Role of NAD+ and ADP-Ribosylation in the Maintenance of the Golgi Structure

We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114–1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065–7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD⁺ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD⁺-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD⁺ triggered Golgi disassembly by BFA. This effect of NAD⁺ was mimicked by the use of pre–ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200–14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre–ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50–enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.     

CD4+Foxp3+ regulatory T cells prolong drug-induced disease remission in (NZBxNZW) F1 lupus mice

Introduction

The ability to ameliorate murine lupus renders regulatory T cells (Treg) a promising tool for the treatment of systemic lupus erythematosus (SLE). In consideration to the clinical translation of a Treg-based immunotherapy of SLE, we explored the potential of CD4+Foxp3+ Treg to maintain disease remission after induction of remission with an established cyclophosphamide (CTX) regimen in lupus-prone (NZBxNZW) F1 mice. As a prerequisite for this combined therapy, we also investigated the impact of CTX on the biology of endogenous Treg and conventional CD4+ T cells (Tcon).

Methods

Remission of disease was induced in diseased (NZBxNZW) F1 mice with an established CTX regimen consisting of a single dose of glucocorticosteroids followed by five day course with daily injections of CTX. Five days after the last CTX injection, differing amounts of purified CD4+Foxp3+CD25+ Treg were adoptively transferred and clinical parameters, autoantibody titers, the survival and changes in peripheral blood lymphocyte subsets were determined at different time points during the study. The influence of CTX on the numbers, frequencies and proliferation of endogenous Treg and Tcon was analyzed in lymphoid organs by flow cytometry.

Results

Apart from abrogating the proliferation of Tcon, we found that treatment with CTX induced also a significant inhibition of Treg proliferation and a decline in Treg numbers in lymphoid organs. Additional adoptive transfer of 1.5 × 10⁶ purified Treg after the CTX regimen significantly increased the survival and prolonged the interval of remission by approximately five weeks compared to mice that received only the CTX regimen. The additional clinical amelioration was associated with an increase in the Treg frequency in the peripheral blood indicating a compensation of CTX-induced Treg deficiency by the Treg transfer.

Conclusions

Treg were capable to prolong the interval of remission induced by conventional cytostatic drugs. This study provides valuable information and a first proof-of-concept for the feasibility of a Treg-based immunotherapy in the maintenance of disease remission in SLE.     

Imaging cell biology in live animals: Ready for prime time

Time-lapse fluorescence microscopy is one of the main tools used to image subcellular structures in living cells. Yet for decades it has been applied primarily to in vitro model systems. Thanks to the most recent advancements in intravital microscopy, this approach has finally been extended to live rodents. This represents a major breakthrough that will provide unprecedented new opportunities to study mammalian cell biology in vivo and has already provided new insight in the fields of neurobiology, immunology, and cancer biology.The discovery of GFP combined with the ability to engineer its expression in living cells has revolutionized mammalian cell biology (Chalfie et al., 1994). Since its introduction, several light microscopy–based techniques have become invaluable tools to investigate intracellular events (Lippincott-Schwartz, 2011). Among them are: time-lapse confocal microscopy, which has been instrumental in studying the dynamics of cellular and subcellular processes (Hirschberg et al., 1998; Jakobs, 2006; Cardarelli and Gratton, 2010); FRAP, which has enabled determining various biophysical properties of proteins in living cells (Berkovich et al., 2011); and fluorescence resonance energy transfer (FRET), which has been used to probe for protein–protein interactions and the local activation of specific signaling pathways (Balla, 2009). The continuous search for improvements in temporal and spatial resolution has led to the development of more sophisticated technologies, such as spinning disk microscopy, which allows the resolution of fast cellular events that occur on the order of milliseconds (Nakano, 2002); total internal reflection microscopy (TIRF), which enables imaging events in close proximity (100 nm) to the plasma membrane (Cocucci et al., 2012); and super-resolution microscopy (SIM, PALM, and STORM), which captures images with resolution higher than the diffraction limit of light (Lippincott-Schwartz, 2011).Most of these techniques have been primarily applied to in vitro model systems, such as cells grown on solid substrates or in 3D matrices, explanted embryos, and organ cultures. These systems, which are relatively easy to maintain and to manipulate either pharmacologically or genetically, have been instrumental in providing fundamental information about cellular events down to the molecular level. However, they often fail to reconstitute the complex architecture and physiology of multicellular tissues in vivo. Indeed, in a live organism, cells exhibit a 3D organization, interact with different cell types, and are constantly exposed to a multitude of signals originated from the vasculature, the central nervous system, and the extracellular environment. For this reason, scientists have been attracted by the possibility of imaging biological processes in live multicellular organisms (i.e., intravital microscopy [IVM]). The first attempt in this direction was in 1839, when Rudolph Wagner described the interaction of leukocytes with the walls of blood vessels in the webbed feet of a live frog by using bright-field transillumination (Wagner, 1839). Since then, this approach has been used for over a century to study vascular biology in thin areas of surgically exposed organs (Irwin and MacDonald, 1953; Zweifach, 1954) or by implanting optical windows in the skin or the ears (Clark and Clark, 1932). In addition, cell migration has also been investigated using transparent tissues, such as the fin of the teleost (Wood and Thorogood, 1984; Thorogood and Wood, 1987). The introduction of epifluorescence microscopy has enabled following in more detail the dynamics of individual cells in circulation (Nuttall, 1987), in tumors (MacDonald et al., 1992), or in the immune system (von Andrian, 1996), and the spatial resolution has been significantly improved by the use of confocal microscopy, which has made it possible to collect serial optical sections from a given specimen (Villringer et al., 1989; O’Rourke and Fraser, 1990; Jester et al., 1991). However, these techniques can resolve structures only within a few micrometers from the surface of optically opaque tissues (Masedunskas et al., 2012a). It was only in the early nineteen nineties, with the development of multiphoton microscopy, that deep tissue imaging has become possible (Denk et al., 1990; Zipfel et al., 2003b), significantly contributing to several fields, including neurobiology, immunology, and cancer biology (Fig. 1; Svoboda and Yasuda, 2006; Amornphimoltham et al., 2011; Beerling et al., 2011). In the last few years, the development of strategies to minimize the motion artifacts caused by the heartbeat and respiration has made it possible to successfully image subcellular structures with spatial and temporal resolutions comparable to those achieved in in vitro model systems, thus providing the opportunity to study cell biology in live mammalian tissues (Fig. 1; Weigert et al., 2010; Pittet and Weissleder, 2011).Open in a separate windowFigure 1.Spatial resolution and current applications of intravital microscopy. IVM provides the opportunity to image several biological processes in live animals at different levels of resolution. Low-magnification objectives (5–10×) enable visualizing tissues and their components under physiological conditions and measuring their response under pathological conditions. Particularly, the dynamics of the vasculature have been one of topic most extensively studied by IVM. Objectives with higher magnification (20–30×) have enabled imaging the behavior of individual cell over long periods of time. This has led to major breakthroughs in fields such as neurobiology, immunology, cancer biology, and stem cell research. Finally, the recent developments of strategies to minimize the motion artifacts caused by the heartbeat and respiration combined with high power lenses (60–100×) have opened the door to image subcellular structures and to study cell biology in live animals.The aim of this review is to highlight the power of IVM in addressing cell biological questions that cannot be otherwise answered in vitro, due to the intrinsic limitations of reductionist models, or by other more classical approaches. Furthermore, we discuss limitations and areas for improvement of this imaging technique, hoping to provide cell biologists with the basis to assess whether IVM is the appropriate choice to address their scientific questions.

Imaging techniques currently used to perform intravital microscopy

Confocal and two-photon microscopy are the most widely used techniques to perform IVM. Confocal microscopy, which is based on single photon excitation, is a well-established technique (Fig. 2 A) that has been extensively discussed elsewhere (Wilson, 2002); hence we will only briefly describe some of the main features of two-photon microscopy and other nonlinear optical techniques.Open in a separate windowFigure 2.Fluorescent light microscopy imaging techniques used for intravital microscopy. (A) Confocal microscopy. (top) In confocal microscopy, a fluorophore absorbs a single photon with a wavelength in the UV-visible range of the spectrum (blue arrow). After a vibrational relaxation (orange curved arrow), a photon with a wavelength shifted toward the red is emitted (green arrow). (center) In thick tissue, excitation and emission occur in a relative large volume around the focal plane (F.P.). The off-focus emissions are eliminated through a pinhole, and the signal from the focal plane is detected via a photomultiplier (PMT). Confocal microscopy enables imaging at a maximal depth to 80–100 µm. (bottom) Confocal z stack of the tongue of a mouse expressing the membrane marker m-GFP (green) in the K14-positive basal epithelial layer, and the membrane marker mTomato in the endothelium (red). The xy view shows a maximal projection of 40 z slices acquired every 2.5 µm, whereas the xz view shows a lateral view of the stack. In blue are the nuclei labeled by a systemic injection of Hoechst. Excitation wavelengths: 450 nm, 488 nm, and 562 nm. (B) Two- and three-photon microscopy. (top) In this process a fluorophore absorbs almost simultaneously two or three photons that have half (red arrow) or a third (dark red arrow) of the energy required for its excitation with a single photon. Two- or three-photon excitations typically require near-IR or IR light (from 690 to 1,600 nm). (center) Emission and excitation occur only at the focal plane in a restricted volume (1.5 fl), and for this reason a pinhole is not required. Two- and three-photon microscopy enable imaging routinely at a maximal depth of 300–500 µm. (bottom) Two-photon z stack of an area adjacent to that imaged in A. xy view shows a maximal projection of 70 slices acquired every 5 µm. xz view shows a lateral view of the stack. Excitation wavelength: 840 nm. (C) SHG and THG. (top) In SHG and THG, photons interact with the specimen and combine to form new photons that are emitted with twice or three times their initial energy without any energy loss. (center) These processes have similar features to those described for two- and three-photon microscopy and enable imaging at a maximal depth of 200–400 µm. (bottom) z stack of a rat heart excited by two-photon microscopy (740 nm) to reveal the parenchyma (green), and SHG (930 nm) to reveal collagen fibers (red). xy shows a maximal projection of 20 slices acquired every 5 µm. xz view shows a lateral view of the stack. Bars: (xy views) 40 µm; (xz views) 50 µm.The first two-photon microscope (Denk et al., 1990) was based on the principle of two-photon excitation postulated by Maria Göppert-Mayer in her PhD thesis (Göppert-Mayer, 1931). In this process a fluorophore is excited by the simultaneous absorption of two photons with wavelengths in the near-infrared (IR) or IR spectrum (from 690 to 1,600 nm; Fig. 2 B). Two-photon excitation requires high-intensity light that is provided by lasers generating very short pulses (in the femtosecond range) and is focused on the excitation spot by high numerical aperture lenses (Zipfel et al., 2003b). There are three main advantages in using two-photon excitation for IVM. First, IR light has a deeper tissue penetration than UV or visible light (Theer and Denk, 2006). Indeed, two-photon microscopy can resolve structures up to a depth of 300–500 µm in most of the tissues (Fig. 2 B), and up to 1.5 mm in the brain (Theer et al., 2003; Masedunskas et al., 2012a), whereas confocal microscopy is limited to 80–100 µm (Fig. 2 A). Second, the excitation is restricted to a very small volume (1.5 fl; Fig. 2 B). This implies that in two-photon microscopy there is no need to eliminate off-focus signals, and that under the appropriate conditions photobleaching and phototoxicity are negligible (Zipfel et al., 2003b). However, confocal microscopy induces out-of-focus photodamage, and thus is less suited for long-term imaging. Third, selected endogenous molecules can be excited, thus providing the contrast to visualize specific biological structures without the need for exogenous labeling (Zipfel et al., 2003a). Some of these molecules can also be excited by confocal microscopy using UV light, although with the risk of inducing photodamage.More recently, other nonlinear optical techniques have been used for IVM, and among them are three-photon excitation, and second and third harmonic generation (SHG and THG; Campagnola and Loew, 2003; Zipfel et al., 2003b; Oheim et al., 2006). Three-photon excitation follows the same principle as two-photon (Fig. 2 B), and can reveal endogenous molecules such as serotonin and melatonin (Zipfel et al., 2003a; Ritsma et al., 2013). In SHG and THG, photons interact with the specimen and combine to form new photons that are emitted with two or three times their initial energy (Fig. 2 C). SHG reveals collagen (Fig. 2 C) and myosin fibers (Campagnola and Loew, 2003), whereas THG reveals lipid droplets and myelin fibers (Débarre et al., 2006; Weigelin et al., 2012). Recently, two other techniques have been used for IVM: coherent anti-Stokes Raman scattering (CARS) and fluorescence lifetime imaging (FLIM). CARS that is based on two laser beams combined to match the energy gap between two vibrational levels of the molecule of interest, has been used to image lipids and myelin fibers (Müller and Zumbusch, 2007; Fu et al., 2008; Le et al., 2010). FLIM, which measures the lifetime that a molecule spends in the excited state, provides quantitative information on cellular parameters such as pH, oxygen levels, ion concentration, and the metabolic state of various biomolecules (Levitt et al., 2009; Provenzano et al., 2009; Bakker et al., 2012).We want to emphasize that two-photon microscopy and the other nonlinear techniques are the obligatory choice when the imaging area is located deep inside the tissue, endogenous molecules have to be imaged, or long-term imaging with frequent sampling is required. However, confocal microscopy is more suited to resolve structures in the micrometer range, because of the possibility of modulating the optical slice (Masedunskas et al., 2012a).

IVM to investigate biological processes at the tissue and the single cell level

The main strength of IVM is to provide information on the dynamics of biological processes that otherwise cannot be reconstituted in vitro or ex vivo. Indeed, IVM has been instrumental in studying several aspects of tissue physiopathology (Fig. 3, A and B). Although other approaches such as classical immunohistochemistry, electron microscopy, and indirect immunofluorescence may provide detailed structural and quantitative information on blood vessels, IVM enables measuring events such as variations of blood flow at the level of the capillaries or local changes in blood vessel permeability. These data have been instrumental in understanding the mechanisms of ischemic diseases and tumor progression, and in designing effective anticancer treatments.

Table 1.

IVM to study tissue physiopathology

Phosphorylation of Serine 1137/1138 of Mouse Insulin Receptor Substrate (IRS) 2 Regulates cAMP-dependent Binding to 14-3-3 Proteins and IRS2 Protein Degradation

Insulin receptor substrate (IRS) 2 as intermediate docking platform transduces the insulin/IGF-1 (insulin like growth factor 1) signal to intracellular effector molecules that regulate glucose homeostasis, β-cell growth, and survival. Previously, IRS2 has been identified as a 14-3-3 interaction protein. 14-3-3 proteins can bind their target proteins via phosphorylated serine/threonine residues located within distinct motifs. In this study the binding of 14-3-3 to IRS2 upon stimulation with forskolin or the cAMP analog 8-(4-chlorophenylthio)-cAMP was demonstrated in HEK293 cells. Binding was reduced with PKA inhibitors H89 or R_p-8-Br-cAMPS. Phosphorylation of IRS2 on PKA consensus motifs was induced by forskolin and the PKA activator N⁶-Phe-cAMP and prevented by both PKA inhibitors. The amino acid region after position 952 on IRS2 was identified as the 14-3-3 binding region by GST-14-3-3 pulldown assays. Mass spectrometric analysis revealed serine 1137 and serine 1138 as cAMP-dependent, potential PKA phosphorylation sites. Mutation of serine 1137/1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding. Application of cycloheximide revealed that forskolin enhanced IRS2 protein stability in HEK293 cells stably expressing IRS2 as well as in primary hepatocytes. Stimulation with forskolin did not increase protein stability either in the presence of a 14-3-3 antagonist or in the double 1137/1138 alanine mutant. Thus the reduced IRS2 protein degradation was dependent on the interaction with 14-3-3 proteins and the presence of serine 1137/1138. We present serine 1137/1138 as novel cAMP-dependent phosphorylation sites on IRS2 and show their importance in 14-3-3 binding and IRS2 protein stability.     

Individual‐ versus group‐optimality in the production of secreted bacterial compounds

How unicellular organisms optimize the production of compounds is a fundamental biological question. While it is typically thought that production is optimized at the individual‐cell level, secreted compounds could also allow for optimization at the group level, leading to a division of labor where a subset of cells produces and shares the compound with everyone. Using mathematical modeling, we show that the evolution of such division of labor depends on the cost function of compound production. Specifically, for any trait with saturating benefits, linear costs promote the evolution of uniform production levels across cells. Conversely, production costs that diminish with higher output levels favor the evolution of specialization–especially when compound shareability is high. When experimentally testing these predictions with pyoverdine, a secreted iron‐scavenging compound produced by Pseudomonas aeruginosa, we found linear costs and, consistent with our model, detected uniform pyoverdine production levels across cells. We conclude that for shared compounds with saturating benefits, the evolution of division of labor is facilitated by a diminishing cost function. More generally, we note that shifts in the level of selection from individuals to groups do not solely require cooperation, but critically depend on mechanistic factors, including the distribution of compound synthesis costs.     

Role of brefeldin A-dependent ADP-ribosylation in the control of intracellular membrane transport

The fungal toxin brefeldin A (BFA) dissociates coat proteins from Golgi membranes, causes the rapid disassembly of the Golgi complex and potently stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa. These proteins have been identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a novel guanine nucleotide binding protein (BARS-50), respectively. The role of ADP-ribosylation in mediating the effects of BFA on the structure and function of the Golgi complex was analyzed by several approaches including the use of selective pharmacological blockers of the reaction and the use of ADP-ribosylated cytosol and/or enriched preparations of the BFA-induced ADP-ribosylation substrates, GAPDH and BARS-50.A series of blockers of the BFA-dependent ADP-ribosylation reaction identified in our laboratory inhibited the effects of BFA on Golgi morphology and, with similar potency, the ADP-ribosylation of BARS-50 and GAPDH. In permeabilized RBL cells, the BFA-dependent disassembly of the Golgi complex required NAD+ and cytosol. Cytosol that had been previously ADP-ribosylated (namely, it contained ADP-ribosylated GAPDH and BARS-50), was instead sufficient to sustain the Golgi disassembly induced by BFA.Taken together, these results indicate that an ADP-ribosylation reaction is part of the mechanism of action of BFA and it might intervene in the control of the structure and function of the Golgi complex.     

Effects of the new adiponectin paralogous protein CTRP-3 and of LPS on cytokine release from monocytes of patients with type 2 diabetes mellitus

Aims/Hypothesis: It was the aim to investigate the hypothesis that the new C1q/TNF-family member CTRP-3 (C1q/TNF-related protein-3) acts anti-inflammatory in human monocytes from healthy controls and patients with type 2 diabetes mellitus (T2D). Methods: Monocytes were isolated from 20 healthy controls and 30 patients with T2D. IL-6 and TNF concentrations were measured by ELISA. CTRP-3 was expressed in insect cells and used for stimulation experiments. Results: Basal IL-6 and TNF were not different in control and in T2D monocytes. LPS-stimulation (1 μg/ml) significantly (p < 0.001) increased IL-6 and TNF in the supernatants of control and in T2D monocytes to a similar extent. CTRP-3 (1 μg/ml) significantly (p = 0.03) inhibited LPS-induced IL-6 in control monocytes but not in T2D monocytes. TNF upon co-stimulation with LPS and CTRP-3 was significantly (p = 0.012) lower in control than in T2D monocytes. LPS-induced TNF concentration was significantly and positively correlated with serum total cholesterol and LDL cholesterol in T2D patients. Conclusions: CTRP-3 inhibits LPS-induced IL-6 and TNF release. This anti-inflammatory effect is lost in T2D. Serum cholesterol concentration affects the pro-inflammatory potential of LPS to induce TNF release from T2D monocytes in the presence or absence of CTRP-3. CTRP-3 might partly account for the pro-inflammatory state in T2D.