44 results on '"Diana Pendin"'
Search Results
2. Dynamic constriction and fission of endoplasmic reticulum membranes by reticulon
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Javier Espadas, Diana Pendin, Rebeca Bocanegra, Artur Escalada, Giulia Misticoni, Tatiana Trevisan, Ariana Velasco del Olmo, Aldo Montagna, Sergio Bova, Borja Ibarra, Peter I. Kuzmin, Pavel V. Bashkirov, Anna V. Shnyrova, Vadim A. Frolov, and Andrea Daga
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Science - Abstract
The endoplasmic reticulum (ER) is an intracellular network characterized by highly dynamic behavior whose control mechanisms are unclear. Here, the authors show that the ER-membrane protein Reticulon (Rtnl1) can constrict ER bilayers and lead to ER fission.
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- 2019
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3. mCerulean3-Based Cameleon Sensor to Explore Mitochondrial Ca2+ Dynamics In Vivo
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Elisa Greotti, Ilaria Fortunati, Diana Pendin, Camilla Ferrante, Luisa Galla, Lorena Zentilin, Mauro Giacca, Nina Kaludercic, Moises Di Sante, Letizia Mariotti, Annamaria Lia, Marta Gómez-Gonzalo, Michele Sessolo, Giorgio Carmignoto, Renato Bozio, and Tullio Pozzan
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Science - Abstract
Summary: Genetically Encoded Ca2+ Indicators (GECIs) are extensively used to study organelle Ca2+ homeostasis, although some available probes are still plagued by a number of problems, e.g., low fluorescence intensity, partial mistargeting, and pH sensitivity. Furthermore, in the most commonly used mitochondrial Förster Resonance Energy Transfer based-GECIs, the donor protein ECFP is characterized by a double exponential lifetime that complicates the fluorescence lifetime analysis. We have modified the cytosolic and mitochondria-targeted Cameleon GECIs by (1) substituting the donor ECFP with mCerulean3, a brighter and more stable fluorescent protein with a single exponential lifetime; (2) extensively modifying the constructs to improve targeting efficiency and fluorescence changes caused by Ca2+ binding; and (3) inserting the cDNAs into adeno-associated viral vectors for in vivo expression. The probes have been thoroughly characterized in situ by fluorescence microscopy and Fluorescence Lifetime Imaging Microscopy, and examples of their ex vivo and in vivo applications are described. : Biological Sciences Tools; Cell Biology; Optical Imaging Subject Areas: Biological Sciences Tools, Cell Biology, Optical Imaging
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- 2019
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4. In vivo Analysis of CRISPR/Cas9 Induced Atlastin Pathological Mutations in Drosophila
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Aldo Montagna, Nicola Vajente, Diana Pendin, and Andrea Daga
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atlastin ,mutation ,CRISPR/Cas9 ,hereditary spastic paraplegia ,endoplasmic reticulum ,Golgi ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
The endoplasmic reticulum (ER) is a highly dynamic network whose shape is thought to be actively regulated by membrane resident proteins. Mutation of several such morphology regulators cause the neurological disorder Hereditary Sp astic Paraplegia (HSP), suggesting a critical role of ER shape maintenance in neuronal activity and function. Human Atlastin-1 mutations are responsible for SPG3A, the earliest onset and one of the more severe forms of dominant HSP. Atlastin has been initially identified in Drosophila as the GTPase responsible for the homotypic fusion of ER membrane. The majority of SPG3A-linked Atlastin-1 mutations map to the GTPase domain, potentially interfering with atlastin GTPase activity, and to the three-helix-bundle (3HB) domain, a region critical for homo-oligomerization. Here we have examined the in vivo effects of four pathogenetic missense mutations (two mapping to the GTPase domain and two to the 3HB domain) using two complementary approaches: CRISPR/Cas9 editing to introduce such variants in the endogenous atlastin gene and transgenesis to generate lines overexpressing atlastin carrying the same pathogenic variants. We found that all pathological mutations examined reduce atlastin activity in vivo although to different degrees of severity. Moreover, overexpression of the pathogenic variants in a wild type atlastin background does not give rise to the loss of function phenotypes expected for dominant negative mutations. These results indicate that the four pathological mutations investigated act through a loss of function mechanism.
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- 2020
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5. Microtubules Stabilization by Mutant Spastin Affects ER Morphology and Ca2+ Handling
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Nicola Vajente, Rosa Norante, Nelly Redolfi, Andrea Daga, Paola Pizzo, and Diana Pendin
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spastin ,drosophila ,microtubules ,endoplasmic reticulum ,calcium ,SOCE ,Physiology ,QP1-981 - Abstract
The endoplasmic reticulum (ER) extends as a network of interconnected tubules and sheet-like structures in eukaryotic cells. ER tubules dynamically change their morphology and position within the cells in response to physiological stimuli and these network rearrangements depend on the microtubule (MT) cytoskeleton. Store-operated calcium entry (SOCE) relies on the repositioning of ER tubules to form specific ER-plasma membrane junctions. Indeed, the tips of polymerizing MTs are supposed to provide the anchor for ER tubules to move toward the plasma membrane, however the precise role of the cytoskeleton during SOCE has not been conclusively clarified. Here we exploit an in vivo approach involving the manipulation of MT dynamics in Drosophila melanogaster by neuronal expression of a dominant-negative variant of the MT-severing protein spastin to induce MT hyper-stabilization. We show that MT stabilization alters ER morphology, favoring an enrichment in ER sheets at the expense of tubules. Stabilizing MTs has a negative impact on the process of SOCE and results in a reduced ER Ca2+ content, affecting the flight ability of the flies. Restoring proper MT organization by administering the MT-destabilizing drug vinblastine, chronically or acutely, rescues ER morphology, SOCE and flight ability, indicating that MT dynamics impairment is responsible for all the phenotypes observed.
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- 2019
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6. Manipulation of Mitochondria Dynamics Reveals Separate Roles for Form and Function in Mitochondria Distribution
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Tatiana Trevisan, Diana Pendin, Aldo Montagna, Sergio Bova, Anna Maria Ghelli, and Andrea Daga
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Biology (General) ,QH301-705.5 - Abstract
Summary: Mitochondria shape is controlled by membrane fusion and fission mediated by mitofusins, Opa1, and Drp1, whereas mitochondrial motility relies on microtubule motors. These processes govern mitochondria subcellular distribution, whose defects are emphasized in neurons because of their polarized structure. We have studied how perturbation of the fusion/fission balance affects mitochondria distribution in Drosophila axons. Knockdown of Marf or Opa1 resulted in progressive loss of distal mitochondria and in a distinct oxidative phosphorylation and membrane potential deficit. Downregulation of Drp1 rescued the lethality and bioenergetic defect caused by neuronal Marf RNAi, but induced only a modest restoration of axonal mitochondria distribution. Surprisingly, Drp1 knockdown rescued fragmentation and fully restored aberrant distribution of axonal mitochondria produced by Opa1 RNAi; however, Drp1 knockdown did not improve viability or mitochondria function. Our data show that proper morphology is critical for proper axonal mitochondria distribution independent of bioenergetic efficiency. The health of neurons largely depends on mitochondria function, but does not depend on shape or distribution. : Trevisan et al. separate the independent contribution of form and function in determining the distribution of mitochondria in axons. They show that morphology is crucial for proper axonal mitochondria distribution, independent of their bioenergetic efficiency. However, the health of neurons depends on mitochondria function, but does not depend on shape or distribution.
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- 2018
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7. ER Morphology in the Pathogenesis of Hereditary Spastic Paraplegia
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Sonia Sonda, Diana Pendin, and Andrea Daga
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endoplasmic reticulum ,hereditary spastic paraplegia ,ER-shaping proteins ,Cytology ,QH573-671 - Abstract
The endoplasmic reticulum (ER) is the most abundant and widespread organelle in cells. Its peculiar membrane architecture, formed by an intricate network of tubules and cisternae, is critical to its multifaceted function. Regulation of ER morphology is coordinated by a few ER-specific membrane proteins and is thought to be particularly important in neurons, where organized ER membranes are found even in the most distant neurite terminals. Mutation of ER-shaping proteins has been implicated in the neurodegenerative disease hereditary spastic paraplegia (HSP). In this review we discuss the involvement of these proteins in the pathogenesis of HSP, focusing on the experimental evidence linking their molecular function to disease onset. Although the precise biochemical activity of some ER-related HSP proteins has been elucidated, the pathological mechanism underlying ER-linked HSP is still undetermined and needs to be further investigated.
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- 2021
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8. Generation and Characterization of a New FRET-Based Ca2+ Sensor Targeted to the Nucleus
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Luisa Galla, Nicola Vajente, Diana Pendin, Paola Pizzo, Tullio Pozzan, and Elisa Greotti
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calcium ,nucleus ,nuclear ,FRET-based probe ,endoplasmic reticulum ,Cameleon ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Calcium (Ca2+) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca2+ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca2+]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca2+] changes. To this aim, as nucleoplasm Ca2+ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca2+ probe. In particular, we modified the previously described nuclear Ca2+ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca2+ measurements. The affinity for Ca2+ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca2+ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca2+ signal dynamics in different subcellular compartments in the very same cells are also presented.
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- 2021
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9. Lighting Up Ca2+ Dynamics in Animal Models
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Nelly Redolfi, Paloma García-Casas, Chiara Fornetto, Sonia Sonda, Paola Pizzo, and Diana Pendin
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calcium imaging ,calcium indicators ,GECI ,animal models ,nervous system ,Cytology ,QH573-671 - Abstract
Calcium (Ca2+) signaling coordinates are crucial processes in brain physiology. Particularly, fundamental aspects of neuronal function such as synaptic transmission and neuronal plasticity are regulated by Ca2+, and neuronal survival itself relies on Ca2+-dependent cascades. Indeed, impaired Ca2+ homeostasis has been reported in aging as well as in the onset and progression of neurodegeneration. Understanding the physiology of brain function and the key processes leading to its derangement is a core challenge for neuroscience. In this context, Ca2+ imaging represents a powerful tool, effectively fostered by the continuous amelioration of Ca2+ sensors in parallel with the improvement of imaging instrumentation. In this review, we explore the potentiality of the most used animal models employed for Ca2+ imaging, highlighting their application in brain research to explore the pathogenesis of neurodegenerative diseases.
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- 2021
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10. Presenilin-2 and Calcium Handling: Molecules, Organelles, Cells and Brain Networks
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Paola Pizzo, Emy Basso, Riccardo Filadi, Elisa Greotti, Alessandro Leparulo, Diana Pendin, Nelly Redolfi, Michela Rossini, Nicola Vajente, Tullio Pozzan, and Cristina Fasolato
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presenilin-2 ,calcium signalling ,Alzheimer’s disease mouse models ,SOCE ,mitochondria ,autophagy ,Cytology ,QH573-671 - Abstract
Presenilin-2 (PS2) is one of the three proteins that are dominantly mutated in familial Alzheimer’s disease (FAD). It forms the catalytic core of the γ-secretase complex—a function shared with its homolog presenilin-1 (PS1)—the enzyme ultimately responsible of amyloid-β (Aβ) formation. Besides its enzymatic activity, PS2 is a multifunctional protein, being specifically involved, independently of γ-secretase activity, in the modulation of several cellular processes, such as Ca2+ signalling, mitochondrial function, inter-organelle communication, and autophagy. As for the former, evidence has accumulated that supports the involvement of PS2 at different levels, ranging from organelle Ca2+ handling to Ca2+ entry through plasma membrane channels. Thus FAD-linked PS2 mutations impact on multiple aspects of cell and tissue physiology, including bioenergetics and brain network excitability. In this contribution, we summarize the main findings on PS2, primarily as a modulator of Ca2+ homeostasis, with particular emphasis on the role of its mutations in the pathogenesis of FAD. Identification of cell pathways and molecules that are specifically targeted by PS2 mutants, as well as of common targets shared with PS1 mutants, will be fundamental to disentangle the complexity of memory loss and brain degeneration that occurs in Alzheimer’s disease (AD).
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- 2020
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11. The Concerted Action of Mitochondrial Dynamics and Positioning: New Characters in Cancer Onset and Progression
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Diana Pendin, Riccardo Filadi, and Paola Pizzo
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mitochondria ,cancer ,mitochondrial shape ,fusion ,fission ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Mitochondria are dynamic organelles whose morphology and activity are extremely variable, depending on the metabolic state of the cell. In particular, their shape and movements within the cell are finely regulated by an increasing number of proteins, which take part in the process of mitochondrial fission/fusion and connect the organelles to the cytoskeleton. As to their activities, mitochondria are considered to be at the crossroad between cell life and death since, on the one hand, they are essential in ATP production and in multiple metabolic pathways but, on the other, they are involved in the intrinsic apoptotic cascade, triggered by different stress conditions. Importantly, the process of mitochondrial Ca2+ uptake, as well as the morphology and the dynamics of these organelles, is known to deeply impact on both pro-survival and pro-death mitochondrial activities. Recently, increasing evidence has accrued on a central role of deregulated mitochondrial functionalities in the onset and progression of different pathologies, ranging from neurodegenerative diseases to cancer. In this contribution, we will present the latest findings connecting alterations in the machineries that control mitochondrial dynamics and localization to specific cancer hallmarks, highlighting the importance of mitochondria for the viability of cancer cells and discussing their role as promising targets for the development of novel anticancer therapies.
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- 2017
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12. Characterization of the ER-Targeted Low Affinity Ca2+ Probe D4ER
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Elisa Greotti, Andrea Wong, Tullio Pozzan, Diana Pendin, and Paola Pizzo
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Calcium ,Endoplasmic reticulum ,Cameleon ,FRET-based probe ,Presenilin ,Chemical technology ,TP1-1185 - Abstract
Calcium ion (Ca2+) is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER) represents the major intracellular Ca2+ store and the free Ca2+ concentration ([Ca2+]) within its lumen ([Ca2+]ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca2+ sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca2+ probes are plagued by different drawbacks, such as a double dissociation constant (Kd) for Ca2+, low dynamic range, and an affinity for the cation that is too high for the levels of [Ca2+] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET)-based, Cameleon probe, named D4ER, characterized by suitable Ca2+ affinity and dynamic range for monitoring [Ca2+] variations within the ER. As an example, resting [Ca2+]ER have been evaluated in a known paradigm of altered ER Ca2+ homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer’s Disease-linked protein Presenilin 2 (PS2). The lower Ca2+ affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca2+ content in cells expressing mutated PS2, compared to controls.
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- 2016
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13. Dynamic constriction and fission of endoplasmic reticulum membranes by reticulon
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Pavel V. Bashkirov, Andrea Daga, Artur Escalada, Aldo Montagna, Sergio Bova, Peter I. Kuzmin, Giulia Misticoni, Vadim A. Frolov, Borja Ibarra, Diana Pendin, Anna V. Shnyrova, Ariana Velasco del Olmo, Javier Espadas, Rebeca Bocanegra, and Tatiana Trevisan
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0301 basic medicine ,Atlastin ,fusion ,Nuclear Envelope ,Fission ,Science ,General Physics and Astronomy ,Optical tweezers ,Endoplasmic Reticulum ,Membrane Fusion ,Article ,General Biochemistry, Genetics and Molecular Biology ,GTP Phosphohydrolases ,Cell membrane ,Membrane biophysics ,03 medical and health sciences ,0302 clinical medicine ,Chlorocebus aethiops ,Organelle ,medicine ,Animals ,Drosophila Proteins ,image ,lcsh:Science ,Nanotubes ,Multidisciplinary ,Chemistry ,Endoplasmic reticulum ,Cell Membrane ,Lipid bilayer fusion ,nanoscale ,General Chemistry ,proteins ,3. Good health ,030104 developmental biology ,medicine.anatomical_structure ,Membrane curvature ,Reticulon ,COS Cells ,network ,reconstitution ,Biophysics ,cells ,lcsh:Q ,Drosophila ,030217 neurology & neurosurgery - Abstract
The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. Here we find that the ER has an embedded fragmentation mechanism based upon the ability of reticulon to produce fission of elongating network branches. In Drosophila, Rtnl1-facilitated fission is counterbalanced by atlastin-driven fusion, with the prevalence of Rtnl1 leading to ER fragmentation. Ectopic expression of Drosophila reticulon in COS-7 cells reveals individual fission events in dynamic ER tubules. Consistently, in vitro analyses show that reticulon produces velocity-dependent constriction of lipid nanotubes leading to stochastic fission via a hemifission mechanism. Fission occurs at elongation rates and pulling force ranges intrinsic to the ER, thus suggesting a principle whereby the dynamic balance between fusion and fission controlling organelle morphology depends on membrane motility., The endoplasmic reticulum (ER) is an intracellular network characterized by highly dynamic behavior whose control mechanisms are unclear. Here, the authors show that the ER-membrane protein Reticulon (Rtnl1) can constrict ER bilayers and lead to ER fission.
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- 2019
14. Mt-fura-2, a Ratiometric Mitochondria-Targeted Ca
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Andrea, De Nadai, Nicola, Vajente, Diana, Pendin, and Andrea, Mattarei
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Cytosol ,Microscopy, Fluorescence ,Cyclosporine ,Humans ,Calcium ,Fura-2 ,Calcium Chelating Agents ,HeLa Cells ,Mitochondria - Abstract
Ca
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- 2021
15. Familial Alzheimer’s disease presenilin-2 mutants affect Ca2+ homeostasis and brain network excitability
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Nelly Redolfi, Paola Pizzo, Diana Pendin, Elisa Greotti, Alessandro Leparulo, Cristina Fasolato, Tullio Pozzan, Elena Scremin, Riccardo Filadi, Chiara Gomiero, Emy Basso, Luisa Galla, and Nicola Vajente
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Aging ,Amyloid beta ,Cell ,Mutant ,Presenilin ,Alzheimer’s disease ,Amyloid-beta ,Brain network ,Ca2+ probes ,Calcium homeostasis ,03 medical and health sciences ,0302 clinical medicine ,mental disorders ,medicine ,Amyloid precursor protein ,Dementia ,030212 general & internal medicine ,biology ,Neurodegeneration ,medicine.disease ,medicine.anatomical_structure ,biology.protein ,Geriatrics and Gerontology ,Age of onset ,Neuroscience ,030217 neurology & neurosurgery - Abstract
Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Few cases are familial (FAD), due to autosomal dominant mutations in presenilin-1 (PS1), presenilin-2 (PS2) or amyloid precursor protein (APP). The three proteins are involved in the generation of amyloid-beta (Aβ) peptides, providing genetic support to the hypothesis of Aβ pathogenicity. However, clinical trials focused on the Aβ pathway failed in their attempt to modify disease progression, suggesting the existence of additional pathogenic mechanisms. Ca2+ dysregulation is a feature of cerebral aging, with an increased frequency and anticipated age of onset in several forms of neurodegeneration, including AD. Interestingly, FAD-linked PS1 and PS2 mutants alter multiple key cellular pathways, including Ca2+ signaling. By generating novel tools for measuring Ca2+ in living cells, and combining different approaches, we showed that FAD-linked PS2 mutants significantly alter cell Ca2+ signaling and brain network activity, as summarized below.
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- 2019
16. Familial Alzheimer’s disease-linked presenilin mutants and intracellular Ca2+ handling: A single-organelle, FRET-based analysis
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Tullio Pozzan, Diana Pendin, Paola Capitanio, Andrea Wong, Elisa Greotti, and Paola Pizzo
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SERCA ,Physiology ,Chemistry ,Endoplasmic reticulum ,STIM1 ,Cell Biology ,Golgi apparatus ,Presenilin ,Cell biology ,symbols.namesake ,Organelle ,symbols ,Signal transduction ,Molecular Biology ,Intracellular - Abstract
An imbalance in Ca2+ homeostasis represents an early event in the pathogenesis of Alzheimer's disease (AD). Presenilin-1 and -2 (PS1 and PS2) mutations, the major cause of familial AD (FAD), have been extensively associated with alterations in different Ca2+ signaling pathways, in particular those handled by storage compartments. However, FAD-PSs effect on organelles Ca2+ content is still debated and the mechanism of action of mutant proteins is unclear. To fulfil the need of a direct investigation of intracellular stores Ca2+ dynamics, we here present a detailed and quantitative single-cell analysis of FAD-PSs effects on organelle Ca2+ handling using specifically targeted, FRET (Fluorescence/Forster Resonance Energy Transfer)-based Ca2+ indicators. In SH-SY5Y human neuroblastoma cells and in patient-derived fibroblasts expressing different FAD-PSs mutations, we directly measured Ca2+ concentration within the main intracellular Ca2+ stores, e.g., Endoplasmic Reticulum (ER) and Golgi Apparatus (GA) medial- and trans-compartment. We unambiguously demonstrate that the expression of FAD-PS2 mutants, but not FAD-PS1, in either SH-SY5Y cells or FAD patient-derived fibroblasts, is able to alter Ca2+ handling of ER and medial-GA, but not trans-GA, reducing, compared to control cells, the Ca2+ content within these organelles by partially blocking SERCA (Sarco/Endoplasmic Reticulum Ca2+-ATPase) activity. Moreover, by using a cytosolic Ca2+ probe, we show that the expression of both FAD-PS1 and -PS2 reduces the Ca2+ influx activated by stores depletion (Store-Operated Ca2+ Entry; SOCE), by decreasing the expression levels of one of the key molecules, STIM1 (STromal Interaction Molecule 1), controlling this pathway. Our data indicate that FAD-linked PSs mutants differentially modulate the Ca2+ content of intracellular stores yet leading to a complex dysregulation of Ca2+ homeostasis, which represents a common disease phenotype of AD.
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- 2019
17. Proteasome dysfunction induces excessive proteome instability and loss of mitostasis that can be mitigated by enhancing mitochondrial fusion or autophagy
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Sentiljana Gumeni, Issidora S. Papassideri, Ioannis P. Trougakos, Vassilis G. Gorgoulis, Konstantinos Vougas, Eleni N. Tsakiri, Andrea Daga, Luca Scorrano, Diana Pendin, and Gábor Juhász
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0301 basic medicine ,Aging ,autophagy ,Proteasome Endopeptidase Complex ,Proteome ,foxo ,Biology ,Mitochondrial Dynamics ,Animals, Genetically Modified ,03 medical and health sciences ,DNM1L ,Sequestosome 1 ,Heat shock protein ,Mitophagy ,Animals ,Drosophila Proteins ,education ,cncC ,Drosophila ,mitostasis ,proteasome ,proteostasis ,Molecular Biology ,education.field_of_study ,030102 biochemistry & molecular biology ,Cell Biology ,Mitochondria ,Cell biology ,Drosophila melanogaster ,030104 developmental biology ,Proteostasis ,mitochondrial fusion ,Proteasome ,Larva ,Proteolysis ,Unfolded protein response ,Research Paper - Abstract
The ubiquitin-proteasome pathway (UPP) is central to proteostasis network (PN) functionality and proteome quality control. Yet, the functional implication of the UPP in tissue homeodynamics at the whole organism level and its potential cross-talk with other proteostatic or mitostatic modules are not well understood. We show here that knock down (KD) of proteasome subunits in Drosophila flies, induced, for most subunits, developmental lethality. Ubiquitous or tissue specific proteasome dysfunction triggered systemic proteome instability and activation of PN modules, including macroautophagy/autophagy, molecular chaperones and the antioxidant cncC (the fly ortholog of NFE2L2/Nrf2) pathway. Also, proteasome KD increased genomic instability, altered metabolic pathways and severely disrupted mitochondrial functionality, triggering a cncC-dependent upregulation of mitostatic genes and enhanced rates of mitophagy. Whereas, overexpression of key regulators of antioxidant responses (e.g., cncC or foxo) could not suppress the deleterious effects of proteasome dysfunction; these were alleviated in both larvae and adult flies by modulating mitochondrial dynamics towards increased fusion or by enhancing autophagy. Our findings reveal the extensive functional wiring of genomic, proteostatic and mitostatic modules in higher metazoans. Also, they support the notion that age-related increase of proteotoxic stress due to decreased UPP activity deregulates all aspects of cellular functionality being thus a driving force for most age-related diseases. Abbreviations: ALP: autophagy-lysosome pathway; ARE: antioxidant response element; Atg8a: autophagy-related 8a; ATPsynβ: ATP synthase, β subunit; C-L: caspase-like proteasomal activity; cncC: cap-n-collar isoform-C; CT-L: chymotrypsin-like proteasomal activity; Drp1: dynamin related protein 1; ER: endoplasmic reticulum; foxo: forkhead box, sub-group O; GLU: glucose; GFP: green fluorescent protein; GLY: glycogen; Hsf: heat shock factor; Hsp: Heat shock protein; Keap1: kelch-like ECH-associated protein 1; Marf: mitochondrial assembly regulatory factor; NFE2L2/Nrf2: nuclear factor, erythroid 2 like 2; Opa1: optic atrophy 1; PN: proteostasis network; RNAi: RNA interference; ROS: reactive oxygen species; ref(2)P: refractory to sigma P; SQSTM1: sequestosome 1; SdhA: succinate dehydrogenase, subunit A; T-L: trypsin-like proteasomal activity; TREH: trehalose; UAS: upstream activation sequence; Ub: ubiquitin; UPR: unfolded protein response; UPP: ubiquitin-proteasome pathway.
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- 2019
18. A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals
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Giulia Zanetti, Tullio Pozzan, Elisa Greotti, Cristina Fasolato, Diana Pendin, Tino Hochepied, and Nelly Redolfi
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EXPRESSION ,INDICATORS ,DYNAMICS ,0301 basic medicine ,Genetically modified mouse ,HOMEOSTASIS ,PROTEINS ,ENDOPLASMIC-RETICULUM ,chemistry.chemical_element ,METABOLISM ,Biology ,Calcium ,CAG PROMOTER ,Cameleon ,calcium imaging ,transgenic mouse ,mitochondria ,ROSA26 ,Cre/loxP ,GECI ,FRET ,mt-Cam ,03 medical and health sciences ,0302 clinical medicine ,Medicine and Health Sciences ,SENSORS ,Biology and Life Sciences ,CA2+ UPTAKE ,Cell biology ,030104 developmental biology ,chemistry ,Line (text file) ,030217 neurology & neurosurgery - Abstract
Mitochondria play a key role in cellular calcium (Ca2+) homeostasis. Dysfunction in the organelle Ca2+ handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) have been developed to study Ca2+ dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse ROSA26 genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a loxP-STOP-loxP sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca2+ imaging experiments performed in vitro and ex vivo in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca2+ dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca2+ concentration ([Ca2+]).
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- 2021
19. Mt-fura-2, a Ratiometric Mitochondria-Targeted Ca2+ Sensor. Determination of Spectroscopic Properties and Ca2+ Imaging Assays
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Nicola Vajente, Andrea De Nadai, Andrea Mattarei, and Diana Pendin
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0301 basic medicine ,Ca ,Fura-2 ,2+ ,probe ,Mitochondrion ,Cell fate determination ,Absorption ,Affinity ,imaging ,indicator ,Fluorescence ,Mitochondria ,mt-fura-2 ,Quantum yield ,Calcium ,Calcium Chelating Agents ,Cyclosporine ,Cytosol ,HeLa Cells ,Humans ,Microscopy, Fluorescence ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Microscopy ,Chemistry ,Coupling (electronics) ,Loading Technique ,030104 developmental biology ,Biophysics ,030217 neurology & neurosurgery ,Mitochondria targeted - Abstract
Ca2+ handling by mitochondria is implicated in energy production, shaping of cytosolic Ca2+ rises, and determination of cell fate. It is therefore of crucial interest for researchers to directly measure mitochondrial Ca2+ concentration [Ca2+] in living cells. Synthetic fluorescent Ca2+ indicators, providing a straightforward loading technique, represents a tempting strategy. Recently, we developed a new highly selective mitochondria-targeted Ca2+ indicator named mt-fura-2 , obtained by coupling two triphenylphosphonium cation-containing groups to the molecular backbone of the cytosolic ratiometric Ca2+ indicator fura-2 .The protocols we describe here cover all the significant steps that are necessary to characterize the probe and apply it to biologically relevant contexts. The procedures reported are referred to mt-fura-2 but could in principle be applied to characterize other mitochondria-targeted Ca2+ probes . More in general, with the due modifications, this chapter can be considered as a handbook for the characterization and/or application of mitochondria-targeted chemical Ca2+ probes .
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- 2021
20. Calcium Imaging in Drosophila melanogaster
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Nicola Vajente, Rosa Pia Norante, Paola Pizzo, and Diana Pendin
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Cell signaling ,ved/biology.organism_classification_rank.species ,Calcium imaging ,Computational biology ,Calcium indicators ,Calcium signaling ,Drosophila ,GECI ,Neurodegenerative diseases ,Animals ,Brain ,Humans ,Models, Animal ,Calcium ,Drosophila melanogaster ,Genome ,03 medical and health sciences ,0302 clinical medicine ,Models ,030212 general & internal medicine ,Model organism ,Gene ,biology ,ved/biology ,Animal ,fungi ,biology.organism_classification ,Genetic redundancy ,Identification (biology) - Abstract
Drosophila melanogaster, colloquially known as the fruit fly, is one of the most commonly used model organisms in scientific research. Although the final architecture of a fly and a human differs greatly, most of the fundamental biological mechanisms and pathways controlling development and survival are conserved through evolution between the two species. For this reason, Drosophila has been productively used as a model organism for over a century, to study a diverse range of biological processes, including development, learning, behavior and aging. Ca2+ signaling comprises complex pathways that impact on virtually every aspect of cellular physiology. Within such a complex field of study, Drosophila offers the advantages of consolidated molecular and genetic techniques, lack of genetic redundancy and a completely annotated genome since 2000. These and other characteristics provided the basis for the identification of many genes encoding Ca2+ signaling molecules and the disclosure of conserved Ca2+ signaling pathways. In this review, we will analyze the applications of Ca2+ imaging in the fruit fly model, highlighting in particular their impact on the study of normal brain function and pathogenesis of neurodegenerative diseases.
- Published
- 2020
21. Presenilin-2 and Calcium Handling: Molecules, Organelles, Cells and Brain Networks
- Author
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Elisa Greotti, Alessandro Leparulo, Nelly Redolfi, Paola Pizzo, Cristina Fasolato, Michela Rossini, Tullio Pozzan, Riccardo Filadi, Nicola Vajente, Emy Basso, and Diana Pendin
- Subjects
autophagy ,calcium signalling ,Bioenergetics ,Cell ,Review ,Mitochondrion ,calcium signaling ,slow-waves ,Alzheimer Disease ,Organelle ,medicine ,Presenilin-1 ,Humans ,Alzheimer’s disease mouse models ,skin and connective tissue diseases ,lcsh:QH301-705.5 ,Calcium signaling ,presenilin-2 ,Chemistry ,Autophagy ,Cell Membrane ,functional connectivity ,Brain ,General Medicine ,brain networks ,Cell biology ,mitochondria ,oscillations ,SOCE ,medicine.anatomical_structure ,lcsh:Biology (General) ,Multiprotein Complexes ,Flavin-Adenine Dinucleotide ,Calcium ,Mutant Proteins ,Amyloid Precursor Protein Secretases ,Homeostasis ,Function (biology) - Abstract
Presenilin-2 (PS2) is one of the three proteins that are dominantly mutated in familial Alzheimer’s disease (FAD). It forms the catalytic core of the γ-secretase complex—a function shared with its homolog presenilin-1 (PS1)—the enzyme ultimately responsible of amyloid-β (Aβ) formation. Besides its enzymatic activity, PS2 is a multifunctional protein, being specifically involved, independently of γ-secretase activity, in the modulation of several cellular processes, such as Ca2+ signaling, mitochondrial function, inter-organelle communication, and autophagy. As for the former, evidence has accumulated that supports the involvement of PS2 at different levels, ranging from organelle Ca2+ handling to Ca2+ entry through plasma membrane channels. Thus FAD-linked PS2 mutations impact on multiple aspects of cell and tissue physiology, including bioenergetics and brain network excitability. In this contribution, we summarize the main findings on PS2, primarily as a modulator of Ca2+ homeostasis, with particular emphasis on the role of its mutations in the pathogenesis of FAD. Identification of cell pathways and molecules that are specifically targeted by PS2 mutants, as well as of common targets shared with PS1 mutants, will be fundamental to disentangle the complexity of memory loss and brain degeneration that occurs in Alzheimer’s disease (AD).
- Published
- 2020
22. Microtubules Stabilization by Mutant Spastin Affects ER Morphology and Ca2+ Handling
- Author
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Diana Pendin, Andrea Daga, Nelly Redolfi, Rosa Pia Norante, Nicola Vajente, and Paola Pizzo
- Subjects
0301 basic medicine ,calcium ,calcium imaging ,drosophila ,endoplasmic reticulum ,microtubules ,SOCE ,spastin ,Physiology ,Mutant ,Spastin ,lcsh:Physiology ,03 medical and health sciences ,0302 clinical medicine ,Calcium imaging ,Microtubule ,Physiology (medical) ,Cytoskeleton ,lcsh:QP1-981 ,biology ,Chemistry ,Endoplasmic reticulum ,biology.organism_classification ,Phenotype ,Cell biology ,030104 developmental biology ,Drosophila melanogaster ,030217 neurology & neurosurgery - Abstract
The endoplasmic reticulum (ER) extends as a network of interconnected tubules and sheet-like structures in eukaryotic cells. ER tubules dynamically change their morphology and position within the cells in response to physiological stimuli and these network rearrangements depend on the microtubule (MT) cytoskeleton. Store-operated calcium entry (SOCE) relies on the repositioning of ER tubules to form specific ER-plasma membrane junctions. Indeed, the tips of polymerizing MTs are supposed to provide the anchor for ER tubules to move toward the plasma membrane, however the precise role of the cytoskeleton during SOCE has not been conclusively clarified. Here we exploit an in vivo approach involving the manipulation of MT dynamics in Drosophila melanogaster by neuronal expression of a dominant-negative variant of the MT-severing protein spastin to induce MT hyper-stabilization. We show that MT stabilization alters ER morphology, favoring an enrichment in ER sheets at the expense of tubules. Stabilizing MTs has a negative impact on the process of SOCE and results in a reduced ER Ca2+ content, affecting the flight ability of the flies. Restoring proper MT organization by administering the MT-destabilizing drug vinblastine, chronically or acutely, rescues ER morphology, SOCE and flight ability, indicating that MT dynamics impairment is responsible for all the phenotypes observed.
- Published
- 2019
23. Mitofusin 2: from functions to disease
- Author
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Riccardo Filadi, Diana Pendin, and Paola Pizzo
- Subjects
0301 basic medicine ,Cancer Research ,Bioenergetics ,Immunology ,Cell ,Mitochondrion ,Biology ,GTP Phosphohydrolases ,Mitochondrial Proteins ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Mitofusin-2 ,Metabolic Diseases ,Neoplasms ,Organelle ,medicine ,Animals ,Humans ,Viability assay ,lcsh:QH573-671 ,Review Paper ,lcsh:Cytology ,Endoplasmic reticulum ,Neurodegenerative Diseases ,Cell Biology ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Cardiomyopathies ,Function (biology) - Abstract
Mitochondria are highly dynamic organelles whose functions are essential for cell viability. Within the cell, the mitochondrial network is continuously remodeled through the balance between fusion and fission events. Moreover, it dynamically contacts other organelles, particularly the endoplasmic reticulum, with which it enterprises an important functional relationship able to modulate several cellular pathways. Being mitochondria key bioenergetics organelles, they have to be transported to all the specific high-energy demanding sites within the cell and, when damaged, they have to be efficiently removed. Among other proteins, Mitofusin 2 represents a key player in all these mitochondrial activities (fusion, trafficking, turnover, contacts with other organelles), the balance of which results in the appropriate mitochondrial shape, function, and distribution within the cell. Here we review the structural and functional properties of Mitofusin 2, highlighting its crucial role in several cell pathways, as well as in the pathogenesis of neurodegenerative diseases, metabolic disorders, cardiomyopathies, and cancer.
- Published
- 2018
24. Lighting Up Ca2+ Dynamics in Animal Models
- Author
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Diana Pendin, Chiara Fornetto, Paloma García-Casas, Sonia Sonda, Nelly Redolfi, and Paola Pizzo
- Subjects
calcium indicators ,Nervous system ,QH301-705.5 ,Context (language use) ,Review ,Neurotransmission ,Biology ,Calcium imaging ,TheoryofComputation_ANALYSISOFALGORITHMSANDPROBLEMCOMPLEXITY ,Neuroplasticity ,medicine ,Animals ,Humans ,Calcium Signaling ,Biology (General) ,Brain function ,Neurons ,nervous system ,Neurodegeneration ,Neurodegenerative Diseases ,General Medicine ,medicine.disease ,animal models ,calcium imaging ,medicine.anatomical_structure ,GECI ,Calcium ,Neuroscience ,Homeostasis - Abstract
Calcium (Ca2+) signaling coordinates are crucial processes in brain physiology. Particularly, fundamental aspects of neuronal function such as synaptic transmission and neuronal plasticity are regulated by Ca2+, and neuronal survival itself relies on Ca2+-dependent cascades. Indeed, impaired Ca2+ homeostasis has been reported in aging as well as in the onset and progression of neurodegeneration. Understanding the physiology of brain function and the key processes leading to its derangement is a core challenge for neuroscience. In this context, Ca2+ imaging represents a powerful tool, effectively fostered by the continuous amelioration of Ca2+ sensors in parallel with the improvement of imaging instrumentation. In this review, we explore the potentiality of the most used animal models employed for Ca2+ imaging, highlighting their application in brain research to explore the pathogenesis of neurodegenerative diseases.
- Published
- 2021
25. Calcium Imaging in Drosophila melanogaster
- Author
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Nicola, Vajente, Rosa, Norante, Paola, Pizzo, and Diana, Pendin
- Subjects
Drosophila melanogaster ,Models, Animal ,Animals ,Brain ,Humans ,Calcium - Abstract
Drosophila melanogaster, colloquially known as the fruit fly, is one of the most commonly used model organisms in scientific research. Although the final architecture of a fly and a human differs greatly, most of the fundamental biological mechanisms and pathways controlling development and survival are conserved through evolution between the two species. For this reason, Drosophila has been productively used as a model organism for over a century, to study a diverse range of biological processes, including development, learning, behavior and aging. Ca
- Published
- 2019
26. Familial Alzheimer's disease presenilin-2 mutants affect Ca
- Author
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Diana, Pendin, Cristina, Fasolato, Emy, Basso, Riccardo, Filadi, Elisa, Greotti, Luisa, Galla, Chiara, Gomiero, Alessandro, Leparulo, Nelly, Redolfi, Elena, Scremin, Nicola, Vajente, Tullio, Pozzan, and Paola, Pizzo
- Subjects
Amyloid beta-Peptides ,Alzheimer Disease ,Presenilin-2 ,Presenilin-1 ,Brain ,Homeostasis ,Humans ,Aged - Abstract
Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Few cases are familial (FAD), due to autosomal dominant mutations in presenilin-1 (PS1), presenilin-2 (PS2) or amyloid precursor protein (APP). The three proteins are involved in the generation of amyloid-beta (Aβ) peptides, providing genetic support to the hypothesis of Aβ pathogenicity. However, clinical trials focused on the Aβ pathway failed in their attempt to modify disease progression, suggesting the existence of additional pathogenic mechanisms. Ca
- Published
- 2019
27. mCerulean3-Based Cameleon Sensor to Explore Mitochondrial Ca2+ Dynamics In Vivo
- Author
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Michele Sessolo, Ilaria Fortunati, Diana Pendin, Moises Di Sante, Fabio Di Lisa, Elisa Greotti, Camilla Ferrante, Mauro Giacca, Lorena Zentilin, Tullio Pozzan, Nina Kaludercic, Giorgio Carmignoto, Letizia Mariotti, Marta Gómez-Gonzalo, Annamaria Lia, Renato Bozio, Luisa Galla, Greotti, Elisa, Fortunati, Ilaria, Pendin, Diana, Ferrante, Camilla, Galla, Luisa, Zentilin, Lorena, Giacca, Mauro, Kaludercic, Nina, Di Sante, Moise, Mariotti, Letizia, Lia, Annamaria, Gómez-Gonzalo, Marta, Sessolo, Michele, Di Lisa, Fabio, Carmignoto, Giorgio, Bozio, Renato, and Pozzan, Tullio
- Subjects
0301 basic medicine ,Fluorescence-lifetime imaging microscopy ,02 engineering and technology ,Article ,Biological Sciences Tool ,03 medical and health sciences ,0302 clinical medicine ,In vivo ,Organelle ,Fluorescence microscope ,lcsh:Science ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,biology ,Chemistry ,Optical Imaging ,Correction ,Cameleon (protein) ,Cell Biology ,021001 nanoscience & nanotechnology ,Fluorescence ,Cytosol ,Förster resonance energy transfer ,030104 developmental biology ,biology.protein ,Biophysics ,lcsh:Q ,Biological Sciences Tools ,0210 nano-technology ,030217 neurology & neurosurgery - Abstract
Summary Genetically Encoded Ca2+ Indicators (GECIs) are extensively used to study organelle Ca2+ homeostasis, although some available probes are still plagued by a number of problems, e.g., low fluorescence intensity, partial mistargeting, and pH sensitivity. Furthermore, in the most commonly used mitochondrial Förster Resonance Energy Transfer based-GECIs, the donor protein ECFP is characterized by a double exponential lifetime that complicates the fluorescence lifetime analysis. We have modified the cytosolic and mitochondria-targeted Cameleon GECIs by (1) substituting the donor ECFP with mCerulean3, a brighter and more stable fluorescent protein with a single exponential lifetime; (2) extensively modifying the constructs to improve targeting efficiency and fluorescence changes caused by Ca2+ binding; and (3) inserting the cDNAs into adeno-associated viral vectors for in vivo expression. The probes have been thoroughly characterized in situ by fluorescence microscopy and Fluorescence Lifetime Imaging Microscopy, and examples of their ex vivo and in vivo applications are described., Graphical Abstract, Highlights • Donor substitution in a mitochondrial Ca2+ sensor improves photo-physical properties • Mitochondria-targeting sequence amelioration enhances the sensor localization • Donor substitution allows FLIM-FRET analysis, with a compensation for pH bias • The performance of the sensor is improved in situ, ex vivo, and in vivo, Biological Sciences Tools; Cell Biology; Optical Imaging
- Published
- 2019
28. A Synthetic Fluorescent Mitochondria-Targeted Sensor for Ratiometric Imaging of Calcium in Live Cells
- Author
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Andrea Mattarei, Gaia Gherardi, Cristina Paradisi, Nina Kaludercic, Andrea De Nadai, Diana Pendin, Nicola Vajente, Tullio Pozzan, Cristina Mammucari, Emy Basso, and Rosa Pia Norante
- Subjects
Cell type ,Ca ,Cell ,2+ ,chemistry.chemical_element ,Mitochondrion ,Calcium ,010402 general chemistry ,01 natural sciences ,Catalysis ,sensor ,fluorescent probes ,medicine ,Humans ,bioimaging ,Fluorescent Dyes ,calcium ,010405 organic chemistry ,mitochondria ,General Medicine ,General Chemistry ,Fluorescence ,In vitro ,0104 chemical sciences ,Dissociation constant ,medicine.anatomical_structure ,chemistry ,Biophysics ,Mitochondria targeted - Abstract
Ca2+ handling by mitochondria is crucial for cell life and the direct measure of mitochondrial Ca2+ concentration in living cells is of pivotal interest. Genetically-encoded indicators greatly facilitated this task, however they require demanding delivery procedures. On the other hand, existing mitochondria-targeted synthetic Ca2+ indicators are plagued by several drawbacks, for example, non-specific localization, leakage, toxicity. Here we report the synthesis and characterization of a new fluorescent Ca2+ sensor, named mt-fura-2, obtained by coupling two triphenylphosphonium cations to the molecular backbone of the ratiometric Ca2+ indicator fura-2. Mt-fura-2 binds Ca2+ with a dissociation constant of ≈1.5 μm in vitro. When loaded in different cell types as acetoxymethyl ester, the probe shows proper mitochondrial localization and accurately measures matrix [Ca2+ ] variations, proving its superiority over available dyes. We describe the synthesis, characterization and application of mt-fura-2 to cell types where the delivery of genetically-encoded indicators is troublesome.
- Published
- 2019
29. Familial Alzheimer's disease-linked presenilin mutants and intracellular Ca
- Author
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Elisa, Greotti, Paola, Capitanio, Andrea, Wong, Tullio, Pozzan, Paola, Pizzo, and Diana, Pendin
- Subjects
Organelles ,Alzheimer Disease ,Mutation ,Presenilin-2 ,Fluorescence Resonance Energy Transfer ,Presenilin-1 ,Tumor Cells, Cultured ,Humans ,Calcium - Abstract
An imbalance in Ca
- Published
- 2018
30. Dynamic constriction and fission of ER membranes by reticulon
- Author
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Giulia Misticoni, Vadim A. Frolov, Tatiana Trevisan, Rebeca Bocanegra, Andrea Daga, Peter I. Kuzmin, Ariana Velasco del Olmo, Sergio Bova, Javier Espadas, Borja Ibarra, Artur Escalada, Anna V. Shnyrova, and Diana Pendin
- Subjects
Atlastin ,0303 health sciences ,Fission ,Chemistry ,Endoplasmic reticulum ,03 medical and health sciences ,0302 clinical medicine ,Membrane ,Membrane curvature ,Reticulon ,Organelle ,Biophysics ,Fragmentation (cell biology) ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been widely associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. We found that the ER network has an embedded fragmentation mechanism based upon the ability of reticulons to produce fission of elongating network branches. In Drosophila, fission is counterbalanced by atlastin-driven fusion, with their imbalance leading to ER fragmentation. Live imaging of ER network dynamics upon ectopic expression of Drosophila reticulon linked fission to augmented membrane friction. Consistently, in vitro analysis revealed that purified reticulon produced velocity-dependent constriction and fission of lipid nanotubes pulled from a flat reservoir membrane. Fission occurred at elongation rates and pulling force ranges intrinsic to the ER network, thus suggesting a novel principle of organelle morphology regulation where the dynamic balance between fusion and fission is governed by membrane motility.
- Published
- 2018
31. Optogenetic control of mitochondrial metabolism and Ca
- Author
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Tatiana, Tkatch, Elisa, Greotti, Gytis, Baranauskas, Diana, Pendin, Soumitra, Roy, Luliaoana I, Nita, Jennifer, Wettmarshausen, Matthias, Prigge, Ofer, Yizhar, Orian S, Shirihai, Daniel, Fishman, Michal, Hershfinkel, Ilya A, Fleidervish, Fabiana, Perocchi, Tullio, Pozzan, and Israel, Sekler
- Subjects
Membrane Potential, Mitochondrial ,Mitochondria, Heart ,Rats ,Optogenetics ,Rats, Sprague-Dawley ,HEK293 Cells ,Oxygen Consumption ,Channelrhodopsins ,PNAS Plus ,Insulin-Secreting Cells ,Animals ,Humans ,Myocytes, Cardiac ,Calcium Signaling ,HeLa Cells - Abstract
Mitochondrial functions depend on the steep H+ electrochemical gradient (ΔμH+) across their inner membrane. The available tools for controlling this gradient are essentially limited to inhibitors of the respiratory chain or of the H+ ATPase or to uncouplers, poisons plagued by important side effects and that lack both cell and spatial specificity. We show here that, by transfecting cells with the cDNA encoding channelrhodopsins specifically targeted to the inner mitochondrial membrane, we can obtain an accurate and spatially confined, light-dependent control of mitochondrial membrane potential and, as a consequence, of a series of mitochondrial activities ranging from electron transport to ATP synthesis and Ca2+ signaling.
- Published
- 2017
32. Optogenetic control of mitochondrial metabolism and Ca 2+ signaling by mitochondria-targeted opsins
- Author
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Tullio Pozzan, Ilya A. Fleidervish, Fabiana Perocchi, Orian S. Shirihai, Matthias Prigge, Michal Hershfinkel, Elisa Greotti, Tatiana Tkatch, Ofer Yizhar, Jennifer Wettmarshausen, Israel Sekler, Gytis Baranauskas, Diana Pendin, Soumitra Roy, Luliaoana I Nita, and Daniel Fishman
- Subjects
0301 basic medicine ,Membrane potential ,Multidisciplinary ,ATP synthase ,Oxidative phosphorylation ,Mitochondrion ,Biology ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,biology.protein ,Inner membrane ,Inner mitochondrial membrane ,Electrochemical gradient ,Ca Signaling 2+ ,Mitochondria ,Mitochondrial Membrane Potential ,Optogenetic ,Calcium signaling - Abstract
Key mitochondrial functions such as ATP production, Ca2+ uptake and release, and substrate accumulation depend on the proton electrochemical gradient (ΔμH+) across the inner membrane. Although several drugs can modulate ΔμH+, their effects are hardly reversible, and lack cellular specificity and spatial resolution. Although channelrhodopsins are widely used to modulate the plasma membrane potential of excitable cells, mitochondria have thus far eluded optogenetic control. Here we describe a toolkit of optometabolic constructs based on selective targeting of channelrhodopsins with distinct functional properties to the inner mitochondrial membrane of intact cells. We show that our strategy enables a light-dependent control of the mitochondrial membrane potential (Δψm) and coupled mitochondrial functions such as ATP synthesis by oxidative phosphorylation, Ca2+ dynamics, and respiratory metabolism. By directly modulating Δψm, the mitochondriatargeted opsinswere used to control complex physiological processes such as spontaneous beats in cardiac myocytes and glucose-dependent ATP increase in pancreatic β-cells. Furthermore, our optometabolic tools allow modulation of mitochondrial functions in single cells and defined cell regions.
- Published
- 2017
33. The Concerted Action of Mitochondrial Dynamics and Positioning: New Characters in Cancer Onset and Progression
- Author
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Paola Pizzo, Riccardo Filadi, and Diana Pendin
- Subjects
0301 basic medicine ,Cancer Research ,fusion ,mitochondrial shape ,Cell ,Review ,Biology ,Mitochondrion ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,lcsh:RC254-282 ,Cell biology ,mitochondria ,03 medical and health sciences ,030104 developmental biology ,medicine.anatomical_structure ,mitochondrial fusion ,Oncology ,Organelle ,Cancer cell ,medicine ,DNAJA3 ,cancer ,fission ,Mitochondrial fission ,Cytoskeleton - Abstract
Mitochondria are dynamic organelles whose morphology and activity are extremely variable, depending on the metabolic state of the cell. In particular, their shape and movements within the cell are finely regulated by an increasing number of proteins, which take part in the process of mitochondrial fission/fusion and connect the organelles to the cytoskeleton. As to their activities, mitochondria are considered to be at the crossroad between cell life and death since, on the one hand, they are essential in ATP production and in multiple metabolic pathways but, on the other, they are involved in the intrinsic apoptotic cascade, triggered by different stress conditions. Importantly, the process of mitochondrial Ca2+ uptake, as well as the morphology and the dynamics of these organelles, are known to deeply impact on both pro-survival and pro-death mitochondrial activities. Recently, increasing evidence has accrued on a central role of deregulated mitochondrial functionalities in the onset and progression of different pathologies, ranging from neurodegenerative diseases to cancer. In this contribution, we will present the latest findings connecting alterations in the machineries that control mitochondrial dynamics and localization to specific cancer hallmarks, highlighting the importance of mitochondria for the viability of cancer cells and discussing their role as promising targets for the development of novel anticancer therapies.
- Published
- 2017
34. Spying on organelle Ca2+ in living cells: the mitochondrial point of view
- Author
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Diana Pendin, Riccardo Filadi, Elisa Greotti, and Tullio Pozzan
- Subjects
Endocrinology, Diabetes and Metabolism ,Green Fluorescent Proteins ,Cell ,Aequorin ,Mitochondrion ,Green fluorescent protein ,Human health ,Endocrinology ,Organelle ,medicine ,Animals ,Humans ,Calcium Signaling ,Calcium ,Calcium indicators ,GECI ,Mitochondria ,Luminescent Measurements ,Organelles ,Medicine (all) ,biology ,Cell biology ,Diabetes and Metabolism ,medicine.anatomical_structure ,Cytoplasm ,biology.protein ,Homeostasis - Abstract
Over the past years, the use of genetically encoded Ca(2+) indicators (GECIs), derived from aequorin and green fluorescent protein, has profoundly transformed the study of Ca(2+) homeostasis in living cells leading to novel insights into functional aspects of Ca(2+) signalling. Particularly relevant for a deeper understanding of these key aspects of cell pathophysiology has been the possibility of imaging changes in Ca(2+) concentration not only in the cytoplasm, but also inside organelles. In this review, we will provide an overview of the ongoing developments in the use of GECIs, with particular focus on mitochondrially targeted probes. Indeed, due to recent advances in organelle Ca(2+) imaging with GECIs, mitochondria are now at the centre of renewed interest: they play key roles both in the physiology of the cell and in multiple pathological conditions relevant to human health.
- Published
- 2014
35. The elusive importance of being a mitochondrial Ca2+ uniporter
- Author
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Tullio Pozzan, Elisa Greotti, and Diana Pendin
- Subjects
Mitochondrial Ca2+ uniporter ,Physiology ,Protein subunit ,MCUR ,Biology ,Mitochondrial Membrane Transport Proteins ,Human disease ,EMRE ,Animals ,Humans ,Missense mutation ,Uniporter ,MCU ,MICU1 ,MICU2 ,Calcium ,Calcium Channels ,Mitochondria ,Protein Subunits ,RNA Interference ,Cell Biology ,Molecular Biology ,Medicine (all) ,Regulation of gene expression ,Cell biology - Abstract
The molecular components of the mitochondrial Ca2+ uptake machinery have been only recently identified. In the last months, in addition to the pore forming subunit and of one regulatory protein (named MCU and MICU1, respectively) other four components of this complex have been described. In addition, a MCU KO mouse model has been generated and a genetic human disease due to missense mutation of MICU1 has been discovered. In this contribution, we will first summarize the recent findings, discussing the roles of the different subunits of the mitochondrial Ca2+ uptake complex, pointing to the current contradictions in the published data, as well as possible explanations. Finally we will speculate on the recent, totally unexpected, results obtained in the MCU knock-out (KO) mice.
- Published
- 2014
36. Ca2+and cAMP cross-talk in mitochondria
- Author
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Diana Pendin, Elisa Greotti, Giulietta Di Benedetto, Paola Pizzo, and Tullio Pozzan
- Subjects
Signalling ,ATP synthase ,biology ,Physiology ,Mitochondrial matrix ,Organelle ,Second messenger system ,biology.protein ,Mitochondrion ,Homeostasis ,Cell biology ,Calcium signaling - Abstract
While mitochondrial Ca2+ homeostasis has been studied for several decades and many of the functional roles of Ca2+ accumulation within the matrix have been at least partially clarified, the possibility of modulation of the organelle functions by cAMP remains largely unknown. In this contribution we briefly summarize the key aspects of Ca2+ and cAMP signalling pathways in mitochondria. In particular, we focus on recent findings concerning the discovery of an autonomous cAMP toolkit within the mitochondrial matrix, its crossroad with mitochondrial Ca2+ signalling and its role in controlling ATP synthesis. The discovery of a cAMP signalling, and the demonstration of a cross-talk between cAMP and Ca2+ inside mitochondria, open the way to a re-evaluation of these organelles as integrators of multiple second messengers. A description of the main methods presently available to measure Ca2+ and cAMP in mitochondria of living cells with genetically encoded probes is also presented.
- Published
- 2013
37. Characterization of the ER-Targeted Low Affinity Ca2+ Probe D4ER
- Author
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Andrea Wong, Tullio Pozzan, Diana Pendin, Elisa Greotti, and Paola Pizzo
- Subjects
0301 basic medicine ,Cell ,chemistry.chemical_element ,Calcium ,lcsh:Chemical technology ,Biochemistry ,Presenilin ,Article ,Analytical Chemistry ,03 medical and health sciences ,medicine ,lcsh:TP1-1185 ,Electrical and Electronic Engineering ,Instrumentation ,biology ,Chemistry ,Endoplasmic reticulum ,FRET-based probe ,Cameleon ,Cameleon (protein) ,Atomic and Molecular Physics, and Optics ,030104 developmental biology ,medicine.anatomical_structure ,Förster resonance energy transfer ,biology.protein ,Biophysics ,Intracellular ,Homeostasis - Abstract
Calcium ion (Ca(2+)) is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER) represents the major intracellular Ca(2+) store and the free Ca(2+) concentration ([Ca(2+)]) within its lumen ([Ca(2+)]ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca(2+) sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca(2+) probes are plagued by different drawbacks, such as a double dissociation constant (Kd) for Ca(2+), low dynamic range, and an affinity for the cation that is too high for the levels of [Ca(2+)] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET)-based, Cameleon probe, named D4ER, characterized by suitable Ca(2+) affinity and dynamic range for monitoring [Ca(2+)] variations within the ER. As an example, resting [Ca(2+)]ER have been evaluated in a known paradigm of altered ER Ca(2+) homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer's Disease-linked protein Presenilin 2 (PS2). The lower Ca(2+) affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca(2+) content in cells expressing mutated PS2, compared to controls.
- Published
- 2016
38. Homotypic fusion of ER membranes requires the dynamin-like GTPase Atlastin
- Author
-
Jessica Tosetto, James A. McNew, Andrea Daga, Tyler J. Moss, Genny Orso, Andrea Martinuzzi, Joseph E. Faust, Diana Pendin, Song Liu, Massimo Micaroni, and Anastasia Egorova
- Subjects
Dynamins ,Atlastin ,Multidisciplinary ,Proteolipids ,Endoplasmic reticulum ,Lipid bilayer fusion ,GTPase ,Biology ,Endoplasmic Reticulum ,Membrane Fusion ,GTP Phosphohydrolases ,Cell biology ,Protein Transport ,Drosophila melanogaster ,Membrane fission ,Reticulon ,Membrane biogenesis ,Animals ,Drosophila Proteins ,Humans ,HeLa Cells ,Dynamin - Abstract
Establishment and maintenance of proper architecture is essential for endoplasmic reticulum (ER) function. Homotypic membrane fusion is required for ER biogenesis and maintenance, and has been shown to depend on GTP hydrolysis. Here we demonstrate that Drosophila Atlastin—the fly homologue of the mammalian GTPase atlastin 1 involved in hereditary spastic paraplegia—localizes on ER membranes and that its loss causes ER fragmentation. Drosophila Atlastin embedded in distinct membranes has the ability to form trans-oligomeric complexes and its overexpression induces enlargement of ER profiles, consistent with excessive fusion of ER membranes. In vitro experiments confirm that Atlastin autonomously drives membrane fusion in a GTP-dependent fashion. In contrast, GTPase-deficient Atlastin is inactive, unable to form trans-oligomeric complexes owing to failure to self-associate, and incapable of promoting fusion in vitro. These results demonstrate that Atlastin mediates membrane tethering and fusion and strongly suggest that it is the GTPase activity that is required for ER homotypic fusion. The endoplasmic reticulum (ER), critical for many cellular functions including membrane biogenesis, vesicle trafficking and protein secretion, is an interconnected network of tubular structures that pervades the eukaryotic cell. The mechanism by which this particular architecture is maintained is unclear, though homotypic membrane fusion dependent on GTP hydrolysis is known to be essential for ER biogenesis and maintenance. A study in Drosophila now shows that a GTPase called Atlastin — a homologue of the human atlastin 1 mutated in hereditary spastic paraplegia — is required for homotypic membrane fusion, and hence for ER formation. The mechanism by which the tubular architecture of the endoplasmic reticulum (ER) is maintained is unclear, although homotypic membrane fusion is known to be required for ER biogenesis and maintenance and this is dependent on GTP hydrolysis. Here it is demonstrated that loss of the GTPase Atlastin in Drosophila causes ER fragmentation, whereas its overexpression induces enlargement of ER profiles.
- Published
- 2009
39. Reduction of endoplasmic reticulum stress attenuates the defects caused by Drosophila mitofusin depletion
- Author
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Valentina Debattisti, Diana Pendin, Luca Scorrano, Andrea Daga, and Elena Ziviani
- Subjects
MFN2 ,Mitochondrion ,Biology ,Phenylbutyrate ,Taurochenodeoxycholic Acid ,Mice ,Report ,medicine ,Animals ,Drosophila Proteins ,Humans ,Research Articles ,Genetics ,Endoplasmic reticulum ,fungi ,Genetic Complementation Test ,food and beverages ,Membrane Proteins ,Cell Biology ,medicine.disease ,Endoplasmic Reticulum Stress ,Phenylbutyrates ,3. Good health ,Cell biology ,Mitochondria ,Drosophila melanogaster ,mitochondrial fusion ,Unfolded protein response ,Optic Atrophy 1 ,RNA Interference ,Drosophila Protein ,Locomotion - Abstract
The developmental and motor defects evident in flies depleted of the mitofusin Marf can be ameliorated by treatments that reduce ER stress, confirming an active role for ER stress in the observed phenotypes., Ablation of the mitochondrial fusion and endoplasmic reticulum (ER)–tethering protein Mfn2 causes ER stress, but whether this is just an epiphenomenon of mitochondrial dysfunction or a contributor to the phenotypes in mitofusin (Mfn)-depleted Drosophila melanogaster is unclear. In this paper, we show that reduction of ER dysfunction ameliorates the functional and developmental defects of flies lacking the single Mfn mitochondrial assembly regulatory factor (Marf). Ubiquitous or neuron- and muscle-specific Marf ablation was lethal, altering mitochondrial and ER morphology and triggering ER stress that was conversely absent in flies lacking the fusion protein optic atrophy 1. Expression of Mfn2 and ER stress reduction in flies lacking Marf corrected ER shape, attenuating the developmental and motor defects. Thus, ER stress is a targetable pathogenetic component of the phenotypes caused by Drosophila Mfn ablation.
- Published
- 2014
40. P2‐149: The ER‐mitochondria connection: New players in Alzheimer's disease
- Author
-
Elisa Greotti, Riccardo Filadi, Diana Pendin, Paola Pizzo, and Enrico Zampese
- Subjects
Psychiatry and Mental health ,Cellular and Molecular Neuroscience ,Developmental Neuroscience ,Epidemiology ,Computer science ,Health Policy ,Neurology (clinical) ,Geriatrics and Gerontology ,Mitochondrion ,Neuroscience ,Connection (mathematics) - Published
- 2012
41. GTP-dependent packing of a three-helix bundle is required for atlastin-mediated fusion
- Author
-
Jessica Tosetto, James A. McNew, Andrea Daga, Camilla Andreazza, Stefano Moro, Tyler J. Moss, and Diana Pendin
- Subjects
Atlastin ,Helix bundle ,Multidisciplinary ,GTP' ,Base Sequence ,Hydrolysis ,Lipid bilayer fusion ,Membrane Proteins ,GTPase ,Biology ,Guanosine triphosphate ,Biological Sciences ,chemistry.chemical_compound ,GTP-binding protein regulators ,Membrane protein ,Biochemistry ,chemistry ,GTP-Binding Proteins ,Biophysics ,Animals ,Humans ,Drosophila ,Guanosine Triphosphate ,DNA Primers ,HeLa Cells - Abstract
The mechanisms governing atlastin-mediated membrane fusion are unknown. Here we demonstrate that a three-helix bundle (3HB) within the middle domain is required for oligomerization. Mutation of core hydrophobic residues within these helices inactivates atlastin function by preventing membrane tethering and the subsequent fusion. GTP binding induces a conformational change that reorients the GTPase domain relative to the 3HB to permit self-association, but the ability to hydrolyze GTP is required for full fusion, indicating that nucleotide binding and hydrolysis play distinct roles. Oligomerization of atlastin stimulates its ability to hydrolyze GTP, and the energy released drives lipid bilayer merger. Mutations that prevent atlastin self-association also abolish oligomerization-dependent stimulation of GTPase activity. Furthermore, increasing the distance of atlastin complex formation from the membrane inhibits fusion, suggesting that this distance is crucial for atlastin to promote fusion.
- Published
- 2011
42. Balancing ER dynamics: Shaping, bending, severing, and mending membranes
- Author
-
James A. McNew, Diana Pendin, and Andrea Daga
- Subjects
Organelles ,Cytoplasm ,Nuclear Envelope ,Endoplasmic reticulum ,Dynamics (mechanics) ,Lipid bilayer fusion ,Biological Transport ,Bending ,Intracellular Membranes ,Cell Biology ,Biology ,Endoplasmic Reticulum ,Membrane Fusion ,Microtubules ,Article ,Cell biology ,Membrane ,Microtubule ,Organelle ,Animals ,Humans - Abstract
The endoplasmic reticulum is a multifunctional organelle composed of functionally and morphologically distinct domains. These include the relatively planar nuclear envelope and the peripheral ER, a network of sheet-like cisternae interconnected with tubules that spread throughout the cytoplasm. The ER is highly dynamic and the shape of its domains as well as their relative content are in constant flux. The multiple forces driving these morphological changes depend on the interaction between the ER and microtubules, membrane fusion and fission events and the action of proteins capable of actively shaping membranes. The interplay between these forces is ultimately responsible for the dynamic morphology of the ER, which in turn is crucial for properly executing the varied functions of this organelle.
- Published
- 2011
43. Erratum: Homotypic fusion of ER membranes requires the dynamin-like GTPase Atlastin
- Author
-
Andrea Daga, James A. McNew, Song Liu, Genny Orso, Anastasia Egorova, Joseph E. Faust, Diana Pendin, Tyler J. Moss, Jessica Tosetto, Andrea Martinuzzi, and Massimo Micaroni
- Subjects
Atlastin ,Fusion ,Multidisciplinary ,Membrane ,Chemistry ,GTPase ,Dynamin ,Cell biology - Published
- 2010
44. S9.5 The mitofusin of D. melanogaster
- Author
-
Andrea Daga, Maria Giovanna Rossetto, Luca Scorrano, Olga Martins de Brito, Diana Pendin, and Valentina Debattisti
- Subjects
biology ,Biophysics ,Melanogaster ,Cell Biology ,biology.organism_classification ,Biochemistry ,Cell biology - Published
- 2008
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