Wenxia Song - US grants

DESCRIPTION (Adapted from Investigator's abstract): The processing and presentation of antigen by B cells to helper T cells is a central event in the initiation of T-cell dependent antibody responses. Processing involves the conversion of protein antigens that are displayed bound to the Major Histocompatibility Complex class II molecules (MHC class II) on the surfaces of B cells for the recognition by T cells. Specific B cells bind native antigen to the B cell antigen receptor (BCR) on the cell surface and the bound antigen is subsequently internalized and delivered to an appropriate subcellular compartment for processing. BCR-mediated antigen processing is highly efficient. The BCR plays at least two crucial roles in antigen processing which are: (1) to capture antigens on the cell surface and deliver them to the subcellular compartments for processing, and (2) upon cross-linking by antigen, to initiate a signal transduction cascade that regulates antigen processing. At present, the molecular basis of both functions of the BCR is not understood. The BCR is a multi-component complex consisting of an antigen binding component, the membrane immunoglobulin (mIg), and a signal transducing component, the disulfide-linked Iga/Igb heterodimer. The goal of this proposal is to define the function of BCR in antigen processing at a molecular level. The aims of this proposal are 1) to characterize the BCR-mediated antigen transport pathway, 2) to identify the discrete steps in the BCR trafficking that are regulated by BCR-mediated signal transduction, 3) to determine which components of BCR are necessary for the BCR-mediated antigen processing and 4) to identify the structural elements carried by BCR complex that are required for antigen transport and for the regulation of this transport. The results of these studies will help us to gain the ability to control antigen processing and provide the strategies for the design of effective vaccines and therapies for autoimmune diseases.

A Fluorescence Image Deconvolution-Reconstruction Microscope will be used for a variety of different applications with living cells. Use will be restricted to observations of living cells that are present singly, or in thin specimens. This microscope generates a stack of fluorescent images at different focal planes, and then employs a set of sophisticated algorithms to determine point-spread functions for sources of fluorescence in the specimen. Out-of focus noise is subtracted from the signals in the image slices, and the slices are restacked to generate a three-dimensional reconstruction of the object at high resolution. Since small, bright objects placed against a dark background are detected as spots, it is possible to image fluorescence sources that are smaller than the theoretical limit of resolution for the microscope.

During the last twenty years, developments in the design of novel indicator fluorescent probes have enabled biologists to attack formerly intractable problems in cell biology and cell physiology. The cellular processes that have been amenable to this kind of analysis include photoreception, neuronal transmission, animal development, hormonal signaling, triggered gene expression, mitotic regulation, and chemotaxis. The developments in fluorescent dye design have moved in parallel with improvements in our ability to visualize and measure low light intensity signals from small numbers of molecules in living cells, with newly-designed optical microscopes and large pixel array CCD camera detectors. It is reasonable to expect that a significant expansion of analysis of processes in living cells will result from combined developments of reporter molecules and digital imaging technologies. It seems clear that the convergence of developments in photochemistry, biochemistry, cell biology, physiology and microscopy are all about to intersect within the living cell, and when accurate assessments of changing abundance and activity of a variety of molecules can be made in vivo and through time.

This microscope will be placed in an imaging facility that provides faculty, postdocs, graduate and undergraduate students with access to several high performance microscopes equipped with the capacity to view small quantities of fluorescent reporter molecules in living cells.

[unreadable] DESCRIPTION (provided by applicant): B cell activation is tightly regulated to ensure the specificity and efficacy of the antibody responses. Abnormalities in this regulation lead to immune deficiency and autoimmune diseases. Conversely, the efficacy of a vaccine depends on its ability to activate B cell responses. The long term goal, of this project is to delineate the cellular and molecular mechanism for the initiation and regulation of B cell activation. B cell activation is initiated by the binding of antigens to the B cell antigen receptor (BCR), which induces signaling cascades and antigen internalization for processing and presentation. The BCR is unique in that it recognizes antigens in their native forms and serves as a signaling transducer as well as an antigen transporter. The BCR coordinates its two functions to achieve an optimal level of activation. An early event in B cell activation is the association of the BCR with the actin cytoskeleton and the actin cytoskeleton reorganization. Recent studies show that the actin cytoskeleton is required for BCR-mediated antigen transport and is involved in regulating BCR signaling. However, the underlying mechanism by which the actin cytoskeleton regulates BCR functions has not been well studied. Mammalian actin-binding protein 1 (mAbp1/SH3P7/HP-55) that has been shown to simultaneously interact with F-actin and proteins of the other cellular systems is one of the potential linkers that bridge the interaction between the actin cytoskeleton and the BCR. The central hypothesis of this proposal is that in response to different antigens, the BCR differentially activates the mAbp1 and induces the interaction of mAbp1 with BCR signaling and antigen- transport machineries, which links the actin cytoskeleton with BCR signaling and antigen-transport apparatus. To test this hypothesis, we propose to define the roles of mAbp1 in the signaling and antigen- transport functions of the BCR, to examine BCR-triggered activation of mAbp1, and to analyze the interaction of mAbp1 with the components of BCR signaling and antigen-transport pathways. The proposed studies combine genetic, cell biological, and biochemical approaches and aim to identify novel functions of mAbp1 in B cell activation and delineate the molecular basis for the functional interaction between the BCR and the actin cytoskeleton. These studies will increase our understanding of underlying mechanisms for regulation of B cell activation and enhance our ability to manipulate and control antibody responses. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Neisseria gonorrhoeae, an obligate human pathogen, causes a common sexually transmitted infection, gonorrhea. While gonococci (GC) cause symptomatic inflammation of the genital tissue in men, GC infections in women are often asymptomatic. The silent infection allows the bacteria to disseminate, which can lead to pelvic inflammatory disease (PID), a leading cause of ectopic pregnancy and infertility. Disseminated gonococcal infection (DGI) may give rise to septic arthritis. Pregnant women can transmit the infection to newborns, causing blindness. Furthermore, silent GC infections increase the susceptibility of patients to HIV. These complications clearly make gonorrhea a significant public health problem, particularly in women. Our long-term goal is to delineate the cellular and molecular mechanisms underlying GC infections in women. The research on the mechanistic basis of GC infection in the female reproductive tract has been hindered by a lack of effective research models. Previous studies have mainly relied on nonpolarized epithelial cells. Because the surface of the female reproductive tract is protected by a layer of polarized epithelial cells, these data can only be applicable to clinical infections f GC interacts with polarized and nonpolarized epithelial cells in the same manner. The central hypothesis to be tested in this proposal is that GC interactions with polarized epithelial cells differ significantly from GC interactions with nonpolarized cells. We have formulated this hypothesis based on our strong preliminary data that show that the actin cytoskeleton is excluded from GC adherent sites at the apical surface of polarized epithelial cells, rather than being recruited to the adherent sites as seen in nonpolarized cells. The goal of this proposal is to define the differences between GC interactions with polarized and nonpolarized endocervical epithelial cells and to investigate the mechanistic links between the unique interaction of GC with the polarized cells and GC infection. To reach this goal, we will use polarized primary and virally immortalized human endocervical epithelial cells to pursue two aims: 1) to examine the nature of GC interactions with polarized endocervical epithelial cells and 2) to investigate how the unique interactions of GC with polarized endocervical epithelia lead to GC infection. Polarized primary and virally immortalized endocervical epithelial cell models will enable us to examine GC-epithelia interactions under the conditions that mimic the in vivo conditions of the female reproductive tract and to discover novel mechanisms that are specific for GC infections in women. Our strong preliminary studies demonstrate the potential of the proposed studies to expand the existing paradigms of GC infections. New mechanistic knowledge derived from the proposed studies will help to generate new strategies for interventions and therapies for gonorrhea and other sexually transmitted infections

  Summary The rapid resealing of plasma membrane wounds is critical for cellular survival. Work supported by previous cycles of this grant showed that plasma membrane repair involves Ca2+-triggered exocytosis of lysosomes followed by massive endocytosis. Injury-induced release of lysosomal acid sphingomyelinase triggers a cholesterol/sphingolipid-dependent, clathrin-independent form of endocytosis, which involves caveolae or tubular invaginations that pinch off from the plasma membrane and carry lesions into cells for degradation. Clathrin-independent endocytosis has been described in many cell types, but there is considerable debate about how many independent pathways exist, and how they are initiated. By demonstrating that plasma membrane injury, Ca2+ influx and secretion of lysosomal acid sphingomyelinase trigger clathrin-independent endocytosis, our results have introduced much needed clarity to this field. The studies we now propose provide a unique opportunity for understanding how clathrin-independent endocytosis is regulated by plasma membrane injury, and how it promotes wound removal. Strikingly, our recent studies in B lymphocytes suggest that endocytosis-dependent plasma membrane repair and BCR-mediated B cell activation interfere with each other because of competition for lipid rafts, the cholesterol/sphingolipid-enriched plasma membrane microdomains that play a central role in clathrin-independent endocytosis and in BCR signaling and internalization after antigen capture. To understand the physiological impact of these findings, we will pursue two specific aims: 1) Characterize the injury-induced clathrin-independent form of endocytosis that promotes plasma membrane repair; 2) Examine the impact of plasma membrane wounding and repair on the regulation of B cell activation. These studies will significantly advance our understanding of clathrin-independent endocytosis and its role in plasma membrane resealing, and demonstrate how plasma membrane injury and repair regulate the function of B lymphocytes, cells that play an essential role in immune protection against infectious agents.

Neisseria gonorrhoeae causes about 800,000 new infections each year in the United States, with health-care costs approaching 2 billion dollars/year. Various surface components, including lipooligosaccharide (LOS) and opacity protein(s) (Opa(s)), are important in mediating disease. The objectives of this proposal are to understand how Opa and LOS function in disease, and determine if their interaction enhance virulence. The central hypothesis of the proposed research is that expression of specific Opa/LOS combinations promotes GC- GC interactions to produce biofilms with different disease potentiating properties and antibiotic resistance profiles. We intend to test our hypotheses by pursuing three specific aims: We will determine how Opa and LOS function cooperatively to promote bacterial-bacterial interactions, how bacterial-bacterial interactions effect the antibiotic resistance properties of GC biofilms and how bacterial aggregation influences adherence, invasion and/or transmigration. The results from our study will allow us to define how LOS and Opa variation contribute to disease pathogenesis. The impact on human health is expected to be significant, because with the new knowledge gained, we will be better positioned to understand what is needed to make a successful vaccine and what challenges we will face in developing new approaches to the treatment of gonorrhea.

Project Summary Sexually transmitted infections (STIs) are a major public health challenge and a serious women's health issue, as women can suffer severe complications from these infections: pelvic inflammatory disease (PID), infertility, and predisposition to life-threatening ectopic pregnancy. However, the majority of these infections in the female reproductive tract (FRT) are asymptomatic. How infection in the FRT causes such a wide range of clinical outcomes in the absence of symptoms remains unknown. The primary obstacle to understanding STIs of the FRT is the lack of a model that reasonably mimics all aspects of human infection. The cervix is the initiation site for STIs in the FRT. The cervical mucosa is not uniform, composed of multilayered non-polarized squamous epithelial cells at the ectocervix, a single layer of polarized columnar cells at the endocervix, and the progressively changing epithelia in the transformation zone. While tissue culture models have contributed significantly to explaining specific host-pathogen interactions, how STI pathogens deal with different epithelia for infection is unclear, as no cell culture model can mimic the varying mucosal surfaces of the human cervix. To overcome these obstacles, we are developing a new infection model using human cervical tissue explants to address our long-term goal: to delineate the mechanisms by which STI pathogens infect the FRT. To pursue this goal, this proposal focuses on the cellular mechanism by which Neisseria gonorrhoeae (GC) modulates the infection process in the human cervix. GC causes gonorrhea that is the second most common STI and a public health crisis worldwide due to the upsurge of multi-drug resistant GC. The surface molecules of GC undergo phase variation, which has been implicated in its broad infection outcomes. We hypothesize that the expression of pili and distinct variants of opacity associated proteins (Opa) allows for changes in GC infectivity, while the properties of epithelial cells of the human cervix determine which regions are vulnerable to GC infections. To test the hypothesis, we will use our human cervical tissue model and isogenic strains of GC that express invariable Opas and pili to define the cellular mechanism by which pili and Opa phase variation and the distinct properties of cervical epithelial cells regulate GC infection in the FRT. Our cervical explant model breaks a major barrier of the field, making it possible for the first time to examine cellular events occurring during GC infection to their in vivo targeted epithelial cells under a physiologically relevant environment. These studies will reveal new mechanisms that can finally explain how GC manipulate signaling and cytoskeleton based on their surface molecules and the type of epithelial cells with which they interact to switch the cervical infection between colonizing and penetrating nature. The new tissue model and infection mechanisms established by this project may fundamentally change our way to pursue the understanding of STIs and open new avenues for interventive drug designs for the prevention of STIs.