Spatial organization of microfilaments and vitronectin receptor, alpha v beta 3, in osteoclasts. A study using confocal laser scanning microscopy

1993 ◽  
Vol 104 (3) ◽  
pp. 663-670 ◽  
Author(s):  
P.T. Lakkakorpi ◽  
M.H. Helfrich ◽  
M.A. Horton ◽  
H.K. Vaananen
Keyword(s):  
Cell Adhesion ◽  
Bone Resorption ◽  
Spatial Organization ◽  
Basolateral Membrane ◽  
Bone Matrix ◽  
Cytoskeletal Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Bone Surface ◽  
Vitronectin Receptor

The primary function of the osteoclast is that of the major cell mediating bone resorption. They are actively migrating cells but during resorption they polarize to form a specialized tight attachment structure, the sealing zone, adjacent to the mineralized bone matrix. The processes of adhesion to, and migration on, bone involves cell adhesion molecules, integrins, interacting with their ligands in bone. We have used confocal microscopy to analyse, in rat osteoclasts cultured on bone and glass substrata, the distribution of vitronectin receptor, the major integrin of osteoclasts, and cytoskeletal proteins that it may be linked to. Double staining for F-actin and vinculin, and for vinculin with talin, revealed that cytoskeletal organization differs at various activation states of osteoclasts. Microfilament structures were flat, of 1.5 microns size, and concentrated near the bone surface. The vitronectin receptor was localized both in the basolateral membrane (away from the bone surface) and in the ruffled border (adjacent to bone) in osteoclasts cultured on bone, but was detected mainly in the basolateral membrane when cultured on glass. The vitronectin receptor appeared to be condensed on small microvilli-like projections on the basolateral membrane of osteoclasts on either bone or glass and may provide a route for alternative signalling pathways to modify osteoclast behaviour, other than by influencing cell adhesion directly. The leading edges of migrating osteoclasts, and the attachment structure, a broad vinculin band, which forms before bone resorption, also expressed vitronectin receptor, particularly when the antibody against the alpha v subunit was used.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered Cytoskeleton in Smooth Muscle of Aganglionic Bowel

2002 ◽  
Vol 126 (6) ◽  
pp. 692-696
Author(s):  
Laszlo Nemeth ◽  
Udo Rolle ◽  
Prem Puri
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Hirschsprung Disease ◽  
Muscle Cells ◽  
Cytoskeletal Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Normal Bowel ◽  
Pull Through

Abstract Context.—Intestinal motility is under the control of smooth muscle cells, enteric plexus, and hormonal factors. In Hirschsprung disease (HD), the aganglionic colon remains spastic or tonically enhanced and unable to relax. The smooth muscle cell's cytoskeleton consists of proteins or structures whose primary function is to link or connect protein filaments to each other or to the anchoring sites. Dystrophin is a subsarcolemmal protein with a double adhesion property, one between the membrane elements and the contractile filaments of the cytoskeleton and the other between the cytoskeletal proteins and the extracellular matrix. Desmin and vinculin are functionally related proteins that are present in the membrane-associated dense bodies in the sarcolemma of the smooth muscle cells. Objective.—To examine the distribution of the cytoskeletal proteins in the smooth muscle of the aganglionic bowel. Design.—Bowel specimens from ganglionic and aganglionic sections of the colon were collected at the time of pull-through surgery from 8 patients with HD. Colon specimens collected from 4 patients at the time of bladder augmentation acted as controls. Anti-dystrophin, anti-desmin, and anti-vinculin antibodies were used for fluorescein immunostaining using confocal laser scanning microscopy. Results.—Moderate to strong dystrophin immunoreactivity was observed at the periphery of smooth muscle fibers in normal bowel and ganglionic bowel from patients with HD, whereas dystrophin immunoreactivity was either absent or weak in the smooth muscle of aganglionic colon. Moderate to strong cytoplasmic immunostaining for vinculin and desmin was seen in the smooth muscle of normal bowel and ganglionic bowel from patients with HD, whereas vinculin and desmin staining in the aganglionic colon was absent or weak. Conclusion.—This study demonstrates that the cytoskeletal proteins are abundant in the smooth muscle of normal bowel, but are absent or markedly reduced in the aganglionic bowel of HD. As cytoskeletal proteins are required for the coordinated contraction of muscle cells, their absence may be responsible for the motility dysfunction in the aganglionic segment.

scholarly journals Software-Based Three-Dimensional Deconvolution Microscopy of Cytoskeletal Proteins in Cultured Fibroblast Using Open-Source Software and Open Hardware

2019 ◽  
Vol 5 (12) ◽  
pp. 88
Author(s):  
Kazuo Katoh
Keyword(s):  
Open Source ◽  
Open Source Software ◽  
Laser Scanning ◽  
Low Cost ◽  
Three Dimensional ◽  
Cytoskeletal Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Actual Measurement ◽  
Open Hardware

As conventional fluorescence microscopy and confocal laser scanning microscopy generally produce images with blurring at the upper and lower planes along the z-axis due to non-focal plane image information, the observation of biological images requires “deconvolution.” Therefore, a microscope system’s individual blur function (point spread function) is determined theoretically or by actual measurement of microbeads and processed mathematically to reduce noise and eliminate blurring as much as possible. Here the author describes the use of open-source software and open hardware design to build a deconvolution microscope at low cost, using readily available software and hardware. The advantage of this method is its cost-effectiveness and ability to construct a microscope system using commercially available optical components and open-source software. Although this system does not utilize expensive equipment, such as confocal and total internal reflection fluorescence microscopes, decent images can be obtained even without previous experience in electronics and optics.

scholarly journals Vitronectin receptor has a role in bone resorption but does not mediate tight sealing zone attachment of osteoclasts to the bone surface.

1991 ◽  
Vol 115 (4) ◽  
pp. 1179-1186 ◽  
Author(s):  
P T Lakkakorpi ◽  
M A Horton ◽  
M H Helfrich ◽  
E K Karhukorpi ◽  
H K Väänänen
Keyword(s):  
Bone Resorption ◽  
Cell Membrane ◽  
Immunoperoxidase Staining ◽  
Bone Surface ◽  
Intense Staining ◽  
Vitronectin Receptor ◽  
Gold Particles ◽  
Light Microscopical Level ◽  
Sealing Zone ◽  
Arginine Glycine Aspartic Acid

During bone resorption, osteoclasts form a tight attachment, the sealing zone, around resorption lacunae. Vitronectin receptor has previously been shown to be expressed in osteoclasts and it has been suggested that it mediates the tight attachment at the sealing zone. In this study we have shown that glycine-arginine-glycine-aspartic acid-serine pentapeptide inhibits bone resorption by isolated osteoclasts and drastically changes the morphology of the osteoclasts. When the vitronectin receptor was localized by immunofluorescence in rat and chicken osteoclasts cultured on bone slices, it was found to be distributed throughout the osteoclast cell membrane except in the sealing zone areas. Immunoperoxidase staining of rat bone sections at the light microscopical level also revealed intense staining of the cell membrane with occasional small unstained areas, probably corresponding to the sealing zones. Immunoelectron microscopy confirmed the results obtained by light microscopy showing specific labeling only at the ruffled borders and basolateral membranes (0.82 and 2.43 gold particles/microns of membrane, respectively), but not at the sealing zone areas (0.06 gold particles/microns of membrane). Both alpha v and beta 3 subunits of the vitronectin receptor were similarly localized. These results strongly suggest that, although the vitronectin receptor is important in the function of osteoclasts, it is not mediating the final sealing zone attachment of the osteoclasts to the mineralized bone surface.

Skeletal-Targeted, Osteolytic-Responsive Drug Delivery System Improves Anti-Tumor Efficacy in Multiple Myeloma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3155-3155
Author(s):  
Xuli Wang ◽  
Ye Yang ◽  
Scott Miller ◽  
Fenghuang Zhan
Keyword(s):  
Drug Delivery ◽  
Multiple Myeloma ◽  
Polymeric Micelles ◽  
Conflicts Of Interest ◽  
Drug Loading ◽  
Bone Matrix ◽  
Cathepsin K ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Tumor Inhibition

Abstract Abstract 3155 Background: Multiple myeloma (MM) cells often directly or indirectly induce the formation of osteoclasts, which enhance bone resorption by increasing secretion of a key protease (cathepsin K) to degrade bone matrix, leading to osteolytic lesions and serious skeletal complications. Hence, bone-targeted, osteolytic-responsive therapeutic modalities are greatly needed to improve clinical outcomes for MM. Methods and Results: A tri-block copolymer of peptide, poly(ethylene glycol) and poly(trimethylene carbonate) (Pep-b-PEG-b-PTMC) has been synthesized as a nanocarrier to improve treatment for MM. The functional peptide with the sequence of CKGHPGGPQAsp8 was designed to possess a bone tropism octapeptide (Asp8), a cathepsin K (CTSK)-cleavable substrate (HPGGPQ), multiple cationic residues and a terminal cysteine for site-specific conjugation. Maleimide-terminated diblock copolymer of PEG-b-PTMC was readily functionalized with the peptide to obtain Pep-b-PEG-b-PTMC that can spontaneously form micelles with the size of 75 nm in diameter. Sixty-six % of polymeric micelles were able to bind to hydroxyl apatite, showing high bone binding capability. The nanoparticles exhibited a negative-to-positive charge conversional profile upon exposure to cathepsin K, an overexpressed enzyme in osteolytic microenvironments. By using doxorubicin as a model drug, Pep-b-PEG-b-PTM showed 7.5 ± 0.5 % and 22.7 ± 1.5% for drug loading content and drug loading efficiency, respectively. More importantly, a unique characteristic of on-demand charge-conversional behaviour in a cathepsin K-rich condition led to enhanced cellular uptake of the nanotherapeutics, as demonstrated by confocal laser scanning microscopy. Enhanced tumor inhibition was observed in 5TGM1 MM cells when nanoparticles were pre-treated with 150 nM cathepsin K, demonstrating enzyme-triggered improved therapy. Efficacy of free DOX or DOX-loaded NPs in 5TGM1 mice bearing myeloma was further preliminarily tested. 5TGM1 mice bearing myeloma were established through injection of 5TGM1 cells (1 × 106 cells in 100 μL PBS) via tail vein, and tumor was allowed to grow for a week before initiating treatment study. Mice (n=3) were injected twice weekly with different therapeutic formulations at equivalent DOX dose (0.75 mg/Kg) or PBS. Tumor burden in the mice was monitored by ELISA measurements of serum IgG2b. Drug-loaded nanoparticles from Pep-b-PEG-b-PTMC were more efficacious in terms of mice survival rate and tumor inhibition when compared to the groups with non-targeted nanoparticles from mPEG-PTMC, free DOX or PBS controls. This improved drug efficacy may be attributed to more selective delivery of DOX to bone metastatic tissues and/or responsiveness of the nanoparticles to cathepsin K, thus improving tumor uptake of DOX, enhancing therapeutic efficacy in terms of tumor reduction as well as MM mouse survival. Conclusions: The promising results from this study may prompt the development of bone-targeted, enzyme-triggered drug delivery systems to improve their affinity to skeletal tissues, enhance selectivity for osteolytic regions and improve efficacy of anti-cancer agents, thus facilitating the development of effective nanotherapeutic modalities for multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Characterization of Binary Biofilms of Listeria monocytogenes and Lactobacillus and Their Response to Chlorine Treatment

2021 ◽  
Vol 12 ◽  
Author(s):  
Magdalena A. Olszewska ◽  
Francisco Diez-Gonzalez
Keyword(s):  
Listeria Monocytogenes ◽  
Laser Scanning ◽  
Spatial Organization ◽  
Volume Ratio ◽  
Single Species ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Interspecies Interactions ◽  
Biofilm Thickness ◽  
Cell Counts

In nature, Listeria may interact competitively and cooperatively with other organisms, resulting in unique spatial organization and functions for cells within the community. This study was undertaken to characterize the biofilm architecture of binary biofilms of Listeria monocytogenes and Lactobacillus species and to assess their effect on the survival of Listeria during exposure to hypochlorite. Three L. monocytogenes strains, ATCC 19115 (Lm5), ATCC 19117 (Lm7), and Coleslaw (LmC), were selected and combined individually with three Lactobacillus strains: L. fermentum (Lf), L. bavaricus (Lb), and L. plantarum (Lp). In binary Lm-Lp biofilms, the Lm cell counts were similar to single-species biofilms (8.5 log CFU/well), and the Lp cell numbers declined by 1.0 log CFU/well. In the presence of Lb, the Lm cell counts were reduced by 1.5 log CFU/well (p < 0.05), whereas the Lf cell counts increased at least by 3.5 log CFU/well. Confocal laser scanning microscopy (CLSM) determined that interspecies interactions significantly affected the spatial organization of three binary biofilms. Biofilm surface-to-volume ratio increased from 0.8 μm2/μm3 for Lm5 in the monoculture to 2.1 μm2/μm3 for Lm5-Lp in the dual-species model (p < 0.05), and was characterized by a thicker structure with a largely increased surface area. Biofilm roughness increased from 0.2 for Lm7 to 1.0 for Lm7-Lb biofilms (p < 0.05), which appeared as interspecific segregation. Biofilm thickness increased from 34.2 μm for LmC to 46.3 μm for LmC–Lf (p < 0.05), which produced flat and compact structures that covered the entire surface available. The biomass of the extracellular matrix was higher in the case of some binary biofilms (p < 0.05); however, this effect was dependent upon the species pair. When treated with hypochlorite, Lm5 in binary biofilms had an approximately 1.5 log CFU/well greater survival than individually. The unique spatial organization and greater protein production may explain the protective effect of Lp after hypochlorite exposure.

Hematopoietic Progenitor Cells Harbouring BCR-ABL Exhibit Increased Adhesiveness but an Impaired Migration and Homing Potential.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4807-4807
Author(s):  
Fernando A. Fierro ◽  
Anja Taubenberger ◽  
Gerhard Ehninger ◽  
Daniel Mueller ◽  
Martin Bornhaeuser ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Progenitor Cells ◽  
Cell Tracking ◽  
Laser Scanning Microscopy ◽  
Transformed Cells ◽  
Confocal Laser ◽  
Special Focus ◽  
Adhesion Assay ◽  
Differentiation Potential ◽  
Adhesion Properties

Abstract Recent studies suggest that one of the most important treatment resistance mechanisms of cells with the BCR-ABL translocation could be the interaction with the hematopoietic niche. During the contact with the respective microenvironment, leukemic stem and progenitor cells acquire both a state of cell adhesion mediated drug resistance (CAM-DR) and may change their proliferation and differentiation potential. Studies on the effect of the BCR-ABL oncogene on cell adhesion and migration have yielded conflicting results due to different cell type models (primary cells vs. transduced cell lines), non-standardized quantification methods and diverse substrates (fibronectin vs. bone marrow stromal cells) studied. In the present work, we investigated the adhesion properties of 32D cells which were stably transformed with the BCR-ABL fusion transcript in comparison to an empty vector transformed control. The commonly used panning-style adhesion assay determining the percentage of hematopoietic cells adhering to a stroma cell line (M2-10B4) clearly demonstrated higher adhesion capacity of BCR-ABL transformed cells. Accordingly, an atomic force microscopy (AFM)-based method to measure adhesion forces of a single leukemic cell over stroma cells, showed an increased ‘stickyness’ of BCR-ABL transformed cells. AFM experiments demonstrated highly different forces that were needed to retract attached 32D-vector cells as compared to 32D/BCR-ABL cells (1000 pN vs. 3500 pN; p<0.01). This higher superficial adhesion of BCR-ABL transformed cells was associated with a decreased migration and homing potential of adhering cells into the stromal layer (quantified by cell tracking movies analysis) and cobblestones formation which was analysed by confocal laser scanning microscopy. The differences found between control and BCR-ABL-expressing cells were reversible by pre-incubation with 0.5 μM Imatinib Mesylate (IM), suggesting that altered adhesion is a tyrosine kinase-activity dependent process. To further gain insight in the mechanisms of adhesion we used a cDNA microarray (Affymetrix-Gene chip 430A 2.0) for the identification of BCR-ABL related gene expression with special focus on adhesion associated genes. P-Selectin upregulation (40 folds) in cells harbouring BCR-ABL was seen as the most dramatic change in adhesion related molecules. Consequently, a monoclonal antibody against P-selectin inhibited adhesion capacity of BCR-ABL cells on M2-10B4. In conclusion, we describe an increased adhesion capacity of 32/BCR-ABL transformed cells which is associated with an up-regulation of P-selectin expression. Moreover, whilst 32D/BCR-ABL cells show a high potential to primarily attach to the stroma layer, physiological functions like cobblestone formation is impaired by a reduced migration and consecutively impaired homing potential of the cells into the supporting niches formed by the feeder cells.

scholarly journals Femtosecond Laser Nano/Micro Textured Ti6Al4V Surfaces—Effect on Wetting and MG-63 Cell Adhesion

Materials ◽  
2019 ◽  
Vol 12 (13) ◽  
pp. 2210 ◽  
Author(s):  
Georg Schnell ◽  
Susanne Staehlke ◽  
Ulrike Duenow ◽  
J. Barbara Nebe ◽  
Hermann Seitz
Keyword(s):  
Cell Adhesion ◽  
Surface Properties ◽  
Laser Scanning ◽  
Surface Characterization ◽  
Cellular Adhesion ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Material Surface ◽  
Structured Surfaces

Nano- and microstructured titanium surfaces have recently attracted attention in the field of regenerative medicine because of the influence which surface characteristics such as roughness and wettability can have on cellular processes. This study focuses on the correlation of surface properties (wettability and nano/micro texture) of laser-structured Ti6Al4V samples with pronounced cell adhesion. Samples were structured with multiple laser parameters in order to create a range of surface properties. Surface characterization was performed by contact angle measurements 1 and 7 days after laser processing. The arithmetic mean roughness of the material surface in an area (Sa) was determined by means of confocal laser scanning microscopy (CLSM). Immediately after wettability tests of the laser-structured surfaces, in vitro experiments with human MG-63 osteoblasts were carried out. For this purpose, the cell morphology and actin cytoskeleton organization were analyzed using CLSM and scanning electron microscopy. On rough microstructures with deep cavities, the cell growth and spreading were inhibited. An improved cellular adhesion and growth on nanostructured and sinusoidal microstructured surfaces could be demonstrated, regardless of hydrophilicity of the surfaces.

scholarly journals Exploring the Diversity of Listeria monocytogenes Biofilm Architecture by High-Throughput Confocal Laser Scanning Microscopy and the Predominance of the Honeycomb-Like Morphotype

2014 ◽  
Vol 81 (5) ◽  
pp. 1813-1819 ◽  
Author(s):  
Morgan Guilbaud ◽  
Pascal Piveteau ◽  
Mickaël Desvaux ◽  
Sylvain Brisse ◽  
Romain Briandet
Keyword(s):  
Listeria Monocytogenes ◽  
High Throughput ◽  
Biofilm Formation ◽  
Spatial Organization ◽  
Confocal Imaging ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Published Data ◽  
Content Type

ABSTRACTListeria monocytogenesis involved in food-borne illness with a high mortality rate. The persistence of the pathogen along the food chain can be associated with its ability to form biofilms on inert surfaces. While most of the phenotypes associated with biofilms are related to their spatial organization, most published data comparing biofilm formation byL. monocytogenesisolates are based on the quantitative crystal violet assay, which does not give access to structural information. Using a high-throughput confocal-imaging approach, the aim of this work was to decipher the structural diversity of biofilms formed by 96L. monocytogenesstrains isolated from various environments. Prior to large-scale analysis, an experimental design was created to improveL. monocytogenesbiofilm formation in microscopic-grade microplates, with special emphasis on the growth medium composition. Microscopic analysis of biofilms formed under the selected conditions by the 96 isolates revealed only weak correlation between the genetic lineages of the isolates and the structural properties of the biofilms. However, a gradient in their geometric descriptors (biovolume, mean thickness, and roughness), ranging from flat multilayers to complex honeycomb-like structures, was shown. The dominant honeycomb-like morphotype was characterized by hollow voids hosting free-swimming cells and localized pockets containing mixtures of dead cells and extracellular DNA (eDNA).

scholarly journals Histomorphometric Evidence for Echistatin Inhibition of Bone Resorption in Mice with Secondary Hyperparathyroidism

Endocrinology ◽  
1998 ◽  
Vol 139 (3) ◽  
pp. 1401-1410 ◽  
Author(s):  
Patricia Masarachia ◽  
Michiko Yamamoto ◽  
Chih-Tai Leu ◽  
Gideon Rodan ◽  
Le Duong
Keyword(s):  
Bone Resorption ◽  
Secondary Hyperparathyroidism ◽  
Basolateral Membrane ◽  
Fold Increase ◽  
Ultrastructural Level ◽  
Bone Surface ◽  
Histological Evidence ◽  
Low Calcium Diet ◽  
Ruffled Border

Echistatin, an RGD-containing peptide, was shown to inhibit the acute calcemic response to exogenous PTH or PTH-related protein (PTH-rP) in thyroparathyroidectomized rats, suggesting that echistatin inhibits bone resorption. In this study: 1) we present histological evidence for echistatin inhibition of bone resorption in mice with secondary hyperparathyroidism, and show that 2) echistatin binds to osteoclasts in vivo, 3) increases osteoclast number, and 4) does not detectably alter osteoclast morphology. Infusion of echistatin (30μ g/kg·min) for 3 days prevented the 2.6-fold increase in tibial cancellous bone turnover and the 36% loss in bone volume, produced by a low calcium diet. At the light microscopy level, echistatin immunolocalized to osteoclasts and megakaryocytes. Echistatin treatment increased osteoclast-covered bone surface by about 50%. At the ultrastructural level, these osteoclasts appeared normal, and the fraction of cells containing ruffled borders and clear zones was similar to controls. Echistatin was found on the basolateral membrane and in intracellular vesicles of actively resorbing osteoclasts. Weak labeling was found in the ruffled border, and no immunoreactivity was detected at the clear zone/bone surface interface. These findings provide histological evidence for echistatin binding to osteoclasts and for inhibition of bone resorption in vivo, through reduced osteoclast efficacy, without apparent changes in osteoclast morphology.

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