Tunneling nanotubes (TNTs) are long intercellular connecting structures providing a special transport route between two neighboring cells. To date TNTs have been reported in different cell types including immune cells such as T-, NK, dendritic cells, or macrophages. Here we report that mature, but not immature, B cells spontaneously form extensive TNT networks under conditions resembling the physiological environment. Live-cell fluorescence, structured illumination, and atomic force microscopic imaging provide new insights into the structure and dynamics of B cell TNTs. Importantly, the selective interaction of cell surface integrins with fibronectin or laminin extracellular matrix proteins proved to be essential for initiating TNT growth in B cells. These TNTs display diversity in length and thickness and contain not only F-actin, but their majority also contain microtubules, which were found, however, not essential for TNT formation. Furthermore, we demonstrate that Ca-dependent cortical actin dynamics exert a fundamental control over TNT growth-retraction equilibrium, suggesting that actin filaments form the TNT skeleton. Non-muscle myosin 2 motor activity was shown to provide a negative control limiting the uncontrolled outgrowth of membranous protrusions. Moreover, we also show that spontaneous growth of TNTs is either reduced or increased by B cell receptor- or LPS-mediated activation signals, respectively, thus supporting the critical role of cytoplasmic Ca in regulation of TNT formation. Finally, we observed transport of various GM/GM vesicles, lysosomes, and mitochondria inside TNTs, as well as intercellular exchange of MHC-II and B7-2 (CD86) molecules which may represent novel pathways of intercellular communication and immunoregulation.

Transglutaminase-2 (TG2) is best known as a Ca 2+ -dependent cross-linking enzyme; however, some of its extracellular matrix-related functions are independent from its catalytic activity and include matrix remodeling, adhesion and migration. S100A4 belongs to the Ca 2+ -binding EF-hand S100 protein family and acts both intra-and extracellularly through binding to various partners. It regulates cell migration and its overexpression is strongly associated with metastasis and poor survival in various cancers. TG2 has recently been suggested to mediate S100A4-dependent tumor cell migration. Here we provide evidence that S100A4 is an interacting partner and also specific amine donor of TG2. TG2 incorporates a glutamine donor peptide to Lys100 in the C-terminal random coil region of S100A4. Importantly, the enzyme activity is not necessary for the interaction: S100A4 also binds to TG2 in the presence of a specific inhibitor that keeps the enzyme in an open conformation, or to an enzymatically inactive mutant. We also found that S100A4 considerably enhances TG2-mediated adhesion of A431 epithelial carcinoma cells to the extracellular matrix. This role is independent of enzyme activity and requires the open conformation of TG2. We propose that S100A4 stabilizes the open conformation of TG2, which binds to its cell surface receptor in this state and increases cell adhesion.Summary: S100A4 and transglutaminase-2 have a role in metastasis. S100A4 is an interaction partner and specific amine substrate of transglutaminase-2, promoting its open conformation and leading to enhanced cell adhesion. Studying their interaction could contribute to the better understanding of metastasis.Running title: S100A4: substrate and binding partner of transglutaminase-2

a b s t r a c tBackground: Age at the final menstrual period is of clinical and public health interest because the age at which natural menopause occurs may be a marker of ageing and health, and in general the menopausal transition increases the risk of many diseases, e.g. redistribution in the pattern of adiposity during the menopausal transition may increase risk of metabolic disease. The purpose of this research was to study the relationship between the menopausal status and body fatness. Subjects and methods: A random sample of 1932Hungarian women was studied. Body composition was estimated by body impedance analysis. In a subsample free estradiol and progesterone levels in saliva were quantified. Results: Body fat mass increased until the late 50s and then had a decrease through senescence. Premenopausal women who were much older than the median age at menopause had a higher amount of fat than their postmenopausal age-peers, while postmenopausal women, whose menopause occurred much earlier than the median age at menopause, had less fat than their premenopausal age-peers. The body fat mass in premenopausal women with low levels of sex hormones was always below the agemedian value of the menopausal status subgroups, while the body fat mass of postmenopausal women with high levels of sex hormone levels was above the age-median values. Conclusions: The analysis of body fatness in the menopausal transition revealed that (1) the rate of reproductive ageing and the body fat pattern were significantly related, and (2) body fat mass of women with unexpected levels of sex hormones was related more to their hormonal levels than to their menopausal status or their age. Thus future epidemiological screenings of women exposed to higher levels of menopause-related health risks should be expanded beyond the estimation of menopausal status based only on menstrual history to include sex hormone level assessment, as well as body composition analysis.

HIGHLIGHTSNovel protein substrate for isopeptidase activity assay of TG2 Confirmation of specific isopeptidase cleavage of new substrate ABSTRACTTransglutaminase 2 (TG2) is a ubiquitously expressed multifunctional protein with Ca 2+ -dependent transamidase activity forming protease resistant Nε-(γ-glutamyl)lysine crosslinks between proteins. It can also function as an isopeptidase cleaving the previously formed crosslinks. The biological significance of this activity has not been revealed yet mainly because of the lack of protein based method for its characterization. Here we report development of a novel kinetic method for measuring isopeptidase activity of human TG2 by monitoring decrease in the fluorescence polarisation of a protein substrate previously formed by crosslinking fluorescently labelled glutamine donor FLpepT26 to S100A4 at a specific lysine residue. The developed method could be applied to test mutant enzymes and compounds which influence isopeptidase activity of TG2.