Role of the extracellular matrix in epithelial morphogenesis

The extracellular matrix (ECM) plays an essential role in organizing tissues, defining their shapes or in presenting growth factors. Their components have been well described in most species, but our understanding of the mechanisms that control ECM remodeling remains limited. Likewise, how the ECM contributes to cellular mechanical responses has been examined in few cases. Here, I review how studies performed in C. elegans have brought several significant advances on those topics. Focusing only on epithelial cells, I discuss basement membrane invasion by the anchor cell during vulva morphogenesis, a process that has greatly expanded our knowledge of ECM remodeling in vivo. I then discuss the ECM role in a novel mechanotransduction process, whereby muscle contractions stimulate the remodeling of hemidesmosome-like junctions in the epidermis, which highlights that these junctions are mechanosensitive. Finally, I discuss progress in defining the composition and potential roles of the apical ECM covering epidermal cells in embryos.     

Nutrient balances and phytoplankton dynamics in two agriculturally loaded shallow lakes

The impact of agriculture was estimated on two shallow, eutrophic lakes, Lake Kotojärvi and Lake Villikkalanjärvi in southern Finland. The main emphasis was on phosphorus and nitrogen budgets and on the phytoplankton dynamics. Special attention was paid to internal P loading and blue-green algal blooms. The mean Tot-P load from agricultural land was 1.2 kg ha^-1 a^-1 in both basins and Tot-N loads were 19 kg ha^-1 a^-1 in L. Villikkalanjärvi and 12 kg ha^-1 a^-1 in L. Kotojärvi. The Tot-P input to L. Kotojärvi was on an average 0.62 g m^-2 a^-1 (per lake surface area), and the Tot-N input 9.1 g m^-2 a^-1. The corresponding inputs to L. Villikkalanjärvi were 3.1 and 57 g m^-2 a^-1, respectively. The annual variation followed the runoff volumes. About half of the Tot-P and one third of the Tot-N load was retained in L. Kotojärvi. In L. Villikkalanjärvi the retention was only 24% for Tot-P and 19% for Tot-N. The difference was very probably due to a longer theoretical retention time in L. Kotojärvi. In L. Villikkalanjärvi the mean concentration of Tot-P was 120 µg 1^-1 and that of Tot-N 1700 µg 1^-1 and the corresponding figures in L. Kotojärvi 67 and 990 µg 1^-1, respectively. The mean chlorophyll a concentration was, however, higher in L. Kotojärvi (26 µg 1^-1) than in L. Villikkalanjärvi (20 µg 1^-1). This was probably due to an internal P load in L. Kotojärvi: in 1988 the internal load of dissolved P was estimated to be as much as twofold the external load. In L. Villikkalanjärvi the internal dissolved P load was only up to 50% of the external input. In L. Kotojärvi the high internal P load coupled with a low DIN:DIP ratio resulted in a strong blue-green algal bloom in the summer of 1988. In L. Villikkalanjärvi blue-green algae were observed only in small amounts. Even in August 1990, when the DIN:DIP ratio was low enough to favor the occurrence of blue-green algae, they contributed only up to 10–15% of the total phytoplankton biomass.     

Nanomechanical Properties of Tenascin-X Revealed by Single-Molecule Force Spectroscopy

Tenascin-X is an extracellular matrix protein and binds a variety of molecules in extracellular matrix and on cell membrane. Tenascin-X plays important roles in regulating the structure and mechanical properties of connective tissues. Using single-molecule atomic force microscopy, we have investigated the mechanical properties of bovine tenascin-X in detail. Our results indicated that tenascin-X is an elastic protein and the fibronectin type III (FnIII) domains can unfold under a stretching force and refold to regain their mechanical stability upon the removal of the stretching force. All the 30 FnIII domains of tenascin-X show similar mechanical stability, mechanical unfolding kinetics, and contour length increment upon domain unfolding, despite their large sequence diversity. In contrast to the homogeneity in their mechanical unfolding behaviors, FnIII domains fold at different rates. Using the 10th FnIII domain of tenascin-X (TNXfn10) as a model system, we constructed a polyprotein chimera composed of alternating TNXfn10 and GB1 domains and used atomic force microscopy to confirm that the mechanical properties of TNXfn10 are consistent with those of the FnIII domains of tenascin-X. These results lay the foundation to further study the mechanical properties of individual FnIII domains and establish the relationship between point mutations and mechanical phenotypic effect on tenascin-X. Moreover, our results provided the opportunity to compare the mechanical properties and design of different forms of tenascins. The comparison between tenascin-X and tenascin-C revealed interesting common as well as distinguishing features for mechanical unfolding and folding of tenascin-C and tenascin-X and will open up new avenues to investigate the mechanical functions and architectural design of different forms of tenascins.     

Phenotypic heterogeneity influences the behavior of rat aortic smooth muscle cells in collagen lattice

Phenotypic modulation of vascular smooth muscle cells (SMCs) in atherosclerosis and restenosis involves responses to the surrounding microenvironment. SMCs obtained by enzymatic digestion from tunica media of newborn, young adult (YA) and old rats and from the thickened intima (TI) and underlying media of young adult rat aortas 15 days after ballooning were entrapped in floating populated collagen lattice (PCL). TI-SMCs elongated but were poor at PCL contraction and remodeling and expressed less alpha2 integrin compared to other SMCs that appeared more dendritic. During early phases of PCL contraction, SMCs showed a marked decrease in the expression of alpha-smooth muscle actin and myosin. SMCs other than TI-SMCs required 7 days to re-express alpha-smooth muscle actin and myosin. Only TI-SMCs in PCL were able to divide in 48 h, with a greater proportion in S and G2-M cell cycle phases compared to other SMCs. Anti-alpha2 integrin antibody markedly inhibited contraction but not proliferation in YA-SMC-PLCs; anti-alpha1 and anti-alpha2 integrin antibodies induced a similar slight inhibition in TI-SMC-PCLs. Finally, TI-SMCs rapidly migrated from PCL on plastic reacquiring their epithelioid phenotype. Heterogeneity in proliferation and cytoskeleton as well the capacity to remodel the extracellular matrix are maintained, when SMCs are suspended in PCLs.     

Modulation of lymphocyte nuclear matrix organization in vivo by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole: an autoradiographic and immunofluorescence study

Assembly of active nuclei in lymphocytes stimulated by mitogen is paralleled by the elaboration of a structurally and biochemically complex nuclear matrix (NM). To examine the dynamics of individual NM polypeptide components during blastogenesis, we have applied immunofluorescence labelling with anti-NM antibodies to concanavalin A-stimulated mouse splenocytes. Whereas peripherin and PI2 antigens did not reorganize during stimulation, labelling of PI1 and small nuclear ribonucleoprotein (snRNP) antigens increased markedly in intensity and redistributed in concert with the previously reported NM restructuring. Double-labelling showed, furthermore, that snRNPs and the internal staining component of PI1 were largely co-localized. As an approach to studying the role of RNA and RNA synthesis in NM organization, we have further examined the effects of the inhibitor of RNA synthesis, 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), on NM antigen distribution. The rapid inhibition of 3H-uridine incorporation by DRB was accompanied by coordinate aggregation of snRNPs and of the internal PI1 component into large, brightly stained patches. Both 3H-uridine incorporation levels and antigen localization were readily reversed upon removal of DRB. We conclude that NM antigens behave independently during nuclear and NM assembly and that NM organization, as reflected by NM antigen distribution, is modulated by con A- and DRB-induced alterations in RNA synthesis. We propose, furthermore, that the PI1 antigen plays a role in RNA metabolism, and is possibly involved in RNA transport to the nuclear periphery.     

Immuno-electron-microscopic localization of types III pN-collagen and IV collagen,laminin and tenascin in developing and adult human spleen

The distribution of the extracellular matrix proteins types III pN-collagen and IV collagen, laminin and tenascin was investigated in fetal, infant, and adult human spleens by using immuno-electron microscopy. The presence of type III pN-collagen was assessed by using an antibody against the aminoterminal propeptide of type III procollagen. All the proteins other than type III pN-collagen were found in reticular fibers throughout development. In the white pulp of the fetus aged 16 gestational weeks, only an occasional type III pN-collagen-containing fibril was present, although type III pN-collagen was abundant in the reticular fibers of the red pulp. Conversely, in adults, most of the reticular fibers of the white pulp, but not of the red pulp, were immunoreactive for type III pN-collagen. Ring fibers, the basement membranes of venous sinuses, were well developed in both infant and adult spleens. The first signs of their formation could be seen as a discontinuous basement membrane, which was immunoreactive for type IV collagen, laminin, and tenascin in the fetus aged 20 gestational weeks. Intracytoplasmic immunoreactivity for all the proteins studied was visible in the mesenchymal cells of the fetus aged 16 gestational weeks and in the reticular cells of the older fetuses, which also showed labeling for type IV collagen and laminin in the endothelial cells. The results suggest that proteins of the extracellular matrix are produced by these stationary cells.     

Prominent collagen type VI expression in juvenile angiofibromas

The extracellular matrix component collagen type VI demonstrates potent growth-stimulatory effects and has been associated with aggressive tumour growth. Although, juvenile angiofibromas (JAs) often exhibit an aggressive growth pattern, the collagen type VI expression of this fibrovascular tumour has not been addressed so far. RT-PCR, Western blot analysis and immunohistochemistry were used in this study to analyse collagen type VI, type VI collagen receptor subunits (integrin α1, α2, α10, α11 and β1) and the type VI collagen receptor NG2 in JAs (N = 15) and nasal mucosa (NM, N = 8) samples. The mRNA expression of all three collagen type VI chains was found to be up-regulated significantly (P < 10⁻³–10⁻⁵, adjusted) in JAs compared to NM tissues. The Western blot analysis proved highly prominent collagen-type VI expression in JAs. The ApoTome technique revealed strong collagen-type VI signals in tumour endothelium. NG2 (P < 10⁻³, adjusted) and α11-integrin (P = 0.04, adjusted) showed a significantly higher mRNA expression levels in JAs than in NM samples. NG2, α1-, α2- and β1-intergin were located to tumour vessels, and additional stromal signals were observed for NG2 and α1-integrin in JAs. This study demonstrates a prominent collagen-type VI expression in JAs. The collagen-type VI may exert an important growth stimulus in this tumour.     

Expression and localization of DNA topoisomerase II during rat spermatogenesis

The potential role(s) of DNA topoiosmerase II (topo II) during chromatin changes that characterize different stages of spermatogenesis was investigated in the rat by an analysis of the expression and localization of topo II mRNA and protein in individual spermatogenic cells. Expression of topo II was restricted to spermatogonia, spermatocytes, and round and early-elongating spermatids. Two protein bands of 177 and 170 kDa were detected in immunoblots of spermatocytes and round spermatids, while bands of 148 and 142 kDa were prominent in preparations of elongating spermatids. Topo II levels and distribution patterns, as observed by immunofluorescent microscopy, exhibited cell type-specific variations. Differences in topo II staining patterns were also apparent when nuclear matrices of spermatogenic cells were prepared with different extraction conditions. In addition to its possible function as a structural component, topo II, associated with nuclear matrix preparations from spermatogenic cells, possessed catalytic activity. These observations indicate that both the 177 and 170 kDa and the 148 and 142 kDa forms of topo II share similar structural and functional properties. Topo IIβ mRNA was transcribed in rat spermatogenic cells at 6.2 kb. Relative levels of topo IIβ mRNA were high in spermatogonia and spermatocytes, and decreased in both round and early-elongating spermatids. Changes in topo II expression levels and localization patterns represent distinct stage-specific markers for the maturation of spermatogenic cells, and are consistent with the involvement of topo II in mediating DNA modifications and chromatin changes during spermatogenesis. © 1996 Wiley-Liss, Inc.