Lamin A/C sustains PcG protein architecture, maintaining transcriptional repression at target genes

Beyond its role in providing structure to the nuclear envelope, lamin A/C is involved in transcriptional regulation. However, its cross talk with epigenetic factors--and how this cross talk influences physiological processes--is still unexplored. Key epigenetic regulators of development and differentiation are the Polycomb group (PcG) of proteins, organized in the nucleus as microscopically visible foci. Here, we show that lamin A/C is evolutionarily required for correct PcG protein nuclear compartmentalization. Confocal microscopy supported by new algorithms for image analysis reveals that lamin A/C knock-down leads to PcG protein foci disassembly and PcG protein dispersion. This causes detachment from chromatin and defects in PcG protein-mediated higher-order structures, thereby leading to impaired PcG protein repressive functions. Using myogenic differentiation as a model, we found that reduced levels of lamin A/C at the onset of differentiation led to an anticipation of the myogenic program because of an alteration of PcG protein-mediated transcriptional repression. Collectively, our results indicate that lamin A/C can modulate transcription through the regulation of PcG protein epigenetic factors

Electrospun Collagen: A Tissue Engineering Scaffold with Unique Functional Properties in a Wide Variety of Applications

Type I collagen and gelatin, a derivative of Type I collagen that has been denatured, can each be electrospun into tissue engineering scaffolds composed of nano- to micron-scale diameter fibers. We characterize the biological activity of these materials in a variety of tissue engineering applications, including endothelial cell-scaffold interactions, the onset of bone mineralization, dermal reconstruction, and the fabrication of skeletal muscle prosthetics. Electrospun collgen (esC) consistently exhibited unique biological properties in these functional assays. Even though gelatin can be spun into fibrillar scaffolds that resemble scaffolds of esC, our assays reveal that electrospun gelatin (esG) lacks intact α chains and is composed of proinflammatory peptide fragments. In contrast, esC retains intact α chains and is enriched in the α 2(I) subunit. The distinct fundamental properties of the constituent subunits that make up esC and esG appear to define their biological and functional properties

Who Needs Microtubules? Myogenic Reorganization of MTOC, Golgi Complex and ER Exit Sites Persists Despite Lack of Normal Microtubule Tracks

A wave of structural reorganization involving centrosomes, microtubules, Golgi complex and ER exit sites takes place early during skeletal muscle differentiation and completely remodels the secretory pathway. The mechanism of these changes and their functional implications are still poorly understood, in large part because all changes occur seemingly simultaneously. In an effort to uncouple the reorganizations, we have used taxol, nocodazole, and the specific GSK3-β inhibitor DW12, to disrupt the dynamic microtubule network of differentiating cultures of the mouse skeletal muscle cell line C2. Despite strong effects on microtubules, cell shape and cell fusion, none of the treatments prevented early differentiation. Redistribution of centrosomal proteins, conditional on differentiation, was in fact increased by taxol and nocodazole and normal in DW12. Redistributions of Golgi complex and ER exit sites were incomplete but remained tightly linked under all circumstances, and conditional on centrosomal reorganization. We were therefore able to uncouple microtubule reorganization from the other events and to determine that centrosomal proteins lead the reorganization hierarchy. In addition, we have gained new insight into structural and functional aspects of the reorganization of microtubule nucleation during myogenesis

Proteins in Enamel Development: Secretome, Transport and Posttranslational Fate

Amelogenesis is a process involving formation of mineralized tooth enamel by ameloblast cells. During amelogenesis, ameloblasts secrete matrix consisting of enamel proteins that facilitate future enamel prism formation. In this study, we focused on understanding the transportation and fate of enamel proteins from the time of secretion in the enamel matrix to the stage where the fragments come in contact with the initial enamel prisms and thereafter. For the first part, we used focused on the transportation part of proteins using Phospho1 knockout mice to understand the role of secretory vesicles in ameloblasts and the transportation of proteins through ameloblasts. Our study demonstrated the presence of PHOSPHO1 protein in ameloblasts including the enamel layer and the ameloblast Tomes’ processes. The effect of disruption of the vesicular coat protein in Phospho1 -/- mice significantly affected physiological enamel mineralization. A loss of enamel prism organization was observed and a 25% increase in enamel layer was noted indicating the retention of enamel proteins in the matrix due to transportation failure. Phospho1 -/- mice indicated that the matrix vesicular protein PHOSPHO 1 is an integral part of enamel mineralization process. For the second part, our intent was to use a mouse molar model to establish a temporo-spatial correlation of the events occurring through the different stages of enamel development and document the consequential changes in the enamel matrix proteins. Our proteomic analysis identified some novel proteins which may also play a key role during amelogenesis. The final part of the study focuses on amelogenin and its fate once it is secreted in the matrix. Our data demonstrates that the protein matrix configuration changes substantially from the time it is transported through the ameloblasts and the amelogenin fragmentation plays a major role in enamel crystal growth. The Western blot analysis of enamel matrix from different layers of porcine enamel tooth organ documented the close association of amelogenin C-terminus with the apatite crystal surface while the N-terminus fragments are likely retained in between the growing enamel crystals indicating perhaps the C-terminal fragments help with prism growth in C-axis direction while N-terminal fragments maintain the interprismatic distance

Change blindness: eradication of gestalt strategies

Arrays of eight, texture-defined rectangles were used as stimuli in a one-shot change blindness (CB) task where there was a 50% chance that one rectangle would change orientation between two successive presentations separated by an interval. CB was eliminated by cueing the target rectangle in the first stimulus, reduced by cueing in the interval and unaffected by cueing in the second presentation. This supports the idea that a representation was formed that persisted through the interval before being 'overwritten' by the second presentation (Landman et al, 2003 Vision Research 43149–164]. Another possibility is that participants used some kind of grouping or Gestalt strategy. To test this we changed the spatial position of the rectangles in the second presentation by shifting them along imaginary spokes (by ±1 degree) emanating from the central fixation point. There was no significant difference seen in performance between this and the standard task [F(1,4)=2.565, p=0.185]. This may suggest two things: (i) Gestalt grouping is not used as a strategy in these tasks, and (ii) it gives further weight to the argument that objects may be stored and retrieved from a pre-attentional store during this task

Selected properties of the cytoskeletal protein synemin and its interactions with proteins at the muscle Z-line

The primary purposes of this study were to: (1) examine selected properties of the intermediate filament-associated protein (IFAP), synemin, (2) identify synemin (or a synemin homolog) in adult mammalian muscle, and (3) examine synemin\u27s ability to interact with proteins located at the myofibrillar Z-line. An integrated biochemical, immunological, and ultrastructural approach was used;Synemin (230 kD) was solubilized from an actomyosin-extracted avian smooth muscle residue and purified by chromatography on two hydroxyapatite columns and one DEAE-Sephacel column in the presence of 6 M urea. Renatured, soluble synemin was obtained by removal of the urea by dialysis against 10 mM Tris-HCl, pH 8.5. Electron microscopy of negatively stained specimens revealed that synemin in 10 mM Tris-HCl, pH 8.5, is spherical in nature (diameter ~11 nm), and chemical crosslinking experiments showed that synemin molecules exist primarily as dimers. Synemin and desmin have similar pH- and ionic strength-dependent solubility properties, but desmin forms intermediate filaments (IFs) and synemin forms complex aggregates under physiological-like conditions. Synemin\u27s wide distribution in adult avian and mammalian skeletal, cardiac, and smooth muscles was shown by Western blot analysis and by immunofluorescence labeling of isolated myofibrils and of muscle cryosections. Double-labeling experiments with conventional immunofluorescence, confocal scanning laser microscopy, and computer-assisted image analysis showed that desmin and synemin colocalize at the myofibrillar Z-lines in a punctate pattern;Synemin\u27s ability to interact with desmin was examined by negative staining and immunogold electron microscopy as well as by immunoblot overlay assays. Purified desmin self-assembles into long (\u3e1 [mu]m) IFs when dialyzed against physiological-like buffers; however, desmin\u27s ability to assemble into these long IFs decreases as the relative amount of synemin to desmin increases. The smallest, full width (~10 nm) IF assembly intermediate formed in the presence of synemin was ~50-70 nm long. Immunogold labeling experiments indicated that synemin binds along the desmin IFs, or at points of filament intersection. Solid-phase binding assays indicated that synemin can bind to desmin and to [alpha]-actinin;These results, taken in toto, indicate that synemin (or a homolog) exists in mammalian muscles, that synemin copolymerizes with, or binds along the length of, desmin IFs, and suggest that synemin may serve as a cytoskeletal crosslinking component between IFs and myofibrils in muscle cells

Aerospace Medicine and Biology: A continuing bibliography with indexes, supplement 171

This bibliography lists 186 reports, articles, and other documents introduced into the NASA scientific and technical information system in August 1977