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Articles by Jonathan S. Marchant in JoVE
JoVE General
Фармакологические и функциональные Генетические анализы манипулировать Регенерация планарии Dugesia японская
Department of Pharmacology and The Stem Cell Institute, University of Minnesota Medical School
Привлекательной моделью для изучения дифференциации стволовых клеток в живых животных планарии плоских червей. Регенерация изучается простых экспериментов ампутации, которые легко выполняются в основной лаборатории и поддаются фармакологических и генетических (
Other articles by Jonathan S. Marchant on PubMed
Structural and Functional Relationships Between Ca2+ Puffs and Mitochondria in Xenopus Oocytes
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.
Intracellular Trafficking and Membrane Targeting Mechanisms of the Human Reduced Folate Carrier in Mammalian Epithelial Cells
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.
Cell Biology of the Human Thiamine Transporter-1 (hTHTR1). Intracellular Trafficking and Membrane Targeting Mechanisms
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.
Polarized Expression of Members of the Solute Carrier SLC19A Gene Family of Water-soluble Multivitamin Transporters: Implications for Physiological Function
Humans lack biochemical pathways for the synthesis of the micro-nutrients thiamine and folate. Cellular requirements are met through membrane transport activity, which is mediated by proteins of the SLC19A gene family. By using live-cell confocal imaging methods to resolve the localization of all SLC19A family members, we show that the two human thiamine transporters are differentially targeted in polarized cells, establishing a vectorial transport system. Such polarization decreases functional redundancy between transporter isoforms and allows for independent regulation of thiamine import and export pathways in cells.
Expression and Functional Contribution of HTHTR-2 in Thiamin Absorption in Human Intestine
The aim of this study was to investigate expression and relative contribution of human thiamin transporter (hTHTR)-2 toward overall carrier-mediated thiamin uptake by human intestinal epithelial cells. Northern blot analysis showed that the message of the hTHTR-2 is expressed along the native human gastrointestinal tract with highest expression being in the proximal part of small intestine. hTHTR-2 protein was found, by Western blot analysis, to be expressed at the brush-border membrane (BBM), but not at the basolateral membrane, of native human enterocytes. This pattern of expression was confirmed in studies using a fusion protein of hTHTR-2 with the enhanced green fluorescent protein (hTHTR2-EGFP) expressed in living Caco-2 cells grown on filter. Pretreating Caco-2 cells (which also express the hTHTR-2 at RNA and protein levels) with hTHTR-2 gene-specific small interfering RNA (siRNA) led to a significant (P < 0.01) and specific inhibition (48%) in carrier-mediated thiamin uptake. Similarly, pretreating Caco-2 cells with siRNA that specifically target hTHTR-1 (which is expressed in Caco-2 cells) also significantly (P < 0.01) and specifically inhibited (by 56%) carrier-mediated thiamin uptake. When Caco-2 cells were pretreated with siRNAs against both hTHTR-2 and hTHTR-1 genes, an almost complete inhibition in carrier-mediated thiamin uptake was observed. These results show that the message of hTHTR-2 is expressed along the human gastrointestinal tract and that expression of its protein in intestinal epithelia is mainly localized to the apical BBM domain. In addition, results show that this transporter plays a significant role in carrier-mediated thiamin uptake in human intestine.
A C-terminal Region Dictates the Apical Plasma Membrane Targeting of the Human Sodium-dependent Vitamin C Transporter-1 in Polarized Epithelia
The human sodium-dependent vitamin C transporter (hSVCT1) mediates sodium-dependent cellular uptake of the essential micronutrient l-ascorbic acid (vitamin C). However, the molecular determinants that control the cell surface expression, subcellular distribution, and dynamics of hSVCT1 remain undefined. To identify molecular determinants involved in hSVCT1 targeting in polarized epithelia, we used live cell imaging approaches to resolve the targeting and trafficking dynamics of hSVCT1 truncation mutants in renal and intestinal cells. Confocal imaging demonstrated that hSVCT1 was expressed at the apical cell surface and video rate measurements revealed hSVCT1 also resided in a heterogeneous population of intracellular organelles with discrete dynamic properties. By progressive truncation of the cytoplasmic C-terminal tail of hSVCT1, we delimited an essential role for an embedded ten amino acid sequence PICPVFKGFS (amino acids 563-572) in defining the physiological targeting of hSVCT1. Intriguingly, this sequence bears significant homology to recently identified apical targeting motifs in two other sodium-dependent transporters, and we suggest this conservation is reflected topologically through the adoption of a beta-turn confirmation in the cytoplasmic C-tail of each transporter. Our results provide the first direct resolution of functional hSVCT1 expression at the apical cell surface of polarized epithelia and define an apical targeting signal of relevance to transporters of diverse substrate specificity.
Calcium-dependent Dephosphorylation Mediates the Hyperosmotic and Lysophosphatidic Acid-dependent Inhibition of Natriuretic Peptide Receptor-B/guanylyl Cyclase-B
C-type natriuretic peptide binding to natriuretic peptide receptor-B (NPR-B) stimulates cGMP synthesis, which regulates vasorelaxation, cell proliferation, and bone growth. Here, we investigated the mechanistic basis for hyperosmotic and lysophosphatidic acid-dependent inhibition of NPR-B. Whole cell cGMP measurements and guanylyl cyclase assays indicated that acute hyperosmolarity decreased NPR-B activity in a reversible, concentration- and time-dependent manner, whereas chronic exposure had no effect. Acute hyperosmolarity elevated intracellular calcium in a concentration-dependent fashion that paralleled NPR-B desensitization. A calcium chelator, but not a protein kinase C inhibitor, blocked both calcium elevations and desensitization. Hyperosmotic medium stimulated NPR-B dephosphorylation, and the receptor was rapidly rephosphorylated and resensitized when the hypertonic media was removed. Lysophosphatidic acid also inhibited NPR-B in a calcium- and phosphorylation-dependent process, consistent with calcium being a universal regulator of NPR-B. The absolute requirement of dephosphorylation in this process was demonstrated by showing that a receptor with glutamates substituted at all known NPR-B phosphorylation sites is unresponsive to hyperosmotic stimuli. This is the first study to measure the phosphorylation state of an endogenous guanylyl cyclase and to link intracellular calcium elevations with its dephosphorylation.
Improved "optical Highlighter" Probes Derived from Discosoma Red Fluorescent Protein
The tetrameric red fluorescent protein, DsRed, undergoes a rapid red to green color change evoked by short wavelength (lambda < 760 nm) femtosecond irradiation--a phenomenon that underpins the application of DsRed as an "optical highlighter" probe for tracking live cells, organelles, and fusion proteins. This color change results from selective bleaching of the "mature" red-emitting species of DsRed and an enhancement of emission from the "immature" green species, likely caused by dequenching of fluorescence resonance energy transfer occurring within the protein tetramer. Here, we have examined the role of residues known to influence the rate and completeness of chromophore maturation on the cellular and biophysical properties of DsRed mutants. Surprisingly, a single amino acid mutation (N42Q) with increased basal green emission yet rapid chromophore maturation displayed a multiphoton-evoked color change that was brighter, more consistent, more vivid, and easier to evoke than DsRed, despite the larger proportion of green chromophores. Rapidly maturing mutants with more complete chromophore maturation, exhibited little color change and increased resistance to multiphoton bleaching. We describe improved optical and cell biological properties for two DsRed-derived variants which we showcase in photolabeling studies, and discuss these data in terms of implications for fluorescence resonance energy transfer-based probes.
IP3 Receptor Activity is Differentially Regulated in Endoplasmic Reticulum Subdomains During Oocyte Maturation
Fertilization competency results from hormone-induced remodeling of oocytes into eggs. The signaling pathways that effect this change exemplify bistability, where brief hormone exposure irrevocably switches cell fate. In Xenopus, changes in Ca(2+) signaling epitomize such remodeling: The reversible Ca(2+) signaling phenotype of oocytes rapidly adapts to support irreversible propagation of the fertilization Ca(2+) wave. Here, we simultaneously resolved IP(3) receptor (IP(3)R) activity with endoplasmic reticulum (ER) structure to optically dissect the functional architecture of the Ca(2+) release apparatus underpinning this reorganization. We show that changes in Ca(2+) signaling correlate with IP(3)R redistribution from specialized ER substructures called annulate lamellae (AL), where Ca(2+) release activity is attenuated, into IP(3)R-replete patches in the cortical ER of eggs that support the fertilization Ca(2+) wave. These data show: first, that IP(3)R sensitivity is regulated with high spatial acuity even between contiguous ER regions; and second, that drastic reorganization of Ca(2+) signaling dynamics can be driven by subcellular redistribution in the absence of changes in channel number or molecular or familial Ca(2+) channel diversity. Finally, these results define a novel role for AL in Ca(2+) signaling. Because AL are prevalent in other scenarios of rapid cell division, further studies of their impact on Ca(2+) signaling are warranted.
Cellular Signalling: STIMulating Calcium Entry
The molecular identity of the signal coupling intracellular Ca(2+) store depletion to the activation of Ca(2+) entry has long resisted exposure. Two recent studies independently implicate the STIM protein family as essential components in this coupling. These data provide new impetus to resolve how store Ca(2+) content is sensed and communicated to store-operated Ca(2+) channels at the cell surface.
Targeting and Trafficking of the Human Thiamine Transporter-2 in Epithelial Cells
Humans lack biochemical pathways for thiamine synthesis, so cellular requirements are met via specific carrier-mediated uptake pathways. Two proteins from the solute carrier SLC19A gene family have been identified as human thiamine transporters (hTHTRs), SLC19A1 (hTHTR1) and SLC19A2 (hTHTR2). Both of these transporters are co-expressed but are differentially targeted in polarized cell types that mediate vectorial thiamine transport (e.g. renal and intestinal epithelia). It is important to understand the domain structure of these proteins, namely which regions within the polypeptide sequence are important for physiological delivery to the cell surface, in order to understand the impact of clinically relevant mutations on thiamine transport. Here we have characterized the mechanisms regulating hTHTR2 distribution by using live cell imaging methods that resolve the targeting and trafficking dynamics of full-length hTHTR2, a series of hTHTR2 truncation mutants, as well as chimeras comprising the hTHTR1 and hTHTR2 sequence. We showed the following: (i) that the cytoplasmic COOH-tail of hTHTR2 is not essential for apical targeting in polarized cells; (ii) that delivery of hTHTR2 to the cell surface is critically dependent on the integrity of the transmembrane backbone of the polypeptide so that minimal truncations abrogate cell surface expression of hTHTR2; and (iii) video rate images of hTHTR2-containing intracellular vesicles displayed rapid bi-directional trafficking events to and from the cell surface impaired by microtubule-disrupting but not microfilament-disrupting agents as well as by overexpression of the dynactin subunit dynamitin (p50). Finally, we compared the behavior of hTHTR2 with that of hTHTR1 and the human reduced folate carrier (SLC19A1) to underscore commonalities in the cell surface targeting mechanisms of the entire SLC19A gene family.
Biotin-responsive Basal Ganglia Disease-linked Mutations Inhibit Thiamine Transport Via HTHTR2: Biotin is Not a Substrate for HTHTR2
The water-soluble micronutrient thiamine is required for normal tissue growth and development in humans. Thiamine is accumulated into cells through the activity of two cell surface thiamine transporters (hTHTR1 and hTHTR2), which are differentially targeted in polarized tissues. Mutational dysfunction of hTHTR1 is associated with the clinical condition of thiamine-responsive megaloblastic anemia: the symptoms of which are alleviated by thiamine supplementation. Recently, two hTHTR2 mutants (G23V, T422A) have been discovered in clinical kindreds manifesting biotin-responsive basal ganglia disease (BBGD): the symptoms of which are alleviated by biotin administration. Why then does mutation of a specific thiamine transporter isoform precipitate a disorder correctable by exogenous biotin? To investigate the suggestion that hTHTR2 can physiologically function as a biotin transporter, we examined 1) the cell biological basis of hTHTR2 dysfunction associated with the G23V and T422A mutations and 2) the substrate specificity of hTHTR2 and these clinically relevant mutants. We show that the G23V and T422A mutants both abrogate thiamine transport activity rather than targeting of hTHTR2 to the cell surface. Furthermore, biotin accumulation was not detectable in cells overexpressing either the full length hTHTR2 or the clinically relevant hTHTR2 mutants, yet was demonstrable in the same assay using cells overexpressing the human sodium-dependent multivitamin transporter, a known biotin transporter. These results cast doubt on the most parsimonious explanation for the BBGD phenotype, namely that hTHTR2 is a physiological biotin transporter.
Calcium Influx: Beyond 'current' Biology
A novel, cell-surface protein essential for Ca(2+) release-activated Ca(2+) (CRAC) channel function has been identified through independent genome-wide screens. This huge advance will enable molecular dissection of the CRAC channel complex, moving the field beyond Icrac signature to structure.
Targeting and Intracellular Trafficking of Clinically Relevant HTHTR1 Mutations in Human Cell Lines
The micronutrient thiamine is required for normal growth and development of human tissues, and is accumulated into cells through the activity of plasma membrane thiamine transporters, e.g. hTHTR1 (human thiamine transporter 1). Recent genetic evidence has linked mutations in hTHTR1 with the manifestation of TRMA (thiamine-responsive megaloblastic anaemia), a condition also associated with diabetes mellitus, sensorineural deafness and retinal disorders. To examine how mutations in hTHTR1 impair thiamine accumulation, we have investigated the targeting and functional properties of several different hTHTR1 mutants in human cell lines derived from epithelia relevant to thiamine absorption or tissues implicated in TRMA pathology. These constructs encompassed two newly identified point mutations (P51L and T158R) and two truncations of hTHTR1 identical with those found in TRMA kindreds (W358X and Delta383fs). Our results reveal a spectrum of mutant phenotypes, underlining that TRMA can result from decreased thiamine transport activity underpinned by changes in hTHTR1 expression levels, cellular targeting and/or protein transport activity.
The Inositol 1,4,5-trisphosphate Receptor (Itpr) Gene Family in Xenopus: Identification of Type 2 and Type 3 Inositol 1,4,5-trisphosphate Receptor Subtypes
Studies in the Xenopus model system have provided considerable insight into the developmental role of intracellular Ca2+ signals produced by activation of IP3Rs (inositol 1,4,5-trisphosphate receptors). However, unlike mammalian systems where three IP3R subtypes have been well characterized, our molecular understanding of the IP3Rs that underpin Ca2+ signalling during Xenopus embryogenesis relate solely to the original characterization of the 'Xenopus IP3R' cloned and purified from Xenopus laevis oocytes several years ago. In the present study, we have identified Xenopus type 2 and type 3 IP3Rs and report the full-length sequence, genomic architecture and developmental expression profile of these additional IP3R subtypes. In the light of the emerging genomic resources and opportunities for genetic manipulation in the diploid frog Xenopus tropicalis, these data will facilitate manipulations to resolve the contribution of IP3R diversity in Ca2+ signalling events observed during vertebrate development.
Vitamin B1 (thiamine) Uptake by Human Retinal Pigment Epithelial (ARPE-19) Cells: Mechanism and Regulation
Retinal abnormality and visual disturbances occur in thiamine-responsive megaloblastic anaemia (TRMA), an autosomal recessive disorder caused by mutations in the human thiamine transporter-1 (hTHTR-1). Human retinal pigment epithelial cells play a pivotal role in supplying thiamine to the highly metabolically active retina but nothing is known about the mechanism, regulation or biological processes involved in thiamine transport in these cells. To address these issues, we used human-derived retinal pigment epithelial ARPE-19 cells to characterize the thiamine uptake process. Thiamine uptake is energy- and temperature-dependent, pH-sensitive, Na+-independent, saturable at both the nanomolar (apparent Km, 30 +/- 5 nM) and the micromolar (apparent Km, 1.72 +/- 0.3 microM) concentration ranges, specific for thiamine and sensitive to sulfhydryl group inhibition. The diuretic amiloride caused a concentration-dependent inhibition in thiamine uptake, whereas the anti-trypanosomal drug, melarsoprol, failed to affect the uptake process. Both hTHTR-1 and hTHTR-2 are expressed in ARPE-19 cells as well as in native human retinal tissue with expression of the former being significantly higher than that of the latter. Uptake of thiamine was adaptively regulated by extracellular substrate level via transcriptionally mediated mechanisms that involve both hTHTR-1 and hTHTR-2; it was also regulated by an intracellular Ca2+-calmodulin-mediated pathway. Confocal imaging of living ARPE-19 cells expressing TRMA-associated hTHTR-1 mutants (D93H, S143F and G172D) showed various expression phenotypes. These results demonstrate for the first time the existence of a specialized and regulated uptake process for thiamine in a cellular model of human retinal pigment epithelia that involves hTHTR-1 and hTHTR-2. Further, clinically relevant mutations in hTHTR-1 lead to impaired cell surface expression or function of the transporter in retinal epithelial ARPE-19 cells.
Molecular Characterization of a Novel Intracellular ADP-ribosyl Cyclase
ADP-ribosyl cyclases are remarkable enzymes capable of catalyzing multiple reactions including the synthesis of the novel and potent intracellular calcium mobilizing messengers, cyclic ADP-ribose and NAADP. Not all ADP-ribosyl cyclases however have been characterized at the molecular level. Moreover, those that have are located predominately at the outer cell surface and thus away from their cytosolic substrates.
Tight Junction Targeting and Intracellular Trafficking of Occludin in Polarized Epithelial Cells
Occludin, a transmembrane (TM)-spanning protein, is an integral component of the tight junctional (TJ) complexes that regulate epithelial integrity and paracellular barrier function. However, the molecular determinants that dictate occludin targeting and delivery to the TJs remain unclear. Here, using live cell imaging of yellow fluorescent protein-labeled occludin fragments, we resolved the intracellular trafficking of occludin-fusion proteins in polarized Madin-Darby canine kidney and Caco-2 cells to delineate the regions within the occludin polypeptide that are important for occludin targeting to the TJs. Live cell confocal imaging showed that complete or partial truncation of the COOH-terminal tail of the occludin polypeptide did not prevent occludin targeting to the TJs in epithelial cell lines. Progressive truncations into the COOH-terminal tail decreased the efficiency of occludin expression; after the removal of the regions proximal to the fourth transmembrane domain (TM4), the efficiency of expression increased. However, further deletions into the TM4 abolished TJ targeting, which resulted in constructs that were retained intracellularly within the endoplasmic reticulum. The full-length occludin polypeptide trafficked to the cell surface within a heterogenous population of intracellular vesicles that delivered occludin to the plasma membrane in a microtubule- and temperature-dependent manner. In contrast, the steady-state localization of occludin at the cell surface was dependent on intact microfilaments but not microtubules.
Apical Membrane Targeting and Trafficking of the Human Proton-coupled Transporter in Polarized Epithelia
The human proton-coupled folate transporter (hPCFT) is a recently discovered intestinal transporter involved in folate uptake in epithelia (and possibly other cells). Little is currently known about the structure-function relationship of the different domains of this transporter, particularly which regions are important for substrate transport as well as targeting of the transporter to the apical cell surface of polarized cells. Here we have investigated the role of the COOH-terminal domain and a well-conserved sequence separating transmembrane (TM) domains TM2 and TM3 (DXXGRR; amino acids 109-114) speculated by others to be important for transport function. Using live cell imaging approaches, we show that 1) an hPCFT-yellow fluorescent protein construct is functionally expressed at the apical membrane domain and is localized differentially to the human reduced folate carrier; 2) the predicted cytoplasmic COOH-terminal region of hPCFT is not essential for apical targeting or transporter functionality; 3) mutations that ablate a consensus beta-turn sequence separating predicted TM2 and TM3 abolished apical [(3)H]folic acid uptake as a consequence of endoplasmic reticulum retention of mutant, likely misfolded, transporters; and 4) cell surface delivery of hPCFT is disrupted by microtubule depolymerization or by overexpression of the dynactin complex dynamitin (p50). For the first time, our data present information regarding structure-function and membrane targeting of the hPCFT polypeptide, as well as the mechanisms that control its steady-state expression in polarized cells.
Enhanced Ca2+ Leak from ER Ca2+ Stores Induced by Hepatitis C NS5A Protein
The hepatitis C non-structural protein 5A (NS5A) is a Zn(2+)-binding phosphoprotein essential for viral replication. Expression of NS5A perturbs intracellular Ca(2+) levels by an undefined mechanism, activating transcription factors implicated in the chronic pathogenesis of hepatitis infections. Here, we demonstrate that regulated expression of NS5A enhanced the passive leak of Ca(2+) from a subset of the endoplasmic reticulum (ER) Ca(2+) stores. This action was not replicated by expression of the amphipathic NH(2)-membrane anchoring domain of NS5A alone, despite targeting to intracellular membranes. Depletion of the NS5A-targeted ER Ca(2+) store was prevented under conditions of ample ATP supply suggesting compensatory Ca(2+) ATPase activity, but observed under conditions of ATP insufficiency and in intact cells expressing NS5A.
Nuclear Pore Disassembly from Endoplasmic Reticulum Membranes Promotes Ca2+ Signalling Competency
The functionality of the endoplasmic reticulum (ER) as a Ca(2+) storage organelle is supported by families of Ca(2+) pumps, buffers and channels that regulate Ca(2+) fluxes between the ER lumen and cytosol. Although many studies have identified heterogeneities in Ca(2+) fluxes throughout the ER, the question of how differential functionality of Ca(2+) channels is regulated within proximal regions of the same organelle is unresolved. Here, we studied the in vivo dynamics of an ER subdomain known as annulate lamellae (AL), a cytoplasmic nucleoporin-containing organelle widely used in vitro to study the mechanics of nuclear envelope breakdown. We show that nuclear pore complexes (NPCs) within AL suppress local Ca(2+) signalling activity, an inhibitory influence relieved by heterogeneous dissociation of nucleoporins to yield NPC-denuded ER domains competent at Ca(2+) signalling. Consequently, we propose a novel generalized role for AL - reversible attenuation of resident protein activity - such that regulated AL (dis)assembly via a kinase/phosphatase cycle allows cells to support rapid gain/loss-of-function transitions in cellular physiology.
N-Glycosylation is Required for Na+-dependent Vitamin C Transporter Functionality
The human sodium-dependent vitamin C transporters (hSVCT1 and hSVCT2) mediate cellular uptake of ascorbic acid. Both these transporters contain potential sites for N-glycosylation in their extracellular domains (Asn-138, Asn-144 [hSVCT1]; Asn-188, Asn-196 [hSVCT2]), however the role of N-glycosylation in transporter function is unexplored. On the basis of the result that tunicamycin decreased (14)C-ascorbic acid uptake in HepG2 cells, we systematically ablated all consensus N-glycosylation sites in hSVCT1 and hSVCT2 to resolve any effects on ascorbic acid uptake, transporter expression and targeting. We show that removal of individual N-glycosylation sites significantly impairs protein expression and consequently ascorbic acid uptake for hSVCT1 mutants (N138Q is retained intracellularly) and for hSVCT2 mutants (all of which reach the cell surface). N-Glycosylation is therefore essential for vitamin C transporter functionality.
Timing in Cellular Ca2+ Signaling
Calcium (Ca2+) signals are generated across a broad time range. Kinetic considerations impact how information is processed to encode and decode Ca2+ signals, the choreography of responses that ensure specific and efficient signaling and the overall temporal amplification such that ephemeral Ca2+ signals have lasting physiological value. The reciprocal importance of timing for Ca2+ signaling, and Ca2+ signaling for timing is exemplified by the altered kinetic profiles of Ca2+ signals in certain diseases and the likely role of basal Ca2+ fluctuations in the perception of time itself.
Molecular Characterization of a Novel Cell Surface ADP-ribosyl Cyclase from the Sea Urchin
The sea urchin is an extensively used model system for the study of calcium signalling by the messenger molecules NAADP and cyclic ADP-ribose. Both are synthesized by ADP-ribosyl cyclases but our molecular understanding of these enzymes in the sea urchin is limited. We have recently reported the cloning of an extended family of sea urchin ADP-ribosyl cyclases and shown that one of these enzymes (SpARC1) is active within the endoplasmic reticulum lumen. These studies suggest that production of messengers is compartmentalized. Here we characterize the properties of SpARC2. SpARC2 catalyzed both NAADP and cyclic ADP-ribose production. Unusually, the NAD surrogate, NGD was a poor substrate. In contrast to SpARC1, heterologously expressed SpARC2 localized to the plasma membrane via a glycosylphosphatidylinositol (GPI)-anchor. Transcripts for SpARC2 were readily detectable in sea urchin eggs and a majority of the endogenous membrane bound activity was found to be GPI-anchored. Our data reveal striking differences in the properties of sea urchin ADP-ribosyl cyclases and provide further evidence that messenger production may occur outside of the cytosol.
Membrane Targeting and Intracellular Trafficking of the Human Sodium-dependent Multivitamin Transporter in Polarized Epithelial Cells
The human sodium-dependent multivitamin transporter (hSMVT) mediates sodium-dependent uptake of biotin in renal and intestinal epithelia. To date, however, there is nothing known about the structure-function relationship or targeting sequences in the hSMVT polypeptide that control its polarized expression within epithelia. Here, we focused on the role of the COOH-terminal tail of hSMVT in the targeting and functionality of this transporter. A full-length hSMVT-green fluorescent protein (GFP) fusion protein was functional and expressed at the apical membrane in renal and intestinal cell lines. Microtubule disrupting agents disrupted the mobility of trafficking vesicles and impaired cell surface delivery of hSMVT, which was also prevented in cells treated with dynamitin (p50), brefeldin, or monensin. Progressive truncation of the COOH-terminal tail impaired the functionality and targeting of the transporter. First, biotin transport decreased by approximately 20-30% on deletion of up to 15 COOH-terminal amino acids of hSMVT, a decrease mimicked solely by deletion of the terminal PDZ motif (TSL). Second, deletions into the COOH-terminal tail (between residues 584-612, containing a region of predicted high surface accessibility) resulted in a further drop in hSMVT transport (to approximately 40% of wild-type). Third, apical targeting was lost on deletion of a helical-prone region between amino acids 570-584. We conclude that the COOH tail of hSMVT contains several determinants important for polarized targeting and biotin transport.
A Novel Biological Activity of Praziquantel Requiring Voltage-operated Ca2+ Channel Beta Subunits: Subversion of Flatworm Regenerative Polarity
Approximately 200 million people worldwide harbour parasitic flatworm infections that cause schistosomiasis. A single drug-praziquantel (PZQ)-has served as the mainstay pharmacotherapy for schistosome infections since the 1980s. However, the relevant in vivo target(s) of praziquantel remain undefined.
Dual Mechanisms of SHA 14-1 in Inducing Cell Death Through Endoplasmic Reticulum and Mitochondria
HA 14-1 is a small-molecule Bcl-2 antagonist that promotes apoptosis in malignant cells, but its mechanism of action is not well defined. We recently reported that HA 14-1 has a half-life of only 15 min in vitro, which led us to develop a stable analog of HA 14-1 (sHA 14-1). The current study characterizes its mode of action. Because of the antiapoptotic function of Bcl-2 family proteins on the endoplasmic reticulum (ER) and mitochondria, the effect of sHA 14-1 on both organelles was evaluated. sHA 14-1 induced ER calcium release in human leukemic cells within 1 min, followed by induction of the ER stress-inducible transcription factor ATF4. Similar kinetics and stronger intensity of ER calcium release were induced by the sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin, accompanied by similar kinetics and intensity of ATF4 induction. sHA 14-1 directly inhibited SERCA enzymatic activity but had no effect on the inositol triphosphate receptor. Evaluation of the mitochondrial pathway showed that sHA 14-1 triggered a loss of mitochondrial transmembrane potential (Delta psi m) and weak caspase-9 activation, whereas thapsigargin had no effect. (R)-4-(3-Dimethylamino-1-phenylsulfanylmethyl-propylamino)-N-{4-[4-(4'-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-3-nitrobenzenesulfonamide (ABT-737), a well established small-molecule Bcl-2 antagonist, rapidly induced loss of Delta psi m and caspase-9 activation but had no effect on the ER. The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone had some protective effect on sHA 14-1-induced cell death. These collective results suggest a unique dual targeting mechanism of sHA 14-1 on the apoptotic resistance machinery of tumor cells that includes antiapoptotic Bcl-2 family proteins and SERCA proteins.
Essential Requirement for Two-pore Channel 1 in NAADP-mediated Calcium Signaling
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a widespread and potent calcium-mobilizing messenger that is highly unusual in activating calcium channels located on acidic stores. However, the molecular identity of the target protein is unclear. In this study, we show that the previously uncharacterized human two-pore channels (TPC1 and TPC2) are endolysosomal proteins, that NAADP-mediated calcium signals are enhanced by overexpression of TPC1 and attenuated after knockdown of TPC1, and that mutation of a single highly conserved residue within a putative pore region abrogated calcium release by NAADP. Thus, TPC1 is critical for NAADP action and is likely the long sought after target channel for NAADP.
Localization and Socialization: Experimental Insights into the Functional Architecture of IP3 Receptors
Inositol 1,4,5-trisphosphate (IP(3))-evoked Ca(2+) signals display great spatiotemporal malleability. This malleability depends on diversity in both the cellular organization and in situ functionality of IP(3) receptors (IP(3)Rs) that regulate Ca(2+) release from the endoplasmic reticulum (ER). Recent experimental data imply that these considerations are not independent, such that-as with other ion channels-the local organization of IP(3)Rs impacts their functionality, and reciprocally IP(3)R activity impacts their organization within native ER membranes. Here, we (i) review experimental data that lead to our understanding of the "functional architecture" of IP(3)Rs within the ER, (ii) propose an updated terminology to span the organizational hierarchy of IP(3)Rs observed in intact cells, and (iii) speculate on the physiological significance of IP(3)R socialization in Ca(2+) dynamics, and consequently the emerging need for modeling studies to move beyond gridded, planar, and static simulations of IP(3)R clustering even over short experimental timescales.
Molecular Determinants Dictating Cell Surface Expression of the Human Sodium-dependent Vitamin C Transporter-2 in Human Liver Cells
The human sodium-dependent vitamin C transporter-2 (hSVCT2) plays an important role in cellular accumulation of ascorbic acid in liver cells. However, little is known about the molecular determinants that direct hSVCT2 to the cell surface in hepatocytes. We addressed this issue using live cell imaging methods to resolve the distribution and trafficking of truncated or mutated hSVCT2 constructs in a cellular model of human hepatocytes, HepG2 cells. Whereas a full-length hSVCT2-yellow fluorescent protein (YFP) fusion protein was functionally expressed at the cell surface in HepG2 cells, serial truncation and mutation analysis demonstrated an essential role for both NH(2)- and COOH-terminal sequence(s) for cell surface expression and function. Video-rate confocal imaging showed evidence of dynamic hSVCT2-YFP containing intracellular trafficking vesicles, the motility of which was impaired following disruption of microtubules using nocodazole. However, in a HepG2 cell line stably expressing hSVCT2-YFP at the cell surface, plasma membrane levels of hSVCT2 were unaffected by inhibition of microtubule-associated motor proteins; rather, surface expression of hSVCT2-YFP was increased following treatment with myosin inhibitors. Together, these results show that 1) both NH(2)- and COOH-terminal sequences are essential for proper localization of hSVCT2, 2) cell surface delivery is dependent on intact microtubules, and 3) peripheral microfilaments regulate insertion and retrieval of hSVCT2 into the plasma membrane.
An Ancestral Deuterostome Family of Two-pore Channels Mediates Nicotinic Acid Adenine Dinucleotide Phosphate-dependent Calcium Release from Acidic Organelles
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent and widespread calcium-mobilizing messenger, the properties of which have been most extensively described in sea urchin eggs. The molecular basis for calcium release by NAADP, however, is not clear and subject to controversy. Recent studies have provided evidence that members of the two-pore channel (TPC) family in mammals are the long sought after target channels for NAADP. Here, we show that the TPC3 gene, which has yet to be functionally characterized, is present throughout the deuterostome lineage but is a pseudogene in humans and other primates. We report the molecular cloning of the complete ancestral TPC gene family from the sea urchin and demonstrate that all three isoforms localize to acidic organelles to mediate NAADP-dependent calcium release. Our data highlight the functional divergence of this novel gene family during deuterostome evolution and provide further evidence that NAADP mediates calcium release from acidic stores through activation of TPCs.
Two-pore Channels: Regulation by NAADP and Customized Roles in Triggering Calcium Signals
NAADP is a potent regulator of cytosolic calcium levels. Much evidence suggests that NAADP activates a novel channel located on an acidic (lysosomal-like) calcium store, the mobilisation of which results in further calcium release from the endoplasmic reticulum. Here, we discuss the recent identification of a family of poorly characterized ion channels (the two-pore channels) as endo-lysosomal NAADP receptors. The generation of calcium signals by these channels is likened to those evoked by depolarisation during excitation-contraction coupling in muscle. We discuss the idea that two-pore channels can mediate a trigger release of calcium which is then amplified by calcium-induced calcium release from the endoplasmic reticulum. This is similar to the activation of voltage-sensitive calcium channels and subsequent mobilisation of sarcoplasmic reticulum calcium stores in cardiac tissue. We suggest that two-pore channels may physically interact with ryanodine receptors to account for more direct release of calcium from the endoplasmic reticulum in analogy with the conformational coupling of voltage-sensitive calcium channels and ryanodine receptors in skeletal muscle. Interaction of two-pore channels with other calcium release channels likely occurs between stores "trans-chatter" and possibly within the same store "cis-chatter". We also speculate that trafficking of two-pore channels through the endo-lysosomal system facilitates interactions with calcium entry channels. Strategic placing of two-pore channels thus provides a versatile means of generating spatiotemporally complex cellular calcium signals.
Inositol (1,4,5)-trisphosphate Receptor Microarchitecture Shapes Ca2+ Puff Kinetics
Inositol (1,4,5)-trisphosphate receptors (IP(3)Rs) release intracellular Ca(2+) as localized Ca(2+) signals (Ca(2+) puffs) that represent the activity of small numbers of clustered IP(3)Rs spaced throughout the endoplasmic reticulum. Although much emphasis has been placed on estimating the number of active Ca(2+) release channels supporting Ca(2+) puffs, less attention has been placed on understanding the role of cluster microarchitecture. This is important as recent data underscores the dynamic nature of IP(3)R transitions between heterogeneous cellular architectures and the differential behavior of IP(3)Rs socialized into clusters. Here, we applied a high-resolution model incorporating stochastically gating IP(3)Rs within a three-dimensional cytoplasmic space to demonstrate: 1), Ca(2+) puffs are supported by a broad range of clustered IP(3)R microarchitectures; 2), cluster ultrastructure shapes Ca(2+) puff characteristics; and 3), loosely corralled IP(3)R clusters (>200 nm interchannel separation) fail to coordinate Ca(2+) puffs, owing to inefficient triggering and impaired coupling due to reduced Ca(2+)-induced Ca(2+) release microwave velocity (<10 nm/s) throughout the channel array. Dynamic microarchitectural considerations may therefore influence Ca(2+) puff occurrence/properties in intact cells, contrasting with a more minimal role for channel number over the same simulated conditions in shaping local Ca(2+) dynamics.
Role of Cysteine Residues in Cell Surface Expression of the Human Riboflavin Transporter-2 (hRFT2) in Intestinal Epithelial Cells
The water-soluble vitamin B2 (riboflavin, RF) is an essential micronutrient for normal cell function and survival. Recent studies have identified a role for the human riboflavin transporter-2 (hRFT2) in normal intestinal RF absorption. However, little is known about the cell biology of this transporter and specifically about the molecular determinant(s) that dictate its cell surface expression in human intestinal epithelial cells. Here we show that the full-length hRFT2 protein fused to green fluorescent protein (GFP) (GFP-hRFT2) is expressed exclusively at the apical membrane domain of Caco-2 cells. COOH-terminal sequence was essential in dictating cell surface expression with a specific role for conserved cysteine residues (C463 and C467). Mutation of C463 and C467 ablated RF uptake, explained by retention of the constructs within the endoplasmic reticulum. Modeling analysis suggested a potential disulfide bridge between C463 and C386. Consistent with this prediction, mutating the C386 site in the context of the full-length transporter resulted in intracellular retention, whereas mutation of another conserved cysteine (C326A) was without effect on hRFT2 targeting. Intracellular trafficking of hRFT2 was also examined and appeared to involve distinct vesicular structures, the motility of vesicles critically dependent on an intact microtubule network. These results demonstrate a potential role for specific cysteine residues in the cell surface expression of the hRFT2 in human intestinal epithelial cells.
The Endo-lysosomal System As an NAADP-sensitive Acidic Ca(2+) Store: Role for the Two-pore Channels
Accumulating evidence suggests that the endo-lysosomal system provides a substantial store of Ca(2+) that is tapped by the Ca(2+)-mobilizing messenger, NAADP. In this article, we review evidence that NAADP-mediated Ca(2+) release from this acidic Ca(2+) store proceeds through activation of the newly described two-pore channels (TPCs). We discuss recent advances in defining the sub-cellular targeting, topology and biophysics of TPCs. We also discuss physiological roles and the evolution of this ubiquitous ion channel family.
Differential Expression of Human Riboflavin Transporters -1, -2, and -3 in Polarized Epithelia: a Key Role for HRFT-2 in Intestinal Riboflavin Uptake
Transport of riboflavin (RF) across both the brush border membrane (BBM) and basolateral membrane (BLM) of the polarized enterocyte occurs via specific carrier-mediated mechanisms. Although, three human riboflavin transporters (hRFTs), i.e., hRFT-1, hRFT-2 and hRFT-3 are expressed in the intestine, little is known about the cell surface domain(s) at which these specific hRFTs are expressed. Here, we used live cell confocal imaging of intestinal epithelial Caco-2 and renal MDCK cells to show that the hRFT-1 is mainly expressed at the BLM, hRFT-2 is exclusively expressed at the apical membrane, while hRFT-3 is mostly localized inside intracellular vesicular structures (with some expression at the BLM). Further the level of hRFT-2 mRNA expression in Caco-2 cells and in native human intestine is significantly higher than that of hRFT-1 and -3; hRFT-2 was also more efficient in transporting 3H-RF than hRFT-1 and -3. These findings implied an important role for hRFT-2 in intestinal RF uptake, a conclusion that was further supported by findings of hRFT-2 gene-specific siRNA knockdown investigation. These results show that members of the hRFT family are differentially expressed in polarized epithelia, and that the apically expressed hRFT-2 plays a key role in intestinal RF accumulation.
Opposing Roles of Voltage-gated Ca2+ Channels in Neuronal Control of Regenerative Patterning
There is intense interest in developing methods to regulate proliferation and differentiation of stem cells into neuronal fates for the purposes of regenerative medicine. One way to do this is through in vivo pharmacological engineering using small molecules. However, a key challenge is identification of relevant signaling pathways and therein druggable targets to manipulate stem cell behavior efficiently in vivo. Here, we use the planarian flatworm as a simple chemical-genetic screening model for nervous system regeneration to show that the isoquinoline drug praziquantel (PZQ) acts as a small molecule neurogenic to produce two-headed animals with integrated CNSs following regeneration. Characterization of the entire family of planarian voltage-operated Ca(2+) channel α subunits (Ca(v)α), followed by in vivo RNAi of specific Ca(v) subunits, revealed that PZQ subverted regeneration by activation of a specific voltage-gated Ca(2+) channel isoform (Ca(v)1A). PZQ-evoked Ca(2+) entry via Ca(v)1A served to inhibit neuronally derived Hedgehog signals, as evidenced by data showing that RNAi of Ca(v)1A prevented PZQ-evoked bipolarity, Ca(2+) entry, and decreases in wnt1 and wnt11-5 levels. Surprisingly, the action of PZQ was opposed by Ca(2+) influx through a closely related neuronal Ca(v) isoform (Ca(v)1B), establishing a novel interplay between specific Ca(v)1 channel isoforms, Ca(2+) entry, and neuronal Hedgehog signaling. These data map PZQ efficacy to specific neuronal Ca(v) complexes in vivo and underscore that both activators (Ca(v)1A) and inhibitors (Ca(v)1B) of Ca(2+) influx can act as small molecule neurogenics in vivo on account of the unique coupling of Ca(2+) channels to neuronally derived polarity cues.
Photoaffinity Labeling of High Affinity Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-Binding Proteins in Sea Urchin Egg
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [(32)P-5-azido]nicotinic acid adenine dinucleotide phosphate ([(32)P-5N(3)]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [(32)P-5N(3)]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N(3)-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [(32)P-5N(3)]NAADP binding was saturable and displayed high affinity (K(d) ∼10 nm) in both binding and photolabeling experiments. [(32)P-5N(3)]NAADP photolabeling was irreversible in a high K(+) buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [(32)P-5N(3)]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs.
Photoaffinity Labeling of Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Targets in Mammalian Cells
Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca(2+) from intracellular acidic Ca(2+) stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca(2+) channels that release organellar Ca(2+) in response to NAADP. However, little is known about the mechanism coupling NAADP binding to calcium release. To identify the NAADP binding site, we employed a photoaffinity labeling method using a radioactive photoprobe based on 5-azido-NAADP ([(32)P-5N(3)]NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca(2+) signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N(3)-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca(2+) release. Surprisingly, we were unable to demonstrate labeling of endogenous, or overexpressed, TPCs. Furthermore, labeling of high affinity NAADP binding sites was preserved in pancreatic samples from TPC1 and TPC2 knock-out mice. These photolabeling data suggest that an accessory component within a larger TPC complex is responsible for binding NAADP that is unique from the core channel itself. This observation necessitates critical evaluation of current models of NAADP-triggered activation of the TPC family.
