https://orcid.org/0000-0002-5573-2167
Abstract
Small GTPases act as molecular switches regulating various cellular processes by cycling between the GDP- and GTP-bound states. Several methods, including radioisotope-based nucleotide exchange assays, effector-binding pull-down assays and fluorescence-based biosensor methods, have been developed to assess the activation of small GTPases. In vitro techniques mainly provide quantitative insights, whereas live-cell imaging approaches facilitate the real-time monitoring of the activation dynamics of small GTPases. Recent advances, such as the development of fluorescence resonance energy transfer-based probes and membrane-localization sensors, have improved the spatial and temporal resolution of small GTPase activation dynamics. Specifically, the small GTPase activity analysing system using a split fluorescent protein to detect membrane recruitment upon activation provides a novel approach to study small GTPases in living cells. This review comprehensively discusses various conventional and emerging small GTPase activation analysis techniques, highlighting their advantages and disadvantages in studying small GTPase activation dynamics under different cellular conditions.
Abbreviations
- ER
endoplasmic reticulum
- ERGIC
ER–Golgi intermediate compartment
- FRET
fluorescence resonance energy transfer
- GAP
GTPase-activating protein
- GEF
guanine nucleotide exchange factor
- HPLC
high-performance liquid chromatography
- SAIYAN
small GTPase activity analysing
- Sar1
secretion-associated Ras-related GTPase
Small GTP-binding proteins act as molecular switches regulating various cellular responses by cycling between the inactive GDP-bound and active GTP-bound states (1–4,). This transition from the inactive to active state is typically facilitated by the guanine nucleotide exchange factors (GEFs), which promote the dissociation of GDP from small GTPases. Upon GDP release, free forms of small GTPases readily bind to the abundant intracellular GTP, thereby becoming activated. Active GTPases hydrolyse GTP to GDP through intrinsic activity and subsequently revert back to the inactive state. Hydrolysis reaction is generally slow and accelerated by the GTPase-activating proteins (GAPs; Fig. 1) (5,). Many methods have been established to assess the small GTPase activation status owing to their crucial roles in regulating various intracellular signalling pathways. Recently, real-time monitoring of small GTPase activation in living cells has garnered increasing research interest. In this review, we discuss various small GTPase activation analysis methods, ranging from conventional isotope-based assays to the recently developed small GTPase activity analysing (SAIYAN) system (6).
In Vitro Methods for Small GTPase Activation Analysis
Isotope-based assays
A key method to assess the activation status of small GTP-binding proteins involves the in vitro addition of isotope-labelled nucleotides to recombinant small GTP-binding proteins, followed by assessment of the nucleotide-binding status or hydrolysis rate (7). Based on the specific objective, various nucleotides, including non-hydrolysable analogues such as GTPγS and GMP-PNP ([35S]GTPγS, GMP-PNP) as well as [3H]GDP and [32P]-GTP, can be used for analysis. The fate of [32P]-GTP upon hydrolysis is determined by the insertion position of radioactive phosphorus, with insertion at the α- or γ-position resulting in the retention of the isotope on GDP or release of radiolabelled inorganic phosphate ([32P]Pi), respectively.
Schematic representation of the GTPase cycle of small GTP-binding proteins. Small GTP-binding proteins cycle between an inactive GDP-bound state and an active GTP-bound state. GEFs facilitate the release of GDP, allowing small GTPases to bind to the abundant intracellular GTP and become activated. Activated small GTPases interact with effector proteins to transmit cellular signals. Due to their intrinsically low ability to hydrolyse GTP, small GTPases rely on GAPs to accelerate GTP hydrolysis, thereby reverting them to the inactive GDP-bound state.
As small GTPases generally interact with Mg2+, their intrinsic GTPase activity can be regulated by controlling Mg2+ concentration using chelating agents, such as EDTA (8,). Their detection methods include the filter-binding assay, which quantifies the isotope-labelled nucleotides bound to small GTP-binding proteins, and charcoal-based method, which measures the release of inorganic phosphate by adsorbing the protein-bound isotopes onto activated charcoal (9, 10).
The above-mentioned methods have been described in detail in various reviews and method collections (ex. Methods Enzymol.; 1995 Vol. 255–257). These in vitro techniques are valuable tools to assess the activities of small GTP-binding proteins, GEFs and GAPs.
Non-isotope fluorescence-based assays
Over the years, various in vitro measurement methods that do not rely on isotopes have been developed. One simple and effective approach taking advantage of the intrinsic properties of small GTPases involves the analysis of nucleotide exchange by monitoring the changes in the fluorescence of tryptophan residues, which exhibit different fluorescence characteristics in GDP- and GTP-bound states (11–13,). However, this method is not applicable to the Ras family, which lacks the well-conserved tryptophan residues found in many small GTPases. Another fluorescence-based approach uses nucleotide analogues, such as Mant-GTP (14, 15,) and boron-dipyrromethene (BODIPY)-GTP (16, 17), which emit fluorescence upon binding to small GTPases, thereby facilitating the detection of activated GTPases.
Cell-Based Methods for Detecting the Activation of Small GTPases
Immunoprecipitation and high-performance liquid chromatography-based nucleotide quantification
One method to assess the nucleotide-binding state of small GTPases in cells involves immunoprecipitating the small GTPases from [32P]-radiolabelled cells and separating the bound nucleotides via thin-layer chromatography to determine the GTP/GDP ratio (18, 19,). This technique facilitates the direct quantification of nucleotides bound to small GTPases in living cells. However, it has some drawbacks, including changes in the nucleotide ratio during cell lysis and immunoprecipitation and the need for relatively high doses of radioisotopes. Recently, a high-performance liquid chromatography (HPLC)-based method has been developed as an improvement over this technique (20, 21). This HPLC-based method eliminates the need for isotope labelling by immunoprecipitating the small GTPases from cells and directly quantifying the bound nucleotides via ion-pair reverse-phase HPLC analysis.
Effector-based pull-down assays
Effector proteins are widely used to assess the activation of small GTPases in cells. They are a class of proteins that bind to small GTP-binding proteins upon activation. In the GTP-bound state, these proteins mediate various cellular responses. Using only the GTPase-binding domains (GBD) of effector proteins, pull-down assays can be performed to isolate and quantify small GTPases in their activated state in cells (22, 23). This method is widely used owing to its relative simplicity and convenience. However, it exhibits the same fundamental limitation as the above-mentioned isotope-based methods, i.e. it requires cell lysate preparation for quantification, thereby preventing live-cell analysis.
Live-Cell Imaging Techniques
Relocation biosensors
All above-mentioned methods rely on either recombinant small GTPases or those purified from cells to quantify their nucleotide-binding state. In contrast, recently developed methods have facilitated the real-time visualization of small GTPase activation in living cells.
Schematic representation of various live-cell imaging techniques to monitor small GTPase activation. (A) Relocation biosensors: A fluorescent protein-fused effector domain or the GBD of an effector protein is expressed in cells. Upon activation of the target small GTPase, the effector domain is recruited to specific cellular regions where fluorescence accumulation is observed. (B) FRET-based biosensors: A molecular construct containing the target small GTPase, its effector domain or GBD, and a FRET sensor is expressed in cells. Upon activation of small GTPases, an intramolecular interaction occurs, bringing the FRET sensor components closer and generating a fluorescence signal.
Relocation biosensor method is among the first approaches developed for this purpose. This method is conceptually similar to the above-mentioned pull-down assay using effector domains but involves fluorescent labelling of the effector domain and tracking of its intracellular localization to detect small GTPase activation (24, 25) (Fig. 2A). Although fluorescence signals are continuously emitted regardless of the activation status, activated small GTPases localize to specific membrane domains, leading to concentrated fluorescence signals. A major advantage of this method is the real-time monitoring of the small GTPase activation dynamics in living cells. However, this method also has some limitations. First, owing to the continuous emission of the fluorescence signal, its intensity does not directly indicate the activation status of small GTPases. Second, because the biosensor must be introduced into the cells before measurement, its expression can possibly alter endogenous signalling. Finally, depending on the affinity of the effector domain for different small GTPases, the biosensor can possibly detect the activation of similar small GTPases instead of the target small GTPase. On the positive side, this property can aid in the visualization of small GTPases sharing the same effector proteins.
Fluorescence resonance energy transfer-based biosensors
Another method to visualize small GTPase activation in living cells is based on fluorescence resonance energy transfer (FRET) signalling (26,). Developed by the Matsuda Lab, this technique was designed to detect the activation of the Ras family using the Ras superfamily and interacting protein chimeric unit (Raichu) (27). A key feature of this method is that the FRET probe introduced into the cells contains both a small GTPase and its effector domain within a single molecular construct. When intracellular signalling activates a small GTPase, the effector and small GTPase domains intramolecularly interact, bringing CFP and YFP into close proximity, thereby generating a FRET signal (Fig. 2B). This approach is widely used to monitor the activation of various small GTPases in real time.
A major advantage of this method is that a signal is generated only upon the activation of small GTPases, thereby facilitating direct small GTPase activity measurement. However, this method has some limitations. The location of the FRET signal may not always reflect the natural activation sites of endogenous small GTPases, as activation can be influenced by the local presence of GEFs or GAPs, potentially leading to ectopic activation. Moreover, introducing this biosensor into cells possibly interferes with the endogenous signalling pathways, thereby affecting the results and warranting careful interpretation of all findings.
SAIYAN System: A Novel Approach
Sar1 is a small GTPase belonging to the Arf family and involved in protein transport from the endoplasmic reticulum (ER) to the Golgi through the formation of COPII vesicles. Upon activation by its GEF, Sec12, Sar1 binds to the ER membrane and forms the pre-budding complex. The pre-budding complex consists of activated Sar1, the inner coat components of COPII, Sec23/Sec24 and cargo proteins bound to Sec24. Cargo proteins associate with Sec24 either directly or through cargo receptors. Once the pre-budding complex is assembled, the outer coat components of COPII, Sec13/31, bind to the complex, leading to the formation of COPII-coated vesicles. This binding also enhances the GAP activity of Sec23, promoting the conversion of Sar1 to its GDP-bound form (28, 29) (Fig. 3).
Schematic representation of Sar1-mediated COPII vesicle formation. Activation of Sar1 by the GEF Sec12 leads to its enhanced binding to the ER membrane. The activated Sar1, along with the inner coat proteins Sec23/24 and cargo, forms the prebudding complex. Binding of the outer coat proteins Sec13/31 to Sec23/24 increases the GAP activity of Sec23, resulting in the inactivation of Sar1 and the subsequent formation of the COPII vesicle.
Protein transport from the ER has traditionally been considered to be exclusively mediated by COPII-coated vesicles, which are formed at specialized ER domains known as ER exit sites. However, even molecules that are too large to fit within conventional COPII vesicles—such as collagen—depend on COPII components for their transport, thereby drawing significant attention to the underlying mechanisms involved (30).
Our previous studies focused on the functional analysis of TANGO1, a cargo receptor essential for collagen secretion (31,). We revealed that the efficient activation of Sar1 is critical for its function (32–34,). However, we mainly focused on the intracellular localization of Sec12, and we did not measure Sar1 activation in real time during collagen secretion (33,). Sec23 is an effector molecule that binds to the GTP-bound form of Sar1, but it also possesses GAP activity. Therefore, conventional methods for detecting small GTPase activation using effector domains were not applicable (35). To overcome this limitation, we established the SAIYAN system, a novel tool for analysing the activation of small GTPases.
The SAIYAN system quantitatively measures the membrane localization of small GTPases in cells using a split fluorescent protein system (36–39,). Sar1, even in its GDP-bound state, weakly associates with the ER membrane; however, upon conversion to its GTP-bound state, its N-terminal amphipathic helix is inserted into the membrane, leading to stronger membrane association, as indicated by structural analyses (40, 41). To exploit this property, we fused beta strands 1–10 of mNG2 (mNG) to the N-terminal region of TANGO1S via a linker, thereby tethering it to the ER membrane. We also established a stable cell line expressing this construct. Subsequently, the 11th beta strand of mNG2 was inserted into the C-terminal region of Sar1 via a linker and transiently expressed. When Sar1 was activated and recruited to the ER membrane, the complete mNG protein was reconstituted and successfully emitted fluorescence (Fig. 4).
Schematic representation of the SAIYAN system. β-strands 1–10 of a split fluorescent protein are pre-expressed on the membranes where the target small GTPase is recruited upon activation. The 11th β-strand is fused to the small GTPase. The complete fluorescent protein is reconstituted upon membrane recruitment and emits fluorescence.
As split fluorescent proteins exhibit self-assembly, it is crucial to fine-tune the linker length to prevent unintended fluorescence in the inactive state. After optimizing the linker length, mNG signals were quantitatively analysed using various Sar1 mutants. For the N-terminal truncated mutant of Sar1 (ΔSar1), a minimal signal was detected, whereas GDP-form mutant Sar1-T39N exhibited a signal approximately half that of Sar1 wild-type or GTP-form mutant Sar1 H79G distributed throughout the ER membrane. In contrast, mNG signals were specifically localized to the ER exit site in cells expressing Sar1 wild-type or Sar1 H79G (Fig. 5). This observation is consistent with our previous report that Sec12 is localized to the ER exit site (34).
SAIYAN signals of various Sar1 mutants in HeLa cells. The Sar1 activation signal by SAIYAN is shown in the left panels. Sec16A is a marker of ER exit sites (right panels). Scale bar = 10 μm. ©2024 Maeda et al. Originally published in JCB. https://doi.org/10.1083/jcb.202403179
To further validate the SAIYAN system, a HeLa cell line stably expressing mNG1–10 was generated, and mNG11 was knocked into the C-terminal region of Sar1 using the CRISPR/Cas9 technique. Notably, SAIYAN signals decreased by approximately half upon Sec12 knockdown but increased upon Sec31 knockdown in these cells. Furthermore, Sec13/Sec31 overexpression decreased the mNG signal, suggesting that the SAIYAN system effectively monitors the Sar1 turnover associated with its inactivation. Interestingly, in Sec23-knockdown cells, the SAIYAN signal was reduced. This result suggests that Sec23 requires its interaction with Sec13/31 to enhance its GAP activity, while primarily functioning as an effector that stabilizes activated Sar1 within the prebudding complex.
Based on the HeLa cell results, we confirmed that the SAIYAN system effectively detects Sar1 activation. Subsequently, SAIYAN-expressing BJ-5ta fibroblasts were generated to assess Sar1 activation in collagen-secreting cells. Interestingly, in contrast to that in HeLa cells, where the SAIYAN signal was restricted to the ER exit site, SAIYAN signal was observed both at the ER exit site and ER–Golgi intermediate compartment (ERGIC) in BJ-5ta fibroblasts. Moreover, SAIYAN signal in the ERGIC region co-localized with Sec23. To determine whether this localization pattern depends on collagen secretion, we treated the cells with the collagen-folding inhibitor, DPD, and observed a decreased SAIYAN signal in ERGIC (42, 43,). These results suggest that Sar1 activation occurs not only at the ER exit site but also in ERGIC in collagen-secreting cells. This finding aligns with recent reports suggesting that ER–Golgi transport occurs via tubular structures extending from ERGIC to the Golgi, rather than exclusively through conventional COPII-coated vesicles (44–46). However, further studies are essential to elucidate the specific underlying mechanisms.
All methods for detecting the activation of small GTPases have unique advantages and limitations, necessitating the careful selection of methods based on the specific research objective.
As the SAIYAN system detects membrane localization as an indicator of the small GTPase activation, it is only applicable to small GTPases exhibiting increased membrane-binding affinity upon activation. Additionally, careful consideration of the self-assembly property of split fluorescent proteins is essential when using this system. Nevertheless, this system shows great potential to detect the activation status of small GTPases associated with multiple membrane compartments and study small GTPases with unknown effectors.
Conclusions and Future Prospects
Small GTPases play essential roles in cellular signalling, and various methods have been developed to assess their activation. Traditional in vitro assays, such as radioisotope-based methods, exhibit high sensitivity but lack spatial and temporal resolution. Pull-down assays detect activation in cell lysates but do not capture the activation dynamics. Live-cell imaging techniques, including those using relocation biosensors and FRET-based probes, offer real-time insights but require careful optimization to avoid interference with the endogenous signalling pathways.
The recently developed SAIYAN system provides a new approach to detect the activation of small GTPases by monitoring the membrane localization in living cells. Although currently optimized for Sar1, this system can be further adapted to expand its applicability to other GTPases.
Future advances in imaging technologies and biosensor methods will further improve the accuracy and resolution of small GTPase activation analysis. Moreover, multiple approaches can be combined to better understand the small GTPase regulation mechanisms under physiological and pathological conditions.
FUNDING
This work was supported by JSPS Grants-in-Aid for Scientific Research (22H02760 and 23K24023 to M.M. and 23H02430, 23K27123 and 24K22065 to K.S.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and Naito Foundation (K.S. and M.M.). K.S. received support from the Takeda Science Foundation, Asahi Glass Foundation and Princess Takamatsu Cancer Research Foundation.
CONFLICT OF INTEREST
The authors declare no conflicts of interest.
