Structure-function relationships in the stem cell's mechanical world A: seeding protocols as a means to control shape and fate of live stem cells

Shape and fate are intrinsic manifestations of form and function at the cell scale. Here we hypothesize that seeding density and protocol affect the form and function of live embryonic murine mesenchymal stem cells (MSCs) and their nuclei. First, the imperative for study of live cells was demonstrated in studies showing changes in cell nucleus shape that were attributable to fixation per se. Hence, we compared live cell and nuclear volume and shape between groups of a model MSC line (C3H10T1/2) seeded at, or proliferated from 5,000 cells/cm2 to one of three target densities to achieve targeted development contexts. Cell volume was shown to be dependent on initial seeding density whereas nucleus shape was shown to depend on developmental context but not seeding density. Both smaller cell volumes and flatter nuclei were found to correlate with increased expression of markers for mesenchymal condensation as well as chondrogenic and osteogenic differentiation but a decreased expression of pre-condensation and adipogenic markers. Considering the data presented here, both seeding density and protocol significantly alter the morphology of mesenchymal stem cells even at very early stages of cell culture. Thus, these design parameters may play a critical role in the success of tissue engineering strategies seeking to recreate condensation events. However, a better understanding of how these changes in cell volume and nucleus shape relate to the differentiation of MSCs is important for prescribing precise seeding conditions necessary for the development of the desired tissue type. In a companion study (Part B, following), we address the effect of concomitant volume and shape changing stresses on spatiotemporal distribution of the cytoskeletal proteins actin and tubulin. Taken together, these studies bring us one step closer to our ultimate goal of elucidating the dynamics of nucleus and cell shape change as tissue templates grow (cell proliferation) and specialize (cell differentiation).     

Propagation of mechanical stress through the actin cytoskeleton toward focal adhesions: model and experiment

We investigate both theoretically and experimentally how stress is propagated through the actin cytoskeleton of adherent cells and consequentially distributed at sites of focal adhesions (FAs). The actin cytoskeleton is modeled as a two-dimensional cable network with different lattice geometries. Both prestrain, resulting from actomyosin contractility, and central application of external force, lead to finite forces at the FAs that are largely independent of the lattice geometry, but strongly depend on the exact spatial distribution of the FAs. The simulation results compare favorably with experiments with adherent fibroblasts onto which lateral force is exerted using a microfabricated pillar. For elliptical cells, central application of external force along the long axis leads to two large stress regions located obliquely opposite to the pulling direction. For elliptical cells pulled along the short axis as well as for circular cells, there is only one region of large stress opposite to the direction of pull. If in the computer simulations FAs are allowed to rupture under force for elliptically elongated and circular cell shapes, then morphologies arise which are typical for migrating fibroblasts and keratocytes, respectively. The same effect can be obtained also by internally generated force, suggesting a mechanism by which cells can control their migration morphologies.     

Structure-function relationships in 3-phosphoglycerate kinase: role of the carboxy-terminal peptide

Yeast 3-phosphoglycerate kinase (PGK) is a monomeric enzyme (Mr approximately 45,000) composed of two globular domains. Each domain corresponds approximately to the amino- and carboxy-terminal halves of the polypeptide chain. The carboxy-terminal end extends over the interdomain hinge region and packs against the amino-terminal domain. It has been proposed that domain movement, resulting in closure of the active site cleft, is essential for the catalytic function of PGK. Large-scale conformational changes have also been postulated to explain activation of the enzyme by sulfate ions. Using site-specific mutagenesis, we have removed a 15-amino-acid carboxy-terminal fragment, in order to probe its role in the substrate- and sulfate-induced conformational changes. The truncated enzyme exhibited approximately 1% of the activity of native PGK and lost the ability to undergo sulfate-induced activation. The Km for ATP was essentially unchanged (Km = 0.23 mM) in comparison to the native enzyme (Km = 0.30 mM), whereas the Km value for 3-phosphoglycerate was increased about eightfold (Km = 3.85 mM and 0.50 mM, respectively). These results suggest that the carboxy-terminal segment is important for the mechanism of the substrate- and sulfate-induced conformational transitions. CD spectra and sedimentation velocity measurements indicate that the carboxy-terminal peptide is essential for structural integrity of PGK. The increased susceptibility of the truncated enzyme to thermal inactivation implies that the carboxy-terminal peptide also contributes to the stability of PGK.     

Structure-function relationships in glucosidase I: amino acids involved in binding the substrate to the enzyme

As the enzyme that initiates the maturation phase of the oligosaccharidemoiety of N-linked glycoproteins, glucosidase I controls theflux of carbohydrate during the biosynthesis of these proteins.In a previous study to elucidate the structure-function relationships,we reported the presence of a cysteine residue at or near theactive site of the enzyme from the bovine mammary gland (Pukazhenthi,BS.,Muniappa,N. and Vijay,I.K., 1993, J. Biol. Chem, 268, 6445–6452).We have now extended this approach to identify the participationof an arginine and a tryptophan residue in the enzyme that mayplay an important role in binding the substrate. The data havebeen combined with the results of the previous study and thecDNA-derived sequence to propose a ERHLDLRCW motif in the activesite of the enzyme in the rat mammary gland that is involvedin binding the incipient glycoprotein substrate for processing. glucosidase I glycoprotein processing active site     

Endocardial endothelium in the rat: cell shape and organization of the cytoskeleton

The cytoskeleton in endocardial endothelium of rat heart was examined by en face confocal scanning laser microscopy. In the ventricular cavity, endocardial endothelial cells had a polygonal shape and F-actin staining was generally restricted to the peripheral junctional actin band. Central F-actin bundles, or stress fibers, in endocardial endothelial cells were found on the tendon end of papillary muscles, especially in the right ventricle, and frequently in the outflow tract of both ventricles; elsewhere, stress fibers were scarce. Many endocardial endothelial cells were elongated in areas of endothelium with stress fibers, but no correlation was found between cell elongation and the number of stress fibers. An inverse correlation was found between the number of stress fibers and the surface area of endocardial endothelial cells. Shear stress as well as mechanical deformation of the surface of the ventricular wall during the cardiac cycle may affect cell shape and the organization of actin filaments in endocardial endothelial cells. Vimentin in endocardial endothelial cells formed a filamentous network with some distinct cytoplasmic and juxtanuclear vimentin bundles. No perinuclear ring of vimentin filaments was observed in endocardial endothelium. Microtubules in endocardial endothelial cells were, in contrast to endothelial cells of rat aorta, not aligned, less closely packed and originated from randomly distributed centriolar regions. The cytoskeleton has been suggested to play an important role in cellular functions of vascular endothelial cells. Accordingly, differences in the cytoskeletal organization between endocardial and vascular endothelial cells may relate to differences in functional properties.     

Structure-function relationships in the IL-17 receptor: implications for signal transduction and therapy

IL-17 is the defining cytokine of a newly-described "Th17" population that plays critical roles in mediating inflammation and autoimmunity. The IL-17/IL-17 receptor superfamily is the most recent class of cytokines and receptors to be described, and until recently very little was known about its function or molecular biology. However, in the last year important new insights into the composition and dynamics of the receptor complex and mechanisms of downstream signal transduction have been made, which will be reviewed here.     

Structure-function relationships in 3alpha-hydroxysteroid dehydrogenases: a comparison of the rat and human isoforms

3alpha-Hydroxysteroid dehydrogenases (3alpha-HSDs) inactivate steroid hormones in the liver, regulate 5alpha-dihydrotestosterone (5alpha-DHT) levels in the prostate, and form the neurosteroid, allopregnanolone in the CNS. Four human 3alpha-HSD isoforms exist and correspond to AKR1C1-AKR1C4 of the aldo-keto reductase (AKR) superfamily. Unlike the related rat 3alpha-HSD (AKR1C9) which is positional and stereospecific, the human enzymes display varying ratios of 3-, 17-, and 20-ketosteroid reductase activity as well as 3alpha-, 17beta-, and 20alpha-hydroxysteroid oxidase activity. Their k(cat) values are 50-100-fold lower than that observed for AKR1C9. Based on their product profiles and discrete tissue localization, the human enzymes may regulate the levels of active androgens, estrogens, and progestins in target tissues. The X-ray crystal structures of AKR1C9 and AKR1C2 (human type 3 3alpha-HSD, bile acid binding protein and peripheral 3alpha-HSD) reveal that the AKR1C2 structure can bind steroids backwards (D-ring in the A-ring position) and upside down (beta-face inverted) relative to the position of a 3-ketosteroid in AKR1C9 and this may account for its functional plasticity. Stopped-flow studies on both enzymes indicate that the conformational changes associated with binding cofactor (the first ligand) are slow; they are similar in both enzymes but are not rate-determining. Instead the low k(cat) seen in AKR1C2 (50-fold less than AKR1C9) may be due to substrate "wobble" at the plastic active site.     

Structure-function relationships in the HAMP and proximal signaling domains of the aerotaxis receptor Aer

Aer, the Escherichia coli aerotaxis receptor, faces the cytoplasm, where the PAS (Per-ARNT-Sim)-flavin adenine dinucleotide (FAD) domain senses redox changes in the electron transport system or cytoplasm. PAS-FAD interacts with a HAMP (histidine kinase, adenylyl cyclase, methyl-accepting protein, and phosphatase) domain to form an input-output module for Aer signaling. In this study, the structure of the Aer HAMP and proximal signaling domains was probed to elucidate structure-function relationships important for signaling. Aer residues 210 to 290 were individually replaced with cysteine and then cross-linked in vivo. The results confirmed that the Aer HAMP domain is composed of two α-helices separated by a structured loop. The proximal signaling domain consisted of two α-helices separated by a short undetermined structure. The Af1503 HAMP domain from Archaeoglobus fulgidus was recently shown to be a four-helix bundle. To test whether the Af1503 HAMP domain is a prototype for the Aer HAMP domain, the latter was modeled using coordinates from Af1503. Several findings supported the hypothesis that Aer has a four-helix HAMP structure: (i) cross-linking independently identified the same residues at the dimer interface that were predicted by the model, (ii) the rate of cross-linking for residue pairs was inversely proportional to the β-carbon distances measured on the model, and (iii) clockwise lesions that were not contiguous in the linear Aer sequence were clustered in one region in the folded HAMP model, defining a potential site of PAS-HAMP interaction during signaling. In silico modeling of mutant Aer proteins indicated that the four-helix HAMP structure was important for Aer stability or maturation. The significance of the HAMP and proximal signaling domain structure for signal transduction is discussed.     

Trophic relationships in the soil microfood-web: predicting the responses to a changing global environment

In this article, we evaluate how global environmental change may affect microfood-webs and trophic interactions in the soil, and the implications of this at the ecosystem level. First we outline how bottom-up (resource control) and top-down (predation-control) forces regulate food-web components. Food-web components can respond either positively or negatively to shifts in NPP resulting from global change, thus creating difficulties in developing general principles about the response of soil biota to global change phenomena. We also demonstrate that top-down effects can be important in soil food-webs, creating negative feed-backs which may partially counter bottom-up effects. Secondly, we determine how soil food-webs and the processes they regulate respond to various global change phenomena. Enhanced atmospheric CO₂ levels can have two main effects on plants which are relevant for the soil food-web, i.e. enhanced NPP (often positive) and diminished organic matter quality (with negative effects, at least in the short term). Climate change effects resulting from elevated CO₂ levels may be mainly secondary through alteration of vegetation, as shown by examples. Intensification of land management is usually associated with greater disturbance, which alters soil food-web composition and key processes; this is particularly apparent in comparisons of conventionally tilled and nontilled agroecosystems. Global change involves shifts in plant species composition and diversity, possibly affecting soil food-webs; we interpret this in terms of theories relating biodiversity to ecosystem function. We conclude that a more detailed understanding of interactions between NPP, soil organic matter and components of the soil food-web, as well as their regulation of biogeochemical processes and ultimately ecosystem-level properties, is essential in better understanding long-term aspects of global change phenomena.     

Differential regulation of cell volume and shape in confluent rat hepatocytes under hypertonic stress.

In confluent primary cultures of rat hepatocytes,hypertonic stress leads to cell shrinkage and activates non-selective cation channels as the main mechanism of regulatory cell volume increase. The process is found to employ the exocytotic insertion of channels into the plasma membrane and (in addition to PKC) PLC, tyrosine kinases and G proteins, but not PI 3-kinase are part of the signalling network. Furthermore, hypertonic stress leads to the formation of stress fibres and significantly alters the activity of RhoA, Rac and Cdc42. These latter effects, however, are likely to reflect the restoration of cell shape rather than the regulation of cell volume, both most probably converging at the level of focal adhesions and integrins.     

Structure-function relationships in the neuropeptide S receptor: molecular consequences of the asthma-associated mutation N107I

Neuropeptide S (NPS) and its receptor (NPSR) are thought to have a role in asthma pathogenesis; a number of single nucleotide polymorphisms within NPSR have been shown to be associated with an increased prevalance of asthma. One such single nucleotide polymorphism leads to the missense mutation N107I, which results in an increase in the potency of NPS for NPSR. To gain insight into structure-function relationships within NPS and NPSR, we first carried out a limited structural characterization of NPS and subjected the peptide to extensive mutagenesis studies. Our results show that the NH(2)-terminal third of NPS, in particular residues Phe-2, Arg-3, Asn-4, and Val-6, are necessary and sufficient for activation of NPSR. Furthermore, part of a nascent helix within the peptide, spanning residues 5 through 13, acts as a regulatory region that inhibits receptor activation. Notably, this inhibition is absent in the asthma-linked N107I variant of NPSR, suggesting that residue 107 interacts with the aforementioned regulatory region of NPS. Whereas this interaction may be at the root of the increase in potency associated with the N107I variant, we show here that the mutation also causes an increase in cell-surface expression of the mutant receptor, leading to a concomitant increase in the maximal efficacy (E(max)) of NPS. Our results identify the key residues of NPS involved in NPSR activation and suggest a molecular basis for the functional effects of the N107I mutation and for its putative pathophysiological link with asthma.     

Wetlands and global climate change: the role of wetland restoration in a changing world

Global climate change is recognized as a threat to species survival and the health of natural systems. Scientists worldwide are looking at the ecological and hydrological impacts resulting from climate change. Climate change will make future efforts to restore and manage wetlands more complex. Wetland systems are vulnerable to changes in quantity and quality of their water supply, and it is expected that climate change will have a pronounced effect on wetlands through alterations in hydrological regimes with great global variability. Wetland habitat responses to climate change and the implications for restoration will be realized differently on a regional and mega-watershed level, making it important to recognize that specific restoration and management plans will require examination by habitat. Floodplains, mangroves, seagrasses, saltmarshes, arctic wetlands, peatlands, freshwater marshes and forests are very diverse habitats, with different stressors and hence different management and restoration techniques are needed. The Sundarban (Bangladesh and India), Mekong river delta (Vietnam), and southern Ontario (Canada) are examples of major wetland complexes where the effects of climate change are evolving in different ways. Thus, successful long term restoration and management of these systems will hinge on how we choose to respond to the effects of climate change. How will we choose priorities for restoration and research? Will enough water be available to rehabilitate currently damaged, water-starved wetland ecosystems? This is a policy paper originally produced at the request of the Ramsar Convention on Wetlands and incorporates opinion, interpretation and scientific-based arguments.     

Regulation of VE-cadherin linkage to the cytoskeleton in endothelial cells exposed to fluid shear stress

Endothelial cells exposed to shear stress realigned and elongated in the direction of flow through the coordinated remodeling of their adherens junctions and actin cytoskeleton. The elaborate networks of VE-cadherin complexes in static cultures became more uniform and compact in response to shear. In contrast, the cortical actin present in static cultures was reorganized into numerous stress fiber bundles distributed parallel to the direction of flow. Exposure to shear did not significantly alter the expression of the junctional proteins VE-cadherin, beta-catenin, and alpha-catenin, but the composition of the junctional complexes did change. We detected a marked decrease in the alpha-catenin associated with VE-cadherin complexes in endothelial monolayers subjected to shear. This loss of alpha-catenin, the protein that links beta-catenin-bound cadherin to the actin cytoskeleton, was not due to decreased quantities of beta-catenin associated with VE-cadherin. Instead, the loss of alpha-catenin from the junctional complexes coincided with the increased tyrosine phosphorylation of beta-catenin associated with VE-cadherin. The change in beta-catenin phosphorylation closely correlated with the shear-induced loss of the protein tyrosine phosphatase SHP-2 from VE-cadherin complexes. Thus, the functional interaction of alpha-catenin with VE-cadherin-bound beta-catenin is regulated by the extent of tyrosine phosphorylation of beta-catenin. This, concomitantly, is regulated by SHP-2 associated with VE-cadherin complexes.