The Rho GTPases are related to the Ras proto-oncogenes and consist of 22 family members. These proteins have important roles in regulating the organization of the actin filament system, and thereby the morphogenesis of vertebrate cells as well as their ability to migrate. In an effort to compare the effects of all members of the Rho GTPase family, active Rho GTPases were transfected into porcine aortic endothelial cells and the effects on the actin filament system were monitored. Cdc42, TCL (TC10-like), Rac1-Rac3 and RhoG induced the formation of lamellipodia, whereas Cdc42, Rac1 and Rac2 also induced the formation of thick bundles of actin filaments. In contrast, transfection with TC10 or Chp resulted in the formation of focal adhesion-like structures, whereas Wrch-1 induced long and thin filopodia. Transfection with RhoA, RhoB or RhoC induced the assembly of stress fibres, whereas Rnd1-Rnd3 resulted in the loss of stress fibres, but this effect was associated with the formation of actin- and ezrin-containing dorsal microvilli. Cells expressing RhoD and Rif had extremely long and flexible filopodia. None of the RhoBTB or Miro GTPases had any major influence on the organization of the actin filament system; instead, RhoBTB1 and RhoBTB2 were present in vesicular structures, and Miro-1 and Miro-2 were present in mitochondria. Collectively, the data obtained in this study to some extent confirm earlier observations, but also allow the identification of previously undetected roles of the different members of the Rho GTPases.
Fluorescent proteins (FPs) have proven to be valuable tools for high-resolution imaging studies of vesicle transport processes, including exo- and endocytosis. Since the pH of the vesicle lumen changes between acidic and neutral during these events, pH-sensitive FPs with near neutral pKa, such as pHluorin, are particularly useful. FPs with pKa>6 are readily available in the green spectrum, while red-emitting pH-sensitive FPs are rare and often not well characterized as reporters of exo- or endocytosis. Here we tested a panel of ten orange/red and two green FPs in fusions with neuropeptide Y (NPY) for use as secreted vesicle marker and reporter of dense core granule exocytosis and release. We report relative brightness, bleaching rate, targeting accuracy, sensitivity to vesicle pH, and their performance in detecting exocytosis in live cells. Tandem dimer (td)-mOrange2 was identified as well-targeted, bright, slowly bleaching and pH-sensitive FP that performed similar to EGFP. Single exocytosis events were readily observed, which allowed measurements of fusion pore lifetime and the dynamics of the exocytosis protein syntaxin at the release site during membrane fusion and cargo release.
Background
Regulatory peptides have previously been detected in epithelial cells of human mammary glands. As these peptides are produced by scattered neuroendocrine cells in the epithelium of other tissues the aim of this study was to investigate whether the mammary glands express molecular markers for neuroendocrine cells.
Material and methods
Specimens from 28 human mammary glands were retrieved. The distribution of immunoreactive cells was determined using immunohistochemistry with antibodies versus a set of endocrine markers including peptide hormones, chromogranins/secretogranins, vesicular monoamine transporters, synaptophysin, serotonin and synaptic vesicle protein 2.
Results
Cells of the luminal epithelium of ducts and lobules of human mammary glands expressed vesicular monoamine transporter 2 and chromogranin B, as well as the previously reported regulatory peptides obestatin, ghrelin, adrenomedullin and apelin. Using consecutive sections, it was revealed that the immunoreactivity patterns of the regulatory peptides and vesicular monoamine transporter 2 were similar. Interestingly, immunoreactivity for secretogranin II, secretogranin III and chromogranin B was identified in myoepithelial cells. No immunoreactivity was detected for chromogranin A or synaptophysin.
Conclusion
Specific cells in the epithelium and myoepithelium of mammary glands express neuroendocrine markers suggesting that mammary glands may have neuroendocrine functions.
The peptide hormone somatostatin, as well as the somatostatin analog octreotide, induces rapid morphological changes in neuroendocrine cells. The effect can be detected in less than 2min: retraction fibers are formed, cells round up and cell–cell contacts are broken. Somatostatin-dependent cell contraction is inhibited by Y-27632, indicating that this effect is dependent on Rho kinase. In BON1 cells, the somatostatin-induced inhibition of forskolin-induced secretion of chromogranin A is not blocked by Y-27632. It is therefore concluded that the inhibitory effect of somatostatin in forskolin-stimulated cells is not dependent on cell contraction.
The Rho family of small GTPases controls many biological processes, including cytoskeletal regulation, membrane trafficking, cell adhesion, cell polarization, transcriptional activity, apoptosis, and cell proliferation. Wrch1, which belongs to the Cdc42 subfamily, is one of the least characterized family member. Despite its homology to other Cdc42-like proteins, we found that Wrch1 has unique characteristics. Biochemical experiments showed that Wrch1 has no detectable GTPase activity in vitro and that its intrinsic nucleotide exchange rate is very high in comparison to Cdc42. Furthermore, NIH3T3 cells transiently transfected with Wrch1 showed an up-rounded, retracted phenotype. In addition, Wrch1 was shown to be more efficient than Cdc42 in triggering the formation of filopodia. Serum stimulation of cells expressing Wrch1 induces vigorous membrane blebbing, a phenomenon dependent on the activity of ROCK. In a search for proteins interacting with Wrch1, PAK1 and NCKbeta were identified as binding partners. Interestingly, the interaction to NCKbeta was shown to be mediated via PxxP motifs present in an N-terminal extension of Wrch1 to the second and third SH3 domains of NCKbeta.
Objective:
Obestatin and ghrelin are derived from the same gene and co-expressed in the same endocrine cells. Vesicular monoamine transporter-2 (VMAT-2), a marker for enterochromaffin-like (ECL) cells, is considered to be expressed in ghrelin cells. The aim was to establish if the two peptides and the transporter are co-expressed, both in normal gastric mucosa and in gastric endocrine tumours.
Design:
An immunohistochemical study was performed on gastric biopsy material and on surgical specimens from 63 patients with gastric endocrine tumours and from individuals with normal gastric mucosa. Cells displaying obestatin immunoreactivity were examined regarding co-localization with ghrelin and VMAT-2. Both single- and double-immunostaining techniques were applied. Obestatin concentration in blood was measured in a subgroup of these patients. The results were correlated to various clinico-pathological parameters.
Results:
In the normal mucosa, obestatin/ghrelin-immunoreactive cells rarely co-expressed VMAT-2. In most tumour tissue specimens, only a fraction of neoplastic cells displayed immunoreactivity to obestatin, and these cells always co-expressed ghrelin. Neoplastic obestatin-/ ghrelin-IR cells invariably expressed VMAT-2, except for two ghrelinomas. The obestatin concentrations in blood were consistently low and did not correlate to clinico-pathological data.
Conclusions:
Obestatin and ghrelin immunoreactivity always occurred in the same endocrine cells in the gastric mucosa but these cells only occasionally co-expressed VMAT-2, opposite to the findings in tumours. These results indicate that endocrine cells expressing obestatin and ghrelin mainly differ from VMAT-2 expressing cells (ECL-cells) and can develop into pure ghrelinomas. Plasma concentrations of obestatin did not correlate to cellular expression.
Syntaxin (stx)-1 is an integral plasma membrane protein that is crucial for two distinct steps of regulated exocytosis, docking of secretory granules at the plasma membrane and membrane fusion. During docking, stx1 clusters at the granule docking site, together with the S/M protein munc18. Here we determined features of stx1 that contribute to its clustering at granules. In live insulin-secreting cells, stx1 and stx3 (but not stx4 or stx11) accumulated at docked granules, and stx1 (but not stx4) rescued docking in cells expressing botulinum neurotoxin-C. Using a series of stx1 deletion mutants and stx1/4 chimeras, we found that all four helical domains (Ha, Hb, Hc, SNARE) and the short N-terminal peptide contribute to recruitment to granules. However, only the Hc domain confers specificity, and it must be derived from stx1 for recruitment to occur. Point mutations in the Hc or the N-terminal peptide designed to interfere with binding to munc18-1 prevent stx1 from clustering at granules, and a mutant munc18 deficient in binding to stx1 does not cluster at granules. We conclude that stx1 is recruited to the docking site in a munc18-1-bound conformation, providing a rationale for the requirement for both proteins for granule docking.