Axonemal Lumen Dominates Cytosolic Protein Diffusion inside the Primary Cilium

Luo, Weimin; Ruba, Andrew; Takao, Daisuke; Zweifel, Ludovit; Lim, Roderick Y. H.; Verhey, Kristen J.; Yang, Weidong

doi:10.1038/s41598-017-16103-z

Cited by 37 publications

(48 citation statements)

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“…For the IFT motor, we determined the transport route for KIF3A-GFP, a component of the heterotrimeric kinesin-2 (KIF3A/KIF3B/KAP) complex that drives anterograde IFT (Engelke et al, 2019, Pazour and Rosenbaum, 2002, Kozminski et al, 1995. In agreement with our previous work in the cilium shaft (Luo et al, 2017), KIF3A was found to have two transport routes at the TZ: an outer route at 92±7 nm along the cilium radius with a route width of 71±17 nm, and an inner route at 0±3 nm along the cilium radius with a 52±2 nm route width (Figure 1 I-K). The IFT cargo α-tubulin-GFP (Hao et al, 2011, Craft et al, 2015 also occupies two distinct transport routes in the TZ: one between the microtubules and ciliary membrane with a radial peak position at 111±1 nm and a route width of 43±3 nm, and the other inside the axonemal lumen with a radial peak position at 0±1 nm and a route width of 98±3 nm (Figure 1 L-N).…”

Section: Speed Microscopy Maps the Transit Routes Of Ciliary Proteinssupporting

confidence: 76%

“…That the outer transport routes of both KIF3A and a-tubulin localize between the microtubules and ciliary membrane was expected as was the microtubule-proximal localization of the motor as compared to IFT proteins and cargo. We hypothesize that the inner route of both proteins corresponds to a passive diffusion route as we have previously observed in the cilium shaft (Luo et al 2017).…”

Section: Speed Microscopy Maps the Transit Routes Of Ciliary Proteinssupporting

confidence: 58%

“…It relies on high spatiotemporal resolution (2 ms, 10-20 nm) capture of 2D single-molecule locations of labeled proteins in live cells and the subsequent back-projection algorithm to obtain the 3D probability spatial density distribution of targeted proteins inside the primary cilium ( Figure S6) (Ma and Yang, 2010, Ma et al, 2016, Ruba et al, 2017. We used this approach to determine the contributions of IFT and free diffusion to the movement of soluble proteins within the lumen of the cilium shaft (Luo et al 2017). Here, we probe the transit of soluble and TM proteins at the TZ and demonstrate that the lumen is used to mediate vesicular trafficking of TM proteins as well as passive diffusion of structural and soluble proteins.…”

Section: Introductionmentioning

confidence: 99%

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The Ciliary Lumen Accommodates Passive Diffusion and Vesicle Trafficking in Cytoplasmic-Ciliary Transport

Ruba

Luo

et al. 2019

Preprint

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Transport of membrane and cytosolic proteins into the primary cilium is essential for its role in cellular signaling. Using single molecule microscopy, we mapped the movement of membrane and soluble proteins at the base of the primary cilium. In addition to the well-known intraflagellar transport (IFT) route, we identified two new pathways within the lumen of the primary cilium -passive diffusional and vesicle transport routes -that are adopted by proteins for cytoplasmic-cilium transport in live cells. Independent of the IFT path, approximately half of IFT motors (KIF3A) and cargo (α-tubulin) take the passive diffusion route and more than half of membrane-embedded G protein coupled receptors (SSTR3 and HTR6) use RAB8A-regulated vesicles to transport into and inside cilia. Furthermore, ciliary lumen transport is the preferred route for membrane proteins in the early stages of ciliogenesis and inhibition of SSTR3 vesicle transport completely blocks ciliogenesis. Furthermore, clathrin-mediated, signaldependent internalization of SSTR3 also occurs through the ciliary lumen. These transport routes were also observed in Chlamydomonas reinhardtii flagella, suggesting their conserved roles in trafficking of ciliary proteins.

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Section: Speed Microscopy Maps the Transit Routes Of Ciliary Proteinssupporting

confidence: 76%

Section: Speed Microscopy Maps the Transit Routes Of Ciliary Proteinssupporting

confidence: 58%

Section: Introductionmentioning

confidence: 99%

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The Ciliary Lumen Accommodates Passive Diffusion and Vesicle Trafficking in Cytoplasmic-Ciliary Transport

Ruba

Luo

et al. 2019

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“…Species-specific differences are also noted when considering the recycling of the kinesin motors after delivery of IFT particles to the tip of the cilium. In mammalian cilia, kinesin motors have been observed to undergo both retrograde transport and free diffusion back to the cell body [45, 61]. In Chlamydomonas cilia, the kinesin motors do not rely on dynein, rather they return to the base of the cilium by diffusion [51, 58, 62, 81].…”

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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“…Besides IFT, two additional, IFT-independent routes within the lumen of PC are known: passive diffusion and vesicle trafficking (Luo et al, 2017;Ruba et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Centrosome and ciliary abnormalities in fetal akinesia deformation sequence human fibroblasts

Jühlen

Martinelli

Vinci

et al. 2019

Preprint

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Ciliopathies are clinical disorders of the primary cilium with widely recognized phenotypic and genetic heterogeneity. Here we found that impaired ciliogenesis in fibroblasts derived from individuals with fetal akinesia deformation sequence (FADS).FADS refers to a broad spectrum of neuromuscular disorders arising from impaired fetal movement. We show that cells derived from two FADS individuals, one with an unknown genetic cause and one with bi-allelic pathogenic variants in RAPSN, have shorter and less primary cilia (PC), in association with alterations in posttranslational modifications in α -tubulin. Similarly, siRNA-mediated depletion of two known FADS proteins, the scaffold protein rapsyn and the nucleoporin NUP88, resulted in defective PC formation. Consistent with a role in ciliogenesis, rapsyn and NUP88 localized to centrosomes and PC. By proximity-ligation assays, we show that rapsyn and NUP88 are interacting and that both proteins are connecting to all three tubulin isoforms (α, ß, and γ ). The rapsyn-NUP88 interface, as well as their contact to microtubules, is perturbed in the examined FADS cells, similarly to their contact to microtubules. We suggest that the perturbed rapsyn-NUP88-tubulin interface leads to defects in PC formation and that defective ciliogenesis contributes to the pleiotropic defects seen in FADS.

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Axonemal Lumen Dominates Cytosolic Protein Diffusion inside the Primary Cilium

Cited by 37 publications

References 50 publications

The Ciliary Lumen Accommodates Passive Diffusion and Vesicle Trafficking in Cytoplasmic-Ciliary Transport

The Ciliary Lumen Accommodates Passive Diffusion and Vesicle Trafficking in Cytoplasmic-Ciliary Transport

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

Centrosome and ciliary abnormalities in fetal akinesia deformation sequence human fibroblasts

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