Uni-directional ciliary membrane protein trafficking by a cytoplasmic retrograde IFT motor and ciliary ectosome shedding

The role of the primary cilium in key signaling pathways depends on dynamic regulation of ciliary membrane protein composition, yet we know little about the motors or membrane events that regulate ciliary membrane protein trafficking in existing organelles. Recently, we showed that cilium-generated signaling in Chlamydomonas induced rapid, anterograde IFT-independent, cytoplasmic microtubule-dependent redistribution of the membrane polypeptide, SAG1-C65, from the plasma membrane to the periciliary region and the ciliary membrane. Here, we report that the retrograde IFT motor, cytoplasmic dynein 1b, is required in the cytoplasm for this rapid redistribution. Furthermore, signaling-induced trafficking of SAG1-C65 into cilia is unidirectional and the entire complement of cellular SAG1-C65 is shed during signaling and can be recovered in the form of ciliary ectosomes that retain signal-inducing activity. Thus, during signaling, cells regulate ciliary membrane protein composition through cytoplasmic action of the retrograde IFT motor and shedding of ciliary ectosomes

Endothelial and cancer cells interact with mesenchymal stem cells via both microparticles and secreted factors

Tightly associated with blood vessels in their perivascular niche, human mesenchymal stem cells (MSCs) closely interact with endothelial cells (ECs). MSCs also home to tumours and interact with cancer cells (CCs). Microparticles (MPs) are cell-derived vesicles released into the extracellular environment along with secreted factors. MPs are capable of intercellular signalling and, as biomolecular shuttles, transfer proteins and RNA from one cell to another. Here, we characterize interactions among ECs, CCs and MSCs via MPs and secreted factors in vitro. MPs and non-MP secreted factors (Sup) were isolated from serum-free medium conditioned by human microvascular ECs (HMEC-1) or by the CC line HT1080. Fluorescently labelled MPs were prepared from cells treated with membrane dyes, and cytosolic GFP-containing MPs were isolated from cells transduced with CMV-GFP lentivirus. MSCs were treated with MPs, Sup, or vehicle controls, and analysed for MP uptake, proliferation, migration, activation of intracellular signalling pathways and cytokine release. Fluorescently labelled MPs fused with MSCs, transferring the fluorescent dyes to the MSC surface. GFP was transferred to and retained in MSCs incubated with GFP-MPs, but not free GFP. Thus, only MP-associated cellular proteins were taken up and retained by MSCs, suggesting that MP biomolecules, but not secreted factors, are shuttled to MSCs. MP and Sup treatment significantly increased MSC proliferation, migration, and MMP-1, MMP-3, CCL-2/MCP-1 and IL-6 secretion compared with vehicle controls. MSCs treated with Sup and MPs also exhibited activated NF-κB signalling. Taken together, these results suggest that MPs act to regulate MSC functions through several mechanisms

Proteomic profiling reveals the transglutaminase-2 externalization pathway in kidneys after unilateral ureteric obstruction

Increased export of transglutaminase-2 (TG2) by tubular epithelial cells (TECs) into the surrounding interstitium modifies the extracellular homeostatic balance, leading to fibrotic membrane expansion. Although silencing of extracellular TG2 ameliorates progressive kidney scarring in animal models of CKD, the pathway through which TG2 is secreted from TECs and contributes to disease progression has not been elucidated. In this study, we developed a global proteomic approach to identify binding partners of TG2 responsible for TG2 externalization in kidneys subjected to unilateral ureteric obstruction (UUO) using TG2 knockout kidneys as negative controls. We report a robust and unbiased analysis of the membrane interactome of TG2 in fibrotic kidneys relative to the entire proteome after UUO, detected by SWATH mass spectrometry. The data have been deposited to the ProteomeXchange with identifier PXD008173. Clusters of exosomal proteins in the TG2 interactome supported the hypothesis that TG2 is secreted by extracellular membrane vesicles during fibrosis progression. In established TEC lines, we found TG2 in vesicles of both endosomal (exosomes) and plasma membrane origin (microvesicles/ectosomes), and TGF-β1 stimulated TG2 secretion. Knockout of syndecan-4 (SDC4) greatly impaired TG2 exosomal secretion. TG2 coprecipitated with SDC4 from exosome lysate but not ectosome lysate. Ex vivo, EGFP-tagged TG2 accumulated in globular elements (blebs) protruding/retracting from the plasma membrane of primary cortical TECs, and SDC4 knockout impaired bleb formation, affecting TG2 release. Through this combined in vivo and in vitro approach, we have dissected the pathway through which TG2 is secreted from TECs in CKD

Bioinspiring Chondrosia reniformis (Nardo, 1847) collagen-based hydrogel: a new extraction method to obtain a sticky and self-healing collagenous material

Collagen is a natural and abundant polymer that serves multiple functions in both invertebrates and vertebrates. As collagen is the natural scaffolding for cells, collagen-based hydrogels are regarded as ideal materials for tissue engineering applications since they can mimic the natural cellular microenvironment. Chondrosia reniformis is a marine demosponge particularly rich in collagen, characterized by the presence of labile interfibrillar crosslinks similarly to those described in the mutable collagenous tissues (MCTs) of echinoderms. As a result single fibrils can be isolated using calcium-chelating and disulphide-reducing chemicals. In the present work we firstly describe a new extraction method that directly produces a highly hydrated hydrogel with interesting self-healing properties. The materials obtained were then biochemically and rheologically characterized. Our investigation has shown that the developed extraction procedure is able to extract collagen as well as other proteins and Glycosaminoglycans (GAG)-like molecules that give the collagenous hydrogel interesting and new rheological properties when compared to other described collagenous materials. The present work motivates further in-depth investigations towards the development of a new class of injectable collagenous hydrogels with tailored specifications.The authors gratefully acknowledge the financial support from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement ERC-2012-ADG 20120216-321266 (ERC Advanced Grant project ComplexiTE), as well as from the European Regional Development Fund (ERDF) under the projects “Accelerating tissue engineering and personalized medicine discoveries by the integration of key enabling nanotechonologies, marine-derived biomaterials and stem cells” (NORTE-01-0145-FEDER-000021), supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, and 0687_NOVOMAR_1_P, co-financed by transborder cooperation programme POCTEP. The authors are also thankful to the Area Marina Protetta Portofino (Italy) for permission to collect sponge specimens and to Daniela Candia (University of Milan, Italy) and Marco Giovine (University of Genoa, Italy) for the logistical support on the sponge sampling and immediate processing. We are grateful to the two anonymous referees for improving the quality of the present article.info:eu-repo/semantics/publishedVersio

Ectosomes as immunomodulators

Considerable progress has been made in recognizing microvesicles as important mediators of intercellular communication rather than irrelevant cell debris. Microvesicles released by budding directly from the cell membrane surface (i.e., ectocytosis) either spontaneously or in response to various stimuli are called shed vesicles or ectosomes. Ectosomes are rightside-out vesicles with cytosolic content, and they expose phosphatidylserine in the outer leaflet of their membrane. Depending on their cellular origin, ectosomes have been associated with a broad spectrum of biological activities. In the light of recent findings, we now know that ectosomes derived from polymorphonuclear leukocytes, erythrocytes, platelets, and tumor cells have profound effects on the innate immune system, as well as on the induction of the adaptive immunity, globally reprogramming cells such as macrophages or dendritic cells toward an immunosuppressive and possibly tolerogenic phenotype. Although the effects observed in the circulation are mainly procoagulant and pro-inflammatory, ectosomes might be anti-inflammatory/immunosuppressive in local inflammatio

Endosymbiotic calcifying bacteria across sponge species and oceans

Este artículo contiene 14 páginas, 7 figuras, 1 tabla.From an evolutionary point of view, sponges are ideal targets to study marine symbioses as they are the most ancient living metazoans and harbour highly diverse microbial communities. A recently discovered association between the sponge Hemimycale columella and an intracellular bacterium that generates large amounts of calcite spherules has prompted speculation on the possible role of intracellular bacteria in the evolution of the skeleton in early animals. To gain insight into this purportedly ancestral symbiosis, we investigated the presence of symbiotic bacteria in Mediterranean and Caribbean sponges. We found four new calcibacteria OTUs belonging to the SAR116 in two orders (Poecilosclerida and Clionaida) and three families of Demospongiae, two additional OTUs in cnidarians and one more in seawater (at 98.5% similarity). Using a calcibacteria targeted probe and CARD-FISH, we also found calcibacteria in Spirophorida and Suberitida and proved that the calcifying bacteria accumulated at the sponge periphery, forming a skeletal cortex, analogous to that of siliceous microscleres in other demosponges. Bacteria-mediated skeletonization is spread in a range of phylogenetically distant species and thus the purported implication of bacteria in skeleton formation and evolution of early animals gains relevance.The research has been funded by MARSYMBIOMICS project (Spanish MINECO, CTM2013-43287-P) BluePharmTrain (FP7 People-INT, Ref. 2013- 667786), and Grup Consolidat SGR-120, to MJU. LG benefited from a fellowship within the Benthomics project (Spanish MICINN, CTM-2010-22218-C02-01).Peer reviewe

The genus <i>Batzella</i>: a chemosystematic problem

Biogenetically unrelated cyclic guanidine alkaloids and pyrroloquinoline alkaloids have been reported from sponges assigned to the genus Batzella. These sponges have been assigned to this genus because of their possession of a simple complement of thin strongyles in irregular plumoreticulate arrangement. Cyclic guanidine alkaloids were first reported from an alleged axinellid species from the Caribbean, Ptilocaulis aff. P. spiculifer, and subsequently from a second Carribean specimen identified as Ptilocaulis spiculifer and at the same time from a Red Sea poecilosclerid, Hemimycale sp. Closely related compounds were described from a Caribbean specimen identified as Batzella sp. and also from the poecilosclerids Crambe crambe (Mediterranean) and Monanchora arbuscula (Brazil). Isobatzellins (pyrroloquinoline alkaloids) were reported from a black deep-water species from the Bahamas identified as Batzella sp. Chemically related pyrroloquinoline alkaloids were found in Pacific representatives of the fistular poecilosclerid genus Zyzzya, the hadromerid genus Latrunculia and the ?haplosclerid genus Prianos. Most of the voucher specimens involved in this puzzle were re-examined and several conclusions can be drawn: when inspected closely it appears, that the cyclic guanidine alkaloids are produced by sponges containing anisostrongyles, often in two categories, a thicker and a thinner one. Monanchora arbuscula, which has been recently discovered to produce these compounds, has monactinal spicules differentiated into a thinner subtylostyle and a thicker (tylo-) style, but many specimens have anisostrongylote modifications. Microscleres in Monanchora can be absent or very rare. By association, all the sponges from which cyclic guanidine alkaloids are known may be united in one family, possibly in a single wider defined genus Monanchora. However, further relationships with Crambe need to be studied. Both have cyclic guanidine alkaloids, both have megascleres of very variable shapes and thickness, differentiated mostly into two overlapping categories, microscleres and other additional spicules are often rare or absent. Relationships with the type of Hemimycale, viz. H. columella remain obscure, but in view of the much larger spicules of that species and the intricate ectosomal specialization (lacking in the above mentioned specimens) it is possible that similarities between the Red Sea Hemimycale and the European species are the product of parallel evolution. The strongyles of sponges producing pyrroloquinoline alkaloids are perfect isostrongyles and in the ectosome these are arranged in a definite ectosomal tangential crust. A good proportion of these strongyles have a faint spination on the apices. Assignment of these sponges to Batzella rest on the properties of its type species Batzella inops. Examination of a type spicule slide of that species did not solve that question, but until further notice Batzella may be used for the deep-water material. A further unsolved problem that remains is the phylogenetic relationships of Batzella with Zyzzya and Latrunculia. The likelyhoods of possible causes for this distribution of compounds are discussed

Memoirs of the Queensland Museum

v.38:pt.2 (1995

Immune complexes in chronic Chagas disease patients are formed by exovesicles from Trypanosoma cruzi carrying the conserved MASP N-terminal region

The exovesicles (EVs) are involved in pathologic host-parasite immune associations and have been recently used as biomarkers for diagnosis of infectious diseases. The release of EVs by Trypanosoma cruzi, the causative agent of Chagas disease, has recently been described, with different protein cargoes including the MASP multigene family of proteins MASPs are specific to this parasite and characterized by a conserved C-terminal (C-term) region and an N-terminal codifying for a signal peptide (SP). In this investigation, we identified immature MASP proteins containing the MASP SP in EVs secreted by the infective forms of the parasite. Those EVs are responsible for the formation of immune complexes (ICs) containing anti-MASP SP IgGs in patients with different (cardiac, digestive and asymptomatic) chronic Chagas disease manifestations. Moreover, purified EVs as well as the MASP SP inhibit the action of the complement system and also show a significant association with the humoral response in patients with digestive pathologies. These findings reveal a new route for the secretion of MASP proteins in T. cruzi, which uses EVs as vehicles for immature and misfolded proteins, forming circulating immune complexes. Such complexes could be used in the prognosis of digestive pathologies of clinical forms of Chagas disease.Fil: Díaz Lozano, Isabel María. Universidad de Granada; EspañaFil: De Pablos, Luis Miguel. Universidad de Granada; España. University Of York;Fil: Longhi, Silvia Andrea. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires; ArgentinaFil: Osuna, Antonio. Universidad de Granada; Españ