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. 2025 Aug 12;36(9):ar109. doi: 10.1091/mbc.E25-02-0079

GAS2L1 facilitates Golgi-derived microtubule growth and Golgi organization

Bill KT Hau a, Khoi TD Le a, Franco KC Au a,†, Yusong Guo a, Robert Z Qi a,b,*
Editor: Xuebiao Yaoc
PMCID: PMC12415604  PMID: 40668599

Abstract

The Golgi complex serves as a major noncentrosomal microtubule-organizing center in animal cells, with Golgi-derived microtubules playing a crucial role in Golgi assembly, structural organization, cell migration, and various Golgi-related cellular functions. In this study, we identify GAS2L1 as a key regulator of Golgi-based microtubule growth and Golgi reassembly. Loss of GAS2L1 leads to significant defects in Golgi morphology and impairs nocodazole washout-induced Golgi reassembly. Notably, microtubule regrowth from the Golgi after nocodazole treatment is markedly inhibited in GAS2L1-knockout cells. Furthermore, we demonstrate that GAS2L1 interacts with CLASP1/2, known regulators of Golgi-derived microtubules, suggesting that GAS2L1 functions in concert with CLASP1/2 to regulate Golgi-associated microtubule dynamics. These findings provide new insights into the molecular mechanisms governing Golgi-derived microtubules, expanding our understanding beyond the established roles of CLASP1/2.

  • Golgi-derived microtubules play a critical role in maintaining Golgi morphology, organization, and assembly, yet the mechanisms governing their regulation remain incompletely understood.

  • The authors identify GAS2L1 as a Golgi-associated protein essential for the growth and stabilization of Golgi-derived microtubules.

  • As a microtubule- and actin-binding protein that also interacts with EB1/3, GAS2L1 promotes Golgi microtubule dynamics by interacting with CLASP1/2, a key regulator of microtubule stability. This interaction is crucial for proper Golgi assembly and morphology.

  • These findings establish GAS2L1 as a critical regulator of Golgi-derived microtubules, providing new insights into their organization and broader implications for Golgi functions.

INTRODUCTION

The Golgi complex is a crucial organelle responsible for protein posttranslational modification, sorting, and transport. In addition to these core functions, it acts as a microtubule-organizing center, regulating microtubule dynamics essential for cellular processes such as cell polarization, directed migration, and intracellular trafficking (Chabin-Brion et al., 2001; Efimov et al., 2007; Lüders and Stearns, 2007; Miller et al., 2009; Hurtado et al., 2011). These microtubules also contribute to maintaining the structural integrity of the Golgi (Vinogradova et al., 2012). Throughout the cell cycle, the Golgi complex undergoes dynamic remodeling, disassembling during mitotic entry, and reassembling upon mitotic exit (Tang et al., 2010). Golgi-derived microtubules play a pivotal role in this process by facilitating the clustering of dispersed Golgi fragments into ministacks. Subsequently, centrosome-derived microtubules guide the recruitment of these ministacks to the periphery of the centrosome, where they coalesce into a fully reassembled Golgi complex (Miller et al., 2009). Thus, Golgi-derived and centrosome-derived microtubules work in concert to ensure the proper assembly and structural maintenance of the Golgi complex.

At the Golgi complex, the initiation of microtubule growth occurs through nucleation by γ-Tubulin ring complex (γTuRC). Once nucleated, these microtubules are stabilized by the proteins CLASP1 (also known as CLASP1α) and CLASP2, which exists in three isoforms α, β, and γ, through binding to conserved TOG (tumor-overexpressed gene) domains (Efimov et al., 2007; Slep, 2009; Grimaldi et al., 2014; Maki et al., 2015; Aher et al., 2018). The minus end of Golgi-derived microtubules is anchored at the Golgi by a protein complex comprising AKAP450, MMG8, and CAMSAP2 (Gillingham and Munro, 2000; Rivero et al., 2009; Wang et al., 2010; 2014; Wu et al., 2016). Additionally, the spectraplakin family of actin-microtubule cross-linking proteins, including the spectraplakin family protein ACF7 (also known as MACF), plays a crucial role in the regulation of Golgi morphology through their interactions with both actin and microtubules (Kodama et al., 2003; Lin et al., 2005). Similarly, GAS2L1, a microtubule- and actin-binding protein, is involved in the regulation of microtubule and actin dynamics and is involved in proximal axon growth and branching (van de Willige et al., 2019). Furthermore, GAS2L1 is also a centriole-associated protein and acts in the attachment of actin and microtubules to centrioles, which generates the forces necessary for centriole motility and for centrosome disjunction that occurs during the late G2 phase of the cell cycle (Au et al., 2017; 2020).

In the present study, we investigated the cellular role of GAS2L1 and found that it localizes to the Golgi complex, where it is involved in the growth of Golgi-derived microtubules. Our observations reveal that the absence of GAS2L1 resulted in significant defects in the regrowth of Golgi-derived microtubules, the assembly of the Golgi after disassembly, and the overall structure integrity of the Golgi. Moreover, we found that GAS2L1 associates with CLASP1/2 to exert its function. These findings indicate that GAS2L1, in conjunction with CLASP1/2, plays a crucial role in the organization of Golgi-derived microtubules, which is essential for the formation and maintenance of the Golgi complex.

RESULTS

GAS2L1 is involved in the Golgi assembly and organization and cell migration

In a previous study, we generated gas2l1−/− hTERT RPE-1 cell lines and derived inducible GAS2L1-expression sublines from these knockout cells (Au et al., 2020). Using immunofluorescence imaging with the Golgi marker TGN46, we observed that gas2l1−/− RPE-1 cells exhibited a higher number of smaller Golgi complexes with more compact, circular morphology compared with parental cells (Figure 1A).

FIGURE 1:

FIGURE 1:

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GAS2L1 knockout impairs Golgi morphology and Golgi assembly. (A) Parental and gas2l1−/− RPE-1 cells were fixed and stained for Golgi networks with an antibody against TGN46, and nuclear DNA was labeled with Hoechst 33258. The size and circularity of the Golgi were determined in three independent experiments, with sample sizes of n = 355 (parental) and 350 (gas2l1−/−). (B) RPE-1 cells were treated with nocodazole to induce Golgi disassembly. Following nocodazole washout, cells were fixed at the indicated timepoints for immunostaining. Arrowheads indicate Golgi particles. The size of Golgi particles was measured based on TGN46 staining in three independent experiments, with sample sizes of n = 62 (0 min), 100 (20 min), 124 (40 min), and 157 (60 min) for parental cells; n = 63 (0 min), 106 (20 min), 100 (40 min), and 129 (60 min) for gas2l1−/− cells. (C) A wound-healing assay was performed for parental and gas2l1−/− RPE-1 cells over a time course. Representative micrographs from three independent experiments are shown. The intermargin distance of the scratch was quantified at each timepoint and plotted. (A‒C) Data are presented as mean ± SD, ***, p < 0.001; ****, p < 0.0001; Student's t test; scale bars, 10 µm.

To further characterize this phenotype, we performed a Golgi reassembly assay: Golgi complexes were disrupted using the microtubule-depolymerizing agent nocodazole, and reassembly was monitored after nocodazole washout. In parental cells, Golgi elements fused into ministacks following nocodazole removal, progressively maturing into intact Golgi complexes (Figure 1B). Conversely, gas2l1−/− cells showed impaired Golgi reassembly: the average size of Golgi ministacks was reduced by 26.7%, 18.6%, and 49.6% at 20, 40, and 60 min of reassembly, respectively, compared with parental cells (Figure 1B). These findings indicate that GAS2L1 regulates Golgi assembly and morphology maintenance.

We also assessed cell migration using a wound-healing assay. gas2l1 knockout significantly attenuated cell migration, delaying wound closure kinetics compared with the parental cells (Figure 1C). Collectively, these results demonstrate that GAS2L1 is essential for directed cell migration.

GAS2L1 is involved in the organization of Golgi-derived microtubules

Golgi-derived microtubules are essential for the assembly and structural organization of the Golgi complex, and disruption of these microtubules impairs the reassembly of the Golgi complex following cell division or treatment with nocodazole (Efimov et al., 2007; Miller et al., 2009). To investigate the role of GAS2L1 in the organization of Golgi-derived microtubules, we conducted microtubule regrowth assays using gas2l1−/− RPE-1 cells alongside their parental counterparts. The cells were treated with nocodazole to depolymerize the microtubules, and regrowth was initiated following the washout of the nocodazole.

Our examination of microtubule regrowth at 30 s postwashout revealed a distinct presence of microtubules emanating from the Golgi particles dispersed throughout the cytoplasm. Notably, we observed a 59.1% reduction in the number of Golgi-derived microtubules in gas2l1−/− cells compared with parental cells (Figure 2A). To confirm the specificity of this knockout effect, we utilized a subline of gas2l1−/− cells that allows for the inducible expression of green fluorescent protein-tagged GAS2L1 (GFP-GAS2L1). The reintroduction of GFP-GAS2L1 in gas2l1−/− cells completely restored the levels of microtubule regrowth (Figure 2A), indicating that the observed defect in regrowth was specifically attributable to the absence of GAS2L1. However, we observed no significant differences in the growth of centrosome-derived microtubules among the parental cells, GAS2L1-deficient cells, and cells in which GAS2L1 expression was restored (Supplemental Figure S1).

FIGURE 2:

FIGURE 2:

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GAS2L1 knockout suppresses microtubule growth at the Golgi complex. (A) Microtubule regrowth after nocodazole treatment was assayed on the following RPE-1 cells: parental, gas2l1−/−, and GFP-GAS2L1-expressing gas2l1−/− cells. The cells were then stained for Golgi complexes (anti-TGN46), microtubules (anti–α-Tubulin), and nuclear DNA (Hoechst 33258). Golgi-derived microtubules were quantified from three independent experiments: n = 228 (parental), 229 (gas2l1−/−), and 204 (GFP-GAS2L1-expressing gas2l1−/−). (B) A RUSH assay was performed on parental and gas2l1−/− RPE-1 cells transfected with the SBP-GFP-Shh plasmid. Cells were fixed at indicated timepoints and immunostained for SBP-GFP-Shh and the Golgi marker TGN46. ER-to-Golgi translocation of SBP-GFP-Shh was quantified using the Manders’ colocalization coefficient, calculated from fluorescence signals of SBP-GFP-Shh and TGN46. (A and B) Data are presented as mean ± SD. ****, p < 0.0001; n.s., not significant; Student's t test; scale bar, 10 µm.

Next, we used the retention using selective hooks (RUSH) assay to investigate the impact of gas2l1 knockout on ER-to-Golgi trafficking (Boncompain et al., 2012). We transiently transfected RPE-1 cells with a plasmid encoding SBP-GFP-ShhN (epitope-tagged sonic hedgehog amino-terminal fragment), which is retained in the endoplasmic reticulum (ER) via a streptavidin-binding peptide (SBP) tag until biotin addition (Boncompain et al., 2012; Tang et al., 2022). Upon biotin treatment, we observed the gradual translocation of GFP fluorescence from the ER to the Golgi in parental and gas2l1−/− cells (Figure 2B). However, quantifying the colocalization of SBP-GFP-ShhN and the Golgi marker TGN46 revealed a significant reduction in the accumulation rate of SBP-GFP-Shh at the Golgi in gas2l1−/− cells compared with parental controls.

Collectively, our results establish GAS2L1 as a critical regulator of Golgi-nucleated microtubule assembly, while confirming its dispensable role in centrosomal microtubule dynamics. The impairment of Golgi-mediated microtubule biogenesis leads to defective ER-to-Golgi transport, thereby defining a specific role for GAS2L1 in intracellular trafficking dependent on Golgi-nucleated microtubules.

GAS2L1 localizes to the Golgi complex

We set out to examine whether GAS2L1 is localized on Golgi complexes. Using an optimized immunostaining protocol developed in previous studies, we enhanced sensitivity for detecting Golgi-resident proteins (e.g., γ-Tubulin, DNA polymerase δ catalytic subunit) (Wang et al., 2014; Shen et al., 2017; Au et al., 2023). Using this method, we observed that GAS2L1 localized to punctate structures on Golgi membranes labeled by GM130, as validated by fluorescence intensity profiling (Figure 3A). The specificity of GAS2L1 staining was confirmed by the absence of detectable signal in gas2l1−/− cells (Figure 3A).

FIGURE 3:

FIGURE 3:

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GAS2L1 localizes to the Golgi complex. (A) RPE-1 cells were preextracted, fixed, and costained with antibodies against GM130 and GAS2L1. DNA was labeled by Hoechst 33258. Boxed areas were enlarged, and intensity profiles along defined linear regions were generated; scale bar, 10 µm. (B) PNS of RPE-1 cells was fractionated by equilibrium centrifugation over a sucrose gradient (0.5–1.6 M). Membranes were pelleted from each gradient fraction for immunoblotting.

To further validate the association of GAS2L1 with the Golgi complex, we performed subcellular fractionation followed by immunoblotting. Using established protocols (Wang et al., 2006; Tang et al., 2010; Shen et al., 2017), we isolated the post-nuclear supernatant (PNS) from RPE-1 cell extracts and fractioned it by centrifugation through a sucrose gradient. We subsequently collected the membranes from each fraction via centrifugation and performed immunoblotting. Our results revealed that GAS2L1 cofractionated with the Golgi proteins CLASP2 and GOLPH3, which showed a slightly overlapping pattern with the ER marker CLIMP63 (Figure 3B). These findings corroborate our immunostaining results, further indicating that GAS2L1 is present on Golgi complexes.

GAS2L1 binds to CLASP1/2

Several proteins have been identified as playing roles in the organization of microtubules on Golgi complexes, including AKAP450, MMG8 (myomegalin variant 8), CDK5RAP2, and CLASP1/2 (CLASP1 and CLASP2) (Efimov et al., 2007; Miller et al., 2009; Wang et al., 2014; Wu et al., 2016). To explore the potential interactions between GAS2L1 and these proteins, we conducted an immunoprecipitation assay using transiently expressed proteins. We found that GAS2L1 specifically coimmunoprecipitated with CLASP1 and CLASP2 (Figure 4A), indicating an interaction between GAS2L1 and the CLASP proteins. To further characterize the interaction, we mapped the CLASP1/2-binding domain of GAS2L1 using a pulldown assay. The carboxy-terminal fragment (residues 369–681) of GAS2L1 specifically interacted with both CLASP1 and CLASP2, as demonstrated by immunoblotting of pulldown products (Figure 4, B and C). In contrast, the GAS2L1 construct 1−304, which comprises the CH and GAR domains, did not bind to the CLASP proteins (Figure 4, B and C). Truncation of 369−681 to 369−540 did not affect the binding, but further truncation to 369−520 abolished the binding activity (Figure 4, B and C). These results indicate that CLASP1 and CLASP2 are associated with GAS2L1 through the 369−540 region.

FIGURE 4:

FIGURE 4:

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GAS2L1 binds to CLASP1/2. (A) HEK293T cells were cotransfected by plasmids encoding GFP-CLASP1α, GFP-CLASP2α, or GFP-CLASP2, along with either FLAG-GAS2L1 or empty FLAG-vector. After anti-FLAG immunoprecipitation, an aliquot of the immunoprecipitates (50%) and cell lysate inputs (5%) was analyzed by anti-GFP and anti-FLAG immunoblotting. (B and C) Bio-TEV-FLAG–tagged GAS2L1 and its fragments were coexpressed with GFP-CLASP1α (B) or GFP-CLASP2 (C) in HEK293T cells for streptavidin pulldown. An aliquot of the pulldowns (50%) and cell lysate inputs (5%) was immunoblotted for GFP and FLAG. (D) Structural analysis of GAS2L1-CLASP2 interaction by ColabFold. The computational model shows GAS2L1 498–540 (red and pink) and CLASP2 645–890 (blue and light blue). Interacting helical regions of GAS2L1 and CLASP2 are highlighted in pink and light blue, respectively.

We next investigated the GAS2L1-binding region of CLASP2 via copulldown assays using GAS2L1(369–681) and truncated CLASP2 fragments. Among tested constructs, only CLASP2(450–930), encompassing the TOG2 domain, retained binding activity, whereas further truncations abolished interaction (Supplemental Figure S2). To characterize this interface structurally, we used ColabFold (Mirdita et al., 2022), an AlphaFold2-based platform optimized for protein complex prediction. The resulting model predicts that residues 511–538 of GAS2L1 form a helical structure that interacts with a helical segment (residues 816–830) within the TOG2 domain of CLASP2 (Figure 4D).

Neither GAS2L1 nor CLASP1/2 are known to localize to mitochondria (Akhmanova et al., 2001; Goriounov et al., 2003; Maiato et al., 2003; Mimori-Kiyosue et al., 2005; 2006; Wittmann and Waterman-Storer, 2005; Pereira et al., 2006; Bratman and Chang, 2007; Efimov et al., 2007; Miller et al., 2009; Beffert et al., 2012; Grimaldi et al., 2014; Au et al., 2017; van de Willige et al., 2019). To test the interaction between GAS2L1 and CLASP2 in cells, we transiently expressed Tom20–GAS2L1 (a fusion protein of the mitochondrial outer membrane receptor Tom20 and GAS2L1) or empty Tom20 vector. Immunostaining revealed that Tom20–GAS2L1 was efficiently targeted to mitochondria. Notably, CLASP2 showed significant enrichment on mitochondria in Tom20–GAS2L1-expressing cells, with fluorescence intensity profiles matching those of Tom20–GAS2L1 (Figure 5), indicating that CLASP2 is recruited to mitochondria through binding to GAS2L1.

FIGURE 5:

FIGURE 5:

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CLASP2 is recruited to mitochondria upon expression of Tom20-GAS2L1. RPE-1 cells transiently expressing the Tom20 vector or Tom20-GAS2L1 were stained for CLASP2 (anti-CLASP2), Tom20-GAS2L1 (anti-GAS2L1), mitochondria (MitoTracker Red CMXRos), and nuclear DNA (Hoechst 33258). Fluorescent intensity profiles along defined linear regions were quantified; scale bar, 10 µm.

GAS2L1–CLASP2 interaction participates in microtubule organization at the Golgi

First, we verified that overexpression of the CLASP1/2-binding fragment (369–540) of GAS2L1 disrupts the association between GAS2L1 and CLASP1/2. Through coimmunoprecipitation assays of ectopically expressed GAS2L1 and CLASP2 in the presence and absence of overexpressed GAS2L1 369–540, we found that the fragment 369–540 specifically interfered with the interaction between GAS2L1 and CLASP2 (Figure 6A).

FIGURE 6:

FIGURE 6:

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Disruption of GAS2L1–CLASP1/2 interaction affects Golgi-associated microtubule growth. Microtubule regrowth following nocodazole washout was performed on RPE-1 cells overexpressing the GAS2L1 fragments 1–304 or 369–540 (FLAG-tagged). The cells were then stained for the Golgi (anti-TGN46), microtubules (anti–α-Tubulin), FLAG (anti-FLAG), and nuclei (Hoechst 33258). Golgi-derived microtubules were counted from four independent experiments: n = 51 (MOCK), 48 (GAS2L1 1–304), and 52 (GAS2L1 369–540). Data are presented as mean ± SD. ****, p < 0.0001; n.s., not significant; Student's t test; scale bar, 10 µm.

Next, we investigated whether disruption of the GAS2L1–CLASP2 complex affects the organization of Golgi-derived microtubules using a regrowth assay. Notably, overexpression of 369–540 reduced the regrowth of Golgi-derived microtubules by 52.3% compared with controls (Figure 6B). As a negative control, we overexpressed the GAS2L1 1–304 fragment, which lacks binding activity for CLASP1/2 (Figure 4, B and C). This control expression did not significantly impact Golgi-based microtubule regrowth (Figure 6B), confirming the specificity of the 369–540 fragment's effect. Collectively, these findings demonstrate that the interaction between GAS2L1 and CLASP1/2 is critical for the proper organization of microtubules at the Golgi complex.

DISCUSSION

Previous studies have demonstrated that GAS2L1 plays a crucial role in centrosome disjunction during late G2, a process regulated by its Nek2-mediated phosphorylation at the centrosome (Au et al., 2017; 2020). In addition to its centrosomal functions, which are governed by its autoinhibitory mechanisms, our current study identifies a previously undescribed role for GAS2L1 in Golgi assembly and morphogenesis, specifically through its regulation of Golgi-derived microtubules. The precise mechanisms underlying the nucleation and stabilization of these microtubules remain poorly understood. Here, we propose a model in which GAS2L1 regulates Golgi-derived microtubules via its interaction with CLASP1/2, thereby promoting their formation and stability (Figure 7). Furthermore, our findings suggest that GAS2L1 executes its Golgi-related functions by localizing to the Golgi membrane, establishing it as a crucial regulator of Golgi organization.

FIGURE 7:

FIGURE 7:

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Schematic model depicting the role of GAS2L1 in Golgi-organized microtubules.

We demonstrate that GAS2L1 is a Golgi-associated protein and regulates Golgi functions by mediating Golgi-derived microtubules through its interaction with CLASP1/2. GAS2L1 contains CH and GAR domains, which enable microtubule and actin binding, respectively (Goriounov et al., 2003). Notably, overexpression of GAS2L1 leads to Golgi fragmentation (Supplemental Figure S3), reminiscent of the overexpression effects of FHDC1 and ACF7, microtubule- and actin-associating proteins that are involved in the control of Golgi morphology by coordinating actin and microtubule dynamics (Kodama et al., 2003; Lin et al., 2005; Copeland et al., 2016). Therefore, GAS2L1 may function similarly to FHDC1 and ACF7 as a mediator of microtubule–actin interactions at the Golgi. Given that GAS2L1 also contains an SxIP (Ser-x-Ile-Pro) motif, which allows it to bind end-binding proteins (EB), it is plausible that GAS2L1 collaborates with EB1 and EB3 to regulate Golgi structural organization by controlling microtubule dynamics at the Golgi (Yang et al., 2017).

Our findings demonstrate that GAS2L1 regulates Golgi-derived microtubules through its association with CLASP1/2. Specifically, the interaction between endogenous GAS2L1 and CLASP2 is disrupted by the overexpression of the CLASP2-binding fragment of GAS2L1 (residues 369–540), indicating that GAS2L1’s function at the Golgi is dependent on its interaction with CLASP1/2 (Figure 6). Furthermore, we identify TOG2 (TOG-like domain 2) of CLASP1/2 as the binding site for GAS2L1 (Supplemental Figure S2). Because TOG2 is known to suppress microtubule catastrophes and is regulated by autoinhibition (Aher et al., 2018), we propose that GAS2L1 stabilizes Golgi-derived microtubules by interacting with TOG2, potentially relieving its autoinhibition.

GAS2L1 and CLASP1/2 share functional similarities in promoting and stabilizing Golgi-derived microtubules (Miller et al., 2009). Both proteins also facilitate cross-talk between the microtubule and actin cytoskeletons (Goriounov et al., 2003; Tsvetkov et al., 2007). Disrupting either of these proteins, through gas2l1 knockout or CLASP1/2 depletion, impairs the growth of Golgi-derived microtubules, resulting in defective Golgi reassembly and impaired ER-to-Golgi trafficking (Figures 1B and 2) (Efimov et al., 2007; Miller et al., 2009). These findings underscore the role of GAS2L1 in association with CLASP1/2 in maintaining Golgi structure and function. Notably, gas2l1 knockout resulted in severe defects in Golgi reassembly, evidenced by the widespread cytoplasmic dispersion of Golgi ministacks at 40 and 60 min (Figure 1B). This suggests that while GAS2L1 collaborates with CLASP1/2 to promote the assembly of Golgi-derived microtubules, GAS2L1 also exerts CLASP1/2-independent functions critical for Golgi assembly. Collectively, these results position GAS2L1 as a dual-mode regulator of Golgi architecture, acting both in concert with CLASP1/2 and through distinct molecular mechanisms.

During the cell cycle, the Golgi apparatus undergoes dynamic remodeling, fragmenting into vesiculated membranes during mitotic entry and reassembling into a perinuclear network upon mitotic exit (Shorter and Warren, 2002; Altan-Bonnet et al., 2004). Integrating prior literature with our findings, we propose a model wherein postmitotic Golgi-associated microtubule nucleation by γTuRC is spatiotemporally coordinated with stabilization by GAS2L1 and CLASP1/2 (Figure 7). GAS2L1- and CLASP1/2-stabilized microtubules facilitate the clustering of Golgi mini-stacks into larger Golgi structures. Concurrently, centrosomal microtubules pull these GAS2L1 and CLASP1/2-bound Golgi clusters toward the centrosome, ultimately leading to the reassembly of an intact Golgi network (Miller et al., 2009).

Defects in Golgi-derived microtubules have been linked to various types of cancer, and diabetes (Petrosyan, 2015; Trogden et al., 2019). Notably, GAS2L1 expression is downregulated in acute myeloid leukemia and intracranial ependymomas (Desmond et al., 2007; de Bont et al., 2008). Based on these findings and the deficits in Golgi-based MT organization observed in our study, it is tempting to propose that GAS2L1 may represent a novel pharmaceutical target for disorders associated with the Golgi complex acting as a microtubule organizing center.

Beyond its role in postmitotic Golgi reassembly, GAS2L1, together with CLASP1/2, plays a critical role in regulating Golgi-derived microtubules, which are essential for Golgi organization, polarized transport, and directional cell migration. Our findings delineate a novel mechanism by which GAS2L1 acts as a regulator of Golgi-derived microtubules and underscore its interplay with CLASP1/2 in preserving Golgi structural integrity and functionality.

MATERIALS AND METHODS

Plasmids

The construction of plasmids for the expression of human GAS2L1 has been described previously (Au et al., 2017; 2020). GAS2L1 fragments were subcloned into FLAG-CMV2, GFP, and biotinylation vectors (Bio-2 × TEV-FLAG-C1) using standard molecular cloning techniques. The expression constructs for GFP-CLASP1 and GFP-CLASP2 were generously provided by Dr. Anna Akhmanova (Utrecht University, the Netherlands) (Akhmanova et al., 2001; Mimori-Kiyosue et al., 2005). The plasmid Str-KDEL_SBP-EGFP-ShhN was constructed as described previously (Huang et al., 2021).

Antibodies

Various primary antibodies were used in this study, including sheep anti-TGN46 (AHP500G; Bio-Rad, 1:500 [IF]), mouse anti-GM130 (610822; BD Biosciences, 1:500 [IF]), rabbit anti-GOLPH3 (ab98023; Abcam, 1:2000 [WB]), mouse anti–α-Tubulin (DM1A; T6199; Sigma-Aldrich, 1:100 [IF]), mouse anti-CLIMP-63 (G1/296; ALX-804-604-C100; ENZO Life Sciences, 1:1000 [WB]), rat anti-CLASP2 (monoclonal KT68; ab95373; Abcam, 1:100 [IF]) and anti-FLAG (mouse M2; F1804 or rabbit polyclonal; F7425; Sigma-Aldrich, 1:2000 [WB], 1:100 [IF]). Polyclonal antibodies against GAS2L1 (1:5000 [WB], 1:100 [IF]), NEDD1 (1:200 [IF]), and GFP were prepared as previously reported (Au et al., 2017), while antibodies against CLASP1 (1:100 [IF]) and CLASP2 (1:100 [IF]) were used as described previously (Akhmanova et al., 2001; Mimori-Kiyosue et al., 2005).

Cell cultures and treatments

Cell lines used in this study were obtained from the ATCC. HEK293T cells were maintained in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS and 100 U/mL penicillin–streptomycin mix (Sigma-Aldrich). hTERT RPE-1 cells were cultured in a 1:1 ratio of DMEM and Ham's F12 (Thermo Fisher Scientific) also supplemented with 10% FBS and 100 U/mL penicillin–streptomycin mix. gas2l1−/− RPE-1 cells and the gas2l1−/− sublines with the tet-on system for inducible expression of GFP-GAS2L1 were generated previously (Au et al., 2017; 2020). All cells were grown at 37°C under 5% CO2 and were plated on glass coverslips for 24 h before conducting experiments. Plasmids were transfected into HEK293T cells using polyethylenimine (Polysciences) and into RPE-1 cells using either Lipofectamine 2000 (Thermo Fisher Scientific) or FuGENE HD transfection reagent (Promega).

To induce microtubule depolymerization and Golgi fragmentation, cells were treated with 2.5 µg/ml nocodazole (Sigma-Aldrich) for 2 h (Miller et al., 2009; Shen et al., 2017). Microtubule regrowth and Golgi reassembly were initiated by removing nocodazole, washing the cells with ice-cold medium, and then transferring the coverslips to 37°C prewarmed medium. Alternatively, microtubule depolymerization was induced by incubating cells on ice-cold water for 1 h and microtubule regrowth was initiated by placing the cells into 37°C prewarmed medium (Au et al., 2023). Cells were subsequently fixed with 4% paraformaldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES, pH 6.9, 10 mM EGTA, and 1 mM MgCl2) containing 0.5% Triton X-100 for 15 min at room temperature (Choi et al., 2010; Shen et al., 2017; Au et al., 2020; 2023).

In Golgi reassembly assays, cells grown on coverslips were incubated at 37°C in medium containing nocodazole (2.5 µg/ml) for 2 h (Miller et al., 2009; Shen et al., 2017). After rinsing with ice-cold medium, Golgi reassembly was initiated by transferring the cells to prewarmed medium. The reassembly process was terminated at various timepoints by fixing the cells with absolute methanol at −20°C.

Immunofluorescence microscopy and image analysis

Cells grown on 18-mm coverslips were fixed with absolute methanol for 10 min at −20°C, unless otherwise stated. Following fixation, the cells were blocked with 3% BSA in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.47 mM KH2PO4, pH 7.4) for 40 min at room temperature or overnight at 4°C. Cells were then stained sequentially with primary antibody and a fluorophore-conjugated secondary antibody (Fluorophores: Alexa-Fluor 488/568/594/647; Invitrogen) at room temperature. DNA was stained with Hoechst 33258 (Sigma-Aldrich), and F-actin was stained with Alexa Fluor 647-coupled phalloidin (Invitrogen). The coverslips were mounted on glass slides, and fluorescence images were captured using a Zeiss Axio Observer Z1 microscope (Carl Zeiss, Jena, Germany) equipped with an ORCA-Flash 4.0 camera (Hamamatsu, Japan), a Leica SP8 confocal microscope paired with HyD Sp GaAsP detectors (Leica Microsystems) or a LSM 980 confocal microscope paired with AiryScan GaAsP detectors (Carl Zeiss). Images were processed and analyzed using the microscope software ZEN 3.1 (Blue edition, Carl Zeiss).

To visualize GAS2L1 at the Golgi complex, RPE-1 cells were extracted in saponin buffer (100 mM PIPES-KOH, pH 6.9, 2 M glycerol, 5 mM MgCl2, 2 mM EGTA, and 1% saponin) for 30 min, followed by fixation in methanol at −20°C for 5 min (Wang et al., 2014; Shen et al., 2017; Au et al., 2023). The cells were stained with a rabbit anti-GAS2L1 antibody, and then sequentially stained with two secondary antibodies: Alexa-Fluor–conjugated goat anti-rabbit and Alexa-Fluor–conjugated donkey anti-goat antibodies (Thermo Fisher Scientific). To visualize mitochondria, cells were incubated with 1 µM MitoTracker Red CMXRos (#9082, Cell Signaling Technology) in warm medium for 30 min before fixation.

The shape of Golgi complexes was analyzed based on TGN46 staining to determine the circularity index using the ImageJ circularity plugin (https://imagej.nih.gov/ij/plugins/circularity.html), where circularity is calculated as 4 × π × (area/perimeter2) (Miller et al., 2009). A circularity index of one corresponds to a perfect circle.

The spreadness of Golgi complexes was analyzed based on TGN46 staining to determine the solidity/packing index using the ImageJ solidity plugin (https://imagej.net/ij/plugins/3d-convex-hull/index.html), where solidity is calculated as area/convex area. A solidity index of one corresponds to a circle without any concave area.

To analyze Golgi reassembly after fragmentation, the size of anti-TGN46–labeled Golgi particles was measured using the ImageJ analyze particles function, following background subtraction and standardized entropy thresholding (Miller et al., 2009).

Golgi membrane isolation

Golgi membranes were isolated at 4°C using an equilibrium gradient method (Wang et al., 2006; Tang et al., 2010; Shen et al., 2017; Au et al., 2023). RPE-1 cells were homogenized in homogenization buffer (10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 5 mM EDTA, and Roche Protease Inhibitor Cocktail). The lysates were clarified by centrifugation at 1000 × g for 10 min at 4°C and the PNS was collected. To prepare a discontinuous sucrose gradient, five layers of sucrose (450 µl of 1.6 M, 450 µl of 1.4 M, 450 µl of 1.2 M, 350 µl of 0.8 M, and 250 µl of 0.5 M sucrose in the buffer of 10 mM Tris-HCl, pH 7.4, and 5 mM EDTA) were sequentially overlaid from the bottom to the top of a centrifuge tube (TLS55 rotor, Beckman). The PNS (250 µl) was carefully overlaid on top of this gradient and centrifuged at 90,000  ×  g for 3.5 h, after which the gradient was collected into 12 fractions from the top to the bottom. Each fraction was diluted threefold in 10 mM Tris-HCl (pH 7.4) and centrifuged at 180,000  ×  g for 1 h, yielding pellets of isolated membranes for subsequent immunoblotting analysis.

Protein-binding assays

Cells were lysed using lysis buffer (50 mM HEPES, pH7.4, 0.5% Triton X-100, 150 mM NaCl, 5 mM MgCl2, 5 mM NaF, 1 mM dithiothreitol, and Roche Complete Protease Inhibitor Cocktail). The lysates were then clarified by centrifugation (16,000 × g, 15 min) at 4°C and subjected to immunoprecipitation using anti-FLAG M2 Affinity Gel (Sigma-Aldrich) for 3 h with rotation at 4°C. The beads were washed extensively with washing buffer (lysis buffer without dithiothreitol and the protease inhibitor Cocktail) and then boiled at 95°C for SDS–PAGE and immunoblotting.

In the biotin pulldown assay, HEK293T cells expressing GAS2L1 tagged with Bio-2 × TEV-FLAG and the biotin ligase BirA were incubated in a 1:1 ratio of DMEM/Ham's F10 medium for 24 h before cell lysis. Biotin-conjugated proteins were captured using Dynabeads M-280 Streptavidin (Thermo Fisher Scientific) by rotating at 4°C for 3 h.

Structural modeling of GAS2L1–CLASP2 interaction

The heterodimeric structure of the GAS2L1–CLASP2 complex was modeled using ColabFold (Mirdita et al., 2022). Protein sequences corresponding to GAS2L1(369–540) and CLASP2(450–930) were input using default parameters for structure prediction. ColabFold generated five structural models, all consistently predicted an identical binding interface between GAS2L1 and CLASP2. A representative model was presented in the paper.

Wound-healing assay

Wound-healing assay was performed as described previously (Shen et al., 2017). In brief, parental and gas2l1−/− RPE-1 cells were cultured in 6-well plates until confluence. The cells were serum-starved in FBS-free medium for 24 h, followed by creating a scratch at the well center using a micropipette tip. After a 20-min recovery period, medium was supplemented with 10% FBS and scratch closure was imaged at specified timepoints under a light microscope.

RUSH assay

The RUSH assay was adapted from a previous publication (Huang et al., 2021), with modifications. Parental and gas2l1−/− RPE-1 cells were cultured on coverslips and transfected with the Str-KDEL_SBP-EGFP-ShhN plasmid. To induce the release of SBP-GFP-Shh from the ER, the cells were treated with 100 ng/µl cycloheximide (Sigma-Aldrich) and 40 µM D-biotin (Sigma-Aldrich), then fixed with 4% paraformaldehyde at specified timepoints. Fixed cells were immunostained with anti-GFP and anti-TGN46 antibodies for fluorescent microscopy analysis.

Statistical analysis

Quantification data were derived from at least three independent experiments. Statistical analyses were performed using GraphPad Prism, with specific tests detailed in the figure legends. For two-group comparisons, unpaired Student's t tests were used to calculate p values. Statistical significance was denoted as p < 0.05, p < 0.01, p < 0.001, or p < 0.0001, as indicated in the figures.

Supplementary Material

mbc-36-ar109-s001.pdf (763.5KB, pdf)

ACKNOWLEDGMENTS

We thank Dr. Anna Akhmanova (Utrecht University, The Netherlands) for providing the DNA constructs of CLASP1/2. The antibodies against CLASP1 and CLASP2 were gifts from Dr. Anna Akhmanova (Utrecht University, The Netherlands), Dr. Niels Galjart (Erasmus Medical Center, The Netherlands), and Dr. Fedor Severin (Moscow State University, Russia). We also thank Dr. Yuehong Shen (The Hong Kong University of Science and Technology) for sharing immunostaining techniques and Zechen Lu (The Hong Kong University of Science and Technology) for the technical support on the RUSH assay. This work was supported by grants from the Research Grants Council (Theme-based Research Scheme T13-605/18-W and General Research Fund) of Hong Kong, HKUST Aging Research Grant (Z1056), Guangdong Provincial Key Lab of Integrated Communication (2023B1212010007), Guangdong Provincial Innovation Team Program for Higher Education Institutions (2024KCXTD008), Guangzhou-HKUST(GZ) Joint Funding Scheme (2025A03J3636), and Shenzhen Science and Technology Committee Research Grant (SGDX20210823103200005). F.K.C.A. was supported by the Postdoctoral Fellowship Scheme (PDFS2021-6S03) and K.T.D.L. was supported by the Hong Kong PhD Fellowship Scheme (PF21-60517) from the Research Grants Council of Hong Kong.

Abbreviations used:

PNS

post-nuclear supernatant

γTuRC

γ-Tubulin ring complex.

Footnotes

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E25-02-0079) on July 16, 2025.

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Supplementary Materials

mbc-36-ar109-s001.pdf (763.5KB, pdf)

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