Changes in the chondrocyte and extracellular matrix proteome during post-natal mouse cartilage development

Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (post-natal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Mol. Cell. Proteomics 9, 1296-1313) identified components involved in both systems, such as Urb, and components with specific roles in vivo, including vitrin and CILP-2 (cartilage intermediate layer protein-2). Immunolocalization of Urb, vitrin, and CILP-2 indicated specific roles at different maturation stages. In addition to ECM-related changes, we provide the first biochemical evidence of changing endoplasmic reticulum function during cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head.     

Production of membrane proteins using cell-free expression systems

Different overexpression systems are widely used in the laboratory to produce proteins in a reasonable amount for functional and structural studies. However, to optimize these systems without modifying the cellular functions of the living organism remains a challenging task. Cell-free expression systems have become a convenient method for the high-throughput expression of recombinant proteins, and great effort has been focused on generating high yields of proteins. Furthermore, these systems represent an attractive alternative for producing difficult-to-express proteins, such as membrane proteins. In this review, we highlight the recent improvements of these cell-free expression systems and their direct applications in the fields of membrane proteins production, protein therapy and modern proteomics.     

Arabinogalactan proteins: actors or spectators during abiotic and biotic stress in plants?

Arabinogalactan proteins (AGPs) are a family of hydroxyproline-rich glycoproteins (HRGP) ubiquitous in the plant kingdom. They are probably one of the most heterogeneous and complex families of macromolecules, making them able to perform different and multiple functions. Located at the plasma membrane–cell wall interface, AGPs are involved in several processes, from plant growth and development to reproduction. An additional function of AGPs in response to biotic and abiotic stress has been suggested by several studies. The purpose of this review is to summarize critically and analytically the available knowledge on the effects of abiotic stress (low and high temperatures, drought, flooding, anoxia and metal deficiency/toxicity) and biotic stress (bacteria, fungi, nematodes and viruses) on AGPs. A deeper understanding of the role of AGPs during these conditions can be an important tool for understanding AGP biology and for the possible development of efficient breeding strategies.     

Single-step production of functional OEP24 proteoliposomes

The pea chloroplastic outer envelope protein OEP24 is a voltage-dependent channel that can function as a general solute channel in plants. OEP24 is a close functional homologue of VDAC which, in mammalian cells, modulates the permeability of the outer mitochondrial membrane. Here, we describe the production in a one-step reaction of active OEP24 in proteoliposomes or in soluble form using a cell-free expression system. We combine evidence from electrophysiological experiments, biophysical characterization, and biochemical analysis demonstrating that OEP24 is present as a functional channel in liposomes. Thus, production of OEP-containing proteoliposomes may provide a helpful tool for deciphering the role of the OEP family members.     

Morphological Analysis of the Transfer of VSV ts-045 G Glycoprotein from the Endoplasmic Reticulum to the Intermediate Compartment in Vero Cells

Vero cells were infected with the ts-045 strain of vesicular stomatitis virus, and the cells were incubated at 39°C to accumulate the mutant G glycoprotein in the ER as a misfolded aggregate. Cycloheximide was added to the culture medium 3.5 h after infection to prevent further protein synthesis, and the temperature was lowered to 10, 15, or 31°C. At these temperatures, the mutant G glycoprotein correctly folds and oligomerizes. Immunofluorescence light microscopy showed that the G glycoprotein was exported to the Golgi complex at 31°C and to the intermediate compartment (IC) at 15°C, but no export was observed at 10°C. However, incubations at 10°C followed by shift to 15 or 31°C resulted in the normal transfer of the glycoprotein to the IC and the Golgi, respectively. Immunoelectron microscopical analysis confirmed all these results, but showed also that the glycoprotein was frequently clustered in the ER at 10°C. Conventional electron microscopy showed that the morphology of the ER, IC, and Golgi complex remained essentially unchanged at all temperatures. The only significant difference detectable in cells incubated at 10°C was the increased number of partially coated ER protrusions, longer than those detected at higher temperatures. These results demonstrate that the transport toward the Golgi complex of G glycoprotein can be arrested at a step preceding the entry into the IC, thus suggesting that ER and IC are separate stations in the exocytic pathway.     

Diversity arrays technology (DArT) for pan-genomic evolutionary studies of non-model organisms

Background

High-throughput tools for pan-genomic study, especially the DNA microarray platform, have sparked a remarkable increase in data production and enabled a shift in the scale at which biological investigation is possible. The use of microarrays to examine evolutionary relationships and processes, however, is predominantly restricted to model or near-model organisms.

Methodology/Principal Findings

This study explores the utility of Diversity Arrays Technology (DArT) in evolutionary studies of non-model organisms. DArT is a hybridization-based genotyping method that uses microarray technology to identify and type DNA polymorphism. Theoretically applicable to any organism (even one for which no prior genetic data are available), DArT has not yet been explored in exclusively wild sample sets, nor extensively examined in a phylogenetic framework. DArT recovered 1349 markers of largely low copy-number loci in two lineages of seed-free land plants: the diploid fern Asplenium viride and the haploid moss Garovaglia elegans. Direct sequencing of 148 of these DArT markers identified 30 putative loci including four routinely sequenced for evolutionary studies in plants. Phylogenetic analyses of DArT genotypes reveal phylogeographic and substrate specificity patterns in A. viride, a lack of phylogeographic pattern in Australian G. elegans, and additive variation in hybrid or mixed samples.

Conclusions/Significance

These results enable methodological recommendations including procedures for detecting and analysing DArT markers tailored specifically to evolutionary investigations and practical factors informing the decision to use DArT, and raise evolutionary hypotheses concerning substrate specificity and biogeographic patterns. Thus DArT is a demonstrably valuable addition to the set of existing molecular approaches used to infer biological phenomena such as adaptive radiations, population dynamics, hybridization, introgression, ecological differentiation and phylogeography.