Victoria E. Centonze

Oxidative stress-induced apoptosis is associated with alterations in mitochondrial caspase activity and Bcl-2-dependent alterations in mitochondrial pH (pHm).

Oxidative stress, the result of cellular production of reactive oxygen species (ROS), has been implicated in a number of diseases of the eye. Exposure of eye tissues (e.g. the cornea and retina) to oxidative stress over time has been hypothesized to underlie the development of age-related macular degeneration (AMD) and maturity onset cataract formation. Light-induced free radicals can damage the eye, and alterations in the antioxidant defenses of the eye have been suggested to play a role in the etiology of glaucoma. Mitochondria are both a major endogenous source and target of ROS, and oxidative stress has been shown to induce apoptotic cell death by targeting the mitochondria directly. Mitochondrial-dependent apoptosis has been shown to require release of cytochrome c from mitochondria and subsequent activation of a specific class of cytoplasmic proteases known as caspases. Bcl-2, an anti-apoptotic protein localized to mitochondria, has been shown to inhibit cytochrome c release and protect against oxidative stress-induced apoptosis. Here we demonstrate that oxidative stress causes activation of mitochondrial matrix caspase-2 and -9 activity that is associated with Bcl-2-inhibitable acidification of mitochondrial pH (pH(m)). In conjunction with recent reports that caspase activation is maximal at acidic pH, these findings have led us to hypothesize that Bcl-2 may modulate cytochrome c release following oxidative stress by modifying the pH-dependent activation of mitochondrial caspase activity. These studies provide an increased understanding of the mechanism(s) by which oxidative stress damages tissues, and may have important therapeutic implications for treatment of opthamological diseases.

Altered Twist1 and Hand2 dimerization is associated with Saethre-Chotzen syndrome and limb abnormalities

Autosomal dominant mutations in the gene encoding the basic helix-loop-helix transcription factor Twist1 are associated with limb and craniofacial defects in humans with Saethre-Chotzen syndrome. The molecular mechanism underlying these phenotypes is poorly understood. We show that ectopic expression of the related basic helix-loop-helix factor Hand2 phenocopies Twist1 loss of function in the limb and that the two factors have a gene dosage–dependent antagonistic interaction. Dimerization partner choice by Twist1 and Hand2 can be modulated by protein kinase A– and protein phosphatase 2A–regulated phosphorylation of conserved helix I residues. Notably, multiple Twist1 mutations associated with Saethre-Chotzen syndrome alter protein kinase A–mediated phosphorylation of Twist1, suggesting that misregulation of Twist1 dimerization through either stoichiometric or post-translational mechanisms underlies phenotypes of individuals with Saethre-Chotzen syndrome.

PKA, PKC, and the protein phosphatase 2A influence HAND factor function: a mechanism for tissue-specific transcriptional regulation.

The bHLH factors HAND1 and HAND2 are required for heart, vascular, neuronal, limb, and extraembryonic development. Unlike most bHLH proteins, HAND factors exhibit promiscuous dimerization properties. We report that phosphorylation/dephosphorylation via PKA, PKC, and a specific heterotrimeric protein phosphatase 2A (PP2A) modulates HAND function. The PP2A targeting-subunit B56delta specifically interacts with HAND1 and -2, but not other bHLH proteins. PKA and PKC phosphorylate HAND proteins in vivo, and only B56delta-containing PP2A complexes reduce levels of HAND1 phosphorylation. During RCHOI trophoblast stem cell differentiation, B56delta expression is downregulated and HAND1 phosphorylation increases. Mutations in phosphorylated residues result in altered HAND1 dimerization and biological function. Taken together, these results suggest that site-specific phosphorylation regulates HAND factor functional specificity.

Development of a multiphoton fluorescence lifetime imaging microscopy system using a streak camera

We report the development and detailed calibration of a multiphoton fluorescence lifetime imaging system (FLIM) using a streak camera. The present system is versatile with high spatial (~0.2 μm) and temporal (~50 psec) resolution and allows rapid data acquisition and reliable and reproducible lifetime determinations. The system was calibrated with standard fluorescent dyes and the lifetime values obtained were in very good agreement with values reported in literature for these dyes. We also demonstrate the applicability of the system to FLIM studies in cellular specimens including stained pollen grains and fibroblast cells expressing green fluorescent protein. The lifetime values obtained matched well with those reported earlier by other groups for these same specimens. Potential applications of the present system include the measurement of intracellular physiology and Fluorescence Resonance Energy Transfer (FRET) imaging which are discussed in the context of live cell imaging.We report the development and detailed calibration of a multiphoton fluorescence lifetime imaging system (FLIM) using a streak camera. The present system is versatile with high spatial (∼0.2 μm) and temporal (∼50 ps) resolution and allows rapid data acquisition and reliable and reproducible lifetime determinations. The system was calibrated with standard fluorescent dyes and the lifetime values obtained were in very good agreement with values reported in the literature for these dyes. We also demonstrate the applicability of the system to FLIM studies in cellular specimens including stained pollen grains and fibroblast cells expressing green florescent protein. The lifetime values obtained matched well with those reported earlier by other groups for these same specimens. Potential applications of the present system include the measurement of intracellular physiology and fluorescence resonance energy transfer imaging, which are discussed in the context of live cell imaging.

Quantitative imaging of protein–protein interactions by multiphoton fluorescence lifetime imaging microscopy using a streak camera

Fluorescence lifetime imaging microscopy (FLIM) using multiphoton excitation techniques is now finding an important place in quantitative imaging of protein-protein interactions and intracellular physiology. Recent developments in multiphoton FLIM methods are reviewed and a novel multiphoton FLIM system using a streak camera is described. An example of a typical application of the system is provided in which the fluorescence resonance energy transfer between a donor-acceptor pair of fluorescent proteins within a cellular specimen is measured.

Time Series Analysis of Transmesothelial Invasion by Endometrial Stromal and Epithelial Cells Using Three-Dimensional Confocal Microscopy

OBJECTIVE To evaluate endometrial adhesion and invasion of peritoneal mesothelium. DESIGN Descriptive study using confocal laser-scanning microscopy. SETTING University-based laboratory. PATIENT(S) Women undergoing surgery for benign conditions. INTERVENTION(S) Fluorescence-labeled peritoneal mesothelial cells (PMCs) were grown on coverslips. Fluorescence-labeled endometrial stromal cells (ESCs) and epithelial cells (EECs) and myometrial cells (Myos) were plated on the PMCs. Cultures were examined at 1, 6, 12, and 24-27 hours with differential interference contrast and confocal laser-scanning microscopy. MAIN OUTCOME MEASURE(S) Demonstration of adherence and invasion of endometrial cells through peritoneal mesothelium. RESULT(S) At 1 hour, there was adherence of the ESCs, EECs, and Myos on the perimeter of PMCs. There was no invasion by the Myos. By 6 hours, ESCs and EECs spread over the surface of the PMCs and extended cell processes through PMC junctions. Extension of pseudopodia under the PMCs followed. By 12 hours, there was vacuolization and lifting of PMCs that had been undermined by endometrial cells. CONCLUSION(S) This is the first time-phase study to demonstrate adherence and the process of invasion of endometrial cells through the mesothelium. The application of three-dimensional confocal laser-scanning microscopy is a novel technique that can be used to further examine mechanisms involved in the pathogenesis of the early endometriotic lesion.

Composition of the extracellular matrix of the peritoneum.

OBJECTIVE To localize the extracellular matrix proteins collagen I, collagen IV, fibronectin, and laminin in the peritoneal membrane. STUDY DESIGN Peritoneal biopsies (n = 13) from the anterior abdominal wall and the uterine serosa (n = 3) were incubated with antibodies to collagen IV, laminin, collagen I, and fibronectin. Specimens were examined using light and confocal laser scanning microscopy. RESULTS All of the extracellular matrix (ECM) proteins were present immediately under the mesothelium. Collagen (Col) IV and laminin (LM) were seen in the smooth muscle of microvascular structures, in the subendothelial basement membrane, and were present in a fascicular pattern in the peritoneal stroma. Collagen I was distributed diffusely in the peritoneal stroma. Fibronectin was also present in the subendothelial basement membrane. CONCLUSIONS The resolution of the confocal microscope allowed for localization of extracellular matrix proteins in relation to the mesothelium. The presence of collagen IV, laminin, collagen I, and fibronectin under the mesothelium suggests that cells invading the peritoneum must have the ability to degrade and remodel this matrix.

Tutorial on Practical Confocal Microscopy and Use of the Confocal Test Specimen

The other chapters in this book give the reader an in-depth description of every important aspect of biological confocal microscopy that we could think of. This chapter is to provide the novice user of this instrument with a basic understanding of the practical information needed to use it effectively. Because the computer interfaces of the various commercial instruments vary greatly, this chapter will stress the important features of microscopical optics and the basics of sampling that are common to all instruments.

Interaction of Mycoplasma genitalium with host cells: evidence for nuclear localization.

Mycoplasma genitalium (Mg) is a mollicute that causes a range of human urogenital infections. A hallmark of these bacteria is their ability to establish chronic infections that can persist despite completion of appropriate antibiotic therapies and intact and functional immune systems. Intimate adherence and surface colonization of mycoplasmas to host cells are important pathogenic features. However, their facultative intracellular nature is poorly understood, partly due to difficulties in developing and standardizing cellular interaction model systems. Here, we characterize growth and invasion properties of two Mg strains (G37 and 1019V). Mg G37 is a high-passage laboratory strain, while Mg 1019V is a low-passage isolate recovered from the cervix. The two strains diverge partially in gene sequences for adherence-related proteins and exhibit subtle variations in their axenic growth. However, with both strains and consistent with our previous studies, a subset of adherent Mg organisms invade host cells and exhibit perinuclear targeting. Remarkably, intranuclear localization of Mg proteins is observed, which occurred as early as 30 min after infection. Mg strains deficient in adherence were markedly reduced in their ability to invade and associate with perinuclear and nuclear sites.

Fluorescence resonance energy transfer imaging microscopy.

Publisher Summary This chapter describes the fluorescence resonance energy transfer (FRET) imaging microscopy. RET is a process by which a donor fluorophore (D) in its excited state may transfer its excitation energy to a nearby chromophore (acceptor, A). The process is nonradiative (not mediated by a photon) and is achieved through dipole–dipole interactions. In principle, for such a transfer to occur, several major criteria must be satisfied. First, the donor molecule must have an emission spectrum that overlaps the absorption spectrum of the acceptor molecule. The second criterion that needs to be satisfied is that the donor and acceptor molecules must be within 10–100 A of each other. The efficiency of energy transfer between donor and acceptor molecules falls off as the sixth power of the distance separating the donor and acceptor molecules. Another criterion that must be met is that the fluorescence lifetime of the donor molecule must be of sufficient duration to allow FRET to occur. Continued research in this area is needed to determine optimal approaches for the measurement of FRET and the development of more refined algorithms for the analysis of data.