High-throughput monitoring of major cell functions by means of lensfree video microscopy.

Quantification of basic cell functions is a preliminary step to understand complex cellular mechanisms, for e.g., to test compatibility of biomaterials, to assess the effectiveness of drugs and siRNAs, and to control cell behavior. However, commonly used quantification methods are label-dependent, and end-point assays. As an alternative, using our lensfree video microscopy platform to perform high-throughput real-time monitoring of cell culture, we introduce specifically devised metrics that are capable of non-invasive quantification of cell functions such as cell-substrate adhesion, cell spreading, cell division, cell division orientation and cell death. Unlike existing methods, our platform and associated metrics embrace entire population of thousands of cells whilst monitoring the fate of every single cell within the population. This results in a high content description of cell functions that typically contains 25,000 - 900,000 measurements per experiment depending on cell density and period of observation. As proof of concept, we monitored cell-substrate adhesion and spreading kinetics of human Mesenchymal Stem Cells (hMSCs) and primary human fibroblasts, we determined the cell division orientation of hMSCs, and we observed the effect of transfection of siCellDeath (siRNA known to induce cell death) on hMSCs and human Osteo Sarcoma (U2OS) Cells.

Confocal bi-protocol: a new strategy for isotropic 3D live cell imaging.

The conventional approach for microscopic 3D cellular imaging is based on axial through-stack image series which has some significant limitations such as anisotropic resolution and axial aberration. To overcome these drawbacks, we have recently introduced an alternative approach based on micro-rotation image series. Unfortunately, this new technique suffers from a huge burden of computation that makes its use quite difficult for current applications. To address these problems we propose a new imaging strategy called bi-protocol, which consists of coupling micro-rotation acquisition and conventional z-stack acquisition. We experimentally prove bi-protocol 3D reconstruction produces similar quality to that of pure micro-rotation, but offers the advantage of reduced computation burden because it uses the z-stack volume to accelerate the registration of the micro-rotation images.

Isotropic high-resolution three-dimensional confocal micro-rotation imaging for non-adherent living cells.

Recently, micro-rotation confocal microscopy has enabled the acquisition of a sequence of micro-rotated images of nonadherent living cells obtained during a partially controlled rotation movement of the cell through the focal plane. Although we are now able to estimate the three-dimensional position of every optical section with respect to the cell frame, the reconstruction of the cell from the positioned micro-rotated images remains a last task that this paper addresses. This is not strictly an interpolation problem since a micro-rotated image is a convoluted two-dimensional map of a three-dimensional reality. It is rather a 'reconstruction from projection' problem where the term projection is associated to the PSF of the deconvolution process. Micro-rotation microscopy has a specific difficulty. It does not yield a complete coverage of the volume. In this paper, experiments illustrate the ability of the classical EM algorithm to deconvolve efficiently cell volume despite of the incomplete coverage. This cell reconstruction method is compared to a kernel-based method of interpolation, which does not take account explicitly the point-spread-function (PSF). It is also compared to the standard volume obtained from a conventional z-stack. Our results suggest that deconvolution of micro-rotation image series opens some exciting new avenues for further analysis, ultimately laying the way towards establishing an enhanced resolution 3D light microscopy.

Use of micro-rotation imaging to study JNK-mediated cell survival in Theileria parva-infected B-lymphocytes.

Lymphocytes infected with the protozoan parasite Theileria parva are transformed to permanently proliferating cells, an event underlying the pathology of the disease. However, the molecular signalling mediating this process is complex and poorly understood. Here, we show that down-regulation of JNK signalling by transient over expression of a dominant-negative mutant of JNK (JNK-APF) significantly increases Annexin-V-phycoerythrin (V-PE) labelling on infected B cell populations observed using flow cytometry. To establish whether this increase was specifically due to apoptosis, we used a novel single-cell imaging method: micro-rotation (MR)-imaging, designed to allow high-resolution 3-dimensional imaging of single cells in suspension. With this method we visualized subcellular patterns of V-PE uptake and chromatin organization in lymphocytes co-transfected with JNK-APF and GFP-tagged histone-H2B. This single-cell approach allowed us to clearly reveal characteristic apoptotic phenotypes, whose patterns reflected progressive states of programmed cell death due to JNK down-regulation. Our results strongly suggest a role for JNK in the survival of Theileria-infected B cells, and demonstrate the powerful utility of a new and unique 3-dimensional imaging method for living cells in suspension.

Pulsatile exocytosis is functionally associated with GnRH gene expression in immortalized GnRH-expressing cells.

Pulsatile release of GnRH is essential for proper reproductive function, but little information is available on the molecular processes underlying this intermittent activity. Recently, GnRH gene expression (GnRH-GE) episodes and exocytotic pulses have been identified separately in individual GnRH-expressing cells, raising the exciting possibility that both activities are linked functionally and are fundamental to the pulsatile process. To explore this, we monitored GnRH-GE (using a GnRH promoter-driven luciferase reporter) and exocytosis (by FM1-43 fluorescence) in the same, living GT1-7 cells. Our results revealed a strong temporal association between exocytotic pulses and GnRH-GE episodes. To determine whether a functional link existed, we blocked one process and evaluated the other. Transcriptional inhibition with actinomycin D had only a modest influence on exocytosis, suggesting that exocytotic pulse activity was not dictated acutely by episodes of gene expression. In contrast, blockage of exocytosis with anti-SNAP-25 (which obstructs secretory granule fusion) abolished GnRH-GE pulse activity, indicating that part of the exocytotic process is responsible for triggering episodes of GnRH-GE. When taken together, our findings suggest that a careful balance is maintained between release and biosynthesis in GT1-7 cells. Such a property may be important in the hypothalamus to ensure that GnRH neurons are in a constant state of readiness to respond to changes in reproductive function.

Synchronized exocytotic bursts from gonadotropin-releasing hormone-expressing cells: dual control by intrinsic cellular pulsatility and gap junctional communication.

Periodic secretion of GnRH from the hypothalamus is the driving force for the release of gonadotropic hormones from the pituitary, but the roles of individual neurons in the context of this pulse generator are not known. In this study we used FM1-43 to monitor the membrane turnover associated with exocytosis in single GT1-7 neurons and found an intrinsic secretory pulsatility (frequency, 1.4 +/- 0.1/h; pulse duration, 17.3 +/- 0.6 min) that, during time in culture, became progressively synchronized among neighboring cells. Voltage-gated calcium channels and gap junctional communication each played a major role in synchronized pulsatility. An L-type calcium channel inhibitor, nimodipine, abolished synchronized pulsatility. In addition, functional gap junction communication among adjacent cells was detected, but only under conditions where pulsatile synchronization was also observed, and the gap junction inhibitor octanol abolished both without affecting pulse frequency or duration. Our results, therefore, provide strong evidence that the GnRH pulse generator in GT1-7 cells arises from a single cell oscillator mechanism that is synchronized through network signaling involving voltage-gated calcium channels and gap junctions.

Spontaneous calcium oscillatory patterns in mammotropes display non-random dynamics.

We previously showed that primary rat mammotropes exhibited four distinct patterns of 'spontaneous' free intracellular calcium ([Ca2+]i) oscillatory behavior: a quiescent state A and three oscillatory states B,C&D, which differed in frequency/amplitude characteristics. When [Ca2+]i was monitored in 10 min windows separated by several hours, these phenotypes were frequently found to interconvert, raising the question about whether these transitions were random or ordered events. We reasoned that if such activity were random, then neither episode duration nor transitional probabilities should differ among phenotypes. We tested this logic in the current study by making long-term, continuous measurements of [Ca2+]i in mammotropes microinjected with Fura-2-dextran and identified by their ability to express a prolactin promoter-driven reporter plasmid. We found that transitions occurred in ~25% of cells (n = 36 from 9 independent experiments) once every 1-5 h and demarcated phenotype episodes of different duration (A, 1.04 +/- 0.2 h; B, 1.64 +/- 0.3 h; C, 2.45 +/- 0.62 h; D, 0.90 +/- 0.2 h, mean +/- SEM). Moreover, some transitions occurred more frequently than others and linked specific phenotypes into a common pattern: C to B to A. Our results demonstrate that the seemingly spontaneous nature of [Ca2+]i phenotype transitions are, in fact, ordered and support the view that they comprise a structured 'code' like that proposed to underlie calcium-dependent regulation of exocytosis and gene expression.

Functional integrity of green fluorescent protein conjugated glycine receptor channels.

The alpha subunit (alphaZ1) of the zebrafish glycine receptor (GlyR) has been N-terminus fused with green fluorescent protein (GFP). We found that both pharmacological and electrophysiological properties of this chimeric alphaZ1-GFP are indistinguishable from those of the wild-type receptor when expressed in Xenopus oocytes and cell lines. The apparent affinities of this receptor for agonists (glycine, taurine and GABA), and the antagonist (strychnine) are unchanged, and single channel kinetics are not altered. In the same expression systems, alphaZ1-GFP was visualized using fluorescence microscopy. Fluorescence was distributed anisotropically across cellular membranes. In addition to the Golgi apparatus and endoplasmic reticulum, its presence was also detected on the plasmalemma, localized at discrete hot-spots which were identified as sites of high membrane turnover. Overall, the preservation in alphaZ1-GFPs of the wild type receptor functional properties makes it a promising new tool for further in situ investigations of GlyR expression, distribution and function.

N-methyl-D-aspartate evokes rapid net depolymerization of filamentous actin in cultured rat cerebellar granule cells.

Filamentous actin (F-actin) was measured in cultured rat cerebellum granule neurons with the use of fluorescently labeled phallotoxin as a site-specific probe for F-actin, and fluorescence microscopy. The averaged apparent intensity of soma-associated F-actin-derived fluorescence (F(app)) was measured from fixed cells after incubation in either 1) normal Krebs solution containing 2 mM extracellular calcium ([Ca2+]ex) or 2) normal Krebs solution plus N-methyl-D-aspartate (NMDA) for 2 min immediately before fixation. NMDA (10, 50, and 100 microM) decreased F(app) to 63 +/- 5% (mean +/- SE), 53 +/- 4%, and 47 +/- 2%, respectively, of that measured from control cells. This effect was mimicked by treatment of cells with ionomycin. The ability of NMDA to reduce the F(app) in the presence of [Ca2+]ex was abolished when cells were maintained in [Ca2+]ex-free medium. Cells first treated with NMDA for 2 min and then left in normal medium for 30 min before fixation gave F(app) fluorescence similar to control values (91 +/- 12%). However, if the F-actin polymerization inhibitor cytochalasin D was added to cells immediately after NMDA was removed, the F(app) did not recover with time (36 +/- 3%). Cells treated for 30 min with cytochalasin D alone showed a small reduction in staining (approximately 20%). It is concluded that the actin polymerization state of rat cerebellar granule neurons is sensitive to changes in intracellular calcium, and that NMDA receptor activation evokes an initial rapid depolymerization of F-actin.

The effect of extracellular polyvalent cations on bovine anterior pituitary cells. Evidence for a Ca(2+)-sensing receptor coupled to release of intracellular calcium stores.

We have investigated the effects of extracellular cations ([ION]ex) on cytosolic free calcium levels ([Ca2+]i) in bovine anterior pituitary (bAP) cells, using single-cell microfluorimetry. Increasing the [Ca2+]ex from 1 mM to 20 mM caused [Ca2+]i to increase in 64 +/- 14% of bAP cells. The [Ca2+]ex-induced [Ca2+]i increase was observed when cells were maintained in the presence of the voltage-gated-calcium-channel antagonist nitrendipine, but not when cells were treated with thapsigargin. Addition of [La3+]ex (5-15 microM) decreased [Ca2+]i, whereas 30 microM-1 mM caused a [Ca2+]i rise in 60.9 +/- 8.8% of bAP cells. [La3+]ex-induced [Ca2+]i changes were abolished by treating bAP-cells with either thapsigargin or ionomycin, but not nitrendipine. [La3+]ex at 15 microM did not increase [Ca2+]i in any cells tested, but when cells were treated with thimerosal, [La3+]ex (15 microM) caused a [Ca2+]i increase in 62.5 +/- 12.2% of bAP cells. In the presence of 1 mM [Ca2+]ex, successive additions of La3+ caused successive [Ca2+]i rises, but in nominally [Ca2+]ex-free medium only the first addition of [La3+]ex caused a [Ca2+]i rise. Addition of thyroliberin (TRH) in the presence of 1 mM [Ca2+]ex, caused [Ca2+]i to increase in 70% of bAP cells; subsequent addition of [La3+]ex (1 mM) only caused [Ca2+]i increases in 75% of those cells which had already responded to TRH. However, all cells which responded to 1 mM [La3+]ex also responded subsequently to TRH. After treatment with TRH in medium that was nominally [Ca2+]ex free, addition of La3+ (0.5-1 mM) did not increase [Ca2+]i in any cells tested. The number of cells which showed [La3+]ex-induced [Ca2+]i increases decreased in culture: only 21.75 +/- 2.2% cells responded after 7-11 days. When cells were cultured for 7-11 days in the presence of tunicamycin, [La3+]ex failed to increase [Ca2+]i in any cells tested. [Mn2+]ex rapidly quenched the Fura-2 signal measured from all bAP cells, but at 10 mM it also triggered a [Ca2+]i rise in about 60% of bAP cells. The Mn(2+)-induced [Ca2+]i rise was specifically abolished in cells cultured in the presence of tunicamycin although quenching was still observed. From these data we suggest that bAP cells may express a polyvalent cation receptor coupled to the release of calcium from intracellular stores.

Simultaneous measurement of [Ca2+]i and secretion-coupled membrane turnover, by single cell fluorescence microscopy.

Thyrotropin releasing hormone (TRH), which stimulates prolactin secretion, increases the fluorescence of cultured bovine anterior pituitary (bAP) cells in the presence of the non-permeant membrane indicator dye FM 1-43 [Stafford SJV. Shorte SL. Schofield JG. (1993) Use of a fluorescent dye to measure secretion from intact bovine anterior pituitary cells. Biosci. Rep., 13, 9-17]. FM 1-43 is non-fluorescent in aqueous solution but becomes fluorescent when incorporated into the plasma membrane. The membrane area accessible to FM 1-43 dye, and therefore cell fluorescence, increases during exocytosis as secretory granules fuse with the plasma membrane, and endocytosis as vesicles formed at the plasma-membrane fuse with intracellular organelle membranes. We have here measured changes in FM 1-43 uptake and the intracellular calcium concentration ([Ca2+]i) concurrently in the same cells on exposure to TRH, phorbol myristate acetate (PMA) or NH4Cl. TRH (0.1-10 microM) caused a transient increase in [Ca2+]i in 70-90% of bAP cells and in 60-90% of the responding cells also caused a sustained increased FM 1-43 fluorescence. TRH increased [Ca2+]i but did not affect FM 1-43 fluorescence in GH3 rat pituitary cells, probably because they contain too few secretory granules to give a detectable increase. The dopamine D2-receptor agonist quinpirole (10 microM) had little effect on the TRH-induced [Ca2+]i rise in bAP cells, but abolished the increase in FM 1-43 fluorescence. The phorbol ester PMA (0.3-3 microM) caused a small, transient increase in [Ca2+]i followed by a fall to levels lower than original resting levels in 40-60% of bAP cells and increased FM 1-43 uptake in cells showing these changes. Extracellular NH4Cl, which mobilises calcium from an ionomycin-insensitive calcium store, caused a transient [Ca2+]i increase in over 90% of the bAP-cells and increased FM 1-43 uptake in a subpopulation (> 50%) of these. The Na+/H+ ionophore monensin prevented the increase in FM 1-43 fluorescence but not the [Ca2+]i rise induced by TRH, prevented the increases in both FM 1-43 fluorescence and [Ca2+]i caused by NH4Cl, and reduced the number of cells showing a rise in FM 1-43 fluorescence in response to PMA from 64% to 34%. The Ca(2+)-ATPase inhibitor thapsigargin reduced the number of bAP cells displaying TRH-induced increases in [Ca2+]i and membrane-turnover from 74% to 18%, but did not affect the changes in [Ca2+]i or FM 1-43 fluorescence caused by PMA or NH4Cl. We discuss the relationships between the secretogogue-induced increases in FM 1-43 fluorescence and changes in intracellular [Ca2+]i under these conditions.

Thyroliberin-induced changes in the fluorescence of a membrane probe in individual bovine anterior pituitary cells.

1. We have investigated the use of TMA-DPH (1-[4-(trimethylammonio) phenyl]-6-phenylhexa-1,3,5-triene) as an indicator of exocytosis in individual bovine anterior pituitary cells using microfluorimetric imaging. 2. TMA-DPH was photolabile in artificial and cell membranes. In cells incubated in TMA-DPH the distribution of fluorescence depended both on the incubation time and the illumination schedule. If the dye was added while the cells were subjected to repeated cycles of 0.36 s light intermittent with 1-15 s dark, the fluorescence of the peripheral annulus and the central region of individual cells rose in parallel and reached a steady state within 200 s; the annulus was always brighter than the central region. However, using long intervening dark periods (200 s), the central region continued to incorporate dye after the annulus had reached a plateau. 3. When the cells were loaded with TMA-DPH using intermittent light with short dark periods, the dye washed out of the central region and the annulus in parallel when external dye was removed. However, if the cells had been loaded using long dark periods, the dye was washed out of the central region more slowly than from the annulus. 4. When cells were incubated in TMA-DPH in the dark for 1 min and then exposed to constant illumination in the presence of external dye, the fluorescence of the central region and the annulus both decayed in parallel to a new steady state. If the cells were incubated in TMA-DPH in the dark for 240 min the fluorescence from each region fell to a steady state but the falls were larger and were not in parallel. 5. We suggest that TMA-DPH fluorescence was derived from plasma membrane-associated and internalized dye and that the amount of fluorescence from the latter varied because TMA-DPH was photobleached. Thus, when illumination was interrupted by short dark intervals, annular fluorescence was high compared to central fluorescence because bleached dye in the plasma membrane was rapidly replaced by unbleached dye from the medium. However, long dark intervals permitted the dye to be internalized before it was bleached and fluorescence was therefore also present in central regions. 6. The total cell fluorescence, observed using 15 s dark intervals, was increased 5-40% (in single cells) in a dose-dependent fashion by addition of TRH (tripeptide thyrotrophin-releasing hormone; 1-200 nM).(ABSTRACT TRUNCATED AT 400 WORDS)

Use of a fluorescent dye to measure secretion from intact bovine anterior pituitary cells.

The fluorescent dye FM1-43 has been used to indicate membrane changes in individual bovine anterior pituitary cells exposed to secretory stimuli. After ten minutes incubation with FM1-43 (2 microM), cells showed three patterns of dye fluorescence: annular, partly filled and uniformly filled. FM1-43 fluorescence was increased in 61% of the cells by TRH (40 nM), a physiological stimulus for prolactin secretion, and in 89% of the cells by 60 mM external K+. The fluorescence also increased when cells incubated in the presence of quinpirole, a dopamine D2-receptor agonist which inhibits prolactin secretion, were exposed to raclopride, a D-2 antagonist. The increases in FM1-43 fluorescence caused by these treatments suggests that the dye acts as an indicator of secretion, possibly through incorporation into secretory vesicle membranes exposed on the cell surface during exocytosis. If the dye was washed away after loading, the fluorescence of partly and uniformly filled cells was retained and a rise in fluorescence could still be seen on stimulation by TRH. This suggests that some dye had been taken up by endocytosis and trapped in an intracellular compartment, which expanded through membrane recapture after TRH stimulation. FM1-43 could therefore be a useful probe for membrane cycling associated with secretory responses.

Thyrotropin-releasing hormone-induced cytosolic calcium transients: characterisation of store refilling in bovine anterior pituitary cells.

The intracellular calcium ion concentration ([Ca2+]i) in individual bovine anterior pituitary cells was measured using fura-2 and ratiometric imaging. Addition of thyrotropin-releasing hormone (TRH) in the presence of external calcium ion ([Ca2+]e; 1 mM) caused a rapid transient increase in [Ca2+]i falling to a plateau which remained above pre-stimulation levels in the continued presence of TRH. Decreasing [Ca2+]e to 0.1 microM decreased [Ca2+]i. At 0.1 microM [Ca2+]e, the first TRH addition caused the rapid transient rise in [Ca2+]i but no plateau phase and a second addition of TRH did not cause a second transient rise. However, the second application of TRH in 0.1 microM [Ca2+]e caused a rise in [Ca2+]i if it was preceded by transient exposure of the cells to 2 mM [Ca2+]e. The presence of nitrendipine, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBHQ), or TRH during the re-exposure to external calcium blocked this recovery of subsequent responses to TRH in the presence of only 0.1 microM [Ca2+]e. We conclude that refilling of the calcium stores depleted by TRH occurred only after the removal of agonist, used a tBHQ-sensitive uptake mechanism, and was mainly sustained by voltage-gated calcium entry into the cells.

Ammonium ions mobilize calcium from an internal pool which is insensitive to TRH and ionomycin in bovine anterior pituitary cells.

The effects of NH4Cl on cytoplasmic free calcium concentration ([Ca2+]i) and pH (pHi) in single bovine anterior pituitary cells were determined using fluorescence imaging microscopy. Addition of NH4Cl (10-40 mM) in the presence of 1 mM extracellular calcium ([Ca2+]e) increased [Ca2+]i to a peak which then fell to a sustained plateau, returning to resting levels upon removal of NH4Cl. In medium containing 0.1 microM [Ca2+]e, or in 1 mM [Ca2+]e medium containing 0.1 microM nitrendipine, the plateau was absent leaving only a transient [Ca2+]i spike. NH4Cl also increased pHi and this, like the [Ca2+]i plateau, remained elevated during the continued presence of NH4Cl. In medium containing only 0.1 microM [Ca2+]e, to preclude refilling of internal stores by entry of external calcium, repeated exposures to NH4Cl induced repeated [Ca2+]i transients. In contrast, only the initial exposure to thyrotropin releasing hormone (TRH; 20-500 nM) caused a [Ca2+]i rise but, after an additional exposure to NH4CI, TRH responses re-emerged in some cells. Pre-treatment with the calcium ionophore ionomycin abolished the rise caused by TRH, but neither TRH nor ionomycin pretreatment affected the response to NH4Cl. Neither acetate removal nor methylamine increased [Ca2+]i in medium containing 0.1 microM [Ca2+]e, although in both cases pHi increased. We conclude that in bovine anterior pituitary cells NH4Cl raises [Ca2+]i by two independent pathways, increasing net calcium entry and mobilizing Ca2+ from a TRH-insensitive calcium store.