BBS4 is required for intraflagellar transport coordination and basal body number in mammalian olfactory cilia

Intraflagellar transport (IFT) is essential for the elongation and maintenance of eukaryotic cilia and flagella. Due to the traffic jam of multiple trains at the ciliary tip, how IFT trains are remodeled in these turnaround zones cannot be determined by conventional imaging. Using PhotoGate, we visualized the full range of movement of single IFT trains and motors in Chlamydomonas flagella. Anterograde trains split apart and IFT complexes mix with each other at the tip to assemble retrograde trains. Dynein-1b is carried to the tip by kinesin-II as inactive cargo on anterograde trains. Unlike dynein-1b, kinesin-II detaches from IFT trains at the tip and diffuses in flagella. As the flagellum grows longer, diffusion delays return of kinesin-II to the basal body, depleting kinesin-II available for anterograde transport. Our results suggest that dissociation of kinesin-II from IFT trains serves as a negative feedback mechanism that facilitates flagellar length control in Chlamydomonas.

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The ciliopathy-associated CPLANE proteins direct basal body recruitment of intraflagellar transport machinery

Nature Genetics ◽

10.1038/ng.3558 ◽

2016 ◽

Vol 48 (6) ◽

pp. 648-656 ◽

Cited By ~ 62

Author(s):

Michinori Toriyama ◽

◽

Chanjae Lee ◽

S Paige Taylor ◽

Ivan Duran ◽

...

Keyword(s):

Basal Body ◽

Intraflagellar Transport

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An Essential Quality Control Mechanism at the Eukaryotic Basal Body Prior to Intraflagellar Transport

Traffic ◽

10.1111/j.1600-0854.2007.00611.x ◽

2007 ◽

Vol 8 (10) ◽

pp. 1323-1330 ◽

Cited By ~ 59

Author(s):

Angela Stephan ◽

Sue Vaughan ◽

Michael K. Shaw ◽

Keith Gull ◽

Paul G. McKean

Keyword(s):

Quality Control ◽

Basal Body ◽

Control Mechanism ◽

Intraflagellar Transport ◽

Essential Quality ◽

Quality Control Mechanism

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Different Effects of Tetrahymena IFT172 Domains on Anterograde and Retrograde Intraflagellar Transport

Molecular Biology of the Cell ◽

10.1091/mbc.e07-05-0403 ◽

2008 ◽

Vol 19 (4) ◽

pp. 1450-1461 ◽

Cited By ~ 39

Author(s):

Che-Chia Tsao ◽

Martin A. Gorovsky

Keyword(s):

Basal Body ◽

Intraflagellar Transport ◽

Retrograde Transport ◽

Terminal Repeat ◽

Anterograde Transport ◽

Multiprotein Complexes ◽

Repeat Domain

Intraflagellar transport (IFT) particles are multiprotein complexes that move bidirectionally along the cilium/flagellum. The Tetrahymena IFT172 gene encodes a protein with an N-terminal WD domain (WDD) and a C-terminal repeat domain (RPD). Epitope-tagged Ift172p localized to the basal body and in cilia along the axoneme, and IFT172 knockout cells lost cilia and motility. Using serial deletion constructs to rescue the knockout cells, we found that neither the WDD nor the RPD alone is sufficient to assemble cilia. Ift172p containing only the WDD or the RPD failed to enter cilia. Constructs with a partial truncation of the RPD still rescued although cilia were assembled less efficiently, indicating that the WDD and a part of the RPD are sufficient for anterograde transport. Partial truncation of the RPD caused the accumulation of truncated Ift172p itself and of Ift88p at ciliary tips, suggesting that IFT turnaround or retrograde transport was affected. These results implicate different regions of Ift172p in different steps of the IFT process.

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Protein turnover dynamics suggest a diffusion-to-capture mechanism for peri-basal body recruitment and retention of intraflagellar transport proteins

Molecular Biology of the Cell ◽

10.1091/mbc.e20-11-0717 ◽

2021 ◽

pp. mbc.E20-11-0717

Author(s):

Jaime V.K. Hibbard ◽

Neftali Vazquez ◽

Rohit Satija ◽

John B. Wallingford

Keyword(s):

Protein Dynamics ◽

Basal Body ◽

Fluorescence Recovery After Photobleaching ◽

Protein Complexes ◽

Intraflagellar Transport ◽

Basal Bodies ◽

Multiciliated Cells ◽

Capture Mechanism ◽

Body Components ◽

Ciliary Signaling

Intraflagellar transport (IFT) is essential for construction and maintenance of cilia. IFT proteins concentrate at the basal body, where they are thought to assemble into trains and bind cargoes for transport. To study the mechanisms of IFT recruitment to this peri-basal body pool, we quantified protein dynamics of eight IFT proteins, as well as five other basal body localizing proteins, using fluorescence recovery after photobleaching in vertebrate multiciliated cells. We found that members of the IFT-A and IFT-B protein complexes show distinct turnover kinetics from other basal body components. Additionally, known IFT sub-complexes displayed shared dynamics, suggesting shared basal body recruitment and/or retention mechanisms. Finally, we evaluated the mechanisms of basal body recruitment by depolymerizing cytosolic MTs, which suggested that IFT proteins are recruited to basal bodies through a diffusion-to-capture mechanism. Our survey of IFT protein dynamics provides new insights into IFT recruitment to basal bodies, a crucial step in ciliogenesis and ciliary signaling.

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Length-dependent disassembly maintains four different flagellar lengths in Giardia

eLife ◽

10.7554/elife.48694 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 9

Author(s):

Shane G McInally ◽

Jane Kondev ◽

Scott C Dawson

Keyword(s):

Mathematical Modeling ◽

Basal Body ◽

Diffusion Barrier ◽

Quantitative Imaging ◽

Intraflagellar Transport ◽

Ideal Model ◽

Parasitic Protist ◽

Length Regulation ◽

Flagellar Assembly ◽

Membrane Bound

With eight flagella of four different lengths, the parasitic protist Giardia is an ideal model to evaluate flagellar assembly and length regulation. To determine how four different flagellar lengths are maintained, we used live-cell quantitative imaging and mathematical modeling of conserved components of intraflagellar transport (IFT)-mediated assembly and kinesin-13-mediated disassembly in different flagellar pairs. Each axoneme has a long cytoplasmic region extending from the basal body, and transitions to a canonical membrane-bound flagellum at the ‘flagellar pore’. We determined that each flagellar pore is the site of IFT accumulation and injection, defining a diffusion barrier functionally analogous to the transition zone. IFT-mediated assembly is length-independent, as train size, speed, and injection frequencies are similar for all flagella. We demonstrate that kinesin-13 localization to the flagellar tips is inversely correlated to flagellar length. Therefore, we propose a model where a length-dependent disassembly mechanism controls multiple flagellar lengths within the same cell.

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Localization of intraflagellar transport protein IFT52 identifies basal body transitional fibers as the docking site for IFT particles

Current Biology ◽

10.1016/s0960-9822(01)00484-5 ◽

2001 ◽

Vol 11 (20) ◽

pp. 1586-1590 ◽

Cited By ~ 294

Author(s):

James A. Deane ◽

Douglas G. Cole ◽

E.Scott Seeley ◽

Dennis R. Diener ◽

Joel L. Rosenbaum

Keyword(s):

Basal Body ◽

Intraflagellar Transport ◽

Transport Protein ◽

Docking Site

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The Intraflagellar Transport Protein IFT20 Is Associated with the Golgi Complex and Is Required for Cilia Assembly

Molecular Biology of the Cell ◽

10.1091/mbc.e06-02-0133 ◽

2006 ◽

Vol 17 (9) ◽

pp. 3781-3792 ◽

Cited By ~ 326

Author(s):

John A. Follit ◽

Richard A. Tuft ◽

Kevin E. Fogarty ◽

Gregory J. Pazour

Keyword(s):

Membrane Proteins ◽

Golgi Complex ◽

Basal Body ◽

Binding Sites ◽

Mammalian Cells ◽

Intraflagellar Transport ◽

Living Cells ◽

Large Protein ◽

Protein Particles ◽

Golgi Structure

Eukaryotic cilia are assembled via intraflagellar transport (IFT) in which large protein particles are motored along ciliary microtubules. The IFT particles are composed of at least 17 polypeptides that are thought to contain binding sites for various cargos that need to be transported from their site of synthesis in the cell body to the site of assembly in the cilium. We show here that the IFT20 subunit of the particle is localized to the Golgi complex in addition to the basal body and cilia where all previous IFT particle proteins had been found. In living cells, fluorescently tagged IFT20 is highly dynamic and moves between the Golgi complex and the cilium as well as along ciliary microtubules. Strong knock down of IFT20 in mammalian cells blocks ciliary assembly but does not affect Golgi structure. Moderate knockdown does not block cilia assembly but reduces the amount of polycystin-2 that is localized to the cilia. This work suggests that IFT20 functions in the delivery of ciliary membrane proteins from the Golgi complex to the cilium.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1901665117 ◽

2020 ◽

Vol 117 (5) ◽

pp. 2496-2505 ◽

Cited By ~ 3

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.

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The FLA3 KAP Subunit Is Required for Localization of Kinesin-2 to the Site of Flagellar Assembly and Processive Anterograde Intraflagellar Transport

Molecular Biology of the Cell ◽

10.1091/mbc.e04-10-0931 ◽

2005 ◽

Vol 16 (3) ◽

pp. 1341-1354 ◽

Cited By ~ 95

Author(s):

Joshua Mueller ◽

Catherine A. Perrone ◽

Raqual Bower ◽

Douglas G. Cole ◽

Mary E. Porter

Keyword(s):

Amino Acid ◽

Basal Body ◽

Intraflagellar Transport ◽

Body Region ◽

Wild Type ◽

Gene Encoding ◽

Flagellar Assembly ◽

Cilia And Flagella ◽

Preferential Incorporation ◽

Dic Microscopy

Intraflagellar transport (IFT) is a bidirectional process required for assembly and maintenance of cilia and flagella. Kinesin-2 is the anterograde IFT motor, and Dhc1b/Dhc2 drives retrograde IFT. To understand how either motor interacts with the IFT particle or how their activities might be coordinated, we characterized a ts mutation in the Chlamydomonas gene encoding KAP, the nonmotor subunit of Kinesin-2. The fla3-1 mutation is an amino acid substitution in a conserved C-terminal domain. fla3-1 strains assemble flagella at 21°C, but cannot maintain them at 33°C. Although the Kinesin-2 complex is present at both 21 and 33°C, the fla3-1 Kinesin-2 complex is not efficiently targeted to or retained in the basal body region or flagella. Video-enhanced DIC microscopy of fla3-1 cells shows that the frequency of anterograde IFT particles is significantly reduced. Anterograde particles move at near wild-type velocities, but appear larger and pause more frequently in fla3-1. Transformation with an epitope-tagged KAP gene rescues all of the fla3-1 defects and results in preferential incorporation of tagged KAP complexes into flagella. KAP is therefore required for the localization of Kinesin-2 at the site of flagellar assembly and the efficient transport of anterograde IFT particles within flagella.

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