Biomedical subjects
Publications and source records attributed to Ian Parker.
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.
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.
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.
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.
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.
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.
BACKGROUND: 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. METHODS: One hundred and three patients suspected of having colorectal pathology underwent CT colonography. RESULTS: CT colonography suggested a diagnosis of colonic carcinoma in 18 patients, and 17 of these underwent surgery. A colorectal neoplasm was not found in only 1 patient who had extrinsic colonic compression by an ovarian cyst. Twenty-one patients had suspected colonic polyps on scanning. Subsequent endoscopy in 19 of these patients confirmed the presence of polyps in only 10. CT colonography also revealed valuable extracolonic pathology: 8 occult noncolonic neoplasms and 163 other incidental findings. CONCLUSIONS: CT colonography has good patient compliance and is a useful diagnostic modality in detecting colorectal neoplasms. Its main advantage over other such investigative tools is its ability to detect extracolonic pathology.
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.
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.
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.
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.
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.
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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.
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.
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.
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.
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.