Fluorescent probes to evaluate the physiological state and activity of microbial biocatalysts: a guide for prokaryotic and eukaryotic investigation

Many fluorescent techniques are employed to evaluate the viability and activity of microbial cells used in biotechnology. These techniques are sometimes complex and the interpretation of results opened to misunderstanding. Moreover, new developments are constantly proposed especially concerning a more accurate evaluation of the state of the cells including eukaryotic microorganisms. This paper aims at presenting to biotechnologists unfamiliar with fluorescence the principles of these methods and the related possible pitfalls. It focuses on probes of the physical (integrity and fluidity) and energetical (intracellular pH and membrane potential) state of the cell membrane (bacterial and yeast cells) and presents also other probes (nucleic acids, respiration...) and new technical trends. The specificities of Gram-negative bacterial cells are also discussed.     

Synthesis,characterization, and cellular localization of a fluorescent probe of the antimalarial 8-aminoquinoline primaquine

Primaquine (PQ) is the only commercially available drug that clears dormant liver stages of malaria and blocks transmission to mosquito vectors. Although an old drug, much remains to be known about the mechanism(s) of action. Herein we develop a fluorescent tagged PQ to discover cellular localization in the human malaria parasite, Plasmodium falciparum. Successful synthesis and characterization of a primaquine-coumarin fluorescent probe (PQCP) demonstrated potency equivalent to the parent drug and the probe was not cytotoxic to HepG2 carcinoma cells. Cellular localization was found primarily in the cytosol of the asexual erythrocytic and gametocyte stages of parasite development.     

Phospholamban binds with differential affinity to calcium pump conformers

To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. Electrical pacing of cardiac myocytes elicited cytoplasmic Ca(2+) elevations, but these increases in Ca(2+) produced only modest changes in SERCA-PLB FRET. The data suggest that the regulatory complex is not disrupted by elevations of cytosolic calcium during cardiac contraction (systole). This conclusion was also supported by parallel experiments in heterologous cells, which showed that FRET was reduced but not abolished by calcium. Thapsigargin also elicited a small decrease in PLB-SERCA binding affinity. We propose that PLB is not displaced from SERCA by high calcium during systole, and relief of functional inhibition does not require dissociation of the regulatory complex. The observed modest reduction in the affinity of the PLB-SERCA complex with Ca(2+) or thapsigargin suggests that the binding interface is altered by SERCA conformational changes. The results are consistent with multiple modes of PLB binding or alternative binding sites.     

Fate of fluorescent microspheres in developing Ictalurus punctatus following prolonged immersion

Particulate antigen uptake by the mucosa of developing channel catfish was determined by immersing larvae and fry [2-day post-hatch (dph), 1-, 2-, 3-, 4-, and 8-week post-hatch (wph)] to two forms of fluorescent microspheres (FMS): blue FMS were carboxylated, and green FMS were coated via conjugation with a crude extract of Edwardsiella ictaluri outer membrane protein (OMP). Phagocytosis, destination, and clearance appeared similar for the two types of FMS used. In the older age classes, primary uptake was observed in epithelial cells of the torso, fins, nares and to a lesser extent the gills. Fluorescent microspheres were less frequently observed within mononuclear phagocytes in the epidermis, dermis and underlying connective tissue of the tissue mentioned above. Limited FMS trafficking was observed from 4- to 24-h post-immersion (hpi). Significantly higher numbers of FMS (blue and green)/mm(3) of tissue were observed in the posterior kidney of the 4- and 8-wph age classes and in the anterior kidney and spleen of the 8-wph age class when compared to younger age classes (p < 0.05). Significantly higher FMS (blue and green)/mm(3) of tissue were observed in the posterior kidney of 4- and 8-wph fish when compared to all other organs (p < 0.05). The present study indicates that FMS uptake increases with age in channel catfish. The younger age classes may possess an increased ability to exclude particulate antigen, or lack the specific mechanisms that needed to take up particulates in the form of FMS.     

Transcriptional suppression of nephrin in podocytes by macrophages: roles of inflammatory cytokines and involvement of the PI3K/Akt pathway

Expression of nephrin, a crucial component of the glomerular slit diaphragm, is downregulated in patients with proteinuric glomerular diseases. Using conditionally immortalized reporter podocytes, we found that bystander macrophages as well as macrophage-derived cytokines IL-1beta and TNF-alpha markedly suppressed activity of the nephrin gene promoter in podocytes. The cytokine-initiated repression was reversible, observed on both basal and inducible expression, independent of Wilms' tumor suppressor WT1, and caused in part via activation of the phosphatidylinositol-3-kinase/Akt pathway. These results indicated a novel mechanism by which activated macrophages participate in the induction of proteinuria in glomerular diseases.     

Preapoptotic chromatin changes induced by ultraviolet B irradiation in human erythroleukemia K562 cells

Exponentially growing human erythroleukemia K562 cells were permeabilized and the dose dependent decrease of DNA synthesis rate was measured after ultraviolet (UV B, 290 nm) irradiation. Cells were able to overcome 2 and 5 J/m2 UV doses, partial recovery was observed at 15 J/m2, while at high (25 J/m2) UV dose replicative DNA synthesis remained suppressed. K562 cells were subjected to synchronization prior to and after UV irradiation (24 J/m2) and 18 fractions were collected by centrifugal elutriation. Cell cycle analysis by flow cytometry did not show early apoptotic cells after UV irradiation. The gradual increase in DNA content typical for non-irradiated cells was contrasted by an early S phase block between 2.2 and 2.4 C-values after UV irradiation. Cell cycle dependent chromatin changes after ultraviolet irradiation were seen as a fine fibrillary network covering the mainly fibrous chromatin structures and incompletely folded primitive chromosomes. Based on observations after UV irradiation and on earlier results with cadmium treatment and gamma irradiation, we confirm that typical chromatin changes characteristic to genotoxic agents can be recognized and classified.     

Use of fluorescent microscopy in defining a small ligule-like ovuliferous scale of a permineralized taxodiaceous cone from the upper cretaceous of Hokkaido,Japan

Fluorescent microscopy was proved to be effective for structural identification of permineralized plant tissues in calcite nodules from the Upper Cretaceous of Hokkaido, Japan. A minute, scale-like projection on the bract of a fossil Taxodiaceous cone is identified as a true ovuliferous scale because it is bordered with a continuous epidermis that exhibits prominent fluorescence. The presence of the ovuliferous scale suggests that the fossil is aTaiwania archetype.     

Fluorescent substrate analog for monitoring chain elongation by undecaprenyl pyrophosphate synthase in real time

Farnesyl pyrophosphate (FPP) is a common substrate for a variety of prenyltransferases for synthesizing isoprenoid compounds. In this study, (2E,6E)-8-O-(N-methyl-2-aminobenzoyl)-3,7-dimethyl-2,6-octandien-1-pyrophosphate (MANT-O-GPP), a fluorescent analog of FPP, was synthesized and demonstrated as a satisfactory substrate for Escherichia coli undecaprenyl pyrophosphate synthase (UPPS) with a K_m of 1.5 μM and a k_cat of 1.2 s⁻¹ based on [¹⁴C]IPP consumption. Interesting, we found that its emission fluorescence intensity at 420 nm increased remarkably during chain elongation, thereby useful for real-time monitoring kinetics of UPPS to yield a K_m of 1.1 μM and a k_cat of 1.0 s⁻¹, consistent with those measured using radiolabeled substrate. Using this assay, the IC₅₀ of a known UPPS inhibitor farnesyl thiopyrophosphate (FsPP) was confirmed. Our studies provide a convenient and environmentally friendly alternative for kinetics and inhibition studies on UPPS drug target.