IFT54 regulates IFT20 stability but is not essential for tubulin transport during ciliogenesis

Abstract

Intraflagellar transport (IFT) is required for ciliogenesis by ferrying ciliary components using IFT complexes as cargo adaptors. IFT54 is a component of the IFT-B complex and is also associated with cytoplasmic microtubules (MTs). Loss of IFT54 impairs cilia assembly as well as cytoplasmic MT dynamics. The N-terminal calponin homology (CH) domain of IFT54 interacts with tubulins/MTs and has been proposed to transport tubulin during ciliogenesis, whereas the C-terminal coiled-coil (CC) domain binds IFT20. However, the precise function of these domains in vivo is not well understood. We showed that in Chlamydomonas, loss of IFT54 completely blocks ciliogenesis but does not affect spindle formation and proper cell cycle progression, even though IFT54 interacts with mitotic MTs. Interestingly, IFT54 lacking the CH domain allows proper flagellar assembly. The CH domain is required for the association of IFT54 with the axoneme but not with mitotic MTs, and also regulates the flagellar import of IFT54 but not IFT81 and IFT46. The C-terminal CC domain is essential for IFT54 to bind IFT20, and for its recruitment to the basal body and incorporation into IFT complexes. Complete loss of IFT54 or the CC domain destabilizes IFT20. ift54 mutant cells expressing the CC domain alone rescue the stability of IFT20 and form stunted flagella with accumulation of both IFT-A component IFT43 and IFT-B component IFT46, indicating that IFT54 also functions in IFT turn-around at the flagellar tip.

FLS2 is a CDK-like kinase that directly binds IFT70 and is required for proper ciliary disassembly in Chlamydomonas

“…Cilia were isolated after deflagellation by pH shock followed by sucrose gradient purification and fractionated into membrane matrix and axonemal fractions by using 1% NP40 [55]. To induce ciliary disassembly in vitro, cell cultures were treated with 20 mM of sodium pyrophosphate (final concentration) for 10 min or indicated time [23,34].…”

“…IB and IF experiments were performed as described previously [55]. The secondary antibodies used for IF are the following: Goat anti-Rat IgG Alexa Fluor 488, Goat anti-Mouse IgG Alexa Fluor 594.…”

Functional Exploration of Heterotrimeric Kinesin-II in IFT and Ciliary Length Control inChlamydomonas

“…Cilia isolation or ciliary regeneration was performed as described previously (Wang et 534 al., 2019;Zhu et al, 2017b). For ciliary regeneration, cells were deflagellated by pH 535 shock to allow ciliary regeneration at the indicated times followed by fixation with 1% 536 glutaraldehyde.…”

Calmodulin regulates a TRP channel (ADF1) and phospholipase C (PLC) to mediate elevation of cytosolic calcium during acidic stress that induces deflagellation inChlamydomonas

“…Several methods were used to induce deflagellation. For mechanical shearing, an Ultra-Turrax homogenizer (T10 basic; IKA Works, Wilmington, NC, USA) was set at scales of 5.5 and 25 ml of cells at a cell density of 1 3 10 7 cells/ml, and cells were processed in a 50-ml conical tube for 90 s. For deflagellation induced by pH shock or dibucaine, previously published protocols were followed (26,27). For deflagellation induced by calcium shock, cells were incubated in 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.0, and 0.15 mM CaCl 2 for 1 h, followed by addition of an equal volume of buffer that contained 150 mM CaCl 2 (28).…”

“…Immunostaining was performed essentially as previously described (26). The samples were viewed on an LSM 780 Meta Observer Z1 confocal laser-scanning microscope (Carl Zeiss, Oberkochen, Germany) for confocal microscopy or an N-SIM/ N-Storm (Nikon, Tokyo, Japan) for structured illumination microscopy (SIM).…”