Biomedical subjects

Robert Zorec

Publications and source records attributed to Robert Zorec.

At least 19 recordsLinked to original sources

PubMed2007

Cytoskeleton and vesicle mobility in astrocytes.

Exocytotic vesicles in astrocytes are increasingly viewed as essential in astrocyte-to-neuron communication in the brain. In neurons and excitable secretory cells, delivery of vesicles to the plasma membrane for exocytosis involves an interaction with the cytoskeleton, in particular microtubules and actin filaments. Whether cytoskeletal elements affect vesicle mobility in astrocytes is unknown. We labeled single vesicles with fluorescent atrial natriuretic peptide and monitored their mobility in rat astrocytes with depolymerized microtubules, actin, and intermediate filaments and in mouse astrocytes deficient in the intermediate filament proteins glial fibrillary acidic protein and vimentin. In astrocytes, as in neurons, microtubules participated in directional vesicle mobility, and actin filaments played an important role in this process. Depolymerization of intermediate filaments strongly affected vesicle trafficking and in their absence the fraction of vesicles with directional mobility was reduced.

Actins↗

PubMed2006

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.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2006

Astrocyte swelling leads to membrane unfolding, not membrane insertion.

The mechanisms mediating the release of chemical transmitters from astrocytes are the subject of intense research. Recent experiments have shown that hypotonic conditions stimulate the release of glutamate and ATP from astrocytes, but a mechanistic understanding of this process is not available. To determine whether hypotonicity activates the process of regulated exocytosis, we monitored membrane capacitance by the whole-cell patch-clamp technique whilst a hypotonic medium was applied to cultured astrocytes. If exocytosis is triggered under hypotonic conditions, as it is following increases in cytosolic calcium, a net increase in membrane surface area, monitored by measuring the whole-cell membrane capacitance, is expected. Simultaneous measurements of cell size and whole-cell membrane conductance and surface area demonstrated that hypotonic medium (210 mOsm for 200 s) resulted in an increase in membrane conductance and in the swelling of cultured astrocytes by an average of 40%, as monitored by cell cross-sectional area, but without any corresponding change in membrane surface area. As we have demonstrated that capacitance measurements have the sensitivity to detect increases in cell surface area as small as 0.5%, we conclude that cell swelling occurs via an exocytosis-independent mechanism, probably involving the unfolding of the plasma membrane.

Adenosine Triphosphate↗

PubMed2005

Subcellular localization of Apaf-1 in apoptotic rat pituitary cells.

A key step in the intrinsic apoptotic pathway is the assembly of the apoptosome complex. The apoptosome components are well known; however, the physiology of the assembly of the apoptosome complex at the cellular level is still poorly defined. The aim of this work was to study the subcellular distribution of the apoptosome scaffold protein apoptotic protease-activating factor 1 (Apaf-1) before and after triggering apoptosis in single somatotrophs. Somatotrophs are the subject of extensive pituitary tissue remodeling in different physiological situations in which the quality and the number of pituitary cells are determined by cell proliferation and apoptosis. We show herein that 2 h after triggering apoptosis with rotenone, Apaf-1 redistributed to the proximity of mitochondria. In addition, the degree of colocalization between Apaf-1 and fluorescently labeled caspase-9 significantly increased during the same period. Furthermore, we show herein for the first time in single cells that the colocalization between Apaf-1 and cytochrome c increases only transiently, indicating a transient interaction between cytochrome c and Apaf-1 during the activation of apoptosis in these cells.

Animals↗

PubMed2005

Vesicle mobility studied in cultured astrocytes.

Astrocytes release many neuroactive substances, which are stored in membrane bound vesicles and may play a role in synapse modulation and in the coupling between neuronal activity and the local blood flow. However, the mobility of these vesicles in astrocytes has not been studied yet. We here used a fluorescently tagged proatrial natriuretic peptide to label single vesicles and dynamic microscopy to monitor their mobility. To track and analyze labeled vesicles, we employed a computer software. We found two modes of vesicle mobility, directional and non-directional. The mobility of non-directional vesicles is likely determined mainly by free diffusion. Only directional vesicles displayed a straight-line motion. The relationship of mean square displacement with time in directional vesicles resembled a quadratic function, indicating that in addition to free diffusion other mechanisms may contribute to vesicle movements in astrocytes, the biophysical properties of which are similar to those of neurons.

Animals↗

PubMed2005

Concentration-dependent staining of lactotroph vesicles by FM 4-64.

Hormones are released from neuroendocrine cells by passing through an exocytotic pore that forms after vesicle and plasma membrane fusion. An elegant way to study this process at the single-vesicle level is to use styryl dyes, which stain not only the membrane, but also the matrix of individual vesicles in some neuroendocrine cells. However, the mechanism by which the vesicle matrix is stained is not completely clear. One possibility is that molecules of the styryl dye in the bath solution dissolve first in the plasma membrane and are then transported into the vesicle by lateral diffusion in the plane of the membrane, and finally the vesicle matrix is stained from the vesicle membrane. On the other hand, these molecules may enter the vesicle lumen and reach the vesicle matrix by permeation through an open aqueous fusion pore. To address these questions, we exposed pituitary lactotrophs to different concentrations of FM 4-64 to monitor the fluorescence increase of single vesicles by confocal microscopy after the stimulation of cells by high K(+). The results show that the membrane and the vesicle matrix exhibit different concentration-dependent properties: the plasma membrane staining by FM 4-64 has a higher affinity in comparison to the vesicle matrix. Moreover, the kinetics of vesicle loading by FM 4-64 exhibited a concentration-dependent process, which indicates that FM 4-64 molecules stain the vesicle matrix by aqueous permeation through an open fusion pore.

Animals↗

PubMed2005

Insulin-induced exocytosis in single, in vitro innervated human muscle fibres: a new approach.

We describe a new approach for studying insulin-induced exocytosis in individual, well-differentiated, innervated human muscle fibres. We used an in vitro system in which motor axons extending from embryonic rat spinal cord explants functionally innervate co-cultured human muscle fibres. Under such conditions, the human muscle fibres reach a high degree of differentiation that is never observed in non-innervated, cultured human muscle fibres. To monitor insulin-induced membrane dynamics, we used confocal microscopy to measure the fluorescence intensity changes of the styryl dye FM1-43, a marker for membrane area. The fluorescence intensity increased after insulin stimulation. This increase, as well as the intensity of staining for the glucose transporter 4 (GLUT4), was significantly higher in the innervated and contracting fibres than in myoblasts and myotubes. This shows that in vitro innervation of human muscle cells not only enhances the differentiation stage but also improves the insulin response. Our approach allows continuous monitoring and quantitative assessment of insulin-induced increase in cumulative exocytosis in individual human muscle fibres at a differentiation level practically corresponding to that of adult muscle. It is therefore a suitable system for studying various parameters affecting the mechanisms underlying insulin-induced GLUT4 translocation in human skeletal muscle.

Animals↗

PubMed2005

Endocytosis-dominated membrane area decrease requires Rab5 protein in rat melanotrophs.

Eukaryotic cells internalize extracellular macromolecules by endocytosis and it was shown that Rab5 protein is required for this process. While it is clear that endocytosis consists of vesicle fission from the plasma membrane, the role of Rab5 protein in the plasma membrane surface area changes is still unclear. Here we studied whether Rab5 is required for membrane surface area changes in rat melanotrophs-cells deriving from the pituitary pars intermedia. The presence of this protein in melanotrophs was probed by immunocytochemistry and its putative role in membrane area dynamics was monitored electrophysiologically with membrane capacitance measurements as this parameter directly reflects changes in membrane surface area. We found that Rab5 protein exists in melanotrophs. At [Ca(2+)](i) < 3 microM, endocytosis-dominated membrane capacitance decrease was found to be blocked by microinjection of specific Rab5 antibody. At high [Ca(2+)](i), Rab5 antibody did not affect the steady-state increase in membrane capacitance, while it elevated the rate of membrane capacitance increase, which is consistent with an inhibition of endocytosis.

Animals↗

PubMed2005

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.

Adenosine Triphosphate↗

PubMed2005

Focus-drift correction in time-lapse confocal imaging.

In long-term time-lapse imaging of living cells, where recording takes several minutes or longer, a drift of focus may be significant. Focus-drift is due to the slippage in the microscope focus mechanism and/or the thermal gradients in the microscope. Software and hardware solutions may be introduced to correct for the focus-drift. Some autofocus techniques measure position of the specimen by sensing the light or sound reflected from a well-defined surface, such as the microscope slide. An autofocusing approach, where a focus measure is computed for images acquired at different objective positions is less appropriate in confocal microscopy, since more than one section is in focus. To correct for the focal-drift in long-term time-lapse confocal imaging, we acquired an image stack of the specimen periodically. The software calculated Pearson's correlation coefficient between each image in the z-stack and the reference image in the stack, which was selected at the beginning of the experiment. The maximal correlation coefficient of pixel intensities was taken to identify the image, which corresponded to the focal plane of the reference image. To test our approach, we used confocal images of living rat lactotroph cells, which discharged preloaded green fluorescent probe from a single secretory granule. Simultaneously, an extracellularly applied FM 4-64 red fluorescent probe loaded the discharging vesicle and the plasma membrane. We show that our approach is appropriate to correct for focal-drift in long term time-lapse imaging and analysis of living cells.

Animals↗

PubMed2004

Ammodytoxin, a neurotoxic secreted phospholipase A(2), can act in the cytosol of the nerve cell.

Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A(2) acts intracellularly. As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them. Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus. The transport of proteins into the nucleus proceeds via the cytosol of a cell, therefore, ammodytoxin passed the cytosol of the neuron on its way to the nucleus. Although it is not yet clear how ammodytoxin is translocated into the cytosol of the neuron, our results demonstrate that its stability in the cytosol is not in question, providing the evidence that the toxin can act in this cellular compartment.

Animals↗

PubMed2004

Defining pathways of loss and secretion of chemical messengers from astrocytes.

It is becoming evident that glia, and astrocytes in particular, are intimately involved in neuronal signaling. Astrocytic modulation of signaling in neurons appears to be mediated by the release of neuroactive compounds such as the excitatory amino acid glutamate. Release of these transmitters appears to be driven by two different processes: (1) a volume regulatory response triggered by hypo-osmotic conditions that leads to the release of osmotically active solutes from the cytoplasm into the extracellular space, and (2) intracellular calcium-dependent vesicle-mediated excytotic release. The regulatory volume decrease may be mediated by any of several different pathways that increase membrane permeability, thus allowing osmolytes to travel down their concentration gradient into the extracellular space. Such pathways include anion channels, hemichannels, P2X receptor channels, and transporters or multidrug resistance proteins. The excytotic release process may use calcium triggered synaptic like vesicle fusion or alterations in constitutive vesicle trafficking to the membrane. Determining the contribution of any of these release mechanisms requires agents that can be used to specifically block pathways of interest. Currently, many of the pharmacological compounds being used exhibit a great deal of cross-reactivity between several of these pathways. For example, the popular anion channel inhibitor 5-nitro-2-(3-phenyl-propylamino)benzoic acid (NPPB) is an efficient blocker of both hemichannels and vesicle loading. This demonstrates the need to more fully characterize the activities of the agents currently available and to choose pathway blockers carefully when designing experiments.

Animals↗

PubMed2004

Slow spontaneous secretion from single large dense-core vesicles monitored in neuroendocrine cells.

Hormones are released from cells by passing through an exocytotic pore that forms after vesicle and plasma membrane fusion. In stimulated exocytosis vesicle content is discharged swiftly. Although rapid vesicle discharge has also been proposed to mediate basal secretion, this has not been studied directly. We investigated basal hormone release by preloading fluorescent peptides into single vesicles. The hormone discharge, monitored with confocal microscopy, was compared with the simultaneous loading of vesicle by FM styryl dye. In stimulated vesicles FM 4-64 (4 microM), loading and hormone discharge occurs within seconds. In contrast, in approximately 50% of spontaneously releasing vesicles, the vesicle content discharge and the FM 4-64 loading were slow (approximately 3 min). These results show that in peptide secreting neuroendocrine cells the elementary vesicle content discharge differs in basal and in stimulated exocytosis. It is proposed that the view dating back for some decades, which is that, at rest, the vesicle discharge of hormones and neurotransmitters is similar to that occurring after stimulation, needs to be extended. In addition to the classical paradigm that secretory capacity of a cell is determined by controlling the probability of occurrence of elementary exocytotic events, one will have to consider activity modulation of elementary exocytotic events as well.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

Voltage-activated Ca(2+) channels and their role in the endocrine function of the pituitary gland in newborn and adult mice.

We have prepared fresh pituitary gland slices from adult and, for the first time, from newborn mice to assess modulation of secretory activity via voltage-activated Ca(2+) channels (VACCs). Currents through VACCs and membrane capacitance have been measured with the whole-cell patch-clamp technique. Melanotrophs in newborns were significantly larger than in adults. In both newborn and adult melanotrophs activation of VACCs triggered exocytosis. All pharmacologically isolated VACC types contributed equally to the secretory activity. However, the relative proportion of VACCs differed between newborns and adults. In newborn cells L-type channels dominated and, in addition, an exclusive expression of a toxin-resistant R-type-like current was found. The expression of L-type VACCs was up-regulated by the increased oestrogen levels observed in females, and was even more emphasized in the cells of pregnant females and oestrogen-treated adult male mice. We suggest a general mechanism modulating endocrine secretion in the presence of oestrogen and particularly higher sensitivity to treatments with L-type channel blockers during high oestrogen physiological states.

Aging↗

PubMed2004

Fusion-related release of glutamate from astrocytes.

Although cell culture studies have implicated the presence of vesicle proteins in mediating the release of glutamate from astrocytes, definitive proof requires the identification of the glutamate release mechanism and the localization of this mechanism in astrocytes at synaptic locales. In cultured murine astrocytes we show an array of vesicle proteins, including SNARE proteins, and vesicular glutamate transporters that are required to fill vesicles with glutamate. Using immunocytochemistry and single-cell multiplex reverse transcription-PCR we demonstrate the presence of these proteins and their transcripts within astrocytes freshly isolated from the hippocampus. Moreover, immunoelectron microscopy demonstrates the presence of VGLUT1 in processes of astrocytes of the hippocampus. To determine whether calcium-dependent glutamate release is mediated by exocytosis, we expressed the SNARE motif of synaptobrevin II to prevent the formation of SNARE complexes, which reduces glutamate release from astrocytes. To further determine whether vesicular exocytosis mediates calcium-dependent glutamate release from astrocytes, we performed whole cell capacitance measurements from individual astrocytes and demonstrate an increase in whole cell capacitance, coincident with glutamate release. Together, these data allow us to conclude that astrocytes in situ express vesicle proteins necessary for filling vesicles with the chemical transmitter glutamate and that astrocytes release glutamate through a vesicle- or fusion-related mechanism.

Amino Acid Motifs↗

PubMed2004

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.

Animals↗

PubMed2003

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.

Animals↗