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1.  5AtRabD2b and AtRabD2c have overlapping functions in pollen development and pollen tube growth 
BMC Plant Biology  2011;11:25.
Rab GTPases are important regulators of endomembrane trafficking, regulating exocytosis, endocytosis and membrane recycling. Many Rab-like proteins exist in plants, but only a subset have been functionally characterized.
Here we report that AtRabD2b and AtRabD2c play important roles in pollen development, germination and tube elongation. AtrabD2b and AtrabD2c single mutants have no obvious morphological changes compared with wild-type plants across a variety of growth conditions. An AtrabD2b/2c double mutant is also indistinguishable from wild-type plants during vegetative growth; however its siliques are shorter than those in wild-type plants. Compared with wild-type plants, AtrabD2b/2c mutants produce deformed pollen with swollen and branched pollen tube tips. The shorter siliques in the AtrabD2b/2c double mutant were found to be primarily due to the pollen defects. AtRabD2b and AtRabD2c have different but overlapping expression patterns, and they are both highly expressed in pollen. Both AtRabD2b and AtRabD2c protein localize to Golgi bodies.
These findings support a partially redundant role for AtRabD2b and AtRabD2c in vesicle trafficking during pollen tube growth that cannot be fulfilled by the remaining AtRabD family members.
PMCID: PMC3040128  PMID: 21269510
2.  Red fluorescent proteins for imaging Zymoseptoria tritici during invasion of wheat☆ 
Fungal Genetics and Biology  2015;79:132-140.
•We investigate brightness and photo-stability of RFPs in live Z. tritici cells.•mCherry is most useful in epi-fluorescence and confocal laser scanning microscopy.•The combination of mCherry and an orange-shifted filter set proves to provide brightest signals.•We provide 4 vectors with various mRFPs for yeast recombination based cloning.•The vectors carry carboxin resistance and integrate as single copies into the sdi1 locus.
The use of fluorescent proteins (FPs) in plant pathogenic fungi provides valuable insight into their intracellular dynamics, cell organization and invasion mechanisms. Compared with green-fluorescent proteins, their red-fluorescent “cousins” show generally lower fluorescent signal intensity and increased photo-bleaching. However, the combined usage of red and green fluorescent proteins allows powerful insight in co-localization studies. Efficient signal detection requires a bright red-fluorescent protein (RFP), combined with a suitable corresponding filter set. We provide a set of four vectors, suitable for yeast recombination-based cloning that carries mRFP, TagRFP, mCherry and tdTomato. These vectors confer carboxin resistance after targeted single-copy integration into the sdi1 locus of Zymoseptoria tritici. Expression of the RFPs does not affect virulence of this wheat pathogen. We tested all four RFPs in combination with four epi-fluorescence filter sets and in confocal laser scanning microscopy, both in and ex planta. Our data reveal that mCherry is the RFP of choice for investigation in Z. tritici, showing highest signal intensity in epi-fluorescence, when used with a Cy3 filter set, and laser scanning confocal microscopy. However, mCherry bleached significantly faster than mRFP, which favors this red tag in long-term observation experiments. Finally, we used dual-color imaging of eGFP and mCherry expressing wild-type strains in planta and show that pycnidia are formed by single strains. This demonstrates the strength of this method in tracking the course of Z. tritici infection in wheat.
PMCID: PMC4502450  PMID: 26092800
FPs, fluorescent proteins; RFP, red fluorescent protein; mCherry, monomeric cherry; TagRFP, monomeric red (orange) fluorescent protein; tub2, α tubulin; mRFP, monomeric red fluorescent protein; eGFP, enhanced green fluorescent protein; tdTomato, tandem dimeric red fluorescent protein; sdi1, succinate dehydrogenase 1; dpi, days post infection; ROI, region of interest; n, sample size; Red fluorescent protein; Colocalization; Protein localization; Wheat pathogenic fungus; Septoria tritici blotch; Mycosphaerella graminicola
3.  The Anaplasma phagocytophilum-occupied vacuole selectively recruits Rab-GTPases that are predominantly associated with recycling endosomes 
Cellular microbiology  2010;12(9):1292-1307.
Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to reside within a host cell-derived vacuole. The A. phagocytophilum-occupied vacuole (ApV) fails to mature along the endocytic pathway and is non-fusogenic with lysosomes. Rab GTPases regulate membrane traffic. To better understand how the bacterium modulates the ApV’s selective fusogencity, we examined the intracellular localization of 20 green fluorescent protein (GFP) or red fluorescent protein (RFP)-tagged Rab GTPases in A. phagocytophilum infected HL-60 cells. GFP-Rab4A, GFP-Rab10, GFP-Rab11A, GFP-Rab14, RFP-Rab22A, and GFP-Rab35, which regulate endocytic recycling, and GFP-Rab1, which mediates endoplasmic reticulum to Golgi apparatus trafficking, localize to the ApV. Fluorescently tagged Rabs are recruited to the ApV upon its formation and remain associated throughout infection. Endogenous Rab14 localizes to the ApV. Tetracycline treatment concomitantly promotes loss of recycling endosome-associated GFP-Rabs and acquisition of GFP-Rab5, GFP-Rab7, and the lysosomal marker, LAMP-1. Wild-type and GTPase-deficient versions, but not GDP-restricted versions of GFP-Rab1, GFP-Rab4A, and GFP-Rab11A localize to the ApV. Strikingly, GFP-Rab10 recruitment to the ApV is guanine nucleotide-independent. These data establish that A. phagocytophilum selectively recruits Rab GTPases that are primarily associated with recycling endosomes to facilitate its intracellular survival and implicate bacterial proteins in regulating Rab10 membrane cycling on the ApV.
PMCID: PMC2923681  PMID: 20345488
4.  Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity 
This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb3+ and from sensitized Tb3+ to acceptor—the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds), pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated.
PMCID: PMC4519970  PMID: 26204836
FRET-sensor; caspase-3; terbium; fluorescent proteins; apoptosis
5.  Extended Stokes Shift in Fluorescent Proteins: Chromophore–Protein Interactions in a Near-Infrared TagRFP675 Variant 
Scientific Reports  2013;3:1847.
Most GFP-like fluorescent proteins exhibit small Stokes shifts (10–45 nm) due to rigidity of the chromophore environment that excludes non-fluorescent relaxation to a ground state. An unusual near-infrared derivative of the red fluorescent protein mKate, named TagRFP675, exhibits the Stokes shift, which is 30 nm extended comparing to that of the parental protein. In physiological conditions, TagRFP675 absorbs at 598 nm and emits at 675 nm that makes it the most red-shifted protein of the GFP-like protein family. In addition, its emission maximum strongly depends on the excitation wavelength. Structures of TagRFP675 revealed the common DsRed-like chromophore, which, however, interacts with the protein matrix via an extensive network of hydrogen bonds capable of large flexibility. Based on the spectroscopic, biochemical, and structural analysis we suggest that the rearrangement of the hydrogen bond interactions between the chromophore and the protein matrix is responsible for the TagRFP675 spectral properties.
PMCID: PMC3654500  PMID: 23677204
6.  Pressure Induced Changes in the Fluorescence Behavior of Red Fluorescent Proteins 
The journal of physical chemistry. B  2012;116(34):10311-10316.
We present an experimental study on the fluorescence behavior of the red fluorescent proteins TagRFP-S, TagRFP-T, mCherry, mOrange2, mStrawberry, and mKO as a function of pressure up to several GPa. TagRFP-S, TagRFP-T, mOrange2, and mStrawberry show an initial increase in fluorescence intensity upon application of pressure above ambient conditions. At higher pressures, the fluorescence intensity decreases dramatically for all proteins under study, probably due to denaturing of the proteins. Small blue shifts in the fluorescence spectra with increasing pressure were seen in all proteins under study, hinting at increased rigidity of the chromophore environment. In addition, mOrange2 and mStrawberry exhibit strong and abrupt changes in their fluorescence spectra at certain pressures. These changes are likely due to structural modifications of the hydrogen bonding environment of the chromophore. The strong differences in behavior between proteins with identical or very similar chromophores highlight how the chromophore environment contributes to pressure-induced behavior of the fluorescence performance.
PMCID: PMC4022145  PMID: 22861177
Red Fluorescent Protein; mFruit; Hydrostatic Pressure; Protein Chromophore; Fluorescence Spectroscopy
7.  Vam7p, a SNAP-25-Like Molecule, and Vam3p, a Syntaxin Homolog, Function Together in Yeast Vacuolar Protein Trafficking 
Molecular and Cellular Biology  1998;18(9):5308-5319.
A genetic screen to isolate gene products required for vacuolar morphogenesis in the yeast Saccharomyces cerevisiae identified VAM7, a gene which encodes a protein containing a predicted coiled-coil domain homologous to the coiled-coil domain of the neuronal t-SNARE, SNAP-25 (Y. Wada and Y. Anraku, J. Biol. Chem. 267:18671–18675, 1992; T. Weimbs, S. H. Low, S. J. Chapin, K. E. Mostov, P. Bucher, and K. Hofmann, Proc. Natl. Acad. Sci. USA 94:3046–3051, 1997). Analysis of a temperature-sensitive-for-function (tsf) allele of VAM7 (vam7tsf) demonstrated that the VAM7 gene product directly functions in vacuolar protein transport. vam7tsf mutant cells incubated at the nonpermissive temperature displayed rapid defects in the delivery of multiple proteins that traffic to the vacuole via distinct biosynthetic pathways. Examination of vam7tsf cells at the nonpermissive temperature by electron microscopy revealed the accumulation of aberrant membranous compartments that may represent unfused transport intermediates. A fraction of Vam7p was localized to vacuolar membranes. Furthermore, VAM7 displayed genetic interactions with the vacuolar syntaxin homolog, VAM3. Consistent with the genetic results, Vam7p physically associated in a complex containing Vam3p, and this interaction was enhanced by inactivation of the yeast NSF (N-ethyl maleimide-sensitive factor) homolog, Sec18p. In addition to the coiled-coil domain, Vam7p also contains a putative NADPH oxidase p40phox (PX) domain. Changes in two conserved amino acids within this domain resulted in synthetic phenotypes when combined with the vam3tsf mutation, suggesting that the PX domain is required for Vam7p function. This study provides evidence for the functional and physical interaction between Vam7p and Vam3p at the vacuolar membrane, where they function as part of a t-SNARE complex required for the docking and/or fusion of multiple transport intermediates destined for the vacuole.
PMCID: PMC109116  PMID: 9710615
8.  HOPS drives vacuole fusion by binding the vacuolar SNARE complex and the Vam7 PX domain via two distinct sites 
Molecular Biology of the Cell  2011;22(14):2601-2611.
The homotypic fusion and protein sorting (HOPS) tethering complex of the yeast vacuole is involved in multiple fusion reactions. We demonstrate that HOPS has two binding sites for SNAREs and that binding to the minimal SNARE complex is necessary for HOPS-stimulated fusion. Our data highlight the dual role of HOPS in Rab-mediated tethering and SNARE-driven fusion.
Membrane fusion within the endomembrane system follows a defined order of events: membrane tethering, mediated by Rabs and tethers, assembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes, and lipid bilayer mixing. Here we present evidence that the vacuolar HOPS tethering complex controls fusion through specific interactions with the vacuolar SNARE complex (consisting of Vam3, Vam7, Vti1, and Nyv1) and the N-terminal domains of Vam7 and Vam3. We show that homotypic fusion and protein sorting (HOPS) binds Vam7 via its subunits Vps16 and Vps18. In addition, we observed that Vps16, Vps18, and the Sec1/Munc18 protein Vps33, which is also part of the HOPS complex, bind to the Q-SNARE complex. In agreement with this observation, HOPS-stimulated fusion was inhibited if HOPS was preincubated with the minimal Q-SNARE complex. Importantly, artificial targeting of Vam7 without its PX domain to membranes rescued vacuole morphology in vivo, but resulted in a cytokinesis defect if the N-terminal domain of Vam3 was also removed. Our data thus support a model of HOPS-controlled membrane fusion by recognizing different elements of the SNARE complex.
PMCID: PMC3135484  PMID: 21613544
9.  F-Actin Dynamics in Neurospora crassa ▿ † 
Eukaryotic Cell  2010;9(4):547-557.
This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.
PMCID: PMC2863416  PMID: 20139238
10.  Mammalian Late Vacuole Protein Sorting Orthologues Participate in Early Endosomal Fusion and Interact with the Cytoskeleton 
Molecular Biology of the Cell  2004;15(3):1197-1210.
In Saccharomyces cerevisiae, the class C vacuole protein sorting (Vps) proteins, together with Vam2p/Vps41p and Vam6p/Vps39p, form a complex that interacts with soluble N-ethylmaleimide-sensitive factor attachment protein receptor and Rab proteins to “tether” vacuolar membranes before fusion. To determine a role for the corresponding mammalian orthologues, we examined the function, localization, and protein interactions of endogenous mVps11, mVps16, mVps18, mVam2p, and mVam6. We found a significant proportion of these proteins localized to early endosome antigen-1 and transferrin receptor-positive early endosomes in Vero, normal rat kidney, and Chinese hamster ovary cells. Immunoprecipitation experiments showed that mVps18 not only interacted with Syntaxin (Syn)7, vesicle-associated membrane protein 8, and Vti1-b but also with Syn13, Syn6, and the Sec1/Munc18 protein mVps45, which catalyze early endosomal fusion events. Moreover, anti-mVps18 antibodies inhibited early endosome fusion in vitro. Mammalian mVps18 also associated with mVam2 and mVam6 as well as with the microtubule-associated Hook1 protein, an orthologue of the Drosophila Hook protein involved in endosome biogenesis. Using in vitro binding and immunofluorescence experiments, we found that mVam2 and mVam6 also associated with microtubules, whereas mVps18, mVps16, and mVps11 associated with actin filaments. These data indicate that the late Vps proteins function during multiple soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated fusion events throughout the endocytic pathway and that their activity may be coordinated with cytoskeletal function.
PMCID: PMC363107  PMID: 14668490
11.  FLIM-FRET Imaging of Caspase-3 Activity in Live Cells Using Pair of Red Fluorescent Proteins 
Theranostics  2012;2(2):215-226.
We report a new technique to detect enzyme activity inside cells. The method based on Fluorescence Lifetime Imaging (FLIM) technology allows one to follow sensor cleavage by proteolytic enzyme caspase-3. Specifically, we use the FLIM FRET of living cells via the confocal fluorescence microscopy. A specially designed lentivector pLVT with the DNA fragment of TagRFP-23-KFP was applied for transduction of A549 cell lines. Computer simulations are carried out to estimate FRET efficiency and to analyze possible steric restrictions of the reaction between the substrate TagRFP-23-KFP and caspase-3 dimer. Successful use of the fuse protein TagRFP-23-KFP to register the caspase-3 activation based on average life-time measurements is demonstrated. We show that the average life-time distribution is dramatically changed for cells with the modified morphology that is typical for apoptosis. Namely, the short-lived component at 1.8-2.1 ns completely disappears and the long-lived component appears at 2.4-2.6 ns. The latter is a fingerprint of the TagRFP molecule released after cleavage of the TagRFP-23-KFP complex by caspase-3. Analysis of life-time distributions for population of cells allows us to discriminate apoptotic and surviving cells within single frame and to peform statistical analysis of drug efficiency. This system can be adjusted for HTS by using special readers oriented on measurements of fluorescence life-time.
PMCID: PMC3287422  PMID: 22375160
FRET; FLIM; red fluorescent proteins (RFP); caspase; molecular dynamics (MD); lentiviral vector.
12.  Structure and Distribution of Organelles and Cellular Location of Calcium Transporters in Neurospora crassa▿  
Eukaryotic Cell  2009;8(12):1845-1855.
We wanted to examine the cellular locations of four Neurospora crassa proteins that transport calcium. However, the structure and distribution of organelles in live hyphae of N. crassa have not been comprehensively described. Therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. We observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated membranes. Proteins of the vacuolar membrane, encoded by vam-3 and vma-1, were in an interconnected network of small tubules and vesicles near the hyphal tip, while in more distal regions they were in large and small spherical vacuoles. Mitochondria, visualized with tagged ARG-4, were abundant in all regions of the hyphae. Similarly, we tagged the four N. crassa proteins that transport calcium with green or red fluorescent protein to examine their cellular locations. NCA-1 protein, a homolog of the SERCA-type Ca2+-ATPase of animal cells, colocalized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca2+-ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They colocalized with markers in the vacuolar membrane, and they also occurred in the plasma membrane in regions of the hyphae more than 1 mm from the tip. The cax gene encodes a Ca2+/H+ exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. We observed, approximately 50 to 100 μm from the tip, a few spherical organelles that had high amounts of tagged CAX protein and tagged subunits of the vacuolar ATPase (VMA-1 and VMA-5). We suggest that this organelle, not described previously in N. crassa, may have a role in sequestering calcium.
PMCID: PMC2794220  PMID: 19801418
13.  Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells 
Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP tagged clathrin light-chain. Analysis of the resulting sptPALM images revealed that single molecule transmembrane proteins, which are internalized into a cell via endocytosis, co-localize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.
PMCID: PMC2866019  PMID: 20394363
14.  Multi-color imaging of the bacterial nucleoid and division proteins with blue, orange, and near-infrared fluorescent proteins 
Studies of the spatiotemporal protein dynamics within live bacterial cells impose a strong demand for multi-color imaging. Despite the increasingly large collection of fluorescent protein (FP) variants engineered to date, only a few of these were successfully applied in bacteria. Here, we explore the performance of recently engineered variants with the blue (TagBFP), orange (TagRFP-T, mKO2), and far-red (mKate2) spectral colors by tagging HU, LacI, MinD, and FtsZ for visualizing the nucleoid and the cell division process. We find that, these FPs outperformed previous versions in terms of brightness and photostability at their respective spectral range, both when expressed as cytosolic label and when fused to native proteins. As this indicates that their folding is sufficiently fast, these proteins thus successfully expand the applicable spectra for multi-color imaging in bacteria. A near-infrared protein (eqFP670) is found to be the most red-shifted protein applicable to bacteria so far, with brightness and photostability that are advantageous for cell-body imaging, such as in microfluidic devices. Despite the multiple advantages, we also report the alarming observation that TagBFP directly interacts with TagRFP-T, causing interference of localization patterns between their fusion proteins. Our application of diverse FPs for endogenous tagging provides guidelines for future engineering of fluorescent fusions in bacteria, specifically: (1) The performance of newly developed FPs should be quantified in vivo for their introduction into bacteria; (2) spectral crosstalk and inter-variant interactions between FPs should be carefully examined for multi-color imaging; and (3) successful genomic fusion to the 5′-end of a gene strongly depends on the translational read-through of the inserted coding sequence.
PMCID: PMC4469896  PMID: 26136737
fluorescent proteins; bacterial cell division; bacterial chromosome; MinD; HU; FtsZ; TagRFP-T; TagBFP
15.  Myosin Va mediates Rab8A-regulated GLUT4 vesicle exocytosis in insulin-stimulated muscle cells 
Molecular Biology of the Cell  2014;25(7):1159-1170.
Skeletal muscle is the main site of insulin-dependent glucose uptake. Insulin inactivation of the Akt substrate Rab-GAP AS160 leads to Rab8A activation in myocytes. The molecular motor myosin Va is a Rab8A effector in this pathway, leading to GLUT4 translocation to the myocyte surface, linking signal transduction to vesicle traffic.
Rab-GTPases are important molecular switches regulating intracellular vesicle traffic, and we recently showed that Rab8A and Rab13 are activated by insulin in muscle to mobilize GLUT4-containing vesicles to the muscle cell surface. Here we show that the unconventional motor protein myosin Va (MyoVa) is an effector of Rab8A in this process. In CHO-IR cell lysates, a glutathione S-transferase chimera of the cargo-binding COOH tail (CT) of MyoVa binds Rab8A and the related Rab10, but not Rab13. Binding to Rab8A is stimulated by insulin in a phosphatidylinositol 3-kinase–dependent manner, whereas Rab10 binding is insulin insensitive. MyoVa-CT preferentially binds GTP-locked Rab8A. Full-length green fluorescent protein (GFP)–MyoVa colocalizes with mCherry-Rab8A in perinuclear small puncta, whereas GFP–MyoVa-CT collapses the GTPase into enlarged perinuclear depots. Further, GFP–MyoVa-CT blocks insulin-stimulated translocation of exofacially myc-tagged GLUT4 to the surface of muscle cells. Mutation of amino acids in MyoVa-CT predicted to bind Rab8A abrogates both interaction with Rab8A (not Rab10) and inhibition of insulin-stimulated GLUT4myc translocation. Of importance, small interfering RNA–mediated MyoVa silencing reduces insulin-stimulated GLUT4myc translocation. Rab8A colocalizes with GLUT4 in perinuclear but not submembrane regions visualized by confocal total internal reflection fluorescence microscopy. Hence insulin signaling to the molecular switch Rab8A connects with the motor protein MyoVa to mobilize GLUT4 vesicles toward the muscle cell plasma membrane.
PMCID: PMC3967978  PMID: 24478457
16.  A Multispecificity Syntaxin Homologue, Vam3p, Essential for Autophagic and Biosynthetic Protein Transport to the Vacuole  
The Journal of Cell Biology  1997;138(3):517-529.
Protein transport in eukaryotic cells requires the selective docking and fusion of transport intermediates with the appropriate target membrane. t-SNARE molecules that are associated with distinct intracellular compartments may serve as receptors for transport vesicle docking and membrane fusion through interactions with specific v-SNARE molecules on vesicle membranes, providing the inherent specificity of these reactions. VAM3 encodes a 283–amino acid protein that shares homology with the syntaxin family of t-SNARE molecules. Polyclonal antiserum raised against Vam3p recognized a 35-kD protein that was associated with vacuolar membranes by subcellular fractionation. Null mutants of vam3 exhibited defects in the maturation of multiple vacuolar proteins and contained numerous aberrant membrane-enclosed compartments. To study the primary function of Vam3p, a temperature-sensitive allele of vam3 was generated (vam3tsf). Upon shifting the vam3tsf mutant cells to nonpermissive temperature, an immediate block in protein transport through two distinct biosynthetic routes to the vacuole was observed: transport via both the carboxypeptidase Y pathway and the alkaline phosphatase pathway was inhibited. In addition, vam3tsf cells also exhibited defects in autophagy. Both the delivery of aminopeptidase I and the docking/ fusion of autophagosomes with the vacuole were defective at high temperature. Upon temperature shift, vam3tsf cells accumulated novel membrane compartments, including multivesicular bodies, which may represent blocked transport intermediates. Genetic interactions between VAM3 and a SEC1 family member, VPS33, suggest the two proteins may act together to direct the docking and/or fusion of multiple transport intermediates with the vacuole. Thus, Vam3p appears to function as a multispecificity receptor in heterotypic membrane docking and fusion reactions with the vacuole. Surprisingly, we also found that overexpression of the endosomal t-SNARE, Pep12p, suppressed vam3Δ mutant phenotypes and, likewise, overexpression of Vam3p suppressed the pep12Δ mutant phenotypes. This result indicated that SNAREs alone do not define the specificity of vesicle docking reactions.
PMCID: PMC2141632  PMID: 9245783
17.  Trans-Golgi network localized small GTPase RabA1d is involved in cell plate formation and oscillatory root hair growth 
BMC Plant Biology  2014;14:252.
Small Rab GTPases are important regulators of vesicular trafficking in plants. AtRabA1d, a member of the RabA1 subfamily of small GTPases, was previously found in the vesicle-rich apical dome of growing root hairs suggesting a role during tip growth; however, its specific intracellular localization and role in plants has not been well described.
The transient expression of 35S::GFP:RabA1d construct in Allium porrum and Nicotiana benthamiana revealed vesicular structures, which were further corroborated in stable transformed Arabidopsis thaliana plants. GFP-RabA1d colocalized with the trans-Golgi network marker mCherry-VTI12 and with early FM4-64-labeled endosomal compartments. Late endosomes and endoplasmic reticulum labeled with FYVE-DsRed and ER-DsRed, respectively, were devoid of GFP-RabA1d. The accumulation of GFP-RabA1d in the core of brefeldin A (BFA)-induced-compartments and the quantitative upregulation of RabA1d protein levels after BFA treatment confirmed the association of RabA1d with early endosomes/TGN and its role in vesicle trafficking. Light-sheet microscopy revealed involvement of RabA1d in root development. In root cells, GFP-RabA1d followed cell plate expansion consistently with cytokinesis-related vesicular trafficking and membrane recycling. GFP-RabA1d accumulated in disc-like structures of nascent cell plates, which progressively evolved to marginal ring-like structures of the growing cell plates. During root hair growth and development, GFP-RabA1d was enriched at root hair bulges and at the apical dome of vigorously elongating root hairs. Importantly, GFP-RabA1d signal intensity exhibited an oscillatory behavior in-phase with tip growth. Progressively, this tip localization dissapeared in mature root hairs suggesting a link between tip localization of RabA1d and root hair elongation. Our results support a RabA1d role in events that require vigorous membrane trafficking.
RabA1d is located in early endosomes/TGN and is involved in vesicle trafficking. RabA1d participates in both cell plate formation and root hair oscillatory tip growth. The specific GFP-RabA1d subcellular localization confirms a correlation between its specific spatio-temporal accumulation and local vesicle trafficking requirements during cell plate and root hair formation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0252-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4180857  PMID: 25260869
Arabidopsis; Cell plate; Cytokinesis; Rab GTPase; Localization; RabA1d; Root hair; Trans-Golgi network; Vesicle
18.  Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells 
Optics Express  2010;18(11):11148-11158.
Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960’s. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 µm and 10 µm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications.
PMCID: PMC3408954  PMID: 20588974
(170.2520) Fluorescence microscopy; (170.2150) Endoscopic imaging; (170.3650 Lifetime-based sensing
19.  SNAP-23 regulates phagosome formation and maturation in macrophages 
Molecular Biology of the Cell  2012;23(24):4849-4863.
Using macrophages overexpressing or reducing SNAP-23, this study shows that SNAP-23 is implicated in phagosome formation and maturation, presumably by mediating SNARE-based membrane traffic. Indeed, a conformational change in SNAP-23 structure based on FRET signal is observed on the phagosome membrane of cells overexpressing the lysosomal SNARE VAMP7.
Synaptosomal associated protein of 23 kDa (SNAP-23), a plasma membrane–localized soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE), has been implicated in phagocytosis by macrophages. For elucidation of its precise role in this process, a macrophage line overexpressing monomeric Venus–tagged SNAP-23 was established. These cells showed enhanced Fc receptor–mediated phagocytosis. Detailed analyses of each process of phagocytosis revealed a marked increase in the production of reactive oxygen species within phagosomes. Also, enhanced accumulation of a lysotropic dye, as well as augmented quenching of a pH-sensitive fluorophore were observed. Analyses of isolated phagosomes indicated the critical role of SNAP-23 in the functional recruitment of the NADPH oxidase complex and vacuolar-type H+-ATPase to phagosomes. The data from the overexpression experiments were confirmed by SNAP-23 knockdown, which demonstrated a significant delay in phagosome maturation and a reduction in uptake activity. Finally, for analyzing whether phagosomal SNAP-23 entails a structural change in the protein, an intramolecular Förster resonance energy transfer (FRET) probe was constructed, in which the distance within a TagGFP2-TagRFP was altered upon close approximation of the N-termini of its two SNARE motifs. FRET efficiency on phagosomes was markedly enhanced only when VAMP7, a lysosomal SNARE, was coexpressed. Taken together, our results strongly suggest the involvement of SNAP-23 in both phagosome formation and maturation in macrophages, presumably by mediating SNARE-based membrane traffic.
PMCID: PMC3521691  PMID: 23087210
20.  A hyperactive piggyBac transposon system is an easy-to-implement method for introducing foreign genes into mouse preimplantation embryos 
Transgenic mice are important tools for genetic analysis. A current prominent method for producing transgenic mice involves pronuclear microinjection into 1-cell embryos. However, the total transgenic efficiency obtained using this method is less than 10%. Here, we demonstrate that highly efficient transgenesis in mice can be achieved by cytoplasmic microinjection using a hyperactive piggyBac system. In embryos in which hyPBase mRNA and pPB-CAG-TagRFP DNA were co-injected into the cytoplasm, TagRFP fluorescence was observed after the 2-cell stage; when 30 ng/µl pPB-CAG-TagRFP DNA and 30 ng/µl hyPBase mRNA were co-injected, 94.4% of blastocysts were TagRFP positive. Furthermore, a high concentration of hyPBase mRNA resulted in creation of mosaic embryos in which the TagRFP signals partially disappeared. However, suitable concentrations of injected DNA and hyPBase mRNA produced embryos in which almost all blastomeres were TagRFP positive. Thus, the hyperactive piggyBac transposon system is an easy-to-implement and highly effective method that can contribute to production of transgenic mice.
PMCID: PMC4498375  PMID: 25740401
Cytoplasmic injection; Hyperactive piggyBac transposase; Transgenic mice
21.  Practical and reliable FRET/FLIM pair of fluorescent proteins 
BMC Biotechnology  2009;9:24.
In spite of a great number of monomeric fluorescent proteins developed in the recent years, the reported fluorescent protein-based FRET pairs are still characterized by a number of disadvantageous features, complicating their use as reporters in cell biology and for high-throughput cell-based screenings.
Here we screened some of the recently developed monomeric protein pairs to find the optimal combination, which would provide high dynamic range FRET changes, along with high pH- and photo-stability, fast maturation and bright fluorescence, and reliable detection in any fluorescent imaging system. Among generated FRET pairs, we have selected TagGFP-TagRFP, combining all the mentioned desirable characteristics. On the basis of this highly efficient FRET pair, we have generated a bright, high contrast, pH- and photo-stable apoptosis reporter, named CaspeR3 (Caspase 3 Reporter).
The combined advantages suggest that the TagGFP-TagRFP is one of the most efficient green/red couples available to date for FRET/FLIM analyses to monitor interaction of proteins of interest in living cells and to generate FRET-based sensors for various applications. CaspeR3 provides reliable detection of apoptosis, and should become a popular tool both for cell biology studies and high throughput screening assays.
PMCID: PMC2678114  PMID: 19321010
22.  Conversion of red fluorescent protein into a bright blue probe 
Chemistry & biology  2008;15(10):1116-1124.
We utilized a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a novel rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic background such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which were converted into blue probes. Further improvement of a blue variant of TagRFP using random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by substantially higher brightness, faster chromophore maturation and higher pH stability than blue fluorescence proteins with a histidine in the chromophore. Detailed biochemical and photochemical analysis indicates mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging as well as the outstanding donor for green fluorescent proteins in FRET applications.
PMCID: PMC2585067  PMID: 18940671
23.  Molecular Mechanism of Membrane Docking by the Vam7p PX Domain*Ⓢ 
The Journal of biological chemistry  2006;281(48):37091-37101.
The Vam7p t-SNARE is an essential component of the vacuole fusion machinery that mediates membrane trafficking and protein sorting in yeast. Vam7p is recruited to vacuoles by its N-terminal PX domain that specifically recognizes PtdIns(3)P in the bilayers, however the precise mechanism of membrane anchoring remains unclear. Here we describe a molecular basis for membrane targeting and penetration by the Vam7p PX domain based on structural and quantitative analysis of its interactions with lipids and micelles. Our results derived from in vitro binding measurements using NMR, monolayer surface tension experiments and mutagenesis reveal a multivalent membrane docking mechanism involving specific PtdIns(3)P recognition that is facilitated by electrostatic interactions and accompanying hydrophobic insertion. Both the hydrophobic and electrostatic components enhance the Vam7p PX domain association with PtdIns(3)P-containing membranes. The inserting Val70, Leu71, and Trp75 residues located next to the PtdIns(3)P binding pocket are surrounded by a basic patch, which is involved in nonspecific electrostatic contacts with acidic lipids, such as PtdSer. Substitution of the insertion residues significantly reduces the binding and penetrating power of the Vam7p PX domain and leads to cytoplasmic redistribution of the EGFP-tagged protein. The affinities of the PX domain for PtdIns(3)P and other lipids reveal a remarkable synergy within the multivalent complex that stably anchors Vam7p at the vacuolar membrane.
PMCID: PMC1838524  PMID: 16984909
24.  Vam3, a resveratrol dimer, inhibits cigarette smoke-induced cell apoptosis in lungs by improving mitochondrial function 
Acta Pharmacologica Sinica  2014;35(6):779-791.
To investigate the effects of Vam3 (a resveratrol dimer extracted from Vitis amurensis Rupr) on cigarette smoke (CS)-induced cell apoptosis in lungs in vitro and in vivo and the underlying mechanisms of action.
Human bronchial epithelial cell line BEAS-2B was exposed to cigarette smoke condensate (CSC, 300 mg/L), and cell apoptosis was determined using flow cytometry and Hoechst staining. Mitochondrial membrane potential was examined with TMRE staining. ROS and ceramide levels were detected with DCFH-DA fluorescence and HPLC-MS/MS, respectively. Cytochrome c release was detected using immunofluorescence. Caspase-9 and neutral sphingomyelinase 2 expression was measured with Western blotting. The breast carcinoma cell line MCF7 stably expressing GFP-tagged Bax was used to elucidate the role of mitochondria in CS-induced apoptosis. For in vivo study, male mice were exposed to CS for 5 min twice a day for 4 weeks. The mice were orally administered Vam3 (50 mg·kg−1·d−1) or resveratrol (30 mg·kg−1·d−1) each day 1 h before the first CS exposure.
Pretreatment of BEAS-2B cells with Vam3 (5 μmol/L) or resveratrol (5 μmol/L) significantly suppressed CSC-induced apoptosis, and prevented CSC-induced Bax level increase in the mitochondria, mitochondrial membrane potential loss, cytochrome c release and caspase-9 activation. Furthermore, pretreatment of BEAS-2B cells with Vam3 or resveratrol significantly suppressed CSC-stimulated intracellular ceramide production, and CSC-induced upregulation of neutral sphingomyelinase 2, the enzyme responsible for ceramide production in bronchial epithelial cells. Similar results were obtained in C6-pyridinium ceramide-induced apoptosis of GFP-Bax-stable MCF7 cells in vitro, and in the lungs of CS-exposed mice that were treated with oral administration of Vam3 or resveratrol.
Vam3 protects bronchial epithelial cells from CS-induced apoptosis in vitro and in vivo by preventing mitochondrial dysfunction.
PMCID: PMC4086386  PMID: 24747163
Vam3; resveratrol; lung; bronchial epithelial cell; cigarette smoking; apoptosis; Bax; mitochondria; ceramide; sphingomyelinase
25.  Human Vam6p promotes lysosome clustering and fusion in vivo 
The Journal of Cell Biology  2001;154(1):109-122.
Regulated fusion of mammalian lysosomes is critical to their ability to acquire both internalized and biosynthetic materials. Here, we report the identification of a novel human protein, hVam6p, that promotes lysosome clustering and fusion in vivo. Although hVam6p exhibits homology to the Saccharomyces cerevisiae vacuolar protein sorting gene product Vam6p/Vps39p, the presence of a citron homology (CNH) domain at the NH2 terminus is unique to the human protein. Overexpression of hVam6p results in massive clustering and fusion of lysosomes and late endosomes into large (2–3 μm) juxtanuclear structures. This effect is reminiscent of that caused by expression of a constitutively activated Rab7. However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7. Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein. Both the CNH and clathrin heavy chain repeat domains are required for induction of lysosome clustering and fusion. This study implicates hVam6p as a mammalian tethering/docking factor characterized with intrinsic ability to promote lysosome fusion in vivo.
PMCID: PMC2196876  PMID: 11448994
lysosome biogenesis; vacuolar protein sorting; vesicle tethering; vesicle docking; lysosome fusion

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