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AlphaFold2-guided engineering of split-GFP technology enables labeling of endogenous tubulins across species while preserving function [1]

['Kaiming Xu', 'Tsinghua-Peking Center For Life Sciences', 'Beijing Frontier Research Center For Biological Structure', 'Mcgovern Institute For Brain Research', 'State Key Laboratory Of Membrane Biology', 'School Of Life Sciences', 'Moe Key Laboratory For Protein Science', 'Tsinghua University', 'Beijing', 'Zhiyuan Li']

Date: 2024-08

Dynamic properties are essential for microtubule (MT) physiology. Current techniques for in vivo imaging of MTs present intrinsic limitations in elucidating the isotype-specific nuances of tubulins, which contribute to their versatile functions. Harnessing the power of the AlphaFold2 pipeline, we engineered a strategy for the minimally invasive fluorescence labeling of endogenous tubulin isotypes or those harboring missense mutations. We demonstrated that a specifically designed 16-amino acid linker, coupled with sfGFP11 from the split-sfGFP system and integration into the H1-S2 loop of tubulin, facilitated tubulin labeling without compromising MT dynamics, embryonic development, or ciliogenesis in Caenorhabditis elegans. Extending this technique to human cells and murine oocytes, we visualized MTs with the minimal background fluorescence and a pathogenic tubulin isoform with fidelity. The utility of our approach across biological contexts and species set an additional paradigm for studying tubulin dynamics and functional specificity, with implications for understanding tubulin-related diseases known as tubulinopathies.

Funding: This work was supported by the National Natural Science Foundation of China (31991190, 31730052, 31525015, 31861143042, 31561130153, 31671444, and 31871352)( https://www.nsfc.gov.cn/ ) and National Key R&D Program of China (2019YFA0508401, 2017YFA0503501, and 2017YFA0102900)( https://www.most.gov.cn/ ) to G.O., and the National Natural Science Foundation of China (323B200173)( https://www.nsfc.gov.cn/ ) to K.X.. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Guided by artificial intelligence–driven AlphaFold2 pipeline [ 32 ], we developed a novel strategy for the functional labeling of endogenous tubulins while preserving their inherent functionalities. We overcame 3 technical obstacles: the optimal site for tubulin labeling, the selection of fluorescent markers, and the constitution of the linker sequence. Employing the nematode C. elegans as a model system, we showed that a 16-amino acid (aa) linker, in conjunction with sfGFP11 from the split-sfGFP system and inserted at the H1-S2 loop of tubulin [ 33 – 35 ], enabled labeling of either α- or β-tubulin without compromising MT assembly, embryogenesis, or ciliogenesis. Extending this technique to tubulins in HeLa cells and murine oocytes, we achieved visualization of MT networks with significantly diminished background fluorescence. This suggests a majority of labeled tubulin is successfully integrated into MT assemblies, thereby implying that our approach is superior to existing technologies. These findings collectively underscore the extensive applicability of our strategy for functional tubulin labeling across diverse cellular milieus and species.

Currently, GFP-tagged tubulins have been widely used to visualize tubulin isotypes across different species [ 22 , 23 ]. However, C-terminal GFP tagging of tubulin impedes its interaction with MAPs or other regulatory elements, whereas N-terminal tagging obstructs the incorporation of tubulin into MT architecture [ 22 , 24 ]. Moreover, endogenous (or knock-in (KI)) GFP labeling of tubulin produces more pronounced disruptions in MT functionality compared to overexpression (OE) [ 24 ]. This could be attributed to the functional redundancy of tubulin isotypes and resilience exhibited by endogenous, nontagged tubulins. Nonetheless, ectopic OE of wild-type GFP-tagged tubulin isotypes has been widely adopted across diverse cellular environments, including the nematode [ 12 ], mammalian cell lines [ 25 ], or mouse embryos [ 26 ], with a fraction of GFP-tagged tubulins providing strong fluorescent signals allowing to visualized MTs. Despite those successful applications to mark wild-type MTs, GFP labeling fails to trace mutated tubulins associated with tubulinopathy with high fidelity. Instead, the visualization of mutated tubulins were accomplished by immunofluorescence (IF) staining in almost all situations [ 27 – 31 ]. Consequently, there is still a lack of unobtrusive, functional imaging of dynamic tubulin isotypes.

Mutations in tubulin-coding genes cause human diseases collectively designated as tubulinopathies such as cortical malformations, manifesting as microcephaly, lissencephaly, or polymicrogyria [ 13 – 15 ]. The symptomatic spectrum of tubulinopathies extends from severe intellectual deficits to nuanced cognitive impairments, accentuating the indispensable role of tubulins in brain development [ 15 ]. Also, tubulinopathies encompass ocular and renal anomalies [ 16 ]. Notably, many of these mutations are de novo [ 16 – 19 ], underscoring the underestimated impact of tubulinopathies in human diseases. Indeed, AlphaMissense-based predictions revealed that over 80% missense mutations in tubulins (81.9% for α-tubulin TUBA1A and 82.5% for β-tubulin TUBB2B) are likely pathogenic, overwhelming those in KRAS (66.0%) or BRAF (57.7%)—2 hotspot proteins in cancer research ( S1A Fig ) [ 20 , 21 ]. Comprehensive exploration of the functional specificities among divergent wild-type and mutant tubulin isotypes holds the promise of elucidating the underlying pathophysiological mechanisms [ 13 ], thereby facilitating the development of therapeutic strategies. Therefore, there is an imminent requirement to monitor the dynamics of individual tubulin isotypes in both wild-type and pathological forms.

Nevertheless, most existing techniques exhibit intrinsic limitations when delving into the functional specificity of tubulin isotypes, an indispensable aspect of MT biology that contributes to their diverse roles in cellular processes [ 1 ]. Metazoan genomes encode an array of α- and β-tubulin genes, each exhibiting unique spatial and temporal expression profiles and specialized posttranslational modifications within cellular compartments [ 1 , 10 ]. For instance, the mammalian tubulin isotypes TUBB2 and TUBB3 are predominantly incorporated into neuronal MTs, playing an indispensable role in neurite outgrowth [ 11 ]. Conversely, others (for instance, Caenorhabditis elegans TBA-5) are more prevalent in cilia and flagella with exclusive localization within specific ciliary segments, contributing to their unique structural and functional roles [ 12 ]. This functional specificity of tubulin isotypes is expected to provide important regulatory mechanisms, empowering the MT cytoskeleton to various functionalities [ 1 ]. Thus, the real-time interrogation of tubulin isotypes within living cells is foundational for deciphering the functional intricacies of MTs.

Over the past several decades, a myriad of methodologies has been developed to monitor MT dynamics in vivo. Conventional techniques have used microinjection of fluorescein- or rhodamine-labeled tubulins into cells or embryos to measure MT dynamics [ 5 – 7 ]. Furthermore, green fluorescent protein (GFP)-tagged MT-associated proteins (MAPs), such as end-binding (EB) protein or the recent “Stable Microtubule-Associated Rigor-Kinesin” (StableMARK) [ 8 , 9 ], have been rigorously validated for noninvasive and in vivo visualization without disrupting native MT behavior. Complementary to these approaches, computational algorithms for quantification and automated trajectory analysis have been developed, thereby facilitating the acquisition of quantified datasets and imparting real-time measurement of MT dynamics in living systems [ 8 ].

The microtubule (MT) cytoskeleton serves as a dynamic framework of protein assemblies, endowing cells with both structural robustness and functional plasticity [ 1 , 2 ]. Assembled through the polymerization of tubulin monomers into cylindrical arrays [ 3 ], MTs display a behavior known as dynamic instability—a meticulously orchestrated equilibrium between polymerization and spontaneous depolymerization phases [ 4 ]. This regulated dynamic is instrumental for fulfilling diverse physiological functions of MTs [ 5 ]. Consequently, the high-resolution imaging of MT dynamics is pivotal for dissecting their mechanistic roles in cellular activities.

Results

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002615

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