Thermal adaptation of A. pompejana fibrillar collagen

Abstracts sponse to a recFN (FNDIII1-7) lacking the first 7 type III repeats. In the present study, we examined the effect of different FN matrices on the growth of two transformed cell lines, CHOa5 and SVT2. CHOa5 cells assemble no endogenous FN matrix while SVT2 cells assemble some endogenous fibrils. Proliferation rates of both cell types decreased during assembly of FNDIII1-7. In addition, initiation of DNA synthesis, measured by BrdU incorporation, was delayed in CHOa5 and SVT2 cells with FNDIII1-7 matrix. Addition of exogenous native FN to CHOa5 cells increased the growth rate and decreased the amount of time required for initiation of DNA synthesis. Growth rates of cells lacking FN matrix were consistently intermediate to that of cells with native FN and FNDIII1-7. Therefore, in these transformed cell lines, native FN matrix stimulates growth while FNDIII1-7 matrix inhibits it. Synchronization with hydroxyurea demonstrated that S and G2/M phase progression are unaltered by FN matrix. The effect on cell growth required the assembly of matrix as proliferation rates of cells plated on native FN and FNDIII1-7 coated dishes were identical. Furthermore, the addition of the N- terminal 70kD fragment of FN which inhibits matrix assembly but not FN-integrin interactions blocked the effects of both FN and FNDIII1-7 matrices on cell growth. These results suggest that the structure of FN matrix plays an important role in controlling cell proliferation.

Thermal Adaptation of A. pompejana Fibrillar Collagen E-X. Sicot ~, M. Masselot 2, J.-Y. Exposito 3, R. Garrone 3, J. Deutsch ~, F. GailP ~Department of Dermatology, Thomas Jefferson University, Philadelphia PA 19107, '-Evolution Moleculaire and Biologie Marine, Universite Paris VI, Paris 75005, France, and qnstitut de Biologie et Chimie des Proteines, Lyon 69007, France The hydrothermal environment is harsh, considering the pressure (260 atmospheres), temperature (350 °C) and toxicity of the hydrothermal fluid (acidic, anoxic, and rich in metallic sulfides). The hot pole of the hydrothermal communities is characterized by a certain type of annelids, the Pompeii worms (Alvinella pompejana), which live on the surface of hydrothermal vents. Capable of withstanding temperatures up to 100 °C, Alvinella are probably the most thermophilic amongst

161

invertebrates, and maybe even eukaryotes. Whereas the collagen of coastal polychaetous annelids (Arenicola marina) is denatured at 28 °C, the collagen of A. pompejana remains stable at 45 °C and is thus the most thermostable among fibrillar collagens knmvn up to now. The preceding biochemical analysis having not been able to find a reason for this behaviour, the object of the present work was to determine the sequences possibly responsible for the increase of the thermal stability of these collagens. Our results show that the increase in the proline rate and of the number of stabilizing triplets result in the outstanding thermostability of interstitial collagen of A. pompajana. Additional phylogenetic analysis shows the common origin of annelid and vestimentiferan collagens, as well as the existence of tvw) groups of vertebrate fibrillar collagens that have evolved independently.

Demonstration of Mutant Fibrillin-1 Production and Assembly into Abnormal Microfibrils in the Tight Skin Mouse C. M. Kielty ~, M. Raghunath 2, L. D. Siracusa ~, M. J. Sherratt ~, R. Peters ~, C. A. Shuttleworth ~and S. A. Jimenez ~ 'University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom, 2University of Muenster, D-48149, Germany, and -~Jefferson Medical College, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107-5541 Mice carrying the Tight skin (Tsk) mutation harbor a genomic duplication within the Fibrillin-1 (Fbnl) gene that results in a larger than normal in-frame Fbnl transcript. In this study, we examined the consequences of the Tsk mutation for fibrillin-containing microfibrils. Dermal fibroblasts from Tsk/+ mice synthesized and secreted both normal fibrillin (-330 kDa) and the mutant oversized Tsk fibrillin-1 (-450 kDa) in comparable amounts, and Tsk fibrillin-1 was stably incorporated into cell layers. Immunohistochemical and ultrastructural analyses of normal and Tsk/÷ mouse skin demonstrated differences in the organization and distribution of microfibrillar arrays. Rotary shadowing of high-Mr preparations from Tsk/÷ skin demonstrated the presence of abundant beaded microfibrils. Some of these microfibrils had normal morphology and periodicity, but others displayed diffuse interbeads, longer periodicity, and tendency to aggregate. The presence of a struc-