Biomedical subjects

Marko Kreft

Publications and source records attributed to Marko Kreft.

18 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

Computer-aided measurement of psoriatic lesion area in a multicenter clinical trial--comparison to physician's estimations.

BACKGROUND: There is a great need for accurate and informative outcome measures in clinical trials. In psoriasis, therapeutic effect is usually assessed by physician's estimation of psoriatic area and other signs. These measures were found to be un-reproducible. Several automated procedure for more reproducible measurement of psoriatic area were developed, but were not suitable for large-scale trials. OBJECTIVES: In a multicenter clinical trial, we tested a method where the advantage of accurate computerized measurement of the area on the digital photograph was combined with physician's proficiency. METHODS: The patients with psoriasis in four study centers were included in a placebo controlled clinical trial. They were examined and photographed before and after the therapy with calcipotriol ointment or placebo. The psoriatic area was manually outlined on the patient's photographs and the area was automatically measured by a computer. The areas estimated by physician and measured by computer were compared. RESULTS: We found that computer-aided measurement of psoriatic lesion area improved the power of the clinical trial, compared to the standard approach, where physician's estimations of the psoriatic lesion area tend to overestimate. We also found that adapted PASI index, where the psoriatic area was not converted into an area grade, but was maintained as a continuous variable, also improved the power of the clinical trial. CONCLUSIONS: Computer-aided measurement and an adapted PASI can be used as a powerful and reliable measure to evaluate the effects of antipsoriatic treatment.

Adolescent↗

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

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

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

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↗

PubMed2004

Frequency of M-cadherin-stained satellite cells declines in human muscles during aging.

To answer the question of whether the satellite cell pool in human muscle is reduced during aging, we detected satellite cells in 30- microm-thick transverse sections under the confocal microscope by binding of M-cadherin antibody. The basal lamina was detected with laminin. Nuclei were stained with bisbenzimide or propidium iodide. Satellite cells were counted by applying the disector method and unbiased sampling design. To determine if there are age-related differences in muscle fiber types, morphometric characteristics of muscle fibers were examined on thin sections stained for myofibrillar ATPase. Autopsy samples of vastus lateralis muscle from six young (28.7 +/- 2.3 years) and six old (70.8 +/- 1.3 years) persons who had suffered sudden death were analyzed. Numbers of satellite cells per fiber length (Nsc/Lfib) and number of satellite cells per total number of nuclei (satellite cell nuclei + myonuclei) (Nsc/Nnucl) were significantly lower in the old group (p < 0.05). We demonstrate the importance of proper sampling and counting in estimation of sparsely distributed structures such as satellite cells. Our results support the hypothesis that the satellite cell fraction declines during aging.

Adult↗

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↗

PubMed2002

Distinct effect of actin cytoskeleton disassembly on exo- and endocytic events in a membrane patch of rat melanotrophs.

We used the cell-attached mode of patch-clamp technique to measure discrete attofarad steps in membrane capacitance (C(m)), reporting area changes in the plasma membrane due to unitary exocytic and endocytic events. To investigate the role of the actin cytoskeleton in elementary exocytic and endocytic events, neuroendocrine rat melanotrophs were treated with Clostridium spiroforme toxin (CST), which specifically depolymerises F-actin. The average amplitude of exocytic events was not significantly different in control and in CST-treated cells. However, the amplitude of endocytic events was significantly smaller in CST-treated cells as compared to controls. The frequency of exocytic events increased by 2-fold in CST-treated cells relative to controls. In control cells the average frequency of exocytic events (upsilon;(exo)) was lower than the frequency of endocytic events (upsilon;(endo)) with a ratio upsilon;(exo)/upsilon;(endo) < 1. In the toxin treated cells, the predominant process was exocytosis with a ratio (upsilon;(exo)/upsilon;(endo) > 1). To study the coupling between the two processes, the slopes of regression lines relating upsilon;(exo) and upsilon;(endo) in a given patch of membrane were studied. The slopes of regression lines were similar, whereas the line intercepts with the y-axis were significantly different. The increased frequency of unitary exocytic events in CST-treated cells is consistent with the view, that the actin cytoskeleton acts as a barrier for exocytosis. While the disassembly of the actin cytoskeleton diminishes the size of unitary endocytic events, suggesting an important role of the actin cytoskeleton in determining the size of endocytic vesicles, the coupling between exocytosis and endocytosis in a given patch of membrane was independent of the state of the actin cytoskeleton.

Actins↗

PubMed2002

Rapid insulin-induced exocytosis in white rat adipocytes.

Insulin is believed to increase glucose permeability of adipocytes by regulating the incorporation of glucose transporters into the plasma membrane by exocytosis. This process involves fusion of membrane-bound cellular compartments with the plasma membrane, thus influencing the plasma membrane area. However, insulin-induced changes in plasma membrane area have not yet been demonstrated. In the present study we monitored fluorescence intensity with a confocal microscope to study the effect of insulin on adipocyte plasma membrane area. After cell isolation and adhesion to a glass cover-slip, adipocytes were stained with the dye FM1-43, a membrane area reporter. At rest, the rate of fluorescence intensity increase was initially high, but gradually stabilized at 2%/min. This steady increase in fluorescence is due to a slow rate of exocytosis coupled to endocytosis, since the removal of FM1-43 from the bath did not abolish FM1-43 fluorescence. Insulin addition caused an abrupt increase of fluorescence intensity of 4%/min, which was significantly higher than in controls. These results suggest rapid, insulin-induced incorporation of new membrane into the plasma membrane by exocytosis.

Adipocytes↗

PubMed2002

Modeling excess retrieval in rat melanotroph membrane capacitance records.

We have used the patch-clamp technique to monitor changes in membrane capacitance (C(m)) elicited by fast and spatially homogeneous rises in cytosolic calcium concentration ([Ca(2+)](i)) using flash photolysis of NP-EGTA. Average peak [Ca(2+)](i) amplitudes of 20-25 microM triggered three different types of responses in C(m): (i) In 42% of cells, a rise in [Ca(2+)](i) activated a monotonic increase in C(m) followed by a slow decline to resting values; (ii) In 30% of cells, the rise in C(m) was clearly characterized by two dynamic components, consisting of a rapid and a slow exo-endocytosis cycle; (iii) In 28% of cells, after the initial rapid rise in C(m), endocytosis exhibited excess retrieval that was characterized by a decline in C(m) below resting C(m). The aim of this work is to develop a unified mathematical model with a minimum number of parameters that would describe all the observed types of responses. Three models were considered: Model A, a model with a single component of exo-endocytosis cycle; model B, a model consisting of a sum of two independent dynamic components; and model C, a model in which, in addition to the two dynamic components as in model B, excess retrieval due to a lipid flow through the reversal closing of the fusion pore during the rapid component of exo-endocytosis cycle was considered. The results show that the latter model describes all the types of responses in C(m) recorded in rat melanotrophs. The association of excess retrieval exclusively with the rapid, but not the slow, exocytosis indicates that some fusing vesicles mediate a lipidic flux during the reversal closing of the fusion pore, whereas those entering the slow phase of exocytosis may fuse with the plasma membrane completely and are retrieved by other endocytic machinery, independent of the lipid flow that might have occurred as the fusion pore opened permanently.

Animals↗