In the integrin family, the collagen receptors form a structurally and functionally distinct subgroup. Two members of this subgroup, ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins, are known to bind to monomeric form of type I collagen. However, in tissues type I collagen monomers are organized into large fibrils immediately after they are released from cells. Here, we studied collagen fibril recognition by integrins. By an immunoelectron microscopy method we showed that integrin ␣ 2 I domain is able to bind to classical D-banded type I collagen fibrils. However, according to the solid phase binding assay, the collagen fibril formation appeared to reduce integrin ␣ 1 I and ␣ 2 I domain avidity to collagen and to lower the number of putative ␣I domain binding sites on it. Respectively, cellular ␣ 1 ␤ 1 integrin was able to mediate cell spreading significantly better on monomeric than on fibrillar type I collagen matrix, whereas ␣ 2 ␤ 1 integrin appeared still to facilitate both cell spreading on fibrillar type I collagen matrix and also the contraction of fibrillar type I collagen gel. Additionally, ␣ 2 ␤ 1 integrin promoted the integrin-mediated formation of long cellular projections typically induced by fibrillar collagen. Thus, these findings suggest that ␣ 2 ␤ 1 integrin is a functional cellular receptor for type I collagen fibrils, whereas ␣ 1 ␤ 1 integrin may only effectively bind type I collagen monomers. Furthermore, when the effect of soluble ␣I domains on type I collagen fibril formation was tested in vitro, the observations suggest that integrin type collagen receptors might guide or even promote pericellular collagen fibrillogenesis.A fibril-forming type I collagen, a ubiquitous protein in all vertebrates, is known to provide mechanical stability for tissues and serve as a functional environment for cells (1). Depending on the physical properties of the tissue, type I collagen fibrils are arranged with different suprafibrillar architectures and diameters. Thus, narrow fibrils (ϳ20 nm) in highly ordered arrangement occur in the cornea, where optical transparency is important, whereas large diameter fibrils (ϳ500 nm) provide high tensile strength in mature tendon (2).The mechanism of type I collagen fibril formation has been under extensive research for decades. In tissues, type I collagen is synthesized as a monomeric precursor, which is secreted by exocytosis into the extracellular space. In addition to the triple helical collagenous domain, the precursor contains noncollagenous C-and N-propeptides, which are linked to the triple helical domain by short sequences called telopeptides (3). After the enzymatic removal of propeptides, the solubility of collagen monomers decreases, and they spontaneously form fibrils, assisted by remaining nonhelical telopeptides (1, 4). Evidently, collagen molecules themselves contain all the information needed for fibril assembly. Therefore, in physiological conditions, acid-solubilized collagen monomers form tissue-type long fibrils with characteristic axial periodic structure also in v...

Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor ␣2␤1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, ␣2␤1 integrin, and caveolin-1 were internalized together in vesicular structures to the perinuclear area. Electron microscopy showed the presence of EV1 particles inside caveolae. Furthermore, infective EV1 could be isolated with anti-caveolin-1 beads 15 min p.i., confirming a close association with caveolin-1. Finally, the expression of dominant negative caveolin in cells markedly inhibited EV1 infection, indicating the importance of caveolae for the viral replication cycle of EV1.

Two collagen receptors, integrins α1β1 and α2β1, can regulate distinct functions in cells. Ligation of α1β1, unlike α2β1, has been shown to result in recruitment of Shc and activation of the Ras/ERK pathway. To identify the downstream signaling molecules activated by α2β1 integrin, we have overexpressed wild-type α2, or chimeric α2 subunit with α1 integrin cytoplasmic domain in human osteosarcoma cells (Saos-2) lacking endogenous α2β1. The chimeric α2/α1 chain formed a functional heterodimer with β1. In contrast to α2/α1 chimera, forced expression of α2 integrin resulted in upregulation of α1 (I) collagen gene transcription in response to three-dimensional collagen, indicating that the cytoplasmic domain of α2 integrin was required for signaling. Furthermore, signals mediated by α2β1 integrin specifically activated the p38α isoform, and selective p38 inhibitors blocked upregulation of collagen gene transcription. Dominant negative mutants of Cdc42, MKK3, and MKK4 prevented α2β1 integrin–mediated activation of p38α. RhoA had also some inhibitory effect, whereas dominant negative Rac was not effective. Our findings show the isoform-specific activation of p38 by α2β1 integrin ligation and identify Cdc42, MKK3, and MKK4 as possible downstream effectors. These observations reveal a novel signaling mechanism of α2β1 integrin that is distinct from ones previously described for other integrins.

A strong link exists between low aerobic exercise capacity and complex metabolic diseases. To probe this linkage, we utilized rat models of low and high intrinsic aerobic endurance running capacity that differ also in the risk for metabolic syndrome. We investigated in skeletal muscle gene-phenotype relationships that connect aerobic endurance capacity with metabolic disease risk factors. The study compared 12 high capacity runners (HCRs) and 12 low capacity runners (LCRs) from generation 18 of selection that differed by 615% for maximal treadmill endurance running capacity. On average, LCRs were heavier and had increased blood glucose, insulin, and triglycerides compared with HCRs. HCRs were higher for resting metabolic rate, voluntary activity, serum high density lipoproteins, muscle capillarity, and mitochondrial area. Bioinformatic analysis of skeletal muscle gene expression data revealed that many genes up-regulated in HCRs were related to oxidative energy metabolism. Seven mean mRNA expression centroids, including oxidative phosphorylation and fatty acid metabolism, correlated significantly with several exercise capacity and disease risk phenotypes. These expression-phenotype correlations, together with diminished skeletal muscle capillarity and mitochondrial area in LCR rats, support the general hypothesis that an inherited intrinsic aerobic capacity can underlie disease risks.—Kivelä, R., Silvennoinen, M., Lehti, M., Rinnankoski-Tuikka, R., Purhonen, T., Ketola, T., Pullinen, K., Vuento, M., Mutanen, N., Sartor, M. A., Reunanen, H., Koch, L. G., Britton, S. L., Kainulainen, H. Gene expression centroids that link with low intrinsic aerobic exercise capacity and complex disease risk.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a prototype member of the Baculoviridae family, has gained increasing interest as a potential vector candidate for mammalian gene delivery applications. AcMNPV is known to enter both dividing and nondividing mammalian cell lines in vitro, but the mode and kinetics of entry as well as the intracellular transport of the virus in mammalian cells is poorly understood. The general objective of this study was to characterize the entry steps of AcMNPV-and green fluorescent protein-displaying recombinant baculoviruses in human hepatoma cells. The viruses were found to bind and transduce the cell line efficiently, and electron microscopy studies revealed that virions were located on the cell surface in pits with an electron-dense coating resembling clathrin. In addition, virus particles were found in larger noncoated plasma membrane invaginations and in intracellular vesicles resembling macropinosomes. In double-labeling experiments, virus particles were detected by confocal microscopy in early endosomes at 30 min and in late endosomes starting at 45 min posttransduction. Viruses were also seen in structures specific for early endosomal as well as late endosomal/lysosomal markers by nanogold preembedding immunoelectron microscopy. No indication of viral entry into recycling endosomes or the Golgi complex was observed by confocal microscopy. In conclusion, these results suggest that AcMNPV enters mammalian cells via clathrinmediated endocytosis and possibly via macropinocytosis. Thus, the data presented here should enable future design of baculovirus vectors suitable for more specific and enhanced delivery of genetic material into mammalian cells.