Fibronectin and laminin in transverse tubules of cardiac myocytes studied by laser confocal microscopy and immunocytochemistry.

The distribution of two non-collagenous glycoproteins of high molecular weight, fibronectin (FN) and laminin (LMN), was investigated in myocardial cells from the ventricle of rats, and from biopsies collected from the auricle of patients undergoing a coronary bypass operation. In order to elucidate the expression of FN and LMN across cells, non-invasive serial sectioning has been carried out by laser scanning confocal microscopy of frozen, immunostained tissue sections. In addition, immunoelectron microscopy was used to study the distribution of these antigens at higher magnifications. These studies show that FN is part of the basement membrane of the surface sarcolemma of both ventricular and atrial cells, in addition to being an abundant protein of the extracellular matrix (ECM). Along transverse tubular(TT)-membranes, FN was only detected in tubules exceeding 200 nm in diameter. Even here, the intensity of labelling varied greatly and was generally low. By contrast, a heavy investment of LMN was organized in the basal lamina along the surface sarcolemma and along ramifications of the entire TT-system in ventricular heart muscle cells. In this way, the network of TT-membrane systems of working heart muscle cells provides a supply of LMN to all depths of the myocardial fibre. In human atrial muscle cells, a regular TT-system appears to be absent. Instead occasional, deep sarcolemmal invaginations occur with diameters of 300-500 nm, the surfaces of which are also invested with LMN. The significance of the present findings has been discussed, with special reference to LMN as a possible component of a series of proteins involved in transmembrane communication between the ECM and the sarcoplasm.     

Expression of fibronectin,laminin and ribosomes in normal and nocodazole-treated neonatal heart cells in culture: a study by laser scanning confocal microscopy and immunocytochemistry

Polyclonal and monoclonal antibodies were used to examine the effects of the synthetic microtubule disruptive drug nocodazole on the subcellular expression of fibronectin, laminin, and ribosomes in primary cultures of neonatal cardiac ventricular cells. Non-invasive serial optical sectioning was carried out by immunolaser scanning confocal microscopy. In addition, fibronectin and laminin were immunolabelled with peroxidase or gold conjugates for electron-microscopic examination. Immunolabelling for the large 60S ribosome subunit in fibroblast-like non-myocytes showed that punctate ribosome structures with a multi-subunit composition were present in perinuclear region. Double immunostaining with antibodies directed against ribosomes and cellular fibronectin indicated that the punctate structures were cisternae of the rough endoplasmic reticulum. No clear effects of nocodazole treatment were detected on the distribution of cytoskeleton-bound ribosomes. Following immunolabelling for both glycoproteins and double immunolabelling for cellular fibronectin and the 60 S ribosome subunit, fibronectin and laminin were found in the perinuclear cisternae of the rough endoplasmic reticulum and in pleomorphic secretory vesicles. The cisternal stacks of the Golgi complex appeared either unstained or were only weakly labelled. When these cells were exposed to nocodazole, fibronectin and laminin accumulated in peripheral parts of the cytoplasm, including cellular processes. These peripheral accumulations of immunostaining for fibronectin and laminin did not reflect Golgi staining, as shown by double labelling experiments versus wheat-germ-agglutinin staining, and, by exposing cultures to a high dose of brefeldin A.     

血管紧张素Ⅱ对大鼠心肌细胞Ca2+信号的影响

目的:在培养的新生大鼠心肌细胞上,观察AngⅡ对Ca2 信号的影响,探讨其时间和空间形式.方法:以Fluo-4/AM荧光指示剂负载培养的心肌细胞,应用激光共聚焦扫描显微镜观察其变化.结果:在激光共聚焦显微镜下,观察到不论静息或受AngⅡ刺激的心肌细胞,均可见钙波在细胞内及相连的另一细胞间彼此传播的现象.正常心肌细胞内核的荧光强度高于核周与细胞浆,且存在小幅度的钙震荡,AngⅡ(10-6 mol/L)引起心肌细胞的核Ca2 和胞浆Ca2 荧光强度升高的同时,其钙震荡幅度明显升高,外源性一氧化氮(NO)供体硝普钠(10-5mol/L)使钙的周期性震荡消失,荧光强度降低.同时还观察到加入AngⅡ(10-6 mol/L)后在部分心肌细胞膜上的不同部位,出现不能传播的钙闪烁团,在此基础上再加入硝普钠(10-5mol/L),不能取消AngⅡ所致的钙闪烁现象.结论:心肌细胞受AngⅡ刺激,可产生多种钙信号形式如钙闪烁、钙波、钙震荡以及瞬时性钙增高,可能在介导细胞功能的调节中起重要作用.     

Neuropeptide co-localisation in the lepidopteran frontal ganglion studied by confocal laser scanning microscopy

A comparative study of the co-localisation of three different families of neuropeptides, viz. allatostatins of the Y/FXFGL-NH(2) type, Manduca sexta allatostatin (Mas-AS) and allatotropin, in the frontal ganglion of lepidopteran larvae has been carried out by means of immunocytochemistry and confocal laser scanning microscopy. The simultaneous application of three types of fluorochrome-conjugated antibodies reveals triple co-localisation in an anterodorsal pair of neurones in the frontal ganglion of the noctuids Heliothis virescens and Lacanobia oleracea. There is no evidence of differential axonal transport, since all parts of these neurones show complete co-localisation of all three peptides. Prominent axons of the ganglionic neurones project in the recurrent nerve to the foregut and stomodeal valve. Over the crop, lateral and sub-lateral branches follow the course of circular muscle fibres and terminate in varicosities. All three neuropeptides have previously been shown to be myoregulatory on the foregut; the Y/FXFGL-NH(2) allatostatins and Mas-AS are inhibitory, whereas allatotropin is excitatory. The morphological evidence of co-localisation of physiologically antagonistic peptides within the same terminals suggests that an extremely complex mechanism controls the contractile activities of the foregut. A posterodorsal pair of neurones in the frontal ganglion have prominent axons projecting via the frontal connectives to the brain and in the recurrent nerve to the stomodeal valve where extensive branching suggests control over the valve movements. Studies of another noctuid, Spodoptera frugiperda, and the sphingid, M. sexta, show interesting variations in the co-localisation phenomenon.     

Surface:volume relationship in cardiac myocytes studied with confocal microscopy and membrane capacitance measurements: species-dependence and developmental effects.

The quantitative analysis of the contribution of ion fluxes through membrane channels to changes of intracellular ion concentrations would benefit from the exact knowledge of the cell volume. It would allow direct correlation of ionic current measurements with simultaneous measurements of ion concentrations in individual cells. Because of various limitations of conventional light microscopy a simple method for accurate cell volume determination is lacking. We have combined the optical sectioning capabilities of fluorescence laser scanning confocal microscopy and the whole-cell patch-clamp technique to study the correlation between cell volume and membrane capacitance. Single cardiac myocytes loaded with the fluorescent dye calcein were optically sectioned to produce a series of confocal images. The volume of cardiac myocytes of three different mammalian species was determined by three-dimensional volume rendering of the confocal images. The calculated cell volumes were 30.4 +/- 7.3 pl (mean +/- SD) in rabbits (n = 28), 30.9 +/- 9.0 pl in ferrets (n = 23), and 34.4 +/- 7.0 pl in rats (n = 21), respectively. There was a positive linear correlation between membrane capacitance and cell volume in each animal species. The capacitance-volume ratios were significantly different among species (4.58 +/- 0.45 pF/pl in rabbit, 5.39 +/- 0.57 pF/pl in ferret, and 8.44 +/- 1.35 pF/pl in rat). Furthermore, the capacitance-volume ratio was dependent on the developmental stage (8.88 +/- 1.14 pF/pl in 6-month-old rats versus 6.76 +/- 0.62 pF/pl in 3-month-old rats). The data suggest that the ratio of surface area:volume of cardiac myocytes undergoes significant developmental changes and differs among mammalian species. We further established that the easily measurable parameters of cell membrane capacitance or the product of cell length and width provide reliable but species-dependent estimates for the volume of individual cells.     

Temperature dependence of intracellular free calcium in cardiac myocytes from rat and ground squirrel measured by confocal microscopy

The temperature-dependence of infraeeliular free caleimn (Ca) was investigated in mdo-1 loaded ventricular myocytes from the ral, a non-hibernator, and from the ground squirrel, a hibernator. The dissociation constant of indo-l at different temperatures was calibrated both al pll-tat and at @-stat . and the result demonstrated that the @-stat ralibration should be prettrred . Analysis of the fluoreseent image showed a striking increase of Ca2 as well as spontaneous caleiuni waves in ral cells, indicating an overloaded cakuum. In contrast, cardiac myocytes of the ground sqnirraf were found to keep a constant (Ca2 ) without caleium overload regardless of temperature variation. It is be-lieved that understanding of the mechanisms underlying the interccllular caleima homeostasis of hibrernators may lead to solutions of some medical questions .     

Temperature dependence of intracellular free calcium in cardiac myocytes from rat and ground squirrel measured by confocal microscopy

The temperature-dependence of intracellular free calcium ([Ca²⁺]_i) was investigated in indo-1 loaded ventricular myocytes from the rat, a non-hibernator, and from the ground squirrel, a hibernator. The dissociation constant of indo-1 at different temperatures was calibrated both at pH-stat and at α-stat, and the result demonstrated that the α-stat calibration should be preferred. Analysis of the fluorescent image showed a striking increase of [Ca²⁺]_i as well as spontaneous calcium waves in rat cells, indicating an overloaded calcium. In contrast, cardiac myocytes of the ground squirrel were found to keep a constant [Ca²⁺]_i without calcium overload regardless of temperature variation. It is believed that understanding of the mechanisms underlying the intercellular calcium homeostasis of hibemators may lead to solutions of some medical questions.     

Membrane skeleton in cultured chick cardiac myocytes revealed by high resolution immunocytochemistry

Distribution of cytoskeletal proteins with emphasis on the membrane-cytoskeleton interface was examined in cultured cardiac myocytes. Using specific antibodies recognizing α-sarcomeric actin, desmin, β-tubulin, spectrin/α-fodrin and ankyrin, respectively, the cellular localization of these cytoskeletal proteins was detected by laser scanning confocal microscopy. In addition, the fine filamentous structure of these proteins was identified by combining silver-enhanced immunogold labelling with electron microscopy. The latter technique employed the sequence of quick-freezing, deep-etching and rotary shadowing of the specimens. Conventional transmission electron microscopy of the spherical cardiac myocytes revealed a filamentous submembranous layer, approximately 100 nm thick. Specific immunolabelling of α-sarcomeric actin and spectrin/α-fodrin as well as ankyrin was seen beneath the plasmalemma. A three-dimensional meshwork of spectrin/α-fodrin was shown. Numerous desmin filaments that exhibited a tortuous course throughout the cells were also observed running in parallel with the surface in the submembranous area, whereas β-tubulin was infrequently detected in these areas. In conclusion, the present study shows that spherical cardiac myocytes contain a distinct and complex three-dimensional membrane skeleton. Major constituents of this distinct submembranous layer were spectrin/α-fodrin fibres as well as actin and desmin filaments. Accepted: 28 July 1999     

Determination of diffusion coefficients in biofilms by confocal laser microscopy

Microbial exopolymer may hinder the diffusion of nutrients, antibiotics, and other materials to the cell surface. Studies of diffusion in biofilms have been limited to indirect measurements. This study demonstrated the use of fluorescein and size-fractionated fluor-conjugated dextrans in conjunction with scanning confocal laser microscopy to directly monitor and determine diffusion coefficients within biofilms. The monitoring approaches were simple and, when combined with computerized image collection, allowed assembly of a data set suitable for calculation of one-dimensional diffusion coefficients for biofilm regions. With these techniques, it was shown that regional variability in the mobility of the dextrans occurred within mixed-species biofilms. Some regions exhibited rapid diffusion of all test molecules, while adjacent regions were only penetrated by the lower-molecular-weight compounds. The effective diffusion coefficients (D(e)) determined in a mixed-species biofilm were a function of the molecular radius of the probe (i.e., fluorescein, D(e) = 7.7 x 10 cm s; 4,000 molecular weight, D(e) = 3.1 x 10 cm s; and 2,000,000 molecular weight, D(e) = 0.7 x 10 cm s). These results demonstrated that diffusion in the biofilm was hindered relative to diffusion in the bulk solution. The study indicated that in situ monitoring by scanning laser microscopy is a useful approach for determining the mobility of fluorescently labeled molecules in biofilms, allowing image acquisition, appropriate scales of study, both xy and xz monitoring, and calculation of D(e) values.     

Visualization of alginate-poly-L-lysine-alginate microcapsules by confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) was used to study the distribution of polymers and cross-linking ions in alginate-poly-L-lysine (PLL) -alginate microcapsules made by fluorescent-labeled polymers. CLSM studies of Ca-alginate gel beads made in the presence and absence of non-gelling sodium ions revealed a more inhomogeneous distribution of alginate in beads formed in the absence of non-gelling ions. In the formation of alginate-PLL capsules, the polymer gradients in the preformed gel core were destabilized by the presence of non-gelling ions in the washing step and in the PLL solution. Ca-alginate gels preserved the inhomogeneous structure by exposure to ion-free solution in contrast to exposure to non-gelling ions (Na(+)). By exchanging Ca(2+) with Ba(2+) (10 mM), extremely inhomogeneous gel beads were formed that preserved their structure during the washing and exposure to PLL in saline. PLL was shown to bind at the very surface of the alginate core, forming a shell-like membrane. The thickness of the PLL-layer increased about 100% after 2 weeks of storage, but no further increase was seen after 2 years of storage. The coating alginate was shown to overlap the PLL layer. No difference in binding could be observed among coating alginates of different composition. This paper shows an easy and novel method to study the distribution of alginate and PLL in intact microcapsules. As the labeling procedures are easy to perform, the method can also be used for a variety of other polymers in other microencapsulation systems.     

Structural analysis of microparticles by confocal laser scanning microscopy

This study demonstrates the potential of conforcal laser scanning microscopy (CLSM) as a characterization tool for different types of microparticles. Microparticles were prepared by various methods including complex coacervation, spray drying, double emulsion solvent evaporation technique, and ionotropic gelation. Protein drugs and particle wall polymers were covalently labeled with a fluorescent marker prior to particle preparation, while low molecular weight drugs were labeled by mixing with a fluorescent marker of similar solubility properties. As was demonstrated in several examples, CLSM allowed visualization of the polymeric particle wall composition and detection of heterogeneous polymer distribution or changes in polymer matrix composition under the influence of the drug. Furthermore, CLSM provides a method for three-dimensional reconstruction and image analysis of the microparticles by imaging several coplanar sections throughout the object. In conclusion, CLSM allows the inspection of internal particle structures without prior sample destruction. It can be used to localize the encapsulated compounds and to detect special structural details of the particle wall composition.     

Monitoring and visualising plant cuticles by confocal laser scanning microscopy

The present work shows the visualisation of phenolics and flavonoids of plant cuticles by confocal laser scanning microscopy (CLSM). Selected isolated fruit and leaf cuticles were monitored on the basis of autofluorescent phenolics and flavonoids which, in most cases, permitted us to obtain three-dimensional images of the cuticular membranes. The utility of this technique in investigations of cuticular translocation and diffusion of exogenous applied chemicals and cuticle degradation has also been explored.     

Leu-callatostatin gene expression in the blowflies Calliphora vomitoria and Lucilia cuprina studied by in situ hybridisation: comparison with Leu-callatostatin confocal laser scanning immunocytochemistry

In situ hybridisation studies using a digoxigenin-labelled DNA probe encoding the Leu-callatostatin prohormone of the blowflies Calliphora vomitoria and Lucilia cuprina have revealed a variety of neurones in the brain and thoracico-abdominal ganglion, peripheral neurosecretory neurones, and endocrine cells of the midgut. With two exceptions, the hybridising cells are the same as those previously identified in immunocytochemical studies of sections and whole-mounts using Leu-callatostatin COOH-terminal-specific antisera. Within the brain and suboesophageal ganglion, there is a variety of neurones ranging from a single pair of large cells situated in the dorsal protocerebrum, to the several pairs of neurones in the tritocerebrum, some of which, in immunocytochemical preparations, can be seen to project via axons in the cervical connective to the thoracico-abdominal ganglion. In the medulla of the optic lobes, numerous small interneurones hybridise with the probe, as do clusters of similar-sized neurones close to the roots of the ocellar nerves. These results indicate that the Leu-callatostatin neuropeptides of the brain play a variety of roles in neurotransmission and neuromodulation. There are only three pairs of Leu-callatostatin-immunoreactive neurones in the thoracico-abdominal ganglion, at least two pairs of which project axons along the median abdominal nerve to provide extensive innervation of the hindgut. The Leu-callatostatin peripheral neurosecretory cells are located in close association with both nerve and muscle fibres in the thorax. In addition to neuronal Leu-callatostatin, the presence of the peptide and its mRNA has been demonstrated in endocrine cells in the posterior part of the midgut. These observations provide an example of a named brain/gut peptide in an insect.     

Fibronectin in rat heart: a link between cardiac myocytes and collagen

Fibronectin, a glycoprotein that binds to collagen and modifies the adhesion properties and motility of cells in culture, is present in the interstitium of rat hearts. To localize fibronectin more precisely and to assess its relationship to the myocyte and to connective tissue elements, we employed a double antibody technique to label myocardial fibronectin with electron-dense ferritin to permit an ultrastructural analysis. Fibronectin was found to be associated with collagen, and in some cases appeared to link collagen fibers. Fibronectin was also found inserted along the surfaces of cardiac myocytes, connecting these cells to perimyocytic collagen. These ultrastructural relationships imply that fibronectin is a major component of the myocardial interstitium, and may affect myocardial compliance and control the motion of myocytes during the contraction and relaxation of the heart.