Mitochondrial junctions with cellular organelles: Ca2+ signalling perspective

Tepikin, Alexei V.

doi:10.1007/s00424-018-2179-z

Cited by 17 publications

(24 citation statements)

References 175 publications

(243 reference statements)

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“…Cytosolic Ca 2+ is a key element in the cell's extensive homeostatic networks, such that Ca 2+ signals are regulated by and play regulatory roles in multiple processes in cells, including mitochondrial respiratory activity [85] as well as mitochondrial protein import [86] and the ion transport activity of TRPM3 itself [87], both of which are activated by Ca 2+ -calmodulin. The mitochondria serve as both a source and a sink for cytosolic Ca 2+ and the mitochondria interact closely with endoplasmic reticulum at sites that mediate exchange of lipids and Ca 2+ signals [88].…”

Section: Links To Mitochondrial Dysfunctionmentioning

confidence: 99%

Pathological Mechanisms Underlying Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Missailidis¹,

Annesley²,

Fisher³

2019

Preprint

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The underlying molecular basis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is not well understood. Characterized by chronic, unexplained fatigue, a disabling payback following exertion ("post-exertional malaise") and variably presenting, multi-system symptoms, ME/CFS is a complex disease which demands a concerted biomedical investigation from disparate fields of expertise. ME/CFS research and patient treatment have been challenged by the lack of diagnostic biomarkers and finding these is a prominent direction of current work. Despite these challenges, modern research demonstrates a tangible biomedical basis for the disorder across many body systems. This evidence is mostly comprised of disturbances to immunological and inflammatory pathways, autonomic and neurological dysfunction, abnormalities in muscle and mitochondrial function, shifts in metabolism, and gut physiology or gut microbiota disturbances. It is possible that these threads are together entangled as parts of an underlying molecular pathology reflecting a farreaching homeostatic shift. Due to the variability of non-overlapping symptom presentation or precipitating events such as infection or other bodily stresses, the initiation of body-wide pathological cascades with similar outcomes stemming from different causes may be implicated in the condition. Patient stratification to account for this heterogeneity is therefore one important consideration during exploration of potential diagnostic developments.compounded by medical guidelines in some developed countries which are out of date regarding ME/CFS clinical practice and require urgent, overdue revision.Case definitions such as the commonly termed Oxford [1] or Fukuda [2] criteria are most often utilized throughout the UK and USA respectively, yet may fail to discriminate between generalized chronic fatigue and ME/CFS which specifically also involves PEM, which aids in characterizing this disorder as a discrete clinical entity. Also in usage are the Canadian Consensus Criteria [3] and International Consensus Criteria [4] which mandate PEM for a diagnosis of ME/CFS and therefore may be considered more specific definitions. While the presence of PEM is an optional component of the Fukuda criteria, PEM is, unfortunately, not required for research participation by all studies using this or other less strict definitions. Consequently, the discovery of a reliable diagnostic biomarker is perhaps the most common recurring theme in modern ME/CFS research. Despite myriad relevant study outcomes [5][6][7][8][9][10][11][12][13][14][15][16], no such discovery has yet been widely validated or implemented as a suitable diagnostic biomarker of ME/CFS. Not only does ME/CFS affect multiple body systems and organs, but it does so with different and time-varying levels of severity and different patterns of comorbidities in different individuals, thereby producing a highly heterogeneous patient population [7,[17][18][19][20][21][22][23]. This complexity represents a major challenge to the task of incrim...

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Section: Links To Mitochondrial Dysfunctionmentioning

confidence: 99%

Pathological Mechanisms Underlying Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Missailidis¹,

Annesley²,

Fisher³

2019

Preprint

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“…Our results suggest that inducible cross-linking using RNab-FKBP or GNab-FKBP identifies native MCS between mitochondria and PM, with each mitochondrion forming only one or two MCS with the PM. We have not explored the functional consequences of these restricted MCS, but we speculate that they may identify sites where proteins involved in communication between the PM and mitochondria are concentrated, facilitating, for example, phospholipid transfer [49], the generation of ATP microdomains [50], or Ca 2+ exchanges between mitochondria and store-operated Ca 2+ entry (SOCE) [51] or PM Ca 2+ -ATPases [52].…”

Section: Resultsmentioning

confidence: 99%

A genetically-encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling

Prole

Taylor

2019

Preprint

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2 Abbreviations 15 BFP, blue fluorescent protein; [Ca 2+ ] c , cytosolic free Ca 2+ concentration; CALI, 16 chromophore-assisted light inactivation; CaM, calmodulin; CFP, cyan fluorescent protein; 17 ER, endoplasmic reticulum; FKBP, FK506-binding protein; FRB, FKBP-rapamycin-binding 18 domain; FP, fluorescent protein; GFP, green fluorescent protein; GNab, GFP-binding 19 nanobody; HBS, HEPES-buffered saline; IP 3 R, inositol 1,4,5-trisphosphate receptor; 20 LAMP1, lysosomal membrane protein 1; mCherry, monomeric Cherry; MCS, membrane 21 contact site; MHBS, modified HBS; MP, multimerizing protein; mRFP, monomeric red 22 fluorescent protein; OMM, outer mitochondrial membrane; PM, plasma membrane; RFP, red 23 fluorescent protein; RNab, RFP-binding nanobody; ROI, region of interest; SOCE, store-24 operated Ca 2+ entry; TIRFM, total internal reflection fluorescence microscopy; YFP, yellow 25 fluorescent protein. 26 3 Abstract 27Background: Intrabodies enable targeting of proteins in live cells, but it remains a huge task 28 to generate specific intrabodies against the thousands of proteins in a proteome. We leverage 29 the widespread availability of fluorescently labelled proteins to visualize and manipulate 30 intracellular signalling pathways in live cells by using nanobodies targeting fluorescent 31 protein tags. 32 Results:We generated a toolkit of plasmids encoding nanobodies against red and green 33 fluorescent proteins (RFP and GFP variants), fused to functional modules. These include 34 fluorescent sensors for visualization of Ca 2+ , H + and ATP/ADP dynamics; oligomerizing or 35 heterodimerizing modules that allow recruitment or sequestration of proteins and 36 identification of membrane contact sites between organelles; SNAP tags that allow labelling 37 with fluorescent dyes and targeted chromophore-assisted light inactivation; and nanobodies 38 targeted to lumenal sub-compartments of the secretory pathway. We also developed two 39 methods for crosslinking tagged proteins: a dimeric nanobody, and RFP-targeting and GFP-40 targeting nanobodies fused to complementary hetero-dimerizing domains. We show various 41 applications of the toolkit and demonstrate, for example, that IP 3 receptors deliver Ca 2+ to the 42 outer membrane of only a subset of mitochondria, and that only one or two sites on a 43 mitochondrion form membrane contacts with the plasma membrane. 44 Conclusions: This toolkit greatly expands the utility of intrabodies for studying cell 45 signalling in live cells. 46 4 Background 47 Visualizing the location of specific proteins within cells and manipulating their function is 48 crucial for understanding cell biology. Antibodies can define protein locations in fixed and 49 permeabilized cells, but antibodies are large protein complexes that are difficult to introduce 50 into live cells [1]. This limits their ability to interrogate the dynamics or affect the function of 51 proteins in live cells. Small protein-based binders, including nanobodies derived from the 52 variable region of the heavy chains ...

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“…TG induced an increase in the fluorescence of LAR-GECO, indicating an increase of Ca 2+ within the host mitochondria (LAR-GECO), as well as Ca 2+ oscillations within the parasite (GCaMP6f). The increase in host mitochondrial Ca 2+ can be accounted for as due to the close, tight interaction between the ER and the mitochondria (23). Interestingly, addition of TG caused Ca 2+ oscillations in the intracellular tachyzoites but they remained intracellular throughout all 10 min of recording ( Fig 2B&D and Supplemental Video 2).…”

Section: A Threshold Of Ca 2+ Is Needed For Parasite Egressmentioning

confidence: 89%

“…Addition of TG induced an increase in the fluorescence of LAR-GECO, indicating an increase of Ca 2+ within the host mitochondria (LAR-GECO), as well as Ca 2+ oscillations within the parasite (GCaMP6f). The increase in host mitochondrial Ca 2+ can be accounted for as due to the close, tight interaction between the ER and the mitochondria [24].…”

Section: A Threshold Of Ca 2+ Is Needed For Parasite Egressmentioning

confidence: 99%

The Role of Potassium and Host Calcium Signaling inToxoplasma gondiiegress

Vella

Moore

et al. 2020

Preprint

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Toxoplasma gondii, an obligate intracellular parasite, is capable of invading virtually any nucleated cell. Active invasion by the parasite is a precise process and intracellular parasites reside and replicate inside a parasitophorous vacuole (PV). The membrane of the PV functions as a molecular sieve allowing for its ionic milieu to be in equilibrium with the host cytosol. The parasite would thus be exposed to the ionic fluctuations of the host cytoplasm, such as increases in host cytosolic Ca 2+ during Ca 2+ signaling events. Ca 2+ is a universal signaling molecule and both the host cell and T. gondii will utilize Ca 2+ signaling to stimulate diverse cellular functions. Using T. gondii tachyzoites and host cells expressing genetically encoded Ca 2+ indicators, we demonstrate that host Ca 2+ signaling impacts intracellular Ca 2+ levels of the parasite. We propose that Ca 2+ influx derived from host Ca 2+ signaling into the parasite is utilized to maintain the intracellular Ca 2+ stores replenished as the parasite replicates within the low Ca 2+ environment of the host cell.Intracellular Ca 2+ store(s) release is needed to reach the initiation spike in Ca 2+ that meets the threshold of Ca 2+ needed to initiate the tightly coordinated process of egress. Post activation of the signal and subsequent microneme secretion, would cause breakdown of the host cell and allow for extracellular Ca 2+ influx, causing a second, larger spike in Ca 2+ and activation of the glideosome, the main motility machinery. To directly deliver precise concentrations of ions needed for egress, we used patch-clamped infected host cells and monitored parasite egress. In doing so, we discovered an alternative role for K + in timing parasite egress. This is the first study using wholecell patch clamp to study the role of ions such as K + and Ca 2+ in T. gondii egress. Author SummaryToxoplasma gondii is an intracellular parasite that causes disease by engaging in multiple rounds of a lytic cycle which consists of invasion, replication inside a parasitophorous vacuole (PV) and egress, resulting in lysis of the host cell. In intracellular parasites, the release of Ca 2+ from intracellular stores forms part of a series of signaling events that culminate in stimulation of motility and egress. This means that intracellular parasites must be able to uptake Ca 2+ while replicating to keep their intracellular stores filled to be used during egress. This is not an insignificant task considering that they are inside a PV surrounded by the host cytosol which contains Ca 2+ below 100 nM. We present data showing that the host Ca 2+ increases are able to 3 reach the parasite cytosol through a Ca 2+ influx mechanism sensitive to nifedipine. This influx causes oscillations that are the result of Ca 2+ increase and re-uptake by intracellular stores. A Ca 2+ threshold was needed for egress, extracellular Ca 2+ influx was relevant for egress and potassium had a modulatory effect on egress. We used infected patched host cells, a novel approach that leaves the host ...

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Mitochondrial junctions with cellular organelles: Ca2+ signalling perspective

Cited by 17 publications

References 175 publications

Pathological Mechanisms Underlying Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Pathological Mechanisms Underlying Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

A genetically-encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling

The Role of Potassium and Host Calcium Signaling inToxoplasma gondiiegress

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