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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.


Uses of CRY2 domains. For all panels, CRY2 and CIB1 are the full-length domains. PHR is the photolyase homology region of CRY2 (a truncated CRY2) and CIBN is the N terminal portion of CIB1 (a truncated CIB1). (A) Light-sensitive receptor tyrosine kinases. Upon excitation with blue light, PHR homodimerizes to activate downstream components in the Trk signaling pathway. (B) Heterodimerization between PHR and CIBN used to produce light-activated Raf1 for optogenetic control of the Raf1/MEK/ERK pathway in PC12 cells. Membrane localization of Raf1 activated downstream kinases and eventually stimulated neurite growth in PC12 cells. (C) Light-activated phosphatidylinositol 3-kinase (PI3K). CIBN is membrane-localized and PHR is fused to the inter-SH2 domain of p85b, the regulatory subunit of PI3K kinase (iSH). The catalytic p110 component of PI3K was supplied endogenously by the cell expressing these two constructs. Blue light excitation recruited the PHR-iSH-p110 complex to the membrane, where it would produce PIP3 from PIP2. (D) Light-induced heterodimerization between CRY2 and CIBN can assemble a functional protein if an appropriate half of the protein is fused to each of CRY2 and CIBN. Fusing half of cre recombinase to CRY2 and CIBN produces light-dependent loxP site recombination. (E) The light-inducible transcriptional effector (LITE) system allows optical control of transcription or chromatin structure. Transcription activator-like effectors (TALEs) serve as modular DNA-binding domains. TALE fused to PHR comprises one component of LITE. An effector domain (TAD or HED, a histone effector domain) fused to CIB1 comprises the other component of LITE.
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Figure 4: Uses of CRY2 domains. For all panels, CRY2 and CIB1 are the full-length domains. PHR is the photolyase homology region of CRY2 (a truncated CRY2) and CIBN is the N terminal portion of CIB1 (a truncated CIB1). (A) Light-sensitive receptor tyrosine kinases. Upon excitation with blue light, PHR homodimerizes to activate downstream components in the Trk signaling pathway. (B) Heterodimerization between PHR and CIBN used to produce light-activated Raf1 for optogenetic control of the Raf1/MEK/ERK pathway in PC12 cells. Membrane localization of Raf1 activated downstream kinases and eventually stimulated neurite growth in PC12 cells. (C) Light-activated phosphatidylinositol 3-kinase (PI3K). CIBN is membrane-localized and PHR is fused to the inter-SH2 domain of p85b, the regulatory subunit of PI3K kinase (iSH). The catalytic p110 component of PI3K was supplied endogenously by the cell expressing these two constructs. Blue light excitation recruited the PHR-iSH-p110 complex to the membrane, where it would produce PIP3 from PIP2. (D) Light-induced heterodimerization between CRY2 and CIBN can assemble a functional protein if an appropriate half of the protein is fused to each of CRY2 and CIBN. Fusing half of cre recombinase to CRY2 and CIBN produces light-dependent loxP site recombination. (E) The light-inducible transcriptional effector (LITE) system allows optical control of transcription or chromatin structure. Transcription activator-like effectors (TALEs) serve as modular DNA-binding domains. TALE fused to PHR comprises one component of LITE. An effector domain (TAD or HED, a histone effector domain) fused to CIB1 comprises the other component of LITE.

Mentions: Following the observation that light-induced clustering of proteins fused to CRY2 can be applied to activate the small GTPase RhoA (Bugaj et al., 2013), Chang et al. (2014) engineered light activatable TrkB by fusion to CRY2 in neurons (Figure 4A). TrkB belongs to the tropomyosin-related kinase family, which activates through brain-derived neurotrophic factor (BDNF)-mediated homodimerization at the plasma membrane. TrkB mediates multiple downstream signaling pathways and contributes to neuronal survival, neurite outgrowth and synaptic plasticity. Chang et al. (2014) fused the photolyase homology region (CRY2PHR) of CRY2 to the intracellular region of TrkB, such that light-induced oligomerization of the CRY2PHR domain induced the dimerization of TrkB and activated the protein. This photoactivatable TrkB, named optoTrkB, was used in primary neurons to induce the formation and growth of filopodia, the actin-structure linked to dendrite creation when new synapses are formed in the brain (Maletic-Savatic et al., 1999). OptoTrkB features rapid, transient, and localized activation of the signaling pathway, and only requires expression of a single protein chain. CRY2 oligomerization was also used to induce clustering of the cytoplasmic domain of a different receptor tyrosine kinase, the fibroblast growth factor receptor FGFR1, to trigger PI3K activation in non-neuronal cells (Kim et al., 2014). Omission of the extracellular domain prevented activation by endogenous ligands.


Investigating neuronal function with optically controllable proteins.

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

Uses of CRY2 domains. For all panels, CRY2 and CIB1 are the full-length domains. PHR is the photolyase homology region of CRY2 (a truncated CRY2) and CIBN is the N terminal portion of CIB1 (a truncated CIB1). (A) Light-sensitive receptor tyrosine kinases. Upon excitation with blue light, PHR homodimerizes to activate downstream components in the Trk signaling pathway. (B) Heterodimerization between PHR and CIBN used to produce light-activated Raf1 for optogenetic control of the Raf1/MEK/ERK pathway in PC12 cells. Membrane localization of Raf1 activated downstream kinases and eventually stimulated neurite growth in PC12 cells. (C) Light-activated phosphatidylinositol 3-kinase (PI3K). CIBN is membrane-localized and PHR is fused to the inter-SH2 domain of p85b, the regulatory subunit of PI3K kinase (iSH). The catalytic p110 component of PI3K was supplied endogenously by the cell expressing these two constructs. Blue light excitation recruited the PHR-iSH-p110 complex to the membrane, where it would produce PIP3 from PIP2. (D) Light-induced heterodimerization between CRY2 and CIBN can assemble a functional protein if an appropriate half of the protein is fused to each of CRY2 and CIBN. Fusing half of cre recombinase to CRY2 and CIBN produces light-dependent loxP site recombination. (E) The light-inducible transcriptional effector (LITE) system allows optical control of transcription or chromatin structure. Transcription activator-like effectors (TALEs) serve as modular DNA-binding domains. TALE fused to PHR comprises one component of LITE. An effector domain (TAD or HED, a histone effector domain) fused to CIB1 comprises the other component of LITE.
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Figure 4: Uses of CRY2 domains. For all panels, CRY2 and CIB1 are the full-length domains. PHR is the photolyase homology region of CRY2 (a truncated CRY2) and CIBN is the N terminal portion of CIB1 (a truncated CIB1). (A) Light-sensitive receptor tyrosine kinases. Upon excitation with blue light, PHR homodimerizes to activate downstream components in the Trk signaling pathway. (B) Heterodimerization between PHR and CIBN used to produce light-activated Raf1 for optogenetic control of the Raf1/MEK/ERK pathway in PC12 cells. Membrane localization of Raf1 activated downstream kinases and eventually stimulated neurite growth in PC12 cells. (C) Light-activated phosphatidylinositol 3-kinase (PI3K). CIBN is membrane-localized and PHR is fused to the inter-SH2 domain of p85b, the regulatory subunit of PI3K kinase (iSH). The catalytic p110 component of PI3K was supplied endogenously by the cell expressing these two constructs. Blue light excitation recruited the PHR-iSH-p110 complex to the membrane, where it would produce PIP3 from PIP2. (D) Light-induced heterodimerization between CRY2 and CIBN can assemble a functional protein if an appropriate half of the protein is fused to each of CRY2 and CIBN. Fusing half of cre recombinase to CRY2 and CIBN produces light-dependent loxP site recombination. (E) The light-inducible transcriptional effector (LITE) system allows optical control of transcription or chromatin structure. Transcription activator-like effectors (TALEs) serve as modular DNA-binding domains. TALE fused to PHR comprises one component of LITE. An effector domain (TAD or HED, a histone effector domain) fused to CIB1 comprises the other component of LITE.
Mentions: Following the observation that light-induced clustering of proteins fused to CRY2 can be applied to activate the small GTPase RhoA (Bugaj et al., 2013), Chang et al. (2014) engineered light activatable TrkB by fusion to CRY2 in neurons (Figure 4A). TrkB belongs to the tropomyosin-related kinase family, which activates through brain-derived neurotrophic factor (BDNF)-mediated homodimerization at the plasma membrane. TrkB mediates multiple downstream signaling pathways and contributes to neuronal survival, neurite outgrowth and synaptic plasticity. Chang et al. (2014) fused the photolyase homology region (CRY2PHR) of CRY2 to the intracellular region of TrkB, such that light-induced oligomerization of the CRY2PHR domain induced the dimerization of TrkB and activated the protein. This photoactivatable TrkB, named optoTrkB, was used in primary neurons to induce the formation and growth of filopodia, the actin-structure linked to dendrite creation when new synapses are formed in the brain (Maletic-Savatic et al., 1999). OptoTrkB features rapid, transient, and localized activation of the signaling pathway, and only requires expression of a single protein chain. CRY2 oligomerization was also used to induce clustering of the cytoplasmic domain of a different receptor tyrosine kinase, the fibroblast growth factor receptor FGFR1, to trigger PI3K activation in non-neuronal cells (Kim et al., 2014). Omission of the extracellular domain prevented activation by endogenous ligands.

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.