Manipulation of Mitochondria Dynamics Reveals Separate Roles for Form and Function in Mitochondria Distribution

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 distributio

Familial Alzheimer's disease-linked presenilin mutants and intracellular Ca2+ handling: A single-organelle, FRET-based analysis

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

Proteasome dysfunction induces excessive proteome instability and loss of mitostasis that can be mitigated by enhancing mitochondrial fusion or autophagy

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.</p

Dynamic constriction andfission of endoplasmicreticulum membranes by reticulon

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.This work was partially supported by NIH R01GM121725 to V.A.F., a 5x1000 grant from the Italian Ministry of Health and Telethon GGP11189 to A.D., Spanish Ministry of Science, Innovation and Universities grants BFU2015-70552-P to V.A.F. and A.V.S., and BFU2015-63714-R and PGC2018-099341-B-I00 to B.I., Basque Government grant IT1196-19, Russian Science Foundation Grant No. 17-75-30064 and Ministry of Science and Higher Education of the Russian Federation

mCerulean3-Based Cameleon Sensor to Explore Mitochondrial Ca2+ Dynamics In Vivo

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 F\uf6rster 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

Homotypic fusion of ER membranes requires the dynamin-like GTPase Atlastin

The endoplasmic reticulum (ER) is a subcellular organelle comprised of interconnecting membrane networks. Formation and maintenance of the intricate ER architecture is essential for implementation of the multiple functions served by this organelle. Homotypic membrane fusion underlies both the biogenesis and maintenance of the ER and depends categorically on GTP hydrolysis but does not require cytosolic components, suggesting that a membrane bound GTPase may be responsible for this activity. Using in vivo analysis in Drosophila we demonstrate that the GTPase Datlastin, the fly homologue of the dynamin superfamily member atlastin-1 whose mutation causes Hereditary Spastic Paraplegia, is specifically localized on ER membranes. Furthermore, loss of Datlastin causes fragmentation of the ER but does not impair secretory pathway traffic. Datlastin 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 Datlastin autonomously drives membrane fusion in a GTP dependent fashion. In contrast, GTPase-deficient Datlastin is inactive, unable to form trans-oligomeric complexes due to failure to self-associate, and incapable of promoting fusion in vitro. These results demonstrate that Datlastin mediates membrane tethering and fusion and strongly suggest that it is the GTPase activity required for ER homotypic fusion.Il reticolo endoplasmatico (RE) è un organello formato da un complesso network di membrane. La formazione e il mantenimento dell’elaborata architettura del RE è essenziale per lo svolgimento delle numerose funzioni di questo organello. La biogenesi e il mantenimento del RE dipendono dalla capacità delle membrane del RE di fondersi in maniera omotipica. La dipendenza di questo processo dall’idrolisi del GTP, ma non da fattori citosolici, suggerisce che la fusione omotipica sia mediata da una GTPasi di membrana. Datlastina è l’omologo in Drosophila di atlastina-1, GTPasi membro della superfamiglia delle dinamine le cui mutazioni causano l’insorgenza di Paraplegia Spastica Ereditaria nell’uomo. Analisi in vivo in Drosophila hanno mostrato che Datlastina è localizzata sulla membrana del reticolo endoplasmatico. L’assenza di Datlastina causa frammentazione del RE, ma non blocca il traffico secretorio. Molecole di Datlastina inserite in membrane distinte sono in grado di associarsi in trans e la sovraespressione della proteina in vivo induce la formazione di profili di RE allargati, compatibili con una eccessiva fusione delle membrane del RE. Esperimenti in vitro hanno confermato che Datlastina è in grado di promuovere autonomamente la fusione di membrane in modo GTP dipendente. Al contrario, Datlastina priva di attività GTPasica è inattiva, incapace di associarsi in trans e di promuovere la fusione delle membrane in vitro. Questi risultati dimostrano che Datlastina è in grado di mediare l’avvicinamento e la fusione delle membrane e suggeriscono che costituisca l’attività GTPasica necessaria per la fusione omotipica delle membrane del RE

Mitofusin 2: from functions to disease

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