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ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 1621–1629 doi:10.1242/jcs.144527

COMMENTARY

ARTICLE SERIES: IMAGING

Chromophore-assisted laser inactivation – towards a spatiotemporal–functional analysis of proteins, and the ablation of chromatin, organelle and cell function Yukimi Sano1, Wataru Watanabe2 and Sachihiro Matsunaga1,*

Chromophore-assisted laser or light inactivation (CALI) has been employed as a promising technique to achieve spatiotemporal knockdown or loss-of-function of target molecules in situ. CALI is performed using photosensitizers as generators of reactive oxygen species (ROS). There are two CALI approaches that use either transgenic tags with chemical photosensitizers, or genetically encoded fluorescent protein fusions. Using spatially restricted microscopy illumination, CALI can address questions regarding, for example, protein isoforms, subcellular localization or phase-specific analyses of multifunctional proteins that other knockdown approaches, such as RNA interference or treatment with chemicals, cannot. Furthermore, rescue experiments can clarify the phenotypic capabilities of CALI after the depletion of endogenous targets. CALI can also provide information about individual events that are involved in the function of a target protein and highlight them in multifactorial events. Beyond functional analysis of proteins, CALI of nuclear proteins can be performed to induce cell cycle arrest, chromatin- or locus-specific DNA damage. Even at organelle level – such as in mitochondria, the plasma membrane or lysosomes – CALI can trigger cell death. Moreover, CALI has emerged as an optogenetic tool to switch off signaling pathways, including the optical depletion of individual neurons. In this Commentary, we review recent applications of CALI and discuss the utility and effective use of CALI to address open questions in cell biology. KEY WORDS: Chromophore, Photosensitizer, Spatiotemporal inactivation, Reactive oxygen species, Imaging

Introduction

Chromophore-assisted laser or light inactivation (CALI) is a powerful technique for microscopic investigation of the functions of a protein of interest in situ and in a spatiotemporally regulated manner. CALI enables the selective inactivation of proteins that are tagged to chromophores by using antibodies (Jay, 1988; Liao, 1994). Since it was first used in the microscopy (Diamond et al., 1993), CALI has been applied in a variety of experimental settings and proved to be a promising tool to dissect a number of complex phenomena in cell biology (reviewed in Hoffman-Kim et al., 2007; Jacobson et al., 2008). 1 Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. 2 Department of Electrical and Electronic Engineering, College of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan.

*Author for correspondence ([email protected])

Through absorption of the laser light, chromophores become excited, react with oxygen and generate reactive oxygen species (ROS) that, in turn, cause damage to DNA, RNA, lipids and proteins. The principle of CALI is straightforward in that photosensitizers are used to harness ROS to eliminate targets of interest. ROS are unique molecules that can act as ‘double-edged swords’, as they are highly oxidative but have a short lifetime; therefore, they only affect the molecules that are in close proximity to their origins. So, by using a focused laser beam and controlling the generation of ROS within a specific subcellular area with micrometer to sub-micrometer accuracy, the spatiotemporal inactivation of targets can be achieved. The first report of CALI described the inactivation of alkaline phosphatase and b-galactosidase by using malachite-greenconjugated antibodies (Jay, 1988). More recently, the CALI tool box has rapidly improved, mainly through the introduction of new photosensitizers (see Box 1) or a refinement of the methodology itself. In this Commentary, we summarize a number of recent investigations that have employed CALI and discuss their findings. In particular, we emphasize effective applications of CALI that make full use of its advantages. By reflecting on previous research achievements, we also provide perspectives for the potential use of CALI in order to address outstanding questions in cellular biology that, hopefully, will inspire interesting future applications. CALI with transgenically encoded tags

The original CALI technique is based on the use of the dye Malachite Green that was coupled to proteins of interest through specific antibodies (Table 1) (Jay and Keshishian, 1990; Mu¨ller et al., 1996; Schro¨der et al., 1996; Schro¨der et al., 1999; Sakurai et al., 2002). This method is still used to date – especially in the field of neuroscience – and a number of studies have examined the roles of various factors in growth cone dynamics (Abe et al., 2008; Higurashi et al., 2012; Iketani et al., 2013; Iketani et al., 2009). However, although these examples clearly demonstrate the practicality of this approach, there are also drawbacks to an antibody-based method, including the necessity to microinject the antibody into the cell and the risk that antibody binding interferes with protein function (Keppler and Ellenberg, 2009). As an alternative to Malachite Green, the fluorescent dye fluorescein isothiocyanate (FITC) and its derivatives have been introduced for the spatiotemporal inactivation of targets, and this technique is often referred to as FALI (fluorophore-assisted light inactivation) (Beck et al., 2002). Fluorescein is 50-times more efficient in generating ROS than Malachite Green (Surrey et al., 1998). Recently, FALI was applied to a screening method in developing mouse brain (Sato et al., 2011). In order to establish an even more precise inactivation of target molecules, several 1621

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ABSTRACT

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Box 1. Photosensitizers Photosensitizers are substances that induce particular reactions or light emission by transmitting energy from the absorbed light to another molecule. For the most part, photosensitizers are activated from the singlet-ground stage into the singlet-activated stage by light absorption, and then quickly enter the triplet-activated state by state transition. A collision between a photosensitizer in the tripletactivated stage and another molecule causes the exchange of energy. The photosensitizer returns to the ground stage and the other molecule enters the triplet-activated stage, which is accompanied by specific reactions – for instance, fluorescent emissions or the generation of ROS at wavelengths that are unique to each molecule. Types of photosensitization Photosensitizers can undergo two types of reaction, whose procedures and final products differ from each other. The type-I reaction involves electron or hydrogen transfer with a reducing substrate. Light irradiation induces electron transfer from one molecule – that becomes oxidized – to a photosensitizer. The photosensitizer, in turn, interacts with oxygen, resulting in the formation of a superoxide anion radical, e.g. O2?2, HO?. The type-II reaction involves the interaction with oxygen. The energy transfer occurs directly to oxygen from a photosensitizer excited trough light absorption, and singlet oxygen (also known as dioxigen) is formed.

approaches have been developed that are based on this potent dye. Instead of using an antibody-based approach for its targeting, fluorescein is mainly used in combination with transgenically encoded tags (Griffin et al., 1998). These approaches utilize the membrane-permeable property of the fluorescein derivative FlAsH-EDT2, which is synthesized from 49,59-bis(1,3,2dithioarsolan-2-yl) fluorescein (Marek and Davis, 2002) (Table 1). FlAsH-EDT2 in its native form is non-fluorescent, but becomes fluorescent upon binding to an exogenous motif that consists of tetra-cysteine residues. However, its use in CALI might be problematic because nonspecific labeling of endogenous cysteine-rich proteins can take place (Stroffekova et al., 2001) unless the target protein is expressed at a high level. Another synthetic fluorophore, red arsenical helix (ReAsH), that also binds to the tetra-cysteine motif, has even greater efficiency in generating ROS (Tour et al., 2003). Although ReAsH can be excited at a longer wavelength – which can reduce the probability of damage caused by light irradiation itself – it still has the major drawback that undesired inactivation effects arise from nonspecific binding (Hearps et al., 2007). This defect can be ameliorated by increasing the affinity between the bi-arsenicaltetracysteine motifs and fluorescent dyes as well as the

concentration of dithiols such as 1,2-ethanedithiol (EDT) that are used in the washing steps (Martin et al., 2005). The fluorescent dyes mentioned above bind to tetracysteine motifs through thiol groups. Therefore, excessive nonspecific binding of these dyes to endogenous proteins can be washed off by using dithiols, which reduces the background. More refined protein tags for the selective labeling of intracellular proteins have recently been developed based on advances in synthetic chemistry (Jing and Cornish, 2011; Uchinomiya et al., 2014). For example, SNAP-tag is a peptide of 182 amino acids that is derived from the human O6-alkylguanineDNA alkyltransferase. By appending the SNAP-tag to the target in advance through gene engineering, the administration of O6benzylguanine bearing a fluorescent dye results in the formation of a tight connection between the protein of interest and the fluorescent dye through the SNAP-tag (Keppler et al., 2002) (Fig. 1). Using such a SNAP-tag CALI approach, the inactivation of both a-tubulin and c-tubulin in mammalian cells could be achieved (Keppler and Ellenberg, 2009). Here, inactivation of atubulin lead to mitotic arrest that is accompanied by aberrant morphology of the mitotic spindle at metaphase. In contrast, inactivation of c-tubulin disrupted nucleation of microtubules drastically and hampered the growth of microtubules emanating from only those centrosomes that had been exposed to light irradiation. Overall, these results not only confirmed a highly specified restriction of localization as well as activation of SNAPtag-based CALI, but also allowed to identify specific roles of the tubulin isoforms in intracellular molecular dynamics. Eosin is a photosensitizing chromophore that generates 11times as much singlet oxygen as fluorescein. Moreover, when compared with fluorescein-labeled systems, the eosin-labeled system exhibits a fivefold greater efficiency in ROS generation, which was measured using anthracene-9,10-dipropionic acid (Takemoto et al., 2013). Eosin is also used in combination with a protein tag, HaloTag7, a haloalkane dehalogenase mutant (Ohana et al., 2009), which has been fused to protein kinase Cc (PKCc). Upon the chemical stimulation, PKCc translocates to the plasma membrane. Membrane-permeable eosin, diAc-eosin-AM, was administrated as a HaloTag ligand to HeLa cells expressing the HaloTag7–PKCc fusion protein. Upon irradiation with intense green light followed by chemical stimulation, HaloTag– PKCc failed to translocate to the plasma membrane, indicating that CALI can be used to inactivate PKCc (Takemoto et al., 2011). HaloTag/eosin-based CALI has been also used to inactivate the mitotic kinase Aurora B, resulting in cell-division arrest and multinuclear formation, consistent with results of analyses of Aurora B knockdown (Takemoto et al., 2011). It should be noted that the laser power required for the CALI-based HaloTag/eosin-labelling system is 85-times lower than that for

Table 1. Properties of photosensitizers for CALI Photosensitizer

Molecular classification

Excitation (nm)

Emission (nm)

References

Malachite Green Fluorescein FlAsH-EDT2 ReAsH-EDT2 EGFP EYFP KillerRed SuperNova MiniSOG

Triarylmethane dye Xanthene derivative Xanthene derivative Xanthene derivative Protein (239 a.a.) Protein (239 a.a.) Protein (273 a.a.) Protein (273 a.a.) Protein (106 a.a.)

620 (pulsed laser) 488 508 540 488 514 585 579 448, 473

– 508 528 635 507 527 610 610 500, 528

Jay, 1988 Surrey et al., 1998 Marek and Davis, 2002 Tour et al., 2003 Rajfur et al., 2002 McLean et al., 2009 Bulina et al., 2006a Takemoto et al., 2013 Qi et al., 2012

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Target protein

Tag (SNAP-tag, Halo-tag)

Dye (Fluorescein, Eosin)

Protein photsensitizer (EGFP, KillerRed, SuperNova, MiniSOG)

Ligand Target protein

Membrane-permeable ligand is administered to target-protein–Tag fusion protein

Tag links fluorescent dye with target protein

Genetically encoded protein photosensitizer is fused to target protein

Light irradiation at appropriate wavelength

Light irradiation at appropriate wavelength

Target inactivation

Target inactivation

the SNAP-tag system, implying a considerably lower risk of nonspecific photodamage. This might be beneficial when investigating photosensitive cellular processes. CALI with EGFP or EYFP

Green fluorescent protein (GFP) and its variants have a chromophore that consists of three amino acid residues within its b-barrel structure (Cody et al., 1993; Tsien, 1998). ROS formed following the chromophore being hit by light induce the aggregation of proteins that is mediated by crosslinking rather than breaking a protein backbone (McLean et al., 2009) (Fig. 1). The first application of CALI with enhanced GFP (EGFP) was to inactivate a-actinin in fibroblasts, which resulted in the detachment of stress fibers from focal adhesions (Rajfur et al., 2002). In contrast to microinjection of antibodies, the most obvious benefit of using genetically encodable photosensitizers is the precise subcellular localization of the fusion protein. CALI with EGFP is particularly useful when wanting to address the effect of cell division and cytokinesis in the embryo, because specific cells within the embryo can be accurately irradiated by the laser. Thus, to determine whether the nonmuscle myosin II (MyoII) cable in Drosophila melanogaster corrects cell mixing through a barrier of cortical tension, CALI was used to specifically inhibit the MyoII regulatory light chain (MRLC) fused to EGFP in the boundary cables, and combined with live imaging of early embryos (Monier et al., 2010). Here, light irradiation of the boundary results in inactivation of MRLC so that dividing cells are no longer pushed back, resulting in compartmental cell mixing. In contrast, if one side of the ingressing furrow in dividing cells is irradiated with light, the cell

fails to divide because MyoII also plays an important role in this process. These results highlight that CALI-mediated protein inactivation can be highly controlled at the subcellular level in live embryos, and used, for instance, to provide insights into localization-specific roles of MyoII in the cell. EGFP variants, including enhanced yellow protein (EYFP) and enhanced cyan protein (ECFP), have also been used in CALI. Their efficiency with regard to use in CALI follows this order: FLAsH.EGFP.EYFP.ECFP (Table 1) (McLean et al., 2009). The main drawback of CALI together with enhanced fluorescent proteins is their relatively low efficiency in generating ROS, as well as the requirement for high-power light irradiation. To address this problem, two-photon- and multiphoton-excitation by using a femtosecond laser has been applied to CALI (Tanabe et al., 2005; Shimada et al., 2005). A nonlinear dependence of the signal intensity on the tightly focused intensity of the excitation light can be achieved by using femtosecond lasers that emit ultrashort optical pulses of a duration below 1 picosecond, i.e. in the order of femtoseconds. This allows for limited interaction regions together with a reduction of detrimental interactions outside the region of focus, such as photo-induced damage and photobleaching (Watanabe et al., 2004; Watanabe et al., 2007; Watanabe et al., 2008; Higashi et al., 2013). Two-photon excitation generates ROS with a high degree of spatial specificity and has been successfully utilized in a number of studies. For instance, CALI-mediated inactivation of connexin 43 – a main component of gap junctions between neighboring cells – fused to EGFP induces a decrease in the junctional current (Tanabe et al., 2005). CALI with femtosecond laser makes it possible to perform time-lapse observations of subcellular organelle dynamics 1623

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Fig. 1. Schematic overview of CALI using genetically encoded tags or protein sensitizers fused to a target protein. CALI is used for the spatiotemporal inactivation of targeted proteins that are microscopically controlled through light irradiation of specific subcellular regions. CALI is mainly performed by using two methods. For one method, the target protein and tag are expressed as a fusion protein. A membrane-permeable photosensitizer is added at a later point (left). For the other method, the target protein and the photosensitizer are expressed as a fusion protein (right). In both cases, following chromophore damage ROS are generated that can then inactivate target proteins in close vicinity.

(Shimada et al., 2005). Here, circularization, and arrest of mitochondrial fusion and fission in a single mitochondrion were observed in response to mitochondrial membrane depolarization caused by inactivation of cytochrome c, when CALI with YFP tagged to sequence that targets cytochrome c oxidase was carried out (Fig. 2). Although CALI that uses the near-infrared femtosecond laser has advantages regarding the inactivation of proteins – such as a high degree of spatial specificity without causing nonspecific photodamage to living cells – it is not that popular because of the high equipment costs. To further improve the use of EGFP-based CALI, any effects that arise from endogenous, unlabeled proteins need to be minimized. Endogenous target proteins that do not contain the EGFP fusion are resistant to CALI, and can compensate for the inactivation of their EGFP-fusion counterparts. To address this issue, rescue experiments using EGFP-fusion proteins can be used to evaluate the degree of loss-of-function due to CALI. In fact, they have shown that CALI is comparable with other conventional methods of abrogating protein function (Vitriol et al., 2007). CALI with KillerRed

KillerRed is derived from anm2CP (Bulina et al., 2006a), a nonfluorescent chromoprotein isolated from hydrozoa Anthomedusae sp., and has a barrel-like shape similar to GFP. The chromophore resides in the internal side of the barrel and connects directly to the surrounding solvent through a long cavity, which has an important role in oxygen and ROS transmission (Carpentier et al., 2009; Pletnev et al., 2009; Roy et al., 2010; de Rosny and Carpentier, 2012). KillerRed is excited by green light; it emits red light (maximum at 585 nm, emission peak at 610 nm) (Table 1) and generates superoxide (Vegh et al., 2011; Wang et al., 2012). KillerRed is the first choice for protein-based CALI because of its high efficiency in generating ROS (1000-fold compared with EGFP). This powerful generation of ROS makes it possible to analyze the effect of proteins of interest on subcellular dynamics. For

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example, CALI with KillerRed bound to b1-integrin – which is involved in regulating invadosomes – was used to investigate whether signaling from b1-integrin is required to either initiate or maintain the ‘rosette’ structures (formed by several invadosomes) as well as their dynamics (Destaing et al., 2010). The authors showed that inactivation of b1-integrin by CALI results in acute destabilization of both individual invadosomes and rosettes, which indicates the importance of b1-integrin to the entire lifespan of the rosette. The same group subsequently reported that CALI-mediated dynamin inactivation results in a rapid disappearance of invadosomes without any changes in focal adhesions, indicating that dynamin specifically regulates the actin dynamics in invadosome (Destaing et al., 2013). KillerRed-based CALI is particularly useful when specific antibodies or nontoxic chemical inhibitors are not available because a protein of interest belongs to the protein family whose members are structurally very similar, has isoforms arising from splicing variants or has closely related paralogs. The ability to inactivate specific isoforms of a protein of interest would be particularly valuable to gain insight into their specific and differential functions. CALI with KillerRed was used to investigate aquaporins (AQPs), a group of water channels with several functions in cell physiology (Baumgart et al., 2012). Their functions, however, are not fully understood because of the lack of nontoxic inhibitors. CALI was used to inhibit water permeability of AQP1 and of two AQP4 isoforms (splicing variants, M1 and M23; AQP4-M1 tetramers freely diffuse in the plasma membrane, whereas AQP4-M23 tetramers are assembled into stationary aggregates). Water permeability of cells that express AQP fused to KillerRed was measured by osmotic swelling-induced dilution of cytoplasmic chloride, in combination with a chloride-sensing fluorescent protein (Galietta et al., 2001). The authors found that reduction in water permeability upon APQ4 inactivation was much more pronounced for the aggregate-forming M23 isoform, implying that intermolecular CALI took place, or that there was a reciprocal inactivation owing to the recognition of the two isoforms.

Fig. 2. Visualization of mitochondrial dynamics after CALI-mediated inactivation of cytochrome c by using a femtosecond laser. Mitochondria of HeLa cells are visualized in yellow by using EYFP tagged with a targeting sequence from subunit VIII of human cytochrome c oxidase (pEYFP-Mito; Clonetech) in green pseudo color, and MitoTracker Red in red (Life Technologies). pEYFP-Mito-expressing mitochondria stained with MitoTracker Red appear yellow. CALI of cytochrome c oxidase that targets the upper region of a single mitochondrion is performed by irradiation with a femtosecond laser (white arrow in the top left image). Time after irradiation is given in the bottom right corner of each image. Immediately after CALI, the red fluorescence of MitoTracker Red disappears from the irradiated mitochondrion, suggesting the depolarization of the mitochondrial membrane because MitoTracker Red only stains membrane that has a membrane potential, resulting in fluorescence of YFP only, as green pseudo color, that remains in mitochondria (20 seconds). The depolarized mitochondrion then becomes circular and does not undergo any fusion and fission (1 min). Depolarization expands (2 min). Depolarized mitochondria become tubular but cannot fuse with adjacent mitochondria (5 min and 7 min). In contrast, the surrounding mitochondria perform fusion and fission repeatedly. The white dashed line denotes the cell membrane. Scale bar: 5 mm.

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A

been depleted allows to reveal the characteristics of exogenous proteins, including of phosphomimetic mutants. KillerRed has also been used as a tool to simply generate ROS in other approaches, including as a chromatin-targeted phototoxic fluorescent protein for the induction of phase-specific arrest of cell division. In vitro, HeLa cells that express tandem KillerRed fused to histone H2B normally divide in the dark, but stop proliferation when they are irradiated with green light (Serebrovskaya et al., 2011). Similarly, we performed CALI by using the cohesion regulator protein RBMX fused to KillerRed to show that G2-phase-specific inactivation of RBMX by CALI results in mitotic delay, demonstrating that RBMX specifically functions in G2-phase nuclei (Matsunaga et al., 2012; Fig. 3). Moreover, in transgenic Xenopus embryos that express H2Btandem KillerRed under control of tissue-specific promoters, tissue development is slowed in tadpoles when illuminated with green-light, suggesting that CALI can be used to elucidate the effect of temporal arrests of cell division during organogenesis (Serebrovskaya et al., 2011). KillerRed fused to either lamin B1, one of the components of inner nuclear membrane, or histone H2A was shown to induce DNA strand breaks dependent on irradiation time and intranuclear chromatin structure (Waldeck et al., 2011). Recently, CALI with KillerRed fused to a Tetrepressor or a transcriptional activator that can bind to integrated DNA cassettes (,90 kb), was shown to cause induction of sitespecific DNA damage (Lan et al., 2013). The authors showed that DNA repair proteins are differentially recruited to euchromatin and heterochromatin. In addition to its use nuclei, KillerRed-based CALI has mostly been performed in organelles by fusing it to signal peptides that are responsible for their subcellular localization. Accordingly, organelle-specific protein inactivation was performed using KillerRed targeted to peroxisomes (Ivashchenko et al., 2011), mitochondria (Bulina et al., 2006a; Bulina et al., 2006b; Choubey et al., 2011; Shibuya and Tsujimoto, 2012) and lysosomes (Serebrovskaya et al., 2014). CALI with mitochondria-targeting KillerRed in human cells and C. elegans induces mitochondrial membrane depolarization and morphological changes, respectively, including fragmentation and swelling, as well as caspasedependent apoptosis (Shibuya and Tsujimoto, 2012). CALI with lysosome-associated KillerRed also induces cell death, either due to necrosis following exposure to light of high intensity or due to

G2

0

9

18

27

36

45

54 min

G2

0

30

60

90

120

150

180 min

DIC

GFP-H1.2

B DIC

Fig. 3. Visualization of cell division arrest upon CALImediated inactivation of RBMX with KillerRed. (A) Nuclei and chromosomes of HeLa cells are visualized by using histone H1.2 fused to GFP (GFP-H1.2). (B) RBMX, a regulatory protein for centromeric protection and cohesion, is labeled with KillerRed (KR-RBMX). When a G2-phase nucleus, in which GFP-H1.2 is expressed, is irradiated with green light, mitosis is typically finished within 60 minutes from nuclear envelope breakdown at 0 min (A, control). In contrast, CALI-mediated inactivation of RMBX in a G2-phase nucleus expressing GFP-H1.2 and KRRBMX leads to considerable delay of mitosis, which is only completed after 180 minutes (B). Fluorescence of KR-RBMX has disappeared following laser irradiation. DIC, differential interference contrast microscopy image. Scale bars: 10 mm.

GFP-H1.2

KR-RBMX

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Another example for the use of KillerRed when the type of protein isoforms is an issue is the mammalian Golgi complex, which consists of several stacks of membrane-bound structures designated as cisternae. The application of CALI with KillerRed previously revealed that formation of cis-cisternae depends on the recycling of a set of cis-Golgi enzymes through the ERGolgi intermediate compartment (ERGIC) (Jarvela and Linstedt, 2012). Lateral networks among membranes are highly optimized and vital for both protein modification and glycan processing, and membranes are tethered by two reassembly stacking proteins, GRASP55 and GRASP65. Although these proteins are located in different cisternae, previous studies have reported that knockdown of either GRASP55 or GRASP65 causes defective glycosylation. Yet there is little evidence of any difference in terms of their localization that might be responsible for compartment integrity. To address this question, CALImediated inactivation of each GRASP was performed by using KillerRed fused to each isoform. This resulted in the loss of Golgi integrity in an isoform-dependent manner, with inactivation of GRASP55 and GRASP65 affecting trans-Golgi and cis-Golgi integrity, respectively (Jarvela and Linstedt, 2014). These results clearly show that each GRASP isoform has a cisternae-specific role, ensuring the integrity of Golgi compartmentalization. CALI is particularly useful to inactivate mutant forms of key factors in dynamic molecular pathways that are regulated in a highly spatiotemporal manner. Without changing the localization of the actin regulator cofilin, CALI was performed separately in cofilin-depleted cells by using KillerRed coupled to either a constitutively active cofilin mutant (cofilinS3A) or a dominantnegative cofilin mutant (cofilinS3E) (Vitriol et al., 2013). Cells that express either cofilinS3A or cofilinS3E fused to KillerRed show no obvious change in the quantity or distribution of lamellipodial F-actin. However, only cells that express cofilinS3AKillerRed show a dramatic increase in F-actin in the lamellipodia and decrease in the rate of retrograde flow of actin after CALI. These results are consistent with previous studies that have shown a role for cofilin in regulating lamellipodia stabilization and in severing and disassembling of actin filaments. Moreover, this study was not only the first report of a real-time regulation of actin turnover by cofilin in the living cell, but also clearly shows that performing CALI in cells in which endogenous protein has

apoptosis following exposure to light of lower intensity (Serebrovskaya et al., 2014). CALI is, therefore, useful in pathological and pharmacological studies that aim to identify mechanisms underlying aging and certain diseases, by eliciting ROS-mediated effects derived from specific organelles or subcellular regions. Because it can disrupt the plasma membrane, KillerRed can kill cells. The generated ROS directly affect membrane lipids and destroy the plasma membrane barrier, together with the accumulation of toxic ROS compounds (Williams et al., 2013). This cell-death causing feature of KillerRed, has been used to disrupt cells when CALI is applied. These include Mu¨ller glia cells, the primary glial cells in mouse retina (Byrne et al., 2013); habenula neurons in zebrafish (Lee et al., 2010); amphid sensory neurons in C. elegans (Kobayashi et al., 2013), GABAergic interneurons in zebrafish (Del Bene et al., 2010); and sensory neurons, interneurons and motor neurons in C. elegans (Williams et al., 2013). Furthermore, taking advantage of the spatiotemporal ROS generation in specific cells, KillerRed-based CALI has been applied to evaluate ROS cytotoxicity and investigate the oxidative stress response of mammalian cultured cells (Wang et al., 2013; Wang et al., 2012), as well as a screening tool (Liu et al., 2010; Liu et al., 2011). Despite these remarkable properties, KillerRed also has some shortcomings. In addition to red fluorescence, KillerRed also emits weak green fluorescence at 480 nm that is difficult to distinguish from green fluorescent probes, such as GFP. Ideally, KillerRed and green fluorescent probes should, therefore, not be used together in the same cell (Nordgren et al., 2012). In addition, dimerization of KillerRed may hamper the mobility of the target protein (Shirmanova et al., 2013). To prevent any problems owing to dimerization, a monomeric KillerRed variant has been developed by introducing several point mutations (Takemoto et al., 2013). This new KillerRed variant, named SuperNova, possesses the same ability to generate ROS and shares similar excitation and emission properties as the original KillerRed. CALI with miniSOG

MiniSOG is a small (106 amino acids) monomeric protein derived from phototropin 2, which is responsible for detecting the direction of light irradiation and mediating phototaxis in Arabidopsis thaliana (Shu et al., 2011). Owing to the presence of bound flavin mononucleotide (FMN), miniSOG absorbs blue light at a maximum of 448 nm (with a ‘shoulder’ absorption peak at 473 nm) and fluoresces green (peaks at 500 and 528 nm) (Ruiz-Gonza´lez et al., 2013). FMN is a cofactor for various enzymes and receptors (Massey, 2000), and generates singlet oxygen by itself (Baier et al., 2006); it binds to the LOV2 (light, oxygen, voltage) domain of A. thaliana phototropin 2 (Salomon et al., 2000). Fluorescence of miniSOG depends on the concentration of FMN; it is reduced when FMN levels decrease (Ryumina et al., 2013). FMN embedded in miniSOG protein enables the photoinduced electron-transfer reaction (type-I reaction), which results in a relatively poor yield of singlet oxygen compared with its free state, instead of generating singlet oxygen (type-II reaction) (see Box 1). Thus, the definition of miniSOG should be changed to ‘mini super oxide generator’ from ‘mini singlet oxygen generator’ as it was originally named because it was thought to generate mainly singlet oxygen. (Pimenta et al., 2013). Although several controversial issues remain with regard to the type or 1626

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quantity of generated ROS, miniSOG has been shown to serve as a probe that generates ROS. MiniSOG is not only a CALI reagent. It has also been used in correlative fluorescence and electron microscopy, in combination with diaminobenzidine – which forms osmiophilic compounds upon reaction with singlet oxygen – and, particularly, in the field of neuroscience, has enabled the detection of barely visible objects (Boassa et al., 2013; Burgers et al., 2012; Ludwig et al., 2013; Shu, et al., 2011). Besides its applications mentioned above miniSOG has been mainly used in the area of neuroscience. Optogenetic techniques provide effective ways to manipulate functions of selected neurons with light. For instance, direct neurotransmitter release upon focused illumination can be induced by using rhodopsins, the microbial opsin channels, which are valuable tools to manipulate selected neurons. Using CALI together with miniSOG to investigate synaptic proteins has helped to achieve this objective. For instance, the fusion of miniSOG to VAMP2 – the SNARE complex protein located at the presynaptic terminal that mediates vesicular synaptic release – or to synaptophysin disrupts presynaptic vesicular release upon irradiation with blue light (i.e. CALI induction) in both cultured neurons and hippocampal organotypic slices (Rambani et al., 2009). Expression of miniSOG–VAMP2 in whole C. elegans neurons caused reduced movement and paralysis after CALI was induced. Consequently, this technique has been named inhibition of synapses with CALI (InSynC) (Lin et al., 2013). In C. elegans, InSynC was used to address the temporal and spatial requirements of the localization of an UNC-13 isoform derived from the splicing variant UNC-13L to the active zone, which features a high density of Ca2+ channels and where neurotransmitter-containing synaptic vesicles are released in response to Ca2+ influx (Zhou et al., 2013). In the presynaptic active zone, UNC-13 isoforms produced by alternative splicing interact with the synaptic vesicle fusion apparatus and mediates exocytosis of synaptic vesicles. Upon irradiation with blue light, worms that express UNC-13L–miniSOG showed impaired rapid movement accompanied by much reduced slow movement. This suggests that the removal of UNC-13L from only the active zone results in strong inhibition of spontaneous as well as fast release of synaptic vesicles, corroborating the idea that UNC-13L at the active zone is necessary for both types of synaptic vesicle release. MiniSOG has also been employed as a tool to kill cells. Lightinducible selective cell ablation was performed using CALI with transgenically expressed miniSOG targeted at mitochondria (mito-miniSOG) in C. elegans neurons (Qi et al., 2012). Upon illumination with blue light, mitochondria-tethered miniSOG caused rapid neuronal death without harming tissues adjacent to the dead neuron. Considerations for the use of CALI

There are two distinct technical precautions that need to be considered when applying CALI in order to determine a true spatiotemporal photo-inactivation of certain targets. The first is the precise location of chromophores for laser targeting. Targeting can be achieved by using one of three methods: dyeconjugated antibodies; chemical dyes that recognize specific tags that are attached to targets in advance; or genetically encoded photosensitizers fused to targets (Fig. 1, Table 1). Using genetically encoded photosensitizers, endogenous target proteins cannot be labeled and remain functional (Vitriol et al., 2007). Therefore, in combination with molecular genetic techniques, including RNA interference (RNAi) and genome

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editing, the pool of endogenous proteins should be eliminated. Dye-conjugated antibodies are ideal in terms of their effect on endogenous proteins, but require antibody microinjection into cells. Therefore, dye-conjugated antibodies are less well-suited when using CALI for proteins that have extracellular parts (reviewed in Buchstaller and Jay, 2000). This prevents the use of CALI in a range of experiments because microinjection is technically challenging, especially in multicellular organisms. The second precaution also involves spatial restriction and should limit nonspecific damage to surrounding molecules or cells, especially when high-intensity light irradiation is used. Indeed, early studies required high-intensity lasers because the efficiency of ROS production of the probes was low. Irradiation of Malachite Green with a 620-nm laser results in reducing the maximal radius ˚ ) (Hoffman-Kim et al., 2007). Using of damage by half (,15 A tetracysteine motifs in combination with chemical dyes, such as ReAsH, often shows a high efficiency in the production of ROS; however, the outcomes of CALI experiments may suffer from excessive inactivation of target proteins when the dye concentration is too high (Tour et al., 2003). In contrast, fusing target proteins to EGFP requires a high-intensity light irradiation to achieve sufficient inactivation of these proteins because EGFP is inferior to chemical dyes in its ability to produce ROS (Rajfur et al., 2002). However, intense laser illumination of genetically encoded proteins produces deleterious amounts of ROS, which in ˚ ) can cause cleavage or crosslinking of close proximity (30–60 A peptide backbones (Tanabe et al., 2005; Horskotte et al., 2005; McLean et al., 2009). The negative effects of ROS vary strongly dependent on molecular crowding, stability of protein complexes and organelle interactions, and CALI should be performed by using appropriate controls. Thus, although the functional analyses of proteins by CALI has been impressive, the elucidation of observed phenotypes needs to be supported by data obtained from other knockdown or knockout approaches to ensure any secondary effects owing to excessive ROS damage are not misinterpreted. Conclusions and perspectives

The functional analysis of proteins has mainly been performed using either RNAi-based knockdown and mutagenesis, or genetic approaches that involve recombination or tagging-based knockout experiments that are based on protein-tagging. However, these approaches are limited as they are unable to provide definitive demonstrations of protein function within living cells. CALI used together with live imaging provides a novel alternative in order to reveal dynamic subcellular function through precise and accurate spatiotemporal inactivation of proteins in situ. Improvements in the spatial resolution of light irradiation to subcellular target regions are anticipated with the acceleration of advances in superresolution microscopy (Lidke and Lidke, 2012; Sauer, 2013). Additionally, gene editing methods that use transcription-activatorlike effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) in association with Cas9 (CRISPR/Cas9) will make it easier to generate cells that are deficient in the endogenous protein of interest (Gaj et al., 2013; Terns and Terns, 2014), which can then be substituted by target proteins linked to photosensitizers. CALI experiments in deficient cells that are rescued with functional chromophore-tagged or fused proteins allow to ascertain the degree of loss-of-function. Thus, CALI has the potential to develop into a powerful technique that provides phenotype ‘snapshots’ through the inactivation distinct functions of multifunctional proteins by using exogenous mutants after an endogenous protein has been depleted. CALI

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could, thereby, help to dissect the various functions and their connectivity within a cell. Beyond protein analyses, CALI can be extended to investigate different areas of cell biology. CALI can be used to change chromatin organization through the inactivation of chromatinbinding and nuclear factors. Although chromatin organization is important for gene expression, its exact regulation remains unknown (Gibcus and Dekker, 2013; Matsunaga et al., 2013). Because chromatin organization dynamically changes depending on cell phase and developmental stage, CALI may be able to reveal temporally relevant stages of chromatin organization. CALI might also provide insight into DNA-repair mechanisms. Because X- or gamma-rays induce random DNA damage, many studies have attempted to induce locus-specific DNA damage that is physiologically more relevant. Enzymatic systems to induce DNA breakage include a system that is based on the rare-cutter endonuclease I-SceI – which has a recognition sequence that occurs only rarely in a genome; the elicited DNA-repair mechanism may, thus, be different from the naturally occurring DNA-damage response (Bryant et al., 2004). A system to induce site-specific oxidative DNA damage based on ROS generators, thus, holds considerable promise to mimick natural DNA-repair mechanisms. Combination with knock-in systems on the basis of genome editing will allow to insert the binding sequence of ROS generators into expected genomic loci, inducing locus-specific sites of DNA damage (Bedell et al., 2012; Auer et al., 2014). Optogenetics is a functional method of protein analysis that utilizes genetically encoded light-sensitive proteins that are activated by light. It has recently also been used in behavioral research to modify signaling in cranial nerves (Deisseroth, 2011), as well as in studying gene regulation by using a light-inducible transcriptional effector as an optical switch to control transcriptional and epigenetic status (Konermann et al., 2013). CALI has also been used as a ‘switch’ in optogenetics because it can disrupt chromatin and proteins in a locus-specific manner, kill specific organelles or a single cell in vivo (Yang and Yang, 2011; Lin et al., 2013). Moreover, CALI is beginning to be exploited in photodynamic therapy of cancer cells (Mironova et al., 2013; Ryumina et al., 2013; Shirmanova et al., 2013; Liao et al., 2014). Therefore, we anticipate that the portfolio of applications of CALI will further expand, and contribute to a series of remarkable biological findings and medical applications in the future. Acknowledgements We are grateful to Hiroshi Ishii, Eri Mizusawa, Kiichi Fukui and Kazuyoshi Itoh for valuable discussion and comments regarding the use of CALI in live cell imaging.

Competing interests The authors declare no competing interests.

Funding This research was supported by SENTAN and CREST grants from the Japan Science and Technology Agency, a Grant-in-Aid for X-ray Free Electron Laser Priority Strategy Program (MEXT), and grants from MEXT/JSPS KAKENHI (20370027, 20061020, 21027023, 23012027, 23370029, 23120518, 25114514, 25120726).

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