Gabriel G. Martins

Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II, depolarization and protein kinase C

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF‐2) and FGFR1, association of FGFR1 with splicing factor‐rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up‐regulation of endogenous TH protein or a transfected TH promoter‐luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK‐mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF‐2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co‐transfected HMW FGF‐2 (which is exclusively nuclear) but not by cytoplasmic FGF‐1 or extracellular FGFs. Promoter transactivation by HMWFGF‐2 was accompanied by an up‐regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK‐) but not by IP6 or suramin. The TH promoter was also transactivated by co‐transfected wild‐type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP‐/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF‐2 was mediated through the cAMP‐responsive element (CRE) and was associated with induction of CREB‐ and CBP/P‐300‐containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.

Nuclear trafficking of FGFR1: A role for the transmembrane domain

Several members of the fibroblast growth factor (FGF) family lack signal peptide (SP) sequences and are present only in trace amounts outside the cell. However, these proteins contain nuclear localization signals (NLS) and accumulate in the cell nucleus. Our studies have shown that full length FGF receptor 1 (FGFR1) accumulates within the nuclear interior in parallel with FGF‐2. We tested the hypothesis that an atypical transmembrane domain (TM) plays a role in FGFR1 trafficking into the nuclear interior. With FGFR1 destined for constitutive fusion with the plasma membrane due to its SP, how the receptor may enter the nucleus is unclear. Sequence analysis identified that FGFR1 has an atypical TM containing short stretches of hydrophobic amino acids (a.a.) interrupted by polar a.a. The β‐sheet is the predicted conformation of the FGFR1 TM, in contrast to the α‐helical conformation of other single TM tyrosine kinase receptors, including FGFR4. Receptor trafficking in live cells was studied by confocal microscopy via C‐terminal FGFR1 fusions to enhanced green fluorescent protein (EGFP) and confirmed by subcellular fractionation and Western immunoblotting. Nuclear entry of FGFR1–EGFP was independent of karyokinessis, and was observed in rapidly proliferating human TE671 cells, in slower proliferating glioma SF763 and post‐mitotic bovine adrenal medullary cells (BAMC). In contrast, a chimeric FGFR1/R4‐EGFP, where the TM of FGFR1 was replaced with that of FGFR4, was associated with membranes (golgi‐ER, plasma, and nuclear), but was absent from the nucleus and cytosol. FGFR1Δ‐EGFP mutants, with hydrophobic TM a.a. replaced with polar a.a., showed reduced association with membranes and increased cytosolic/nuclear accumulation with an increase in TM hydrophilicity. FGFR1(TM−)‐EGFP (TM deleted), was detected in the golgi‐ER vesicles, cytosol, and nuclear interior; thus demonstrating that the FGFR1 TM does not function as a NLS. To test whether cytosolic FGFR1 provides a source of nuclear FGFR1, cells were transfected with FGFR1(SP−) (SP was deleted), resulting in cytosolic, non‐membrane, protein accumulation in the cytosol and the cell nucleus. Our results indicate that an unstable association with cellular membranes is responsible for the release of FGFR1 into the cytosol and cytosolic FGFR1 constitutes the source of the nuclear receptor. J. Cell. Biochem. 88: 1273–1291, 2003.

In vitro and in vivo evaluation of electrospun nanofibers of PCL, chitosan and gelatin: a comparative study.

Many polymers have been investigated with respect to their use in skin tissue engineering. However, directly comparable data on the role played by different polymers in assisting skin wound healing requires their in vitro and in vivo evaluation under the same conditions. Therefore, we performed a study in order to compare the performance of electrospun nanofiber mats from three different polymers concerning cell-scaffold interaction and wound healing promotion. A polyester (polycaprolactone, PCL), a protein (gelatin from cold water fish skin, GEL) and a polysaccharide (chitosan, CS) were the polymers chosen. Gelatin nanofibers were crosslinked with glutaraldehyde vapor. The scaffolds were characterized physico-chemically, in vitro by seeding with human fetal fibroblasts, HFFF2, and used in vivo as skin substitutes in a rat wound model with total skin removal. In vitro tests revealed that cells adhered and proliferated in all scaffolds. However, cells deep into the scaffold were only observed in the PCL and CS scaffolds. In in vivo tests CS scaffolds had the highest impact on the healing process by decreasing the extent of wound contraction and enhancing the production of a neodermis and re-epithelialization of the wound.

OpenSpinMicroscopy: an open-source integrated microscopy platform

acKnoWLedGments We thank V. Surendranath for help with photography, H. Bellen (Baylor College of Medicine, Texas) for the Csp-sGFP transgene, and S. Singh, Sonal and S. Simmert for seeding the wiki with material during the Dresden International PhD Program course. We thank B. Cox and G. Petry of the Morgridge Institute for Research for assistance with 3D printing and parts fabrication. J.S., L.S. and K.W.E. were supported by US National Institutes of Health grants RC2GM092519 and R01CA136590. S.P. was supported by the Human Frontier Science Program Postdoctoral Fellowship. P.T. and P.G.P. were supported by the European Research Council Community’s Seventh Framework Programme (FP7/2007-2013) grant agreement 260746.

Integrin α6β1-laminin interactions regulate early myotome formation in the mouse embryo

We addressed the potential role of cell-laminin interactions during epaxial myotome formation in the mouse embryo. Assembly of the myotomal laminin matrix occurs as epaxial myogenic precursor cells enter the myotome. Most Myf5-positive and myogenin-negative myogenic precursor cells localise near assembled laminin, while myogenin-expressing cells are located either away from this matrix or in areas where it is being assembled. In Myf5nlacZ/nlacZ (Myf5-null) embryos, laminin, collagen type IV and perlecan are present extracellularly near myogenic precursor cells, but do not form a basement membrane and cells are not contained in the myotomal compartment. Unlike wild-type myogenic precursor cells, Myf5-null cells do not express the α6β1 integrin, a laminin receptor, suggesting that integrin α6β1-laminin interactions are required for myotomal laminin matrix assembly. Blockingα 6β1-laminin binding in cultured wild-type mouse embryo explants resulted in dispersion of Myf5-positive cells, a phenotype also seen in Myf5nlacZ/nlacZ embryos. Furthermore, inhibition ofα 6β1 resulted in an increase in Myf5 protein and ectopic myogenin expression in dermomyotomal cells, suggesting that α6β1-laminin interactions normally repress myogenesis in the dermomyotome. We conclude that Myf5 is required for maintaining α6β1 expression on myogenic precursor cells, and that α6β1 is necessary for myotomal laminin matrix assembly and cell guidance into the myotome. Engagement of laminin byα 6β1 also plays a role in maintaining the undifferentiated state of cells in the dermomyotome prior to their entry into the myotome.

Dynamic 3D Cell Rearrangements Guided by a Fibronectin Matrix Underlie Somitogenesis

Somites are transient segments formed in a rostro-caudal progression during vertebrate development. In chick embryos, segmentation of a new pair of somites occurs every 90 minutes and involves a mesenchyme-to-epithelium transition of cells from the presomitic mesoderm. Little is known about the cellular rearrangements involved, and, although it is known that the fibronectin extracellular matrix is required, its actual role remains elusive. Using 3D and 4D imaging of somite formation we discovered that somitogenesis consists of a complex choreography of individual cell movements. Epithelialization starts medially with the formation of a transient epithelium of cuboidal cells, followed by cell elongation and reorganization into a pseudostratified epithelium of spindle-shaped epitheloid cells. Mesenchymal cells are then recruited to this medial epithelium through accretion, a phenomenon that spreads to all sides, except the lateral side of the forming somite, which epithelializes by cell elongation and intercalation. Surprisingly, an important contribution to the somite epithelium also comes from the continuous egression of mesenchymal cells from the core into the epithelium via its apical side. Inhibition of fibronectin matrix assembly first slows down the rate, and then halts somite formation, without affecting pseudopodial activity or cell body movements. Rather, cell elongation, centripetal alignment, N-cadherin polarization and egression are impaired, showing that the fibronectin matrix plays a role in polarizing and guiding the exploratory behavior of somitic cells. To our knowledge, this is the first 4D in vivo recording of a full mesenchyme-to-epithelium transition. This approach brought new insights into this event and highlighted the importance of the extracellular matrix as a guiding cue during morphogenesis.

Bringing Dicynodonts Back to Life: Paleobiology and Anatomy of a New Emydopoid Genus from the Upper Permian of Mozambique

Dicynodontia represent the most diverse tetrapod group during the Late Permian. They survived the Permo-Triassic extinction and are central to understanding Permo-Triassic terrestrial ecosystems. Although extensively studied, several aspects of dicynodont paleobiology such as, neuroanatomy, inner ear morphology and internal cranial anatomy remain obscure. Here we describe a new dicynodont (Therapsida, Anomodontia) from northern Mozambique: Niassodon mfumukasi gen. et sp. nov. The holotype ML1620 was collected from the Late Permian K5 formation, Metangula Graben, Niassa Province northern Mozambique, an almost completely unexplored basin and country for vertebrate paleontology. Synchrotron radiation based micro-computed tomography (SRµCT), combined with a phylogenetic analysis, demonstrates a set of characters shared with Emydopoidea. All individual bones were digitally segmented allowing a 3D visualization of each element. In addition, we reconstructed the osseous labyrinth, endocast, cranial nerves and vasculature. The brain is narrow and the cerebellum is broader than the forebrain, resembling the conservative, “reptilian-grade” morphology of other non-mammalian therapsids, but the enlarged paraflocculi occupy the same relative volume as in birds. The orientation of the horizontal semicircular canals indicates a slightly more dorsally tilted head posture than previously assumed in other dicynodonts. In addition, synchrotron data shows a secondary center of ossification in the femur. Thus ML1620 represents, to our knowledge, the oldest fossil evidence of a secondary center of ossification, pushing back the evolutionary origins of this feature. The fact that the specimen represents a new species indicates that the Late Permian tetrapod fauna of east Africa is still incompletely known.

Fibronectin promotes migration, alignment and fusion in an in vitro myoblast cell model

Myogenesis is a complex process in which committed myogenic cells differentiate and fuse into myotubes that mature into the muscle fibres of adult organisms. This process is initiated by a cascade of myogenic regulatory factors expressed upon entry of the cells into the myogenic differentiation programme. However, external signals such as those provided by the extracellular matrix (ECM) are also important in regulating muscle differentiation and morphogenesis. In the present work, we have addressed the role of various ECM substrata on C2C12 myoblast behaviour in vitro. Cells grown on fibronectin align and fuse earlier than cells on laminin or gelatine. Live imaging of C2C12 myoblasts on fibronectin versus gelatine has revealed that fibronectin promotes a directional collective migratory behaviour favouring cell-cell alignment and fusion. We further demonstrate that this effect of fibronectin is mediated by RGD-binding integrins expressed on myoblasts, that N-cadherin contributes to this behaviour, and that it does not involve enhanced myogenic differentiation. Therefore, we suggest that the collective migration and alignment of cells seen on fibronectin leads to a more predictable movement and a positioning that facilitates subsequent fusion of myoblasts. This study highlights the importance of addressing the role of fibronectin, an abundant component of the interstitial ECM during embryogenesis and tissue repair, in the context of myogenesis and muscle regeneration.

In vitro evaluation of crosslinked electrospun fish gelatin scaffolds.

Gelatin from cold water fish skin was electrospun, crosslinked and investigated as a substrate for the adhesion and proliferation of cells. Gelatin was first dissolved in either water or concentrated acetic acid and both solutions were successfully electrospun. Cross-linking was achieved via three different routes: glutaraldehyde vapor, genipin and dehydrothermal treatment. Solutions properties (surface tension, electrical conductivity and viscosity) and scaffolds properties (chemical bonds, weight loss and fiber diameters) were measured. Cellular viability was analyzed culturing 3T3 fibroblasts plated on the scaffolds and grown up to 7 days. The cells were fixed and observed with SEM or stained for DNA and F-actin and observed with confocal microscopy. In all scaffolds, the cells attached and spread with varying degrees. The evaluation of cell viability showed proliferation of cells until confluence in scaffolds crosslinked by glutaraldehyde and genipin; however the rate of growth in genipin crosslinked scaffolds was slow, recovering only by day five. The results using the dehydrothermal treatment were the less satisfactory. Our results show that glutaraldehyde treated fish gelatin is the most suitable substrate, of the three studied, for fibroblast adhesion and proliferation.

Evaluation of nanofibrous scaffolds obtained from blends of chitosan, gelatin and polycaprolactone for skin tissue engineering

Polymer blending is a strategy commonly used to obtain hybrid materials possessing properties better than those of the individual constituents regarding their use in scaffolds for Tissue Engineering. In the present work, the scaffolds produced by electrospinning solutions of polymeric blends obtained using a polyester (polycaprolactone, PCL), a polysaccharide (chitosan, CS) and a protein (gelatin extracted from cold water fish skin, GEL), were investigated. Solutions conductivity, shear viscosity and surface tension were determined. GEL-containing scaffolds were crosslinked with vapour phase glutaraldehyde (GTA). The scaffolds were characterized physico-chemically regarding fibre morphology, porosity, water contact angle, mechanical properties, chemical bonds and fibre and dimensional stability upon immersion in water and cell culture medium. The scaffolds were further tested in vitro for cell adhesion, growth and morphology of human foetal fibroblasts (cell line HFFF2). Results show that the nanofibrous scaffolds are hydrophilic and display the typical porosity of non-woven fibre mats. The CS/PCL and CS/PCL/GEL scaffolds have the highest elastic modulus (48MPa). Dimensional stability is best for the CS/PCL/GEL scaffolds. FTIR spectra confirm the occurrence of cross-linking reactions of GTA with both GEL and CS. Cell adhesion ratio ranked from excellent (close to 100%) to satisfactory (around 50%) in the order PCL/GEL>CS/GEL>CS/PCL/GEL>CS/PCL. Cell populations show an extended lag phase in comparison with the controls but cell proliferation occurs on all scaffolds until confluence is reached. In conclusion, all scaffolds studied possess characteristics that enable them to be used in skin tissue engineering but the CS/PCL/GEL scaffolds have better physical properties whereas the PCL/GEL scaffolds support a higher cell adhesion.