ARL3 Mediates BBSome Ciliary Turnover by Promoting Its Outward Diffusion through the Transition Zone

Liu, Yanxia; Sun, Wei-Yue; Xue, Bin; Zhang, Rui-Kai; Li, Wenjuan; Xie, Xixian; Fan, Zhen‐Chuan

doi:10.1101/2021.11.19.469163

Cited by 3 publications

(29 citation statements)

References 72 publications

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“…To discern if this BBSome buildup was caused by its altered IFT behavior, we generated strains rabl2 bbs8 RABL2-HA BBS8-YFP and rabl2 bbs8 RABL2 D76G -HA BBS8-YFP ( SI Appendix , Fig. S3 F ) (33). Two strains had BBS8 replaced with BBS8-YFP ( SI Appendix , Fig.…”

Section: Resultsmentioning

confidence: 99%

“…5 B and SI Appendix , Fig. S4 C ) (33). We noticed that the loss of RABL2 in cilia as shown by rabl2 RABL2 D76G -HA-YFP caused the BBSome to accumulate at the ciliary base (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…5 B and C ). We have known previously that ARL3 GDP , upon diffusing into cilia by binding the ciliary membrane, rapidly converts into ARL3 GTP for detaching from the ciliary membrane so as to reside in the ciliary matrix (33). Since ARL3 resided in the ciliary matrix of CC-5325 and rabl2 RABL2-HA-YFP but remained to bind the ciliary membrane of rabl2 RABL2 D76G -HA-YFP and rabl2 RABL2 Q83L -HA-YFP , we conclude that the loss of RABL2 in cilia and the replacement of RABL2 with RABL2 Q83L both can prevent ARL3 GDP from converting to ARL3 GTP in cilia (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In the presence of DTT that separates the BBSome from IFT-B1 (33), GTPγS addition failed to confer RABL2-HA-YFP to immunoprecipitate the BBSome and ARL3 (Fig. 7B).…”

Section: Rabl2 Gdp Promotes Outward Tz Passage Of the Bbsome Via Acti...mentioning

confidence: 99%

“…At the ciliary tip, BBSomes remodel prior to loading onto retrograde IFT trains for trafficking to the ciliary base (19,(26)(27)(28)(29)(30)(31)(32). Upon reaching the proximal ciliary region right above the transition zone (TZ), cargo-laden BBSomes shed from retrograde IFT trains and diffuse through the TZ and out of cilia (33). Therefore, defects in BBSome assembly and ciliary cycling cause loss or retention of signaling proteins in the ciliary membrane (22,23,(34)(35)(36)(37).…”

Section: Introductionmentioning

confidence: 99%

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RABL2 Regulates Ciliation via Controlling IFT-B1 Basal Body Recruitment and ARL3-mediated BBSome Ciliary Retrieval

Zhang

Liu

Sun

et al. 2022

Preprint

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Highly conserved intraflagellar transport (IFT) trains and certain small GTPases coordinate to direct ciliation and to maintain the ciliary dynamics of signaling molecules via the IFT cargo adaptor BBSome. Unlike murine Rab-like 2 (RABL2) GTPase that enters cilia to drive outward transition zone (TZ) passage of the BBSome, human orthologue fails to enter cilia but resides at the ciliary base, indispensable for ciliation. However, mechanisms underlying how RABL2 regulates ciliation and BBSome barrier passage remain elusive. Here, we show that Chlamydomonas RABL2 regulates basal body targeting of the IFT-B1 subcomplex component of IFT trains as a RABL2-specific effector, mediating ciliation via controlling IFT-B1 basal body amount available for assembling anterograde IFT trains. RABL2GTP binds IFT-B1 to perform IFT; sheds from retrograde IFT trains at the proximal ciliary region right above the TZ; and converts to RABL2GDP rapidly. Next, RABL2GDP activates the ciliary membrane anchored Arf-like 3 (ARL3) GTPase (ARL3GDP) as a ARL3-specific guanine nucleotide exchange factor. Upon detaching from the ciliary membrane, the active ARL3GTP recruits its BBSome effector, autonomous of retrograde IFT train association, to move cross the TZ for ciliary retrieval. This ensures proper BBSome ciliary turnover for maintaining phototactic response of Chlamydomonas cells. For finishing RABL2 ciliary cycle, RABL2GDP passes the TZ for ciliary retrieval by loading onto the ARL3GTP/BBSome as a BBSome cargo. Our data thus propose that RABL2 mediates ciliation and BBSome ciliary retrieval simultaneously but via distinct molecular pathways.Significance statementIntraflagellar transport (IFT) and its cargo adaptor BBSome are indispensable for ciliation and ciliary singling. Rab-like 2 (RABL2) GTPase mediates ciliation and outward transition zone (TZ) passage of BBSomes with mechanisms yet to be determined. Here, we report that RABL2 decides ciliation by controlling the basal body amount of its effector IFT-B1 available for the assembly of anterograde IFT trains. RABL2GTP cycles through cilia as an IFT-B1 cargo; sheds from IFT at the ciliary base; and undergoes nucleotide exchange for activating ARL3 as an ARL3-specfic guanine nucleotide exchange factor. ARLGTP recruits IFT-shed BBSomes to pass the TZ for ciliary retrieval. RABL2GDP exists cilia via ARL3GTP/BBSome as a BBSome cargo. Therefore, RABL2 functions both outside and inside cilia for initiating IFT and BBSome ciliary retrieval, respectively.

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Section: Resultsmentioning

confidence: 99%

“…5 B and SI Appendix , Fig. S4 C ) (33). We noticed that the loss of RABL2 in cilia as shown by rabl2 RABL2 D76G -HA-YFP caused the BBSome to accumulate at the ciliary base (Fig.…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…In the presence of DTT that separates the BBSome from IFT-B1 (33), GTPγS addition failed to confer RABL2-HA-YFP to immunoprecipitate the BBSome and ARL3 (Fig. 7B).…”

Section: Rabl2 Gdp Promotes Outward Tz Passage Of the Bbsome Via Acti...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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RABL2 Regulates Ciliation via Controlling IFT-B1 Basal Body Recruitment and ARL3-mediated BBSome Ciliary Retrieval

Zhang

Liu

Sun

et al. 2022

Preprint

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Mechanisms underlying morphological and functional changes of cilia in fibroblasts derived from patients bearing ARL3^T31A and ARL3^T31A/C118F mutations

Zhang,

Yao,

Zhang

et al. 2024

The FASEB Journal

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ARL3 is essential for cilia development, and mutations in ARL3 are closely associated with ciliopathies. In a previous study, we observed distinct phenotypes of retinal dystrophy in patients with heterozygous ARL3T31A and compound heterozygous ARL3T31A/C118F mutations, indicating that different mutation types may exert diverse effects on their functions. Here, we generated transformed immortal fibroblast cells from patients carrying heterozygous ARL3T31A and compound heterozygous ARL3T31A/C118F mutations, and systematically evaluated their cilia morphology and function, which were further validated in ARPE‐19 cells. Results showed that both ARL3T31A and ARL3T31A/C118F mutations led to a decrease in cilium formation. The ARL3T31A/C118F mutations caused significantly elongated cilia and impaired retrograde transport, whereas the ARL3T31A mutation did not induce significant changes in fibroblasts. RNA‐sequencing results indicated that compared to ARL3T31A, ARL3T31A/C118F fibroblasts exhibited a higher enrichment of biological processes related to neuron projection development, tissue morphogenesis, and extracellular matrix (ECM) organization, with noticeable alterations in pathways such as ECM‐receptor interaction, focal adhesion, and TGF‐β signaling. Similar changes were observed in the proteomic results in ARPE‐19 cells. Core regulated genes including IQUB, UNC13D, RAB3IP, and GRIP1 were specifically downregulated in the ARL3T31A/C118F group, and expressions of IQUB, NPM2, and SLC38A4 were further validated. Additionally, IQUB showed a rescuing effect on the overlong cilia observed in ARL3T31A/C118F fibroblasts. Our results not only enhance our understanding of ARL3‐related diseases but also provide new insights into the analysis of heterozygous and compound heterozygous mutations in genetics.

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Chlamydomonas RABL4/IFT27 Mediates Phototaxis via Promoting BBSome-dependent Ciliary Export of Phospholipase D

Liu

Xue

Fan

2022

Preprint

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Phospholipase D (PLD) interacts with the BBSome for loading onto retrograde intraflagellar transport (IFT) trains to exit cilia in Chlamydomonas reinhardtii. PLD ciliary retention and depletion of Rab-like 4 (RABL4) GTPase IFT27 cause the same non-phototactic phenotype but not impair IFT and ciliation. Here, we show that the IFT-B1 subunit IFT27 binds its partner IFT25 to form the heterodimeric IFT25/27 in an IFT27 nucleotide state-independent manner. IFT25/27, IFT-A, and IFT-B are irrelevant for maintaining the stability of one another. GTP-loading onto IFT27 enhances the IFT25/27 affinity for IFT-B1 in cytoplasm, while GDP-loaded IFT27 does not prevent IFT25/27 from entering and cycling through cilia by integrating into IFT-B1. Upon at the ciliary tip, IFT25/27 cycles on and off IFT-B1 and this process is irrelevant with the nucleotide state of IFT27. During BBSome remodeling at the ciliary tip, IFT25/27 promotes BBSome reassembly independent of IFT27 nucleotide state, making post-remodeled BBSomes available for PLD to interact with. Therefore, IFT25/27 facilitates BBSome-dependent PLD export from cilia via controlling availability of intact BBSomes at the ciliary tip, providing a regulatory mechanism for IFT27 to mediate phototaxis in C. reinhardtii.

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ARL3 Mediates BBSome Ciliary Turnover by Promoting Its Outward Diffusion through the Transition Zone

Cited by 3 publications

References 72 publications

RABL2 Regulates Ciliation via Controlling IFT-B1 Basal Body Recruitment and ARL3-mediated BBSome Ciliary Retrieval

RABL2 Regulates Ciliation via Controlling IFT-B1 Basal Body Recruitment and ARL3-mediated BBSome Ciliary Retrieval

Mechanisms underlying morphological and functional changes of cilia in fibroblasts derived from patients bearing ARL3^T31A and ARL3^T31A/C118F mutations

Chlamydomonas RABL4/IFT27 Mediates Phototaxis via Promoting BBSome-dependent Ciliary Export of Phospholipase D

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ARL3 Mediates BBSome Ciliary Turnover by Promoting Its Outward Diffusion through the Transition Zone

Cited by 3 publications

References 72 publications

RABL2 Regulates Ciliation via Controlling IFT-B1 Basal Body Recruitment and ARL3-mediated BBSome Ciliary Retrieval

RABL2 Regulates Ciliation via Controlling IFT-B1 Basal Body Recruitment and ARL3-mediated BBSome Ciliary Retrieval

Mechanisms underlying morphological and functional changes of cilia in fibroblasts derived from patients bearing ARL3T31A and ARL3T31A/C118F mutations

Chlamydomonas RABL4/IFT27 Mediates Phototaxis via Promoting BBSome-dependent Ciliary Export of Phospholipase D

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Mechanisms underlying morphological and functional changes of cilia in fibroblasts derived from patients bearing ARL3^T31A and ARL3^T31A/C118F mutations