Modification of rhodamine staining allows identification of hematopoietic stem cells with preferential short-term or long-term bone marrow-repopulating ability.

We have developed a modified rhodamine (Rho) staining procedure to study uptake and efflux in murine hematopoietic stem cells. Distinct populations of Rho++ (bright), Rho+ (dull), and Rho- (negative) cells could be discriminated. Sorted Rho- cells were subjected to a second Rho staining procedure with the P-glycoprotein blocking agent verapamil (VP). Most cells became Rho positive [Rho-/Rho(VP)+ cells] and some remained Rho negative [Rho-/Rho(VP)- cells]. These cell fractions were characterized by their marrow-repopulating ability in a syngeneic, sex-mismatch transplantation model. Short-term repopulating ability was determined by recipient survival for at least 6 weeks after lethal irradiation and transplantation--i.e., radioprotection. Long-term repopulating ability at 6 months after transplantation was measured by fluorescence in situ hybridization with a Y-chromosome-specific probe, by graft function and recipient survival. Marrow-repopulating cells were mainly present in the small Rho- cell fraction. Transplantation of 30 Rho- cells resulted in 50% radioprotection and > 80% donor repopulation in marrow, spleen, and thymus 6 months after transplantation. Cotransplantation of cells from both fractions in individual mice directly showed that within this Rho- cell fraction, the Rho-/Rho(VP)+ cells exhibited mainly short-term and the Rho-/Rho(VP)- cells exhibited mainly long-term repopulating ability. Our results indicate that hematopoietic stem cells have relatively high P-glycoprotein expression and that the cells responsible for long-term repopulating ability can be separated from cells exhibiting short-term repopulating ability, probably by a reduced mitochondrial Rho-binding capacity.

Conformational changes couple Na+ and glucose transport

The mechanism by which cotransport proteins couple their substrates across cell membranes is not known. A commonly proposed model is that cotransport results from ligand-induced conformational transitions that change the accessibility of ligand-binding sites from one side of the membrane to the other. To test this model, we have measured the accessibility of covalent probes to a cysteine residue (Q457C) placed in the putative sugar-translocation domain of the Na+/glucose cotransporter (SGLT1). The mutant protein Q457C was able to transport sugar, but transport was abolished after alkylation by methanethiosulfonate reagents. Alkylation blocked sugar translocation but not sugar binding. Accessibility of Q457C to alkylating reagents required external Na+ and was blocked by external sugar and phlorizin. The voltage dependence of accessibility was directly correlated with the presteady–state charge movement of SGLT1. Voltage-jump experiments with rhodamine-6-maleimide-labeled Q457C showed that the time course and level of changes in fluorescence closely followed the presteady–state charge movement. We conclude that conformational changes are responsible for the coupling of Na+ and sugar transport and that Q457 plays a critical role in sugar translocation by SGLT1.

Probes bound to myosin Cys-707 rotate during length transients in contraction

It is widely conjectured that muscle shortens because portions of myosin molecules (the “cross-bridges”) impel the actin filament to which they transiently attach and that the impulses result from rotation of the cross-bridges. Crystallography indicates that a cross-bridge is articulated–consisting of a globular catalytic/actin-binding domain and a long lever arm that may rotate. Conveniently, a rhodamine probe with detectable attitude can be attached between the globular domain and the lever arm, enabling the observer to tell whether the anchoring region rotates. Well-established signature effects observed in shortening are tension changes resulting from the sudden release or quick stretch of active muscle fibers. In this investigation we found that closely correlated with such tension changes are changes in the attitude of the rhodamine probes. This correlation strongly supports the conjecture about how shortening is achieved.

Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR

A sensitive, labor-saving, and easily automatable nonradioactive procedure named APEX-FCS (amplified probe extension detected by fluorescence correlation spectroscopy) has been established to detect specific in vitro amplification of pathogen genomic sequences. As an example, Mycobacterium tuberculosis genomic DNA was subjected to PCR amplification with the Stoffel fragment of Thermus aquaticus DNA polymerase in the presence of nanomolar concentrations of a rhodamine-labeled probe (third primer), binding to the target in between the micromolar amplification primers. The probe becomes extended only when specific amplification occurs. Its low concentration avoids false-positives due to unspecific hybridization under PCR conditions. With increasing portion of extended probe molecules, the probe’s average translational diffusion properties gradually change over the course of the reaction, reflecting amplification kinetics. Following PCR, this change from a stage of high to a stage of low mobility can directly be monitored during a 30-s measurement using a fluorescence correlation spectroscopy device. Quantitation down to 10 target molecules in a background of 2.5 μg unspecific DNA without post-PCR probe manipulations could be achieved with different primer/probe combinations. The assay holds the promise to concurrently perform amplification, probe hybridization, and specific detection without opening the reaction chamber, if sealable foils are used.

Arp2/3 Complex from Acanthamoeba Binds Profilin and Cross-links Actin Filaments

The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 μM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of α-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.

Memory landscapes of single-enzyme molecules

Immobilized single horseradish peroxidase enzymes were observed by confocal fluorescence spectroscopy during catalysis of the oxidation reaction of the nonfluorescent dihydrorhodamine 6G substrate into the highly fluorescent product rhodamine 6G. By extracting only the non-Markovian behavior of the spectroscopic two-state process of enzyme-product complex formation and release, memory landscapes were generated for single-enzyme molecules. The memory landscapes can be used to discriminate between different origins of stretched exponential kinetics that are found in the first-order correlation analysis. Memory landscapes of single-enzyme data shows oscillations that are expected in a single-enzyme system that possesses a set of transient states. Alternative origins of the oscillations may not, however, be ruled out. The data and analysis indicate that substrate interaction with the enzyme selects a set of conformational substates for which the enzyme is active.

A multidrug resistance transporter from human MCF-7 breast cancer cells

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 663-aa member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.

Heterogeneous transcription of an indoleacetic acid biosynthetic gene in Erwinia herbicola on plant surfaces

We investigated the spatial pattern of expression of ipdC, a plant inducible gene involved in indoleacetic acid biosynthesis in Erwinia herbicola, among individual cells on plants to gain a better understanding of the role of this phenotype in the epiphytic ecology of bacteria and the factors involved in the regulation of ipdC. Nonpathogenic E. herbicola strain 299R harboring a transcriptional fusion of ipdC to gfp was inoculated onto bean plants, recovered from individual leaves 48 h after inoculation, and subjected to fluorescence in situ hybridization using a 16S rRNA oligonucleotide probe specific to strain 299R. Epifluorescence images captured through a rhodamine filter were used to distinguish the 5carboxytetramethylrhodamine-labeled cells of strain 299R from other leaf microflora. Quantification of the green fluorescence intensity of individual cells by analysis of digital images revealed that about 65% of the 299R cells recovered from bean leaves had higher ipdC expression than in culture. Additionally, 10% of the cells exhibited much higher levels of green fluorescence than the median fluorescence intensity, indicating that they are more heterogeneous with respect to ipdC expression on plants than in culture. Examination of 299R cells in situ on leaf surfaces by confocal laser scanning microscopy after fluorescence in situ hybridization of cells on leaf samples showed that even cells that were in close proximity exhibited dramatically different green fluorescence intensities, and thus, were in a physical or chemical microenvironment that induced differential expression of ipdC.

Cell Cycle-Dependent Changes in Microtubule Dynamics in Living Cells Expressing Green Fluorescent Protein-α TubulinV⃞

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-α tubulin construct and a cell line permanently expressing GFP-α tubulin was established (LLCPK-1α). The mitotic index and doubling time for LLCPK-1α were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1α cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1α and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1α cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.

Actin-Bundling Protein Isolated from Pollen Tubes of Lily : Biochemical and Immunocytochemical Characterization

A 135-kD actin-bundling protein was purified from pollen tubes of lily (Lilium longiflorum) using its affinity to F-actin. From a crude extract of the pollen tubes, this protein was coprecipitated with exogenously added F-actin and then dissociated from F-actin by treating it with high-ionic-strength solution. The protein was further purified sequentially by chromatography on a hydroxylapatite column, a gel-filtration column, and a diethylaminoethyl-cellulose ion-exchange column. In the present study, this protein is tentatively referred to as P-135-ABP (Plant 135-kD Actin-Bundling Protein). By the elution position from a gel-filtration column, we estimated the native molecular mass of purified P-135-ABP to be 260 kD, indicating that it existed in a dimeric form under physiological conditions. This protein bound to and bundled F-actin prepared from chicken breast muscle in a Ca2+-independent manner. The binding of 135-P-ABP to actin was saturated at an approximate stoichiometry of 26 actin monomers to 1 dimer of P-135-ABP. By transmission electron microscopy of thin sections, we observed cross-bridges between F-actins with a longitudinal periodicity of 31 nm. Immunofluorescence microscopy using rhodamine-phalloidin and antibodies against the 135-kD polypeptide showed that P-135-ABP was colocalized with bundles of actin filaments in lily pollen tubes, leading us to conclude that it is the factor responsible for bundling the filaments.

Single-molecule spectroscopy of the β2 adrenergic receptor: Observation of conformational substates in a membrane protein

Single-molecule studies of the conformations of the intact β2 adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the β2 adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.

TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors

To achieve an efficient intracellular drug and DNA delivery, attempts were made to target microparticulate drug carriers into cytoplasm bypassing the endocytotic pathway. TAT peptides derived from the HIV-1 TAT protein facilitate intracellular delivery of proteins and small colloidal particles. We demonstrated that relatively large drug carriers, such as 200-nm liposomes, can also be delivered into cells by TAT peptide attached to the liposome surface. Liposomes were fluorescently labeled with membranotropic rhodamine-phosphatidylethanolamine or by entrapping FITC-dextran. Incubation of fluorescent TAT liposomes with mouse Lewis lung carcinoma cells, human breast tumor BT20 cells, and rat cardiac myocyte H9C2 results in intracellular localization of certain liposomes. Steric hindrances for TAT peptide⋅cell interaction (attachment of TAT directly to the liposome surface without spacer or the presence of a high MW polyethylene glycol on the liposome surface) abolish liposome internalization, evidencing the importance of direct contact of TAT peptide with the cell surface. Low temperature or metabolic inhibitors, sodium azide or iodoacetamide, have little influence on the translocation of TAT liposomes into cells, confirming the energy-independent character of this process. The approach may have important implications for drug delivery directly into cell cytoplasm.

Mechanism of oligonucleotide release from cationic liposomes.

We propose a mechanism for oligonucleotide (ODN) release from cationic lipid complexes in cells that accounts for various observations on cationic lipid-nucleic acid-cell interactions. Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/ fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures. ODN displacement from the complex was studied by fluorescent resonance energy transfer. Anionic liposome compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex at about a 1:1 (-/+) charge ratio. Release was independent of ionic strength and pH. Physical separation of the F-ODN from monovalent and multivalent cationic lipids was confirmed by gel electrophoresis. Fluid but not solid phase anionic liposomes are required, whereas the physical state of the cationic lipids does not effect the release. Water soluble molecules with a high negative linear charge density, dextran sulfate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or histone did not release ODN, even at 100-fold charge excess (-/+). Based upon these results, we propose that the complex, after internalization by endocytosis, induces flip-flop of anionic lipids from the cytoplasmic facing monolayer. Anionic lipids laterally diffuse into the complex and form a charged neutralized ion-pair with the cationic lipids. This leads to displacement of the ODN from the cationic lipid and its release into the cytoplasm.

Increased liposome extravasation in selected tissues: effect of substance P.

We have used a pharmacologic mediator to open intercellular connections in selected vessels to allow liposomes to escape from the blood stream and to extravasate into tissues that have appropriate receptors. We have examined the effects of substance P (SP), a peptide known to increase vascular permeability in selected tissues, such as trachea, esophagus, and urinary bladder in rats. We used quantitative fluorescence analysis of tissues to measure two fluorescent markers, one attached to the lipid (rhodamine-phosphatidylethanolamine) and another, doxorubicin (an anti-tumor drug), encapsulated within the aqueous interior. We have also examined the deposition of liposomes microscopically by the use of encapsulated colloidal gold and silver enhancement. Analysis of the biochemical and morphological observations indicate the following: (i) Injection of SP produces a striking increase in both liposome labels, but only in tissues that possess receptors for SP in postcapillary venules; (ii) liposome material in these tissues has extravasated and is found extracellularly near a variety of cells beyond the endothelial layer over the first few hours; (iii) 24 h following injection of liposomes and SP, liposome material is found in these tissues, localized intracellularly in both endothelial cells and macrophages. We propose that appropriate application of tissue-specific mediators can result in liposome extravasation deep within tissues that normally do not take up significant amounts of liposomes from the blood. Such liposomes are able to carry a variety of pharmacological agents that can be released locally within selected target tissues for therapeutic purposes.

Functional expression of mouse Mdr1 in an outer membrane permeability mutant of Escherichia coli.

Functional expression of the multidrug resistance protein P-glycoprotein (P-gp) in Escherichia coli is providing an appropriate system for structure/function studies and might provide an invaluable tool to screen potential P-gp substrates and inhibitors. The major problem encountered in such studies, however, is the impermeability of the outer membrane of Gram-negative bacteria, which protects microorganisms against the cytotoxic effects of many lipophilic cancer drugs and blocks accessibility of P-gp reversal agents. In the present study we have constructed, by mutagenesis, a "leaky" (containing a permeable outer membrane) strain of E. coli, which is significantly more susceptible to the toxic effect of known P-gp substrates and cytotoxic agents. Expression of mouse Mdr1 in the mutant confers cross-resistance to daunomycin, quinidine, chloroquine, rhodamine 6G, and puromycin. Most importantly, reserpine and doxorubicin completely abolish Mdr1-mediated rhodamine resistance. The results provide strong support for previous observations, suggesting that Mdr1 can be expressed functionally in E. coli and indicate that the leaky mutant will be useful for further structure/function studies of the heterologously expressed eukaryotic drug efflux protein.