Journal of Molecular Biology
ReviewApplications of Optobiology in Intact Cells and Multicellular Organisms
Graphical abstract
Introduction
Despite the variety of cellular processes occurring during embryonic development, only a handful of signaling pathways, namely, the Notch, Wnt/β-catenin, Sonic Hedgehog, transforming growth factor β, bone morphogenetic protein, and fibroblast growth factor (FGF) signaling pathways, are repeatedly used to regulate early embryonic development and differentiation [1]. How can a limited number of signaling pathways regulate such diverse cell behavior? Mounting evidence suggests that spatial and temporal regulation of these signaling pathways is crucial to cell fate determination during development. The same pathways can be turned on or off at different times and locations to regulate distinct cell functions.
Circadian oscillators play a critical role in coordinating temporal kinetics of gene expression and signal transduction, responding to signals with a periodicity of 10–24 h. In contrast, oscillation with a shorter periodicity, referred to as ultradian oscillation, determines many biological events at shorter timescales [2]. Temporal coordination of ultradian oscillation often correlates with the formation of spatial patterns of tissue structures during development. For instance, cyclical activation of the Notch pathway is crucial for the formation of a “salt and pepper” pattern of ciliated cells during ciliogenesis [3]. Similarly, oscillation of Notch activation is required for the formation of somites, the precursors to a variety of segmental structures such as the peripheral spinal nerves, vertebrae, axial muscles, and early blood vessels [4], [5]. The period of the ultradian oscillations in somitogenesis varies from 20 min in zebrafish to 4–6 h in humans [6]. Besides oscillation, variations in the duration of signals can also lead to distinct cell fates. For instance, transient and sustained activation of the extracellular signal-regulated kinase (ERK) pathway leads to PC12 cell proliferation and differentiation, respectively [7], [8]. In pairs of genetically identical MCF-10A sister cells treated with EGF, cells entering S phase experience sustained ERK activity, whereas the lagging sister cells exhibit pulsatile ERK activity [9]. In cultured rat hippocampal neurons, acute delivery of brain-derived neurotrophic factor (BDNF) elicits transient TrkB signaling and promotes neurite elongation, whereas gradual delivery of BDNF elicits sustained TrkB signaling and promotes neurite branching [10]. Interestingly, high-frequency neuronal stimulation can convert a transient BDNF–TrkB activity into a sustained one [11]. Thus, spatiotemporal coordination of gene expression and signal transduction provides a fundamental molecular mechanism to regulate cell fates.
Both biochemical feedback and molecular stability influence signal oscillation and duration. For instance, negative feedback between the Hes1 protein and its mRNA leads to oscillatory or sustained Hes1 protein expression, resulting in the proliferation and differentiation of neural progenitor cells, respectively [12], [13]. Negative feedback loops in the mitogen-activated protein kinase pathway can lead to sustained oscillation in kinase activity [14], [15], [16]. In the case of molecular stability, an increase in the half-life of Hes1 mRNA leads to an extension of the Hes1 protein oscillation period [17]. Unraveling such intricate signaling mechanisms demands tools to probe these dynamic regulatory processes with spatiotemporal precision.
Our current understanding of gene expression and signaling mechanisms has primarily relied on conventional genetic and pharmacological approaches. Commonly used genetic approaches such as gain- and loss-of-function mutagenesis often lead to constitutive activation or inactivation of signaling activity. To address this issue, alternative chemical and genetic tools have been developed. For instance, several inducible systems are available to activate or repress protein expression in yeast [18]. Chemical regulation of gene expression has been achieved using promoters that respond to molecules such as galactose, methionine, and copper. Alternatively, engineered gene regulatory systems that employ estrogen or doxycycline can be utilized for orthogonal gene transcription control. While these inducible systems have greatly facilitated the control of gene expression, their off-target effects, ineffective delivery, and limited reversibility have restricted their use in live cells and multicellular organisms [19], [20], [21]. Moreover, it remains challenging for these conventional approaches to precisely perturb gene expression and signal transduction at a resolution that matches the spatial and temporal scales of endogenous developmental events, which can occur in minutes within space of several microns.
Light can be confined to a sub-micron space and precisely tuned in time, with minimal invasiveness to biological organisms. These advantages initially enabled the interrogation of neuronal firing by light-sensitive synthetic ion channels [22] and channelrhodopsin [23], [24] in genetically dissected circuits, which led to the coining of the term “optogenetics” [25]. By modulating neuronal firing, optogenetics has provided a new way to investigate neuronal circuits and to establish causal relationships between brain activity and health and disease [26], [27]. These powerful tools not only help researchers in basic science understand signaling circuits in brain functions such as learning and memory, but also show promising preclinical and clinical potential for rewiring neuronal circuits to amplify or override specific neuronal phenotypes. Indeed, optogenetics has been extended to basic and preclinical research on a wide spectrum of topics such as Parkinson's disease [28], sleep [29], cardiac function [30], [31], neuropsychiatric diseases [32], epilepsy [33], and sight-restoring therapy [34], [35]. This new modality provided by light has been transforming neuroscience research, as evidenced by its explosive growth in recent years.
The power of light to interrogate cell functions, however, is not limited to the modulation of the membrane potential of excitable cells [36]. Light has also been used to control a variety of signaling processes [37], [38], [39], [40], [41], [42], [43]. As pointed out earlier by Kim and Lin [44], we believe that it is more appropriate to refer to this broader emerging field as optobiology, in which light enables new modalities to study biological processes in intact cells and multicellular organisms, including in situations where genetic dissection is not essential. In optobiology, light serves not only as an observational tool to follow biological events but also as a manipulative device to modulate activation states of signaling components with high spatial and temporal resolution.
Over the past decade, a portfolio of optobiological tools has emerged to regulate protein–protein interactions in live cells. The core components of optobiology are photoactivatable proteins, which respond to visible or infrared (IR) light stimulation by changing their conformations [45]. These light-triggered conformational changes can induce inter- or intra-molecular interactions. When linked to specific signaling components, photoactivatable proteins allow for light-controlled activation or inactivation of target signal transduction. To date, light has been used to control cellular processes such as gene transcription, translation, protein degradation, differentiation, apoptosis, and migration, as discussed in the following sections. Photocaged small molecules or amino acids also enable light-mediated control of cell functions, a process often referred to as photopharmacological or optochemical control.
Recent optobiological work has expanded its host system from cultured cells to multicellular organisms. Compared to cultured cells, multicellular organisms require better quality control in genetic and protein engineering, as well as material and light delivery. In this review, we outline recent advances in optobiology, focusing primarily on accomplishments made in the past 3 years. For earlier work, interested readers are encouraged to refer to previous reviews [46], [47], [48], [49], [50], [51], [52], [53], [54]. In the following sections, we describe optobiological tools with new photophysical properties, update new optobiological applications in intact cells and multicellular organisms, discuss challenges in optobiology, and present our outlook. Because many reviews have covered optogenetic control of neuronal activity based on channelrhodopsin and their derivatives [55], [56], [57], [58], [59], [60], [61], we will not repeat that aspect of optobiology in this work.
Section snippets
New features of second-generation optobiological tools
Optobiology offers a repertoire of light responsive modules. Because the commonly used phytochrome, cryptochrome, and light-oxygen-voltage (LOV) domains have been extensively reviewed [45], [46], [47], [48], [49], we only list new members of photoactivatable proteins discovered within the past 3 years (Table 1). We summarize key characteristics of these proteins, including their sizes and photophysical properties, namely, the excitation wavelength, association and dissociation kinetics, and
Optobiological control of cellular functions
Parallel to the development of new photoactivatable proteins, optobiology continues finding new applications in the modulation of cellular and organismal functionality though light-induced uncaging, protein translocation, modulation of concentration and avidity, and allosteric control (Fig. 2). In this section, we summarize recent advances in optobiological control of cellular functions. We use subcellular locations (plasma membrane, cytoplasm, and nucleus) to group these cases, highlighting
Challenges and emerging new technologies
A major challenge in optobiology, both in basic sciences and in clinical research, is light delivery. Most photoactivatable proteins respond to visible light. The penetration depth for red light in biological tissues is around 0.2–2 mm [154]. Furthermore, as the light wavelength decreases, its penetration depth is reduced, severely limiting the use of visible light-responsive photoactivatable modules in deep tissues. Most in vivo optobiological applications have required the insertion of optical
Outlook
Using photoactivatable moieties derived from nature, optobiology utilizes photoactivatable molecules in conjunction with proteins of interest to genetically engineer live cell systems. Parallel to the emerging field of synthetic biology, optobiology offers attractive approaches to customize cell and tissue responses. Orthogonal and multiplexed control of signaling circuits would help delineate endogenous signaling mechanisms. In addition, novel functionality can potentially be created by
Acknowledgments
This work was supported by the University of Illinois at Urbana–Champaign (K.Z.). We thank L. Hanson (UC Berkeley) for help with figure design.
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