Characterizing the intracellular distribution of metabolites in intact Chlamydia-infected cells by Raman and two-photon microscopy

Chlamydia species are obligate intracellular pathogens that proliferate only within infected cells. Currently, there are no known techniques or systems that can probe the spatial distribution of metabolites of interest within intact Chlamydia-infected cells. Here we investigate the ability of Raman microscopy to probe the chemical composition of different compartments (nucleus, inclusion, and cytoplasm) of Chlamydia trachomatis-infected epithelial cells. The overall intensity of the Raman spectrum is greatest in the inclusions and lowest in the cytoplasm in fixed cells. Difference spectra generated by normalizing to the intensity of the strong 1004 cm−1 phenylalanine line show distinct differences among the three compartments. Most notably, the concentrations of adenine are greater in both the inclusions and the nucleus than in the cytoplasm, as seen by Raman microscopy. The source of the adenine was explored through a complementary approach, using two-photon microscopy imaging. Autofluorescence measurements of living, infected cells show that the adenine-containing molecules, NAD(P)H and FAD, are present mainly in the cytoplasm, suggesting that these molecules are not the source of the additional adenine signal in the nucleus and inclusions. Experiments of infected cells stained with a DNA-binding dye, Hoechst 33258, reveal that most of the DNA is present in the nucleus and the inclusions, suggesting that DNA/RNA is the main source of the additional Raman adenine signal in the nucleus and inclusions. Thus, Raman and two-photon microscopy are among the few non-invasive methods available to investigate cells infected with Chlamydia and, together, should also be useful for studying infection by other intracellular pathogens that survive within intracellular vacuoles

Local convergence of the symmetric rank-one iteration

SIGLEAvailable from TIB Hannover: RR 1843(95-07) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman

Spatiotemporally Regulated Protein Kinase A Activity Is a Critical Regulator of Growth Factor-Stimulated Extracellular Signal-Regulated Kinase Signaling in PC12 Cells▿

PC12 cells exhibit precise temporal control of growth factor signaling in which stimulation with epidermal growth factor (EGF) leads to transient extracellular signal-regulated kinase (ERK) activity and cell proliferation, whereas nerve growth factor (NGF) stimulation leads to sustained ERK activity and differentiation. While cyclic AMP (cAMP)-mediated signaling has been shown to be important in conferring the sustained ERK activity achieved by NGF, little is known about the regulation of cAMP and cAMP-dependent protein kinase (PKA) in these cells. Using fluorescence resonance energy transfer (FRET)-based biosensors localized to discrete subcellular locations, we showed that both NGF and EGF potently activate PKA at the plasma membrane, although they generate temporally distinct activity patterns. We further show that both stimuli fail to induce cytosolic PKA activity and identify phosphodiesterase 3 (PDE3) as a critical regulator in maintaining this spatial compartmentalization. Importantly, inhibition of PDE3, and thus perturbation of the spatiotemporal regulation of PKA activity, dramatically increases the duration of EGF-stimulated nuclear ERK activity in a PKA-dependent manner. Together, these findings identify EGF and NGF as potent activators of PKA activity specifically at the plasma membrane and reveal a novel regulatory mechanism contributing to the growth factor signaling specificity achieved by NGF and EGF in PC12 cells