Network Of Organelles Research Articles

From systems biology to fuel— Chlamydomonas reinhardtii as a model for a systems biology approach to improve biohydrogen production

With economic wealth the need for energy is rising. Hence we are facing two problems: to satisfy the increasing energy demand and concomitantly deliver emission-free energy to avoid global warming. The process of photosynthesis offers a natural and highly efficient method to produce emission-neutral biofuels. However, using higher plants for such purposes causes several problems which are difficult to overcome and includes competition with food producing agriculture in terms of arable land, the need for fresh water, low process efficiency and the application of energy-intensive fertilizer in order to enhance growth performance. Photosynthetic microorganisms and, in particular, microalgae offer an alternative approach. In this case production sites in photo-bioreactors can be located on cheap, rural land and the organisms can be cultured in sea water rather than fresh water. However microorganisms are not naturally adapted as efficient producers of biofuels. Due to the complex regulatory network and mutual interaction of physiological processes and organelles, identifying the optimal production strategy is impossible without a greater understanding of the complex interplay of all cellular processes. Systems biology has emerged recently as a discipline to gain an understanding of these networks and their translation into a mathematical in silico model. An in silico model allows simulating optimization steps and, therefore, provides a useful method to identify targets for directed genetic/physiological modification to optimize the system for a biotechnological approach.

Visualizing bacterial surfaces in real time

The study of the network of proteins, protein complexes, sugars and surfaces organelles has been frustrated by the complexity of bacterial surfaces and the necessity to rely on molecular coordinates. The logical consequence to develop crystallographic methods for large cellular components and the alternative use of cryo‐electron microscopy to solve organelles structure (flagellum, Type IV pili) has been fundamental but inherently slow. Development of new vaccines may also depend on the identification of complexes located at the surface and exposed protective molecules. Prediction of both is part of routine genome analysis using dedicated algorithms. Antibody staining and FACS analysis is a potent tool to visualize exposed molecules while it is heavily dependent from antibody specificity, affinity and outer layer penetration effect (detection of hidden structures by antibody penetration during preparation of the sample). We suggest it is now possible to merge high‐resolution fluorescence and scanning confocal microscopy with sortase tagging (Popp et al., 2007) to generate native molecules bearing a chromophore. This will potentially allow image rendering of the bacterial surface to visualize the dynamics of protein topology during growth and infection in real time. Science and Nature have both expressed their wonder in 2008 about recent advances in light microscopy (Chi, 2008). In addition, white light sources are moving from lasers to inexpensive mass production of LED (http://www.lens.unifi.it). White light is a necessary step to pulse a sample with a wide range of light wavelengths to simultaneously collect signals in the visible spectrum from excited dyes. The resulting live image can integrate all labelled proteins in a single picture and with a different colour tag providing realistic 3D coordinates. The crucial step in closing the loop is in vivo tagging of a target protein. This in theory can provide a repertoire of one strain‐one tagged protein with the final goal to colour‐code all the proteins of a bacterial species. Sortases are bacterial enzymes that predominantly catalyse the attachment of surface proteins to the bacterial cell wall (Telford et al., 2006; Popp et al., 2007). Other sortases polymerize pilin subunits for the construction of the covalently attached pili of the Gram‐positive bacteria (Telford et al., 2006). The sortase recognition sequence of Staphylococcus aureus sortase A, LPXTG, when engrafted near the C‐terminus of proteins without natural sortase specificity, should be part of a sortase‐catalysed transpeptidation reaction using artificial glycine‐based nucleophiles. The chemical modification of such substrates with fluorophores allows modifications of proteins in in vitro and in vivo conditions. This method can be efficiently scaled‐up for high‐throughput data capture. Once the expected wave of new microscopes will be available and tagging with fluorophores will be pervasive, this technology should be ready also for fast and unexpensive real‐time expression analyses.

Intermediate Organelles of the Plant Secretory Pathway: Identity and Function

The secretory pathway of eukaryotic cells comprises a network of organelles that connects three large membranes, the plasma membrane, the vacuole and the endoplasmic reticulum. The Golgi apparatus and the various post-Golgi organelles that control vacuolar sorting, secretion and endocytosis can be regarded as intermediate organelles of the endocytic and biosynthetic routes. Many processes in the secretory pathway have evolved differently in plants and cannot be studied using yeast or mammalian cells as models. The best characterized organelles are the Golgi apparatus and the prevacuolar compartment, but recent work has shed light on the role of the trans Golgi network, which has to be regarded as a separate organelle in plants. In this study, we wish to highlight recent findings regarding the late secretory pathway and its crosstalk with the early secretory pathway as well as the endocytic route in plants. Recently published findings and suggested models are discussed within the context of known features of the equivalent pathway in other eukaryotes.

Reactive oxygen species generated in mitochondria induce fragmentation of the mitochondrial reticulum in HeLa cells

221 Mitochondria form a ramified network of organelles in cells of human carcinoma and many other eukaryotic cells. This structure was discovered by Bakeeva et al. [1] and is referred to as the mitochondrial reticulum. The processes of fission and fusion of mitochondria determine the state of dynamic equilibrium of the mitochondrial reticulum. The mitochondrial reticulum is fragmented during cell division, apoptosis, and different stresses. There are a number of proteins (e.g., Drp1 and Fis1) involved in this process, however, the details of the mechanism are unclear yet (for review, see [2, 3]). The pathway of signal transmission from various stimuli that induce oxidative stress or apoptosis to the machinery of mitochondrial fission is of interest. The central role in this pathway may be played by reactive oxygen species (ROSs), because their content increases during oxidative stress and apoptosis. The main source of ROSs under these conditions is mitochondria (for review, see [4]).

Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis

In cells, mitochondria are organized as a network of interconnected organelles that fluctuate between fission and fusion events (mitochondrial dynamics). This process is associated with cell death. We investigated whether activation of apoptosis with ceramides affects mitochondrial dynamics and promotes mitochondrial fission in cardiomyocytes. Neonatal rat cardiomyocytes were incubated with C(2)-ceramide or the inactive analog dihydro-C(2)-ceramide for up to 6 h. Three-dimensional images of cells loaded with mitotracker green were obtained by confocal microscopy. Dynamin-related protein-1 (Drp-1) and mitochondrial fission protein 1 (Fis1) distribution and levels were studied by immunofluorescence and western blot. Mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c (cyt c) distribution were used as indexes of early activation of apoptosis. Cell viability and DNA fragmentation were determined by propidium iodide staining/flow cytometry, whereas cytotoxicity was evaluated by lactic dehydrogenase activity. To decrease the levels of the mitochondrial fusion protein mitofusin 2, we used an antisense adenovirus (AsMfn2). C(2)-ceramide, but not dihydro-C(2)-ceramide, promoted rapid fragmentation of the mitochondrial network in a concentration- and time-dependent manner. C(2)-ceramide also increased mitochondrial Drp-1 and Fis1 content, Drp-1 colocalization with Fis1, and caused early activation of apoptosis. AsMfn2 accentuated the decrease in DeltaPsi(m) and cyt c redistribution induced by C(2)-ceramide. Doxorubicin, which induces cardiomyopathy and apoptosis through ceramide generation, also stimulated mitochondrial fragmentation. Ceramides stimulate mitochondrial fission and this event is associated with early activation of cardiomyocyte apoptosis.

Cellular networks and the aging process

The most important interactions between cellular molecules have a high affinity, are unique and specific, and require a network approach for a detailed description. After a brief introduction to cellular networks (protein – protein interaction networks, metabolic networks, gene regulatory networks, signalling networks and membrane – organelle networks) an overview is given on the network aspects of the theories on aging. The most important part of the review summarizes our knowledge on the aging of networks. The effects of aging on the general network models are described, as well as the initial findings on the effects of aging on the cellular networks. Finally we suggest a ‘weak link theory of aging’ linking the random damage of the network constituents to the overwhelming majority of the low affinity, transient interactions (weak links) in the cellular networks. We show that random damage of weak links may lead to an increase of noise and an increased vulnerability of cellular networks, and make a comparison between these predictions and the observed behaviour of the emergent properties of cellular networks in aged organisms.

The apoptotic process of human bladder carcinoma T24 cells induced by retinoid.

Breakdown of the cytoskeletal network and redistribution of cytoplasmic organelles are early events of programmed cell death. Previous studies showed that retinoic acid induces programmed cell death in many tumor cell lines and that cytokeratins, particularly cytokeratin 18, are affected in the early events of apoptosis. In this study, patterns of cytoplasmic intermediate filaments (cytokeratin 18), actin filaments, and microtubules, as well as Bax and Bcl-2 proteins in human bladder carcinoma T24 cells were examined before and after retinoic acid treatment by immunocytochemistry and conventional electron microscopy. Our results demonstrate that the redistribution of Bax and Bcl-2 proteins in the subcellular compartment of T24 cells is correlated with reorganization of the cytoplasmic intermediate filament network and that cytokeratins are cleaved by caspases, as revealed by the M30 antibody which recognizes a specific caspase cleavage site within cytokeratin 18. The cytoskeletal architectures of microtubules are not significantly affected in the early apoptotic process, from our observations. We suggest that the breakdown in the intermediate filament network associated with the aggregation of mitochondria and lysosome may be a crucial event in the apoptotic process and that aggregation of cytoplasmic Bax may accelerate apoptotic death.

Protein transport within the plant cell endomembrane system: an update

The secretory pathway plays a central role in plant development and morphogenesis. Storage protein deposition, plant cell division and the expansion of the plasma membrane and extracellular matrix all require the synthesis and trafficking of membranes, proteins and polysaccharides through this network of organelles. Increasing evidence demonstrates that the plant secretory pathway is more complex than previously appreciated and that its formation and maintenance are guided/regulated by many different mechanisms.

The search for the endogenous digitalis: an alternative hypothesis

The universal presence of a binding site for cardiac glycosides on Na+-K+-ATPase has engendered speculation as to whether it also serves as a receptor for an endogenous digitalis-like hormone or autacoid. If such a hormone were to exist, it could play a role in sodium homeostasis and in the pathophysiology of primary hypertension and uremia. However, we believe that this hypothesis rests on unproven assumptions. Although typical of many toxins and drugs, binding to a single protein that acts as both its receptor and effector mechanism at the cell membrane, thereby directly affecting transmembrane ion flux, would be unusual for a hormone or autacoid. As an alternative hypothesis for the evolutionary conservation of the cardiac glycoside binding site, we suggest that its endogenous ligand may exist within the cell. After cotranslational insertion of the alpha- and beta-subunits into the membrane of the rough endoplasmic reticulum, Na+-K+-ATPase, like most integral membrane proteins, 1) must be targeted through a complex network of intracellular organelles to the correct plasmalemmal domain, 2) must be monitored for appropriate protein conformation and subunit assembly, and perhaps 3) could have its catalytic function regulated before insertion in the cell membrane. Because the lumina of the endoplasmic reticulum, Golgi, and other organelles and vesicles are topologically equivalent to the outside of the cell, all three functions could be subserved by an intraorganellar ligand for the cardiac glycoside binding site.

Morphology of astroglial cells is controlled by beta-adrenergic receptors.

Astroglial cells in vivo and in vitro respond to hormones, growth factors, and neurotransmitters by changing from an epithelial-like to stellate morphology. We have studied the temporal relationship between receptor activation, second messenger mobilization, and morphological changes using LRM55 astroglial cells. Maintenance of an altered morphology required continuous beta-adrenergic receptor activation. These changes appeared to be mediated by cAMP since they were elicited by its analogue, dibutyryl cAMP, and by forskolin, a direct activator of adenylate cyclase. Changes in cell morphology may require a relatively small increase in intracellular cAMP, since receptor-stimulated changes in cAMP levels were transient and peaked approximately 5 min after receptor activation while changes in morphology took at least 30 min to reach a new steady state. Time-lapse videomicroscopy and high voltage electron microscopy indicated that receptor activation resulted in a sequence of morphological events. Time-lapse observations revealed the development and enlargement of openings through the cytoplasm associated with cytoplasmic withdrawal to the perinuclear region and process formation. Higher resolution high voltage electron microscopy indicated that the transition to a stellate morphology was preceded by the appearance of two distinct cytoplasmic domains. One contained an open network of filaments and organelles. The other was characterized by short broad cytoplasmic filaments. The first domain was similar to cytoplasm in control cells while the second was associated with the development and enlargement of openings through the cytoplasm and regions of obvious cytoplasmic withdrawal.