PMC

A proposed model of the functional pathways of PIH preassembly proteins in Chlamydomonas adapted from []. The bold lines indicate the primary pathways, showing the dynein species that were reduced more than 30% in spectral numbers in each PIH mutant compared to the wild-type. The dotted thin lines indicate relatively secondary pathways, showing the dynein species that were reduced more than 30% in spectral numbers in the double PIH-mutants compared to the single PIH-mutants. The pathways are more complicated than previously thought []. Since the three-headed ODA of Chlamydomonas cannot be assembled in axonemes in the absence of ODA β or γ HC [, ], in this study we could not assess the direct contribution of each PIH protein in the cytoplasmic preassembly of each ODA HC since the amount of ODA α in cilia could be reduced simply in the absence of ODA β or γ HC. Thus, we categorized ODA HCs into one group in this model (asterisk). This is also the case for the two-headed IDA f/I1, in which this species cannot be assembled without each HC (f/I1α or f/I1β)[, ], thus we also categorized IDA f/I1 HCs into one group (asterisk). In this study, we could not find any primary pathway of the PIH proteins for the preassembly of IDAs a and f/I1. These dynein species may be preassembled by factors other than PIH proteins (e.g. PF22 or PF23, see ), or can assemble spontaneously in cytoplasm to some extent without the help of preassembly factors.

Without intervention, structure and material properties follow directly from building block attributes (green “subway line” above). Alternate material properties require an alternate route, such as preassembly (blue subway line) to create mesoscale structures that are not found in bulk.

Biphasic expression of cilia preassembly gene during differentiation. (A) mRNA levels of cilia preassembly genes and transcription factors MCIDAS and FOXJ1 detected by RT-PCR. The hTECs were cultured at the ALI, and mRNA was measured at the indicated day. Levels are shown relative to each gene, normalized to FOXJ1 expression at ALI day 0. (B) Cilia preassembly gene mRNA levels following induced MCIDAS expression during ALI culture. Lung epithelial cells were derived from iPSCs that expressed MCIDAS controlled by a tetracycline-regulated element for treatment with (dox+) or without (dox−) doxycycline initiated at ALI day 0. Data are normalized to Actin, and expressed as the fold change relative to ALI day 0. (C) Immunoblot analysis of early preassembly factors compared with FOXJ1 and DNAI1. (D) HEATR2, SPAG1, and DNAAF2 in hTECs at indicated day ALI. (E) HEATR2 (red) and DNAI1 (green) at ALI day 13, detected by immunofluorescence. (F) HEATR2 (red) relative to centriolar replication marked by CETN2 (green) in hTECs at ALI day 13. HEATR2 presents in cells before centriolar replication. The arrow points to a centrosome, and the arrowhead points to amplified centrioles. Nuclei were stained with DAPI (blue). (Scale bars: D–F, 10 μM.) A and B are the mean ± SEM of three independent preparations. In A, the expression of the indicated preassembly genes (*) is partitioned from other genes by K-means cluster analysis (sample size: 44–64 data points per day ALI).

(a) Gα_q-V fluorescence recovery in cells expressing C-TM (n=30), C-M3R (n=30), a chimeric C-M3R with the C terminus of M4R (C-M3R(M4ct); n=30) or C-M4R (n=24). (b) Unrecovered fluorescence for the same cells as panel a; **, P<0.001 compared to C-TM. (c) Gα_q-V fluorescence recovery in cells expressing C-TM (n=16), C-M3R (n=18), a chimeric C-M4R with the C terminus of M3R (C-M4R(M3ct); n=37) or C-M4R (n=30). (d) Unrecovered fluorescence for the same cells as panel c; **, P<0.001 compared to C-TM. Unrecovered fluorescence for C-M3R and C-M4R(M3ct) were not significantly different (P=0.14). Data represent mean values ± s.e.m..