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The following is a modification of a Scherz R., 2001 method forcolony embedding. The protocol described here is from: Sarah Piccirillo,et. al. The Rim101p/PacC Pathway and Alkaline pH Regulate Pattern Formation in Yeast Colonies(2010) Genetics184:707-716; doi:10.1534/genetics.109.113480 Please see the full manuscript for additional details.

Bone acidic glycoprotein-75 is expressed very early during in vivo models of intramembranous bone formation, highly enriched in condensing osteogenic mesenchyme after marrow ablation and the osteoprogenitor layer of tibial periosteum. Bone sialoprotein accumulates within bone acidic glycoprotein-75-enriched matrix areas at a later stage in both models. Decalcification of initial sites of mineralization consistently revealed focal immunostaining for bone acidic glycoprotein-75 underneath these sites suggesting that mineralization occurs within bone acidic glycoprotein-75-enriched matrix areas. Ultrastructural immunolocalization of bone acidic glycoprotein-75 does not support a direct association with banded collagen fibrils, but rather suggests it is a component of a separate, amorphous scaffold occupying interfibrillar spaces. Double immunogold labeling demonstrated that a sizeable proportion of bone sialoprotein particles were located within a 50-nm radius of bone acidic glycoprotein-75. These results define bone acidic glycoprotein-75 as the earliest bone-restricted, extracellular marker of osteogenic mesenchyme. Based on this early bone-restricted expression pattern and a previously documented propensity of bone acidic glycoprotein-75 to form supramolecular complexes through self-association, bone acidic glycoprotein-75 may serve a key structural role in setting boundary limits of condensing osteogenic mesenchyme.

The regions of mouse nebulin extending from the ends of the super repeats to the C-terminus and N-terminus were cloned and sequenced. Comparison of the mouse sequence with the previously published human sequence shows that the terminal regions of nebulin are highly conserved. The four phosphorylation motifs and SH3 domain found at the C-terminus of mouse nebulin are identical to those found in human nebulin, with the exception of four conservative substitutions. The modules linking this C-terminal region to the super repeats have deletions relative to both fetal and adult human nebulins that correspond to integral numbers of modules, making the mouse C-terminal simple repeat region among the shortest observed to date. The N-terminal region and the C-terminal modules were expressed in Escherichia coli and used for antibody production. Immunofluorescent labeling of these regions of nebulin in isolated myofibrils demonstrates that they are located near the center of the sarcomere and near the Z-line, respectively. Immunogold labeling with antibodies raised against the N-terminal nebulin sequence localizes this region in the A-band near the tips of the thin filaments. Nebulin localization is complementary to that of N-RAP, another muscle-specific protein containing nebulin-like super repeats; nebulin is exclusively found in the sarcomeres, while N-RAP is confined to the terminal bundles of actin filaments at the myotendinous junction. Cell Motil. Cytoskeleton 3:211-222, 2000 Published 2000 Wiley-Liss, Inc.

Heparan sulphate proteoglycans on cell surfaces have been shown to mediate the degradation or recycling of several ligands. Since the interaction with ligand may affect proteoglycan catabolism once the complex is internalized, this could alter the cellular pool of heparan sulphate chains, with possible consequences for heparan sulphate-mediated cellular processes. We have recently demonstrated that the specific binding of basic fibroblast growth factor (bFGF) to heparan sulphate chains prevents the glycosaminoglycan from being degraded by partially purified heparanases from Chinese hamster ovary (CHO) cells [Tumova and Bame (1997) J. Biol. Chem. 272, 9078–9085]. The present study examines the effect of bFGF on heparan sulphate catabolism in intact cells. The distribution and size of the heparan sulphate degradation products in CHO cells was analysed in the presence and absence of bFGF using pulse–chase protocols. Although heparan sulphate molecules and bFGF are internalized through the same pathway, even relatively high concentrations of the growth factor do not have any inhibitory effects on glycosaminoglycan degradation. However, the interaction with the growth factor alters the distribution of heparan sulphate-degradation products, presumably by preventing secretion of the short heparanase-derived species. Our findings show that most of the free and bFGF-bound heparan sulphate chains are destined for lysosomes, which would be consistent with a recent hypothesis that the primary role of proteoglycan-mediated internalization of the growth factor is to remove bFGF from its site of action at the cell surface. However, in the presence of bFGF, a fraction of intracellular, heparanase-degraded heparan sulphate chains is delivered to the nucleus, suggesting that the glycosaminoglycan accompanies the growth factor to the organelle. It may be important for bFGF activity that the growth factor is protected from proteolytic degradation by its interaction with heparan sulphate. This work demonstrates that the internalization of a ligand along with the proteoglycan can affect the sorting of heparan sulphate-degradation products in endosomes, and the ultimate destination of the short glycosaminoglycan. It also provides evidence that formation of heparan sulphate–ligand complexes may regulate the recycling and degradation of both ligands and heparan sulphate chains and, consequently, affect their biological activities.

Saccharomyces cerevisiae grown on plastic surfaces formed organized structures, termed minicolonies, that consisted of a core of round (yeast-like) cells surrounded by chains of filamentous cells (pseudohyphae). Minicolonies had a much higher affinity for plastic than unstructured yeast communities growing on the same surface. Pseudohyphae at the surface of these colonies developed further into chains of asci. These structures suggest that pseudohyphal differentiation and sporulation are sequential processes in minicolonies. Consistent with this idea, minicolonies grown under conditions that stimulated pseudohyphal differentiation contained higher frequencies of asci. Furthermore, a flo11Δ mutant, which fails to form pseudohyphae, yielded normal sporulation in cultures, but was defective for minicolony sporulation. When minicolonies were dispersed in water and cells were then allowed to settle on the plastic surface, these cells sporulated very efficiently. Taken together, our results suggest that sporulation in minicolonies is stimulated by pseudohyphal differentiation because these pseudohyphae are dispersed from the core of the colony.

We have purified and generated antisera to a 95 kDa skeletal muscle protein that constitutes the largest mass fraction of gelatin-agarose binding proteins in skeletal muscle. Preliminary results indicated that this 95 kDa chicken skeletal muscle protein bound strongly to gelatin-agarose and type IV collagen-agarose, suggesting a possible function in muscle cell adhesion to collagen. However, N-terminal sequencing of proteolytic fragments of the 95 kDa protein indicates that it is the chicken skeletal muscle form of glycogen phosphorylase, the binding of which to gelatin-agarose is unlikely to be biologically relevant. Further characterization showed that the skeletal muscle form of glycogen phosphorylase is immunologically distinct from the liver and brain forms in the chicken, and suggests that, unlike mammalian skeletal muscle, chicken skeletal muscle may have two phosphorylase isoforms. Furthermore, immunolocalization data and solubility characteristics of glycogen phosphorylase in muscle extraction experiments suggest the enzyme may interact strongly with an unidentified component of the muscle cytoskeleton. Thus, this study yields a novel purification technique for skeletal muscle glycogen phosphorylase, provides new information on the distribution and isoforms of glycogen phosphorylase, and provides a caveat for using gelatin affinity chromatography as a primary step in purifying collagen-binding proteins from skeletal muscle.

Multicellular organisms utilize cell-to-cell signals to build patterns of cell types within embryos, but the ability of fungi to form organized communities has been largely unexplored. Here we report that colonies of the yeast Saccharomyces cerevisiae formed sharply divided layers of sporulating and nonsporulating cells. Sporulation initiated in the colony's interior, and this region expanded upward as the colony matured. Two key activators of sporulation, IME1 and IME2, were initially transcribed in overlapping regions of the colony, and this overlap corresponded to the initial sporulation region. The development of colony sporulation patterns depended on cell-to-cell signals, as demonstrated by chimeric colonies, which contain a mixture of two strains. One such signal is alkaline pH, mediated through the Rim101p/PacC pathway. Meiotic-arrest mutants that increased alkali production stimulated expression of an early meiotic gene in neighboring cells, whereas a mutant that decreased alkali production (cit1Δ) decreased this expression. Addition of alkali to colonies accelerated the expansion of the interior region of sporulation, whereas inactivation of the Rim101p pathway inhibited this expansion. Thus, the Rim101 pathway mediates colony patterning by responding to cell-to-cell pH signals. Cell-to-cell signals coupled with nutrient gradients may allow efficient spore formation and spore dispersal in natural environments.

Duchenne muscular dystrophy is a progressive muscle disease characterized by increasing muscle weakness and death by the third decade. mdx mice exhibit the underlying muscle disease but appear physically normal with ordinary lifespans, possibly due to compensatory expression of utrophin. In contrast, double mutant mice ( mdx/utrn −/− ), deficient for both dystrophin and utrophin die by ∼3 months and suffer from severe muscle weakness, growth retardation, and severe spinal curvature. The capacity of human retinal dystrophin (Dp260) to compensate for the missing 427kDa muscle dystrophin was tested in mdx/utrn −/− mice. Functional outcomes were assessed by histology, EMG, MRI, mobility, weight and longevity. MCK-driven transgenic expression of Dp260 in mdx/utrn −/− mice converts their disease course from a severe, lethal muscular dystrophy to a viable, mild myopathic phenotype. This finding is relevant to the design of exon-skipping therapeutic strategies since Dp260 lacks dystrophin exons 1–29.

Addition of an organophosphate source to UMR osteoblastic cultures activates a mineralization program in which BSP localizes to extracellular matrix sites where hydroxyapatite crystals are subsequently nucleated (Wang, A., Martin, J. A., Lembke, L. A., and Midura, R. J. (2000) J. Biol. Chem. 275, 11082–11091). This study identifies for the first time novel extracellular spherical structures, termed biomineralization foci (BMF), containing bone acidic glycoprotein-75 (BAG-75), bone sialoprotein (BSP), and alkaline phosphatase that are the exclusive sites of initial nucleation of hydroxyapatite crystals in the UMR model. Importantly, in the absence of added phosphate, UMR cultures after reaching confluency contain two size populations of morphologically identifiable BMF precursors enriched in BAG-75 (15–25 and 150–250 μm in diameter). The shape and size of the smaller population are similar to structures assembled in vitro through self-association of purified BAG-75 protein (Gorski, J. P., Kremer, E. A., Chen, Y., Ryan, S., Fullenkamp, C., Delviscio, J., Jensen, K., and McKee, M. D. (1997) J. Cell. Biochem. 64, 547–564). After organophosphate addition, BSP accumulates within these BAG-75-containing BMF precursors, with hydroxyapatite crystal nucleation occurring subsequently. In summary, BAG-75 is the earliest detectable biomarker that accurately predicts the extracellular sites of de novo biomineralization in UMR cultures. We hypothesize that BAG-75 may perform a key structural role in the assembly of BMF precursors and the recruitment of other proteins such as alkaline phosphatase and BSP. Furthermore, we propose a hypothetical mechanism in which BAG-75 and BSP function actively in nucleation of apatite within BMF.

N-RAP is a recently discovered muscle-specific protein found at cardiac intercalated disks. Double immunogold labeling of mouse cardiac muscle reveals that vinculin is located immediately adjacent to the fascia adherens region of the intercalated disk membrane, while N-RAP extends approximately 100 nm further toward the interior of the cell. We partially purified cardiac intercalated disks using low- and high-salt extractions followed by density gradient centrifugation. Immunoblots show that this preparation is highly enriched in desmin and junctional proteins, including N-RAP, talin, vinculin, beta1-integrin, N-cadherin, and connexin 43. Electron microscopy and immunolabeling demonstrate that N-RAP and vinculin are associated with the large fragments of intercalated disks that are present in this preparation, which also contains numerous membrane vesicles. Detergent treatment of the partially purified intercalated disks removed the membrane vesicles and extracted vinculin and beta1-integrin. Further separation on a sucrose gradient removed residual actin and myosin and yielded a fraction morphologically similar to fasciae adherentes that was highly enriched in N-RAP, N-cadherin, connexin 43, talin, desmin, and alpha-actinin. The finding that N-RAP copurifies with detergent-extracted intercalated disk fragments even though beta-integrin and vinculin have been completely removed suggests that N-RAP association with the adherens junction region is mediated by the cadherin system. Consistent with this hypothesis, we found that recombinant N-RAP fragments bind alpha-actinin in a gel overlay assay. In addition, immunofluorescence shows that N-RAP remains bound at the ends of isolated, detergent-treated cardiac myofibrils. These results demonstrate that N-RAP remains tightly bound to myofibrils and fasciae adherentes during biochemical purification and may be a key constituent in the mechanical link between these two structures.

The vertebrate myotendinous junction contains junctional microfibrils, located in the lamina lucida of the basement membrane. The junctional microfibrils are thought to transmit muscular force across the junctional lamina lucida, also called the connecting domain. If true, deformation of the terminal muscle cell processes and connecting domain during force transmission would be detected as a change in spacing and/or orientation of the junctional microfibrils. This study compared connecting domain morphology in frog semitendinosus muscles fixed in two extremes of resting tension, to elucidate the mechanical properties of the myotendinous junction. An initial study of connecting domain ultrastructure revealed that junctional microfibrils are punctate or spinelike in shape, and that they are distributed in a linear, helically-oriented array on the muscle cell surface. The rows in the surface lattice are 10-15 nm in thickness, have a centre-to-centre distance between rows of approximately 24 nm, and are oriented at approximately 41 degrees with respect of the long axis of the muscle fibre. Comparison of slack and highly stretched myotendinous junctions shows no significant changes in spacing or orientation of either individual junctional microfibrils or rows in the helical surface lattice. Thus, both the connecting domain and terminal cell processes at the myotendinous junction are essentially inextensible under the loading conditions used in this study.

Junctional microfibrils, which span the lamina lucida of the vertebrate myotendinous junction, are thought to function in force transmission at the junction. This hypothesis has been tested by disrupting junctional microfibrils through elimination of extracellular divalent cations, and determining the effects of this treatment on the ultrastructure and mechanics of whole frog skeletal muscles passively stretched to failure. Muscles incubated in divalent cation-free solution failed exclusively in the lamina lucida of the myotendinous junction, while control muscles all failed within the muscle fibres, several millimetres away from the junction. Failure sites from divalent cation-free muscles incubated with antibodies against collagen type IV, laminin, and tenascin showed no labelling of the avulsed ends of the muscle fibres, indicating that remnants of junctional microfibrils observed on the cell surface are not composed of any of these extracellular proteins. All three proteins were present on the tendon side of the failure site, confirming that the lamina densa remains attached to the tendon. Breaking stress for control muscles was 3.47 x 10(5) N m-2, and for divalent cation-free muscles, 1.84 x 10(5) N m-2, or approximately half the control value. Breaking strain averaged 1.17 for divalent cation-free muscles and 1.39 for controls, although the difference was not significant. We conclude that junctional microfibrils are components of a divalent cation-dependent adhesion mechanism at the myotendinous junction. In addition, ultrastructural analysis of divalent cation-free fibres stretched just short of failure suggests that a second, divalent cation-independent mechanism persists along the non-junctional cell surface, and can transmit substantial passive tension from myofibrils laterally to the extracellular matrix, bypassing the failed myotendinous junction.

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The tension that develops when relaxed muscles are stretched is the resting (or passive) tension. It has recently been shown that the resting tension of intact skeletal muscle fibers is equivalent to that of mechanically skinned skeletal muscle fibers. Laser diffraction measurements of sarcomere length have now been used to show that the exponential relation between resting tension and sarcomere length for whole frog semitendinosus muscle is similar to that of single fibers. Slack sarcomere lengths and the rates of stress relaxation in these muscles were similar to those in skinned fibers, and sarcomere length remained unchanged during stress relaxation, as in skinned fibers. Thus, in intact semitendinosus muscle of the frog up to a sarcomere length of about 3.8 micrometers, resting tension arises, not in the connective tissue as is commonly thought, but in the elastic resistance of the myofibrils.

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