Molecular insights into AGS3's role in spindle orientation: a biochemical perspective

Abstract

The intrinsic regulation of spindle orientation during asymmetric cell division depends on the evolutionarily conserved protein complex LGN (Pins)/NuMA (Mud)/Gα⋅GDP. While the role of LGN and its Drosophila orthologue Pins is well-established, the function of AGS3, the paralogue of LGN, in spindle orientation during cell division remains controversial. This study substantiates the contentious nature of AGS3's function through systematic biochemical characterizations. The results confirm the high conservation of AGS3 in its functional structural domains, similar to LGN, and its comparable ability to bind to partners including NuMA, Insc, and Gαi3⋅GDP. However, in contrast to LGN, AGS3 and the microtubule-binding protein NuMA are unable to form stable hetero-hexamers or higher-order oligomeric complexes that are pivotal for effective regulation of spindle orientation. It was found that this notable difference between AGS3 and LGN stems from the N-terminal sequence preceding the conserved TPR motifs, which spans ∼20 residues. Furthermore, our findings substantiate the disruptive effect of Insc on the oligomeric AGS3/NuMA complex, while showing no impact on the oligomeric LGN/NuMA complex. Consequently, Insc emerges as an additional regulatory factor that distinguishes the functional roles of AGS3 and LGN, leading to the impairment of AGS3's ability to actively reorient the mitotic spindle. These results elucidate the molecular basis underlying the observed functional disparity in spindle orientation between LGN and AGS3, providing valuable insights into the regulation of cell division at the molecular level.

Keywords: AGS3; LGN; NuMA; asymmetric cell division; spindle orientation.

Figure 1

AGS3-TPR and AGS3-GL interact with their partners similarly to LGN. (A) The domain organizations of LGN and AGS3 isoforms. (B) ITC-based binding affinities of the TPR domains of AGS3 and LGN to Insc and NuMA. The raw data and fits are shown in Supplementary Figure S1. (C) SEC analysis of the mixture of AGS3-TPR, NuMA (1886–1912), and Insc (18–66) at a 1:2:2 ratio. (D) ITC measurement of the binding of Insc (18–66) to the mixture of AGS3-TPR and NuMA (1886–1912). (E) ITC measurement of the binding of NuMA (1886–1912) to the mixture of AGS3-TPR and Insc (18–66). (F) ITC-based binding affinities of Gα_i3⋅GDP to the GL motifs and GoLoco domains of AGS3 and LGN. The raw data and fits are shown in Supplementary Figure S2.

Figure 2

Intramolecular interactions and auto-inhibited conformation of AGS3. (A) ITC measurement of the interaction between AGS3-TPR and AGS3-GL1234. (B) Schematic diagram of the domain organizations of FL-AGS3 and FL-LGN. (C) ITC measurement of the interaction between AGS3-GL1234 and FL-AGS3. (D) ITC measurement of the interaction between LGN-GL1234 and FL-LGN. (E) Sequence alignment of the GL motifs of AGS3 and LGN. The red triangles indicate the conserved phenylalanines. (F) Schematic diagram of the domain organizations of FL-AGS3-4E and FL-LGN-4E. (G) ITC-based binding affinities of FL-AGS3-4E and FL-LGN-4E mutants to Insc (18–66) and NuMA (1886–1912). The raw data and fits are shown in Supplementary Figure S4.

Figure 3

Long-form AGS3-TPR with additional N-terminal residues could form a multimeric complex with NuMA. (A) Schematic diagram of the domain organizations of various fragments of AGS3, LGN, and NuMA. (B) SEC analyses of the complexes formed between LGN-TPR, AGS3-TPR, or AGS3-L and NuMA (1847–1914). The dashed line indicates the elution volume of a 150-kDa globular protein marker. (C–E) SLS characterization of the MW of the LGN-TPR/NuMA (1847–1914) complex (C), AGS3-TPR/NuMA (1847–1914) complex (D), or AGS3-L/NuMA (1847–1914) complex (E). (F) SEC analyses of the complexes formed between LGN-TPR or AGS3-L and NuMA-CC. (G) SEC analyses of the complex formed between AGS3-L, LGN-TPR, and NuMA (1847–1914). Coomassie-stained SDS–PAGE gel to the right shows the protein composition of the elution profile.

Figure 4

Full-length LGN and AGS3 have lower capacity to form oligomeric complexes with NuMA. (A) Schematic diagram of the domain organizations of long isoforms FL-AGS3-L and FL-AGS3-L-4E. (B) SEC analyses of the complexes formed between FL-LGN or FL-LGN-4E and NuMA (1808–2001). The dashed line indicates the elution volume of a 150-kDa globular protein marker. (C) SLS characterization of the MW of the FL-LGN-4E/NuMA (1808–2001) complex. (D) SEC analyses of the complexes formed between FL-AGS3-L or FL-AGS3-L-4E and NuMA (1808–2001). (E) SLS characterization of the MW of the FL-AGS3-L-4E/NuMA (1808–2001) complex.

Figure 5

The impact of Insc on the oligomeric complexes formed between LGN or AGS3 and NuMA. (A) SEC analyses of the complexes formed between LGN-TPR or AGS3-L and NuMA (1847–1914) in the presence or absence of Insc (18–66). Coomassie-stained SDS–PAGE gels to the right show the protein composition of two elution profiles. (B) SEC analyses of the complexes formed between LGN-TPR or AGS3-L and NuMA-CC in the presence or absence of Insc (18–66). Coomassie-stained SDS–PAGE gels to the right show the protein composition of two elution profiles.

Figure 6

Different N-terminal sequences determine different capacities of AGS3 and LGN in forming multimeric complexes with NuMA. (A) Schematic diagram of the domain organizations of chimeric proteins AGS3^LGN and LGN^AGS3. (B) SEC analyses of the complexes formed between AGS3^LGN and NuMA (1847–1914) in the presence or absence of Insc (18–66). The gray box shows the void volume. (C) SEC analyses of the complexes formed between AGS3^LGN and NuMA-CC in the presence or absence of Insc (18–66). (D) SEC analyses of the complex formed between LGN^AGS3 and NuMA (1847–1914).

Figure 7

AGS3-L S−2Y mutant enhances hydrophobic interaction, facilitating the formation of a stable hexamer with NuMA. (A) Multisequence alignment of N-terminal sequences of LGN and AGS3 from five species, including human, mouse, Rattus norvegicus, Danio rerio, and Xenopus tropicalis. The red stars indicate the specific sequences of LGN and AGS3 utilized in this study, while the blue star denotes the sequence of LGN in the crystal structure of the LGN/NuMA hexamer (PDB: 6HC2). (B) Schematic representation of the domains of the mutant proteins AGS3-L S−2Y and LGN Y11S. (C) SEC analyses of the complexes formed between AGS3-L S−2Y and NuMA (1847–1914) in the presence or absence of Insc (18–66). (D) SEC analyses of the complexes formed between AGS3-L S−2Y and NuMA-CC in the presence or absence of Insc (18–66). The gray box denotes the void volume. (E) SEC analyses of the complex formed between LGN Y11S and NuMA (1847–1914). Coomassie-stained SDS–PAGE gels show the protein composition of the corresponding elution profiles.

Figure 8

A model illustrating the molecular mechanism underlying the distinct roles of LGN and AGS3 in regulating oriented cell divisions. (A) The paralogues LGN and AGS3 share a high degree of sequence similarity and exhibit similar characteristics in their interactions with NuMA and Insc. (B) The multimeric AGS3/NuMA complex exhibits reduced stability compared to the LGN/NuMA complex and is more susceptible to disruption by Insc.