Extensive Membrane Remodeling Research Articles

Two-pore channels regulate endomembrane tension to enable remodeling and resolution of phagolysosomes

Phagocytes promptly resolve ingested targets to replenish lysosomes and maintain their responsiveness. The resolution process requires that degradative hydrolases, solute transporters, and proteins involved in lipid traffic are delivered and made active in phagolysosomes. It also involves extensive membrane remodeling. We report that cation channels that localize to phagolysosomes were essential for resolution. Specifically, the conductance of Na+ by two-pore channels (TPCs) and the presence of a Na+ gradient between the phagolysosome lumen and the cytosol were critical for the controlled release of membrane tension that permits deformation of the limiting phagolysosome membrane. In turn, membrane deformation was a necessary step to efficiently transport the cholesterol extracted from cellular targets, permeabilizing them to hydrolases. These results place TPCs as regulators of endomembrane remodeling events that precede target degradation in cases when the target is bound by a cholesterol-containing membrane. The findings may help to explain lipid metabolism dysfunction and autophagic flux impairment reported in TPC KO mice and establish stepwise regulation to the resolution process that begins with lysis of the target.

A Novel Nanosafety Approach Using Cell Painting, Metabolomics, and Lipidomics Captures the Cellular and Molecular Phenotypes Induced by the Unintentionally Formed Metal-Based (Nano)Particles.

Additive manufacturing (AM) or industrial 3D printing uses cutting-edge technologies and materials to produce a variety of complex products. However, the effects of the unintentionally emitted AM (nano)particles (AMPs) on human cells following inhalation, require further investigations. The physicochemical characterization of the AMPs, extracted from the filter of a Laser Powder Bed Fusion (L-PBF) 3D printer of iron-based materials, disclosed their complexity, in terms of size, shape, and chemistry. Cell Painting, a high-content screening (HCS) assay, was used to detect the subtle morphological changes elicited by the AMPs at the single cell resolution. The profiling of the cell morphological phenotypes, disclosed prominent concentration-dependent effects on the cytoskeleton, mitochondria, and the membranous structures of the cell. Furthermore, lipidomics confirmed that the AMPs induced the extensive membrane remodeling in the lung epithelial and macrophage co-culture cell model. To further elucidate the biological mechanisms of action, the targeted metabolomics unveiled several inflammation-related metabolites regulating the cell response to the AMP exposure. Overall, the AMP exposure led to the internalization, oxidative stress, cytoskeleton disruption, mitochondrial activation, membrane remodeling, and metabolic reprogramming of the lung epithelial cells and macrophages. We propose the approach of integrating Cell Painting with metabolomics and lipidomics, as an advanced nanosafety methodology, increasing the ability to capture the cellular and molecular phenotypes and the relevant biological mechanisms to the (nano)particle exposure.

Structural, Metabolic and Evolutionary Comparison of Bacterial Endospore and Exospore Formation.

Sporulation is a specialized developmental program employed by a diverse set of bacteria which culminates in the formation of dormant cells displaying increased resilience to stressors. This represents a major survival strategy for bacteria facing harsh environmental conditions, including nutrient limitation, heat, desiccation, and exposure to antimicrobial compounds. Through dispersal to new environments via biotic or abiotic factors, sporulation provides a means for disseminating genetic material and promotes encounters with preferable environments thus promoting environmental selection. Several types of bacterial sporulation have been characterized, each involving numerous morphological changes regulated and performed by non-homologous pathways. Despite their likely independent evolutionary origins, all known modes of sporulation are typically triggered by limited nutrients and require extensive membrane and peptidoglycan remodeling. While distinct modes of sporulation have been observed in diverse species, two major types are at the forefront of understanding the role of sporulation in human health, and microbial population dynamics and survival. Here, we outline endospore and exospore formation by members of the phyla Firmicutes and Actinobacteria, respectively. Using recent advances in molecular and structural biology, we point to the regulatory, genetic, and morphological differences unique to endo- and exospore formation, discuss shared characteristics that contribute to the enhanced environmental survival of spores and, finally, cover the evolutionary aspects of sporulation that contribute to bacterial species diversification.

HDL mediates reverse cholesterol transport from ram spermatozoa and induces hyperactivated motility.

Reverse cholesterol transport or cholesterol efflux is part of an extensive plasma membrane remodeling process in spermatozoa that is imperative for fertilization. For ram spermatozoa, sheep serum is well known to support in vitro fertilization (IVF), but knowledge of its explicit role is limited. Though, it is postulated to elicit cholesterol efflux owing to the presence of high-density lipoproteins (HDLs) that interact with transmembrane cholesterol transporters, such as adenosinetriphosphate (ATP)-binding cassette transporter A1 (ABCA1) and scavenger receptor class B, type I (SR-BI). In this study, we report that both sheep serum and HDLs were able to elicit cholesterol efflux alone by up to 20–40% (as measured by the boron dipyrromethene (BODIPY)-cholesterol assay). Furthermore, when the antagonists glibenclamide and valspodar were used to inhibit the function of ABCA1 and SR-BI or ABCA1 alone, respectively, cholesterol efflux was only marginally reduced (8–15%). Nevertheless, it is likely that in ram spermatozoa, a specific facilitated pathway of cholesterol efflux is involved in the interaction between cholesterol acceptors and transporters. Interestingly, exposure to HDLs also induced hyperactivated motility, another critical event required for successful fertilization. Taken together, this study details the first report of the dual action of HDLs on ram spermatozoa, providing both an insight into the intricacy of events leading up to fertilization in vivo as well as demonstrating the possible application of HDL supplementation in media for IVF.

Membrane Remodeling and Stimulation of Aggregation Following α-Synuclein Adsorption to Phosphotidylserine Vesicles.

α-Synuclein is an intrinsically disordered protein abundant in presynaptic terminals in neurons and in synaptic vesicles. α-Synuclein's interaction with lipid bilayers is important not only for its normal physiological function but also in its pathological aggregation and deposition as Lewy bodies in Parkinson's disease. α-Synuclein binds preferentially to lipids with acidic head groups and to high-curvature vesicles and can modulate membrane curvature. The relationship between the protein's role as a membrane curvature sensor and generator and the role of membranes in facilitating its aggregation remains unknown. We investigated the interaction of α-synuclein with vesicles of 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) or 1,2-dilauroyl-sn-glycero-3-phospho-l-serine (DLPS). Using nanoparticle tracking along with electron microscopy, we demonstrate that α-synuclein induces extensive vesicle disruption and membrane remodeling into discoids, tubules, and ribbons with DLPS vesicles but not DOPS. Coarse-grained molecular dynamics simulations revealed that adsorption of α-synuclein to DLPS but not DOPS vesicles induced vesicle elongation and redistribution of protein to regions of higher curvature, a process that could drive protein aggregation. In agreement with this hypothesis, DLPS but not DOPS strongly stimulated α-synuclein aggregation. Our results provide new insights into the critical contribution of bilayer stability in the membrane response to α-synuclein adsorption and in stimulation of aggregation.

SRRF-Stream Imaging of Optogenetically Controlled Furrow Formation Shows Localized and Coordinated Endocytosis and Exocytosis Mediating Membrane Remodeling

Cleavage furrow formation during cytokinesis involves extensive membrane remodeling. In the absence of methods to exert dynamic control over these processes, it has been a challenge to examine the basis of this remodeling. Here we used a subcellular optogenetic approach to induce this at will and found that furrow formation is mediated by actomyosin contractility, retrograde plasma membrane flow, localized decrease in membrane tension, and endocytosis. FRAP, 4-D imaging, and inhibition or upregulation of endocytosis or exocytosis show that ARF6 and Exo70 dependent localized exocytosis supports a potential model for intercellular bridge elongation. TIRF and Super Resolution Radial Fluctuation (SRRF) stream microscopy show localized VAMP2-mediated exocytosis and incorporation of membrane lipids from vesicles into the plasma membrane at the front edge of the nascent daughter cell. Thus, spatially separated but coordinated plasma membrane depletion and addition are likely contributors to membrane remodeling during cytokinetic processes.

Role of a patatin-like phospholipase in Plasmodium falciparum gametogenesis and malaria transmission

Transmission of Plasmodium falciparum involves a complex process that starts with the ingestion of gametocytes by female Anopheles mosquitoes during a blood meal. Activation of gametocytes in the mosquito midgut triggers "rounding up" followed by egress of both male and female gametes. Egress requires secretion of a perforin-like protein, PfPLP2, from intracellular vesicles to the periphery, which leads to destabilization of peripheral membranes. Male gametes also develop flagella, which assist in binding female gametes for fertilization. This process of gametogenesis, which is key to malaria transmission, involves extensive membrane remodeling as well as vesicular discharge. Phospholipase A2 enzymes (PLA2) are known to mediate membrane remodeling and vesicle secretion in diverse organisms. Here, we show that a P. falciparum patatin-like phospholipase (PfPATPL1) with PLA2 activity plays a key role in gametogenesis. Conditional deletion of the gene encoding PfPATPL1 does not affect P. falciparum blood stage growth or gametocyte development but reduces efficiency of rounding up, egress, and exflagellation of gametocytes following activation. Interestingly, deletion of the PfPATPL1 gene inhibits secretion of PfPLP2, reducing the efficiency of gamete egress. Deletion of PfPATPL1 also reduces the efficiency of oocyst formation in mosquitoes. These studies demonstrate that PfPATPL1 plays a role in gametogenesis, thereby identifying PLA2 phospholipases such as PfPATPL1 as potential targets for the development of drugs to block malaria transmission.

VPS4 triggers constriction and cleavage of ESCRT-III helical filaments.

Many cellular processes such as endosomal vesicle budding, virus budding, and cytokinesis require extensive membrane remodeling by the endosomal sorting complex required for transport III (ESCRT-III). ESCRT-III protein family members form spirals with variable diameters in vitro and in vivo inside tubular membrane structures, which need to be constricted to proceed to membrane fission. Here, we show, using high-speed atomic force microscopy and electron microscopy, that the AAA-type adenosine triphosphatase VPS4 constricts and cleaves ESCRT-III CHMP2A-CHMP3 helical filaments in vitro. Constriction starts asymmetrically and progressively decreases the diameter of CHMP2A-CHMP3 tubular structure, thereby coiling up the CHMP2A-CHMP3 filaments into dome-like end caps. Our results demonstrate that VPS4 actively constricts ESCRT-III filaments and cleaves them before their complete disassembly. We propose that the formation of ESCRT-III dome-like end caps by VPS4 within a membrane neck structure constricts the membrane to set the stage for membrane fission.

Characterizing How Acidic pH Conditions Affect the Membrane-Disruptive Activities of Lauric Acid and Glycerol Monolaurate.

Fatty acids and monoglycerides are single-chain lipid amphiphiles that interact with phospholipid membranes as part of various biological activities. For example, they can exhibit membrane-disruptive behavior against microbial pathogens on the human skin surface. Supported lipid bilayers (SLBs) provide a useful experimental platform to characterize these membrane-disruptive behaviors, although related studies have been limited to neutral pH conditions. Herein, we investigated how lauric acid (LA) and glycerol monolaurate (GML) interact with SLBs and cause membrane morphological changes under acidic pH conditions that are representative of the human skin surface. Although LA induces tubule formation under neutral pH conditions, we discovered that LA causes membrane phase separation under acidic pH conditions. By contrast, GML induced membrane budding in both pH environments, although there was more extensive membrane remodeling under acidic pH conditions. We discuss these findings in the context of how solution pH affects the ionization states and micellar aggregation properties of LA and GML as well as its effect on the bending stiffness of lipid bilayers. Collectively, the findings demonstrate that solution pH plays an important role in modulating the interaction of fatty acids and monoglycerides with phospholipid membranes, and hence influences the scope and potency of their membrane-disruptive activities.

Amyloid-β Peptide Triggers Membrane Remodeling in Supported Lipid Bilayers Depending on Their Hydrophobic Thickness.

Amyloid-β (Aβ) peptide has been implicated in Alzheimer's disease, which is a leading cause of death worldwide. The interaction of Aβ peptides with the lipid bilayers of neuronal cells is a critical step in disease pathogenesis. Recent evidence indicates that lipid bilayer thickness influences Aβ membrane-associated aggregation, while understanding how Aβ interacts with lipid bilayers remains elusive. To address this question, we employed supported lipid bilayer (SLB) platforms composed of different-length phosphatidylcholine (PC) lipids (C12:0 DLPC, C18:1 DOPC, C18:1-C16:0 POPC), and characterized the resulting interactions with soluble Aβ monomers. Quartz crystal microbalance-dissipation (QCM-D) experiments identified concentration-dependent Aβ peptide adsorption onto all tested SLBs, which was corroborated by fluorescence recovery after photobleaching (FRAP) experiments indicating that higher Aβ concentrations led to decreased membrane fluidity. These commonalities pointed to strong Aβ peptide-membrane interactions in all cases. Notably, time-lapsed fluorescence microscopy revealed major differences in Aβ-induced membrane morphological responses depending on SLB hydrophobic thickness. For thicker DOPC and POPC SLBs, membrane remodeling involved the formation of elongated tubule and globular structures as a passive means to regulate membrane stress depending on Aβ concentration. In marked contrast, thin DLPC SLBs were not able to accommodate extensive membrane remodeling. Taken together, our findings reveal that membrane thickness influences the membrane morphological response triggered upon Aβ adsorption.

PIKfyve activity regulates reformation of terminal storage lysosomes from endolysosomes.

The protein complex composed of the kinase PIKfyve, the phosphatase FIG4 and the scaffolding protein VAC14 regulates the metabolism of phosphatidylinositol 3,5-bisphosphate, which serves as both a signaling lipid and the major precursor for phosphatidylinositol 5-phosphate. This complex is involved in the homeostasis of late endocytic compartments, but its precise role in maintaining the dynamic equilibrium of late endosomes, endolysosomes and lysosomes remains to be determined. Here, we report that inhibition of PIKfyve activity impairs terminal lysosome reformation from acidic and hydrolase-active, but enlarged endolysosomes. Our live-cell imaging and electron tomography data show that PIKfyve activity regulates extensive membrane remodeling that initiates reformation of lysosomes from endolysosomes. Altogether, our findings show that PIKfyve activity is required to maintain the dynamic equilibrium of late endocytic compartments by regulating the reformation of terminal storage lysosomes.

Calorie restriction promotes cardiolipin biosynthesis and distribution between mitochondrial membranes

Calorie restriction (CR) has been amply demonstrated to modify mitochondrial function. However, little is known regarding the effects of this dietary regimen on mitochondrial membranes. We isolated phospholipids from rat liver mitochondria from animals on CR or ad libitum diets and found that mitochondria from ad libitum animals present an increased content of lipoperoxides and the content of cardiolipin. Cardiolipin is the main anionic phospholipid present in mitochondrial membranes, and plays a key role in mitochondrial function, signaling and stress response. Expression levels of the enzymes involved in cardiolipin biosynthesis and remodeling were quantified and found to be upregulated in CR animals. Interestingly, when mitochondrial membranes were fractionated, the outer membrane presented a higher content of cardiolipin, indicating a redistribution of this phospholipid mediated by a phospholipid scramblase in CR. This change is associated with Drp1-mediated mitochondrial fragmentation and autophagy. Overall, we find that CR promotes extensive mitochondrial membrane remodeling, decreasing oxidatively damaged lipids, and increasing cardiolipin levels and redistributing cardiolipin. These changes in membrane properties are consistent with and may be causative of changes in mitochondrial morphology, function and turnover previously found to occur in CR.

Analyzing Endosomal Docking, Fusion, Sorting, and Budding Mechanisms in Isolated Organelles.

Due to their central role in the reception and sorting of newly internalized material, early endosomes undergo extensive membrane remodeling. They dock and fuse with endocytic carrier vesicles originating from the plasma membrane, sort the internalized material in internal microdomains, and allow the budding of new carrier vesicles from their membrane, destined to fuse with the plasma membrane (recycling) or other organelles. Early endosomal compartments might also be involved in the recycling of synaptic vesicles in nerve terminals. The present protocol describes a technique allowing to assess the mechanistic and molecular aspects of the membrane remodeling processes of docking, fusion, sorting, and budding in early endosomes of neuron-like (and other) cells. It involves the fluorescent labeling and isolation of endosomal organelles, the setup of assays allowing for docking/fusion or sorting/budding in vitro, and finally the assessment and quantification of the membrane remodeling events by fluorescent microscopy. The technique can be easily manipulated by the addition of inhibitors or activators, and can be combined with other techniques, such as immunostaining and high-resolution microscopy, expanding the experimental possibilities in the investigation of early endosomal characteristics.

Clathrin localization and dynamics in Aspergillus nidulans.

Cell growth necessitates extensive membrane remodeling events including vesicle fusion or fission, processes that are regulated by coat proteins. The hyphal cells of filamentous fungi concentrate both exocytosis and endocytosis at the apex. This investigation focuses on clathrin in Aspergillus nidulans, with the aim of understanding its role in membrane remodeling in growing hyphae. We examined clathrin heavy chain (ClaH-GFP) which localized to three distinct subcellular structures: late Golgi (trans-Golgi equivalents of filamentous fungi), which are concentrated just behind the hyphal tip but are intermittently present throughout all hyphal cells; the region of concentrated endocytosis just behind the hyphal apex (the "endocytic collar"); and small, rapidly moving puncta that were seen trafficking long distances in nearly all hyphal compartments. ClaH localized to distinct domains on late Golgi, and these clathrin "hubs" dispersed in synchrony after the late Golgi marker PHOSBP . Although clathrin was essential for growth, ClaH did not colocalize well with the endocytic patch marker fimbrin. Tests of FM4-64 internalization and repression of ClaH corroborated the observation that clathrin does not play an important role in endocytosis in A. nidulans. A minor portion of ClaH puncta exhibited bidirectional movement, likely along microtubules, but were generally distinct from early endosomes.

Desmosterol Increases Lipid Bilayer Fluidity during Hepatitis C Virus Infection.

Hepatitis C virus (HCV) uniquely affects desmosterol homeostasis by increasing its intracellular abundance and affecting its localization. These effects are important for productive viral replication because the inhibition of desmosterol synthesis has an antiviral effect that can be rescued by the addition of exogenous desmosterol. Here, we use subgenomic replicons to show that desmosterol has a major effect on the replication of HCV JFH1 RNA. Fluorescence recovery after photobleaching (FRAP) experiments performed with synthetic supported lipid bilayers demonstrate that the substitution of desmosterol for cholesterol significantly increases the lipid bilayer fluidity, especially in the presence of saturated phospholipids and ceramides. We demonstrate using LC-MS that desmosterol is abundant in the membranes upon which genome replication takes place and that supported lipid bilayers derived from these specialized membranes also exhibit significantly higher fluidity compared to that of negative control membranes isolated from cells lacking HCV. Together, these data suggest a model in which the fluidity-promoting effects of desmosterol on lipid bilayers play a crucial role in the extensive membrane remodeling that takes place in the endoplasmic reticulum during HCV infection. We anticipate that the supported lipid bilayer system described can provide a useful model system in which to interrogate the effects of lipid structure and composition on the biophysical properties of lipid membranes as well as their function in viral processes such as genome replication.

Mitotic Membrane Turnover Coordinates Differential Induction of the Heart Progenitor Lineage

In response to microenvironmental cues, embryonic cells form adhesive signaling compartments that influence survival and patterning. Dividing cells detach from the surrounding matrix and initiate extensive membrane remodeling, but the in vivo impact of mitosis on adhesion-dependent signaling remains poorly characterized. We investigate in vivo signaling dynamics using the invertebrate chordate, Ciona intestinalis. In Ciona, matrix adhesion polarizes fibroblast growth factor (FGF)-dependent heart progenitor induction. Here, we show that adhesion inhibits mitotic FGF receptor internalization, leading to receptor enrichment along adherent membranes. Targeted disruption of matrix adhesion promotes uniform FGF receptor internalization and degradation while enhanced adhesion suppresses degradation. Chimeric analysis indicates that integrin β chain-specific impacts on induction are dictated by distinct internalization motifs. We also found that matrix adhesion impacts receptor enrichment through Caveolin-rich membrane domains. These results redefine the relationship between cell division and adhesive signaling, revealing how mitotic membrane turnover orchestrates adhesion-dependent signal polarization.

How to recruit membrane trafficking machinery

Structural Biology PI4KIIIβ is a lipid kinase that underlies Golgi function and is enlisted in biological responses that require rapid delivery of membrane vesicles, such as during the extensive membrane remodeling that occurs at the end of cell division. Burke et al. determined the structure of

Abstract PR12: Bringing the outside in: Macropinocytosis and cancer therapeutics

Abstract Oncogenic Ras mutations are prevalent in a variety of tumor types, including adenocarcinomas of the pancreas, colon, and lung. One of the most overt phenotypes associated with the expression of oncogenic Ras mutants is the stimulation of macropinocytosis, an endocytic process that involves extensive membrane remodeling and the internalization of extracellular fluid via large membrane-bound vesicles called macropinosomes. The functional consequences of this stimulation in oncogenic Ras-expressing cancer cells were unknown prior to our recent work where we linked macropinocytic uptake to nutrient delivery and amino acid supply in tumor cells. We found that Ras-transformed cells utilize macropinocytosis to internalize extracellular albumin, which is then lysosomally degraded releasing the constituent amino acids intracellularly. These protein-derived amino acids have the capacity to enter central carbon metabolism and fuel tumor cell proliferation even in a nutrient-depleted environment. Of particular relevance is the finding that the pharmacological inhibition of macropinocytosis compromises the growth of Ras-driven pancreatic xenograft tumors. This discovery raises the question of whether the inhibition of macropinocytosis can be utilized as a therapeutic intervention in a subset of cancers. Moreover, because macropinocytosis is an established mechanism of drug delivery for nanoparticles, our work highlights the possibility of exploiting this feature of Ras-induced tumors for the effective delivery of nanoscale therapeutics. Our recent studies focused on identifying novel modulators of macropinocytosis and examining macropinocytosis as a delivery mechanism for nab-paclitaxel, an albumin-based nanoparticle therapy, will be discussed. Citation Format: Cosimo Commisso, Venugopal Chenna, Elda Grabocka, Craig Ramirez, Maitra Anirban, Dafna Bar-Sagi. Bringing the outside in: Macropinocytosis and cancer therapeutics. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR12.

Plasma membrane tension orchestrates membrane trafficking, cytoskeletal remodeling, and biochemical signaling during phagocytosis

Phagocytes clear the body of undesirable particles such as infectious agents and debris. To extend pseudopods over the surface of targeted particles during engulfment, cells must change shape through extensive membrane and cytoskeleton remodeling. We observed that pseudopod extension occurred in two phases. In the first phase, pseudopods extended rapidly, with actin polymerization pushing the plasma membrane forward. The second phase occurred once the membrane area from preexisting reservoirs was depleted, leading to increased membrane tension. Increased tension directly altered the small Rho GTPase Rac1, 3'-phosphoinositide, and cytoskeletal organization. Furthermore, it activated exocytosis of vesicles containing GPI-anchored proteins, increasing membrane area and phagocytosis efficiency for large particles. We thus propose that, during phagocytosis, membrane remodeling, cytoskeletal organization, and biochemical signaling are orchestrated by the mechanical signal of membrane tension. These results put a simple mechanical signal at the heart of understanding immunological responses.

Mobilization of lipids and fortification of cell wall and cuticle are important in host defense against Hessian fly.

BackgroundWheat – Hessian fly interaction follows a typical gene-for-gene model. Hessian fly larvae die in wheat plants carrying an effective resistance gene, or thrive in susceptible plants that carry no effective resistance gene.ResultsGene sets affected by Hessian fly attack in resistant plants were found to be very different from those in susceptible plants. Differential expression of gene sets was associated with differential accumulation of intermediates in defense pathways. Our results indicated that resources were rapidly mobilized in resistant plants for defense, including extensive membrane remodeling and release of lipids, sugar catabolism, and amino acid transport and degradation. These resources were likely rapidly converted into defense molecules such as oxylipins; toxic proteins including cysteine proteases, inhibitors of digestive enzymes, and lectins; phenolics; and cell wall components. However, toxicity alone does not cause immediate lethality to Hessian fly larvae. Toxic defenses might slow down Hessian fly development and therefore give plants more time for other types of defense to become effective.ConclusionOur gene expression and metabolic profiling results suggested that remodeling and fortification of cell wall and cuticle by increased deposition of phenolics and enhanced cross-linking were likely to be crucial for insect mortality by depriving Hessian fly larvae of nutrients from host cells. The identification of a large number of genes that were differentially expressed at different time points during compatible and incompatible interactions also provided a foundation for further research on the molecular pathways that lead to wheat resistance and susceptibility to Hessian fly infestation.