Live imaging of intra-lysosome pH in cell lines and primary neuronal culture using a novel genetically encoded biosensor

Ponsford, Amy H.; Ryan, Thomas W.; Raimondi, Andrea; Cocucci, Emanuele; Wycislo, Susanne A.; Fröhlich, Florian; Swan, Laura E.; Stagi, Massimiliano

doi:10.1080/15548627.2020.1771858

Cited by 65 publications

(55 citation statements)

References 106 publications

(119 reference statements)

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“…We would like to acknowledge that recently, two related sensors containing mCherry-pHluorin-LAMP1 (Ponsford et al, 2020) and sfGFP-LAMP1-mCherry fusions (Webb et al, 2020) have been published. The described probes have a reported pKa of ~6.5 and ~5.0, 20 respectively.…”

Section: Discussionmentioning

confidence: 99%

A genetically encoded, pH-sensitive mTFP1 biosensor for probing lysosomal pH

Chin¹,

Patwardhan

Ang³

et al. 2020

Preprint

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Lysosomes are important sites for macromolecular degradation, defined by a profoundly acidic lumenal pH of ~4.5. To better understand lysosomal pH, we designed a novel, genetically encoded, fluorescent protein-based pH biosensor called FIRE-pHLy (Fluorescence Indicator REporting pH in Lysosomes). This sensor was targeted to lysosomes with LAMP1 and reported lumenal pH between 3.0 and 6.0 with monomeric teal fluorescent protein 1 (mTFP1), a bright cyan FP variant with a pKa of 4.3. Ratiometric quantification was enabled with cytosolically oriented mCherry using high-content imaging. We expressed FIRE-pHLy in several cellular models as well as the multicellular organism C. elegans and quantified its alkalinizing response to bafilomycin A1, a specific V-ATPase inhibitor. In summary, we have engineered FIRE-pHLy, a specific, robust and versatile lysosomal pH biosensor that has broad applications for investigating pH dynamics in aging and lysosome-related diseases, as well as in lysosome-based drug discovery.

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Section: Discussionmentioning

confidence: 99%

A genetically encoded, pH-sensitive mTFP1 biosensor for probing lysosomal pH

Chin¹,

Patwardhan

Ang³

et al. 2020

Preprint

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“…Using pH-dependent properties, pHluorin was also utilized in various studies to measure the pH of organelles such as lysosomes and the endoplasmic reticulum (Reifenrath and Boles 2018). pHluorin also measures autophagy flux as autophagosomes are fused to acidic lysosomes to form autophagolysosomes during the autophagy process (Oliva Trejo et al 2020), and pHluorin measures lysosome acidity that is required for protease activity in living cells (Ponsford et al 2020). pHluorin fused to optogenetic probes measures changes in pH in organelles in response to optogenetic acidification (Rost et al 2015).…”

Section: Ph-sensitive Optical Probesmentioning

confidence: 99%

Methods of measuring presynaptic function with fluorescence probes

2021

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Synaptic vesicles, which are endogenous to neurotransmitters, are involved in exocytosis by active potentials and release neurotransmitters. Synaptic vesicles used in neurotransmitter release are reused via endocytosis to maintain a pool of synaptic vesicles. Synaptic vesicles show different types of exo- and endocytosis depending on animal species, type of nerve cell, and electrical activity. To accurately understand the dynamics of synaptic vesicles, direct observation of synaptic vesicles is required; however, it was difficult to observe synaptic vesicles of size 40–50 nm in living neurons. The exo-and endocytosis of synaptic vesicles was confirmed by labeling the vesicles with a fluorescent agent and measuring the changes in fluorescence intensity. To date, various methods of labeling synaptic vesicles have been proposed, and each method has its own characteristics, strength, and drawbacks. In this study, we introduce methods that can measure presynaptic activity and describe the characteristics of each technique.

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“…Moreover, in at least one study, it has been reported that the peripheral pool of lysosomes is less acidic and less accessible to biosynthetic cargo (such as cathepsins) (Johnson et al, 2016). However, a subsequent report has shown that peripheral and perinuclear lysosomes have a similar pH (~ 4.4) (Ponsford et al, 2020).…”

Section: Depletion Of Rufy3 Reduces Lysosome Sizementioning

confidence: 99%

RUFY3 links Arl8b and JIP4-Dynein complex to regulate lysosome size and positioning

Kumar¹,

Chawla²,

Chadha³

et al. 2021

Preprint

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The whole-cell scale spatial organization of lysosomes is regulated by their bidirectional motility on microtubule tracks. Small GTP-binding (G) protein, Arl8b, stimulates the anterograde transport of lysosomes by recruiting adaptor protein SKIP (also known as PLEKHM2), which in turn couples the microtubule motor kinesin-1. Here, we have identified an Arl8b effector, RUN and FYVE domain-containing protein family member 3, RUFY3, which drives the retrograde transport of lysosomes. Artificial targeting of RUFY3 to the surface of mitochondria was sufficient to drive their perinuclear positioning. We find that RUFY3 interacts with the JIP4-Dynein-Dynactin complex and mediates Arl8b association with the retrograde motor complex. The mobile fraction of the total lysosomes per cell was significantly enhanced upon RUFY3 depletion, suggesting that RUFY3 maintains the lysosomes clustering within the perinuclear cloud. Expectedly, RUFY3 knockdown disrupted the perinuclear positioning of lysosomes upon nutrient starvation and/or serum depletion, although lysosome continued to undergo fusion with autophagosomes. Interestingly, lysosome fission events were more frequent in RUFY3-depleted cells and accordingly, there was a striking reduction in lysosome size, an effect that was also observed in dynein and JIP4 depleted cells. These findings indicate that the dynein-dependent “perinuclear cloud” arrangement of lysosomes also regulates the size of these proteolytic compartments and, likely, their cellular roles.

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Live imaging of intra-lysosome pH in cell lines and primary neuronal culture using a novel genetically encoded biosensor

Cited by 65 publications

References 106 publications

A genetically encoded, pH-sensitive mTFP1 biosensor for probing lysosomal pH

A genetically encoded, pH-sensitive mTFP1 biosensor for probing lysosomal pH

Methods of measuring presynaptic function with fluorescence probes

RUFY3 links Arl8b and JIP4-Dynein complex to regulate lysosome size and positioning

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