in hepcidin as breast cells develop a three-dimensional architecture increases intracellular iron, as indicated by an increase in the iron storage protein ferritin. Immunohistochemical staining of human breast tumors confirms that both GDF-15 and hepcidin are expressed in breast cancer specimens. Further, levels of GDF-15 are significantly correlated with levels of hepcidin at both the mRNA and protein level in patient samples, consistent with a role for GDF-15 in control of hepcidin in human breast tumors. Inclusion of tumor-associated fibroblasts in breast cancer spheroids further induces hepcidin. This induction is mediated by fibroblast-dependent secretion of IL-6. Breast cancer cells grown as spheroids are uniquely receptive to IL-6-dependent induction of hepcidin by tumor-associated fibroblasts, since IL6 does not induce hepcidin in cells grown as monolayers. Collectively, our results suggest a new paradigm for tumormediated control of iron through the control of hepcidin by tumor architecture and the breast tumor microenvironment.

Polar auxin transport, facilitated by the combined activities of auxin influx and efflux carriers to maintain asymmetric auxin distribution, is essential for plant growth and development. Here, we show that Arabidopsis (Arabidopsis thaliana) RopGEF1, a guanine nucleotide exchange factor and activator of Rho GTPases of plants (ROPs), is critically involved in polar distribution of auxin influx carrier AUX1 and differential accumulation of efflux carriers PIN7 and PIN2 and is important for embryo and early seedling development when RopGEF1 is prevalently expressed. Knockdown or knockout of RopGEF1 induces embryo defects, cotyledon vein breaks, and delayed root gravity responses. Altered expression from the auxin response reporter DR5rev: GFP in the root pole of RopGEF1-deficient embryos and loss of asymmetric distribution of DR5rev: GFP in their gravistimulated root tips suggest that auxin distribution is affected in ropgef1 mutants. This is reflected by the polarity of AUX1 being altered in ropgef1 embryos and roots, shifting from the normal apical membrane location to a basal location in embryo central vascular and root protophloem cells and also reduced PIN7 accumulation at embryos and altered PIN2 distribution in gravistimulated roots of mutant seedlings. In establishing that RopGEF1 is critical for AUX1 localization and PIN differential accumulation, our results reveal a role for RopGEF1 in cell polarity and polar auxin transport whereby it imapcts auxin-mediated plant growth and development.

Iron is an essential nutrient for breast cancer cell growth and proliferation. Our laboratory has demonstrated that ferroportin (Fpn), an iron efflux pump, plays a critical role in maintenance of intracellular iron levels. Fpn is controlled by hepcidin, a secreted peptide hormone that binds to Fpn and triggers its degradation. We have observed that Fpn expression is down‐regulated and hepcidin expression is up‐regulated in breast cancer cells, promoting increased accumulation of iron in cancer cells to fuel their proliferative demands. Since expression of Fpn/hepcidin predicts outcome in breast cancer patients, we explored pathways that control these proteins. We used a 3D spheroid culture system to model in vivo breast tumor architecture, microenvironment and metabolism. We found that hepcidin expression is significantly up‐regulated from 2D to 3D culture of MCF‐7 and patient‐derived breast cancer cells, suggesting that hepcidin may be regulated by processes that exceed the realm of 2D culture. Culturing MCF‐7 cells as spheroids in the presence of Matrigel repressed the induction of hepcidin, suggesting that exogenous extracellular matrix (ECM) proteins present in Matrigel may negatively regulate hepcidin. Further analysis of the effect of exogenous ECM on hepcidin expression revealed that collagen IV, one of the most abundant basement membrane (BM) proteins in Matrigel, negatively regulates hepcidin expression in MCF‐7 spheroids. On the other hand, endogenous production of ECM components, such as collagen I and collagen II, matrix components frequently up‐regulated in breast cancer, was increased in MCF‐7 spheroids compared to monolayer culture. It is known that there is a modified microenvironement during breast tumorigenesis that includes breakdown of the structural BM proteins and deregulation of matrix components, including those that support tumor progression and promote metastatic spread. Together, our data suggest that components of the ECM play a dynamic role in both positive and negative regulation of hepcidin expression in breast cancer spheroids. This finding suggests that the ECM is an integral component in regulation of hepcidin within the tumor microenvironment. Understanding mechanisms of hepcidin regulation by ECM components will provide a greater understanding of the crosstalk between the ECM and iron metabolism in breast cancer. Support or Funding Information This research is supported by NIH R01 CA171101 and NIH R01 CA188025.

The Arabidopsis receptor kinase FERONIA (FER) is a multifunctional regulator for plant growth and reproduction. Here we report that the female gametophyte-expressed glycosylphosphatidylinositol-anchored protein (GPI-AP) LORELEI and the seedling-expressed LRE-like GPI-AP1 (LLG1) bind to the extracellular juxtamembrane region of FER and show that this interaction is pivotal for FER function. LLG1 interacts with FER in the endoplasmic reticulum and on the cell surface, and loss of LLG1 function induces cytoplasmic retention of FER, consistent with transport of FER from the endoplasmic reticulum to the plasma membrane in a complex with LLG1. We further demonstrate that LLG1 is a component of the FER-regulated RHO GTPase signaling complex and that fer and llg1 mutants display indistinguishable growth, developmental and signaling phenotypes, analogous to how lre and fer share similar reproductive defects. Together our results support LLG1/LRE acting as a chaperone and co-receptor for FER and elucidate a mechanism by which GPI-APs enable the signaling capacity of a cell surface receptor.

BACKGROUND:Monoclonal antibodies have been used to effectively treat various tumors. We previously established a unique strategy to identify tumor specific antibodies by capturing B-cell response against breast tumor antigens from patient-derived sentinel lymph nodes. Initial application of this approach led to identification of a tumor specific single domain antibody. In this paper we optimized our previous strategy by generating heavy chain antibodies (HCAbs) to overcome the deficiencies of single domain antibodies. Here we identified and characterized a heavy chain antibody (HCAb2) that targets cell surface HSP90 antigen on breast tumor cells but not normal cells.METHODS:Eight HCAbs derived from 4 breast cancer patients were generated using an in vitro expression system. HCAbs were screened against normal breast cells (MCF10A, HMEC) and tumor cell lines (MCF7, MDA-MB-231) to identify cell surface targeting and tumor specific antibodies using flow cytometry and immunofluorescence. Results observed with cell lines were validated by screening a cohort of primary human breast normal and tumor tissues using immunofluorescence. Respective antigens for two HCAbs (HCAb1 and HCAb2) were identified using immunoprecipitation followed by mass spectrometry. Finally, we generated MDA-MB-231 xenograft tumors in NOD scid gamma mice and performed in vivo tumor targeting analysis of HCAb1 and HCAb2.RESULTS:Flow cytometry screen revealed that HCAb2 selectively bound to the surface of MDA-MB-231 cells in comparison to MCF10A and MCF7 cells. HCAb2 showed punctate membrane staining on MDA-MB-231 cells and preferential binding to human breast tumor tissues in comparison to normal breast tissues. In primary breast tumor tissues, HCAb2 showed positive binding to both E-cadherin positive and negative tumor cells. We identified and validated the target antigen of HCAb2 as Heat shock protein 90 (HSP90). HCAb2 also selectively targeted MDA-MB-231 xenograft tumor cells in vivo with little targeting to mouse normal tissues. Finally, HCAb2 specifically targeted calnexin negative xenograft tumor cells.CONCLUSIONS:From our screening methodology, we identified HCAb2 as a breast tumor specific heavy chain antibody targeting cell surface HSP90. HCAb2 also targeted MDA-MB-231 tumor cells in vivo suggesting that HCAb2 could be an ideal tumor targeting antibody.

Abstract The emergence of tumor-targeting antibodies such as Trastuzumab, Rituximab and anti-CD47 antibody has highlighted the significant role of antibodies for effective cancer therapy. The most common approach to generate tumor specific antibodies is to select an established tumor specific antigen and generate antibodies to that target. The drawback with this approach is that it does not lead to identification of novel antigens or antigenic epitopes. Therefore, we developed a strategy that will lead to the identification of both tumor specific antibodies and novel tumor antigens. The approach uses the patient's immune system to guide us to define tumor specific antigens. In order to achieve this, we identified reactive germinal centers in the tumor draining sentinel lymph nodes from breast cancer patients. Immunohistochemical analysis of reactive germinal centers defined the B-cell regions for mRNA isolation and generation of cDNA molecules for the variable heavy chain segment (VH) of antibodies. The VH cDNA molecules were cloned into a mammalian expression vector to generate bivalent heavy chain antibodies with a C-terminus mouse Fc tag. At present, we have generated about 250 antigen-driven variable heavy chain clones from 27 breast cancer patient lymph node samples. We screened 46 heavy chain antibodies against a panel of breast cancer cell lines using immunofluorescence and identified 8 antibodies that showed strong and specific staining. Of these 8 antibodies, one of them (VH6-93) showed membrane staining preferentially on MDA-MB-231 cells in comparison to MCF7 cells. VH6-93 antibody also showed strong staining on conditionally reprogrammed tumor cells derived from a breast cancer patient with only moderate staining on normal breast epithelial cells derived from the same patient. VH6-93 also revealed preferential binding to breast tumor tissues as compared to normal ductal tissues evidenced by immunofluorescence and immunohistochemistry. To identify the target antigens, a multi-tiered approach was used, which included immunoprecipitation of target antigen from cell lysates followed by peptide sequencing with mass spectrometry as well as utilizing Nucleic Acid Programmable Protein Array (NAPPA) technology. A comprehensive analysis of two patient-derived antibodies has produced a panel of potential antigen targets, which are undergoing specificity and validation analysis. In summary, this methodology of using activated B-cells from sentinel lymph nodes is a unique way to enrich for tumor-specific antibody clones. The findings from this research will not only lead to tumor specific antibodies but will also reveal novel tumor specific antigens. Identification of these novel antigens can give rise to additional therapeutic strategies such as peptide or protein vaccination. Citation Format: Charan Kumar V. Devarakonda, Daniel Kita, Kathryn N. Phoenix, Dewey M. Magee, Kevin P. Claffey. Patient-derived breast cancer targeting heavy chain antibodies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2661. doi:10.1158/1538-7445.AM2014-2661

In flowering plants, sperm are transported inside pollen tubes to the female gametophyte for fertilization. The female gametophyte induces rupture of the penetrating pollen tube, resulting in sperm release and rendering them available for fertilization. Here we utilize the Arabidopsis FERONIA (FER) receptor kinase mutants, whose female gametophytes fail to induce pollen tube rupture, to decipher the molecular mechanism of this critical male-female interactive step. We show that FER controls the production of high levels of reactive oxygen species at the entrance to the female gametophyte to induce pollen tube rupture and sperm release. Pollen tube growth assays in vitro and in the pistil demonstrate that hydroxyl free radicals are likely the most reactive oxygen molecules, and they induce pollen tube rupture in a Ca(2+)-dependent process involving Ca(2+) channel activation. Our results provide evidence for a RHO GTPase-based signalling mechanism to mediate sperm release for fertilization in plants.

Plant growth and development are controlled by a delicate balance of hormonal cues. Growth-promoting hormones and growth-inhibiting counterparts often antagonize each other in their action, but the molecular mechanisms underlying these events remain largely unknown. Here, we report a cross-talk mechanism that enables a receptor-like kinase, FERONIA (FER), a positive regulator of auxin-promoted growth, to suppress the abscisic acid (ABA) response through activation of ABI2, a negative regulator of ABA signaling. The FER pathway consists of a FER kinase interacting with guanine exchange factors GEF1, GEF4, and GEF10 that, in turn, activate GTPase ROP11/ARAC10. Arabidopsis mutants disrupted in any step of the FER pathway, including fer, gef1gef4gef10, or rop11/arac10, all displayed an ABA-hypersensitive response, implicating the FER pathway in the suppression mechanism. In search of the target for the FER pathway, we found that the ROP11/ARAC10 protein physically interacted with the ABI2 phosphatase and enhanced its activity, thereby linking the FER pathway with the inhibition of ABA signaling.

Plant RHO GTPases (RAC/ROPs) mediate multiple extracellular signals ranging from hormone to stress and regulate diverse cellular processes important for polarized cell growth, differentiation, development, reproduction, and responses to the environment. They shuttle between the GDP-bound inactive state and the GTP-bound activated state and their activation is predominantly mediated by a family of guanine nucleotide exchange factors (GEFs) referred to as ROPGEFs. Using the Arabidopsis ROPGEF1 as bait, we identified members of a receptor-like kinase (RLK) family as potential upstream regulators for RAC/ROP signaling. NADPH oxidase-derived reactive oxygen species (ROS) are emerging as important regulators for growth and development and play a crucial role in mediating RAC/ROP-regulated root hair development, a polarized cell growth process. We therefore screened T-DNA insertion mutants in these RLKs for root hair defects and found that mutations in one of them, At3g51550 encoding the FERONIA (FER) receptor-like kinase, induced severe root hair defects. We show that the fer phenotypes correlated with reduced levels of active RAC/ROPs and NADPH oxidase-dependent, auxin-regulated ROS accumulation in roots and root hairs and that up-regulating RAC/ROP signaling in fer countered the mutant phenotypes. Taken together, these observations strongly support FER as an upstream regulator for the RAC/ROP-signaled pathway that controls ROS-mediated root hair development. Moreover, FER was pulled down by ROP2 GTPase in a guanine nucleotide-regulated manner implying a dynamic signaling complex involving FER, a ROPGEF, and a RAC/ROP.

Mounting evidence supports that RAC/ROP GTPases are central regulators for diverse signaling pathways for plant growth, development, and interactions with the environment. Their regulatory activities for key intracellular process, such as control of actin dynamics, membrane trafficking, and several hormone signal transduction pathways, suggest inevitable functional roles for RAC/ROPs in regulating cellular activities that underlie important growth and developmental events, especially those that involve cellular and morphological asymmetry. RAC/ROPs are well established as a regulator for the two most polarized cell growth processes in plants, pollen tube tip growth, and root hair elongation, and are known to be crucial for the differentiation of leaf epidermal cell patterning, which depends substantially on differential cell expansion around its periphery and asymmetric cell division. We focus here on discussing recent findings, especially those that relate to upstream regulators and downstream effectors of RAC/ROPs that illuminate how these small GTPases and their interactors together contribute to polarity-dependent processes in plants.