scholarly journals Intraflagellar transport protein RABL5/IFT22 recruits the BBSome to the basal body through the GTPase ARL6/BBS3

2020 ◽  
Vol 117 (5) ◽  
pp. 2496-2505 ◽  
Author(s):  
Bin Xue ◽  
Yan-Xia Liu ◽  
Bin Dong ◽  
Jenna L. Wingfield ◽  
Mingfu Wu ◽  
...  
Keyword(s):  
Basal Body ◽  
In Vivo Imaging ◽  
Bound States ◽  
Intraflagellar Transport ◽  
Signaling Proteins ◽  
Gtpase Activity ◽  
Guanosine Triphosphate ◽  
Bardet Biedl Syndrome ◽  
Intrinsic Gtpase Activity

Bardet-Biedl syndrome (BBS) is a ciliopathy caused by defects in the assembly or distribution of the BBSome, a conserved protein complex. The BBSome cycles via intraflagellar transport (IFT) through cilia to transport signaling proteins. How the BBSome is recruited to the basal body for binding to IFT trains for ciliary entry remains unknown. Here, we show that the Rab-like 5 GTPase IFT22 regulates basal body targeting of the BBSome in Chlamydomonas reinhardtii. Our functional, biochemical and single particle in vivo imaging assays show that IFT22 is an active GTPase with low intrinsic GTPase activity. IFT22 is part of the IFT-B1 subcomplex but is not required for ciliary assembly. Independent of its association to IFT-B1, IFT22 binds and stabilizes the Arf-like 6 GTPase BBS3, a BBS protein that is not part of the BBSome. IFT22/BBS3 associates with the BBSome through an interaction between BBS3 and the BBSome. When both IFT22 and BBS3 are in their guanosine triphosphate (GTP)-bound states they recruit the BBSome to the basal body for coupling with the IFT-B1 subcomplex. The GTP-bound BBS3 likely remains to be associated with the BBSome upon ciliary entry. In contrast, IFT22 is not required for the transport of BBSomes in cilia, indicating that the BBSome is transferred from IFT22 to the IFT trains at the ciliary base. In summary, our data propose that nucleotide-dependent recruitment of the BBSome to the basal body by IFT22 regulates BBSome entry into cilia.

scholarly journals Bardet-Biedl Syndrome 3 protein promotes ciliary exit of the signaling protein phospholipase D via the BBSome

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Yan-Xia Liu ◽  
Bin Xue ◽  
Wei-Yue Sun ◽  
Jenna L Wingfield ◽  
Jun Sun ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Phospholipase D ◽  
Basal Body ◽  
Regulatory Mechanism ◽  
Intraflagellar Transport ◽  
Bound State ◽  
Signaling Proteins ◽  
Bardet Biedl Syndrome ◽  
Signaling Protein ◽  
Ciliary Signaling

Certain ciliary signaling proteins couple with the BBSome, a conserved complex of Bardet-Biedl syndrome (BBS) proteins, to load onto retrograde intraflagellar transport (IFT) trains for their removal out of cilia in Chlamydomonas reinhardtii. Here, we show that loss of the Arf-like 6 (ARL6) GTPase BBS3 causes the signaling protein phospholipase D (PLD) to accumulate in cilia. Upon targeting to the basal body, BBSomes enter and cycle through cilia via IFT, while BBS3 in a GTP-bound state separates from BBSomes, associates with the membrane, and translocates from the basal body to cilia by diffusion. Upon arriving at the ciliary tip, GTP-bound BBS3 binds and recruits BBSomes to the ciliary membrane for interacting with PLD, thus making the PLD-laden BBSomes available to load onto retrograde IFT trains for ciliary exit. Therefore, BBS3 promotes PLD exit from cilia via the BBSome providing a regulatory mechanism for ciliary signaling protein removal out of cilia.

scholarly journals Chlamydomonas LZTFL1 mediates phototaxis via controlling BBSome recruitment to the basal body and its reassembly at the ciliary tip

2021 ◽  
Vol 118 (35) ◽  
pp. e2101590118
Author(s):  
Wei-Yue Sun ◽  
Bin Xue ◽  
Yan-Xia Liu ◽  
Rui-Kai Zhang ◽  
Rong-Chao Li ◽  
...  
Keyword(s):  
Basal Body ◽  
Leucine Zipper ◽  
Intraflagellar Transport ◽  
G Protein Coupled Receptors ◽  
Signaling Proteins ◽  
Dual Mode ◽  
Bardet Biedl Syndrome ◽  
Cellular Processes ◽  
G Protein Coupled ◽  
Ciliary Signaling

Many G protein–coupled receptors and other signaling proteins localize to the ciliary membrane for regulating diverse cellular processes. The BBSome composed of multiple Bardet–Biedl syndrome (BBS) proteins is an intraflagellar transport (IFT) cargo adaptor essential for sorting signaling proteins in and/or out of cilia via IFT. Leucine zipper transcription factor-like 1 (LZTFL1) protein mediates ciliary signaling by controlling BBSome ciliary content, reflecting how LZTFL1 mutations could cause BBS. However, the mechanistic mechanism underlying this process remains elusive thus far. Here, we show that LZTFL1 maintains BBSome ciliary dynamics by finely controlling BBSome recruitment to the basal body and its reassembly at the ciliary tip simultaneously in Chlamydomonas reinhardtii. LZTFL1 directs BBSome recruitment to the basal body via promoting basal body targeting of Arf-like 6 GTPase BBS3, thus deciding the BBSome amount available for loading onto anterograde IFT trains for entering cilia. Meanwhile, LZTFL1 stabilizes the IFT25/27 component of the IFT-B1 subcomplex in the cell body so as to control its presence and amount at the basal body for entering cilia. Since IFT25/27 promotes BBSome reassembly at the ciliary tip for loading onto retrograde IFT trains, LZTFL1 thus also directs BBSome removal out of cilia. Therefore, LZTFL1 dysfunction deprives the BBSome of ciliary presence and generates Chlamydomonas cells defective in phototaxis. In summary, our data propose that LZTFL1 maintains BBSome dynamics in cilia by such a dual-mode system, providing insights into how LZTFL1 mediates ciliary signaling through maintaining BBSome ciliary dynamics and the pathogenetic mechanism of the BBS disorder as well.

scholarly journals Structure of the human BBSome core complex

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Björn Udo Klink ◽  
Christos Gatsogiannis ◽  
Oliver Hofnagel ◽  
Alfred Wittinghofer ◽  
Stefan Raunser
Keyword(s):  
High Resolution ◽  
Protein Complex ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Underlying Mechanism ◽  
Signaling Proteins ◽  
Core Complex ◽  
Bardet Biedl Syndrome ◽  
Cargo Proteins ◽  
Positively Charged

The BBSome is a heterooctameric protein complex that plays a central role in primary cilia homeostasis. Its malfunction causes the severe ciliopathy Bardet-Biedl syndrome (BBS). The complex acts as a cargo adapter that recognizes signaling proteins such as GPCRs and links them to the intraflagellar transport machinery. The underlying mechanism is poorly understood. Here we present a high-resolution cryo-EM structure of a human heterohexameric core subcomplex of the BBSome. The structure reveals the architecture of the complex in atomic detail. It explains how the subunits interact with each other and how disease-causing mutations hamper this interaction. The complex adopts a conformation that is open for binding to membrane-associated GTPase Arl6 and a large positively charged patch likely strengthens the interaction with the membrane. A prominent negatively charged cleft at the center of the complex is likely involved in binding of positively charged signaling sequences of cargo proteins.

In vivo imaging shows continued association of several IFT A, B and dynein complexes while IFT trains U-turn at the tip

2021 ◽  
Author(s):  
Jenna L. Wingfield ◽  
Betlehem Mekonnen ◽  
Ilaria Mengoni ◽  
Peiwei Liu ◽  
Mareike Jordan ◽  
...  
Keyword(s):  
Simple Model ◽  
In Vivo Imaging ◽  
Fluorescent Protein ◽  
Intraflagellar Transport ◽  
Entry And Exit ◽  
Protein Carriers ◽  
Data Support ◽  
Flagellar Assembly ◽  
Protein Entry

Flagellar assembly depends on intraflagellar transport (IFT), a bidirectional motility of protein carriers, the IFT trains. The trains are periodic assemblies of IFT-A and IFT-B subcomplexes and the motors kinesin-2 and IFT dynein. At the tip, anterograde trains are remodeled for retrograde IFT, a process that in Chlamydomonas involves kinesin-2 release and train fragmentation. However, the degree of train disassembly at the tip remains unknown. Two-color imaging of fluorescent protein-tagged IFT components indicates that IFT-A and IFT-B proteins from a given anterograde train usually return in the same set of retrograde trains. Similarly, concurrent turnaround was typical for IFT-B proteins and the IFT dynein subunit D1bLIC-GFP but severance was observed as well. Our data support a simple model of IFT turnaround, in which IFT-A, IFT-B, and IFT dynein typically remain associated at the tip and anterograde trains convert directly into retrograde trains without disassembly but for possible splitting into strings of IFT complexes. Continuous association of IFT-A, IFT-B and IFT dynein during tip remodeling could balance protein entry and exit preventing the build-up of IFT material in flagella.

scholarly journals Structure of the human BBSome core complex in the open conformation

2019 ◽  
Author(s):  
Björn U. Klink ◽  
Christos Gatsogiannis ◽  
Oliver Hofnagel ◽  
Alfred Wittinghofer ◽  
Stefan Raunser
Keyword(s):  
High Resolution ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Underlying Mechanism ◽  
Signaling Proteins ◽  
Core Complex ◽  
Bardet Biedl Syndrome ◽  
Open Conformation ◽  
Cargo Proteins ◽  
Positively Charged

AbstractThe BBSome is a heterooctameric protein complex that plays a central role in primary cilia homeostasis. Its malfunction causes the severe ciliopathy Bardet-Biedl syndrome (BBS). The complex acts as a cargo adapter that recognizes signaling proteins such as GPCRs and links them to the intraflagellar transport machinery. The underlying mechanism is poorly understood. Here we present a high-resolution cryo-EM structure of a human heterohexameric core subcomplex of the BBSome. The structure reveals the architecture of the complex in atomic detail. It explains how the subunits interact with each other and how disease-causing mutations hamper this interaction. The complex adopts a conformation that is open for binding to membrane-associated GTPase Arl6 and a large positively charged patch likely strengthens the interaction with the membrane. A prominent negatively charged cleft at the center of the complex is likely involved in binding of positively charged signaling sequences of cargo proteins.

scholarly journals Differential regulation of cellular activities by GTPase-activating protein and NF1.

1993 ◽  
Vol 13 (4) ◽  
pp. 2497-2503 ◽  
Author(s):  
N al-Alawi ◽  
G Xu ◽  
R White ◽  
R Clark ◽  
F McCormick ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Biological Effects ◽  
Type I ◽  
Type Ii ◽  
Gtpase Activity ◽  
Ras Proteins ◽  
Ras Gtpase ◽  
Intrinsic Gtpase Activity

The regulation of the GTPase activity of the Ras proteins is thought to be a key element of signal transduction. Ras proteins have intrinsic GTPase activity and are active in signal transduction when bound to GTP but not following hydrolysis of GTP to GDP. Three cellular Ras GTPase-activating proteins (Ras-gaps) which increase the GTPase activity of wild-type (wt) Ras but not activated Ras in vitro have been identified: type I and type II GAP and type I NF1. Mutations of wt Ras resulting in lowered intrinsic GTPase activity or loss of response to cellular Ras-gap proteins are thought to be the primary reason for the transforming properties of the Ras proteins. In vitro assays show type I and type II GAP and the GAP-related domain of type I NF1 to have similar biochemical properties with respect to activation of the wt Ras GTPase, and it appears as though both type I GAP and NF1 can modulate the GTPase function of Ras in cells. Here we report the assembling of a full-length coding clone for type I NF1 and the biological effects of microinjection of Ras and Ras-gap proteins into fibroblasts. We have found that type I GAP, type II GAP, and type I NF1 show markedly different biological activities in vivo. Coinjection of type I GAP or type I NF1, but not type II GAP, with wt Ras abolished the ability of wt Ras to induce expression from an AP-1-controlled reporter gene. We also found that serum-stimulated DNA synthesis was reduced by prior injection of cells with type I GAP but not type II GAP or type I NF1. These results suggest that type I GAP, type II GAP, and type I NF1 may have different activities in vivo and support the hypothesis that while type I forms of GAP and NF1 may act as negative regulators of wt Ras, they may do so with differential efficiencies.

scholarly journals The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella

2009 ◽  
Vol 187 (7) ◽  
pp. 1117-1132 ◽  
Author(s):  
Karl-Ferdinand Lechtreck ◽  
Eric C. Johnson ◽  
Tsuyoshi Sakai ◽  
Deborah Cochran ◽  
Bryan A. Ballif ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Intraflagellar Transport ◽  
Full Length ◽  
Signaling Proteins ◽  
Related Disorder ◽  
Bardet Biedl Syndrome ◽  
Insertional Mutants ◽  
Integral Component ◽  
Evolutionarily Conserved ◽  
Conserved Genes

In humans, seven evolutionarily conserved genes that cause the cilia-related disorder Bardet-Biedl syndrome (BBS) encode proteins that form a complex termed the BBSome. The function of the BBSome in the cilium is not well understood. We purified a BBSome-like complex from Chlamydomonas reinhardtii flagella and found that it contains at least BBS1, -4, -5, -7, and -8 and undergoes intraflagellar transport (IFT) in association with a subset of IFT particles. C. reinhardtii insertional mutants defective in BBS1, -4, and -7 assemble motile, full-length flagella but lack the ability to phototax. In the bbs4 mutant, the assembly and transport of IFT particles are unaffected, but the flagella abnormally accumulate several signaling proteins that may disrupt phototaxis. We conclude that the BBSome is carried by IFT but is an adapter rather than an integral component of the IFT machinery. C. reinhardtii BBS4 may be required for the export of signaling proteins from the flagellum via IFT.

Differential regulation of cellular activities by GTPase-activating protein and NF1

1993 ◽  
Vol 13 (4) ◽  
pp. 2497-2503
Author(s):  
N al-Alawi ◽  
G Xu ◽  
R White ◽  
R Clark ◽  
F McCormick ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Biological Effects ◽  
Type I ◽  
Type Ii ◽  
Gtpase Activity ◽  
Ras Proteins ◽  
Ras Gtpase ◽  
Intrinsic Gtpase Activity

The regulation of the GTPase activity of the Ras proteins is thought to be a key element of signal transduction. Ras proteins have intrinsic GTPase activity and are active in signal transduction when bound to GTP but not following hydrolysis of GTP to GDP. Three cellular Ras GTPase-activating proteins (Ras-gaps) which increase the GTPase activity of wild-type (wt) Ras but not activated Ras in vitro have been identified: type I and type II GAP and type I NF1. Mutations of wt Ras resulting in lowered intrinsic GTPase activity or loss of response to cellular Ras-gap proteins are thought to be the primary reason for the transforming properties of the Ras proteins. In vitro assays show type I and type II GAP and the GAP-related domain of type I NF1 to have similar biochemical properties with respect to activation of the wt Ras GTPase, and it appears as though both type I GAP and NF1 can modulate the GTPase function of Ras in cells. Here we report the assembling of a full-length coding clone for type I NF1 and the biological effects of microinjection of Ras and Ras-gap proteins into fibroblasts. We have found that type I GAP, type II GAP, and type I NF1 show markedly different biological activities in vivo. Coinjection of type I GAP or type I NF1, but not type II GAP, with wt Ras abolished the ability of wt Ras to induce expression from an AP-1-controlled reporter gene. We also found that serum-stimulated DNA synthesis was reduced by prior injection of cells with type I GAP but not type II GAP or type I NF1. These results suggest that type I GAP, type II GAP, and type I NF1 may have different activities in vivo and support the hypothesis that while type I forms of GAP and NF1 may act as negative regulators of wt Ras, they may do so with differential efficiencies.

scholarly journals In vivo imaging of radial spoke proteins reveals independent assembly and turnover of the spoke head and stalk

2017 ◽  
Author(s):  
Karl F. Lechtreck ◽  
Ilaria Mengoni ◽  
Batare Okivie ◽  
Kiersten B. Hilderhoff
Keyword(s):  
Exchange Rate ◽  
Cell Body ◽  
In Vivo Imaging ◽  
Fluorescent Protein ◽  
De Novo ◽  
Intraflagellar Transport ◽  
Wild Type ◽  
Radial Spokes ◽  
Head Protein

AbstractRadial spokes (RSs) are multiprotein complexes regulating dynein activity. In the cell body and ciliary matrix, RS proteins are present in a 12S precursor, which is converted into axonemal 20S spokes consisting of a head and stalk. To study RS assembly in vivo, we expressed fluorescent protein (FP)-tagged versions of the head protein RSP4 and the stalk protein RSP3 to rescue the corresponding Chlamydomonas mutants pfl, lacking spoke heads, and pf14, lacking RSs entirely. RSP3 and RSP4 mostly co-migrated by intraflagellar transport (IFT). Transport was elevated during ciliary assembly. IFT of RSP4-FP depended on RSP3. To study RS assembly independently of ciliogenesis, strains expressing FP-tagged RS proteins were mated to untagged cells with, without, or with partial RSs. RSP4-FP is added a tip-to-base fashion to preexisting pf1 spoke stalks while de novo RS assembly occurred lengthwise. In wild-type cilia, the exchange rate of head protein RSP4 exceeded that of the stalk protein RSP3 suggesting increased turnover of spoke heads. The data indicate that RSP3 and RSP4 while transported together separate inside cilia during RS repair and maintenance. The 12S RS precursor encompassing both proteins could represent transport form of the RS ensuring stoichiometric delivery by IFT. (196 of 200)

scholarly journals Mechanism of transport of IFT particles in C. elegans cilia by the concerted action of kinesin-II and OSM-3 motors

2006 ◽  
Vol 174 (7) ◽  
pp. 1035-1045 ◽  
Author(s):  
Xiaoyu Pan ◽  
Guangshuo Ou ◽  
Gul Civelekoglu-Scholey ◽  
Oliver E. Blacque ◽  
Nicholas F. Endres ◽  
...  
Keyword(s):  
Motor Coordination ◽  
Intraflagellar Transport ◽  
Quantitative Modeling ◽  
Concerted Action ◽  
Action Mechanism ◽  
Bardet Biedl Syndrome ◽  
C Elegans ◽  
And Function

The assembly and function of cilia on Caenorhabditis elegans neurons depends on the action of two kinesin-2 motors, heterotrimeric kinesin-II and homodimeric OSM-3–kinesin, which cooperate to move the same intraflagellar transport (IFT) particles along microtubule (MT) doublets. Using competitive in vitro MT gliding assays, we show that purified kinesin-II and OSM-3 cooperate to generate movement similar to that seen along the cilium in the absence of any additional regulatory factors. Quantitative modeling suggests that this could reflect an alternating action mechanism, in which the motors take turns to move along MTs, or a mechanical competition, in which the motors function in a concerted fashion to move along MTs with the slow motor exerting drag on the fast motor and vice versa. In vivo transport assays performed in Bardet-Biedl syndrome (BBS) protein and IFT motor mutants favor a mechanical competition model for motor coordination in which the IFT motors exert a BBS protein–dependent tension on IFT particles, which controls the IFT pathway that builds the cilium foundation.

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