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Investigating neuronal function with optically controllable proteins.

Zhou XX, Pan M, Lin MZ - Front Mol Neurosci (2015)

Bottom Line: For example, neurite extension and synaptogenesis both involve localized and transient activation of cytoskeletal and signaling proteins, allowing changes in microarchitecture to occur rapidly and in a localized manner.These tools have demonstrated versatility in controlling various proteins and thereby cellular functions, and possess enormous potential for future applications in nervous systems.Just as optogenetic control of neuronal firing using opsins has changed how we investigate the function of cellular circuits in vivo, optical control may yet yield another revolution in how we study the circuitry of intracellular signaling in the brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Stanford University Stanford, CA, USA.

ABSTRACT
In the nervous system, protein activities are highly regulated in space and time. This regulation allows for fine modulation of neuronal structure and function during development and adaptive responses. For example, neurite extension and synaptogenesis both involve localized and transient activation of cytoskeletal and signaling proteins, allowing changes in microarchitecture to occur rapidly and in a localized manner. To investigate the role of specific protein regulation events in these processes, methods to optically control the activity of specific proteins have been developed. In this review, we focus on how photosensory domains enable optical control over protein activity and have been used in neuroscience applications. These tools have demonstrated versatility in controlling various proteins and thereby cellular functions, and possess enormous potential for future applications in nervous systems. Just as optogenetic control of neuronal firing using opsins has changed how we investigate the function of cellular circuits in vivo, optical control may yet yield another revolution in how we study the circuitry of intracellular signaling in the brain.

No MeSH data available.


Related in: MedlinePlus

Uses of LOV domains. (A) Photoactivation of PA-Rac1 by blue light has been shown to prevent cocaine-induced increase in spine elongation and, separately, to increase synaptic currents. (B) Blue light-mediated LOV2-PDZ interaction can recruit motor proteins for locomotion of targeted organelles. PDZ-kinesin would produce anterograde movement of the organelle, while PDZ-dynein would produce retrograde movement of the organelle. (C) Lumitoxins are fusions of a channel-blocking peptide toxin, a flexible linker, the LOV2 domain, and a transmembrane helix. When excited by blue light, unfolding of the Jα helix is believed to lengthen the linkage to the membrane, decreasing the local concentration of the toxin near the ion channels. (D) The LightON transcription system fuses a truncated Gal4 DNA-binding domain (DBD) to the Vivid (VVD) LOV domain to produce a construct that homodimerizes upon excitation with blue light and binds to Gal4’s cognate DNA, activating downstream transcription. TAD is a transcription-activating domain. (E) A similar homodimerizing optobiological tool for transcription control. Bacterial transcription factor EL222’s light-sensitive LOV domain and helix-turn-helix (HTH) DBD are conjugated to a nuclear localizing sequence and VP16 transcription activating domain (TAD). The compound protein homodimerizes upon excitation with blue light to bind cognate C120 DNA sequences and activate downstream transcription. (F) Interaction between FKF1 and GIGANTEA (GI) domains can produce blue light-activatable transcription employing the VP16 TAD and the Gal4 DBD. (G) Proteins fused to miniSOG, a singlet oxygen-generating mutant of LOV2, can be destroyed by chromophore-assisted light inactivation (CALI). In the case of a VAMP2-miniSOG fusion, light results in blockade of synaptic transmission.
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Figure 3: Uses of LOV domains. (A) Photoactivation of PA-Rac1 by blue light has been shown to prevent cocaine-induced increase in spine elongation and, separately, to increase synaptic currents. (B) Blue light-mediated LOV2-PDZ interaction can recruit motor proteins for locomotion of targeted organelles. PDZ-kinesin would produce anterograde movement of the organelle, while PDZ-dynein would produce retrograde movement of the organelle. (C) Lumitoxins are fusions of a channel-blocking peptide toxin, a flexible linker, the LOV2 domain, and a transmembrane helix. When excited by blue light, unfolding of the Jα helix is believed to lengthen the linkage to the membrane, decreasing the local concentration of the toxin near the ion channels. (D) The LightON transcription system fuses a truncated Gal4 DNA-binding domain (DBD) to the Vivid (VVD) LOV domain to produce a construct that homodimerizes upon excitation with blue light and binds to Gal4’s cognate DNA, activating downstream transcription. TAD is a transcription-activating domain. (E) A similar homodimerizing optobiological tool for transcription control. Bacterial transcription factor EL222’s light-sensitive LOV domain and helix-turn-helix (HTH) DBD are conjugated to a nuclear localizing sequence and VP16 transcription activating domain (TAD). The compound protein homodimerizes upon excitation with blue light to bind cognate C120 DNA sequences and activate downstream transcription. (F) Interaction between FKF1 and GIGANTEA (GI) domains can produce blue light-activatable transcription employing the VP16 TAD and the Gal4 DBD. (G) Proteins fused to miniSOG, a singlet oxygen-generating mutant of LOV2, can be destroyed by chromophore-assisted light inactivation (CALI). In the case of a VAMP2-miniSOG fusion, light results in blockade of synaptic transmission.

Mentions: Light-oxygen-voltage domains undergo versatile light dependent interactions. In the best-studied LOV domain, LOV2 from Avena sativa phototropin, light-induced thioeither bond formation between a cysteine residue and the FMN chromophore leads to partial unfolding of the C-terminal α-helix (named Jα) from the rest of the LOV2 domain (Harper et al., 2004). This conformation change has been widely used to construct light-controllable proteins in allosteric or steric manners (Lee et al., 2008; Strickland et al., 2008; Moglich et al., 2009; Wu et al., 2009; Ohlendorf et al., 2012). Wu et al. (2009) constructed photoactivatable small GTPase Rac1 (PA-Rac1; Figure 3A), which has since been widely used (Walters et al., 2010; Wang et al., 2010; Yoo et al., 2010; Dietz et al., 2012; Ramel et al., 2013; Schwechter et al., 2013). Wu et al. (2009) screened different linkages of the LOV2 domain to the N-terminus of Rac1 and selected the construct that showed light-mediated protein activation. The resulting construct, PA-Rac1, optically controlled membrane ruffling and migration of animal cells. A crystal structure of PA-Rac1 in the dark state revealed that Rac1’s binding sites for downstream effectors are blocked by close interaction with LOV2. The light-triggered unwinding of Jα likely releases Rac1 from LOV2 interaction, leading to the binding of Rac1 to its effectors and activation of downstream signaling proteins.


Investigating neuronal function with optically controllable proteins.

Zhou XX, Pan M, Lin MZ - Front Mol Neurosci (2015)

Uses of LOV domains. (A) Photoactivation of PA-Rac1 by blue light has been shown to prevent cocaine-induced increase in spine elongation and, separately, to increase synaptic currents. (B) Blue light-mediated LOV2-PDZ interaction can recruit motor proteins for locomotion of targeted organelles. PDZ-kinesin would produce anterograde movement of the organelle, while PDZ-dynein would produce retrograde movement of the organelle. (C) Lumitoxins are fusions of a channel-blocking peptide toxin, a flexible linker, the LOV2 domain, and a transmembrane helix. When excited by blue light, unfolding of the Jα helix is believed to lengthen the linkage to the membrane, decreasing the local concentration of the toxin near the ion channels. (D) The LightON transcription system fuses a truncated Gal4 DNA-binding domain (DBD) to the Vivid (VVD) LOV domain to produce a construct that homodimerizes upon excitation with blue light and binds to Gal4’s cognate DNA, activating downstream transcription. TAD is a transcription-activating domain. (E) A similar homodimerizing optobiological tool for transcription control. Bacterial transcription factor EL222’s light-sensitive LOV domain and helix-turn-helix (HTH) DBD are conjugated to a nuclear localizing sequence and VP16 transcription activating domain (TAD). The compound protein homodimerizes upon excitation with blue light to bind cognate C120 DNA sequences and activate downstream transcription. (F) Interaction between FKF1 and GIGANTEA (GI) domains can produce blue light-activatable transcription employing the VP16 TAD and the Gal4 DBD. (G) Proteins fused to miniSOG, a singlet oxygen-generating mutant of LOV2, can be destroyed by chromophore-assisted light inactivation (CALI). In the case of a VAMP2-miniSOG fusion, light results in blockade of synaptic transmission.
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Related In: Results  -  Collection

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Figure 3: Uses of LOV domains. (A) Photoactivation of PA-Rac1 by blue light has been shown to prevent cocaine-induced increase in spine elongation and, separately, to increase synaptic currents. (B) Blue light-mediated LOV2-PDZ interaction can recruit motor proteins for locomotion of targeted organelles. PDZ-kinesin would produce anterograde movement of the organelle, while PDZ-dynein would produce retrograde movement of the organelle. (C) Lumitoxins are fusions of a channel-blocking peptide toxin, a flexible linker, the LOV2 domain, and a transmembrane helix. When excited by blue light, unfolding of the Jα helix is believed to lengthen the linkage to the membrane, decreasing the local concentration of the toxin near the ion channels. (D) The LightON transcription system fuses a truncated Gal4 DNA-binding domain (DBD) to the Vivid (VVD) LOV domain to produce a construct that homodimerizes upon excitation with blue light and binds to Gal4’s cognate DNA, activating downstream transcription. TAD is a transcription-activating domain. (E) A similar homodimerizing optobiological tool for transcription control. Bacterial transcription factor EL222’s light-sensitive LOV domain and helix-turn-helix (HTH) DBD are conjugated to a nuclear localizing sequence and VP16 transcription activating domain (TAD). The compound protein homodimerizes upon excitation with blue light to bind cognate C120 DNA sequences and activate downstream transcription. (F) Interaction between FKF1 and GIGANTEA (GI) domains can produce blue light-activatable transcription employing the VP16 TAD and the Gal4 DBD. (G) Proteins fused to miniSOG, a singlet oxygen-generating mutant of LOV2, can be destroyed by chromophore-assisted light inactivation (CALI). In the case of a VAMP2-miniSOG fusion, light results in blockade of synaptic transmission.
Mentions: Light-oxygen-voltage domains undergo versatile light dependent interactions. In the best-studied LOV domain, LOV2 from Avena sativa phototropin, light-induced thioeither bond formation between a cysteine residue and the FMN chromophore leads to partial unfolding of the C-terminal α-helix (named Jα) from the rest of the LOV2 domain (Harper et al., 2004). This conformation change has been widely used to construct light-controllable proteins in allosteric or steric manners (Lee et al., 2008; Strickland et al., 2008; Moglich et al., 2009; Wu et al., 2009; Ohlendorf et al., 2012). Wu et al. (2009) constructed photoactivatable small GTPase Rac1 (PA-Rac1; Figure 3A), which has since been widely used (Walters et al., 2010; Wang et al., 2010; Yoo et al., 2010; Dietz et al., 2012; Ramel et al., 2013; Schwechter et al., 2013). Wu et al. (2009) screened different linkages of the LOV2 domain to the N-terminus of Rac1 and selected the construct that showed light-mediated protein activation. The resulting construct, PA-Rac1, optically controlled membrane ruffling and migration of animal cells. A crystal structure of PA-Rac1 in the dark state revealed that Rac1’s binding sites for downstream effectors are blocked by close interaction with LOV2. The light-triggered unwinding of Jα likely releases Rac1 from LOV2 interaction, leading to the binding of Rac1 to its effectors and activation of downstream signaling proteins.

Bottom Line: For example, neurite extension and synaptogenesis both involve localized and transient activation of cytoskeletal and signaling proteins, allowing changes in microarchitecture to occur rapidly and in a localized manner.These tools have demonstrated versatility in controlling various proteins and thereby cellular functions, and possess enormous potential for future applications in nervous systems.Just as optogenetic control of neuronal firing using opsins has changed how we investigate the function of cellular circuits in vivo, optical control may yet yield another revolution in how we study the circuitry of intracellular signaling in the brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Stanford University Stanford, CA, USA.

ABSTRACT
In the nervous system, protein activities are highly regulated in space and time. This regulation allows for fine modulation of neuronal structure and function during development and adaptive responses. For example, neurite extension and synaptogenesis both involve localized and transient activation of cytoskeletal and signaling proteins, allowing changes in microarchitecture to occur rapidly and in a localized manner. To investigate the role of specific protein regulation events in these processes, methods to optically control the activity of specific proteins have been developed. In this review, we focus on how photosensory domains enable optical control over protein activity and have been used in neuroscience applications. These tools have demonstrated versatility in controlling various proteins and thereby cellular functions, and possess enormous potential for future applications in nervous systems. Just as optogenetic control of neuronal firing using opsins has changed how we investigate the function of cellular circuits in vivo, optical control may yet yield another revolution in how we study the circuitry of intracellular signaling in the brain.

No MeSH data available.


Related in: MedlinePlus