The Application of KillerRed for Acute Protein Inactivation in Living Cells

Jarvela, Timothy S.; Linstedt, Adam D.

doi:10.1002/0471142956.cy1235s69

Cited by 4 publications

(5 citation statements)

References 23 publications

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“…This feature is utilized in chromophore-assisted laser inactivation ( Jacobson et al. , 2008 ; Jarvela and Linstedt, 2014 ; Sano et al. , 2014 ; Wojtovich and Foster, 2014 ) and has led to the design of fluorescent proteins with higher yield of ROS such as KillerRed ( Bulina et al.…”

Section: Discussionmentioning

confidence: 99%

“…Fluorescent proteins, including GFP species, have the capacity to generate ROS when illuminated and can act as genetically-encoded photosensitizers to produce singlet oxygen and superoxide upon illumination (Trewin et al, 2018). This feature is utilized in chromophore-assisted laser inactivation (Jacobson et al, 2008;Jarvela and Linstedt, 2014;Sano et al, 2014;Wojtovich and Foster, 2014) and has led to the design of fluorescent proteins with higher yield of ROS such as KillerRed (Bulina et al, 2006) and SuperNova (Takemoto et al, 2013). It is therefore possible that GFP in our experiments produces ROS during imaging illumination, particularly within biofilm cells.…”

Section: Subcellular Modifications During Biofilm Developmentmentioning

confidence: 99%

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Aspergillus nidulans biofilm formation modifies cellular architecture and enables light-activated autophagy

Lingo

Shukla

Osmani

et al. 2021

MBoC

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After growing on surfaces, including those of medical and industrial importance, fungal biofilms self-generate internal microenvironments. We previously reported that gaseous microenvironments around founder Aspergillus nidulans cells change during biofilm formation causing microtubules (MTs) to disassemble under control of the hypoxic transcription factor SrbA. Here we investigate if biofilm formation might also promote changes to structures involved in exocytosis and endocytosis. During biofilm formation the ER remained intact but ER exit sites and the Golgi apparatus were modified as were endocytic actin patches. The biofilm driven changes required the SrbA hypoxic transcription factor and could be triggered by nitric oxide, further implicating gaseous regulation of biofilm cellular architecture. By tracking GFP-Atg8 dynamics, biofilm founder cells were also observed to undergo autophagy. Most notably, biofilm cells that had undergone autophagy were triggered into further autophagy by spinning disc confocal light. Our findings indicate that fungal biofilm formation modifies the secretory and endocytic apparatus and show biofilm cells can also undergo autophagy that is reactivated by light. The findings provide new insights into the changes occurring in fungal biofilm cell biology that potentially impact their unique characteristics, including antifungal drug resistance. [Media: see text]

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Section: Discussionmentioning

confidence: 99%

Section: Subcellular Modifications During Biofilm Developmentmentioning

confidence: 99%

Aspergillus nidulans biofilm formation modifies cellular architecture and enables light-activated autophagy

Lingo

Shukla

Osmani

et al. 2021

MBoC

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“…Therefore, we developed a vector system that allows us to generate a spatiotemporally controlled insult within presynaptic boutons. Here, we made use of the fact that free radicals trigger the damage of proteins in vivo (Jarvela and Linstedt, 2014) and tagged synaptic proteins with the genetically encoded photosensitizer SN (Takemoto et al, 2013). With similar approaches, it has been possible to acutely damage mitochondria and induce mitophagy (Yang and Yang, 2011;Wang et al, 2012;Ashrafi et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

“…These latter data imply that the synaptic increase in ROS rapidly induces presynaptic autophagy and that subsequent flux carries the autophagosomal membranes into axons. Although bleaching of SN is described to coincide with ROS generation (Jarvela and Linstedt, 2014), we additionally made use of the superoxide indicator DHE to monitor for ROS production ( Fig. 4 A, B).…”

Section: Light-activated Ros Generation Triggers Presynaptic Autophagymentioning

confidence: 99%

Light-Activated ROS Production Induces Synaptic Autophagy

et al. 2019

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The regulated turnover of synaptic vesicle (SV) proteins is thought to involve the ubiquitin-dependent tagging and degradation through endo-lysosomal and autophagy pathways. Yet, it remains unclear which of these pathways are used, when they become activated, and whether SVs are cleared en masse together with SV proteins or whether both are degraded selectively. Equally puzzling is how quickly these systems can be activated and whether they function in real-time to support synaptic health. To address these questions, we have developed an imaging-based system that simultaneously tags presynaptic proteins while monitoring autophagy. Moreover, by tagging SV proteins with a light-activated ROS generator, Supernova, it was possible to temporally control the damage to specific SV proteins and assess their consequence to autophagy-mediated clearance mechanisms and synaptic function. Our results show that, in mouse hippocampal neurons of either sex, presynaptic autophagy can be induced in as little as 5-10 min and eliminates primarily the damaged protein rather than the SV en masse. Importantly, we also find that autophagy is essential for synaptic function, as light-activated damage to, for example, Synaptophysin only compromises synaptic function when autophagy is simultaneously blocked. These data support the concept that presynaptic boutons have a robust highly regulated clearance system to maintain not only synapse integrity, but also synaptic function.

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“…Therefore we developed a vector system that allows us to generate a spatiotemporally controlled insult within presynaptic boutons. We made use of the fact that free radicals trigger the damage of proteins in vivo (Jarvela and Linstedt, 2014) and tagged synaptic proteins with a genetically encoded photosensitizer Supernova (Takemoto et al, 2013). With similar approaches it has earlier been possible to damage mitochondria and induce mitophagy (Ashrafi et al, 2014; Wang et al, 2012; Yang and Yang, 2011).…”

Section: Discussionmentioning

confidence: 99%

Light induced synaptic vesicle autophagy

Hoffmann

Orlando

Andrzejak

et al. 2018

Preprint

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2The regulated turnover of synaptic vesicle (SV) proteins is thought to involve the ubiquitin 3 dependent tagging and degradation through endo-lysosomal and autophagy pathways. Yet, it remains 4 unclear which of these pathways are used, when they become activated and whether SVs are cleared 5 en-mass together with SV proteins or whether both are degraded selectively. Equally puzzling is how 6 quickly these systems can be activated and whether they function in real time to support synaptic 7 health. To address these questions, we have developed an imaging based system that simultaneously 8 tags presynaptic proteins while monitoring autophagy. Moreover, by tagging SV proteins with a light 9 activated reactive oxygen species (ROS) generator, Supernova, it was possible to temporally control 10 the damage to specific SV proteins and assess their consequence to autophagy mediated clearance 11 mechanisms and synaptic function. Our results show that, in mouse hippocampal neurons, presynaptic 12 autophagy can be induced in as little as 5-10 minutes and eliminates primarily the damaged protein 13 rather than the SV en-mass. Importantly, we also find that autophagy is essential for synaptic function, 14as light-induced damage to e.g. Synaptophysin only compromises synaptic function when autophagy is 15 simultaneously blocked. These data support the concept that presynaptic boutons have a robust highly 16 regulated clearance system to maintain not only synapse integrity, but also synaptic function. 17 18 Significance Statement 19 20 The real-time surveillance and clearance of synaptic proteins is thought to be vital to the health, 21 functionality and integrity of vertebrate synapses and is compromised in neurodegenerative 22disorders, yet the fundamental mechanisms regulating these systems remain enigmatic. Our analysis 23 reveals that presynaptic autophagy is a critical part of a real-time clearance system at glutamatergic 24 synapses capable of responding to local damage of synaptic vesicle proteins within minutes and to be 25 critical for the ongoing functionality of these synapses. These data indicate that synapse autophagy is 26 2 not only locally regulated but also crucial for the health and functionality of vertebrate presynaptic 27 boutons. 28 29 75 Moreover, the selective damage of SV proteins allowed us to show that presynaptic autophagy is 76 critical for the real-time maintenance of synaptic transmission. 77 78 79 80 5 Material and Methods 81 82 Construction of vectors: Monitoring of autophagy within presynaptic boutons was achieved by creating 83 a set of lentiviral expression vectors. All vectors are based on the commercially available vector FUGW 84 (Addgene). In order to co-express mCherry-tagged Synaptophysin (Syp) and eGFP-LC3, 85 Synaptophysin-mCherry (Synaptophysin, NM_012664.3) was synthesized by Eurofins Genomics with a 86 downstream glycine linker that was fused to a self-cleaving 2A peptide (Kim et al., 2011). This element 87 was then exchanged with GFP in the FUGW vector by ligation. Subsequently, t...

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The Application of KillerRed for Acute Protein Inactivation in Living Cells

Cited by 4 publications

References 23 publications

Aspergillus nidulans biofilm formation modifies cellular architecture and enables light-activated autophagy

Aspergillus nidulans biofilm formation modifies cellular architecture and enables light-activated autophagy

Light-Activated ROS Production Induces Synaptic Autophagy

Light induced synaptic vesicle autophagy

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