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Development of new tools to explore organelle calcium dynamics in vivo: a new fret-based calcium sensor and a mitochondria targeted channelrhodopsin
Full text linkThe first part of this thesis aims to develop new tools to explore mitochondrial Ca2+ dynamic in vivo by the improvement of a mitochondria targeting Cameleon probes and by creating a mitochondria targeted Channelrhodopsin.
Genetically encoded calcium indicators (GECIs) allow quantitative Ca2+ measurements in different experimental models. Organelle-specific targeting signals are fused with the GECI’s sequence, achieving selective targeting to a specific organelle or cytoplasmic domain. Moreover, GECI’s coding sequences can be placed under the control of tissue specific or inducible promoters, allowing spatial and temporal control of their expression. Different types of GECIs have been created: in our laboratory we use a class of FRET-based Ca2+ sensors, called Cameleons. FRET (Förster resonance energy transfer) microscopy detects the direct transfer of energy from a donor to an acceptor fluorescence protein (FP) in a living cell. Cameleon structure consists of two Ca2+-responsive elements that alter the efficiency of FRET between two FPs: a cyan fluorescent protein (CFP), the donor, and a yellow fluorescent protein (cpV), the acceptor. The two Ca2+-responsive elements are Calmodulin (CaM) and the CaM-binding domain of myosin light chain kinase.
As reported in the beginning, the aim of this study is to develop novel molecular sensors and new methodologies to express the probes in vivo in intact tissues as well as in organisms in order to explore organelle Ca2+ dynamics in vivo (in particularly in brain and heart).
One of the limitations of Cameleon probes, especially critical for in vivo applications, is the low fluorescence of CFP, reducing the maximal obtainable signal-to-noise ratio, and a multi-exponential lifetime, indicating the presence of multiple excited-state decay pathways. Recently, a brighter and more stable FP, compared to CFP, has been developed: mCerulean3. It has high fluorescence quantum yield and high photostability, making this protein a good donor candidate in Cameleon probes. For this reason, CFP has been replaced with mCerulean3 in two different Cameleons: the cytosolic D3cpv and the mitochondria-targeted probe named 4mtD3cpv. The new probes have been tested in different cell types: HeLa, neonatal rat cardiomyocytes and neonatal mouse neurons. The brightness, the photostability, the pH-sensitivity and the dissociation constant (Kd) of the new probes have also been measured in situ and the data show a clear improvement in brightness and in photostability, compared to the original Cameleons, in both cytosolic and mitochondrial probes. The only drawback of the new probes is a reduced amplitude (about 20-30%) in the maximum change in the fluorescence emission ratio due to Ca2+ binding (dynamic range). In order to extend the dynamic range different approaches have been used. The addition of 16 glycines between the two Ca2+ responsive elements allowed us to generate a new cytosolic probe (D3mCerulean3+16) with an increased dynamic range (+ 20%). Similarly, we have modified the mitochondrial probe, starting from the targeting sequence, each N-terminal sequence of COX-VIII (subunit VIII of human cytochrome C oxidase) was elongated of 5 aminoacids. Indeed, 24 h after transfection a significant mis-localization in the cytosol was observed with the original probe, while the mis-targeting of the modified targeting sequence containing probe was decreased. Moreover, the reduction in the dynamic range due to the mCerulean3 presence was almost fully recovered adding the 16 glycines linker. We than analysed the effect of pH, observing a general stability of all the probe variants in the pH range tested. Finally, we evaluated the Ca2+ affinity of all the mitochondrial variants generated so far, obtaining a Kd of 0.06 µM and one of 10.84 µM in 4mtD3cpV, a Kd of 6,5 µM in 4mtD3mCerulean3 and a Kd of 0.03 µM and one of 10.7 µM in 4mtD3mCerulean3+16. To better characterize the new cytosolic and mitochondrial Cameleon probes, we purified the proteins expressed in E.Coli in order to measure also in vitro all the parameters described above. Up to now only preliminary data have been obtained, however a comparison of in situ and in vitro data suggests that the DR calculated for cytosolic probes is about 3, while that for a mitochondria like environment is about 2.5. Concerning the Ca2+ affinity, just few [Ca2+] concentrations have been tested so far, therefore all the mitochondrial and cytosolic probes display a single Kd. However, further experiments are required to confirm the in vitro calculated Kd.
In parallel, we are also assessing another method to measure Ca2+ with FRET-GECIs, as an alternative of the classical intensity-based method. We are indeed employing FLIM technique that measures the lifetime of FPs (the time the molecule spend in the exited state), which is totally independent from phenomena such as probe photobleaching, expression level, image shading. Preliminary results in cells expressing all the Camelon probes described so far, suggest the presence of homo-FRET phenomena and mCerulean3-based Ca2+ sensors seem to be less affected by them.
Finally, to express the new probes in vivo we are testing three strategies: i) adeno-associated virus (serotype 9) with cardiac tropism viral vectors; ii) adeno-associated virus (serotype 9) with neuron-specific promoters (synapsin promoter) for intracranial injection; iii) transgenic mice. These strategies will allow us to perform in vivo Ca2+ measurements in different tissues and subcellular compartments. We have just generated the AAV9 for the expression of the D3mCerulean3 and 4mD3mCerulean3 under the control of CMV promoter. A good expression levels in heart was obtained through intraperiotneal injection of neonatal mice.
In conclusion, the results obtained so far make the mCerulean3-based Cameleons an attractive choice for in vivo experiments.
To study the role of mitochondria in situ and in vivo, we also took advantage of an optogenetic tool: Channel Rhodopsins (ChRs). ChRs represent the only type of ion channels directly gated by light. When excited by light, ChRs open and depolarize the plasma membrane. For activation, opsins require binding of retinal, a vitamin A–related organic cofactor. In collaboration with Prof. Sekler’s group (Ben-Gurion University-Israel), we have developed a new mitochondria-targeted ChR, called mitochondrial Stabilized Step Function Opsin (4mt-SSFO). This ChR variant was modified to stabilize the conducting state of the channel: SSFO deactivation occurs in 30 minutes after a brief pulse of activating blue light (460-480 nm). It has also been reported the possibility to terminate the photocurrents with red light. To create a non-functional control, a truncated form Chr2(TR) lacking retinal binding site was generated. The targeting of proteins to the inner mitochondrial membrane (IMM) was obtained fusing four signal sequences, derived from human cytochrome C oxidase subunit VIII sequence, to the N-terminus of ChR. To verify the correct topology of the probe, confocal microscopy, elecrtophisiological recordings and fluorescence quenching experiments were carried out. When 4mtSSFO-YFP is properly inserted in the IMM, its C-terminal YFP tag should face the mitochondrial matrix. Indeed, the application of proteinase K to Digitonin permeabilized cells did not cause a significant reduction of 4mtChR2B-YFP signal, while that of N33D3cpv (a probe in which the YFP is located on the cytoplasmic surface of the OMM) is completely abolished. Trypan blue addition, that is permeable across the OMM, but not the IMM, did not affect the fluorescence of 4mtSSFO-YFP or that of the matrix located Cameleon 4mtD3cpv. The 4mtD3cpv fluorescence however was totally lost after alamethicin application that permeabilizes both mitochondrial membranes and releases all matrix proteins into the medium, while the fluorescence of 4mtSSFO-YFP was not affected by the latter treatment, as expected for a membrane bound protein. Taken together these data are consistent with a proper IMM localization.
The new mitochondrial 4mtSSFO-YFP constructs were then tested in situ for their ability to change the mitochondrial membrane potential in response to light, resulting in a significant organelle depolarization in cells expressing 4mtSSFO-YFP, while no effect was induced by blue light in control cells. We then analysed in more details the effects of mitochondrial depolarization on mitochondrial Ca2+ uptake using two genetically encoded probes, 4mtD3cpv and mt-aequorin. Both approaches demonstrate that the depolarization of mitochondria triggered by photo-activation of the channel causes, as predicted, a significant reduction in the amplitude of the mitochondrial Ca2+ rise observed upon stimulation of HeLa cells with an IP3-generating agonist. Thus, we generated a tool able to modulate diverse mitochondria activities in a temporary-controlled, reversible (at least in principle) and cell-specific manner, offering an approach to quantitatively investigate mitochondrial role in a large variety of critical cellular processes.
In the second part of the thesis, the investigation was focused on the role of Presenilins in Ca2+dyshomeostasis associated to Alzheimer's Disease (AD) using a single cell analysis approach and focusing on the role of AD Presenilin 1 and Presenilin 2 mutations in the modulation of Capacitative Calcium Entry (CCE).
AD is the most frequent form of dementia. A small percentage of cases is inherited (Familial AD, FAD) and is due to dominant mutations on three genes, coding for Amyloid Precursor Protein (APP), Presenilin-1 (PS1) and Presenilin-2 (PS2). Mutations in these genes cause alterations in the cleavage of APP by a PS1- or PS2- containing enzyme, named y-secretase complex, thus leading to an increase in the ratio between Abeta42 and Abeta40, the two main peptides eventually derived from APP maturation. This event, in turn, leads to an increased deposition of amyloid plaques, one of the main histopathological feature of AD. To date, the generation of A42 peptides, its oligomers and amyloid plaques is the core of the most widely accepted pathogenic hypothesis for AD, the “Amyloid Cascade Hypothesis”. PS1 and PS2 are ubiquitous 9 transmembrane domains homologous proteins localized mainly in intracellular membranes (Endoplasmic Reticulum, ER, Golgi apparatus, and endosomes) and plasma membrane. Despite being the catalytic core of y-secretase complex, PSs display also some specialized, y-secretase independent activities. On this line, numerous studies reported a role for FAD-linked PS mutations in cellular Ca2+ alterations. Ca2+ is a key second messenger in living cells and it regulates a multitude of cell functions; thus, alterations in its signalling cascade can be detrimental for cell fate. Ca2+ mishandling has been proposed as a causative mechanism for different neurodegenerative diseases and in particular for AD. Although supported by several groups for many years, the Ca2+ hypothesis for AD pathogenesis has never been undisputedly accepted, since some data were clearly conflicting, especially those considering FAD-PS2 mutations. One of the Ca2+ pathway reported to be modulated by different FAD-PSs mutation is the so called CCE or Store Operated Calcium Entry (SOCE). CCE is the mechanism responsible for Ca2+ entry in response to ER Ca2+ depletion. The key molecules responsible for this Ca2+ entry have been identified only recently: STIM and Orai. Basically, Orai forms the channels located in the PM, while STIM is the protein that can “sense” the [Ca2+] in the ER lumen. Upon store depletion, STIM1 changes its distribution from diffuse to clusterized “puncta” and interacts with plasma membrane-located Orai1. Employing a cytosolic Cameleon probe (D3cpV), the CCE variation in SH-SY5Y cells overexpressing PSs or in human FAD and control fibroblasts was investigated. In particular, measuring the effect of PS1-A246, PS2-T122R (in overexpression) and PS1-A246 and PS2N141I (in fibroblasts) FAD-linked mutations, a decrease in both peak and rate of CCE was observed. This phenomenon could be explained by a decrease in STIM1 protein levels, while Orai1 level was not analysed because no antibody sufficiently specific is available. In the over-expression system of SH-SY5Y cells also wild type forms of PS1 and PS2 cause a decrease in CCE and this could be due to the accumulation of the full-length form of the proteins that is thought to be the mediator of the effec
The elusive importance of being a mitochondrial Ca2+ uniporter
No full textAbstractThe 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
Calsequestrin in Purkinje cells of mammalian cerebellum
Full text link: Cerebellum is devoted to motor coordination and cognitive functions. Endoplasmic reticulum is the largest intracellular calcium store involved in all neuronal functions. Intralumenal calcium binding proteins play a pivotal role in calcium storage and contribute to both calcium release and uptake. Calsequestrin, a key calcium binding protein of sarco-endoplasmic reticulum in skeletal and cardiac muscles, was identified in chicken and fish cerebellum Purkinje cells, but its expression in mammals and human counterpart has not been studied in depth. Aim of the present paper was to investigate expression and localization of Calsequestrin in mammalian cerebellum. Calsequestrin was found to be expressed at low level in cerebellum, but specifically concentrated in Calbindin D28- and zebrin- immunopositive-Purkinje cells. Two additional fundamental calcium store markers, sarco-endoplasmic calcium pump isoform 2, SERCA2, and Inositol-trisphosphate receptor isoform 1, IP3R1, were found to be co-expressed in the region, with some localization peculiarities. In conclusion, a new marker was identified for Purkinje cells in adult mammals, including humans. Such a marker might help in staminal neuronal cells specification and in dissection of still unknown neurodegeneration and physio-pathological effects of dysregulated calcium homeostasis
Exploiting Cameleon Probes to Investigate Organelles Ca2+ Handling
No full textCalcium ion (Ca2+) is a ubiquitous intracellular messenger able to generate versatile intracellular signals that modulate a large variety of functions in virtually every cell type. Chemical and genetic biosensors, targeted to different subcellular compartments, have been developed and continuously improved to monitor Ca2+ dynamics in living cells. Here we describe the usage of Förster resonance energy transfer (FRET)-based Cameleon probes to investigate Ca2+ influx across the plasma membrane (PM) or Ca2+ release from the main intracellular Ca2+ store, the endoplasmic reticulum (ER)
Highlighting the Endoplasmic Reticulum-Mitochondria connection: focus on Mitofusin 2
No full textThe endoplasmic reticulum (ER) and the mitochondrial network are two highly interconnected cellular structures. By proteinaceous tethers, specialized membrane domains of the ER are tightly associated with the outer membrane of mitochondria, allowing the assembly of signaling platforms where different cell functions take place or are modulated, such as lipid biosynthesis, Ca2+ homeostasis, inflammation, autophagy and apoptosis. The ER-mitochondria coupling is highly dynamic and contacts between the two organelles can be modified in their number, extension and thickness by different stimuli. Importantly, several pathological conditions, such as cancer, neurodegenerative diseases and metabolic syndromes show alterations in this feature, underlining the key role of ER-mitochondria crosstalk in cell physiology. In this contribution, we will focus on one of the major modulator of ER-mitochondria apposition, Mitofusin 2, discussing the structure of the protein and its debated role on organelles tethering. Moreover, we will critically describe different techniques commonly used to investigate this crucial issue, highlighting their advantages, drawbacks and limits
FLIM-FRET analysis using Ca<sup>2+</sup> sensors in HeLa cells
No full textIn this work, FLIM-FRET experiments are performed in HeLa cells expressing a genetically encoded Ca2+ indicator (GECIs) based on FRET, called Cameleon probes. These sensors allow the analysis of Ca2+ dynamics in different cell compartments, such as mitochondria and cytosol. Cameleons are composed by two fluorescent proteins (FPs, donor and acceptor moieties) and two Ca2+ responsive elements that alter the FRET efficiency between the FPs, as a function of intracellular Ca2+ concentration. In particular, we focus on new generated Cameleon sensors where the Cyan Fluorescent Protein donor moiety has been replaced by mCerulean3. Since it is characterized by single fluorescence lifetime decay and higher brightness, mCerulean3 based sensors has permitted the application of FLIM approach for the determination of FRET efficiency. FLIM measurements, compared to intensity-based FRET analysis, have the advantages of being independent of fluorophore concentration, excitation intensity fluctuation, sample thickness, or photobleaching
Familial Alzheimer’s Disease-linked Presenilin mutants and intracellular Ca2+ handling: a single-organelle, FRET-based analysis
No full textAn imbalance in Ca 2+ 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 Ca 2+ signaling pathways, in particular those handled by storage compartments. However, FAD-PSs effect on organelles Ca 2+ 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 Ca 2+ dynamics, we here present a detailed and quantitative single-cell analysis of FAD-PSs effects on organelle Ca 2+ handling using specifically targeted, FRET (Fluorescence/Förster Resonance Energy Transfer)-based Ca 2+ indicators. In SH-SY5Y human neuroblastoma cells and in patient-derived fibroblasts expressing different FAD-PSs mutations, we directly measured Ca 2+ concentration within the main intracellular Ca 2+ 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 Ca 2+ handling of ER and medial-GA, but not trans-GA, reducing, compared to control cells, the Ca 2+ content within these organelles by partially blocking SERCA (Sarco/Endoplasmic Reticulum Ca 2+ -ATPase) activity. Moreover, by using a cytosolic Ca 2+ probe, we show that the expression of both FAD-PS1 and -PS2 reduces the Ca 2+ influx activated by stores depletion (Store-Operated Ca 2+ 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 Ca 2+ content of intracellular stores yet leading to a complex dysregulation of Ca 2+ homeostasis, which represents a common disease phenotype of AD
Ca2+ and cAMP cross-talk in mitochondria.
No full textWhile mitochondrial Ca(2+) homeostasis has been studied for several decades and many of the functional roles of Ca(2+) 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 Ca(2+) 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 Ca(2+) signalling and its role in controlling ATP synthesis. The discovery of a cAMP signalling, and the demonstration of a cross-talk between cAMP and Ca(2+) 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 Ca(2+) and cAMP in mitochondria of living cells with genetically encoded probes is also presented
Mitofusin 2 ablation increases endoplasmic reticulum–mitochondria coupling
Full text linkThe organization and mutual interactions between endoplasmic reticulum (ER) and mitochondria modulate key aspects of cell pathophysiology. Several proteins have been suggested to be involved in keeping ER and mitochondria at a correct distance. Among them, in mammalian cells, mitofusin 2 (Mfn2), located on both the outer mitochondrial membrane and the ER surface, has been proposed to be a physical tether between the two organelles, forming homotypic interactions and heterocomplexes with its homolog Mfn1. Recently, this widely accepted model has been challenged using quantitative EM analysis. Using a multiplicity of morphological, biochemical, functional, and genetic approaches, we demonstrate that Mfn2 ablation increases the structural and functional ER-mitochondria coupling. In particular, we show that in different cell types Mfn2 ablation or silencing increases the close contacts between the two organelles and strengthens the efficacy of inositol trisphosphate (IP3)-induced Ca(2+) transfer from the ER to mitochondria, sensitizing cells to a mitochondrial Ca(2+) overload-dependent death. We also show that the previously reported discrepancy between electron and fluorescence microscopy data on ER-mitochondria proximity in Mfn2-ablated cells is only apparent. By using a different type of morphological analysis of fluorescent images that takes into account (and corrects for) the gross modifications in mitochondrial shape resulting from Mfn2 ablation, we demonstrate that an increased proximity between the organelles is also observed by confocal microscopy when Mfn2 levels are reduced. Based on these results, we propose a new model for ER-mitochondria juxtaposition in which Mfn2 works as a tethering antagonist preventing an excessive, potentially toxic, proximity between the two organelles
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