Release of insulin granules by simultaneous, high-speed correlative SICM-FCM

Exocytosis of peptides and steroids stored in a dense core vesicular (DCV) form is the final step of every secretory pathway, indispensable for the function of nervous, endocrine and immune systems. The lack of live imaging techniques capable of direct, label‐free visualisation of DCV release makes many aspects of the exocytotic process inaccessible to investigation. We describe the application of correlative scanning ion conductance and fluorescence confocal microscopy (SICM‐FCM) to study the exocytosis of individual granules of insulin from the top, nonadherent, surface of pancreatic β‐cells. Using SICM‐FCM, we were first to directly follow the topographical changes associated with physiologically induced release of insulin DCVs. This allowed us to report the kinetics of the full fusion of the insulin vesicle as well as the subsequent solubilisation of the released insulin crystal

Photon Geodesics in FRW Cosmologies

The Hubble radius is a particular manifestation of the Universe's gravitational horizon, R_h(t_0)=c/H_0, the distance beyond which physical processes remain unobservable to us at the present epoch. Based on recent observations of the cosmic microwave background (CMB) with WMAP, and ground-based and HST searches for Type Ia supernovae, we now know that R_h(t_0)~13.5 Glyr. This coincides with the maximum distance (ct_0~13.7 Glyr) light could have traveled since the big bang. However, the physical meaning of R_h is still not universally understood or accepted, though the minimalist view holds that it is merely the proper distance at which the rate of cosmic recession reaches the speed of light c. Even so, it is sometimes argued that we can see light from sources beyond R_h, the claim being that R_h lies at a redshift of only ~2, whereas the CMB was produced at a much greater redshift (~1100). In this paper, we build on recent developments with the gravitational radius by actually calculating null geodesics for a broad range of FRW cosmologies, to show---at least in the specific cases we consider here, including LCDM---that no photon trajectories reaching us today could have ever crossed R_h(t_0). We therefore confirm that the current Hubble radius, contrary to a commonly held misconception, is indeed the limit to our observability. We find that the size of the visible universe in LCDM, measured as a proper distance, is approximately 0.45ct_0.Comment: Accepted for publication in MNRA

Endocytic pathways: combined scanning ion conductance and surface confocal microscopy study

We introduce a novel high resolution scanning surface confocal microscopy technique that enables imaging of endocytic pits in apical membranes of live cells for the first time. The improved topographical resolution of the microscope together with simultaneous fluorescence confocal detection produces pairs of images of cell surfaces sufficient to identify single endocytic pits. Whilst the precise position and size of the pit is detected by the ion conductance microscope, the molecular nature of the pit, e.g. clathrin coated or caveolae, is determined by the corresponding green fluorescent protein fluorescence. Also, for the first time, we showed that flotillin 1 and 2 can be found co-localising with ~200-nm indentations in the cell membrane that supports involvement of this protein in endocytosis

PKCθ Regulates T-Cell Leukemia-Initiating Activity via Reactive Oxygen Species

Reactive oxygen species (ROS), a by-product of cellular metabolism, damage intracellular macromolecules and, in excess, can promote normal hematopoietic stem cell differentiation and exhaustion1–3. However, mechanisms that regulate ROS levels in leukemia-initiating cells (LICs) and the biological role of ROS in these cells remain largely unknown. We show here the ROSlow subset of CD44+ cells in T-cell acute lymphoblastic leukemia (T-ALL), a malignancy of immature T-cell progenitors, to be highly enriched in the most aggressive LICs, and that ROS are maintained at low levels by downregulation of protein kinase C theta (PKCθ). Strikingly, primary mouse T-ALLs lacking PKCθ show improved LIC activity whereas enforced PKCθ expression in both mouse and human primary T-ALLs compromised LIC activity. We also demonstrate that PKCθ is positively regulated by RUNX1, and that NOTCH1, which is frequently activated by mutation in T-ALL4–6 and required for LIC activity in both mouse and human models7,8, downregulates PKCθ and ROS via a novel pathway involving induction of RUNX3 and subsequent repression of RUNX1. These results reveal key functional roles for PKCθ and ROS in T-ALL and suggest that aggressive biological behavior in vivo could be limited by therapeutic strategies that promote PKCθ expression/activity or ROS accumulation

Scanning ion conductance microscopy: a convergent high-resolution technology for multi-parametric analysis of living cardiovascular cells

Cardiovascular diseases are complex pathologies that include alterations of various cell functions at the levels of intact tissue, single cells and subcellular signalling compartments. Conventional techniques to study these processes are extremely divergent and rely on a combination of individual methods, which usually provide spatially and temporally limited information on single parameters of interest. This review describes scanning ion conductance microscopy (SICM) as a novel versatile technique capable of simultaneously reporting various structural and functional parameters at nanometre resolution in living cardiovascular cells at the level of the whole tissue, single cells and at the subcellular level, to investigate the mechanisms of cardiovascular disease. SICM is a multimodal imaging technology that allows concurrent and dynamic analysis of membrane morphology and various functional parameters (cell volume, membrane potentials, cellular contraction, single ion-channel currents and some parameters of intracellular signalling) in intact living cardiovascular cells and tissues with nanometre resolution at different levels of organization (tissue, cellular and subcellular levels). Using this technique, we showed that at the tissue level, cell orientation in the inner and outer aortic arch distinguishes atheroprone and atheroprotected regions. At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio. We also demonstrated the capability of SICM to measure the entire cell volume as an index of cellular hypertrophy. This method can be further combined with fluorescence to simultaneously measure cardiomyocyte contraction and intracellular calcium transients or to map subcellular localization of membrane receptors coupled to cyclic adenosine monophosphate production. The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents. In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies

Nanoscale live-cell imaging using hopping probe ion conductance microscopy,

We describe hopping mode scanning ion conductance microscopy that allows noncontact imaging of the complex three-dimensional surfaces of live cells with resolution better than 20 nm. We tested the effectiveness of this technique by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allowed examination of nanoscale phenomena on the surface of live cells under physiological conditions. There is a great interest in developing methods to image live cells at nanoscale resolution. Scanning probe microscopy (SPM) is one approach to this problem and both atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM) have been used to image live cells 1,2 . However, deformation of the soft and responsive cell by the AFM cantilever, particularly when imaging eukaryotic cells, represents a substantial problem for AFM. SECM, in contrast, involves no physical contact with the sample, but true topographic imaging of the convoluted surface of living cells with nanoscale resolution has not been reported. Scanning ion conductance microscopy (SICM) 3 is another form of SPM, which allows imaging of the cell surface under physiological conditions without physical contact and with a resolution of 3-6 nm 4,5 . Until now, SICM has been restricted to imaging relatively flat surfaces, as all other SPM techniques. This is because when the probe encounters a vertical structure, it inevitably collides with the specimen SICM is based on the phenomenon that the ion flow through a sharp fluid-filled nanopipette is partially occluded when the pipette approaches the surface of a cell 3 . In conventional SICM, a nanopipette is mounted on a three-dimensional piezoelectric translation stage and automatic feedback control moves the pipette up or down to keep the pipette current constant (the set point) while the sample is scanned in x and y directions. Thus, a pipette-sample separation, typically equal to the pipette's inner radius, is maintained during imaging. In hopping probe ion conductance microscopy (HPICM), we no longer use continuous feedback. Instead, at each imaging point, the pipette approaches the sample from a starting position that is above any of the surface features We illustrate the benefits of HPICM in In contrast to conventional raster scanning, HPICM has the additional advantage that the order of imaging pixels is not predetermined. Therefore, we divided the entire image into equal-sized square

High-resolution label-free 3D mapping of extracellular pH of single living cells

Abstract: Dynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H+ in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity > 0.01 units, 2 ms response time, and 50 nm spatial resolution. The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependance via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes