IFT trains in different stages of assembly queue at the ciliary base for consecutive release into the cilium

Wingfield, Jenna L.; Mengoni, Ilaria; Bomberger, Heather E.; Jiang, Yu-Yang; Walsh, Jonathon; Brown, Jason; Picariello, Tyler; Cochran, Deborah A.; Zhu, Bing; Pan, Junmin; Eggenschwiler, Jonathan; Gaertig, Jacek; Witman, George B.; Kner, Peter; Lechtreck, Karl‐Ferdinand

doi:10.7554/elife.26609

Cited by 100 publications

(150 citation statements)

References 53 publications

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“…IFT particles (trains) are specialized for specific molecular cargos needed to build the growing axoneme. IFT trains accumulate in the transition zone, where they are loaded with axonemal structural material and transported to the distal flagellar tip by the heterotrimeric kinesin-2 complex (Deane, Cole, Seeley, Diener, & Rosenbaum, 2001; Wingfield et al, 2017). Retrograde trains are returned from the distal tip back to the cell body by cytoplasmic dynein (Pazour, Dickert, & Witman, 1999).…”

Section: Discussionmentioning

confidence: 99%

Length-dependent disassembly maintains four different flagellar lengths in Giardia

McInally

Kondev

Dawson

2019

Preprint

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1How flagellar length regulation is achieved in multiciliated eukaryotic cells with flagella of 2 different equilibrium lengths is unknown. The protist Giardia lamblia is an ideal model to 3 evaluate length regulation as it has flagella of four different lengths. Giardia axonemes have 4 both non-membrane-bound and membrane-bound regions, but lack transition zones. Here we 5 quantified the contributions of intraflagellar transport (IFT)-mediated assembly and kinesin-13-6 mediated disassembly to length control. IFT particles assemble and inject at Giardia's flagellar 7 pore complexes, which act as diffusion barriers functionally analogous to the transition zone to 8 compartmentalize the membrane-bound regions of flagella. IFT-mediated assembly is length-9 independent as train size, speed, and injection frequencies are similar between flagella of 10 different lengths. In Giardia, kinesin-13 mediates a length-dependent disassembly mechanism 11 of length regulation to balance length-independent IFT-mediated assembly, resulting in 12 different lengths. We anticipate that similar control mechanisms are widespread in multiciliated 13 cells where cytoplasmic precursor pools are not limiting. 14 15 16 17

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Section: Discussionmentioning

confidence: 99%

Length-dependent disassembly maintains four different flagellar lengths in Giardia

McInally

Kondev

Dawson

2019

Preprint

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“…Because IFT trains enter at the ciliary base, a component of IFT trains would need to be reused in the subsequent IFT round, to convey a length-dependent feedback of the axoneme-anchored LF4/MOK. While many of the IFT components are replaced by fresh IFT proteins that arrive from the cell body, some (including IFT54) are partially recycled [103].…”

Section: Discussionmentioning

confidence: 99%

LF4/MOK and a CDK-related kinase regulate the number and length of cilia inTetrahymena

Jiang

Maier

Baumeister

et al. 2019

Preprint

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22The length of cilia is controlled by a poorly understood mechanism that involves 23 members of the conserved RCK kinase group, and among them, the LF4/MOK 24 kinases. In Tetrahymena, a loss of an LF4/MOK ortholog, LF4A, lengthened the 25 locomotory cilia, but also reduced their total number per cell. Without LF4A, cilia 26 assembled faster and showed signs of increased intraflagellar transport (IFT). 27Consistently, overproduced LF4A shortened cilia and downregulated the IFT. GFP-28 tagged LF4A, expressed in the native locus and imaged by total internal reflection 29 microscopy, was enriched at the basal bodies and distributed along the shafts of 30 cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or 31 moved by IFT. A forward genetic screen identified a CDK-related kinase, CDKR1, 32 whose loss-of-function suppressed the shortening of cilia caused by overexpression 33 of LF4A, by reducing its kinase activity. A loss of CDKR1 alone lengthened both the 34 locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: 35 LF2 of Chlamydomonas, mammalian CCRK and DYF-18 of C. elegans, in lacking the 36 cyclin-binding motif and acting upstream of RCKs. We propose that the total 37 LF4/MOK activity per cilium is dependent on both its activation by an upstream CDK-38 related kinase and cilium length. Previous studies showed that the rate of assembly 39 is high in growing cilia and decreases as cilia elongate to achieve the steady-state 40 length. We propose that in a longer cilium, the IFT components, which travel from 41 the base to the tip, are subjected to a higher dose of inhibition by the uniformly 42 3 distributed LF4/MOK. Thus, in a feedback loop, LF4/MOK may translate cilium length 43 into proportional inhibition of IFT, to balance the rates of assembly and disassembly 44 at steady-state. 45 46Author summary 47 Cilia are conserved organelles that generate motility and mediate vital sensory 48 functions, including olfaction and vision. Cilia that are either too short or too long fail 49 to generate proper forces or responses to extracellular signals. Several cilia-based 50 diseases (ciliopathies) are associated with defects in cilia length. Here we use the 51 multiciliated model protist Tetrahymena, to study a conserved protein kinase whose 52 activity shortens cilia, LF4/MOK. We find that cells lacking an LF4/MOK kinase of 53Tetrahymena, LF4A, have excessively long, but also fewer cilia. We show that LF4A 54 decreases the intraflagellar transport, a motility that shuttles ciliary precursors from 55 the cilium base to the tip. Live imaging revealed that LF4A is distributed along cilium 56 length and remains mostly immobile, likely due to its anchoring to ciliary 57 microtubules. We proposed that in longer cilia, the intraflagellar transport machinery 58 is exposed to a higher dose of inhibition by LF4A, which could decrease the rate of 59 cilium assembly, to balance the rate of cilium disassembly in mature cilia that 60 maintain stable length. 61 62 63T...

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“…Our data supports a requirement of at least one form of Chlamydomonas actin for flagellar length maintenance and full flagellar assembly. Anterograde flagellar motor complexes, which are required for assembly of the flagellum, utilize newly recruited proteins from the cell body pool (Wingfield et al, 2017). With constant turnover and a demand for continuous recruitment to the base of flagella it appears that actin plays a role in this recruitment (Avasthi et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

Partially redundant actin genes in Chlamydomonas control flagellum-directed traffic and transition zone organization

Jack

Mueller

Fee

et al. 2017

Preprint

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Flagella of the unicellular green alga Chlamydomonas reinhardtii are nearly identical to cilia of mammalian cells and provide an excellent model to study ciliogenesis. These biflagellated cells have two actin genes: one encoding a conventional actin (IDA5) and the other encoding a divergent novel actin-like protein (NAP1). Previously, we described a role for actin in regulation of flagella-building intraflagellar transport machinery and now probe how actin redundancy contributes to this process using a nap1 mutant Chlamydomonas strain. Treatment with Latrunculin B, a potent actin polymerization inhibitor on the nap1 mutant background acutely disrupts all filamentous actins in the cell. We find that actins are an absolute requirement for flagellar growth when the preexisting pool of flagellar precursors is depleted. Nap1 mutants treated with Latrunculin B also showed reduced protein synthesis during regeneration. Finally, loss of functional actins reduced the incorporation of existing flagellar proteins as well as caused mislocalization of a key transition zone gating protein, NPHP-4. These experiments demonstrate that each stage of flagellar biogenesis requires redundant actin function to varying degrees, with an absolute requirement for actin in incorporation of newly synthesized flagellar proteins.

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IFT trains in different stages of assembly queue at the ciliary base for consecutive release into the cilium

Cited by 100 publications

References 53 publications

Length-dependent disassembly maintains four different flagellar lengths in Giardia

Length-dependent disassembly maintains four different flagellar lengths in Giardia

LF4/MOK and a CDK-related kinase regulate the number and length of cilia inTetrahymena

Partially redundant actin genes in Chlamydomonas control flagellum-directed traffic and transition zone organization

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