Histological Skin Remodeling Following Autologous Fibroblast Application.

The aim of this study was to quantify the effectiveness of intradermal application of autologous fibroblasts on lean tissue structures. The histological sections of the skin were analysed and evaluated for the expansion potential of autologous fibroblasts in the control skin patch area and the nearby pre-treated skin patch into which we had injected expanded autologous fibroblasts nine month earlier. The results show that the pre-injection of fibroblasts into the dermis leads to a long-term rejuvenation of the skin, as evaluated from the histological appearance and from the significantly increased density of fibroblasts in the pre-injected skin vs. controls, from around 60% to over 80%, determined as the percent of lean tissue by a novel image analysis approach. Interestingly, the rate of the in vitro fibroblast expansion from the pre-injected area of the skin was reduced in comparison with the controls, consistent with the view that fibroblasts exhibit a limited cell-division potential and that fibroblasts from the pre-injected skin already experienced expansion nine month earlier prior to the injection into the skin. We conclude that autologous fibroblast application results in a significant long-term augmentation of the lean tissue elements of the skin.

Munc18-1 tuning of vesicle merger and fusion pore properties.

The release of hormones and neurotransmitters, mediated by regulated exocytosis, can be modified by regulation of the fusion pore. The fusion pore is considered stable and narrow initially, eventually leading to the complete merger of the vesicle and the plasma membranes. By using the high-resolution patch-clamp capacitance technique, we studied single vesicles and asked whether the Sec1/Munc18 proteins, interacting with the membrane fusion-mediating SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, affect fusion pore properties. Munc18-1 mutants were transfected into lactotrophs to affect the interaction of Munc18-1 with syntaxin1 (Synt1) (R39C), Rab3A (E466K), and Mints (P242S). Compared with wild-type, Munc18-1 E466K increased the frequency of the fusion event. The latter two mutants increased the fusion pore dwell-time. All the mutants stabilized narrow fusion pores and increased the amplitude of fusion events, likely via preferential fusion of larger vesicles, since overexpression of Munc18-1 R39C did not affect the average size of vesicles, as determined by stimulated emission depletion (STED) microscopy. Single-molecule atomic force microscopy experiments revealed that wild-type Munc18-1, but not Munc18-1 R39C, abrogates the interaction between synaptobrevin2 (Syb2) and Synt1 binary trans-complexes. However, neither form of Munc18-1 affected the interaction of Syb2 with the preformed binary cis-Synt1A-SNAP25B complexes. This indicates that Munc18-1 performs a proofing function by inhibiting tethering of Syb2-containing vesicles solely to Synt1 at the plasmalemma and favoring vesicular tethering to the preformed binary cis-complex of Synt1A-SNAP25B. The association of Munc18-1 with the ternary SNARE complex leads to tuning of fusion pores via multiple and converging mechanisms involving Munc18-1 interactions with Synt1A, Rab3A, and Mints.

Intermediate filaments attenuate stimulation-dependent mobility of endosomes/lysosomes in astrocytes.

Intermediate filament (IF) proteins upregulation is a hallmark of astrocyte activation and reactive gliosis, but its pathophysiological implications remain incompletely understood. A recently reported association between IFs and directional mobility of peptidergic vesicles allows us to hypothesize that IFs affect vesicle dynamics and exocytosis-mediated astrocyte communication with neighboring cells. Here, we ask whether the trafficking of recycling vesicles (i.e., those fused to and then retrieved from the plasma membrane) and endosomes/lysosomes depends on IFs. Recycling vesicles were labeled by antibodies against vesicle glutamate transporter 1 (VGLUT1) and atrial natriuretic peptide (ANP), respectively, and by lysotracker, which labels endosomes/lysosomes. Quantitative fluorescence microscopy was used to monitor the mobility of labeled vesicles in astrocytes, derived from either wild-type (WT) mice or mice deficient in glial fibrillary acidic protein and vimentin (GFAP(-/-)Vim(-/-)), the latter lacking astrocyte IFs. Stimulation with ionomycin or ATP enhanced the mobility of VGLUT1-positive vesicles and reduced the mobility of ANP-positive vesicles in WT astrocytes. In GFAP(-/-)Vim(-/-) astrocytes, both vesicle types responded to stimulation, but the relative increase in mobility of VGLUT1-positive vesicles was more prominent compared with nonstimulated cells, whereas the stimulation-dependent attenuation of ANP-positive vesicles mobility was reduced compared with nonstimulated cells. The mobility of endosomes/lysosomes decreased following stimulation in WT astrocytes. However, in GFAP(-/-)Vim(-/-) astrocytes, a small increase in the mobility of endosomes/lysosomes was observed. These findings show that astrocyte IFs differentially affect the stimulation-dependent mobility of vesicles. We propose that upregulation of IFs in pathologic states may alter the function of astrocytes by deregulating vesicle trafficking.

Analysis of confocal images using variable-width line profiles.

A line profile of fluorescent intensities in confocal images is frequently examined. We have developed the computer software tool to analyse the profiles of intensities of fluorescent probes in confocal images. The software averages neighbouring pixels, adjacent to the central line, without reducing the spatial resolution of the image. As an experimental model, we have used the skeletal muscle fibre isolated from the rat skeletal muscle extensor digitorum brevis. As a marker of myofibrils' structure, we have used phalloidin-rhodamine staining and the anti-TIM antibody to label mitochondria. We also tested the distribution of the protein kinase B/Akt. Since signalling is ordered in modules and large protein complexes appear to direct signalling to organelles and regulate specific physiological functions, a software tool to analyse such complexes in fluorescent confocal images is required. The software displays the image, and the user defines the line for analysis. The image is rotated by the angle of the line. The line profile is calculated by averaging one dimension of the cropped rotated image matrix. The spatial resolution in averaged line profile is not decreased compared with single-pixel line profile, which was confirmed by the discrete Fourier transform computed with a fast Fourier transform algorithm. We conclude that the custom software tool presented here is a useful tool to analyse line profiles of fluorescence intensities in confocal images.

The fusion pore and vesicle cargo discharge modulation.

Exocytosis, the merger of the vesicle membrane with the plasma membrane, is thought to mediate the release of hormones and neurotransmitters from secretory vesicles. The work of Bernard Katz and colleagues decades ago considered that vesicle cargo discharge initially requires the delivery of secretory vesicles to the plasma membrane where vesicles dock and are primed for fusion with the plasma membrane. Then, upon stimulation, the vesicle and the plasma membranes fuse to form a transient fusion pore through which cargo molecules diffuse out of the vesicle lumen into the extracellular space. Katz and colleagues considered this process to occur in an all-or-none fashion. However, recent studies show that this may not be so simple. The aim of this overview is to highlight the novel findings that indicate that fusion pores are subject to regulations, which affect the release competence of a single vesicle. Here we discuss the elementary properties of spontaneous and stimulated peptidergic vesicle discharge, which appears to be modulated, at least in pituitary lactotrophs, by fusion pore conductance (pore diameter) and fusion pore gating (kinetics).

Rosiglitazone modulates insulin-induced plasma membrane area changes in single 3T3-L1 adipocytes.

In this study we hypothesized that rosiglitazone, an antidiabetic high-affinity agonist for the peroxisome proliferator-activated receptor gamma, affects the plasma membrane (PM) turnover in single 3T3-L1 adipocytes. To study the PM turnover, the patch-clamp electrophysiological method was used to measure changes in membrane capacitance (Cm), a parameter linearly related to the PM area. Microscopy results show that the presence of rosiglitazone in the differentiating medium significantly increased the differentiation of 3T3-L1 adipocytes in cell culture, based on oil red O-stained area (11.4 +/- 1.2%) vs. controls (3.1 +/- 0.5%). Moreover, rosiglitazone treatment significantly reduced the size of single 3T3-L1 adipocytes; their average radius of 21.1 +/- 1.1 microm in controls was reduced to 17.5 +/- 0.5 microm in rosiglitazone-treated cells. Consistent with this, insulin application increased the rate of Cm increase to 2.34 +/- 0.10%/min, which was significantly different from controls (0.12 +/- 0.08%/min). However, pretreatment of cells with rosiglitazone prior to the treatment with insulin resulted in an attenuated rate of Cm increase. These data support the involvement of insulin in the modulation of membrane area and show that treatment by rosiglitazone reduced the insulin-mediated membrane area increase in 3T3-L1 adipocytes.

Prolactin secretion sites contain syntaxin-1 and differ from ganglioside monosialic acid rafts in rat lactotrophs.

In neuroendocrine cells, discharge of hormones follows the fusion of exocytotic vesicles with the plasma membrane at confined sites; however, the molecular nature of these distinct sites remains poorly understood. We studied intact pituitary lactotrophs and plasma membrane lawns by confocal microscopy in conjunction with antibodies against rat prolactin (rPRL), soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins (syntaxin-1 and synaptobrevin-2,) and fluorescent cholera toxin subunit B (CT-B), a marker of ganglioside monosialic acid (GM1) lipid rafts, to examine 1) whether rPRL vesicles discharge cargo at GM1 rafts, 2) whether discharging rPRL vesicles interact with SNAREs, and 3) to examine the overlap of GM1 rafts, rPRL, and syntaxin-1 sites in plasma membrane lawns. In intact cells, immunofluorescently labeled rPRL poorly colocalized (<6%) with CT-B. In conditions favoring endocytotic trafficking, vesicle SNARE synaptobrevin-2 modestly colocalized (35%) with CT-B, whereas it highly colocalized (58%) with retrieved rPRL. Although partial mixing between rPRL and CT-B intracellular trafficking pathways is likely, our results indicated that rPRL discharge involves interactions with plasma membrane SNAREs, but not with GM1 rafts. In support of this, the plasma membrane SNARE syntaxin-1 poorly colocalized with CT-B (<5%), whereas it highly colocalized (75%) with rPRL in inside-out plasma membrane lawns. Spontaneous and stimulated rPRL discharge in live lactotrophs is thus associated with plasma membrane sites enriched with SNARE proteins, however, spatially confined to plasma membrane areas other than GM1 rafts.

EAAT2 density at the astrocyte plasma membrane and Ca(2 + )-regulated exocytosis.

We studied whether regulated exocytosis affects the glutamate transporter density in cultured astrocytes, in which the expression of a fluorescently labeled excitatory amino acid transporter 2 (EAAT2-EGFP) predominantly labeled the plasma membrane. The addition of ionomycin that elevates cytosolic Ca(2+) strongly increased the fluorescence of FM 4-64 membrane area dye, confirming the presence of regulated exocytosis in transfected astrocytes. However, concomitant with Ca(2+)-dependent FM 4-64 fluorescence increase, ionomycin induced a significant steady-state decrease in EAAT2-EGFP fluorescence. This is likely due to a secondary inner filter effect since,(i) in the absence of FM 4-64, ionomycin stimulation was ineffective in changing the EAAT2-EGFP fluorescence, and (ii) fluorescence changes in FM 4-64 and EAAT2-EGFP were inversely correlated. To test whether subcellular EAAT2-EGFP structures are translocated from the cytoplasm to the plasma membrane during ionomycin stimulation, EAAT2-EGFP fluorescence was monitored locally at the plasma membrane and a few microns away in the adjacent cytoplasm. Measurements revealed sites with an increase in EAAT2-EGFP plasma membrane fluorescence correlated with a fluorescence decrease beneath the plasma membrane, and sites with plasma membrane fluorescence decrease correlated with fluorescence increase within the adjacent cytoplasm. The sites of rapid translocation/retrieval of EAAT2-EGFP structures to/from the plasma membrane appeared to be distributed in a punctuate pattern around the cell perimeter. The density of EAAT2-EGFP was regulated in a Ca(2+)-dependent manner, since in the absence of extracellular Ca(2+) local translocation/retrieval events were absent, revealing rapid surface density regulation of EAAT2 in astrocytes by regulated exo/endocytosis.

Ca2+-dependent mobility of vesicles capturing anti-VGLUT1 antibodies.

Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca(2+)-dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowly between the plasma membrane and the cytoplasm. When the cytosolic Ca(2+) level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca(2+)-triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. Our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles.

Glutamate stimulation increases hormone release in rat melanotrophs.

In melanotrophs, neuroendocrine cells from the intermediate lobe of the rat pituitary gland, glutamate causes a rise in intracellular [Ca2+] suggesting the presence of ionotropic NMDA and non-NMDA AMPA/K receptors. However, the Ca(2+)-dependent release of the major peptide hormone, alpha-melanocyte stimulating hormone (alpha-MSH), in response to glutamate stimulation has not been studied yet in this cell model. Significant spontaneous secretion of the peptide, which results in hormone deposits on the perimeter of the cells, has been confirmed by using confocal microscopy. Co-staining with a membrane area marker FM 1-43, which co-localized with the immunocytochemically marked hormone deposits, showed that fusion-competent sites on the plasma membrane coincided with secretion-competent sites. Stimulation of the cells with glutamate and high K+ saline induced a significant increase in the plasma membrane area covered with alpha-MSH deposits compared to control cells incubated with glutamate and CNQX, a glutamate channel blocker. The optical approach to monitor the secretory activity of a single neuroendocrine cell revealed that glutamate stimulates the release of alpha-MSH at distinct exocytotic membrane domains only.

Correlated ATP-induced changes in membrane area and membrane conductance in single rat adipocytes.

In the past few years it has been shown that, like many other non-neuroendocrine cells, adipocytes possess a mechanism for triggered exocytosis. Endocytosis and exocytosis affect the plasma membrane surface area, which can be directly monitored with electrophysiological patch-clamp techniques by measuring membrane capacitance, a parameter linearly related to the plasma membrane area. In this study we used the whole-cell mode of the patch-clamp technique to measure changes in membrane capacitance to monitor the effect of extracellular adenosine triphosphate (ATP) on the dynamics of membrane area changes in single adipocytes. Experimental evidence shows that extracellular application of ATP (100 microM) increases membrane capacitance for 30 +/- 2%. In controls a significantly smaller increase of 3 +/- 2% was measured, which is due to a slow exocytic-endocytic membrane cycling rate of 0.3%/min. We found that ATP induces a transient increase in membrane current, temporally associated with the peak rate in membrane capacitance increase. These results show directly the presence of ATP-induced increase in membrane area correlated to the increase in membrane current in single adipocytes.

Properties of Ca(2+)-dependent exocytosis in cultured astrocytes.

Astrocytes, a subtype of glial cells, have numerous characteristics that were previously considered exclusive for neurons. One of these characteristics is a cytosolic [Ca2+] oscillation that controls the release of the chemical transmitter glutamate and atrial natriuretic peptide. These chemical messengers appear to be released from astrocytes via Ca(2+)-dependent exocytosis. In the present study, patch-clamp membrane capacitance measurements were used to monitor changes in the membrane area of a single astrocyte, while the photolysis of caged calcium compounds by a UV flash was used to elicit steps in [Ca2+]i to determine the exocytotic properties of astrocytes. Experiments show that astrocytes exhibit Ca(2+)-dependent increases in membrane capacitance, with an apparent Kd value of approximately 20 microM [Ca2+]i. The delay between the flash delivery and the peak rate in membrane capacitance increase is in the range of tens to hundreds of milliseconds. The pretreatment of astrocytes by the tetanus neurotoxin, which specifically cleaves the neuronal/neuroendocrine type of SNARE protein synaptobrevin, abolished flash-induced membrane capacitance increases, suggesting that Ca(2+)-dependent membrane capacitance changes involve tetanus neurotoxin-sensitive SNARE-mediated vesicular exocytosis. Immunocytochemical experiments show distinct populations of vesicles containing glutamate and atrial natriuretic peptide in astrocytes. We conclude that the recorded Ca(2+)-dependent changes in membrane capacitance represent regulated exocytosis from multiple types of vesicles, about 100 times slower than the exocytotic response in neurons.

Quantification of cell hybridoma yields with confocal microscopy and flow cytometry.

The fusion of antigen presenting and cancer cells leads to the formation of hybrid cells, which are considered a potential vaccine for treating cancer. The quality assessment of hybrid cell vaccines is crucial for the introduction of this new treatment. Flow cytometry was the method used recently, since it is faster in comparison to classical microscopy. Here we describe a rapid confocal microscopy based approach to quantify hybrid cell yields. The extent of fusion rate was determined by confocal microscopy by counting dual fluorescent cells and by measuring the area of co-localized pixels. Results of both methods showed high degree of correlation. The same samples were also analyzed by flow cytometry. Fusion rates determined with both techniques showed significant correlation. In conclusion, using confocal microscopy we developed a sensitive and a rapid method to assess the yield of hybridomas in a large number of electrofused cells.

Calcium-dependent exocytosis of atrial natriuretic peptide from astrocytes.

Astrocytes are non-neuronal cells in the CNS, which, like neurons, are capable of releasing neuroactive molecules. However, the mechanism of release is ill defined. In this study, we investigated the mechanism of release of atrial natriuretic peptide (ANP) from cultured cortical astrocytes by confocal microscopy. To study the discharge of this hormone, we transfected astrocytes with a construct to express pro-ANP fused with the emerald green fluorescent protein (ANP.emd). The transfection of cells with ANP.emd resulted in fluorescent puncta in the cytoplasm that represent secretory organelles. If ANP is released by exocytosis, in which the vesicle fuses with the plasma membrane, then the total intensity of the green fluorescing probe should decrease, whereas the vesicle membrane is incorporated into the plasma membrane. To monitor exocytosis, we labeled the membrane with the fluorescent styryldye FM 4-64, a reporter of cumulative exocytosis. The application of ionomycin to elevate cytoplasmic [Ca(2+)] increased the fluorescence intensity of FM 4-64, whereas that of ANP.emd decreased. These effects were not observed in the absence of extracellular Ca(2+), suggesting that ANP is released by regulated Ca(2+)-dependent exocytosis from astrocytes.