Optogenetic Reconstitution: Light-Induced Assembly of Protein Complexes and Simultaneous Visualization of Their Intracellular Functions
Dynamic behaviors of cells require optimized functions and regulations of protein complexes within a cell. In vitro reconstitution is a powerful method to dissect the molecular functions and structure of reconstituted protein complexes. However, owing to the difficulties associated with in vitro reconstitution of complicated supra-molecular complexes, such as the mitotic spindle and cortical force-generating machinery, key molecular features underlying their functions and regulations are not fully understood. Recently, several light-inducible dimerization tools have been developed to control localization of target proteins within a cell in a spatiotemporally controlled manner. In this chapter, I have summarized the recent progress in optogenetic tools and fluorescent proteins/probes and have determined strategies to achieve light-induced in cell reconstitution of protein complexes coupled with visualization of its downstream events. Moreover, I have discussed the advantages and potential of the optogenetic reconstitution system in diverse cell and developmental biology fields.
KeywordsLight-induced heterodimerization In cell reconstitution system Cortical force-generating machinery
To understand the basis of life, it is critical to visualize the dynamic behaviors of molecules within a cell. Since the discovery of green fluorescent protein (GFP) (Shimomura 1979), multiple fluorescent proteins or dyes have been developed (Rodriguez et al. 2017), and it has become possible to simultaneously visualize intracellular dynamics of multiple proteins in living cells. In addition, by combining recently developed genome editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR) (Ran et al. 2013), it is possible to monitor the dynamic behaviors of endogenous proteins even in animal cells, including human cultured cells.
In the last few decades, many genetic approaches, such as mutant screens and gene disruption, have been used in combination with live cell imaging to identify genes that code key proteins required for cellular functions (Hartwell 1978; Yanagida 2014; Goshima et al. 2007; Neumann et al. 2010). In addition, biochemical and proteomic approaches have defined functional protein complexes that underlie complicated cellular functions (Hutchins et al. 2010; Cheeseman et al. 2004; Obuse et al. 2004). Once key molecules or complexes are identified, biophysical and structural studies are performed to reveal the detailed molecular properties sufficient for their functions (Cheeseman et al. 2006; Dimitrova et al. 2016; Zhang et al. 2017; McKenney et al. 2014; Schlager et al. 2014). Furthermore, by combining mathematical simulations (Kimura and Onami 2005), nanodevices (Thery et al. 2005), and synthetic approaches (Good et al. 2013; Laan et al. 2012; Nguyen et al. 2014), novel molecular features that underlie complicated dynamic cellular events have been uncovered. However, it is still difficult to fully reconstitute macro-molecular complexes, such as the mitotic spindle, which consists of hundreds of proteins (Goshima et al. 2007; Sauer et al. 2005), and to manipulate their functions under physiological condition.
Recently, several light-induced tools have been developed to manipulate intracellular localization of target proteins with a spatiotemporal precision in living cells (Levskaya et al. 2009; Kennedy et al. 2010; Strickland et al. 2012; Guntas et al. 2015). In combination with other techniques, this optogenetic technology has great potential to reconstitute functional protein complexes, which are otherwise difficult to reconstitute in vitro, and to directly assess their functions within a cell. Here, I have presented an optogenetic reconstitution system to achieve light-induced in cell reconstitution of protein complexes coupled with visualization and manipulation of their cellular functions.
6.2 Light-Induced Heterodimerization Tools
These photoactivatable proteins and chemicals exhibit different characters and requirement for activation (Fig. 6.1; reviewed in (Zhang and Cui 2015)). Therefore, these characters must be considered for the experimental design. For example, to locally assemble protein complexes at the plasma membrane, optogenetic dimerizers with slow dissociation rate of target proteins may diffuse on the membrane following membrane targeting and fail to assemble the protein complexes at the specific site on the membrane. In contrast, to stably recruit signaling molecules on a specific organelle, such as kinetochore or centrosomes, optogenetic tools with slow dissociation rate would be more appropriate to generate robust cell signaling. Finally, simultaneous manipulation of two different target proteins is now possible by using different light-responsible tools such as blue- and near-infrared light-driven dimerizers (Kaberniuk et al. 2016).
6.3 Visualization Tools Compatible with Optogenetic Manipulation
For simultaneous visualization of target protein and its downstream targets or events in response to light illumination, exciting fluorescent proteins or dyes without activating light-responsible proteins is required. For instance, when blue light is used to activate photoactivatable proteins, red or far-red light must be selected for visualization because shorter UV light also activates blue light-responsible elements (Fig. 6.2). To monitor the responses of the target protein and its downstream effectors following light illumination, at least two fluorescent proteins or dyes are required. Importantly, several far-red or near-infrared fluorescent proteins or cell permeable fluoregenic probes/dyes have been recently developed (Fig. 6.1), such as near-infrared fluorescent protein (iRFP) (Filonov et al. 2011), infrared fluorescent protein mutant (IFP2.0) (Yu et al. 2014), silicon–rhodamine-based fluorophore (SiR)-647 (Lukinavicius et al. 2014), SiR-700 (Lukinavicius et al. 2016), and HaloTag STELLA Fluor™ (http://www.promega.co.jp/halotag_imaging/). These fluoregenic probes/dyes are used in combination with SNAP-tag or HaloTag, or these dyes are directly conjugated with chemicals that bind to DNA, tubulin, or actin to visualize chromosomes (SiR–DNA) (Lukinavicius et al. 2015), tubulin (SiR–tubulin) (Lukinavicius et al. 2014), and actin cytoskeleton (SiR–actin) (https://spirochrome.com/). Thus, by combining these novel tools, simultaneous visualization of its downstream targets is now possible in parallel with light-induced manipulation of photoactivatable proteins. In contrast, when photocaged chemical dimerizer or near-infrared-activated proteins are used, GFP and other red fluorescent proteins or dyes can be used for multicolor imaging to visualize its downstream events and phenotypes.
6.4 Light-Induced Assembly/Reconstitution of Force-Generating Complexes During Mitosis
Previous studies have identified key macro-molecular complexes that play critical roles in diverse cellular functions (Cheeseman et al. 2004; Obuse et al. 2004; Kiyomitsu and Cheeseman 2012; Goshima et al. 2008). Hence, it is definitely important to investigate their precise functions and structures in in vitro reconstitution systems. However, in many cases, it is difficult to fully reconstitute these macro-molecular complexes and their targets in vitro. Recent innovation of optogenetic tools has led to the development of in vivo reconstitution systems to understand the functional properties and structure of reconstituted sub-complexes within a cell. Light-induced targeting of proteins and their mutants is a powerful method to define both sufficiency and requirement of their molecular functions under more physiological conditions. Importantly, in cell reconstitution can be also used to manipulate cellular dynamics in a spatially and temporally controlled manner. For example, temporal reconstitution of force-generating sub-complex at specific cortical region during mitosis can induce spindle orientation or displacement and convert cell division mode from symmetric to asymmetric or vice versa in symmetrically or asymmetrically dividing cells. Such manipulation may lead to unexpected phenotypes and open new directions in the field of cell and developmental biology. Because light-based strategies have high penetrance, low toxicity, and high spatial and temporal precision, these characters and convenience are great advantages to manipulate target molecules in complicated in vivo situations, such as tissues in multicellular organisms.
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