Pier Alberto Benedetti

Modulation of mitochondrial respiration by nitric oxide: investigation by single cell fluorescence microscopy

With the electro‐driven import of rhodamine 123, we used single cell fluorescence microscopy to single out the contribution of nitric oxide (NO) in controlling mitochondrial membrane potential expressed by (stationary growing) rhabdomyosarcoma and neuroblastoma cells in culture. The experimental design and the computer‐aided image analysis detected and quantitated variations of fluorescence signals specific to mitochondria. We observed that 1) the two cell lines display changes of fluorescence dependent on mitochondrial energization states; 2) mitochondrial fluorescence decreases after exposure of the cells to a NO releaser; 4) the different fluorescence intensity measured under stationary growing conditions, or after activation and inhibition of constitutive NO synthase, is consistent with a steady‐state production of NO. Direct comparison of single cell fluorescence with bulk cytofluorimetry proved that the results obtained by the latter method may be misleading because of the intrinsic‐to‐measure lack of information about distribution of fluorescence within different cell compartments. The kinetic parameters describing the reactions between cytochrome oxidase, NO, and O2 may account for the puzzling (20‐fold) increase of the KM for O2 reported for cells and tissues as compared to purified cytochrome c oxidase, allowing an estimate of in vivo NO flux.—Sarti, P., Lendaro, E., Ippoliti, R., Bellelli, A., Benedetti, P. A., Brunori, M. Modulation of mitochondrial respiration by nitric oxide: investigation by single cell fluorescence microscopy. FASEB J. 13, 191–197 (1999)

Endocytosis of a chimera between human pro-urokinase and the plant toxin saporin: an unusual internalization mechanism

A fluorescent derivative of a chimeric toxin between human pro‐urokinase and the plant ribo‐some‐inactivating protein saporin (p‐uPA‐SapTRITC), has been prepared in order to study the endocytosis of this potentially antimetastatic conjugate in the murine model cell line LB6 clone19 (Cl19) transfected with the human urokinase receptor gene. The physiological internalization of urokinase‐inhibitor complexes is triggered by the interaction of plasminogen inhibitors (PAIs) with receptors belonging to the low density lipoprotein‐related receptor protein (LRP) family, and involves a macro‐quaternary structure including uPAR, LRP, and PAIs. However, in contrast to this mechanism, we observed a two‐step process: first, the urokinase receptor (uPAR) acts as the anchoring factor on the plasma membrane; subsequently, LRP acts as the endocytic trigger. Once the chimera is bound to the plasma membrane by interaction with uPAR, we suggest that a possible exchange may occur to transfer the toxin to LRP via the saporin moiety and begin the internalization. So an unusual endocytic process is described, where the toxin enters the cell via a receptor different from that used to bind the plasma membrane.—Ippoliti, R., Lendaro, E., Benedetti, P. A., Torrisi, M. R., Belleudi, F., Carpani, D., Soria, M. R., Fabbrini, M. S. Endocytosis of a chimera between human pro‐urokinase and the plant toxin saporin: an unusual internalization mechanism. FASEB J. 14, 1335–1344 (2000)

Intracellular dynamics of ricin followed by fluorescence microscopy on living cells reveals a rapid accumulation of the dimeric toxin in the Golgi apparatus.

The intracellular dynamics of fluorescent conjugates of the toxic lectin ricin was followed by video fluorescence microscopy on living CHO cells, demonstrating that the ricin heterodimer and its isolated B chain, after binding to the plasma membrane receptors, migrate to and accumulate in the Golgi apparatus following internalization. A ricin derivative labelled with fluorescein on the A chain and rhodamine on the B chain did not display significant splitting of the A‐B heterodimer during translocation of the toxin to the Golgi; this novel finding provides support for the hypothesis that further processing of ricin takes place in this cellular compartment.

Kinetics of the reaction of intraerythrocytic haemoglobin by single cell microspectroscopy: effect of shape and osmolarity.

Microspectroscopy Camel hemoglobin Red blood cell Diffusion Osmolarity Cell volume

Single cell microspectroscopy reveals that erythrocytes containing hemoglobin S retain a ‘memory’ of previous sickling cycles

Red blood cells from patients homozygotes for hemoglobin S (HbS) have been studied using a computer‐controlled microspectrophotometer, which allows measurements of spectra and dynamics to be undertaken in a single erythrocyte. Complete photodissociation of HbCO results in polymerization of intracellular deoxyhemoglobin S and deformation of the cell. This is associated with a delayed optical change, which, for the same cell, was found to be highly reproducible between repeated cycles of sickling. Comparison of photographic records and absorbance time courses indicates that an erythrocyte, once having undergone a photochemically induced sickling event, always deforms along the same axis during subsequent cycles. This behaviour implies that the cell retains a ‘memory’ of its previous cycle(s) possibly via slow relaxations of the membrane. In addition, rebinding of CO to intracellular hemoglobin was found to be slower if measured after deformation of the cell, with possible important implications for the pathological mechanism of sickling.

A saporin-insulin conjugate: Synthesis and biochemical characterization

Saporin, a single-chain, non-cytotoxic, ribosome-inactivating protein from Saponaria officinalis, was chemically linked to the hormone insulin in a 1:1 complex. To follow by dynamic video microscopy the endocytosis and intracellular transport in vivo, a second covalent conjugate with a saporin derivative labelled with fluorescein isothiocyanate was also prepared. Both conjugates were characterized with reference to homogeneity, stoichiometry, optical spectroscopy and toxicity. Both were found to exhibit scarce toxicity toward both CHO and HEP G2 cells; optical video microscopy on living cells indicates that reduced toxicity may be (partly) due to a very limited binding of the saporin-insulin conjugate to membrane receptors. These results suggest a strategy for new possible covalent conjugates of saporin with alternative and specific macromolecular carriers.

Measurement of spatial variation of responsiveness in solid-state imager

An experimental setup that provides the measurement of the spatial variation of the responsiveness of a single photodiode belonging to a linear metal-oxide-semiconductor (MOS) photoarray is described. An optical microscanning technique has been employed to sequentially illuminate a small area of a single photoelement in order to achieve the mapping of its spatial responsiveness. Experimental results for a single pixel are graphically presented.

Single cell observations of gas reactions and shape changes in normal and sickling erythrocytes.

Microspectrophotometry has been applied to single red blood cells to reinvestigate the linked processes of diffusion of gases inside the erythrocyte and their combination with hemoglobin. The experiments took advantage of the photosensitivity of the cabron monoxide derivative of hemoglobin, which allows ligand release from the CO-saturated red cells under strong illumination and recombination when the light is switched off. The photochemical method was also used to study the kinetics of sickling on ligand removal in single erythrocytes of Hb S carriers. The results give new information on the mechanism of the sickling process.