A Preliminary Study of the Spreading of AKD in the Presence of Capillary Structures

Journal of Colloid and Interface Science. Aug, 2001  |  Pubmed ID: 11446799

There may be several mechanisms at work in the process of migration or redistribution of alkyl ketene dimers (AKD) on cellulose fiber surfaces during paper sizing and curing. This work is the second part of a continuing investigation of the spreading behavior of AKD on the surfaces of hydrophilic substrates. Paper sheets, single cotton, and cotton lint fibers and smooth cellulose film were used as substrates. These represent samples that have pores, V-shaped grooves, and no capillary structure at all. A very simple and effective testing method for studying the AKD migration behavior through these substrates was designed. AFM was used to study the surface capillary structures of cotton and cotton lint fibers. The results of this study provide hard evidence supporting our finding that capillary structures in the form of either interfiber pores in a paper sheet or V-shaped grooves on the surface of single fibers are essential in order for the spreading of molten AKD on a cellulose substrate to occur. Some preliminary results on the existence and the surface diffusion of an autophobic precursor of AKD are also presented. The results support the conclusion we reached in the first part of this investigation; i.e., the molten AKD wets but does not spread on smooth, capillary-free hydrophilic surfaces such as glass and cellulose. The driving force from interfacial energy alone does not cause spontaneous "flow-like" spreading of molten AKD on these surfaces. This is possibly associated with the formation of an autophobic precursor in front of an AKD droplet. The results in this study do not support the perception that molten AKD forms a single molecular layer on the surface of cellulose fibers by spreading during heat treatment, although the autophobic precursor in front of an AKD droplet could theoretically be of a monolayer thickness and the surface diffusion of this precursor may contribute to the sizing development after heat treatment. Copyright 2001 Academic Press.

Structural and Functional Relationships Between Ca2+ Puffs and Mitochondria in Xenopus Oocytes

American Journal of Physiology. Cell Physiology. Jun, 2002  |  Pubmed ID: 11997252

Ca2+ uptake and release from endoplasmic reticulum (ER) and mitochondrial Ca2+ stores play important physiological and pathological roles, and these processes are shaped by interactions that depend on the structural intimacy between these organelles. Here we investigate the morphological and functional relationships between mitochondria, ER, and the sites of intracellular Ca2+ release in Xenopus laevis oocytes by combining confocal imaging of local Ca2+ release events ("Ca2+ puffs") with mitochondrial localization visualized using vital dyes and subcellularly targeted fluorescent proteins. Mitochondria and ER are localized in cortical bands approximately 6-8 microm wide, with the mitochondria arranged as densely packed "islands" interconnected by discrete strands. The ER is concentrated more superficially than mitochondria, and the mean separation between Ca2+ puff sites and mitochondria is approximately 2.3 microm. However, a subpopulation of Ca2+ puff sites is intimately associated with mitochondria (approximately 28% within <600 nm), a greater number than expected if Ca2+ puff sites were randomly distributed. Ca2+ release sites close to mitochondria exhibit lower Ca2+ puff activity than Ca2+ puff sites in regions with lower mitochondrial density. Furthermore, Ca2+ puff sites in close association with mitochondria rarely serve as the sites for Ca2+ wave initiation. We conclude that mitochondria play important roles in regulating local ER excitability, Ca2+ wave initiation, and, thereby, spatial patterning of global Ca2+ signals.

Two-photon Imaging of Lymphocyte Motility and Antigen Response in Intact Lymph Node

Science (New York, N.Y.). Jun, 2002  |  Pubmed ID: 12016203

Lymphocyte motility is vital for trafficking within lymphoid organs and for initiating contact with antigen-presenting cells. Visualization of these processes has previously been limited to in vitro systems. We describe the use of two-photon laser microscopy to image the dynamic behavior of individual living lymphocytes deep within intact lymph nodes. In their native environment, T cells achieved peak velocities of more than 25 micrometers per minute, displaying a motility coefficient that is five to six times that of B cells. Antigenic challenge changed T cell trajectories from random walks to "swarms" and stable clusters. Real-time two-photon imaging reveals lymphocyte behaviors that are fundamental to the initiation of the immune response.

Intracellular Trafficking and Membrane Targeting Mechanisms of the Human Reduced Folate Carrier in Mammalian Epithelial Cells

The Journal of Biological Chemistry. Sep, 2002  |  Pubmed ID: 12087110

The major pathway for cellular uptake of the water-soluble vitamin folic acid in mammalian cells is via a plasma membrane protein known as the reduced folate carrier (RFC). The molecular determinants that dictate plasma membrane expression of RFC as well as the cellular mechanisms that deliver RFC to the cell surface remain poorly defined. Therefore, we designed a series of fusion proteins of the human RFC (hRFC) with green fluorescent protein to image the targeting and trafficking dynamics of hRFC in living epithelial cells. We show that, in contrast to many other nutrient transporters, the molecular determinants that dictate hRFC plasma membrane expression reside within the hydrophobic backbone of the polypeptide and not within the cytoplasmic NH(2)- or COOH-terminal domains of the protein. Further, the integrity of the hRFC backbone is critical for export of the polypeptide from the endoplasmic reticulum to the cell surface. This trafficking is critically dependent on intact microtubules because microtubule disruption inhibits motility of hRFC-containing vesicles as well as final expression of hRFC in the plasma membrane. For the first time, these data define the mechanisms that control the intracellular trafficking and cell surface localization of hRFC within mammalian epithelia.

Two-photon Imaging in Intact Lymphoid Tissue

Advances in Experimental Medicine and Biology. 2002  |  Pubmed ID: 12405205

Two-photon Tissue Imaging: Seeing the Immune System in a Fresh Light

Nature Reviews. Immunology. Nov, 2002  |  Pubmed ID: 12415310

Many lymphocyte functions, such as antigen recognition, take place deep in densely populated lymphoid organs. Because direct in vivo observation was not possible, the dynamics of immune-cell interactions have been inferred or extrapolated from in vitro studies. Two-photon fluorescence excitation uses extremely brief (<1 picosecond) and intense pulses of light to 'see' directly into living tissues, to a greater depth and with less phototoxicity than conventional imaging methods. Two-photon microscopy, in combination with newly developed indicator molecules, promises to extend single-cell approaches to the in vivo setting and to reveal in detail the cellular collaborations that underlie the immune response.

Cell Biology of the Human Thiamine Transporter-1 (hTHTR1). Intracellular Trafficking and Membrane Targeting Mechanisms

The Journal of Biological Chemistry. Feb, 2003  |  Pubmed ID: 12454006

The human thiamine transporter hTHTR1 is involved in the cellular accumulation of thiamine (vitamin B1) in many tissues. Thiamine deficiency disorders, such as thiamine-responsive megaloblastic anemia (TRMA), which is associated with specific mutations within hTHTR1, likely impairs the functionality and/or intracellular targeting of hTHTR1. Unfortunately, nothing is known about the mechanisms that control the intracellular trafficking or membrane targeting of hTHTR1. To identify molecular determinants involved in hTHTR1 targeting, we generated a series of hTHTR1 truncations fused with the green fluorescent protein and imaged the targeting and trafficking dynamics of each construct in living duodenal epithelial cells. Whereas the full-length fusion protein was functionally expressed at the plasma membrane, analysis of the truncated mutants demonstrated an essential role for both NH(2)-terminal sequence and the integrity of the backbone polypeptide for cell surface expression. Most notably, truncation of hTHTR1 within a region where several TRMA truncations are clustered resulted in intracellular retention of the mutant protein. Finally, confocal imaging of the dynamics of intracellular hTHTR1 vesicles revealed a critical role for microtubules, but not microfilaments, in hTHTR1 trafficking. Taken together, these results correlate hTHTR1 structure with cellular expression profile and reveal a critical dependence on hTHTR1 backbone integrity and microtubule-based trafficking processes for functional expression of hTHTR1.

Ca2+ Signaling in Mouse Cortical Neurons Studied by Two-photon Imaging and Photoreleased Inositol Triphosphate

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Feb, 2003  |  Pubmed ID: 12574404

IP(3)-mediated Ca(2+) release is a crucial neuronal signaling mechanism that has not been extensively characterized in the mammalian cerebral cortex. We used two-photon, video-rate microscopy to image Ca(2+) signals evoked by photoreleased IP(3) in pyramidal neurons of mouse prefrontal cortex. Ca(2+) responses to photoreleased IP(3) varied greatly between different neurons; however, within IP(3)-responsive neurons, the soma invariably showed highest sensitivity, with signals increasing nonlinearly with [IP(3)]. Responses to paired photorelease displayed inhibition, whereas IP(3)-evoked Ca(2+) liberation was potentiated by Ca(2+) entry during action potentials and vice versa. IP(3)-mediated Ca(2+) signals strongly inhibited spike firing through activation of K(+) membrane conductance. Metabotropic signaling via the phosphoinositide pathway thus serves as a powerful and sustained modulator of excitability in cortical neurons and displays complex reciprocal interactions between electrical and chemical signals.

Autonomous T Cell Trafficking Examined in Vivo with Intravital Two-photon Microscopy

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2003  |  Pubmed ID: 12601158

The recirculation of T cells between the blood and secondary lymphoid organs requires that T cells are motile and sensitive to tissue-specific signals. T cell motility has been studied in vitro, but the migratory behavior of individual T cells in vivo has remained enigmatic. Here, using intravital two-photon laser microscopy, we imaged the locomotion and trafficking of naive CD4(+) T cells in the inguinal lymph nodes of anesthetized mice. Intravital recordings deep within the lymph node showed T cells flowing rapidly in the microvasculature and captured individual homing events. Within the diffuse cortex, T cells displayed robust motility with an average velocity of approximately 11 microm x min(-1). T cells cycled between states of low and high motility roughly every 2 min, achieving peak velocities >25 microm x min(-1). An analysis of T cell migration in 3D space revealed a default trafficking program analogous to a random walk. Our results show that naive T cells do not migrate collectively, as they might under the direction of pervasive chemokine gradients. Instead, they appear to migrate as autonomous agents, each cell taking an independent trafficking path. Our results call into question the role of chemokine gradients for basal T cell trafficking within T cell areas and suggest that antigen detection may result from a stochastic process through which a random walk facilitates contact with antigen-presenting dendritic cells.

Photonics for Biologists

Methods in Enzymology. 2003  |  Pubmed ID: 12622159

Video-rate Confocal Microscopy

Methods in Enzymology. 2003  |  Pubmed ID: 12622163

The CLSM described here can be built with relatively little electronic or optical experience and with a budget of approximately $20,000-$30,000 (excluding microscope and table). This cost is substantially less than that of commercial counterparts. However, this CLSM has excellent spatial and temporal resolution and the convenience of digital recording and playback. By building the CLSM, the investigator ensures long-term support and reliability of the instrument as well as the potential for future modifications and improvements. Finally, the sense of accomplishment of building your own instrument should not to be underestimated.

Real-time Imaging of Lymphocytes in Vivo

Current Opinion in Immunology. Aug, 2003  |  Pubmed ID: 12900266

New preparations, fluorescent probes and imaging techniques are providing the means to observe the behavior of cells in the tissue environment of lymphoid organs. In particular, when combined with two-photon laser microscopy, intravital imaging of surgically exposed lymph nodes provides a unique view of lymphocyte migration and antigen presentation as it occurs within the living animal. The view is emerging that lymphocytes migrate randomly within lymphoid organs, and that lymphocyte contact with antigen-presenting cells may be a stochastic process rather than one guided by chemokine gradients.

A Stochastic View of Lymphocyte Motility and Trafficking Within the Lymph Node

Immunological Reviews. Oct, 2003  |  Pubmed ID: 12969316

Two-photon microscopy is providing literal insight into the cellular dynamics of lymphoid organs and, guided by analysis of three-dimensional images, into mechanisms that underlie cell migration and antigen recognition in vivo. This review describes lymphocyte motility and antigen recognition in the native tissue environment and compares these results with a much more extensive literature on lymphocyte motility, signaling, and chemotaxis in vitro. We discuss the in vitro literature on dynamic aspects of lymphocyte motility, chemotaxis, and the response to antigen and present the view that random migration of lymphocytes may drive a stochastic mechanism of antigen recognition in lymphoid organs, rather than being guided by chemotaxis.

Buffer Kinetics Shape the Spatiotemporal Patterns of IP3-evoked Ca2+ Signals

The Journal of Physiology. Dec, 2003  |  Pubmed ID: 14555715

Ca2+ liberation through inositol 1,4,5-trisphosphate receptors (IP3Rs) plays a universal role in cell regulation, and specificity of cell signalling is achieved through the spatiotemporal patterning of Ca2+ signals. IP3Rs display Ca2+-induced Ca2+ release (CICR), but are grouped in clusters so that regenerative Ca2+ signals may remain localized to individual clusters, or propagate globally between clusters by successive cycles of Ca2+ diffusion and CICR. We used confocal microscopy and photoreleased IP3 in Xenopus oocytes to study how these properties are modulated by mobile cytosolic Ca2+ buffers. EGTA (a buffer with slow 'on-rate') speeded Ca2+ signals and 'balkanized' Ca2+ waves by dissociating them into local signals. In contrast, BAPTA (a fast buffer with similar affinity) slowed Ca2+ responses and promoted 'globalization' of spatially uniform Ca2+ signals. These actions are likely to arise through differential effects on Ca2+ feedback within and between IP3R clusters, because Ca2+ signals evoked by influx through voltage-gated channels were little affected. We propose that cell-specific expression of Ca2+-binding proteins with distinct kinetics may shape the time course and spatial distribution of IP3-evoked Ca2+ signals for specific physiological roles.

Optical Single-channel Recording: Imaging Ca2+ Flux Through Individual N-type Voltage-gated Channels Expressed in Xenopus Oocytes

Cell Calcium. Dec, 2003  |  Pubmed ID: 14572808

Functional studies of single membrane ion channels were made possible by the introduction of the patch-clamp technique, which allows single-channel currents to be measured with unprecedented resolution. Nevertheless, patch clamping has some limitations: including the need for physical access of the patch pipette, possible disruption of local cellular architecture, inability to monitor multiple channels, and lack of spatial information. Here, we demonstrate the use of confocal fluorescence microscopy as a non-invasive technique to optically monitor the gating of individual Ca2+ channels. Near-membrane fluorescence signals track the gating of N-type Ca2+ channels with a kinetic resolution of about 10ms, provide a simultaneous and independent readout from several channels, and allow their locations to be mapped with sub-micrometer spatial resolution. Optical single-channel recording should be applicable to diverse voltage- and ligand-gated Ca2+-permeable channels, and has the potential for high-throughput functional analysis of single channels.

T Cell Repertoire Scanning is Promoted by Dynamic Dendritic Cell Behavior and Random T Cell Motility in the Lymph Node

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2004  |  Pubmed ID: 14722354

Dendritic cells (DCs) ingest antigens in peripheral tissues and migrate to lymph nodes where they present MHC class II-bound antigen to CD4(+) T cells. We used two-photon microscopy to image the single-cell dynamics of interactions between DCs and T cells within intact lymph nodes in the absence of relevant antigen. DCs were fluorescently labeled in vivo by cutaneous injection of alum adjuvant including carboxyfluorescein diacetate succinimidyl ester (CFSE). CFSE-positive DCs (CD11c(+), CD11b(+), and low-to-intermediate CD8(+)) were observed in draining lymph nodes 24-72 h later. Labeled DCs meandered slowly (2-3 microm x min(-1)) in the T cell zone near B cell follicles but vigorously extended long agile dendrites. Encounters between T cells and DCs arose as T cells moved autonomously along random paths. Moreover, T cells did not accumulate around DCs, and their relative velocities approaching and departing DCs were equivalent, implying that T cells are not attracted toward DCs by chemotactic gradients but rather encounter them by chance. T cell/DC contacts occurred primarily on dendrites at arm's length from the DC soma and typically lasted approximately 3 min, enabling an individual DC to interact with up to 5000 T cells per hour. We conclude that dynamic DC gesticulation and random T cell motility together enhance the stochastic scanning of the T cell repertoire, thereby enabling rapid initiation of the immune response.

Dysregulated IP3 Signaling in Cortical Neurons of Knock-in Mice Expressing an Alzheimer's-linked Mutation in Presenilin1 Results in Exaggerated Ca2+ Signals and Altered Membrane Excitability

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jan, 2004  |  Pubmed ID: 14724250

Disruptions in intracellular Ca2+ signaling are proposed to underlie the pathophysiology of Alzheimer's disease (AD), and it has recently been shown that AD-linked mutations in the presenilin 1 gene (PS1) enhance inositol triphosphate (IP3)-mediated Ca2+ liberation in nonexcitable cells. However, little is known of these actions in neurons, which are the principal locus of AD pathology. We therefore sought to determine how PS1 mutations affect Ca2+ signals and their subsequent downstream effector functions in cortical neurons. Using whole-cell patch-clamp recording, flash photolysis, and two-photon imaging in brain slices from 4-5-week-old mice, we show that IP3-evoked Ca2+ responses are more than threefold greater in PS1(M146V) knock-in mice relative to age-matched nontransgenic controls. Electrical excitability is thereby reduced via enhanced Ca2+ activation of K+ conductances. Action potential-evoked Ca2+ signals were unchanged, indicating that PS1(M146V) mutations specifically disrupt intracellular Ca2+ liberation rather than reduce cytosolic Ca2+ buffering or clearance. Moreover, IP3 receptor levels are not different in cortical homogenates, further suggesting that the exaggerated cytosolic Ca2+ signals may result from increased store filling and not from increased flux through additional IP3-gated channels. Even in young animals, PS1 mutations have profound effects on neuronal Ca2+ and electrical signaling: cumulatively, these disruptions may contribute to the long-term pathophysiology of AD.

Spatiotemporal Patterning of IP3-mediated Ca2+ Signals in Xenopus Oocytes by Ca2+-binding Proteins

The Journal of Physiology. Apr, 2004  |  Pubmed ID: 14755000

Ca(2+)-binding proteins (CaBPs) are expressed in a highly specific manner across many different cell types, yet the physiological basis underlying their selective distribution patterns remains unclear. We used confocal line-scan microscopy together with photo-release of IP(3) in Xenopus oocytes to investigate the actions of mobile cytosolic CaBPs on the spatiotemporal properties of IP(3)-evoked Ca(2+) signals. Parvalbumin (PV), a CaBP with slow Ca(2+)-binding kinetics, shortened the duration of IP(3)-evoked Ca(2+) signals and 'balkanized' global responses into discrete localized events (puffs). In contrast, calretinin (CR), a presumed fast buffer, prolonged Ca(2+) responses and promoted 'globalization' of spatially uniform Ca(2+) signals at high [IP(3)]. Oocytes loaded with CR or PV showed Ca(2+) puffs following photolysis flashes that were subthreshold in controls, and the spatiotemporal properties of these localized events were differentially modulated by PV and CR. In comparison to results we previously obtained with exogenous Ca(2+) buffers, PV closely mimicked the actions of the slow buffer EGTA, whereas CR showed important differences from the fast buffer BAPTA. Most notably, puffs were never observed after loading BAPTA, and this exogenous buffer did not show the marked sensitization of IP(3) action evident with CR. The ability of Ca(2+) buffers and CaBPs with differing kinetics to fine-tune both global and local intracellular Ca(2+) signals is likely to have significant physiological implications.

Early Experiences with Computed Axial Tomography Colonography

American Journal of Surgery. Apr, 2004  |  Pubmed ID: 15041501

Computed axial tomography (CT) colonography is the latest radiologic technique to be used to image the large bowel. We studied its role as a diagnostic tool in colorectal practice.

Zymogen Granule Exocytosis is Characterized by Long Fusion Pore Openings and Preservation of Vesicle Lipid Identity

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2004  |  Pubmed ID: 15090649

The dynamics of the fusion pore that forms between a secretory vesicle and the plasma membrane are important in the regulation of both exocytosis and endocytosis. Here, we describe characteristics of fusion during zymogen granule exocytosis in exocrine pancreatic acinar cells. By using fluorescence recovery after photobleaching techniques, we show that the fusion pore remains open to allow free aqueous exchange with the vesicle lumen. There is no lipid interchange between the plasma and granule membranes during this time, and at the end of its life, the intact granule shrinks in situ, probably by a gradual pinching off of membrane patches. We propose that the protracted fusion pore lifetime is adapted to permit compound exocytosis, whereby the lingering primary granule acts as a conduit through which the contents of a secondary granule can be released. The lack of lipid intermixing may then facilitate selective recycling of granule membrane and preservation of apical membrane integrity.

Imaging the Activity and Localization of Single Voltage-gated Ca(2+) Channels by Total Internal Reflection Fluorescence Microscopy

Biophysical Journal. May, 2004  |  Pubmed ID: 15111438

The patch-clamp technique has enabled functional studies of single ion channels, but suffers limitations including lack of spatial information and inability to independently monitor currents from more than one channel. Here, we describe the use of total internal reflection fluorescence microscopy as an alternative, noninvasive approach to optically monitor the activity and localization of multiple Ca(2+)-permeable channels in the plasma membrane. Images of near-membrane Ca(2+) signals were obtained from >100 N-type channels expressed within restricted areas (80 x 80 micro m) of Xenopus oocytes, thereby permitting simultaneous resolution of their gating kinetics, voltage dependence, and localization. Moreover, this technique provided information inaccessible by electrophysiological means, demonstrating that N-type channels are immobile in the membrane, show a patchy distribution, and display diverse gating kinetics even among closely adjacent channels. Total internal reflection fluorescence microscopy holds great promise for single-channel recording of diverse voltage- and ligand-gated Ca(2+)-permeable channels in the membrane of neurons and other isolated or cultured cells, and has potential for high-throughput functional analysis of single channels.

Imaging the Single Cell Dynamics of CD4+ T Cell Activation by Dendritic Cells in Lymph Nodes

The Journal of Experimental Medicine. Oct, 2004  |  Pubmed ID: 15466619

The adaptive immune response is initiated in secondary lymphoid organs by contact between antigen-bearing dendritic cells (DCs) and antigen-specific CD4+ T cells. However, there is scant information regarding the single cell dynamics of this process in vivo. Using two-photon microscopy, we imaged the real-time behavior of naive CD4+ T cells and in vivo-labeled DCs in lymph nodes during a robust T cell response. In the first 2 h after entry into lymph nodes, T cells made short-lived contacts with antigen-bearing DCs, each contact lasting an average of 11-12 min and occurring mainly on dendrites. Altered patterns of T cell motility during this early stage of antigen recognition promoted serial engagement with several adjacent DCs. Subsequently, T cell behavior progressed through additional distinct stages, including long-lived clusters, dynamic swarms, and finally autonomous migration punctuated by cell division. These observations suggest that the immunological synapse in native tissues is remarkably fluid, and that stable synapses form only at specific stages of antigen presentation to T cells. Furthermore, the serial nature of these interactions implies that T cells activate by way of multiple antigen recognition events.

Imaging Single-channel Calcium Microdomains by Total Internal Reflection Microscopy

Biological Research. 2004  |  Pubmed ID: 15709697

The microdomains of Ca2+ in the cytosol around the mouth of open Ca2+ channels are the basic 'building blocks' from which cellular Ca2+ signals are constructed. Moreover, the kinetics of local [Ca2+] closely reflect channel gating, so their measurement holds promise as an alternative to electrophysiological patch-clamp recording as a means to study single channel behavior. We have thus explored the development of optical techniques capable of imaging single-channel Ca2+ signals with good spatial and temporal resolution, and describe results obtained using total internal reflection fluorescence microscopy to monitor Ca2+ influx through single N-type channels expressed in Xenopus oocytes.

Calcium Dysregulation and Membrane Disruption As a Ubiquitous Neurotoxic Mechanism of Soluble Amyloid Oligomers

The Journal of Biological Chemistry. Apr, 2005  |  Pubmed ID: 15722360

Increasing evidence suggests that amyloid peptides associated with a variety of degenerative diseases induce neurotoxicity in their intermediate oligomeric state, rather than as monomers or fibrils. To test this hypothesis and investigate the possible involvement of Ca2+ signaling disruptions in amyloid-induced cytotoxicity, we made homogeneous preparations of disease-related amyloids (Abeta, prion, islet amyloid polypeptide, polyglutamine, and lysozyme) in various aggregation states and tested their actions on fluo-3-loaded SH-SY5Y cells. Application of oligomeric forms of all amyloids tested (0.6-6 microg ml-1) rapidly (approximately 5 s) elevated intracellular Ca2+, whereas equivalent amounts of monomers and fibrils did not. Ca2+ signals evoked by Abeta42 oligomers persisted after depletion of intracellular Ca2+ stores, and small signals remained in Ca2+-free medium, indicating contributions from both extracellular and intracellular Ca2+ sources. The increased membrane permeability to Ca2+ cannot be attributed to activation of endogenous Ca2+ channels, because responses were unaffected by the potent Ca2+-channel blocker cobalt (20 microm). Instead, observations that Abeta42 and other oligomers caused rapid cellular leakage of anionic fluorescent dyes point to a generalized increase in membrane permeability. The resulting unregulated flux of ions and molecules may provide a common mechanism for oligomer-mediated toxicity in many amyloidogenic diseases, with dysregulation of Ca2+ ions playing a crucial role because of their strong trans-membrane concentration gradient and involvement in cell dysfunction and death.

Optical Single-channel Recording by Imaging Ca2+ Flux Through Individual Ion Channels: Theoretical Considerations and Limits to Resolution

Cell Calcium. Apr, 2005  |  Pubmed ID: 15755490

Recent developments in microscopy and fluorescent indicators now make it possible to monitor the activity and localization of membrane ion channels by imaging Ca(2+) flux through individual channels. Such optical approaches have advantages over electrophysiological single-channel techniques in that they are less invasive, provide spatial information and can simultaneously and independently monitor hundreds of channels. However, their kinetic resolution does not yet approach that of patch-clamp recordings. To help understand the processes that determine the temporal resolution and noise level of single-channel Ca(2+) fluorescence signals (SCCaFTs), we simulated the microdomains of Ca(2+) ions and Ca(2+)-bound indicator dye that exist around the mouth of an open channel. Further, as an aid to development of improved optical techniques, we modeled the dependence of the amplitude and kinetics of SCCaFTs on parameters such as the imaging volume, the indicator concentration, affinity and mobility, and the presence of endogenous and exogenous Ca(2+) buffers. The results indicate that under optimal conditions, including the use of confocal or total-internal reflection microscopy to image from sub-femtolitre volumes, SCCaFTs should resolve channel openings as brief as 1ms with a signal-to-noise ratio >10.

Optical Single-channel Recording: Imaging Ca2+ Flux Through Individual Ion Channels with High Temporal and Spatial Resolution

Journal of Biomedical Optics. Jan-Feb, 2005  |  Pubmed ID: 15847568

Developments in imaging technology now enable visualization of the functioning of individual ion channels in living cells: something previously possible only by the electrophysiological patch-clamp technique. We review techniques that track channel gating via changes in intracellular [Ca2+] resulting from openings of Ca(2+)-permeable channels. Spatial and temporal resolution are optimized by monitoring Ca2+ close to the channel mouth, and we describe the use of two imaging modalities: confocal laser scan microscopy (linescan CLSM) and total internal reflection fluorescence microscopy (TIRFM). Both currently achieve a kinetic resolution of <10 ms, provide a simultaneous and independent readout from many channels, and enable their locations to be mapped with submicrometer resolution. TIRFM provides 2-D images from a very thin (approximately 100 nm) optical section, but it is restricted to channels in the plasma membrane of cells adhering close to a cover glass. In contrast, CLSM can image channels in intracellular membranes but, to achieve good temporal resolution, has been utilized only in a linescan mode with limited spatial information. We anticipate that imaging techniques will develop as a useful adjunct to patch-clamping for single-channel studies, with capabilities including simultaneous readout from multiple channels, high-resolution mapping of channel location, and mobility that is inaccessible by electrophysiological means. Optical single-channel recording is applicable to diverse voltage- and ligand-gated Ca(2+)-permeable channels and has potential for high-throughput functional analysis.

Antigen-engaged B Cells Undergo Chemotaxis Toward the T Zone and Form Motile Conjugates with Helper T Cells

PLoS Biology. Jun, 2005  |  Pubmed ID: 15857154

Interactions between B and T cells are essential for most antibody responses, but the dynamics of these interactions are poorly understood. By two-photon microscopy of intact lymph nodes, we show that upon exposure to antigen, B cells migrate with directional preference toward the B-zone-T-zone boundary in a CCR7-dependent manner, through a region that exhibits a CCR7-ligand gradient. Initially the B cells show reduced motility, but after 1 d, motility is increased to approximately 9 microm/min. Antigen-engaged B cells pair with antigen-specific helper T cells for 10 to more than 60 min, whereas non-antigen-specific interactions last less than 10 min. B cell-T cell conjugates are highly dynamic and migrate extensively, being led by B cells. B cells occasionally contact more than one T cell, whereas T cells are strictly monogamous in their interactions. These findings provide evidence of lymphocyte chemotaxis in vivo, and they begin to define the spatiotemporal cellular dynamics associated with T cell-dependent antibody responses.

In Situ Characterization of CD4+ T Cell Behavior in Mucosal and Systemic Lymphoid Tissues During the Induction of Oral Priming and Tolerance

The Journal of Experimental Medicine. Jun, 2005  |  Pubmed ID: 15928201

The behavior of antigen-specific CD4+ T lymphocytes during initial exposure to antigen probably influences their decision to become primed or tolerized, but this has not been examined directly in vivo. We have therefore tracked such cells in real time, in situ during the induction of oral priming versus oral tolerance. There were marked contrasts with respect to rate and type of movement and clustering between naive T cells and those exposed to antigen in immunogenic or tolerogenic forms. However, the major difference when comparing tolerized and primed T cells was that the latter formed larger and longer-lived clusters within mucosal and peripheral lymph nodes. This is the first comparison of the behavior of antigen-specific CD4+ T cells in situ in mucosal and systemic lymphoid tissues during the induction of priming versus tolerance in a physiologically relevant model in vivo.

"Optical Patch-clamping": Single-channel Recording by Imaging Ca2+ Flux Through Individual Muscle Acetylcholine Receptor Channels

The Journal of General Physiology. Sep, 2005  |  Pubmed ID: 16103278

We describe an optical technique using total internal reflection fluorescence (TIRF) microscopy to obtain simultaneous and independent recordings from numerous ion channels via imaging of single-channel Ca2+ flux. Muscle nicotinic acetylcholine (ACh) receptors made up of alphabetagammadelta subunits were expressed in Xenopus oocytes, and single channel Ca2+ fluorescence transients (SCCaFTs) were imaged using a fast (500 fps) electron-multiplied c.c.d. camera with fluo-4 as the indicator. Consistent with their arising through openings of individual nicotinic channels, SCCaFTs were seen only when a nicotinic agonist was present in the bathing solution, were blocked by curare, and increased in frequency as roughly the second power of [ACh]. Their fluorescence amplitudes varied linearly with membrane potential and extrapolated to zero at about +60 mV. The rise and fall times of fluorescence were as fast as 2 ms, providing a kinetic resolution adequate to characterize channel gating kinetics; which showed mean open times of 7.9 and 15.8 ms when activated, respectively, by ACh or suberyldicholine. Simultaneous records were obtained from >400 channels in the imaging field, and we devised a novel "channel chip" representation to depict the resultant large dataset as a single image. The positions of SCCaFTs remained fixed (<100 nm displacement) over tens of seconds, indicating that the nicotinic receptor/channels are anchored in the oocyte membrane; and the spatial distribution of channels appeared random without evidence of clustering. Our results extend single-channel TIRFM imaging to ligand-gated channels that display only partial permeability to Ca2+, and demonstrate an order-of-magnitude improvement in kinetic resolution. We believe that functional single-channel imaging opens a new approach to ion channel study, having particular advantages over patch-clamp recording in that it is massively parallel, and provides high-resolution spatial information that is inaccessible by electrophysiological techniques.

Close Encounters of the First and Second Kind: T-DC and T-B Interactions in the Lymph Node

Seminars in Immunology. Dec, 2005  |  Pubmed ID: 16263308

Cellular interactions in lymphoid organs initiate the immune response and determine its outcome. Using two-photon microscopy in the lymph node, several groups have begun to investigate the motility characteristics and interactions among T lymphocytes, B lymphocytes, and dendritic cells (DC) in lymphoid organs. In the first "close encounter", T cells of a particular antigen specificity interact with antigen-bearing dendritic cells and begin to activate. Activation of both CD4+ and CD8+ T cells evolves through several stages; from transient interactions to stable clusters and later to dissociation and proliferation of T cells (clonal expansion). The second "close encounter" requires that antigen-engaged B cells become accessible to T cells by directed migration to the edge of the follicle. T cells and B cells then pair up and waltz together for an extended period, while helper T cells provide signals for B cells to differentiate into plasma cells. In this topical review, we compare the activation choreography of CD4+ T cells interacting first with dendritic cells, and then with B cells, during initiation of the humoral immune response.

Sphingosine 1-phosphate Type 1 Receptor Agonism Inhibits Transendothelial Migration of Medullary T Cells to Lymphatic Sinuses

Nature Immunology. Dec, 2005  |  Pubmed ID: 16273098

Sphingosine 1-phosphate type 1 (S1P(1)) receptor agonists cause sequestration of lymphocytes in secondary lymphoid organs by a mechanism that is not well understood. One hypothesis proposes that agonists act as 'functional antagonists' by binding and internalizing S1P(1) receptors on lymphocytes; a second hypothesis proposes instead that S1P(1) agonists act on endothelial cells to prevent lymphocyte egress from lymph nodes. Here, two-photon imaging of living T cells in explanted lymph nodes after treatment with S1P(1) agonists or antagonists has provided insight into the mechanism by which S1P(1) agonists function. The selective S1P(1) agonist SEW2871 caused reversible slowing and 'log-jamming' of T cells between filled medullary cords and empty sinuses, whereas motility was unaltered in diffuse cortex. Removal or antagonist competition of SEW2871 permitted recovery of T cell motility in the parenchyma of the medulla and resumption of migration across the stromal endothelial barrier, leading to refilling of sinuses. Our results provide visualization of transendothelial migration of T cells into lymphatic sinuses and suggest that S1P(1) agonists act mainly on endothelial cell S1P(1) receptors to inhibit lymphocyte migration.

Two Phases of Zymogen Granule Lifetime in Mouse Pancreas: Ghost Granules Linger After Exocytosis of Contents

The Journal of Physiology. Mar, 2005  |  Pubmed ID: 15637100

Different cell types show widely divergent mechanisms and kinetics of exocytosis. We investigated these processes in pancreatic acinar cells by using video-rate 2-photon microscopy to image entry of extracellular dye into individual zymogen granules undergoing exocytosis. Fluorescence signals display two distinct phases; an initial peak that then decays over several seconds to a prolonged plateau. Several observations suggest that the first component reflects the binding of dye to the granule contents and their subsequent release into the acinar duct. These observations include: the peak/plateau fluorescence ratio differs between different dyes; the initial fluorescence decay mirrors the loss of granule contents as monitored by differential interference contrast microscopy; and the fall in vesicular fluorescence is accompanied by a rise in fluorescence in the adjacent duct lumen. We thus propose the use of extracellular fluorescent probes as a convenient means to monitor the kinetics of loss of proteinaceous content from secretory granules. In pancreatic acinar cells the fusion pore remains open much longer than required to ensure secretion of the granule contents, and instead the persistent empty 'ghost-granule' may act as a conduit to which secondary granules can fuse and release their contents by compound exocytosis.

Dynamic Multiphoton Imaging: a Live View from Cells to Systems

Physiology (Bethesda, Md.). Feb, 2005  |  Pubmed ID: 15653835

Leaps in scientific technology often occur at the interface of seemingly disparate disciplines. This holds true with the recent application of multiphoton microscopy to the biological sciences, leading to a new generation of imaging-based studies extending from the tracking of individual molecules within living cells to the observation of whole organisms.

A Model-independent Algorithm to Derive Ca2+ Fluxes Underlying Local Cytosolic Ca2+ Transients

Biophysical Journal. Apr, 2005  |  Pubmed ID: 15681645

Local intracellular Ca(2+) signals result from Ca(2+) flux into the cytosol through individual channels or clusters of channels. To gain a mechanistic understanding of these events we need to know the magnitude and spatial distribution of the underlying Ca(2+) flux. However, this is difficult to infer from fluorescence Ca(2+) images because the distribution of Ca(2+)-bound dye is affected by poorly characterized processes including diffusion of Ca(2+) ions, their binding to mobile and immobile buffers, and sequestration by Ca(2+) pumps. Several methods have previously been proposed to derive Ca(2+) flux from fluorescence images, but all require explicit knowledge or assumptions regarding these processes. We now present a novel algorithm that requires few assumptions and is largely model-independent. By testing the algorithm with both numerically generated image data and experimental images of sparklets resulting from Ca(2+) flux through individual voltage-gated channels, we show that it satisfactorily reconstructs the magnitude and time course of the underlying Ca(2+) currents.

Analysis of Puff Dynamics in Oocytes: Interdependence of Puff Amplitude and Interpuff Interval

Biophysical Journal. Jun, 2006  |  Pubmed ID: 16533853

Puffs are localized Ca(2+) signals that arise in oocytes in response to inositol 1,4,5-trisphosphate (IP(3)). They are analogous to the sparks of myocytes and are believed to be the result of the liberation of Ca(2+) from the endoplasmic reticulum through the coordinated opening of IP(3) receptor/channels clustered at a functional release site. In this article, we analyze sequences of puffs that occur at the same site to help elucidate the mechanisms underlying puff dynamics. In particular, we show a dependence of the interpuff time on the amplitude of the preceding puff, and of the amplitude of the following puff on the preceding interval. These relationships can be accounted for by an inhibitory role of the Ca(2+) that is liberated during puffs. We construct a stochastic model for a cluster of IP(3) receptor/channels that quantitatively replicates the observed behavior, and we determine that the characteristic time for a channel to escape from the inhibitory state is of the order of seconds.

Enhanced Ryanodine Receptor Recruitment Contributes to Ca2+ Disruptions in Young, Adult, and Aged Alzheimer's Disease Mice

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. May, 2006  |  Pubmed ID: 16687509

Neuronal Ca2+ signaling through inositol triphosphate receptors (IP3R) and ryanodine receptors (RyRs) must be tightly regulated to maintain cell viability, both acutely and over a lifetime. Exaggerated intracellular Ca2+ levels have been associated with expression of Alzheimer's disease (AD) mutations in young mice, but little is known of Ca2+ dysregulations during normal and pathological aging processes. Here, we used electrophysiological recordings with two-photon imaging to study Ca2+ signaling in nontransgenic (NonTg) and several AD mouse models (PS1KI, 3xTg-AD, and APPSweTauP301L) at young (6 week), adult (6 months), and old (18 months) ages. At all ages, the PS1KI and 3xTg-AD mice displayed exaggerated endoplasmic reticulum (ER) Ca2+ signals relative to NonTg mice. The PS1 mutation was the predominant "calciopathic" factor, because responses in 3xTg-AD mice were similar to PS1KI mice, and APPSweTauP301L mice were not different from controls. In addition, we uncovered powerful signaling interactions and differences between IP3R- and RyR-mediated Ca2+ components in NonTg and AD mice. In NonTg mice, RyR contributed modestly to IP3-evoked Ca2+, whereas the exaggerated signals in 3xTg-AD and PS1KI mice resulted primarily from enhanced RyR-Ca2+ release and were associated with increased RyR expression across all ages. Moreover, IP3-evoked membrane hyperpolarizations in AD mice were even greater than expected from exaggerated Ca2+ signals, suggesting increased coupling efficiency between cytosolic [Ca2+] and K+ channel regulation. We conclude that lifelong ER Ca2+ disruptions in AD are related to a modulation of RyR signaling associated with PS1 mutations and represent a discrete "calciumopathy," not merely an acceleration of normal aging.

Plasmalemmal Ca2+ Signaling in Arterial Smooth Muscle: It's Elementary!

The Journal of General Physiology. Jun, 2006  |  Pubmed ID: 16702356

Imaging Ca2+ Signals in Xenopus Oocytes

Methods in Molecular Biology (Clifton, N.J.). 2006  |  Pubmed ID: 16739719

Xenopus oocytes have become a favored preparation in which to study the spatiotemporal dynamics of intracellular Ca2+ signaling. Advantages of the oocyte as a model cell system include its large size, lack of intracellular Ca2+ release channels other than the type 1 inositol trisphosphate receptor, and ease of expression of foreign receptors and channels. We describe the use of high-resolution fluorescence imaging techniques to visualize Ca2+ signals in Xenopus oocytes at levels ranging from global Ca2+ waves to single-channel Ca2+ microdomains.

Imaging the Choreography of Lymphocyte Trafficking and the Immune Response

Current Opinion in Immunology. Aug, 2006  |  Pubmed ID: 16765574

The functioning of the immune system depends upon exquisitely choreographed interactions between its cellular constituents. Two-photon microscopy now enables us to visualize cell motility and cell-cell interactions deep within intact tissues and organs, both in explanted preparations and in vivo. Real-time immunoimaging techniques have illuminated the roles of random and chemokine-driven motility for cellular search strategies, the complex dynamics of cellular interactions, and the micro-anatomical localization and control of lymphocyte trafficking. Recently, advances have been made in these areas of research, as exemplified by studies investigating T cell-dendritic cell interactions, T cell-B cell interactions, and the regulation of lymphocyte egress from the lymph node.

Enhancement of Capillary Leakage and Restoration of Lymphocyte Egress by a Chiral S1P1 Antagonist in Vivo

Nature Chemical Biology. Aug, 2006  |  Pubmed ID: 16829954

Sphingosine 1-phosphate (S1P, 1) regulates vascular barrier and lymphoid development, as well as lymphocyte egress from lymphoid organs, by activating high-affinity S1P1 receptors. We used reversible chemical probes (i) to gain mechanistic insights into S1P systems organization not accessible through genetic manipulations and (ii) to investigate their potential for therapeutic modulation. Vascular (but not airway) administration of the preferred R enantiomer of an in vivo-active chiral S1P1 receptor antagonist induced loss of capillary integrity in mouse skin and lung. In contrast, the antagonist did not affect the number of constitutive blood lymphocytes. Instead, alteration of lymphocyte trafficking and phenotype required supraphysiological elevation of S1P1 tone and was reversed by the antagonist. In vivo two-photon imaging of lymph nodes confirmed requirements for obligate agonism, and the data were consistent with the presence of a stromal barrier mechanism for gating lymphocyte egress. Thus, chemical modulation reveals differences in S1P-S1P1 'set points' among tissues and highlights both mechanistic advantages (lymphocyte sequestration) and risks (pulmonary edema) of therapeutic intervention.

'Trigger' Events Precede Calcium Puffs in Xenopus Oocytes

Biophysical Journal. Dec, 2006  |  Pubmed ID: 16980363

The liberation of calcium ions sequestered in the endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors/channels (IP(3)Rs) results in a spatiotemporal hierarchy of calcium signaling events that range from single-channel openings to local Ca(2+) puffs believed to arise from several to tens of clustered IP(3)Rs to global calcium waves. Using high-resolution confocal linescan imaging and a sensitive Ca(2+) indicator dye (fluo-4-dextran), we show that puffs are often preceded by small, transient Ca(2+) elevations that we christen "trigger events". The magnitude of triggers is consistent with their arising from the opening of a single IP(3) receptor/channel, and we propose that they initiate puffs by recruiting neighboring IP(3)Rs within the cluster by a regenerative process of Ca(2+)-induced Ca(2+) release. Puff amplitudes (fluorescence ratio change) are on average approximately 6 times greater than that of the triggers, suggesting that at least six IP(3)Rs may simultaneously be open during a puff. Trigger events have average durations of approximately 12 ms, as compared to 19 ms for the mean rise time of puffs, and their spatial extent is approximately 3 times smaller than puffs (respective widths at half peak amplitude 0.6 and 1.6 micro m). All these parameters were relatively independent of IP(3) concentration, although the proportion of puffs showing resolved triggers was greatest (approximately 80%) at low [IP(3)]. Because Ca(2+) puffs constitute the building blocks from which cellular IP(3)-mediated Ca(2+) signals are constructed, the events that initiate them are likely to be of fundamental importance for cell signaling. Moreover, the trigger events provide a useful yardstick by which to derive information regarding the number and spatial arrangement of IP(3)Rs within clusters.

The Number and Spatial Distribution of IP3 Receptors Underlying Calcium Puffs in Xenopus Oocytes

Biophysical Journal. Dec, 2006  |  Pubmed ID: 16980372

Calcium puffs are local Ca(2+) release events that arise from a cluster of inositol 1,4,5-trisphosphate receptor channels (IP(3)Rs) and serve as a basic "building block" from which global Ca(2+) waves are generated. Important questions remain as to the number of IP(3)Rs that open during a puff, their spatial distribution within a cluster, and how much Ca(2+) current flows through each channel. The recent discovery of "trigger" events-small Ca(2+) signals that immediately precede puffs and are interpreted to arise through opening of single IP(3)R channels-now provides a useful yardstick by which to calibrate the Ca(2+) flux underlying puffs. Here, we describe a deterministic numerical model to simulate puffs and trigger events. Based on confocal linescan imaging in Xenopus oocytes, we simulated Ca(2+) release in two sequential stages; representing the trigger by the opening of a single IP(3)R in the center of a cluster for 12 ms, followed by the concerted opening of some number of IP(3)Rs for 19 ms, representing the rising phase of the puff. The diffusion of Ca(2+) and Ca(2+)-bound indicator dye were modeled in a three-dimensional cytosolic volume in the presence of immobile and mobile Ca(2+) buffers, and were used to predict the observed fluorescence signal after blurring by the microscope point-spread function. Optimal correspondence with experimental measurements of puff spatial width and puff/trigger amplitude ratio was obtained assuming that puffs arise from the synchronous opening of 25-35 IP(3)Rs, each carrying a Ca(2+) current of approximately 0.4 pA, with the channels distributed through a cluster 300-800 nm in diameter.

Imaging Single-channel Calcium Microdomains

Cell Calcium. Nov-Dec, 2006  |  Pubmed ID: 17067668

The Ca(2+) microdomains generated around the mouth of open ion channels represent the basic building blocks from which cytosolic Ca(2+) signals are constructed. Recent improvements in optical imaging techniques now allow these microdomains to be visualized as single channel calcium fluorescence transients (SCCaFTs), providing information about channel properties that was previously accessible only by electrophysiological patch-clamp recordings. We review recent advances in single channel Ca(2+) imaging methodologies, with emphasis on total internal reflection fluorescence microscopy (TIRFM) as the technique of choice for recording SCCaFTs from voltage- and ligand-gated plasmalemmal ion channels. This technique of 'optical patch-clamp recording' is massively parallel, permitting simultaneous imaging of hundreds of channels; provides millisecond resolution of gating kinetics together with sub-micron spatial resolution of channel locations; and is applicable to diverse families of membrane channels that display partial permeability to Ca(2+) ions.

Psychoanalytic Cyberspace, Beyond Psychology

Psychoanalytic Review. Feb, 2007  |  Pubmed ID: 17367277

Loss of MMP-2 Disrupts Skeletal and Craniofacial Development and Results in Decreased Bone Mineralization, Joint Erosion and Defects in Osteoblast and Osteoclast Growth

Human Molecular Genetics. May, 2007  |  Pubmed ID: 17400654

The 'vanishing bone' or inherited osteolysis/arthritis syndromes represent a heterogeneous group of skeletal disorders characterized by mineralization defects of affected bones and joints. Differing in anatomical distribution, severity and associated syndromic features, gene identification in each 'vanishing bone' disorder should provide unique insights into genetic/molecular pathways contributing to the overall control of skeletal growth and development. We previously described and then demonstrated that the novel autosomal recessive osteolysis/arthritis syndrome, multicentric osteolysis with arthritis (MOA) (MIM #605156), was caused by inactivating mutations in the MMP2 gene [Al Aqeel, A., Al Sewairi, W., Edress, B., Gorlin, R.J., Desnick, R.J. and Martignetti, J.A. (2000) Inherited multicentric osteolysis with arthritis: A variant resembling Torg syndrome in a Saudi family. Am. J. Med. Genet., 93, 11-18.]. These in vivo results were counterintuitive and unexpected since previous in vitro studies suggested that MMP-2 overexpression and increased activity, not deficiency, would result in the bone and joint features of MOA. The apparent lack of a murine model [Itoh, T., Ikeda, T., Gomi, H., Nakao, S., Suzuki, T. and Itohara, S. (1997) Unaltered secretion of beta-amyloid precursor protein in gelatinase A (matrix metalloproteinase 2)-deficient mice. J. Biol. Chem., 272, 22389-22392.] has hindered studies on disease pathogenesis and, more fundamentally, in addressing the paradox of how functional loss of a single proteolytic enzyme results in an apparent increase in bone loss. Here, we report that Mmp2-/- mice display attenuated features of human MOA including progressive loss of bone mineral density, articular cartilage destruction and abnormal long bone and craniofacial development. Moreover, these changes are associated with markedly and developmentally restricted decreases in osteoblast and osteoclast numbers in vivo. Mmp2-/- mice have approximately 50% fewer osteoblasts and osteoclasts than control littermates at 4 days of life but these differences have nearly resolved by 4 weeks of age. In addition, despite normal cell numbers in vivo at 8 weeks of life, Mmp2-/- bone marrow cells are unable to effectively support osteoblast and osteoclast growth and differentiation in culture. Targeted inhibition of MMP-2 using siRNA in human SaOS2 and murine MC3T3 osteoblast cell lines resulted in decreased cell proliferation rates. Taken together, our findings suggest that MMP-2 plays a direct role in early skeletal development and bone cell growth and proliferation. Thus, Mmp2-/- mice provide a valuable biological resource for studying the pathophysiological mechanisms underlying the human disease and defining the in vivo physiological role of MMP-2.

Enhanced Ryanodine-mediated Calcium Release in Mutant PS1-expressing Alzheimer's Mouse Models

Annals of the New York Academy of Sciences. Feb, 2007  |  Pubmed ID: 17413028

Intracellular Ca(2+) signaling involves Ca(2+) liberation through both inositol triphosphate and ryanodine receptors (IP(3)R and RyR). However, little is known of the functional interactions between these Ca(2+) sources in either neuronal physiology, or during Ca(2+) disruptions associated with Alzheimer's disease (AD). By the use of whole-cell recordings and 2-photon Ca(2+) imaging in cortical slices we distinguished between IP(3)R- and RyR-mediated Ca(2+) components in nontransgenic (non-Tg) and AD mouse models and demonstrate powerful signaling interactions between these channels. Ca(2+)-induced Ca(2+) release (CICR) through RyR contributed modestly to Ca(2+) signals evoked by photoreleased IP(3) in cortical neurons from non-Tg mice. In contrast, the exaggerated signals in 3xTg-AD and PS1(KI) mice resulted primarily from enhanced CICR through RyR, rather than through IP(3)R, and were associated with increased RyR expression levels. Moreover, membrane hyperpolarizations evoked by IP(3) in neurons from AD mouse models were even greater than expected simply from the exaggerated Ca(2+) signals, pointing to an increased coupling efficiency between cytosolic [Ca(2+)] and K(+) channel regulation. Our results highlight the critical roles of RyR-mediated Ca(2+) signaling in both neuronal physiology and pathophysiology, and point to presenilin-linked disruptions in RyR signaling as an important genetic factor in AD.

A Kinetic Model of Single and Clustered IP3 Receptors in the Absence of Ca2+ Feedback

Biophysical Journal. Aug, 2007  |  Pubmed ID: 17526578

Ca2+ liberation through inositol 1,4,5-trisphosphate receptor (IP3R) channels generates complex patterns of spatiotemporal cellular Ca2+ signals owing to the biphasic modulation of channel gating by Ca2+ itself. These processes have been extensively studied in Xenopus oocytes, where imaging studies have revealed local Ca2+ signals ("puffs") arising from clusters of IP3R, and patch-clamp studies on isolated oocyte nuclei have yielded extensive data on IP3R gating kinetics. To bridge these two levels of experimental data, we developed an IP3R model and applied stochastic simulation and transition matrix theory to predict the behavior of individual and clustered IP3R channels. The channel model consists of four identical, independent subunits, each of which has an IP3-binding site together with one activating and one inactivating Ca2+-binding site. The channel opens when at least three subunits undergo a conformational change to an "active" state after binding IP3 and Ca2+. The model successfully reproduces patch-clamp data; including the dependence of open probability, mean open duration, and mean closed duration on [IP3] and [Ca2+]. Notably, the biexponential distribution of open-time duration and the dependence of mean open time on [Ca2+] are explained by populations of openings involving either three or four active subunits. As a first step toward applying the single IP3R model to describe cellular responses, we then simulated measurements of puff latency after step increases of [IP3]. Assuming that stochastic opening of a single IP3R at basal cytosolic [Ca2+] and any given [IP3] has a high probability of rapidly triggering neighboring channels by calcium-induced calcium release to evoke a puff, optimal correspondence with experimental data of puff latencies after photorelease of IP3 was obtained when the cluster contained a total of 40-70 IP3Rs.

Natural Killer Cells Actively Patrol Peripheral Lymph Nodes Forming Stable Conjugates to Eliminate MHC-mismatched Targets

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2007  |  Pubmed ID: 17609379

Natural killer (NK) cells are known to reject MHC-mismatched targets within blood organs, yet their role in peripheral lymphoid tissue remains unresolved. Here we address the capacity of NK cells to migrate within lymph nodes (LN) using two-photon microscopy to characterize cell velocities and interaction dynamics within the native lymphoid-tissue environment. Adoptively transferred unmanipulated NK cells were highly motile (6-7 microm/min) and capable of forming transient contacts with both syngeneic and allogeneic B cells. Stable conjugate interactions (lasting >5 min) formed preferentially with allogeneic cells, resulting in diminished motility and subsequent elimination of the target cell. In marked contrast to unmanipulated cells, NK cells purified by CD49b-positive selection exhibited only limited motility (2-3 microm/min). This velocity impairment arose largely because CD49b cross-linking enhanced NK cell adhesion to collagen fibers within the node. Moreover, CD49b cross-linking prevented NK cells from reconstituting effector cytolytic function in vivo, inhibited target cell lysis in vitro, and augmented IFN-gamma responses to IL-2 activation in vitro. Taken together our data demonstrate that NK cells are a functionally important component of the LN microenvironment, and that cell motility and effector function are strongly modulated via CD49b manipulation.

Ca2+ Signals in CD4+ T Cells During Early Contacts with Antigen-bearing Dendritic Cells in Lymph Node

Journal of Immunology (Baltimore, Md. : 1950). Aug, 2007  |  Pubmed ID: 17641025

T cell activation by APC requires cytosolic Ca(2+) ([Ca(2+)](i)) elevation. Using two-photon microscopy, we visualized Ca(2+) signaling and motility of murine CD4(+) T cells within lymph node (LN) explants under control, inflammatory, and immunizing conditions. Without Ag under basal noninflammatory conditions, T cells showed infrequent Ca(2+) spikes associated with sustained slowing. Inflammation reduced velocities and Ca(2+) spiking in the absence of specific Ag. During early Ag encounter, most T cells engaged Ag-presenting dendritic cells in clusters, and showed increased Ca(2+) spike frequency and elevated basal [Ca(2+)](i). These Ca(2+) signals persisted for hours, irrespective of whether T cells were in contact with visualized dendritic cells. We propose that sustained increases in basal [Ca(2+)](i) and spiking frequency constitute a Ca(2+) signaling modality that, integrated over hours, distinguishes immunogenic from basal state in the native lymphoid environment.

Swingers: Bonobos Are Celebrated As Peace-loving, Matriarchal, and Sexually Liberated. Are They?

New Yorker (New York, N.Y. : 1925). Jul, 2007  |  Pubmed ID: 17650578

Class IA Phosphoinositide 3-kinase Modulates Basal Lymphocyte Motility in the Lymph Node

Journal of Immunology (Baltimore, Md. : 1950). Aug, 2007  |  Pubmed ID: 17675487

Recruitment of PI3K to the cell membrane is an indispensable step in normal lymphocyte proliferation and activation. In this study we identify PI3K as an important signaling molecule for maintaining basal T and B lymphocyte motility and homing in the intact lymph node. Pharmacological inhibition of PI3K catalytic isoforms exerted broad effects on basal lymphocyte motility, including changes in homing kinetics, localization of B cells within the lymph node, and reduced cell velocities. Lymphocytes deficient in either or both of the class IA PI3K regulatory subunits p85alpha and p85beta also exhibited reduced velocities, with the magnitude of reduction depending upon both cell type and isoform specificity. B cells deficient in p85alpha exhibited gross morphological abnormalities that were not evident in cells treated with a PI3K inhibitor. Our results show, for the first time, that class IA PI3Ks play an important role in regulating basal lymphocyte motility and that p85alpha regulatory subunit expression is required to maintain B cell morphology in a manner independent of PI3K catalytic function. Moreover, we demonstrate distinct roles for catalytic domain function and class IA PI3K regulatory domain activity in lymphocyte motility, homing, and homeostatic localization of mature resting B cells.

Mode Switching is the Major Mechanism of Ligand Regulation of InsP3 Receptor Calcium Release Channels

The Journal of General Physiology. Dec, 2007  |  Pubmed ID: 17998395

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) plays a critical role in generation of complex Ca(2+) signals in many cell types. In patch clamp recordings of isolated nuclei from insect Sf9 cells, InsP(3)R channels were consistently detected with regulation by cytoplasmic InsP(3) and free Ca(2+) concentrations ([Ca(2+)](i)) very similar to that observed for vertebrate InsP(3)R. Long channel activity durations of the Sf9-InsP(3)R have now enabled identification of a novel aspect of InsP(3)R gating: modal gating. Using a novel algorithm to analyze channel modal gating kinetics, InsP(3)R gating can be separated into three distinct modes: a low activity mode, a fast kinetic mode, and a burst mode with channel open probability (P(o)) within each mode of 0.007 +/- 0.002, 0.24 +/- 0.03, and 0.85 +/- 0.02, respectively. Channels reside in each mode for long periods (tens of opening and closing events), and transitions between modes can be discerned with high resolution (within two channel opening and closing events). Remarkably, regulation of channel gating by [Ca(2+)](i) and [InsP(3)] does not substantially alter channel P(o) within a mode. Instead, [Ca(2+)](i) and [InsP(3)] affect overall channel P(o) primarily by changing the relative probability of the channel being in each mode, especially the high and low P(o) modes. This novel observation therefore reveals modal switching as the major mechanism of physiological regulation of InsP(3)R channel activity, with implications for the kinetics of Ca(2+) release events in cells.

Choreography of Cell Motility and Interaction Dynamics Imaged by Two-photon Microscopy in Lymphoid Organs

Annual Review of Immunology. 2008  |  Pubmed ID: 18173372

The immune system is the most diffuse cellular system in the body. Accordingly, long-range migration of cells and short-range communication by local chemical signaling and by cell-cell contacts are vital to the control of an immune response. Cellular homing and migration within lymphoid organs, antigen recognition, and cell signaling and activation are clearly vital during an immune response, but these events had not been directly observed in vivo until recently. Introduced to the field of immunology in 2002, two-photon microscopy is the method of choice for visualizing living cells deep within native tissue environments, and it is now revealing an elegant cellular choreography that underlies the adaptive immune response to antigen challenge. We review cellular dynamics and molecular factors that contribute to basal motility of lymphocytes in the lymph node and cellular interactions leading to antigen capture and recognition, T cell activation, B cell activation, cytolytic effector function, and antibody production.

SERCA Pump Activity is Physiologically Regulated by Presenilin and Regulates Amyloid Beta Production

The Journal of Cell Biology. Jun, 2008  |  Pubmed ID: 18591429

In addition to disrupting the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins also alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca(2+) is maintained at low levels by pumping Ca(2+) into stores in the endoplasmic reticulum (ER) via the sarco ER Ca(2+)-ATPase (SERCA) pumps. We show that SERCA activity is diminished in fibroblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steady-state levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca(2+), whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca(2+) clearance and exaggerating inositol 1,4,5-trisphosphate-mediated Ca(2+) liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimer's disease is underscored by our findings that modulating SERCA activity alters amyloid beta production. Our results point to a physiological role for the presenilins in Ca(2+) signaling via regulation of the SERCA pump.

Modeling Ca2+ Feedback on a Single Inositol 1,4,5-trisphosphate Receptor and Its Modulation by Ca2+ Buffers

Biophysical Journal. Oct, 2008  |  Pubmed ID: 18641077

The inositol 1,4,5-trisphosphate receptor/channel (IP(3)R) is a major regulator of intracellular Ca(2+) signaling, and liberates Ca(2+) ions from the endoplasmic reticulum in response to binding at cytosolic sites for both IP(3) and Ca(2+). Although the steady-state gating properties of the IP(3)R have been extensively studied and modeled under conditions of fixed [IP(3)] and [Ca(2+)], little is known about how Ca(2+) flux through a channel may modulate the gating of that same channel by feedback onto activating and inhibitory Ca(2+) binding sites. We thus simulated the dynamics of Ca(2+) self-feedback on monomeric and tetrameric IP(3)R models. A major conclusion is that self-activation depends crucially on stationary cytosolic Ca(2+) buffers that slow the collapse of the local [Ca(2+)] microdomain after closure. This promotes burst-like reopenings by the rebinding of Ca(2+) to the activating site; whereas inhibitory actions are substantially independent of stationary buffers but are strongly dependent on the location of the inhibitory Ca(2+) binding site on the IP(3)R in relation to the channel pore.

SERCA Pump Activity is Physiologically Regulated by Presenilin and Regulates Amyloid Beta Production

The Journal of General Physiology. Aug, 2008  |  Pubmed ID: 18663130

The CRAC Channel Consists of a Tetramer Formed by Stim-induced Dimerization of Orai Dimers

Nature. Nov, 2008  |  Pubmed ID: 18820677

Ca(2+)-release-activated Ca(2+) (CRAC) channels underlie sustained Ca(2+) signalling in lymphocytes and numerous other cells after Ca(2+) liberation from the endoplasmic reticulum (ER). RNA interference screening approaches identified two proteins, Stim and Orai, that together form the molecular basis for CRAC channel activity. Stim senses depletion of the ER Ca(2+) store and physically relays this information by translocating from the ER to junctions adjacent to the plasma membrane, and Orai embodies the pore of the plasma membrane calcium channel. A close interaction between Stim and Orai, identified by co-immunoprecipitation and by Förster resonance energy transfer, is involved in the opening of the Ca(2+) channel formed by Orai subunits. Most ion channels are multimers of pore-forming subunits surrounding a central channel, which are preassembled in the ER and transported in their final stoichiometry to the plasma membrane. Here we show, by biochemical analysis after cross-linking in cell lysates and intact cells and by using non-denaturing gel electrophoresis without cross-linking, that Orai is predominantly a dimer in the plasma membrane under resting conditions. Moreover, single-molecule imaging of green fluorescent protein (GFP)-tagged Orai expressed in Xenopus oocytes showed predominantly two-step photobleaching, again consistent with a dimeric basal state. In contrast, co-expression of GFP-tagged Orai with the carboxy terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca(2+) store depletion or clustering of Orai into punctae yielded mostly four-step photobleaching, consistent with a tetrameric stoichiometry of the active Orai channel. Interaction with the C terminus of Stim thus induces Orai dimers to dimerize, forming tetramers that constitute the Ca(2+)-selective pore. This represents a new mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule.

Quantum Dots for Tracking Dendritic Cells and Priming an Immune Response in Vitro and in Vivo

PloS One. 2008  |  Pubmed ID: 18820727

Dendritic cells (DCs) play a key role in initiating adaptive immune response by presenting antigen to T cells in lymphoid organs. Here, we investigate the potential of quantum dots (QDs) as fluorescent nanoparticles for in vitro and in vivo imaging of DCs, and as a particle-based antigen-delivery system to enhance DC-mediated immune responses. We used confocal, two-photon, and electron microscopies to visualize QD uptake into DCs and compared CD69 expression, T cell proliferation, and IFN-gamma production by DO11.10 and OT-II T cells in vivo in response to free antigen or antigen-conjugated to QDs. CD11c(+) DCs avidly and preferentially endocytosed QDs, initially into small vesicles near the plasma membrane by an actin-dependent mechanism. Within 10 min DCs contained vesicles of varying size, motion, and brightness distributed throughout the cytoplasm. At later times, endocytosed QDs were compartmentalized inside lysosomes. LPS-induced maturation of DCs reduced the rate of endocytosis and the proportion of cells taking up QDs. Following subcutaneous injection of QDs in an adjuvant depot, DCs that had endocytosed QDs were visualized up to 400 microm deep within draining lymph nodes. When antigen-conjugated QDs were used, T cells formed stable clusters in contact with DCs. Antigen-conjugated QDs induced CD69 expression, T cell proliferation, and IFN-gamma production in vivo with greater efficiency than equivalent amounts of free antigen. These results establish QDs as a versatile platform for immunoimaging of dendritic cells and as an efficient nanoparticle-based antigen delivery system for priming an immune response.

Imaging of Effector Memory T Cells During a Delayed-type Hypersensitivity Reaction and Suppression by Kv1.3 Channel Block

Immunity. Oct, 2008  |  Pubmed ID: 18835197

Effector memory T (Tem) cells are essential mediators of autoimmune disease and delayed-type hypersensitivity (DTH), a convenient model for two-photon imaging of Tem cell participation in an inflammatory response. Shortly (3 hr) after entry into antigen-primed ear tissue, Tem cells stably attached to antigen-bearing antigen-presenting cells (APCs). After 24 hr, enlarged Tem cells were highly motile along collagen fibers and continued to migrate rapidly for 18 hr. Tem cells rely on voltage-gated Kv1.3 potassium channels to regulate calcium signaling. ShK-186, a specific Kv1.3 blocker, inhibited DTH and suppressed Tem cell enlargement and motility in inflamed tissue but had no effect on homing to or motility in lymph nodes of naive and central memory T (Tcm) cells. ShK-186 effectively treated disease in a rat model of multiple sclerosis. These results demonstrate a requirement for Kv1.3 channels in Tem cells during an inflammatory immune response in peripheral tissues. Targeting Kv1.3 allows for effector memory responses to be suppressed while central memory responses remain intact.

Multi-dimensional Resolution of Elementary Ca2+ Signals by Simultaneous Multi-focal Imaging

Cell Calcium. Apr, 2008  |  Pubmed ID: 17716727

Elementary events such as puffs and sparks are cytosolic microdomains of Ca2+ from which cellular Ca2+ signals are constructed. Because of the tight localization and fast kinetics of elementary events, imaging studies have been hindered by instrumental limitations of confocal and deconvolution fluorescence microscopy which necessitate compromises between spatial and temporal resolution. Here, we describe a novel, yet simple 'multi-focal' fluorescence microscopy system that employs three high-speed cameras focused at different axial depths to enable 4-dimensional imaging with millisecond resolution. We demonstrate the utility of this system for studies of puffs in Xenopus oocytes by mapping the axial distribution of puff sites, by obtaining measurements of puff amplitudes undistorted by focus error, and by deriving deblurred images that reveal novel sub-micron jumps of Ca2+ release sites.

CADPR Stimulates SERCA Activity in Xenopus Oocytes

Cell Calcium. Mar, 2009  |  Pubmed ID: 19131109

The intracellular second messenger cyclic ADP-ribose (cADPR) induces Ca(2+) release through the activation of ryanodine receptors (RyRs). Moreover, it has been suggested that cADPR may serve an additional role to modulate sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump activity, but studies have been complicated by concurrent actions on RyR. Here, we explore the actions of cADPR in Xenopus oocytes, which lack RyRs. We examined the effects of cADPR on the sequestration of cytosolic Ca(2+) following Ca(2+) transients evoked by photoreleased inositol 1,4,5-trisphosphate (InsP(3)), and by Ca(2+) influx through expressed nicotinic acetylcholine receptors (nAChR) in the oocytes membrane. In both cases the decay of the Ca(2+) transients was accelerated by intracellular injection of a non-metabolizable analogue of cADPR, 3-Deaza-cADPR, and photorelease of cADPR from a caged precursor demonstrated that this action is rapid (a few s). The acceleration was abolished by pre-treatment with thapsigargin to block SERCA activity, and was inhibited by two specific antagonists of cADPR, 8-NH(2)-cADPR and 8-br-cADPR. We conclude that cADPR serves to modulate Ca(2+) sequestration by enhancing SERCA pump activity, in addition to its well-established action on RyRs to liberate Ca(2+).

Imaging the Quantal Substructure of Single IP3R Channel Activity During Ca2+ Puffs in Intact Mammalian Cells

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2009  |  Pubmed ID: 19332787

The spatiotemporal patterning of Ca(2+) signals regulates numerous cellular functions, and is determined by the functional properties and spatial clustering of inositol trisphosphate receptor (IP(3)R) Ca(2+) release channels in the endoplasmic reticulum membrane. However, studies at the single-channel level have been hampered because IP(3)Rs are inaccessible to patch-clamp recording in intact cells, and because excised organelle and bilayer reconstitution systems disrupt the Ca(2+)-induced Ca(2+) release (CICR) process that mediates channel-channel coordination. We introduce here the use of total internal reflection fluorescence microscopy to image single-channel Ca(2+) flux through individual and clustered IP(3)Rs in intact mammalian cells. This enables a quantal dissection of the local calcium puffs that constitute building blocks of cellular Ca(2+) signals, revealing stochastic recruitment of, on average, approximately 6 active IP(3)Rs clustered within <500 nm. Channel openings are rapidly ( approximately 10 ms) recruited by opening of an initial trigger channel, and a similarly rapid inhibitory process terminates puffs despite local [Ca(2+)] elevation that would otherwise sustain Ca(2+)-induced Ca(2+) release indefinitely. Minimally invasive, nano-scale Ca(2+) imaging provides a powerful tool for the functional study of intracellular Ca(2+) release channels while maintaining the native architecture and dynamic interactions essential for discrete and selective cell signaling.

Chapter 16. Two-photon Microscopy and Multidimensional Analysis of Cell Dynamics

Methods in Enzymology. 2009  |  Pubmed ID: 19480927

Two-photon (2P) microscopy is a high-resolution imaging technique that was initially applied by neurobiologists and developmental cell biologists but has subsequently been broadly adapted by immunologists. The value of 2P microscopy is that it affords an unparalleled view of single-cell spatiotemporal dynamics deep within intact tissues and organs. As the technology develops and new transgenic mice and fluorescent probes become available, 2P microscopy will serve as an increasingly valuable tool for assessing cell function and probing molecular mechanisms. Here we discuss the technical aspects related to 2P microscope design, explain in detail various tissue imaging preparations, and walk the reader through the often daunting process of analyzing multidimensional data sets and presenting the experimental results.

Localization of Puff Sites Adjacent to the Plasma Membrane: Functional and Spatial Characterization of Ca2+ Signaling in SH-SY5Y Cells Utilizing Membrane-permeant Caged IP3

Cell Calcium. Jan, 2009  |  Pubmed ID: 18639334

The Xenopus oocyte has been a favored model system in which to study spatio-temporal mechanisms of intracellular Ca2+ dynamics, in large part because this giant cell facilitates intracellular injections of Ca2+ indicator dyes, buffers and caged compounds. However, the recent commercial availability of membrane-permeant ester forms of caged IP3 (ci-IP3) and EGTA, now allows for facile loading of these compounds into smaller mammalian cells, permitting control of [IP3]i and cytosolic Ca2+ buffering. Here, we establish the human neuroblastoma SH-SY5Y cell line as an advantageous experimental system for imaging Ca2+ signaling, and characterize IP3-mediated Ca2+ signaling mechanisms in these cells. Flash photo-release of increasing amounts of i-IP3 evokes Ca2+ puffs that transition to waves, but intracellular loading of EGTA decouples release sites, allowing discrete puffs to be studied over a wide range of [IP3]. Puff activity persists for minutes following a single photo-release, pointing to a slow rate of i-IP3 turnover in these cells and suggesting that repetitive Ca2+ spikes with periods of 20-30s are not driven by oscillations in [IP3]. Puff amplitudes are independent of [IP3], whereas their frequencies increase with increasing photo-release. Puff sites in SH-SY5Y cells are not preferentially localized near the nucleus, but instead are concentrated close to the plasma membrane where they can be visualized by total internal reflection microscopy, offering the potential for unprecedented spatio-temporal resolution of Ca2+ puff kinetics.

Ca(2+) Puffs Originate from Preestablished Stable Clusters of Inositol Trisphosphate Receptors

Science Signaling. 2009  |  Pubmed ID: 19934435

Intracellular calcium ion (Ca(2+)) signaling crucially depends on the clustered organization of inositol trisphosphate receptors (IP(3)Rs) in the endoplasmic reticulum (ER) membrane. These ligand-gated ion channels liberate Ca(2+) to generate local signals known as Ca(2+) puffs. We tested the hypothesis that IP(3) itself elicits rapid clustering of IP(3)Rs by using flash photolysis of caged IP(3) in conjunction with high-resolution Ca(2+) imaging to monitor the activity and localization of individual IP(3)Rs within intact mammalian cells. Our results indicate that Ca(2+) puffs arising with latencies as short as 100 to 200 ms after photorelease of IP(3) already involve at least four IP(3)R channels, and that this number does not subsequently grow. Moreover, single active IP(3)Rs show limited mobility, and stochastic simulations suggest that aggregation of IP(3)Rs at puff sites by a diffusional trapping mechanism would require many seconds. We thus conclude that puff sites represent preestablished, stable clusters of IP(3)Rs and that functional IP(3)Rs are not readily diffusible within the ER membrane.

NK Cell Patrolling and Elimination of Donor-derived Dendritic Cells Favor Indirect Alloreactivity

Journal of Immunology (Baltimore, Md. : 1950). Mar, 2010  |  Pubmed ID: 20139277

Direct presentation of foreign MHC molecules expressed by donor-derived dendritic cells (DCs) has generally been considered the dominant pathway of allorecognition in acute transplant rejection. However, recent studies implicate preferential activation of the indirect pathway by host DCs. The respective importance of each pathway and the mechanisms that determine their relative contributions remain to be clearly established. In this study, using two-photon microscopy, we visualized host NK cell interactions with syngeneic and allogeneic DCs within intact lymph nodes of mice. Upon contact with allogeneic DCs, NK cells formed prolonged interactions that led directly to target cell lysis. This rapid elimination limited the ability of allogeneic DCs to stimulate primary and recall T cell responses. To discriminate whether donor or host DCs are principally involved in presenting Ag derived from allografts, we used CD11c-diphtheria toxoid receptor mice to conditionally ablate CD11c(+) DCs and to show that direct presentation by donor DCs is alone insufficient to elicit acute allograft rejection. We thus propose that rapid elimination of allogeneic DCs limits direct Ag presentation and thereby favors the indirect pathway of alloreactivity.

Calcium Signaling and Amyloid Toxicity in Alzheimer Disease

The Journal of Biological Chemistry. Apr, 2010  |  Pubmed ID: 20212036

Intracellular Ca(2+) signaling is fundamental to neuronal physiology and viability. Because of its ubiquitous roles, disruptions in Ca(2+) homeostasis are implicated in diverse disease processes and have become a major focus of study in multifactorial neurodegenerative diseases such as Alzheimer disease (AD). A hallmark of AD is the excessive production of beta-amyloid (Abeta) and its massive accumulation in amyloid plaques. In this minireview, we highlight the pathogenic interactions between altered cellular Ca(2+) signaling and Abeta in its different aggregation states and how these elements coalesce to alter the course of the neurodegenerative disease. Ca(2+) and Abeta intersect at several functional levels and temporal stages of AD, thereby altering neurotransmitter receptor properties, disrupting membrane integrity, and initiating apoptotic signaling cascades. Notably, there are reciprocal interactions between Ca(2+) pathways and amyloid pathology; altered Ca(2+) signaling accelerates Abeta formation, whereas Abeta peptides, particularly in soluble oligomeric forms, induce Ca(2+) disruptions. A degenerative feed-forward cycle of toxic Abeta generation and Ca(2+) perturbations results, which in turn can spin off to accelerate more global neuropathological cascades, ultimately leading to synaptic breakdown, cell death, and devastating memory loss. Although no cause or cure is currently known, targeting Ca(2+) dyshomeostasis as an underlying and integral component of AD pathology may result in novel and effective treatments for AD.

Selective and Site-specific Mobilization of Dermal Dendritic Cells and Langerhans Cells by Th1- and Th2-polarizing Adjuvants

Proceedings of the National Academy of Sciences of the United States of America. May, 2010  |  Pubmed ID: 20404167

Dendritic cells (DCs) initiate and polarize adaptive immune responses toward varying functional outcomes. By means of intravital two-photon microscopy, we report that dermal dendritic cells (DDCs) and Langerhans cells (LCs) are differentially mobilized during contact sensitization and by adjuvants such as unmethylated CpG oligonucleotide (CpG) and LPS that induce T helper type 1 (Th1) responses, or papain that induces T helper type 2 (Th2) responses. In ear pinna, contact sensitization, CpG, LPS, and papain all mobilized DDCs in three distinct phases: increased motility and dendritic probing, directed migration, and entry into lymphatic vessels. During the same treatments, the adjacent LCs in ear pinna remained immotile over a 48-hr period of observation. In contrast, footpads lacked DDCs and Th1-polarizing adjuvants selectively induced a delayed mobilization of LCs after 48 hr. Th1 polarization of CD4(+) T cells was independent of the immunization site, whereas ear immunization favored Th2 polarization, correlating with site-specific DC distribution and dynamics. Our results provide an initial description of peripheral DC dynamics in response to adjuvants and imply that LC mobilization enhances a Th1 response and is not sufficient to trigger a Th2 response, whereas mobilization of DDCs alone is sufficient to trigger T-cell proliferation and to polarize initial T-cell activation toward a Th2 response.

Modulation of Endoplasmic Reticulum Ca2+ Store Filling by Cyclic ADP-ribose Promotes Inositol Trisphosphate (IP3)-evoked Ca2+ Signals

The Journal of Biological Chemistry. Aug, 2010  |  Pubmed ID: 20538594

In addition to its well established function in activating Ca(2+) release from the endoplasmic reticulum (ER) through ryanodine receptors (RyR), the second messenger cyclic ADP-ribose (cADPR) also accelerates the activity of SERCA pumps, which sequester Ca(2+) into the ER. Here, we demonstrate a potential physiological role for cADPR in modulating cellular Ca(2+) signals via changes in ER Ca(2+) store content, by imaging Ca(2+) liberation through inositol trisphosphate receptors (IP(3)R) in Xenopus oocytes, which lack RyR. Oocytes were injected with the non-metabolizable analog 3-deaza-cADPR, and cytosolic [Ca(2+)] was transiently elevated by applying voltage-clamp pulses to induce Ca(2+) influx through expressed plasmalemmal nicotinic channels. We observed a subsequent potentiation of global Ca(2+) signals evoked by strong photorelease of IP(3), and increased numbers of local Ca(2+) puffs evoked by weaker photorelease. These effects were not evident with cADPR alone or following cytosolic Ca(2+) elevation alone, indicating that they did not arise through direct actions of cADPR or Ca(2+) on the IP(3)R, but likely resulted from enhanced ER store filling. Moreover, the appearance of a new population of puffs with longer latencies, prolonged durations, and attenuated amplitudes suggests that luminal ER Ca(2+) may modulate IP(3)R function, in addition to simply determining the size of the available store and the electrochemical driving force for release.

A Novel Postsynaptic Mechanism for Heterosynaptic Sharing of Short-term Plasticity

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2010  |  Pubmed ID: 20592201

Postsynaptic release of Ca(2+) from intracellular stores is an important means of cellular signaling that mediates numerous forms of synaptic plasticity. Previous studies have identified a postsynaptic intracellular Ca(2+) requirement for a form of short-term plasticity, post-tetanic potentiation (PTP) at sensory neuron (SN)-motor neuron synapses in Aplysia. Here, we show that postsynaptic IP(3)-mediated Ca(2+) release in response to a presynaptic tetanus in an SN that induces PTP can confer transient plasticity onto a neighboring SN synapse receiving subthreshold activation. This heterosynaptic sharing of plasticity represents a dynamic, short-term synaptic enhancement of synaptic inputs onto a common postsynaptic target. Heterosynaptic sharing is blocked by postsynaptic disruption of Ca(2+)- and IP(3)-mediated signaling, and, conversely, it is mimicked by postsynaptic injection of nonhydrolyzable IP(3), and by photolysis of caged IP(3) in the MN. The molecular mechanism for heterosynaptic sharing involves metabotropic glutamate receptors and Homer-dependent interactions, indicating that Homer can facilitate the integration of Ca(2+)-dependent plasticity at neighboring postsynaptic sites and provides a postsynaptic mechanism for the spread of plasticity induced by presynaptic activation. Our results support a model in which postsynaptic summation of IP(3) signals from suprathreshold and subthreshold inputs results in molecular coincidence detection that gives rise to a novel form of heterosynaptic plasticity.

Superresolution Localization of Single Functional IP3R Channels Utilizing Ca2+ Flux As a Readout

Biophysical Journal. Jul, 2010  |  Pubmed ID: 20643061

The subcellular localization of membrane Ca2+ channels is crucial for their functioning, but is difficult to study because channels may be distributed more closely than the resolution of conventional microscopy is able to detect. We describe a technique, stochastic channel Ca2+ nanoscale resolution (SCCaNR), employing Ca2+-sensitive fluorescent dyes to localize stochastic openings and closings of single Ca2+-permeable channels within <50 nm, and apply it to examine the clustered arrangement of inositol trisphosphate receptor (IP3R) channels underlying local Ca2+ puffs. Fluorescence signals (blips) arising from single functional IP3Rs are almost immotile (diffusion coefficient<0.003 microm2 s(-1)), as are puff sites over prolonged periods, suggesting that the architecture of this signaling system is stable and not subject to rapid, dynamic rearrangement. However, rapid stepwise changes in centroid position of fluorescence are evident within the durations of individual puffs. These apparent movements likely result from asynchronous gating of IP3Rs distributed within clusters that have an overall diameter of approximately 400 nm, indicating that the nanoscale architecture of IP3R clusters is important in shaping local Ca2+ signals. We anticipate that SCCaNR will complement superresolution techniques such as PALM and STORM for studies of Ca2+ channels as it obviates the need for photoswitchable labels and provides functional as well as spatial information.

Essential Regulation of Cell Bioenergetics by Constitutive InsP3 Receptor Ca2+ Transfer to Mitochondria

Cell. Jul, 2010  |  Pubmed ID: 20655468

Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role for constitutive InsP(3)R-mediated Ca(2+) release in maintaining cellular bioenergetics. Macroautophagy provides eukaryotes with an adaptive response to nutrient deprivation that prolongs survival. Constitutive InsP(3)R Ca(2+) signaling is required for macroautophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to diminished mitochondrial Ca(2+) uptake. Mitochondrial uptake of InsP(3)R-released Ca(2+) is fundamentally required to provide optimal bioenergetics by providing sufficient reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer results in enhanced phosphorylation of pyruvate dehydrogenase and activation of AMPK, which activates prosurvival macroautophagy. Thus, constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics.

Recording Single-channel Activity of Inositol Trisphosphate Receptors in Intact Cells with a Microscope, Not a Patch Clamp

The Journal of General Physiology. Aug, 2010  |  Pubmed ID: 20660654

Optical single-channel recording is a novel tool for the study of individual Ca2+-permeable channels within intact cells under minimally perturbed physiological conditions. As applied to the functioning and spatial organization of IP3Rs, this approach complements our existing knowledge, which derives largely from reduced systems - such as reconstitution into lipid bilayers and patch clamping of IP3Rs on the membrane of excised nuclei - where the spatial arrangement and interactions among IP3Rs via CICR are disrupted. The ability to image the activity of single IP3R channels with millisecond resolution together with localization of their positions with a precision of a few tens of nanometers both raises several intriguing questions and holds promise of answers. In particular, what mechanism underlies the anchoring of puffs and blips to static locations; why do these Ca2+ release events appear to involve only a very small fraction of the IP3Rs within a cell; and how can we reconcile the relative immotility of functional IP3Rs with numerous studies reporting free diffusion of IP3R protein in the ER membrane?

Acquisition of Chemiluminescent Signals from Immunoblots with a Digital Single-lens Reflex Camera

Analytical Biochemistry. Feb, 2010  |  Pubmed ID: 19788886

We found that certain mid-range consumer-level digital single-lens reflex (SLR) cameras using full-frame complementary metal oxide semiconductor (CMOS) sensors outperform X-ray film in acquiring signals from immunoblots that use enhanced chemiluminescence for detection. These cameras exhibit a sensitivity comparable to X-ray film, yet they provide a 3-fold increase in linear dynamic range and substantial cost savings over time, are more convenient to use, and eliminate the chemical waste associated with processing film.

General Approach to Adoptive Transfer and Cell Labeling for Immunoimaging

Cold Spring Harbor Protocols. Jan, 2011  |  Pubmed ID: 21285265

Induction of an Immune Response for Imaging Antigen-presenting Cell/T-cell Interactions

Cold Spring Harbor Protocols. Jan, 2011  |  Pubmed ID: 21285266

In Situ Lymph Node Imaging

Cold Spring Harbor Protocols. Jan, 2011  |  Pubmed ID: 21285267

In Vivo Lymph Node Imaging

Cold Spring Harbor Protocols. Jan, 2011  |  Pubmed ID: 21285268

Immunoimaging: Studying Immune System Dynamics Using Two-photon Microscopy

Cold Spring Harbor Protocols. Jan, 2011  |  Pubmed ID: 21285279

Active Generation and Propagation of Ca2+ Signals Within Tunneling Membrane Nanotubes

Biophysical Journal. Apr, 2011  |  Pubmed ID: 21504718

A new mechanism of cell-cell communication was recently proposed after the discovery of tunneling nanotubes (TNTs) between cells. TNTs are membrane protrusions with lengths of tens of microns and diameters of a few hundred nanometers that permit the exchange of membrane and cytoplasmic constituents between neighboring cells. TNTs have been reported to mediate intercellular Ca(2+) signaling; however, our simulations indicate that passive diffusion of Ca(2+) ions alone would be inadequate for efficient transmission between cells. Instead, we observed spontaneous and inositol trisphosphate (IP(3))-evoked Ca(2+) signals within TNTs between cultured mammalian cells, which sometimes remained localized and in other instances propagated as saltatory waves to evoke Ca(2+) signals in a connected cell. Consistent with this, immunostaining showed the presence of both endoplasmic reticulum and IP(3) receptors along the TNT. We propose that IP(3) receptors may actively propagate intercellular Ca(2+) signals along TNTs via Ca(2+)-induced Ca(2+) release, acting as amplification sites to overcome the limitations of passive diffusion in a chemical analog of electrical transmission of action potentials.

Enhanced ER Ca2+ Store Filling by Overexpression of SERCA2b Promotes IP3-evoked Puffs

Cell Calcium. Jul, 2011  |  Pubmed ID: 21616533

Liberation of Ca(2+) from the endoplasmic reticulum (ER) through inositol trisphosphate receptors (IP(3)R) is modulated by the ER Ca(2+) content, and overexpression of SERCA2b to accelerate Ca(2+) sequestration into the ER has been shown to potentiate the frequency and amplitude of IP(3)-evoked Ca(2+) waves in Xenopus oocytes. Here, we examined the effects of SERCA overexpression on the elementary IP(3)-evoked puffs to elucidate whether ER [Ca(2+)] may modulate IP(3)R function via luminal regulatory sites in addition to simply determining the size of the available store and electrochemical driving force for Ca(2+) release. SERCA2b and Ca(2+) permeable nicotinic plasmalemmal channels were expressed in oocytes, and hyperpolarizing pulses were delivered to induce Ca(2+) influx and thereby load ER stores. Puffs evoked by photoreleased IP(3) were significantly potentiated in terms of numbers of responding sites, frequency and amplitude following transient Ca(2+) influx in SERCA-overexpressing cells, whereas little change was evident with SERCA overexpression alone or following Ca(2+) influx in control cells not overexpressing SERCA. Intriguingly, we observed the appearance of a new population of puffs that arose after long latencies and had prolonged durations supporting the notion of luminal regulation of IP(3)R gating kinetics.

Discursive Social Psychology Now

The British Journal of Social Psychology / the British Psychological Society. Jul, 2011  |  Pubmed ID: 21790666

This paper reviews the progress of discourse-analytic approaches in social psychology from the late 1980s to the present day, with a particular focus on the way conceptual and methodological contributions from within the Discourse and Rhetoric Group at Loughborough University have negotiated a positive role for innovative studies of language in the discipline of psychology. Social psychology has become a key site for the accumulation of a series of empirical studies that have seen the flourishing of a distinctive form of 'discursive social psychology' that has succeeded in moving from the margins of the discipline to a more accepted position. The paper traces this trajectory of discourse analysis from the limits to the centre of social psychology attending to five features that now characterise its contribution to psychology; an emphasis on everyday conversation, a concern with interpersonal interaction, explication of formal sequences; an insistence on empirical claims; and fidelity to the ethos of its host discipline. The paper concludes with some comments on the wider context of this new approach inside psychology today.

Subunit Stoichiometry of Human Orai1 and Orai3 Channels in Closed and Open States

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2011  |  Pubmed ID: 21987805

We applied single-molecule photobleaching to investigate the stoichiometry of human Orai1 and Orai3 channels tagged with eGFP and expressed in mammalian cells. Orai1 was detected predominantly as dimers under resting conditions and as tetramers when coexpressed with C-STIM1 to activate Ca(2+) influx. Orai1 was also found to be tetrameric when coexpressed with STIM1 and evaluated following fixation. We show that fixation rapidly causes release of Ca(2+), redistribution of STIM1 to the plasma membrane, and STIM1/Orai1 puncta formation, and may cause the channel to be in the activated state. Consistent with this possibility, Orai1 was found predominantly as a dimer when coexpressed with STIM1 in living cells under resting conditions. We further show that Orai3, like Orai1, is dimeric under resting conditions and is predominantly tetrameric when activated by C-STIM1. Interestingly, a dimeric Orai3 stoichiometry was found both before and during application of 2-aminoethyldiphenyl borate (2-APB) to activate a nonselective cation conductance in its STIM1-independent mode. We conclude that the human Orai1 and Orai3 channels undergo a dimer-to-tetramer transition to form a Ca(2+)-selective pore during store-operated activation and that Orai3 forms a dimeric nonselective cation pore upon activation by 2-APB.

Single-channel Ca(2+) Imaging Implicates Aβ1-42 Amyloid Pores in Alzheimer's Disease Pathology

The Journal of Cell Biology. Oct, 2011  |  Pubmed ID: 22024165

Oligomeric forms of Aβ peptides are implicated in Alzheimer's disease (AD) and disrupt membrane integrity, leading to cytosolic calcium (Ca(2+)) elevation. Proposed mechanisms by which Aβ mediates its effects include lipid destabilization, activation of native membrane channels, and aggregation of Aβ into Ca(2+)-permeable pores. We distinguished between these using total internal reflection fluorescence (TIRF) microscopy to image Ca(2+) influx in Xenopus laevis oocytes. Aβ1-42 oligomers evoked single-channel Ca(2+) fluorescence transients (SCCaFTs), which resembled those from classical ion channels but which were not attributable to endogenous oocyte channels. SCCaFTs displayed widely variable open probabilities (P(o)) and stepwise transitions among multiple amplitude levels reminiscent of subconductance levels of ion channels. The proportion of high P(o), large amplitude SCCaFTs grew with time, suggesting that continued oligomer aggregation results in the formation of highly toxic pores. We conclude that formation of intrinsic Ca(2+)-permeable membrane pores is a major pathological mechanism in AD and introduce TIRF imaging for massively parallel single-channel studies of the incorporation, assembly, and properties of amyloidogenic oligomers.

Timescales of IP(3)-Evoked Ca(2+) Spikes Emerge from Ca(2+) Puffs Only at the Cellular Level

Biophysical Journal. Dec, 2011  |  Pubmed ID: 22261051

The behavior of biological systems is determined by the properties of their component molecules, but the interactions are usually too complex to understand fully how molecular behavior generates cellular behavior. Ca(2+) signaling by inositol trisphosphate receptors (IP(3)R) offers an opportunity to understand this relationship because the cellular behavior is defined largely by Ca(2+)-mediated interactions between IP(3)R. Ca(2+) released by a cluster of IP(3)R (giving a local Ca(2+) puff) diffuses and ignites the behavior of neighboring clusters (to give repetitive global Ca(2+) spikes). We use total internal reflection fluorescence microscopy of two mammalian cell lines to define the temporal relationships between Ca(2+) puffs (interpuff intervals, IPI) and Ca(2+) spikes (interspike intervals) evoked by flash photolysis of caged IP(3). We find that IPI are much shorter than interspike intervals, that puff activity is stochastic with a recovery time that is much shorter than the refractory period of the cell, and that IPI are not periodic. We conclude that Ca(2+) spikes do not arise from oscillatory dynamics of IP(3)R clusters, but that repetitive Ca(2+) spiking with its longer timescales is an emergent property of the dynamics of the whole cluster array.