Regulation of ciliary membrane protein trafficking and signalling by kinesin motor proteins

Morthorst, Stine K.; Christensen, Søren Tvorup; Pedersen, Lotte B.

doi:10.1111/febs.14583

Cited by 42 publications

(49 citation statements)

References 333 publications

(541 reference statements)

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“…One of these antibodies was able to induce clustering and movement (capping) of its target protein, indicating the ability of at least one other membrane protein, in addition to FMG-1B, to interact with force transduction machinery within the flagellum (data not shown). Similarly, morerecent studies have demonstrated the role of IFT-A complexes in the movement of peripheral as well as integral membrane proteins into the flagellum; the latter include the polycystin 1 and 2 complex, TRPV channels, and various classes of G-protein-coupled receptors (Qin et al, 2005;Huang et al, 2007;Shih et al, 2013;Hirano et al, 2017;Morthorst et al, 2018). There is also evidence that at least some ciliary membrane proteins are not dependent upon IFT for entry into and movement within the ciliary membrane (Ye et al, 2013;Belzile et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

“…Primers flanking the predicted insertion sites were designed using SnapGene (http:// www.snapgene.com/) and synthesized by Integrated DNA Technologies (Skokie, IL). Genomic DNA was isolated from wt, 47, and 56 strains using phenol:chloroform (Newman et al, 1990). Modifications to the protocol were as described at the Chlamydomonas Resource Center (https://www.…”

Section: Genomic Analysismentioning

confidence: 99%

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The Chlamydomonas flagellar membrane glycoprotein FMG-1B is necessary for expression of force at the flagellar surface

Bloodgood

Tetreault

Sloboda

2019

Journal of Cell Science

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In addition to bend propagation for swimming, Chlamydomonas cells use their flagella to glide along a surface. When polystyrene microspheres are added to cells, they attach to and move along the flagellar surface, thus serving as a proxy for gliding that can be used to assay for the flagellar components required for gliding motility. Gliding and microsphere movement are dependent on intraflagellar transport (IFT). Circumstantial evidence suggests that mechanical coupling of the IFT force-transducing machinery to a substrate is mediated by the flagellar transmembrane glycoprotein FMG-1B. Here, we show that cells carrying an insertion in the 5′-UTR of the FMG-1B gene lack FMG-1B protein, yet assemble normal-length flagella despite the loss of the major protein component of the flagellar membrane. Transmission electron microscopy shows a complete loss of the glycocalyx normally observed on the flagellar surface, suggesting it is composed of the ectodomains of FMG-1B molecules. Microsphere movements and gliding motility are also greatly reduced in the 5′-UTR mutant. Together, these data provide the first rigorous demonstration that FMG-1B is necessary for the normal expression of force at the flagellar surface in Chlamydomonas.

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Section: Discussionmentioning

confidence: 99%

Section: Genomic Analysismentioning

confidence: 99%

The Chlamydomonas flagellar membrane glycoprotein FMG-1B is necessary for expression of force at the flagellar surface

Bloodgood

Tetreault

Sloboda

2019

Journal of Cell Science

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“…Future work with the i3A/i3B motor will allow us to directly determine the ciliary cargoes that depend on KIF3A/KIF3B/KAP activity for import into cilia and/or transport along the axoneme. We will also be able to directly test the roles of kinesin-2 in the steps that lead to ciliogenesis, such as the assembly of the subdistal appendages and the positioning of the basal body [83]. Furthermore, the i3A/i3B motor will be a valuable tool to dissect other dynamic cellular processes in which KIF3A/KIF3B/KAP has been implicated, such as neurite outgrowth in neuronal cells [84].…”

Section: Discussionmentioning

confidence: 99%

Acute inhibition of heterotrimeric kinesin-2 function reveals mechanisms of intraflagellar transport in mammalian cilia

Engelke

Waas

Kearns

et al. 2018

Preprint

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The trafficking of components within cilia, called intraflagellar transport (IFT), is powered by kinesin-2 and dynein-2 motors. Loss of function in any subunit of the heterotrimeric KIF3A/KIF3B/KAP kinesin-2 motor prevents ciliogenesis in mammalian cells and has hindered an understanding of how kinesin-2 motors function in IFT. We used a chemicalgenetic approach to engineer an inhibitable KIF3A/KIF3B (i3A/i3B) kinesin-2 motor that is capable of rescuing WT motor function in Kif3a/Kif3b double-knockout cells. Inhibitor addition blocks ciliogenesis or, if added to ciliated cells, blocks IFT within two minutes, which leads to a complete loss of primary cilia within six hours. The kinesin-2 family members KIF3A/KIF3C and KIF17 cannot rescue ciliogenesis in Kif3a/Kif3b double-knockout cells nor delay the disassembly of full-formed cilia upon i3A/i3B inhibition.These data suggest that KIF3A/KIF3B/KAP is the sole and essential motor for cilia assembly and function in mammalian cells, indicating a species-specific adaptation of kinesin-2 motors for IFT function. E n g e l k e e t a l . 2 Δ 11 ), 12 (i3A Δ 12 ), or 13 (i3A Δ 13 ) amino acids. In similar fashion, we fused the DmrB domain to the N-terminus of KIF3B truncated by five (i3B Δ 5 ), six (i3B Δ 6 ), or seven (i3B Δ 7) amino acids (Fig. 2b). We compared the ability of each i3A construct to pair with each i3B construct and generate primary cilia in the absence but not in the presence of B/B inhibitor. Fusion of the Δ 12 /i3B Δ 6 motor resulted in ~200-fold luciferase induction (Fig. 3c). Addition of B/B inhibitor resulted in a greater increase in luciferase activity induction only in cells expressing the i3A Δ 12 /i3B Δ 6 motor, reflecting pathway hyper-activation only in cells that lack a functional primary cilium (Fig. 3c).In summary, we find that expression of the i3A Δ 12 and i3B Δ 6 constructs in Kif3a -/-;Kif3b -/cells results in a bona fide inhibitable kinesin-2 motor. The engineered i3A Δ 12 /i3B Δ 6 motor is referred to as i3A/i3B throughout the rest of the manuscript. Inhibition of KIF3A/KIF3B results in the stalling of IFT trains and their exclusion from ciliaThe generation of an inhibitable kinesin-2 motor enables us, for the first time, to directly examine the role of KIF3A/KIF3B/KAP motors during IFT in fully-formed cilia. To investigate this, Kif3a -/-;Kif3b -/cells were transfected with plasmids for expressing the inhibitable i3A/i3 motor together with a fluorescently-tagged subunit of the IFT-B complex (IFT88-mNG; mNeonGreen) as in previous studies [34, 35]. Analysis of kymographs generated from live-cell imaging experiments revealed that IFT88-marked IFT trains moved processively towards the tip of the cilium, paused for variable durations, and then trafficked back towards the base (Fig. 4a), similar to what has been observed previously [34, 35]. Anterograde and retrograde speeds were on the order of 0.7 μ m/s, consistent with previously E n g e l k e e t a l . 5

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“…KIF13B/Gakin is a member of the large Kinesin‐3 family which was previously involved in endosomal transport [Hirokawa et al ., ] as well as in caveolae‐mediated trafficking [Kanai et al ., ]. A role at PC was identified based on the high expression in serum‐starved fibroblasts and was recently extensively reviewed elsewhere [Morthorst et al ., ]. Briefly, KIF13B localises to the BB and axoneme and has been shown to interact with NPHP4, a key component of the transition zone.…”

Section: Pc‐specific Kifmentioning

confidence: 99%

Ciliary kinesins beyond IFT: Cilium length, disassembly, cargo transport and signalling

Reilly

Benmerah

2019

Biology of the Cell

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Cilia and flagella are microtubule-based antenna which are highly conserved among eukaryotes. In vertebrates, primary and motile cilia have evolved to exert several key functions during development and tissue homoeostasis. Ciliary dysfunction in humans causes a highly heterogeneous group of diseases called ciliopathies, a class of genetic multisystemic disorders primarily affecting kidney, skeleton, retina, lung and the central nervous system. Among key ciliary proteins, kinesin family members (KIF) are microtubule-interacting proteins involved in many diverse cellular functions, including transport of cargo (organelles, proteins and lipids) along microtubules and regulating the dynamics of cytoplasmic and spindle microtubules through their depolymerising activity. Many KIFs are also involved in diverse ciliary functions including assembly/disassembly, motility and signalling. We here review these ciliary kinesins in vertebrates and focus on their involvement in ciliopathy-related disorders.

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Regulation of ciliary membrane protein trafficking and signalling by kinesin motor proteins

Cited by 42 publications

References 333 publications

The Chlamydomonas flagellar membrane glycoprotein FMG-1B is necessary for expression of force at the flagellar surface

The Chlamydomonas flagellar membrane glycoprotein FMG-1B is necessary for expression of force at the flagellar surface

Acute inhibition of heterotrimeric kinesin-2 function reveals mechanisms of intraflagellar transport in mammalian cilia

Ciliary kinesins beyond IFT: Cilium length, disassembly, cargo transport and signalling

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