The Bone Sialoprotein RGD Domain Modulates and Maintains Periodontal Development.

Bone sialoprotein (gene: Ibsp; protein: BSP) is a multifunctional extracellular matrix protein present in bone, cementum, and dentin. Accumulating evidence supports BSP as a key regulator of mineralized tissue formation via evolutionarily conserved functional domains, including a C-terminal integrin-binding Arg-Gly-Asp (RGD) domain implicated in extracellular matrix-cell signaling. Ablation of Ibsp in mice (Ibsp-/-) results in impaired bone growth and mineralization and defective osteoclastogenesis, with effects in the craniofacial region including reduced acellular cementum formation, detachment of the periodontal ligament (PDL), alveolar bone hypomineralization, and severe periodontal breakdown. We hypothesized that BSP-RGD plays an important role in cementum and alveolar bone formation and mineralization, as well as periodontal function. This hypothesis was tested by replacing the RGD motif with a nonfunctional Lys-Ala-Glu (KAE) sequence in (IbspKAE/KAE) mice and OCCM.30 murine (IbspKAE) cementoblasts. The RGD domain was not critical for acellular or cellular cementum formation in IbspKAE/KAE mice. However, PDL volume and thickness were increased, and significantly more tartrate-resistant acid phosphatase-positive osteoclasts were found on alveolar bone surfaces of IbspKAE/KAE mice versus wild type mice. PDL organization was disrupted as indicated by picrosirius red stain, second harmonic generation imaging, dynamic mechanical analysis, and decreased asporin proteoglycan localization. In vitro studies implicated RGD functions in cell migration, adhesion, and mineralization, and this was confirmed by an ossicle implant model where cells lacking BSP-RGD showed substantial defects as compared with controls. In total, the BSP-RGD domain is implicated in periodontal development, though the scale and scope of changes indicated by in vitro studies indicate that other factors may partially compensate for and reduce the phenotypic severity of mice lacking BSP-RGD in vivo.

Attachment of Algal Cells to Zwitterionic Self-Assembled Monolayers Comprised of Different Anionic Compounds.

The influence of zwitterionic self-assembled monolayers on settlement and removal of algae was studied. The monolayers were constructed either from zwitterionic thiols or from solutions of positively and negatively charged thiols. The cationic component was composed of quaternary ammonium terminated thiols and the anionic component contained sulfate or carboxylate termination. During assembly, all surfaces showed a strong tendency for equilibration of the surface charge. Settlement and adhesion assays with zoospores of Ulva linza and the diatom Navicula incerta, and field tests of the initial surface colonization revealed the relevance of charge equilibration for the biological inertness of the prepared surfaces.

Resistance of Amphiphilic Polysaccharides against Marine Fouling Organisms.

Amphiphilic coatings are promising candidates for fouling-release applications. As hydrophilic components, polysaccharides are interesting and environmentally benign building blocks. We used covalently coupled alginic acid (AA) and hyaluronic acid (HA) and postmodified them with a hydrophobic fluorinated amine. The surfaces showed good stability under marine conditions and fluorination led to a decreased uptake of Ca(2+) ions after modification. In single species settlement assays (bacteria, diatoms, barnacle cypris larvae), the modification decreased the settlement density and/or the adhesion strength of many of the tested species. Field studies supported findings of the laboratory experiments, as hydrophobic modification of AA and HA decreased diatom colonization.

Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence.

The large size of the multinucleated muscle fibers of skeletal muscle makes their examination for structural and pathological defects a challenge. Sections and single fibers are accessible to antibodies and other markers but imaging of such samples does not provide a three-dimensional view of the muscle. Regrettably, bundles of fibers cannot be stained or imaged easily. Two-photon microscopy techniques overcome these obstacles. Second harmonic generation (SHG) by myosin filaments and two-photon excited fluorescence (2PEF) of mitochondrial and lysosomal components provides detailed structural information on unstained tissue. Furthermore, the infrared exciting light can penetrate several layers of muscle fibers and the minimal processing is particularly valuable for fragile biopsies. Here we demonstrate the usefulness of SHG, combined with 2PEF, to reveal enlarged lysosomes and accumulations of non-contractile material in muscles from the mouse model for the lysosomal storage disorder Pompe disease (PD), and in biopsies from adult and infant PD patients. SHG and 2PEF also detect sarcomeric defects that may presage the loss of myofibrils in atrophying muscle and signify loss of elasticity. The combination of SHG and 2PEF should be useful in the analysis and diagnosis of a wide range of skeletal muscle pathologies.

Blood vessels and desmin control the positioning of nuclei in skeletal muscle fibers.

Skeletal muscle fibers contain hundreds to thousands of nuclei which lie immediately under the plasmalemma and are spaced out along the fiber, except for a small cluster of specialized nuclei at the neuromuscular junction. How the nuclei attain their positions along the fiber is not understood. Here we show that the nuclei are preferentially localized near blood vessels (BV), particularly in slow-twitch, oxidative fibers. Thus, in rat soleus muscle fibers, 81% of the nuclei appear next to BV. Lack of desmin markedly perturbs the distribution of nuclei along the fibers but does not prevent their close association with BV. Consistent with a role for desmin in the spacing of nuclei, we show that denervation affects the organization of desmin filaments as well as the distribution of nuclei. During chronic stimulation of denervated muscles, new BV form, along which muscle nuclei align themselves. We conclude that the positioning of nuclei along muscle fibers is plastic and that BV and desmin intermediate filaments each play a distinct role in the control of this positioning.

Golgi complex reorganization during muscle differentiation: visualization in living cells and mechanism.

During skeletal muscle differentiation, the Golgi complex (GC) undergoes a dramatic reorganization. We have now visualized the differentiation and fusion of living myoblasts of the mouse muscle cell line C2, permanently expressing a mannosidase-green fluorescent protein (GFP) construct. These experiments reveal that the reorganization of the GC is progressive (1-2 h) and is completed before the cells start fusing. Fluorescence recovery after photobleaching (FRAP), immunofluorescence, and immunogold electron microscopy demonstrate that the GC is fragmented into elements localized near the endoplasmic reticulum (ER) exit sites. FRAP analysis and the ER relocation of endogenous GC proteins by phospholipase A2 inhibitors demonstrate that Golgi-ER cycling of resident GC proteins takes place in both myoblasts and myotubes. All results support a model in which the GC reorganization in muscle reflects changes in the Golgi-ER cycling. The mechanism is similar to that leading to the dispersal of the GC caused, in all mammalian cells, by microtubule-disrupting drugs. We propose that the trigger for the dispersal results, in muscle, from combined changes in microtubule nucleation and ER exit site localization, which place the ER exit sites near microtubule minus ends. Thus, changes in GC organization that initially appear specific to muscle cells, in fact use pathways common to all mammalian cells.

Glycogen synthase localization and activity in rat skeletal muscle is strongly dependent on glycogen content.

1. The influence of muscle glycogen content on glycogen synthase (GS) localization and GS activity was investigated in skeletal muscle from male Wistar rats. 2. Two groups of rats were obtained, preconditioned with a combination of exercise and diet to obtain either high (HG) or low (LG) muscle glycogen content. The cellular distribution of GS was studied using subcellular fractionation and confocal microscopy of immunostained single muscle fibres. Stimulation of GS activity in HG and LG muscle was obtained with insulin or contractions in the perfused rat hindlimb model. 3. We demonstrate that GS translocates from a glycogen-enriched membrane fraction to a cytoskeleton fraction when glycogen levels are decreased. Confocal microscopy supports the biochemical observations that the subcellular localization of GS is influenced by muscle glycogen content. GS was not found in the nucleus. 4. Investigation of the effect of glycogen content on GS activity in basal and insulin- and contraction-stimulated muscle shows that glycogen has a strong inhibitory effect on GS activity. Our data demonstrate that glycogen is a more potent regulator of glycogen synthase activity than insulin. Furthermore we show that the contraction-induced increase in GS activity is merely a result of a decrease in muscle glycogen content. 5. In conclusion, the present study shows that GS localization is influenced by muscle glycogen content and that not only basal but also insulin- and contraction-stimulated GS activity is strongly regulated by glycogen content in skeletal muscle.

Golgi complex, endoplasmic reticulum exit sites, and microtubules in skeletal muscle fibers are organized by patterned activity.

The Golgi complex of skeletal muscle fibers is made of thousands of dispersed elements. The distributions of these elements and of the microtubules they associate with differ in fast compared with slow and in innervated compared with denervated fibers. To investigate the role of muscle impulse activity, we denervated fast extensor digitorum longus (EDL) and slow soleus (SOL) muscles of adult rats and stimulated them directly with patterns that resemble the impulse patterns of normal fast EDL (25 pulses at 150 Hz every 15 min) and slow SOL (200 pulses at 20 Hz every 30 sec) motor units. After 2 weeks of denervation plus stimulation, peripheral and central regions of muscle fibers were examined by immunofluorescence microscopy with regard to density and distribution of Golgi complex, microtubules, glucose transporter GLUT4, centrosomes, and endoplasmic reticulum exit sites. In extrajunctional regions, fast pattern stimulation preserved normal fast characteristics of all markers in EDL type IIB/IIX fibers, although inducing changes toward the fast phenotype in originally slow type I SOL fibers, such as a 1.5-fold decrease of the density of Golgi elements at the fiber surface. Slow pattern stimulation had converse effects such as a 2.2-fold increase of the density of Golgi elements at the EDL fiber surface. In junctional regions, where fast and slow fibers are similar, both stimulation patterns prevented a denervation-induced accumulation of GLUT4. The results indicate that patterns of muscle impulse activity, as normally imposed by motor neurons, play a major role in regulating the organization of Golgi complex and related proteins in the extrajunctional region of muscle fibers.

The organization of the Golgi complex and microtubules in skeletal muscle is fiber type-dependent.

Skeletal muscle has a nonconventional Golgi complex (GC), the organization of which has been a subject of controversy in the past. We have now examined the distribution of the GC by immunofluorescence and immunogold electron microscopy in whole fibers from different rat muscles, both innervated and experimentally denervated. The total number of GC elements, small polarized stacks of cisternae, is quite similar in all fibers, but their intracellular distribution is fiber type-dependent. Thus, in slow-twitch, type I fibers, approximately 75% of all GC elements are located within 1 micrometer from the plasma membrane, and each nucleus is surrounded by a belt of GC elements. In contrast, in the fast-twitch type IIB fibers, most GC elements are in the fiber core, and most nuclei only have GC elements at their poles. Intermediate, type IIA fibers also have an intermediate distribution of GC elements. Interestingly, the distribution of microtubules, with which GC elements colocalize, is fiber type-dependent as well. At the neuromuscular junction, the distribution of GC elements and microtubules is independent of fiber type, and junctional nuclei are surrounded by GC elements in all fibers. After denervation of the hindlimb muscles, GC elements as well as microtubules converge toward a common pattern, that of the slow-twitch fibers, in all fibers. Our data suggest that innervation regulates the distribution of microtubules, which in turn organize the Golgi complex according to muscle fiber type.

A simple method to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences.

A simple modification to standard binary vector design has been utilized to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences. A lethal gene was incorporated into the non-T-DNA portion of a binary vector, along with a screenable marker. The resulting class of vectors is designated as NTL T-DNA vectors (non-T-DNA lethal gene-containing T-DNA vectors). The lethal gene used here is a CaMV 35S-barnase gene with an intron in the coding sequence (barnase-INT); the screenable marker is a pMAS-luciferase gene with an intron in the coding sequence (LUC-int). To evaluate the utility of this vector design, tobacco plants were transformed with either the NTL T-DNA vector or a control vector from which most of the barnase-INT gene was deleted. Populations of 50 transgenic plants were scored for LUC expression. The results indicated a dramatic reduction in the presence of non-T-DNA sequences in the transgenic population using the NTL T-DNA vector. Only one transgenic plant was found to be LUC+ using the NTL vector, compared with 42 of 50 plants using the control vector. Importantly, the efficiency with which transformed tobacco plants was obtained was reduced by no more than 30%. The reduction in LUC+ transgenics was partially reversed when a barstar-expressing tobacco line was transformed, indicating that barnase expression was responsible for the reduced frequency of incorporating non-T-DNA sequences. Similar transformation results were obtained with tomato and grape. The incorporation of a barnase-INT gene outside the left border appears to provide a generally applicable tool for enriching an Agrobacterium-transformed population for plants containing only T-DNA sequences.

Caveolin-3 is associated with the T-tubules of mature skeletal muscle fibers.

Caveolae are abundant in skeletal muscle and their coat contains a specific isoform of caveolin, caveolin-3. It has been suggested that during muscle development, caveolin-3 is associated with the T-tubules, but that in adult muscle it is found on the plasma membrane only. We have studied the distribution of caveolin-3 in single skeletal muscle fibers from adult rat soleus by confocal immunofluorescence and by immunogold electron microscopy. We found that caveolin-3 occurs at the highest density on the plasma membrane but is also present in the core of the fibers, at the I-band/A-band interface where it is associated with the T-tubules. In neither domain of the muscle surface does caveolin-3 colocalize with the glucose transporter GLUT4 and there is no evidence for internalization of the caveolae in muscle.

Molecular mechanisms involved in GLUT4 translocation in muscle during insulin and contraction stimulation.

Studies in mammalian cells have established the existence of numerous intracellular signaling cascades that are critical intermediates in the regulation of various biological functions. Over the past few years considerable research has shown that many of these signaling proteins are expressed in skeletal muscle. However, the detailed mechanisms involved in the regulation of glucose transporter (GLUT4) translocation from intracellular compartments to the cell surface membrane in response to insulin and contractions in skeletal muscle are not well understood. In the present essay we report three different approaches to unravel the GLUT4 translocation mechanism: 1. specific pertubation of the insulin and/or contraction signaling pathways; 2. characterization of the protein composition of GLUT4-containing vesicles with the expectation that knowledge of the constituent proteins of the vesicles may help in understanding their trafficking; 3. degree of co-immunolocalization of the GLUT4 glucose transporters with other membrane marker proteins assessed by immunofluorescense and electron microscopy.

Analysis of GLUT4 distribution in whole skeletal muscle fibers: identification of distinct storage compartments that are recruited by insulin and muscle contractions.

The effects of insulin stimulation and muscle contractions on the subcellular distribution of GLUT4 in skeletal muscle have been studied on a preparation of single whole fibers from the rat soleus. The fibers were labeled for GLUT4 by a preembedding technique and observed as whole mounts by immunofluorescence microscopy, or after sectioning, by immunogold electron microscopy. The advantage of this preparation for cells of the size of muscle fibers is that it provides global views of the staining from one end of a fiber to the other and from one side to the other through the core of the fiber. In addition, the labeling efficiency is much higher than can be obtained with ultracryosections. In nonstimulated fibers, GLUT4 is excluded from the plasma membrane and T tubules. It is distributed throughout the muscle fibers with approximately 23% associated with large structures including multivesicular endosomes located in the TGN region, and 77% with small tubulovesicular structures. The two stimuli cause translocation of GLUT4 to both plasma membrane and T tubules. Quantitation of the immunogold electron microscopy shows that the effects of insulin and contraction are additive and that each stimulus recruits GLUT4 from both large and small depots. Immunofluorescence double labeling for GLUT4 and transferrin receptor (TfR) shows that the small depots can be further subdivided into TfR-positive and TfR-negative elements. Interestingly, we observe that colocalization of TfR and GLUT4 is increased by insulin and decreased by contractions. These results, supported by subcellular fractionation experiments, suggest that TfR-positive depots are only recruited by contractions. We do not find evidence for stimulation-induced unmasking of resident surface membrane GLUT4 transporters or for dilation of the T tubule system (Wang, W., P.A. Hansen, B.A. Marshall, J.O. Holloszy, and M. Mueckler. 1996. J. Cell Biol. 135:415-430).

Ribosome association contributes to restricting mRNAs to the cell body of hippocampal neurons.

In neurons, mRNAs are differentially sorted to axons, dendrites, and the cell body. Recently, regions of certain mRNAs have been identified that target those mRNAs for translocation to the processes. However, the mechanism by which many, if not most mRNAs are retained in the cell body is not understood. Total inhibition of translation, by puromycin or cycloheximide, results in the mislocalization of cell body mRNAs to dendrites. We have examined the effect of translational inhibitors on the localization of ferritin mRNA, the translation of which can also be inhibited specifically by reducing iron levels. Using nonisotopic in situ hybridization, ferritin mRNA is found restricted to the cell body of cultured rat hippocampal neurons. Following treatment with either puromycin or cycloheximide, it migrates into dendrites. Control experiments reveal that the drugs affect neither the viability of the neuronal cultures, nor the steady-state level of ferritin mRNA. When transcription and protein synthesis are inhibited simultaneously, ferritin mRNA is found in the dendrites of puromycin, but not of cycloheximide-treated neurons. However, the localization of ferritin mRNA is unaffected by changes in iron concentration that regulate its translation rate specifically. We propose a model whereby cell body-restricted mRNAs are maintained in that location by association with ribosomes and with another cell component, which traps mRNAs when they are freed of ribosome association. The release of all mRNA species, as happens after total protein synthesis inhibition, floods the system and allows cell body mRNAs to diffuse into dendrites. In contrast, the partial release of the single ferritin mRNA species does not saturate the trapping system and the mRNA is retained in the cell body.

Altered subcellular localization patterns of ferritin and beta-actin mRNAs in muscle cultures, resulting from incomplete penetration of digoxigenin-labelled riboprobes.

Protocols for in situ hybridization (ISH) of cultured cells often include storage in alcohol at -20 degrees C between fixation of the cultures and the ISH procedure. In experiments aimed at localizing ferritin mRNA in C2 muscle cultures by ISH with digoxigenin-labelled riboprobes, we have noticed that omission of the ethanol storage dramatically changed the pattern of mRNA localization. In cultures stored in 50%, 70%, or 90% ethanol for at least 15 min, ferritin signal was stronger on myotubes than myoblasts but was uniformly distributed over both. In untreated cultures, the signal was patchy, concentrated on the extremities of the elongated myoblasts and very sparse in myotubes. Similar results were obtained with a probe to beta-actin used as a control, except that signal was higher in myoblasts in all conditions. When the probes were reduced in size to approximately 100 bases from 561 for ferritin and 1150 for actin, the pattern became uniform, regardless of prehybridization treatment. The patchy pattern disappeared when cells were treated with RNase A following hybridization, suggesting that it is non-specific, despite its absence in cultures hybridized with a sense probe. We conclude that incomplete access of RNA probes can result not only in a reduced ISH signal but also in artefactual patterns of mRNA localization.

Nuclear domains in skeletal myotubes: the localization of transferrin receptor mRNA is independent of its half-life and restricted by binding to ribosomes.

The retention of mRNAs near the nuclei that synthesize them may be an important feature of the organization of multinucleated skeletal myotubes. Here, we assess the possible role of two factors in this localization. First, we examine the role of mRNA half-life, by studying the distribution of the mRNA for the transferrin receptor (TfR), whose half-life can be manipulated in culture by changing the availability of iron. In situ hybridization of myotubes of the mouse muscle cell line C2 shows that TfR mRNA is concentrated in the core of the myotubes. Its distribution around the nuclei is often asymmetric and its concentration changes abruptly. Stable transcripts display the same asymmetric localization as unstable ones, suggesting that half-life does not determine subcellular localization of TfR mRNA. Differential effects of the protein synthesis inhibitors puromycin and cycloheximide suggest that the mRNA is retained in position by its association with ribosomes. We then examine the distribution of the rough endoplasmic reticulum (RER) and find it to be broader than the distribution of TfR mRNA. In contrast to TfR mRNA, the mRNA for a secreted immunoglobulin kappa light chain has a more uniform distribution. Taken together, the results suggest that TfR mRNA may associate with RER subdomains by specific targeting.

Expression of the adrenoleukodystrophy protein in the human and mouse central nervous system.

The gene mutated in X-linked adrenoleukodystrophy (ALD), a progressive demyelinating disease, codes for a protein (ALDP) involved in very-long-chain fatty acid (VLCFA) transport. The expression of ALDP and of two peroxisomal enzymes involved in beta-oxidation of VLCFA, acyl-CoA oxidase, and catalase was studied in human and mouse brain. The pattern of expression was similar in both species. While acyl-CoA oxidase and catalase are found in all types of CNS cells, including neurons and oligodendrocytes, ALDP expression is restricted mostly to the white matter and endothelial cells. ALDP is highly expressed in astrocytes and microglial cells in vivo and in regenerating oligodendrocytes in vitro. In contrast, in vivo, ALDP is detected in much fewer oligodendrocytes and quantitative Western blot analysis confirmed the lower abundance of ALDP in these cells than in astrocytes. Only oligodendrocytes localized in corpus callosum, internal capsules, and anterior commissure express ALDP at levels comparable to those seen in astrocytes. In ALD, demyelination is first detected in these white matter regions, suggesting that the ALD gene mutation selectively affects those oligodendrocytes strongly expressing ALDP. Because of their failure to express ALDP, microglia and astrocytes may also contribute to demyelination in ALD patients.

GLUT4 in cultured skeletal myotubes is segregated from the transferrin receptor and stored in vesicles associated with TGN.

There is little consensus on the nature of the storage compartment of the glucose transporter GLUT4, in non-stimulated cells of muscle and fat. More specifically, it is not known whether GLUT4 is localized to unique, specialized intracellular storage vesicles, or to vesicles that are part of the constitutive endosomal-lysosomal pathway. To address this question, we have investigated the localization of the endogenous GLUT4 in non-stimulated skeletal myotubes from the cell line C2, by immunofluorescence and immunoelectron microscopy. We have used a panel of antibodies to markers of the Golgi complex (alpha mannosidase II and giantin), of the trans-Golgi network (TGN38), of lysosomes (lgp110), and of early and late endosomes (transferrin receptor and mannose-6-phosphate receptor, respectively), to define the position of their subcellular compartments. By immunofluorescence, GLUT4 appears concentrated in the core of the myotubes. It is primarily found around the nuclei, in a pattern suggesting an association with the Golgi complex, which is further supported by colocalization with giantin and by immunogold electron microscopy. GLUT4 appears to be in the trans-most cisternae of the Golgi complex and in vesicles just beyond, i.e. in the structures that constitute the trans-Golgi network (TGN). In myotubes treated with brefeldin A, the immunofluorescence pattern of GLUT4 is modified, but it differs from both Golgi complex markers and TGN38. Instead, it resembles the pattern of the transferrin receptor, which forms long tubules. In untreated cells, double staining for GLUT4 and transferrin receptor by immunofluorescence shows similar but distinct patterns. Immunoelectron microscopy localizes transferrin receptor, detected by immunoperoxidase, to large vesicles, presumably endosomes, very close to the GLUT4-containing tubulo-vesicular elements. In brefeldin A-treated cells, a network of tubules of approximately 70 nm diameter, studded with varicosities, stains for both GLUT4 and transferrin receptor, suggesting that brefeldin A has caused fusion of the transferrin receptor and GLUT4-containing compartments. The results suggest that GLUT4 storage vesicles constitute a specialized compartment that is either a subset of the TGN, or is very closely linked to it. The link between GLUT4 vesicles and transferrin receptor containing endosomes, as revealed by brefeldin A, may be important for GLUT4 translocation in response to muscle stimulation.