Imaging of caspase-3 activation in HeLa cells stimulated with etoposide using a novel fluorescent probe.

Microscopic visualization of intracellular enzyme activity can provide information about the physiological role of the enzyme. Caspases are cysteine proteases that have critical roles in the execution of apoptosis. General fluorometric substrates of caspase-3, such as DEVD-MCA, are unsuitable for imaging because they are excited at short wavelength, so we designed and synthesized novel fluorescent probes that are excited at suitable wavelengths for detecting caspase-3 activity in living cells. Using one of these probes, we succeeded in microscopic visualization of caspase-3-like activity within HeLa cells treated with etoposide. The caspase-3-like activity was increased in the cytosol at first, then expanded to the whole cell.     

Membrane fluidity aspects in endocytosis; a study with the fluorescent probe trimethylamino-diphenylhexatriene in L929 cells

The fluorescent hydrophobic plasma membrane probe, trimethylamino-diphenylhexatriene (TMA-DPH) was previously shown to follow the plasma membrane throughout its internalization and recycling process and thus to behave as a marker for endo- and exocytosis in living cell systems. In this paper, we made use of these properties to investigate membrane fluidity effects associated with endocytosis in L929 cells. For that purpose we performed TMA-DPH fluorescence anisotrophy measurements which showed that endocytosis starts from particularly rigid regions of the plasma membrane (probably coated pits). The fluorescence anisotropy then continuously decreases to a lower limit corresponding to the membrane fluidity of the probe in the lysosomial membrane. Strikingly, the value of this limit is identical to the average anisotropy value in the peripheral membrane, which suggests that lysosomes and plasma membrane may have a similar phospholipidic composition and a possible common origin.     

The lipopolysaccharide barrier: correlation of antibiotic susceptibility with antibiotic permeability and fluorescent probe binding kinetics

Lipopolysaccharide (LPS), the primary lipid on the surface of Gram-negative bacteria, is thought to act as a permeability barrier, making the outer membrane relatively impermeable to hydrophobic antibiotics, detergents, and host proteins. Mutations in the LPS biosynthetic apparatus increase bacterial susceptibility to such agents. To determine how this increased susceptibility is mediated, we have correlated antibiotic susceptibilities of rough (antibiotic resistant) and deep rough (antibiotic susceptible) bacterial strains with antibiotic permeabilities and fluorescent probe binding kinetics for bilayers composed of LPS purified from the same strains. Bilayer permeabilities of two hydrophobic beta-lactam antibiotics were measured by encapsulating the appropriate beta-lactamases in large unilamellar vesicles. In the presence of MgCl(2), permeabilities of LPS bilayers from rough and deep rough bacteria were similar and significantly lower than those of bacterial phospholipids (BPL). Addition of BPL to the LPS bilayers increased their antibiotic permeability to approximately the level of the BPL bilayers. Binding rates of the fluorescent probe bis-aminonaphthylsulfonic acid (BANS) were 2 orders of magnitude slower for both rough and deep rough LPS bilayers compared to that of bilayers composed of BPL or mixtures of LPS and BPL. On the basis of these results and the observation that deep rough bacteria have higher levels of phospholipid on their surface than do rough bacteria (Kamio, Y., and Nikaido, H. (1976) Biochemistry 15, 2561-2569), we argue that the high susceptibility of deep rough bacteria is due to the presence of phospholipids on their surface. Experiments with phospholipid bilayers showed that the addition of PEG-lipids (containing covalently attached hydrophilic polymers) had little effect on permeability and binding rates, whereas the addition of cholesterol reduced permeability and slowed binding to levels approaching those of LPS. Therefore, we argue that the barrier provided by LPS is primarily due to its tight hydrocarbon chain packing (Snyder et al., (1999) Biochemistry 38, 10758-10767) rather than to its polysaccharide headgroup.     

Secretion of endoplasmic reticulum aminopeptidase 1 is involved in the activation of macrophages induced by lipopolysaccharide and interferon-gamma

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme with an important role in processing antigenic peptides presented to class I major histocompatibility complex in the endoplasmic reticulum. In this study, we found that endoplasmic reticulum-retained ERAP1 was secreted from macrophages in response to activation by treatment with lipopolysaccharide (LPS) and interferon (IFN)-γ and enhanced their phagocytic activity. Enhancement of the phagocytic activity of murine macrophage RAW264.7 cells induced by LPS/IFN-γ was inhibited by a potent aminopeptidase inhibitor, amastatin. The addition of recombinant wild-type but not inactive mutant ERAP1 to culture medium enhanced phagocytosis. These results suggest that enhancement of phagocytic activity is at least in part mediated by secreted ERAP1 through the generation of active peptides processed by the enzyme. Our data reveal ERAP1-mediated activation of macrophages for the first time and will provide new insights into the role of this enzyme in innate immunity.     

Lysosomal accumulation and recycling of lipopolysaccharide to the cell surface of murine macrophages, an in vitro and in vivo study.

In this study, we detailed in a time-dependent manner the trafficking, the recycling, and the structural fate of Brucella abortus LPS in murine peritoneal macrophages by immunofluorescence, ELISA, and biochemical analyses. The intracellular pathway of B. abortus LPS, a nonclassical endotoxin, was investigated both in vivo after LPS injection in the peritoneal cavity of mice and in vitro after LPS incubation with macrophages. We also followed LPS trafficking after infection of macrophages with B. abortus strain 19. After binding to the cell surface and internalization, Brucella LPS is routed from early endosomes to lysosomes with unusual slow kinetics. It accumulates there for at least 24 h. Later, LPS leaves lysosomes and reaches the macrophage cell surface. This recycling pathway is also observed for LPS released by Brucella S19 following in vitro infection. Indeed, by 72 h postinfection, bacteria are degraded by macrophages and LPS is located inside lysosomes dispersed at the cell periphery. From 72 h onward, LPS is gradually detected at the plasma membrane. In each case, the LPS present at the cell surface is found in large clusters with the O-chain facing the extracellular medium. Both the antigenicity and heterogenicity of the O-chain moiety are preserved during the intracellular trafficking. We demonstrate that LPS is not cleared by macrophages either in vitro or in vivo after 3 mo, exposing its immunogenic moiety toward the extracellular medium.     

IFN-gamma/lipopolysaccharide activation of macrophages is associated with protein kinase C-dependent down-modulation of the colony-stimulating factor-1 receptor.

IFN gamma/LPS treatment increases macrophage tumoricidal and microbicidal activity and inhibits CSF-1-induced macrophage proliferation. The mechanism underlying the latter effect was investigated in the CSF-1-dependent mouse macrophage cell line, BAC-1.2F5. IFN-gamma and LPS together dramatically reduced the total number of CSF-1 receptors (CSF-1R) via selective degradation of the cell surface form. Processing and transport of intracellular CSF-1R to the cell surface were unaffected. IFN-gamma alone had no effect but significantly enhanced LPS-induced CSF-1R down-regulation. The reduction in CSF-1R number was protein kinase C-dependent and involved changes in serine phosphorylation of the receptor at different sites. CSF-1R down-modulation by this mechanism may be important in switching off the energy-consuming processes of CSF-1R-mediated proliferation and chemotaxis in activated macrophages.     

Internalization of the lipophilic fluorescent probe trimethylamino-diphenylhexatriene follows the endocytosis and recycling of the plasma membrane in cells

The lipophilic fluorescent probe trimethylamino-diphenylhexatriene (TMA-DPH) has been shown previously to behave as a marker of plasma membrane in living cell systems, and it has therefore been widely used in membrane fluidity studies via fluorescence anisotropy measurements. However, progressive internalization of this probe in cells could lead to unsuitable interferences, when long incubations times were required. The mechanism of this internalization had not yet been elucidated. We present here fluorescence-intensity kinetic results and fluorescence micrographic data on L929 cells and on mouse bone-marrow macrophages, which allow us to identify the mechanism as fluid-phase pinocytosis: the probe remains associated with the plasma membrane throughout its internalization-recycling flow and it is finally concentrated in lysosomes. The study was facilitated by the partition equilibrium property of TMA-DPH between plasma membranes and the external aqueous medium, which allowed to immediately distinguish the internalized fraction of the probe from the peripheral labelling, by simply washing cells. This conclusion is confirmed by the features of the influence of temperature on TMA-DPH internalization.     

Development of a fluorescent probe for the study of nucleosome assembly and dynamics

To develop a probe for use in real-time dynamic studies of nucleosomes, core histones (from Drosophila) were conjugated to a DNA-intercalating dye, thiazole orange, by a reaction targeting Cys 110 of histone H3. In the absence of DNA, the conjugated histones are only very weakly fluorescent. However, upon reconstitution into nucleosomes by standard salt dialysis procedures, the probe fluoresces strongly, reflecting its ability to intercalate into the nucleosomal DNA. The probe is also sensitive to the nature of the DNA-histone interaction. Nucleosomes reconstituted by stepwise salt dialysis give a fluorescence signal quite different from that of the species formed when DNA and histones are simply mixed in low salt. In addition, changing either the DNA length or the type of sequence (nucleosome positioning sequences versus random DNA of the same size) used in the reconstitution alters the resulting fluorescence yield. The results are all consistent with the conclusion that a more rigid, less flexible nucleosome structure results in less fluorescence than a looser structure, presumably due to structural constraints on dye intercalation. This probe should be well suited to analyzing nucleosome dynamics and to following factor-mediated assembly and remodeling of nucleosomes in real time, particularly at the single-molecule level.     

Highly fluorescent GFPm2+‐based genome integration‐proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages

Study of activity of cloned promoters in slow‐growing Mycobacterium tuberculosis during long‐term growth conditions in vitro or inside macrophages, requires a genome‐integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate‐independent, easily scorable and highly sensitive reporter gene. In order to meet this requirement, we constructed pAKMN2, which contains mycobacterial codon‐optimized gfp_m²⁺ gene, coding for GFP_m²⁺ of highest fluorescence reported till date, mycobacteriophage L5 attP‐int sequence for genome integration, and a multiple cloning site. pAKMN2 showed stable integration and expression of GFP_m²⁺ from M. tuberculosis and M. smegmatis genome. Expression of GFP_m²⁺, driven by the cloned minimal promoters of M. tuberculosis cell division gene, ftsZ (MtftsZ), could be detected in the M. tuberculosis/pAKMN2‐promoter integrants, growing at exponential phase in defined medium in vitro and inside macrophages. Stable expression from genome‐integrated format even without antibiotic, and high sensitivity of detection by flow cytometry and fluorescence imaging, in spite of single copy integration, make pAKMN2 useful for the study of cloned promoters of any mycobacterial species under long‐term in vitro growth or stress conditions, or inside macrophages.     

Interleukin-1beta isolated from a marine fish reveals up-regulated expression in macrophages following activation with lipopolysaccharide and lymphokines.

The gilthead seabream IL-1beta gene consists of five exons/four introns. The complete coding sequence contains a 102 bp 5' untranslated region (UTR), a single open reading frame of 762 bp which translates into a 253 amino acid molecule, and a 407 bp 3'UTR with a polyadenylation signal 14 nucleotides upstream of the poly(A)tail. The seabream sequence has the highest degree of nucleotide (61.7%) and amino acid (53%) identity with the trout IL-1beta sequences. The IL-1beta message was detected by RT-PCR in head-kidney, blood, spleen, liver, gill and peritoneal exudate of both non-infected and Vibrio anguillarum-challenged fish. More importantly, IL-1beta was highly expressed by purified macrophage monolayers and was up-regulated by lipopolysaccharide and lymphocyte-derived macrophage-activating factor stimulation.     

Characterization of the receptor for lymphotoxin; a spontaneous internalization without recycling and ligand-induced downregulation in HL-60 cells

The receptor system for lymphotoxin (LT) was investigated by use of the human promyelocytic HL-60 cell line. Utilizing subcellular fractionation, internalization of cell surface bound recombinant LT (rLT) and transfer to lysosomes followed by degradation was demonstrated. Binding of rLT to its cell surface receptor induced a downregulation of the receptor which was persistent for at least 6 h. Downregulation of the receptors from the cell surface by activation of protein kinase C with the diacylglycerol OAG was completely reversible but the recovery of the binding capacity was dependent on protein synthesis as it was inhibited by cycloheximide. Treatment with cycloheximide alone resulted in a loss of binding capacity with a half life of approximately 2 h, suggesting a spontaneous consumption of receptors. Affinity cross-linking revealed three ligand-receptor complexes with Mr of 85, 105 and 125 kDa. Because of a strong tendency for ligand polymerization these results suggest oligomer binding of the ligand to a single receptor molecule with an apparent Mr of 70 kDa. N-linked glycosylation constituted 4 to 5 kDa of the total molecular weight. In conclusion, we demonstrated a spontaneous internalization of the receptor for LT without recycling, and that ligand binding resulted in an irreversible downregulation of the receptor.     

Activation of calpain I and calpain II: a comparative study using terbium as a fluorescent probe for calcium-binding sites

The present study demonstrates the activation of calpain I and calpain II by micromolar levels of terbium and has utilized the enhancement in the fluorescence of protein-bound terbium to study and compare the calcium binding sites of the two enzymes. Calpain I and calpain II were isolated from bovine erythrocytes and brain, respectively. While the rates of activation of calpain I by terbium and calcium are comparable, the rate of activation of calpain II was much greater in the presence of terbium than in the presence of calcium. Binding of terbium ions to calpains was monitored by the enhanced terbium fluorescence and by the changes in the intrinsic protein fluorescence of calpains. Stoichiometric titrations indicated that calpain I and calpain II bound four and six molar equivalents of terbium ion, respectively. During the titration, the intrinsic protein fluorescence of calpain II was successively quenched whereas that of calpain I showed an abrupt drop just prior to the saturation. The association constants (Ka) increased from 10(5) to 10(7) M-1 for calpain I and from 10(4) to 10(6) M-1 for calpain II with addition of increasing molar equivalents of terbium. Titration of enzymatic activities with calcium showed that the activation of calpain I required fewer molar equivalents of metal ions than were necessary for the activation of calpain II, in agreement with stoichiometric titration with terbium.     

The efflux of a fluorescent probe is catalyzed by an ATP-driven extrusion system in Lactococcus lactis.

Many bacteria, both gram positive and gram negative, extrude in an energy-dependent manner the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5[and -6]-carboxyfluorescein (BCECF) (D. Molenaar, T. Abee, and W. N. Konings, Biochim. Biophys. Acta 1115:75-83, 1991). This efflux was studied in detail in Lactococcus lactis, and several indications that a transport system is involved were found. This transport system is most likely driven by ATP or a related compound. The evidence is that BCECF extrusion (i) occurs against a BCECF gradient, (ii) is strictly correlated with ATP concentration and not with the proton motive force, and (iii) is inhibited by vanadate and to a lesser extent by N,N'-dicyclohexylcarbodiimide. Most convincingly, a UV mutant with a strongly reduced efflux rate was isolated. Such a mutant was isolated from a BCECF-loaded and lactose-energized population by selection of highly fluorescent cells in a flow cytometer-cell sorter. The physiological function of this extrusion system is unknown, but its characteristics classify it among the traffic ATPases.     

The interaction of egg yolk and ram spermatozoa studied with a fluorescent probe.

The flourescent membrane marker, 1-anilinoaphtalene-8-sulphonate (ANS) was used to investigate the attachment of egg-yolk to the plasma membranes of ram spermatozoa. The degree of fluorescence was assessed using a subjective scoring system. It was found that egg yolk competes with ANS for sites on the plasma membrane. When the diluent contained 10% egg yolk, no ANS could be detected on the membranes. Egg yolk attached to the plasma membrane could be removed by washing twice with a yolk-free diluent. Loss of sperm motility in the presence of ANS was observed but some spermotozoa remained motile after incubation at 37 degrees C for 15 min with 2mM-ANS. Egg yolk protected spermatozoa against this loss of motility. It is suggested that egg yolk protects spermatozoa during chilling and freezing by its attachment to the sperm plasma membrane.     

Distance measurements between the metal-binding sites in thermolysin using terbium ion as a fluorescent probe.

A single terbium ion has been introduced into thermolysin replacing two of the four calcium ions, and the fluorescence properties of the protein-bound terbium have been studied. The fluorescence of Tb⁺³ is tremendously enhanced (～7 × 10³) upon binding and is significantly quenched when divalent cobalt is substituted for the zinc ion normally found in the enzyme. By use of the Förster equation for energy transfer the distance between the protein-bound Tb⁺³ and Co⁺² in the active site was calculated to be 13.6±0.5 A. This agrees closely with the value of 13.9 A obtained from the crystal structure and suggests that energy transfer between the two metal ions bound to the protein takes place by a dipole-dipole mechanism.