Tetraspanins constitute a family of widely expressed integral membrane proteins that associate extensively with one another and with other membrane proteins to form specific membrane microdomains distinct from conventional lipid rafts. So far, because of the lack of appropriate tools, the functionality of these microdomains has remained largely unknown. Here, using a new monoclonal antibody that only binds to the tetraspanin CD81 associated with other tetraspanins, we show that membrane cholesterol contributes to the organization of tetraspanin microdomains on the surface of live cells. Furthermore, our data demonstrate involvement of host membrane cholesterol during infection by Plasmodium yoelii and Plasmodium falciparum sporozoites, which both depend on host CD81 expression for invasion, but not during CD81-independent infection by Plasmodium berghei sporozoites. Our results unravel a functional link between CD81 and cholesterol during infection by malaria parasites, and illustrate that tetraspanin microdomains constitute a novel type of membrane microdomains that could be used by pathogens for infection.

The migration of polymorphonuclear leukocytes from the blood to sites of infection in tissues is a hallmark of the innate immune response. Formylated peptides produced as a byproduct of bacterial protein synthesis are powerful chemoattractants for leukocytes. Formyl peptides bind to two different G protein-coupled receptors (formyl peptide receptor (FPR) and the low affinity formyl peptide receptor-like-1 (FPRL1)) to initiate a signal transduction cascade leading to cell activation and migration. Our analysis of expressed sequences from many cDNA libraries draws attention to the fact that FPRs are widely expressed in nonlymphoid tissues. Here we demonstrate that FPRs are expressed by normal human lung and skin fibroblasts and the human fibrosarcoma cell line HT-1080. The expression on fibroblasts of receptors for bacteria-derived peptides raises questions about the possible function of these receptors in nonleukocyte cells. We studied the function of FPRs on fibroblasts and find that stimulation with fMLP triggers dose-dependent migration of these cells. Furthermore, fMLP induces signal transduction including intracellular calcium flux and a transient increase in F-actin. The fMLP-induced adhesion and motility of fibroblasts on fibronectin require functional protein kinase C and phosphatidylinositol 3-kinase. This first report of a functional formyl peptide receptor in cells of fibroblast origin opens new possibilities for the role of fibroblasts in innate immune responses.

CD81 exerts a range of interesting effects on T cells including early thymocyte differentiation, LFA-1 activation, and provision of costimulation. To better understand the mechanisms by which CD81 influences T cell function we evaluated CD81 molecular complexes on T cells. The most prominent CD81-associated cell surface protein on thymocytes as well as a number of T cell and B cell lines has an apparent molecular mass of 75 kDa. The 75-kDa protein was purified and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry followed by postsource-decay profiling. p75 is a novel type I transmembrane protein of the Ig superfamily which is most similar to FPRP. We cloned and sequenced both human and mouse PG regulatory-like protein (PGRL) and characterized mouse PGRL expression in both lymphocytes and nonlymphoid tissues. The discovery of PGRL allows for the clustering of a small family of related proteins including PGRL, FPRP, V7/CD101, and IGSF3. Expression constructs containing various domains of PGRL with an epitope tag were coexpressed with CD81 and used to determine that the interaction of CD81 with PGRL requires the membrane distal Ig3–Ig4 domains of PGRL. Although it remains to be determined whether PGRL possesses PG regulatory functions, transwell chamber experiments show that PGs and CD81 coordinately regulate T cell motility.

Alternatives and/or supplements to animal dissection are being explored by educators of human anatomy at different academic levels. Clay modeling is one such alternative that provides a kinesthetic, three-dimensional, constructive, and sensory approach to learning human anatomy. The present study compared two laboratory techniques, clay modeling of human anatomy and dissection of preserved cat specimens, in the instruction of muscles, peripheral nerves, and blood vessels. Specifically, we examined the effect of each technique on student performance on low-order and high-order questions related to each body system as well as the student-perceived value of each technique. Students who modeled anatomic structures in clay scored significantly higher on low-order questions related to peripheral nerves; scores were comparable between groups for high-order questions on peripheral nerves and for questions on muscles and blood vessels. Likert-scale surveys were used to measure student responses to statements about each laboratory technique. A significantly greater percentage of students in the clay modeling group "agreed" or "strongly agreed" with positive statements about their respective technique. These results indicate that clay modeling and cat dissection are equally effective in achieving student learning outcomes for certain systems in undergraduate human anatomy. Furthermore, clay modeling appears to be the preferred technique based on students' subjective perceptions of value to their learning experience.

As a member of the tetraspanin superfamily of proteins, CD81 has been linked to a number of biologic functions including cellular proliferation, differentiation, activation, and degranulation. As a co-receptor for hepatitis C virus, and a requirement for hepatocytes for infectivity of human Plasmodium falciparum and rodent P. yoelii sporozoite infectivity, CD81 may also play a vital role in pathology. Despite the importance of CD81 in multiple cellular functions, the molecular mechanism of action of CD81 in these processes has remained elusive. Here we report an association between CD81 and the epsilon isoform of 14-3-3, a serine/threonine-binding intracellular signaling protein. Furthermore, we provide evidence that in human, this association is influenced by the palmitoylation state of the CD81 cytoplasmic tails. We have generated a series of CD81 cysteine mutants to identify palmitoylated intracellular motifs of CD81, and reveal palmitoylation on the N-and C-terminal tails as well as the intracellular loop between transmembrane domains 2 and 3. One of these mutants lacks all five of its intracellular cysteines and therefore cannot be palmitoylated. This unpalmitoylated version of CD81 shows constitutive association with 14-3-3. Interestingly, we find that under oxidative conditions, CD81 palmitoylation is inhibited and that condition correlates with the association of CD81 and 14-3-3. These finding suggest that CD81 signaling events could be mediated by 14-3-3 adapter proteins, and these signals may be dependent on cellular redox.CD81 is a member of the tetraspanin family of integral membrane proteins. CD81 was first discovered in 1990 as the target of an antibody with reversible antiproliferative effects on a human B cell lymphoma line (1-4). There are currently 26 membrane proteins classified as members of the tetraspanin family. Members must meet the following criteria: four highly conserved hydrophobic transmembrane domains, two extracellular loops, short intracellular amino and carboxyl tails, and three motifs, CCG, PXSC, and EGC, that contain conserved cysteine residues in the major extracellular domain (5). The smaller extracellular loop is between transmembrane 1 and 2, and the larger extracellular loop, which is likely to mediate interactions with other cell surface proteins, lies between transmembrane 3 and 4.Many of the initial discoveries of CD81 function were focused in the immune system. On the surface of B cells, CD81 is associated with the CD19⅐CD21⅐Leu 13 protein complex (6 -8). This complex is involved in regulation of B cell receptor signal transduction through recruitment of phosphatidylinositol 3-kinase (PI3K). The CD21⅐CD19⅐CD81 complex functions by bringing together required signaling molecules to lower the B cell threshold of activation (9, 10). Similarly, on T cells, CD81 associates with the T cell co-receptors CD4 and CD8 and provides a costimulatory signal with the CD3 subunit of the T cell receptor (11). CD81 may play a role in early T cell development in the thymus (12). The roles...