Biology subjects

Fan, Z.-C.

Publications and source records attributed to Fan, Z.-C..

6 recordsLinked to original sources

bioRxiv2025

RAB23 Modulates Signaling Protein Ciliary Homeostasis through Promoting RAB18-mediated Inward BBSome Transition Zone Passage

As an intraflagellar transport (IFT) cargo adaptor, the BBSome targets the basal body region located just below the transition zone (TZ) before proceeding to cross the TZ for ciliary entry. This process of TZ crossing crucially governs BBSome presence and quantity available for signaling protein coupling within cilia, influencing the dynamic behavior of signaling proteins in cilia by controlling their ciliary removal. Our previous study revealed that the GTPase RAB18 binds to its BBSome effector in the basal bodies right below the TZ and recruits the BBSome to cross the TZ for ciliary entry by lateral transport between the plasma and ciliary membranes. However, the mechanism by which RAB18 is activated to accomplish this function has remained elusive. This study uncovers that, under physiological conditions, Chlamydomonas RAB23 in its GDP-bound state (RAB23-GDP) predominantly localizes to the same basal body region as RAB18-GDP and activates RAB18-GDP as a RAB18-specific guanine nucleotide exchange factor. RAB18-GDP attaches to the membrane and binds to the BBSome. This interaction occurs independently of anterograde IFT association, allowing the BBSome to diffuse through the TZ and enter cilia. Subsequently, RAB23-GDP facilitates its own ciliary entry by binding to the RAB18-GTP/BBSome entity as a BBSome cargo. Our findings establish a model where the BBSome enters cilia through the RAB23-RAB18 module-mediated inward BBSome TZ diffusion pathway. According to this model, hedgehog signaling defects caused by rab23 mutations could induce Carpenter syndrome in humans, providing a mechanistic understanding behind the inward BBSome TZ passage required for proper ciliary signaling. Significance statementCertain ciliary signaling proteins are removed from cilia through the outward BBSome transition zone (TZ) diffusion pathway. The inward BBSome TZ crossing is orchestrated by RAB18. The current study unveils that RAB23 in its GDP-bound state (RAB23-GDP) and RAB18-GDP predominantly localize to the same basal body region located just below the TZ. RAB23-GDP activates RAB18-GDP as a RAB18-specific guanine nucleotide exchange factor. RAB18-GTP subsequently recruits the BBSome as its effector to cross the TZ for ciliary entry. Following this, RAB23-GDP enters cilia as a BBSome cargo. The regulatory interplay between RAB23 and RAB18, with RAB23 promoting inward BBSome TZ crossing via RAB18, establishes a molecular mechanism for the BBSome-dependent retrieval of signaling proteins from cilia.

cell biology↗

bioRxiv2025

RAB18 Orchestrates Signaling Protein Ciliary Homeostasis by Facilitating BBSome Diffusion through the Transition Zone for Ciliary Entry

The BBSome acts as an intraflagellar transport (IFT) cargo adaptor that connects transmembrane and membrane-anchored signaling proteins to the process of IFT within cilia. This function is critical for ciliary homeostasis maintenance of signaling proteins, allowing for successfully sensing and transducing extracellular stimuli inside the cell. It is established that, upon reaching the proximal ciliary region just above the transition zone (TZ), a diffusion barrier, via retrograde IFT, the BBSome, carrying signaling proteins, disengages from IFT and diffuses through the TZ for ciliary retrieval. This TZ passage event is promoted by the Arf-like 3 GTPase, which utilizes the BBSome autonomously of IFT association as its major effector. While it is known that the intact BBSome is recruited to the basal bodies before entering cilia, the precise mechanism by which it crosses the TZ to access cilia remains elusive. In our study on Chlamydomonas reinhardtii, we disclose that the Ras-associated binding (Rab) GTPase RAB18 is concentrated in a basal body region that overlaps with the BBSome. Positioned at this location, RAB18 in a GTP-bound state (RAB18-GTP) is anchored to the membrane, allowing it to diffuse into cilia. Throughout this process, the BBSome functions as a RAB18 effector, binding to RAB18-GTP and subsequently being recruited to diffuse through the TZ for ciliary entry in an IFT-independent manner. This collaboration between the BBSome and RAB18 is pivotal in maintaining the signaling protein phospholipase D ciliary homeostasis, providing a precise mechanistic understanding of the inward BBSome TZ passage essential for proper ciliary signaling. Significance statementAlthough certain ciliary signaling proteins cross the transition zone (TZ) for ciliary removal via the RABL2-ARL3 module-mediated outward BBSome TZ diffusion pathway, how the BBSome passes the TZ for ciliary entry remains unclear. Here, we show that RAB18, in its GTP-bound state, anchors to the membrane at the basal bodies. As a RAB18 effector, the BBSome is recruited to cross the TZ for ciliary entry via lateral transport between plasma and ciliary membranes. Once inside cilia, the BBSome integrates into anterograde IFT trains and subsequently functions as a phospholipase D (PLD) adaptor, ensuring PLD removal from cilia. This study highlights RAB18s pivotal role in facilitating BBSome entry into cilia, unveiling a regulatory mechanism crucial for maintaining ciliary signaling protein homeostasis.

cell biology↗

bioRxiv2022

Unraveling the Intricate Cargo-BBSome Coupling Mechanism at the Ciliary Tip

Certain ciliary transmembrane and membrane-tethered signaling proteins migrate from the ciliary tip to base via retrograde intraflagellar transport (IFT), essential for maintaining their ciliary dynamics to enable cells to sense and transduce extracellular stimuli inside the cell. During this process, the BBSome functions as an adaptor between retrograde IFT trains and these signaling protein cargoes. The Arf-like 13 (ARL13) small GTPase resembles ARL6/BBS3 in facilitating these signaling cargoes to couple with the BBSome at the ciliary tip prior to loading onto retrograde IFT trains for transporting towards the ciliary base, while the molecular basis for how this intricate coupling event happens remains elusive. Here, we report that Chlamydomonas ARL13 only in a GTP-bound form (ARL13GTP) anchors to the membrane for diffusing into cilia. Upon entering cilia, ARL13 undergoes GTPase cycle for shuttling between the ciliary membrane (ARL13GTP) and matrix (ARL13GDP). To achieve this goal, the ciliary membrane-anchored BBS3GTP binds and activates the ciliary matrix-residing ARL13GDP as an ARL13 guanine nucleotide exchange factor. At the ciliary tip, ARL13GTP binds and recruits the ciliary matrix-residing and post-remodeled BBSome as an ARL13 effector to anchor to the ciliary membrane. This makes the BBSome spatiotemporally become available for the ciliary membrane-tethered phospholipase D (PLD) to couple with. Afterward, ARL13GTP hydrolyzes GTP for releasing the PLD-laden BBSome to load onto retrograde IFT trains. According to this model, hedgehog signaling defects associated with ARL13b and BBS3 mutations in humans could be satisfactorily explained, providing us a mechanistic understanding behind BBSome-cargo coupling required for proper ciliary signaling. Significance statementCertain signaling proteins export in a BBSome-dependent manner from cilia via retrograde IFT. As IFT cargoes, their coupling with the BBSome is mediated by ARL13 and BBS3. Here, we report that ARL13 in a GTP-bound state binds the membrane for diffusing into cilia followed by running GTPase cycle with BBS3 acting as an ARL13-specific guanine nucleotide exchange factor. ARL13GTP recruits the IFT-shed BBSome as its effector to anchor to the membrane of the ciliary tip, making it spatiotemporally available for the ciliary membrane-tethered PLD to bind. Afterward, ARL13 GTP hydrolysis releases the PLD-laden BBSome for retrograde IFT train loading. ARL13 and BBS3 facilitate the cargo-BBSome coupling in such a way providing a regulatory mechanism for signaling protein export from cilia.

cell biology↗

bioRxiv2022

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

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.

cell biology↗

bioRxiv2022

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

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.

cell biology↗

bioRxiv2021

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

Ciliary receptors and their certain downstream signaling components undergo intraflagellar transport (IFT) as BBSome cargoes to maintain their ciliary dynamics for sensing and transducing extracellular stimuli inside the cell. Cargo laden BBSomes shed from retrograde IFT at the proximal ciliary region above the transition zone (TZ) followed by diffusing through the TZ for ciliary retrieval, while how the BBSome barrier passage is controlled remains elusive. Here, we show that the BBSome is a major effector of the Arf-like 3 (ARL3) GTPase in Chlamydomonas. Under physiological condition, ARL3GDP binds the membrane for diffusing into and residing in cilia. Following a nucleotide conversion, ARL3GTP dissociates with the ciliary membrane and binds and recruits the IFT-detached and cargo (phospholipase D, PLD)-laden BBSome at the proximal ciliary region to diffuse through the TZ and out of cilia. ARL3 deficiency impairs ciliary signaling, e.g. phototaxis of Chlamydomonas cells, by disrupting BBSome ciliary retrieval, providing a mechanistic understanding behind BBSome ciliary turnover required for ciliary signaling.

cell biology↗