Golgi duplication in Trypanosoma brucei

Duplication of the single Golgi apparatus in the protozoan parasite Trypanosoma brucei has been followed by tagging a putative Golgi enzyme and a matrix protein with variants of GFP. Video microscopy shows that the new Golgi appears de novo, near to the old Golgi, about two hours into the cell cycle and grows over a two-hour period until it is the same size as the old Golgi. Duplication of the endoplasmic reticulum (ER) export site follows exactly the same time course. Photobleaching experiments show that the new Golgi is not the exclusive product of the new ER export site. Rather, it is supplied, at least in part, by material directly from the old Golgi. Pharmacological experiments show that the site of the new Golgi and ER export is determined by the location of the new basal body.     

Post-translational modification detection using metastable ions in reflector matrix-assisted laser desorption/ionization-time of flight mass spectrometry

In addition to protein identification, characterization of post-translational modifications (PTMs) is an essential task in proteomics. PTMs represent the major reason for the variety of protein isoforms and they can influence protein structure and function. Upon matrix-assisted laser desorption/ionization (MALDI) most post-translationally modified peptides form a fraction of labile molecular ions, which lose PTM-specific residues only after acceleration. Compared to fully accelerated ions these fragment ions are defocused and show in reflector mass spectra reduced resolution. A short time Fourier transform using a Hanning window function now uses this difference in resolution to detect the metastable fragments. Its application over the whole mass range yields frequency distributions and amplitudes as a function of mass, where an increased low frequency proportion is highly indicative for metastable fragments. Applications on the detection of metastable losses originating from carboxamidomethylated cysteines, oxidized methionines, phosphorylated and glycosylated amino acid residues are presented. The metastable loss of mercaptoacetamide detected with this procedure represents a new feature and its integration in search algorithms will improve the specificity of MALDI peptide mass fingerprinting.     

Sequence elements in both subunits of the DNA fragmentation factor are essential for its nuclear transport

DNA cleavage is a biochemical hallmark of apoptosis. In humans, apoptotic DNA cleavage is executed by DNA fragmentation factor (DFF) 40. In proliferating cells DFF40 is expressed in the presence of its chaperone and inhibitor DFF45, which results in the formation of the DFF complex. Here, we present a systematic analysis of the nuclear import of the DFF complex. Our in vitro experiments demonstrate that the importin alpha/beta-heterodimer mediates the translocation of the DFF complex from the cytoplasm to the nucleus. Both DFF subunits interact directly with the importin alpha/beta-heterodimer. However, importin alpha/beta binds more tightly to the DFF complex compared with the individual subunits. Additionally, the isolated C-terminal regions of both DFF subunits together bind importin alpha/beta more strongly than the individual C termini. Our results from in vivo studies reveal that the C-terminal regions of both DFF subunits harbor nuclear localization signals. Furthermore, nuclear import of the DFF complex requires the C-terminal regions of both subunits. In more detail, one basic cluster in the C-terminal region of each subunit, DFF40 (RLKRK) and DFF45 (KRAR), is essential for nuclear accumulation of the DFF complex. Based on these findings two alternative models for the interaction of importin alpha/beta with the DFF complex are presented.     

Regulation of cardiac hypertrophy by intracellular signalling pathways

The mammalian heart is a dynamic organ that can grow and change to accommodate alterations in its workload. During development and in response to physiological stimuli or pathological insults, the heart undergoes hypertrophic enlargement, which is characterized by an increase in the size of individual cardiac myocytes. Recent findings in genetically modified animal models implicate important intermediate signal-transduction pathways in the coordination of heart growth following physiological and pathological stimulation.     

A long-term porcine model for measurement of gastrointestinal motility

Animal models have become an essential tool in the investigations of gut motility under experimental conditions. To determine the influence of various anaesthetic drugs on the motility pattern of the gastroduodenal tract, a new long-term model has had to be developed for allowing measurements in conscious and unrestrained as well as in sedated and analgosedated pigs. Since mechanical ventilation influences gut motility, it was necessary that this animal model enabled the investigation of the effect of drugs causing sedation and analgosedation during spontaneous breathing. Seven male, castrated pigs, German landrace, 32-40 kg bodyweight (BW) were investigated in this study. After habituation of the pigs to local housing conditions over 5 days, the animals were trained over 4 days to prepare for experimental situations and investigators. Pigs were inserted with a central venous catheter and with percutaneous enterogastrostomy (PEG) under general anaesthesia. Intestinal motility was measured by intraluminal impedancometry. The catheter was introduced over the PEG into the stomach and positioned into the duodenum by duodenoscopy. Measurements were done in conscious, unrestrained pigs and with sedated, and analgosedated animals on subsequent days. The habituation and training of the pigs to the investigators and for the laboratory conditions took between 7 and 9 days. The initial anaesthesia protocol for the instrumentation using remifentanil/propofol led to pyloric spasm and was thus unsuitable for duodenal intubation with an endoscope. In contrast, a combination of ketamine/propofol enabled this procedure. It was practicable to measure gut motility in conscious, unrestrained pigs. Spontaneous breathing was sufficient under propofol sedation and analgosedation using fentanyl-propofol. Systematically local application of polividon iodine in the area of the subcutaneous catheters avoided the necessity of using systemic prophylactic antibiotics. In conclusion, the habituation and training for 9 days enabled the measurement of gut motility by intraluminal impedancometry in conscious pigs. The insertion of the catheter was done during general anaesthesia using a combination of propofol and ketamine. For the future determination of gut motility performed under general anaesthesia, each sedation and analgosedation concept has to be evaluated to see whether it allows spontaneous breathing or whether mechanical ventilation is necessary.     

Regulation of Plant Glycine Decarboxylase by S-Nitrosylation and Glutathionylation

Mitochondria play an essential role in nitric oxide (NO) signal transduction in plants. Using the biotin-switch method in conjunction with nano-liquid chromatography and mass spectrometry, we identified 11 candidate proteins that were S-nitrosylated and/or glutathionylated in mitochondria of Arabidopsis (Arabidopsis thaliana) leaves. These included glycine decarboxylase complex (GDC), a key enzyme of the photorespiratory C₂ cycle in C3 plants. GDC activity was inhibited by S-nitrosoglutathione due to S-nitrosylation/S-glutathionylation of several cysteine residues. Gas-exchange measurements demonstrated that the bacterial elicitor harpin, a strong inducer of reactive oxygen species and NO, inhibits GDC activity. Furthermore, an inhibitor of GDC, aminoacetonitrile, was able to mimic mitochondrial depolarization, hydrogen peroxide production, and cell death in response to stress or harpin treatment of cultured Arabidopsis cells. These findings indicate that the mitochondrial photorespiratory system is involved in the regulation of NO signal transduction in Arabidopsis.Nitric oxide (NO) has emerged as a new chemical messenger in plant biology. It can interact with a variety of intracellular and extracellular targets, acting as either a cytotoxic or a cytoprotective agent. NO stimulates seed germination in different species, and a decrease in NO levels has been associated with fruit maturation and senescence of flowers (Beligni and Lamattina, 2001). NO production has been observed in response to several biotic and abiotic stimuli, such as pathogen infection, bacterial elicitors, high temperature, osmotic stress, and UV-B light (Durner et al., 1998; Barroso et al., 1999; Krause and Durner, 2004; Zeidler et al., 2004; Shapiro, 2005; Corpas et al., 2008; Kolbert et al., 2008; Zhao et al., 2009).Despite the proven importance of NO, little is known about signaling pathways downstream from it. During both programmed cell death and defense responses, NO requires cGMP and cADP Rib as secondary messengers (Wendehenne et al., 2001). Furthermore, NO activates mitogen-activated protein kinases in different plant species during stress signaling (Nakagami et al., 2005). However, direct biological activity of NO arises from chemical reactions between proteins and NO itself (Foster and Stamler, 2004; Dahm et al., 2006). S-Nitrosylation is a labile posttranslational modification with a half-life of seconds to a few minutes and represents a very sensitive mechanism for regulating cellular processes (Hess et al., 2005). More than 100 candidate S-nitrosylated proteins were identified from extracts of Arabidopsis (Arabidopsis thaliana) cultured cells treated with the NO donor S-nitrosoglutathione (GSNO) and from Arabidopsis leaves treated with gaseous NO (Lindermayr et al., 2005). Using the same proteomic approach, changes were characterized in S-nitrosylated proteins in Arabidopsis leaves undergoing a hypersensitive response (Romero-Puertas et al., 2008).In animals, mitochondria play a crucial role in S-nitrosylation-dependent NO signaling (Foster and Stamler, 2004). The mitochondrion is an essential organelle for normal cellular function, being an important site of ATP synthesis and an integrator for apoptotic signaling (Skulachev, 1999). Mitochondria interact with NO at several levels. One particularly well-characterized example is the inhibition of complex IV (cytochrome c oxidase) via binding of NO to its binuclear CuB/heme a₃ site (Cleeter et al., 1994). There are several reasons why S-nitrosylation may be an important mitochondrial regulatory mechanism. For example, mitochondria contain sizeable pools of thiols and transition metals, all of which are known to modulate nitrosothiol (SNO) biochemistry (Foster and Stamler, 2004). In addition, mitochondria are highly membranous and accumulate lipophilic molecules such as NO. Interesting in this respect is the fact that the formation of the S-nitrosylating intermediate N₂O₃ is enhanced within membranes (Burwell et al., 2006).The role of mitochondria in stress-related responses has been investigated in both animals and plants. Endogenous nitrosylation of the catalytic Cys site of a subset of mitochondrial caspases serves as an on/off switch regulating caspase activity during apoptosis (Mannick et al., 2001). Moreover, cytochrome c, which is modified by NO at its heme iron during apoptosis, is released from mitochondria into the cytoplasm, which plays a critical role in many forms of apoptosis by stimulating apoptosome formation and subsequent caspase activation (Schonhoff et al., 2003). We previously showed that a prime target of NO in plants is the mitochondrial apparatus, causing an inhibition of KCN-sensitive respiration and an activation of alternative respiration via alternative oxidase (AOX; Huang et al., 2002; Krause and Durner, 2004; Livaja et al., 2008).The aim of this study was to identify possible targets for S-nitrosylation in mitochondria of Arabidopsis leaves in order to gain more insight into the regulatory function of NO at the protein level. Using a proteomic approach involving the highly specific biotin-switch method for detection and purification of S-nitrosylated proteins (Jaffrey and Snyder, 2001) in conjunction with liquid chromatography and tandem mass spectrometry (nanoLC/MS/MS), we could identify 11 mitochondrial proteins as targets for S-nitrosylation. Among these identified proteins, we focused our attention on the P-subunit of the Gly decarboxylase complex (GDC), which is an integral part of the photorespiratory system. Since the release of apoptotic factors from mitochondria may be a result of inhibition of respiration, transition of mitochondrial permeability, and formation of reactive oxygen species (ROS; Saviani et al., 2002; Taylor et al., 2004; Chen and Gibson, 2008), we investigated the molecular mechanism and the function of GDC-Cys modification in Arabidopsis.     

New cationic lipids form channel-like pores in phospholipid bilayers

Two representatives of a new class of cationic lipids were found to have high pore-forming activity in planar bilayer membranes. These molecules, called BHHD-TADC and BHTD-TADC, have qualitatively similar effects on phospholipid membranes. Addition of 2.5-5 micro M of either of them to the membrane bathing solutions resulted in formation of long-lived anion-selective pores with conductance in the range 0.1-2 nS in 0.1 M KCl. Pore formation was found to be dependent on the potential applied to the membrane. When negative potential was applied to membrane at the side of addition, the rate of pore formation was much lower compared to when the positive potential was applied. Dependence of pore formation on compound concentration was highly nonlinear, indicating that this process requires assembly of molecules in the membrane. Addition of any of these compounds on both sides of the membrane increased the efficiency of pore formation by one to two orders of magnitude. Pore formation was strongly pH dependent. Although pores were formed with high efficiency at pH 6.5, only occasional fluctuations of membrane conductance were observed at pH 7.5. Possible mechanisms of new compounds biological activity are discussed.     

Lrig1: a new master regulator of epithelial stem cells

Nat Cell Biol 14 4, 401–408 March042012The intestine represents the most vigorously renewing, adult epithelial tissue that makes maintenance of its homeostasis a delicate balance between proliferation, cell cycle arrest, migration, differentiation, and cell death. These processes are precisely controlled by a network of developmental signalling cascades, which include Wnt, Notch, BMP/TGFβ, and Hedgehog pathways. A new, elegant study by Wong et al (2012) now adds Lrig1 as a key player in the control of intestinal homeostasis. As for epidermal stem cells, Lrig1 limits the size of the intestinal progenitor compartment by dampening EGF/ErbB-triggered stem cell expansion.The epithelium of the small intestine is separated into two distinct compartments: a proliferative crypt, containing tissue-specific stem cells, and a villus with differentiated, short-lived cells, which are replenished by a constant stream of cell migration from the underlying crypt (Scoville et al, 2008). In particular, the canonical Wnt pathway in combination with Notch signals control stem cell maintenance and proliferation in the crypt. In addition, both pathways direct differentiation into the Paneth and the absorptive cell lineage, respectively. Intensive cross-talk between the epithelium and the underlying mesenchyme helps to define the crypt–villus boundary. This relies on epithelial-derived Hedgehog and Wnt ligands that trigger stromal BMP production, which in turn signals back to the epithelium to restrict proliferation to the crypt. A gradient of BMP antagonists produced by mesenchymal cells at the bottom of the crypts supports compartmentalization. In addition, a Wnt gradient in the crypt defines EphB expression and establishes repulsion-mediated separation into Paneth cell, proliferative, and differentiation zones along the crypt–villus axis (Figure 1A).Open in a separate windowFigure 1(A) The epithelium of the small intestine contains two populations of multipotent stem cells that reside at the bottom of the crypts. These give rise to transit-amplifying progenitors, which rapidly divide while migrating upwards. Cell cycle arrest and functional differentiation occur when these cells pass from the upper part of the crypt into the villus where they continue their upward movement until they finally undergo apoptosis. Only long-living Paneth cells follow a different path as they migrate downwards to populate the base of the crypt. Control of proliferation and lineage specification of all intestinal epithelial cells is directed in a self-organizing, dynamically regulated process based on cell–cell and cell–environment interactions. Among them, Wnt and Notch signalling have been defined as major determinants for stem cell maintenance, for proliferation of stem cells in the crypt and lineage specification. Epithelial-derived Hedgehog ligands and reciprocal stromal BMP ligands establish a connection between the epithelium and the stroma that regulates the crypt–villus boundary. In addition, repulsive interactions mediated by the Eph/ephrin family allow establishment of stable compartments. Importantly, ErbB signalling, which is partially suppressed by Lrig1 at the base of the crypt, is now shown to be a new key player in the control of stem and progenitor cell expansion. (B) Cross-talk of signalling pathways in intestinal homeostasis with an emphasis on ErbB signalling. A negative feedback loop via Lrig1 helps to fine-tune population size and proliferative activity of intestinal progenitor cells. Lrig1 has been identified as a direct target of Myc and is known to repress ErbB signalling. Myc itself is a main target of the ErbB and Wnt pathways implicated in intestinal stem and progenitor cell expansion. Moreover, Lrig1 has been found to promote BMP signalling, which interferes with intestinal proliferation by restricting AKT activation via PTEN.In the small intestine, two stem cell (SC) populations coexist: Lgr5⁺crypt base columnar cells (CBCs) that cycle every 24 h and are interspersed between Paneth cells, and slower dividing SCs concentrated above (around position +4 relative to the crypt bottom) the Lgr5⁺position (Takeda et al, 2011). The localization of these Hopx⁺mTert⁺slowly cycling SCs partly overlaps with that of quiescent cells, which show long-term label retention upon irradiation damage and pulse labelling with BrdU. Lgr5⁺CBCs are, however, dispensable (Tian et al, 2008) and can be replaced by the second stem cell population, which also shows greater activity during damage repair. The relationship between these two stem cell populations, which can reciprocally generate each other, and the mechanisms that govern quiescence are being elucidated. Importantly, leucine-rich repeats and Ig-like domains 1 (Lrig1), a transmembrane protein that interacts with ErbBs and promotes its degradation, has now been found to be enriched at the crypt base and in the progenitor compartment of the small intestine and colon (Wong et al, 2012). Lrig1 is highly expressed in Lgr5⁺, Musashi1⁺, Ascl2⁺, and Olfm4⁺CBCs, and shows an inverse relation to the pattern of activated, phosphorylated EGFR above the crypt base (Figure 1A). In line with these patterns, deletion of Lrig1 in the mouse causes a dramatic crypt expansion and increased numbers of CBCs, transit-amplifying and Paneth cells. Whether the increase of Paneth cells, which actually do not express Lrig1, is a secondary effect due to the progenitor expansion remains open. Importantly, reduction of EGFR signalling by pharmacological (Gefitinib) and genetic modulation (Egfr^wa-2 mice) is able to partially normalize all Lrig1 phenotypes. These data establish EGF/ErbB signalling, as an important regulator of the crypt compartment, and suggest Lrig1 as a central control that dampens the expansion of stem cells during normal intestinal homeostasis.Lrig1 was initially identified in the skin and proposed to maintain epidermal stem cells in a quiescent state (Watt and Jensen, 2009). Lrig1 marks human interfollicular epidermal stem cells, which can give rise to all epithelial lineages including hair follicle cells in skin reconstitution assays. However, during normal homeostasis, these cells are only bipotent, contributing to the sebaceous gland and the interfollicular epidermis. In contrast to quiescent Lrig1⁺SCs in the skin, Lrig1⁺ intestinal SCs are rapidly dividing and Lrig1 appears to only reduce their proliferative capacity. However, similar to the situation in the skin, Lrig1 and EGF signalling may play an important role during damage repair. Earlier experiments analysed the phenotype of mice lacking major EGF family members (Egger et al, 1997; Troyer et al, 2001). While these mice display some duodenal lesions during normal homeostasis, further experiments established EGF signalling as a key protective component that ameliorates mucosal damage. It remains to be seen whether activation of intestinal SCs during damage repair involves mitigation of Lrig1 dampening.Lrig1 is known to repress ErbB signalling by mediating ubiquitinylation and degradation of activated receptors, thereby limiting the amplitude of EGF signalling (Watt and Jensen, 2009). Consequently, Lrig1 deletion in the intestine induced upregulation of EGFR, ErbB2, and ErbB3, promoting downstream activation of c-Myc within intestinal stem and progenitor cells (Wong et al, 2012). Importantly, Lrig1 is a direct Myc target gene, and thereby part of a negative feedback loop that helps to fine-tune the population size and proliferative activity of intestinal progenitor cells (Figure 1B).Since the rescue of the Lrig1^−/− phenotype by EGFR deficiency was only partial (Wong et al, 2012), other mechanisms may contribute. Intriguingly, Lrig1 has been shown to promote BMP signalling by direct binding to Type I (ALK6) and Type II (ALK1, ALK2, ALK3, and ActRIB) BMP receptors (Gumienny et al, 2010). BMPR1A inactivation, deficiency of its downstream effector PTEN, and transgenic overexpression of the BMP inhibitor Noggin display crypt expansion and increased SC numbers. Inhibition of BMP signalling in these genetic models enhanced AKT activation and increased Wnt signalling, promoting proliferation and adenoma formation (Figure 1B; Scoville et al, 2008). Future work will reveal a potential involvement of BMP and Wnt signalling in the Lrig1 knockout phenotype.The ErbB pathway has been linked to inflammatory bowel disease, and progression and metastatic potential of colorectal cancer. EGFR inhibition blocks adenoma formation in preclinical models, and ErbB pathway inhibition is currently being evaluated in clinical trials with colorectal cancer patients, where promising results have been reported (Cunningham et al, 2004). In contrast, Lrig1 is expressed at low levels in several cancer types but is overexpressed in some prostate and colorectal tumours (Hedman and Henriksson, 2007). Given this heterogeneity, the Lrig1 function in tumours appears to be cell- and context-dependent. Due to early postnatal lethality of Lrig1 knockout mice, the exciting possibility that Lrig1 may act as an intestinal tumour suppressor could not be answered by the current study but clearly deserves further attention.