Biomedical subjects

Maura Francolini

Publications and source records attributed to Maura Francolini.

16 recordsLinked to original sources

PubMed2006

Dynamic and reversible restructuring of the ER induced by PDMP in cultured cells.

In many cells, the endoplasmic reticulum (ER) contains segregated smooth and rough domains, but the mechanism of this segregation is unclear. Here, we used a HeLa cell line, inducibly expressing a GFP fusion protein [GFP-b(5)tail] anchored to the ER membrane, as a tool to investigate factors influencing ER organisation. Induction of GFP-b(5)tail expression caused proliferation of the ER, but its normal branching polygonal meshwork architecture was maintained. Experiments designed to test the effects of drugs that alter ceramide levels revealed that treatment of these cells with Phenyl-2-decanoyl-amino-3-morpholino-1-propanol-hydrocholride (PDMP) generated patches of segregated smooth ER, organised as a random tubular network, which rapidly dispersed after removal of the drug. The effect of PDMP was independent of its activity as sphingolipid synthesis inhibitor, but could be partially reversed by a membrane-permeant Ca(2+) chelator. Although the smooth ER patches maintained connectivity with the remaining ER, they appeared to represent distinct domains differing in protein and lipid composition from the remaining ER. PDMP did not cause detachment of membrane-bound ribosomes, indicating that smooth ER patch generation was due to a reorganisation of pre-existing ribosome-free areas. Our results demonstrate a dynamic relationship between smooth and rough ER and have implications for the mechanisms regulating ER architecture.

Animals↗

PubMed2006

Endoplasmic reticulum architecture: structures in flux.

The endoplasmic reticulum (ER) is a dynamic pleiomorphic organelle containing continuous but distinct subdomains. The diversity of ER structures parallels its many functions, including secretory protein biogenesis, lipid synthesis, drug metabolism and Ca2+ signaling. Recent studies are revealing how elaborate ER structures arise in response to subtle changes in protein levels, dynamics, and interactions as well as in response to alterations in cytosolic ion concentrations. Subdomain formation appears to be governed by principles of self-organization. Once formed, ER subdomains remain malleable and can be rapidly transformed into alternative structures in response to altered conditions. The mechanisms that modulate ER structure are likely to be important for the generation of the characteristic shapes of other organelles.

Animals↗

PubMed2006

Cell culture models to investigate the selective vulnerability of motoneuronal mitochondria to familial ALS-linked G93ASOD1.

Mitochondrial damage induced by superoxide dismutase (SOD1) mutants has been proposed to have a causative role in the selective degeneration of motoneurons in amyotrophic lateral sclerosis (ALS). In order to investigate the basis of the tissue specificity of mutant SOD1 we compared the effect of the continuous expression of wild-type or mutant (G93A) human SOD1 on mitochondrial morphology in the NSC-34 motoneuronal-like, the N18TG2 neuroblastoma and the non-neuronal Madin-Darby Canine Kidney (MDCK) cell lines. Morphological alterations of mitochondria were observed in NSC-34 expressing the G93A mutant (NSC-G93A) but not the wild-type SOD1, whereas a ten-fold greater level of total expression of the mutant had no effect on mitochondria of non-motoneuronal cell lines. Fragmented network, swelling and cristae remodelling but not vacuolization of mitochondria or other intracellular organelles were observed only in NSC-G93A cells. The mitochondrial alterations were not explained by a preferential localization of the mutant within NSC-G93A mitochondria, as a higher amount of the mutant SOD1 was found in mitochondria of MDCK-G93A cells. Our results suggest that mitochondrial vulnerability of motoneurons to G93ASOD1 is recapitulated in NSC-34 cells, and that peculiar features in network dynamics may account for the selective alterations of motoneuronal mitochondria.

Amyotrophic Lateral Sclerosis↗

PubMed2005

Synaptobrevin2-expressing vesicles in rat astrocytes: insights into molecular characterization, dynamics and exocytosis.

The SNARE-dependent exocytosis of glutamate-containing vesicles in astrocytes is increasingly viewed as an important signal at the basis of the astrocyte-to-neurone communication system in the brain. Here we provide further insights into the molecular features and dynamics of vesicles in cultured astrocytes. We found that immunoisolated synaptobrevin2 vesicles are clear vesicles quite heterogenous in size and contain the vesicular glutamate transporter v-Glut-2. Moreover, they are immunopositive for synaptotagmin IV, for AMPA receptor subunits GluR2,3 and, to a lesser extent, for GluR1. We also provide direct evidence for the functional expression of v-Glut-2 in astrocytes and demonstrate that synaptobrevin2-positive vesicles can specifically take up (3H)L-glutamate via a bafilomycin-sensitive mechanism. Finally, by time lapse confocal microscopy, we show that a subpopulation of vesicles (tagged with a synaptobrevin2-EGFP chimera) is highly mobile and can fuse with the plasma membrane, preferentially at the level of the astrocyte processes, in a Ca2+-dependent manner. These latter observations, together with the evidence reported here for the expression of functional v-Glut-2 in synaptobrevin2-positive vesicles, provide a molecular basis for regulated exocytosis in astrocyte.

Animals↗

PubMed2005

Two tail-anchored protein variants, differing in transmembrane domain length and intracellular sorting, interact differently with lipids.

C-tail-anchored (TA) proteins often require a transmembrane domain of moderate hydrophobicity to maintain their endoplasmic reticulum residence, but the suggested role of protein-lipid interactions in this phenomenon has not been established. Here, we studied the interaction of TA proteins with lipids by differential scanning calorimetry by using a model system consisting of liposomes embedding either of two forms of cytochrome b(5): the endoplasmic reticulum-resident wild-type (b(5)wt) and a mutant thereof (b(5)ext), that contains five extra nonpolar amino acids in its transmembrane domain and, therefore, reaches the plasma membrane. The proteins were incorporated into liposomes of palmitoyl-oleyl-phosphatidylcholine (POPC) or POPC mixed with either distearoyl-phosphatidylserine (DSPS), palmitoyl-oleyl-phosphatidylserine (POPS), distearoyl-phosphatidylcholine (DSPC), or C16-ceramide (CER). POPC liposomes displayed a single thermotropic transition centered at -3.4 degrees C. When present, the second lipid formed a domain within the POPC bilayer, as indicated by the appearance of an additional peak. This peak was centered at temperatures close to 0 degrees C in the case of liposomes containing 10% CER, DSPS, and POPS and at 23 degrees C in the case of DSPC, likely reflecting a higher degree of molecular packing for DSPC domains. In DSPS/POPC, POPS/POPC, or CER/POPC, but not in DSPC/POPC liposomes, the insertion of b(5)wt increased, whereas b(5)ext decreased, the relative contribution to the total enthalpy of the higher temperature, phase-separated component. These results were confirmed with fluorescence measurements by using pyrene-labeled phospholipids. The dissimilar interaction with lipids of these two differently localized TA proteins could have implications for their intracellular sorting.

Amino Acid Sequence↗

PubMed2004

Mitochondrial biogenesis by NO yields functionally active mitochondria in mammals.

We recently found that long-term exposure to nitric oxide (NO) triggers mitochondrial biogenesis in mammalian cells and tissues by activation of guanylate cyclase and generation of cGMP. Here, we report that the NO/cGMP-dependent mitochondrial biogenesis is associated with enhanced coupled respiration and content of ATP in U937, L6, and PC12 cells. The observed increase in ATP content depended entirely on oxidative phosphorylation, because ATP formation by glycolysis was unchanged. Brain, kidney, liver, heart, and gastrocnemius muscle from endothelial NO synthase null mutant mice displayed markedly reduced mitochondrial content associated with significantly lower oxygen consumption and ATP content. In these tissues, ultrastructural analyses revealed significantly smaller mitochondria. Furthermore, a significant reduction in the number of mitochondria was observed in the subsarcolemmal region of the gastrocnemius muscle. We conclude that NO/cGMP stimulates mitochondrial biogenesis, both in vitro and in vivo, and that this stimulation is associated with increased mitochondrial function, resulting in enhanced formation of ATP.

Adenosine Triphosphate↗

PubMed2003

Role of lipid microdomains in P/Q-type calcium channel (Cav2.1) clustering and function in presynaptic membranes.

Lipid microdomains can selectively include or exclude proteins and may be important in a variety of functions such as protein sorting, cell signaling, and synaptic transmission. The present study demonstrates that two different voltage-gated calcium channels, which both interact with soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins but have distinct subcellular distributions and roles in synaptic transmission, are differently distributed in lipid microdomains; presynaptic P/Q (Cav2.1) but not Lc (Cav1.2) calcium channel subtypes are mainly accumulated in detergent-insoluble complexes. The immunoisolation of multiprotein complexes from detergent-insoluble or detergent-soluble fractions shows that the alpha1A subunits of Cav2.1 colocalize and interact with SNARE complexes in lipid microdomains. The altered organization of these microdomains caused by saponin and methyl-beta-cyclodextrin treatment largely impairs the buoyancy and distribution of Cav2.1 channels and SNAREs in flotation gradients. On the other hand, cholesterol reloading partially reverses the drug effects. Methyl-beta-cyclodextrin treatment alters the colocalization of Cav2.1 with the proteins of the exocytic machinery and also impairs calcium influx in nerve terminals. These results show that lipid microdomains in presynaptic terminals are important in organizing membrane sites specialized for synaptic vesicle exocytosis. The cholesterol-enriched microdomains contribute to optimizing the compartmentalization of exocytic machinery and the calcium influx that triggers synaptic vesicle exocytosis.

Animals↗

PubMed2003

Formation of stacked ER cisternae by low affinity protein interactions.

The endoplasmic reticulum (ER) can transform from a network of branching tubules into stacked membrane arrays (termed organized smooth ER [OSER]) in response to elevated levels of specific resident proteins, such as cytochrome b(5). Here, we have tagged OSER-inducing proteins with green fluorescent protein (GFP) to study OSER biogenesis and dynamics in living cells. Overexpression of these proteins induced formation of karmellae, whorls, and crystalloid OSER structures. Photobleaching experiments revealed that OSER-inducing proteins were highly mobile within OSER structures and could exchange between OSER structures and surrounding reticular ER. This indicated that binding interactions between proteins on apposing stacked membranes of OSER structures were not of high affinity. Addition of GFP, which undergoes low affinity, antiparallel dimerization, to the cytoplasmic domains of non-OSER-inducing resident ER proteins was sufficient to induce OSER structures when overexpressed, but addition of a nondimerizing GFP variant was not. These results point to a molecular mechanism for OSER biogenesis that involves weak homotypic interactions between cytoplasmic domains of proteins. This mechanism may underlie the formation of other stacked membrane structures within cells.

Animals↗

PubMed2003

Oxytocin receptor elicits different EGFR/MAPK activation patterns depending on its localization in caveolin-1 enriched domains.

We have recently shown that oxytocin inhibits cell proliferation when the vast majority of oxytocin receptors are excluded from caveolin-1-enriched microdomains, and that, on the contrary, it has a mitogenic effect when the receptors are targeted to these plasma membrane domains. In this study, we investigated whether the receptors located inside and outside caveolar microdomains initiate different signalling pathways and how this may lead to opposite effects on cell proliferation. Our data indicate that, depending on their localization, oxytocin receptors transactivate EGFR and activate ERK1/2 using different signalling intermediates. The final outcome is a different temporal pattern of EGFR and ERK1/2 phosphorylation, which is more persistent when the receptors are located outside caveolar microdomains and inhibit cell growth, and very transient when they are located in caveolar microdomains and stimulate cell growth. Finally, only the activation of receptors located outside caveolar microdomains correlates with the activation of the cell cycle inhibitor p21(WAF1/CIP1), thus suggesting that the antiproliferative OTR effects may, in this case, be achieved by a sustained activation of EGFR and MAPK leading to the induction of this cell cycle regulator.

Caveolae↗

PubMed2003

Mitochondrial biogenesis in mammals: the role of endogenous nitric oxide.

Nitric oxide was found to trigger mitochondrial biogenesis in cells as diverse as brown adipocytes and 3T3-L1, U937, and HeLa cells. This effect of nitric oxide was dependent on guanosine 3',5'-monophosphate (cGMP) and was mediated by the induction of peroxisome proliferator-activated receptor gamma coactivator 1alpha, a master regulator of mitochondrial biogenesis. Moreover, the mitochondrial biogenesis induced by exposure to cold was markedly reduced in brown adipose tissue of endothelial nitric oxide synthase null-mutant (eNOS-/-) mice, which had a reduced metabolic rate and accelerated weight gain as compared to wild-type mice. Thus, a nitric oxide-cGMP-dependent pathway controls mitochondrial biogenesis and body energy balance.

3T3 Cells↗

PubMed2002

Neuronal calcium sensor 1 and phosphatidylinositol 4-OH kinase beta interact in neuronal cells and are translocated to membranes during nucleotide-evoked exocytosis.

Neuronal calcium sensor 1 (NCS-1) belongs to a family of EF-hand calcium-binding proteins and is mainly expressed in neurons and neuroendocrine cells, where it causes facilitation of neurotransmitter release through unknown mechanisms. The yeast homologue of NCS-1 has been demonstrated to interact with and regulate the activity of yeast phosphatidylinositol 4-OH kinase beta (PI4Kbeta). However, in neurons and neurosecretory cells NCS-1 has not unequivocally been shown to interact with PI4Kbeta. Here we have compared the subcellular distribution of NCS-1 and PI4Kbeta and investigated whether they are capable of forming complexes. In neurons, both proteins are widely distributed and are present in perikarya and, to a lesser extent, in nerve terminals. A consistent portion of NCS-1 and PIK4beta is cytosolic, whereas a portion of both proteins appears to be associated with the membranes of the endoplasmic reticulum and the Golgi complex. Very small amounts of NCS-1 and PI4Kbeta are present in synaptic vesicles. Our results further demonstrate that in neurosecretory cells, endogenous NCS-1 and PIK4beta interact to form a complex that can be immunoisolated from membrane as well as from cytosolic fractions. Moreover, both proteins can be recruited to membranes when cells are treated with nucleotide receptor agonists known to increase polyphosphoinositide turnover and concomitantly induce exocytosis of secretory vesicles. Finally, in PC12 cells overexpressing NCS-1, the amount of PI4Kbeta associated with the membranes is increased concomitantly with the increased levels of NCS-1 detected in the same membrane fractions. Together, these findings demonstrate that mammalian NCS-1 and PI4Kbeta interact under physiological conditions, which suggest a possible role for NCS-1 in the translocation of PI4Kbeta to target membranes.

1-Phosphatidylinositol 4-Kinase↗

PubMed2002

Early oxidative damage in primary cultured trout hepatocytes: a time course study.

The aim of this study was to evaluate the influence of the two-step hepatocyte isolation procedure on primary cultured trout (Oncorhynchus mykiss) hepatocytes over time. We characterised the possible changes of a variety of some cellular parameters within the first 24-48 h after seeding. We followed the time dependent changes of these parameters during subsequent culture times in order to see if the cells maintained a differentiated status. Scanning electron microscopy revealed bleb formation and 20% cell damage in freshly isolated hepatocytes. During subsequent culture times the bleb dimension appear to be reduced. Heat shock proteins 70 and 50 (HSP70, HSP50) were induced by hepatocyte isolation. During the first 4 h of culture, the hepatocytes showed a variation in mitochondrial activity, an increase in free radical species (ROS), and a decrease in both glutathione (GSH) content and catalase (CAT) activity; the generation of free radicals led to an increase in the formation of 8-hydroxydeoxyguanosine (8-OHdG) in the DNA. The cells showed detectable ethoxyresorufin-O-deethylase activity after 4 h of culture, which had rapidly increased by the 24th hour. After 24 h, mitochondrial and CAT activity, free radical production, and the content of GSH and 8-OHdG returned to their original levels. P450 activity was retained for at least 48 h after seeding. Our data show that trout hepatocytes suffer significant cell injury as a result of the isolation procedure, but primary cultured cells metabolically recover from this stress after a few hours: they are capable of repairing their damaged surfaces, recovering their antioxidant defences and retaining their ability to repair DNA. Our results also confirm that trout hepatocytes in a primary culture maintain their in vivo-like metabolic activities for 3-8 days.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2002

Invasive behaviour of glioblastoma cell lines is associated with altered organisation of the cadherin-catenin adhesion system.

As little is known about the role of cadherin-mediated cell-cell adhesion in astrocytes and its alteration in migrating and invasive glioblastomas, we investigated its molecular composition and organisation in primary cultured astrocytes and the T98G and U373MG glioblastoma cell lines. Biochemical and morphological analysis indicated that all three cell types express all of the structural components of the adhesion system, including the LIN-7 PDZ protein, a novel component involved in the organisation of the junctional domain in epithelia and neurons. However, only the astrocytes and T98G cells generated and maintained mature adhesive junctional domains to which LIN-7 was recruited. Alterations in the junctional domain of U373MG cells were associated with higher motility in a poly-L-lysine migration assay. When the T98G cells were cultured on Matrigel matrix, they acquired invasive properties but, despite unchanged cadherin adhesion system protein levels, the invasive T98G cell-cell contacts failed to accumulate LIN-7 and failed to mature. These results identify the LIN-7 PDZ protein as a marker of cell adhesion maturity and cell invasion and indicate that instability and disorganisation of cadherin-mediated junctions rather than reduced expression of cadherin-catenin system components are required to promote migration and invasiveness in glioblastoma cell lines.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Neuronal calcium sensor-1 binds to regulated secretory organelles and functions in basal and stimulated exocytosis in PC12 cells.

Neuronal calcium sensor-1 (NCS-1) and its non-mammalian homologue, frequenin, have been implicated in a spectrum of cellular processes, including regulation of stimulated exocytosis of synaptic vesicles and secretory granules (SGs) in neurons and neuroendocrine cells and regulation of phosphatidylinositol 4-kinase beta activity in yeast. However, apart from these intriguing putative functions, NCS-1 and frequenin are relatively poorly understood. Here, the distribution, dynamics and function of NCS-1 were studied using PC12 cells that stably express NCS-1-EYFP (NCS-1 fused to enhanced yellow fluorescent protein) or that stably overexpress NCS-1. Fluorescence and electron microscopies show that NCS-1-EYFP is absent from SGs but is present on small clear organelles, some of which are just below the plasma membrane. Total internal reflection fluorescence microscopy shows that NCS-1-EYFP is associated with synaptic-like microvesicles (SLMVs) in growth cones. Overexpression studies show that NCS-1 enhances exocytosis of synaptotagmin-labeled regulated secretory organelles (RSOs) under basal conditions and during stimulation by UTP. Significantly, these studies implicate NCS-1 in the enhancement of both basal and stimulated phosphoinositide-dependent exocytosis of RSOs in PC12 cells, and they show that NCS-1 is distributed strategically to interact with putative targets on the plasma membrane and on SLMVs. These studies also reveal that SLMVs undergo both fast directed motion and highly hindered diffusive motion in growth cones, suggesting that cytoskeletal constituents can both facilitate and hinder SLMV motion. These results also reveal interesting similarities and differences between transport organelles in differentiated neuroendocrine cells and neurons.

Animals↗

PubMed2002

Mechanisms underlying the neuronal calcium sensor-1-evoked enhancement of exocytosis in PC12 cells.

Neuronal calcium sensor-1 (NCS-1) or the originally identified homologue frequenin belongs to a superfamily of EF-hand calcium binding proteins. Although NCS-1 is thought to enhance synaptic efficacy or exocytosis mainly by activating ion channel function, the detailed molecular basis for the enhancement is still a matter of debate. Here, mechanisms underlying the NCS-1-evoked enhancement of exocytosis were investigated using PC12 cells overexpressing NCS-1. NCS-1 was found to have a broad distribution in the cells being partially distributed in the cytosol and associated to vesicles and tubular-like structures. Biochemical and immunohistochemical studies indicated that NCS-1 partially colocalized with the light synaptic vesicle marker synaptophysin. When stimulated with UTP or bradykinin, agonists to phospholipase C-linked receptors, NCS-1 enhanced the agonist-mediated elementary and global Ca2+ signaling and increased the levels of downstream signals of phosphatidylinositol 4-kinase. NCS-1 enhanced the UTP-evoked exocytosis but not the depolarization-evoked Ca2+ responses or exocytosis, suggesting that the enhancement by NCS-1 should involve phospholipase C-linked receptor-mediated signals rather than the Ca2+ channels or exocytotic machinery per se. Taken together, NCS-1 enhances phosphoinositide turnover, resulting in enhancement of Ca2+ signaling and exocytosis. This is a novel regulatory mechanism of exocytosis that might involve the activation of phosphatidylinositol 4-kinase.

Animals↗

PubMed2002

Localization of the human oxytocin receptor in caveolin-1 enriched domains turns the receptor-mediated inhibition of cell growth into a proliferative response.

In this study, we investigated the functional role of the localization of human OTR in caveolin-1 enriched membrane domains. Biochemical fractionation of MDCK cells stably expressing the WT OTR-GFP indicated that only minor quantities of receptor are partitioned in caveolin-1 enriched domains. However, when fused to caveolin-2, the OTR protein proved to be exclusively localized in caveolin-1 enriched fractions, where it bound the agonist with increased affinity and efficiently coupled to Galpha(q/11). Interestingly, the chimeric protein was unable to undergo agonist-induced internalization and remained confined to the plasma membrane even after prolonged agonist exposure (120 min). A striking difference in receptor stimulation was observed when the OT-induced effect on cell proliferation was analysed: stimulation of the human WT OTR inhibited cell growth, whereas the chimeric protein had a proliferative effect. These data indicate that the localization of human OTR in caveolin-1 enriched microdomains radically alters its regulatory effects on cell growth; the fraction of OTR residing in caveolar structures may therefore play a crucial role in regulating cell proliferation.

Animals↗