Chlamydomonas LZTFL1 mediates phototaxis via controlling BBSome recruitment to the basal body and its reassembly at the ciliary tip

Significance

Bardet–Biedl syndrome (BBS) characteristic of blindness, obesity, and kidney anomalies is a rare human ciliopathy. BBS could result from disrupted ciliary dynamics of the BBSome, a conserved octamer of BBS proteins, which facilitates intraflagellar transport with the ciliary entry and/or removal of signaling proteins. Here, we show that LZTFL1 mediates phototaxis through balancing BBSomes available for transporting into and out of cilia. LZTFL1 controls the BBSome basal body amount available for entering cilia by promoting BBS3 targeting to the basal bodies. LZTFL1 simultaneously promotes BBSome removal out of cilia by stabilizing IFT25/27, a regulator essential for BBSome reassembly at the ciliary tip. LZTFL1 applies this dual-mode system to maintain BBSome ciliary dynamics, providing a mechanistic mechanism for BBS disorder.

Abstract

Many G protein–coupled receptors and other signaling proteins localize to the ciliary membrane for regulating diverse cellular processes. The BBSome composed of multiple Bardet–Biedl syndrome (BBS) proteins is an intraflagellar transport (IFT) cargo adaptor essential for sorting signaling proteins in and/or out of cilia via IFT. Leucine zipper transcription factor-like 1 (LZTFL1) protein mediates ciliary signaling by controlling BBSome ciliary content, reflecting how LZTFL1 mutations could cause BBS. However, the mechanistic mechanism underlying this process remains elusive thus far. Here, we show that LZTFL1 maintains BBSome ciliary dynamics by finely controlling BBSome recruitment to the basal body and its reassembly at the ciliary tip simultaneously in Chlamydomonas reinhardtii. LZTFL1 directs BBSome recruitment to the basal body via promoting basal body targeting of Arf-like 6 GTPase BBS3, thus deciding the BBSome amount available for loading onto anterograde IFT trains for entering cilia. Meanwhile, LZTFL1 stabilizes the IFT25/27 component of the IFT-B1 subcomplex in the cell body so as to control its presence and amount at the basal body for entering cilia. Since IFT25/27 promotes BBSome reassembly at the ciliary tip for loading onto retrograde IFT trains, LZTFL1 thus also directs BBSome removal out of cilia. Therefore, LZTFL1 dysfunction deprives the BBSome of ciliary presence and generates Chlamydomonas cells defective in phototaxis. In summary, our data propose that LZTFL1 maintains BBSome dynamics in cilia by such a dual-mode system, providing insights into how LZTFL1 mediates ciliary signaling through maintaining BBSome ciliary dynamics and the pathogenetic mechanism of the BBS disorder as well.

Flagella and cilia are interchangeable terms referring to the membrane-surrounded and microtubule-based axoneme that protrudes from the cell surface of nearly all types of eukaryotic cells (1). As cellular hubs, cilia transduce extracellular signals inside the cell through ciliary transmembrane G protein–coupled receptors and ion channels (2–6). These signaling proteins enter and/or remove from cilia via intraflagellar transport (IFT) for maintaining their ciliary dynamics. For instance, somatostatin receptor 1, dopamine receptor 1, smoothened (Smo), patched 1, and GPR161 rely on IFT for exiting cilia (4, 7–9). In contrast, somatostatin receptor 3 and melanin-concentrating hormone receptor 1 depend on IFT for entering cilia (2, 10). During these processes, signaling proteins couple with IFT through binding the IFT cargo adaptor, the BBSome complex composed of eight Bardet–Biedl syndrome (BBS) proteins (BBS1/2/4/5/7/8/9/18) (11) (seven BBS proteins, including BBS1/2/4/5/7/8/9 in Chlamydomonas reinhardtii) (12). Therefore, defects in BBSome assembly and ciliary cycling cause loss and/or abnormal buildup of signaling proteins in cilia (11–16). This eventually impairs ciliary signaling and causes BBS, a human inherited disease characterized by obesity, blindness, kidney failure, and polydactyly (17), and phototaxis defects in C. reinhardtii (7, 12).

IFT is executed by motor-driven IFT trains consisting of repeating units of the complexes IFT-A and IFT-B (composed of IFT-B1 and -B2 subcomplexes) and trafficking along the axoneme (18–26). The BBSome loads onto anterograde IFT trains at the ciliary base followed by entry and anterograde traffic from the ciliary base to tip. At the ciliary tip, the BBSome remodels, followed by loading onto retrograde IFT trains for exiting cilia (8, 27–33). In the cell body, Rab-like 5 (RABL5) GTPase IFT22 binds and stabilizes Arf-like 6 GTPase BBS3 (34). BBS3/IFT22 binds and targets the BBSome to the ciliary base only when they both are in a GTP-bound state (34). In mammalian cells, the GTP-bound BBS3 enters cilia and undergoes GTPase cycling with the aid of the Rab-like 4 GTPase IFT27 as a BBS3-specific guanine nucleotide exchange factor (GEF) (8). Upon finishing a GTPase cycle at the ciliary tip, BBS3 reloaded with GTP, in turn, binds and loads the cargo-laden BBSome onto retrograde IFT trains for ciliary exit, thus contributing to maintain dynamics of signaling proteins in the ciliary membrane (8). In C. reinhardtii, BBS3 promotes ciliary signaling protein(s) (e.g., phospholipase D [PLD]) to associate with the BBSome at the ciliary tip (35). However, it does not facilitate BBSome loading onto retrograde IFT trains (35). Meanwhile, BBS3 promotes the PLD association with the BBSome at the ciliary tip, even at a GTP-locked configuration, excluding GTPase cycling of BBS3 from mediating this process (35).

Defects in leucine zipper transcription factor-like 1 (LZTFL1) protein (also known as BBS17) causes BBS (36, 37). As reflected in both human and murine cells, LZTFL1 interacts with the BBSome through binding the BBSome subunit BBS9 and maintains Smo abundance in cilia by negatively controlling the ciliary removal of the BBSome (27, 38). Underlying this process, LZTFL1 was proposed to bridge the BBSome to retrograde IFT trains through directly binding the IFT-B1 subcomplex IFT25/27 in cilia of murine cells (27). Other than those limited knowledges, how LZTFL1 regulates ciliary signaling by maintaining BBSome ciliary dynamics has not been established. In this study, we explored the interplay among LZTFL1, BBSome/BBS3, and IFT at a molecular level by using a combination of functional, biochemical, and single-particle in vivo imaging assays on C. reinhardtii. We show that LZTFL1 promotes phototaxis by balancing the BBSome basal body amount available for ciliary entry and its ciliary removal through BBS3 and IFT25/27 pathways, respectively.

Results

LZTFL1 Resides in the Ciliary Matrix at Low Abundance.

Chlamydomonas LZTFL1 shares significant homology with its orthologs in ciliated species and is more closely related to homologs of ciliated protists than vertebrates phylogenetically (SI Appendix, Fig. S1 A and B). LZTFL1 resides in cilia of MEF, NIH 3T3, and mouse kidney tissue cells but not hTERT-RPE1, HEK293T, and IMCD3 cells, as determined by immunostaining (27, 38, 39). Ciliary proteomic analysis, however, identified LZTFL1 to be present at low abundance in cilia of IMCD3 and Chlamydomonas cells (40, 41). These findings are contradictory about whether LZTFL1 conserves cross-species to be a ciliary protein. To clarify this, we examined ciliary positioning of LZTFL1 using our newly developed anti-LZTFL1 antiserum that specifically recognizes LZTFL1 as a single band with a molecular weight of ∼36 kDa in Chlamydomonas cells (CC-125), as determined by immunoblotting (SI Appendix, Fig. S2). Of note, the endogenous LZTFL1 was not detectable in ciliary extracts of CC-125 cells until 30 times more ciliary extracts than whole-cell extracts were loaded, indicating that LZTFL1 enters cilia but remains at low abundance in cilia (Fig. 1A). The anti-LZTFL1 antiserum recognized an additional band of ∼62 kDa in LZTFL1::YFP cells overexpressing LZTFL1 fused at its C terminus to yellow fluorescence protein (YFP) (LZTFL1::YFP) (Fig. 1B). In ciliary extracts of LZTFL1::YFP cells, neither the native LZTFL1 nor the LZTFL1::YFP fusion protein was detectable until 30 times more ciliary extracts than whole-cell extracts were loaded, confirming the presence of LZTFL1 at low abundance in cilia (Fig. 1B). Chlamydomonas LZTFL1 has been reported to reside in the membrane and matrix fraction of cilia (40). We further defined LZTFL1 to reside in the matrix fraction of cilia, as both the native LZTFL1 and its C-terminal YFP-tagged version were only detectable in the matrix fraction but not in the membrane and axonemal fractions of cilia (Fig. 1C). Furthermore, the native LZTFL1 copositioned with the IFT-B1 subunit IFT81 both at the basal body and in cilia and so did LZTFL1::YFP and the IFT-B1 subunit IFT46 (Fig. 1 D and E). By combining these biochemical and immunostaining evidence together, we conclude that LZTFL1 is delivered to the ciliary base, enters cilia, and resides in the matrix fraction of cilia at low abundance in C. reinhardtii.

Fig. 1.

LZTFL1 resides in the ciliary matrix at low abundance. (A and B) Immunoblots of whole-cell extracts (W) and ciliary extracts (C) of CC-125 (A) and LZTFL1::YFP (B) cells probed with α-LZTFL1. Around 10 and 30 times more proteins of ciliary extracts than whole-cell extracts were also loaded in the SDS–polyacrylamide-based discontinuous gel. α-tubulin was used for adjusting the loading. MW stands for molecular weight. (C) Immunoblots of ciliary extracts and ciliary fractions of CC-125 and LZTFL1::YFP cells probed with antibodies against LZTFL1 (CC-125 cells), YFP (LZTFL1::YFP cells), the ciliary matrix marker IFT57, the ciliary membrane marker PLD, and the matrix and axonemal marker α-tubulin. (D and E) Immunostaining of CC-125 (D) and LZTFL1::YFP (E) cells. Both at the basal bodies and in cilia, LZTFL1 copositioned with the IFT-B1 subunit IFT81 (green: α-IFT81 and red: α-LZTFL1) (D) and LZTFL1::YFP copositioned with the IFT-B1 subunit IFT46 (green: α-YFP and red: α-IFT46) (E). The inset shows the basal bodies. White arrows show ciliary staining. (Scale bars, 10 µm.)

LZTFL1 Diffuses in Cilia and Binds the IFT-Separated BBSome at the Ciliary Tip.

Considering that LZTFL1 distributes to the basal body and the matrix fraction of cilia, where the IFT and BBSome proteins reside, we wondered whether Chlamydomonas LZTFL1 mimics its mammalian counterpart to bridge in between the IFT cargo adaptor BBSome and IFT-B1 and thus cycles through cilia via IFT (27). To test this notion, we first performed immunoprecipitation assays on ciliary extracts of LZTFL1::YFP-, BBS5::YFP-, IFT46::YFP-, and YFP-expressing CC-125 cells (strains LZTFL1::YFP, BBS5::YFP, IFT46::YFP, and HR-YFP, respectively) (34, 42, 43). Immunoprecipitation with LZTFL1::YFP recovered the BBSome subunits BBS1 and BBS5 but not the IFT-B1 subunits IFT46 and IFT70 (Fig. 2A); BBS5::YFP immunoprecipitated IFT46, IFT70, BBS1, and LZTFL1 (Fig. 2B); and IFT46::YFP recovered IFT70, BBS1, and BBS5 but not LZTFL1 (Fig. 2C). Since LZTFL1::YFP and IFT46::YFP did not recover one another but both pull down BBS1 and BBS5, and BBS5::YFP immunoprecipitated LZTFL1, IFT46, and IFT70, BBSomes likely reside in cilia either by binding IFT-B1 or by interacting with LZTFL1, and LZTFL1 does not bind IFT-B1 directly nor through interacting with the BBSome in cilia. Supportive of this notion, sucrose density gradient centrifugation assay identified that the majority of LZTFL1 exists as a free form separated from the BBSome and IFT-B1 in ciliary extracts of CC-125 cells, while a minority cosedimented with the BBSome and IFT-B1 (Fig. 2D). In ciliary extracts of BBS1-null mutant bbs1-1 cells (12), BBSome absence did not alter the IFT-B1 content (checked for IFT46 and IFT70) nor caused IFT-B1 to shift to the right of the fractions in sucrose density gradient, excluding IFT-B1 from associating with the BBSome at the experimental condition performed (Fig. 2 E and F). Of note, LZTFL1 existed completely as a free form separated from IFT-B1 in ciliary extracts of bbs1-1 cells (Fig. 2F). Therefore, our data confirm that LZTFL1 interacts with the BBSome but does not bind IFT-B1 directly or indirectly through interacting with the BBSome in cilia.

Fig. 2.

LZTFL1 diffuses in cilia and binds the IFT-separated BBSome at the ciliary tip. (A–C) Immunoblots of α-YFP–captured proteins from ciliary extracts (CE) of LZTFL1::YFP (A), BBS5::YFP (BBS5::YFP-expressing CC-125 cells) (B), and IFT46::YFP (IFT46::YFP-expressing CC-125 cells) (C) cells probed with α-IFT46, α-IFT70, α-BBS1, α-BBS5, or α-LZTFL1, as shown. The input was adjusted with α-YFP by immunoblotting. (D) Immunoblots of sucrose density gradient of ciliary extracts (CE) of CC-125 cells probed with α-IFT46, α-IFT70, α-BBS1, α-BBS5, and α-LZTFL1. (E) Immunoblots of CE of BBS1-null bbs1-1 cells probed with α-IFT46, α-IFT70, α-BBS1, α-BBS5, and α-LZTFL1. Acetylated α-tubulin (Ac-tubulin) was used for adjusting the loading. (F). Immunoblots of sucrose density gradient of CE of bbs1-1 cells probed with α-IFT46, α-IFT70, and α-LZTFL1. (G) Immunoblots of whole-cell extracts (WCE) of LZTFL1::YFP, BBS5::YFP, and IFT46::YFP cells probed with α-YFP. α-tubulin was used for adjusting the loading. (H) TIRF images and corresponding kymograms of LZTFL1::YFP, BBS5::YFP, and IFT46::YFP cells (Movies S1–S3, 15 frames per second). The time and transport lengths are indicated on the right and on the bottom, respectively. (I) Speeds and frequencies of the YFP-tagged particles to traffic inside cilia of the listed cells. Error bar indicates SD. n: number of cilia analyzed. n.s.: nonsignificance, *: significance at P < 0.005. The actual P value was listed. Ant. and Ret. stand for anterograde and retrograde, respectively. For panels A–C and G, MW stands for molecular weight.

To better understand the dynamics of LZTFL1 in cilia, we examined the LZTFL1::YFP, BBS5::YFP, and IFT46::YFP cells, in which LZTFL1::YFP was expressed at a higher level than BBS5::YFP and IFT46::YFP, while BBS5::YFP and IFT46::YFP were expressed at the same level (Fig. 2G). Total internal reflection fluorescence (TIRF) imaging of living cells observed LZTFL1::YFP to diffuse in cilia (Fig. 2H and Movie S1). In contrast, BBS5::YFP, as expected, underwent bidirectional movements at speeds similar to those for the YFP-tagged IFT46 in cilia (Fig. 2 H and I and Movies S2 and S3 and Table 1) (34, 44, 45). Consistent with the previous observation that only partial IFT trains are loaded with BBSomes (12), BBS5::YFP moved in two directions in cilia both with dramatically reduced frequencies, as compared to IFT46::YFP (Fig. 2 H and I and Movies S2 and S3 and Table 1). By combining these biochemical and motility data, we conclude that, differing from the IFT/BBS system that undergoes IFT in cilia, LZTFL1 diffuses in cilia, suggesting that LZTFL1 does not interact with IFT/BBS during transportation between the ciliary base and tip. It was known that IFT/BBS undergoes remodeling at the ciliary tip (30, 31). During this procedure, the BBSome transiently separates from IFT (33, 35). Therefore, LZTFL1 binds the BBSome, most likely at the ciliary tip where the BBSome dissociates from IFT trains during IFT/BBS remodeling.

Table 1.

Transport of fluorescence protein–tagged particles in cilia

Group	Strain	Frequency		Speed
		Anterograde (particles/s)	Retrograde (particles/s)	Anterograde (µm/s)	Retrograde (µm/s)
1	BBS5::YFP	0.71 ± 0.13	0.47 ± 0.12	2.53 ± 0.35	3.53 ± 0.36
	IFT46::YFP	1.17 ± 0.11	0.93 ± 0.18	2.60 ± 0.35	3.61 ± 0.31
2	BBS5::YFP	0.69 ± 0.15	0.41 ± 0.12	2.22 ± 0.25	3.24 ± 0.42
	BBS5::YFPLZ-miRNA	0.29 ± 0.06	0.15 ± 0.07	2.16 ± 0.19	3.27 ± 0.37
3	IFT22::HA::GFP	1.09 ± 0.12	0.85 ± 0.11	2.26 ± 0.28	3.34 ± 0.33
	IFT22LZ-miRNA	1.12 ± 0.12	0.84 ± 0.12	2.20 ± 0.18	3.30 ± 0.29
4	BBS5::YFP	0.71 ± 0.10	0.47 ± 0.09	2.26 ± 0.20	3.26 ± 0.22
	BBS5::YFP27-miRNA	0.70 ± 0.11	0.24 ± 0.08	2.22 ± 0.19	3.28 ± 0.26

LZTFL1 Promotes the BBSome to Enter Cilia.

The BBSome accumulates in cilia of human and murine LZTFL1-null mutants (38, 39). To have a comprehensive review on this process in C. reinhardtii, we used vector-based microRNA (miRNA) to deplete the endogenous LZTFL1 to ∼7% of wild-type (WT) level; we referred to this strain as LZTFL1^miRNA (Fig. 3A). Reflecting its cellular reduction, LZTFL1 was strongly reduced to ∼12% of WT level in LZTFL1^miRNA cilia (Fig. 3B). Partial depletion of LZTFL1 did not appear to affect cell morphology, flagellar length, cell growth, and cell swimming behavior, thus excluding LZTFL1 from assembling cilia (SI Appendix, Fig. S3 A–C and Movies S4–S6). To elucidate the role of LZTFL1 within the BBSome pathway, we surveyed ciliary presence of BBS1 and BBS5 and observed a markable reduction of two proteins in cilia (Fig. 3B). Of note, LZTFL1 knockdown did not alter their cellular contents, indicating that LZTFL1 positively maintains BBSome abundance in cilia (Fig. 3A). This was confirmed as the rescue of LZTFL1 with LZTFL1::YFP to WT level (resulting strain LZTFL1^Res) did not alter cellular contents of BBS1 and BBS5 but restored them to WT levels in cilia (Fig. 3 A and B).

Fig. 3.

LZTFL1 promotes the BBSome to enter cilia. (A and B) Immunoblots of whole-cell extracts (WCE) (A) and ciliary extracts (CE) (B) of CC-125, the LZTFL1-knockdown LZTFL1^miRNA, and the LZTFL1-rescuing LZTFL1^Res cells probed with α-LZTFL1, α-BBS1, and α-BBS5. (C and D) Immunoblots of WCE (C) and CE (D) of CC-125, BBS5::YFP, and BBS5::YFP^LZ-miRNA (BBS5::YFP-expressing LZTFL1^miRNA cells) cells probed with α-BBS5. Protein levels of BBS5 and BBS5::YFP were quantified and presented as fold change, relative to CC-125 protein (D). (E) TIRF images and corresponding kymograms of BBS5::YFP and BBS5::YFP^LZ-miRNA cells (Movies S7 and S8, 15 frames per second [fps]). (F) Speeds and frequencies of BBS5::YFP molecules to traffic inside cilia of the listed cells. (G and H) Immunoblots of WCE (G) and CE (H) of IFT22::HA::GFP (IFT22::HA::GFP-expressing CC-125 cells) and IFT22^LZ-miRNA (IFT22::HA::GFP-expressing LZTFL1^miRNA cells) cells probed with α-IFT22 and α-LZTFL1. (I) TIRF images and corresponding kymograms of IFT22::HA::GFP and IFT22^LZ-miRNA cells (Movies S9 and S10, 15 fps). (J) Speeds and frequencies of IFT22::HA::GFP molecules to traffic inside cilia of the listed cells. For panels A–D, G, and H, α-tubulin or acetylated α-tubulin (Ac-tubulin) was used as a loading control. MW stands for molecular weight. For panels E and I, the time and transport lengths are indicated on the right and on the bottom, respectively. For panels F and J, error bar indicates SD. n: number of cilia analyzed. n.s.: nonsignificance, *: significance at P < 0.005. **: significance at P < 0.001. The actual P value was listed. Ant. and Ret. stand for anterograde and retrograde, respectively.

To determine whether BBSome reduction in cilia is caused by its decreased ciliary entry, increased ciliary removal, or both, we expressed BBS5::YFP in LZTFL1^miRNA cells (resulting strain BBS5::YFP^LZ-miRNA) to the same level as in the BBS5::YFP-expressing CC-125 cells (BBS5::YFP) (Fig. 3C) (34). As expected, BBS5::YFP^LZ-miRNA cells contained BBS5 (BBS5::YFP plus the endogenous BBS5) in cilia ∼81% less than BBS5::YFP cells (Fig. 3D). TIRF imaging of living cells identified that BBS5::YFP undergoes IFT with similar anterograde and retrograde speeds in two strains (Fig. 3 E and F and Movies S7 and S8 and Table 1). Anterograde and retrograde IFT of BBS5::YFP was frequently observed in BBS5::YFP cells, while its frequencies for both directions were significantly reduced in BBS5::YFP^LZ-miRNA cells (Fig. 3 E and F and Movies S7 and S8 and Table 1). This observation indicated that LZTFL1 knockdown disrupts the efficiency of BBSome entry into cilia. To exclude LZTFL1 from affecting IFT dynamics in cilia, we expressed the IFT-B1 subunit IFT22 fused at its C terminus to hemagglutinin (HA) and GFP (IFT22::HA::GFP) in LZTFL1^miRNA cells (resulting strain IFT22^LZ-miRNA) to the same level as in IFT22::HA::GFP-expressing CC-125 cells (IFT22::HA::GFP) (Fig. 3G) (34). Both strains contained IFT22 (IFT22::HA::GFP plus the endogenous IFT22) in cilia at the same level (Fig. 3H). TIRF imaging of living cells identified that IFT22::HA::GFP undergoes IFT with similar anterograde and retrograde speeds and frequencies in two strains, indicating that IFT trains cycle through cilia normally in the absence of LZTFL1 in cilia (Fig. 3 I and J and Movies S9 and S10 and Table 1). Therefore, LZTFL1 promotes the BBSome to enter cilia.

LZTFL1 Targets the BBSome to the Basal Body via BBS3.

The BBSome is recruited to the basal body for loading onto anterograde IFT trains for entering cilia via IFT (34). To investigate the interplay among the BBSome, IFT-B1, and LZTFL1 in the cell body, we performed immunoprecipitation assays on cell body extracts of LZTFL1::YFP, BBS5::YFP, IFT46::YFP, and HR-YFP cells (34, 42, 43). As expected, BBS5::YFP immunoprecipitated BBS1, IFT46, and IFT70 and IFT46::YFP recovered BBS1, BBS5, and IFT70, reflecting that IFT-B1 and the BBSome interact with one another (Fig. 4 A and B) (34). However, they both did not recover LZTFL1, and LZTFL1::YFP did not immunoprecipitate IFT46 and IFT70 nor BBS1 and BBS5, suggesting that LZTFL1 does not interact with IFT/BBS in the cell body (Fig. 4 A–C). Our previous study has shown that the loss of even an individual BBSome subunit deprives the rest of BBSome subunits of assembling into a BBSome entity, and this eventually leads to a lack of intact BBSomes for being recruited to the basal body for entering cilia (35). In the bbs1-1 mutant, knockout of BBS1 did not affect the cellular contents of IFT46, IFT70, and LZTFL1, excluding the BBSome from affecting LZTFL1 and IFT-B1 levels (Fig. 4D). In agreement with the immunoprecipitation results, LZTFL1 existed in a free form separated from the BBSome and IFT-B1 in cell body extracts of CC-125 cells (Fig. 4E). Even in bbs1-1 cells that cannot assemble BBSome, LZTFL1 remained to exist as a free form separated from IFT-B1 in the cell body, excluding LZTFL1 from binding IFT/BBS in the cell body (Fig. 4E).

Fig. 4.

LZTFL1 targets the BBSome to the basal body via BBS3. (A–C) Immunoblots of α-YFP–captured proteins from cell body extracts (CBE) of BBS5::YFP (A), IFT46::YFP (B), and LZTFL1::YFP (C) cells probed with α-IFT46, α-IFT70, α-BBS1, α-BBS5, or α-LZTFL1. The input was adjusted with α-YFP by immunoblotting. (D) Immunoblots of CBE of CC-125 and bbs1-1 cells probed with α-IFT46, α-IFT70, α-BBS1, α-BBS5, and α-LZTFL1. (E) Immunoblots of sucrose density gradient of CBE of CC-125 and bbs1-1 cells probed for IFT46, IFT70, BBS1, BBS4, BBS5, BBS7, BBS8, and LZTFL1. (F) CC-125, LZTFL1^miRNA, and LZTFL1^Res cells stained with α-LZTFL1, α-IFT46, α-IFT70, α-BBS1, and α-BBS5. (G) Immunoblots of sucrose density gradient of CBE of LZTFL1^miRNA cells probed for BBS1, BBS4, BBS5, BBS7, and BBS8. (H and I) Immunoblots of whole-cell extracts (WCE) (H) and ciliary extracts (CE) (I) of CC-125, LZTFL1^miRNA, and LZTFL1^Res cells probed for BBS3. (J) CC-125, LZTFL1^miRNA, and LZTFL1^Res cells stained with α-BBS3. For panels A–C, MW stands for molecular weight. For panels D, H, and I, α-tubulin or acetylated α-tubulin (Ac-tubulin) was used as a loading control. For panels F and J, the inset shows the basal body staining. The fluorescence intensity of the stained proteins was quantified and shown on the right. Error bar indicates SD. n: number of cells analyzed. n.s.: nonsignificance. **: significance at P < 0.001. (Scale bars, 10 µm.)

The BBSome and IFT-B1 enrich and couple with one another at the basal body for entering cilia via IFT (34). Immunostaining showed that LZTFL1 enriches at the basal body of CC-125 cells as well (Figs. 1D and 4F). Reflecting its reduced cellular abundance, LZTFL1 exhibited an ∼89% reduction at the basal body of LZTFL1^miRNA cells, as compared to CC-125 cells (Fig. 4F). This observation was confirmed as LZTFL1 was restored to WT level at the basal body of LZTFL1^Res cells (Fig. 4F). In agreement with the fact that LZTFL1 does not affect IFT dynamics in cilia (Fig. 3 G–J and Movies S9 and S10 and Table 1), IFT46 and IFT70 enriched at the basal body of LZTFL1^miRNA cells at levels the same as in CC-125 and LZTFL1^Res cells (Fig. 4F). Remarkably, BBS1 and BBS5 enriched at the basal body of LZTFL1^miRNA cells ∼90% less than in CC-125 cells, suggesting that the BBSome is defective in targeting to the basal body without LZTFL1 (Fig. 4F). After this notion was confirmed as BBS1 and BBS5 were restored to WT levels at the basal body of LZTFL1^Res cells (Fig. 4F), and the intact BBSome was indeed assembled in the cell body of LZTFL1^miRNA cells (Fig. 4G), we conclude that LZTFL1 promotes the BBSome to the basal body so as to positively regulate the presence of the BBSome in cilia.

Our previous study has identified that BBS3 binds the BBSome in the cell body and, only when in its GTP-bound form, recruits the BBSome to the basal body (34). BBS3 did not affect LZTFL1 abundance in whole-cell and ciliary extracts (SI Appendix, Fig. S4 A and B). In contrast, LZTFL1 knockdown does not alter cellular BBSome content but blocks BBSome targeting to the basal body, hinting that LZTFL1 might recruit the BBSome to the basal body via BBS3. To test this notion, we examined the LZTFL1^miRNA cells and found that LZTFL1 knockdown does not affect cellular BBS3 abundance but causes remarkable BBS3 reduction in cilia, identifying that LZTFL1 positively maintains ciliary BBS3 content (Fig. 4 H and I). This notion was verified as the LZTFL1^Res cells contained BBS3 at WT level in both whole-cell sample and cilia (Fig. 4 H and I). We next quantified the BBS3 content at the basal body by immunostaining and identified that BBS3 exhibits an ∼88% reduction at the basal body of LZTFL1^miRNA cells, as compared to CC-125 and LZTFL1^Res cells, revealing that BBS3 is defective in targeting to the basal body without LZTFL1, which eventually hampers BBSome trafficking to the basal body (Fig. 4J) (34).

To survey if LZTFL1 overexpression affects basal body and the ciliary distribution of BBS3 and the BBSome, we quantified the cellular and ciliary abundance of LZTFL1 (LZTFL1::YFP plus the endogenous LZTFL1), BBS3, BBS1, and BBS5 in LZTFL1::YFP cells. As reflected by immunoblotting assays, this strain contained cellular and ciliary BBS3, BBS1, and BBS5 at WT levels (SI Appendix, Fig. S5 A and B). LZTFL1::YFP cells also harbored LZTFL1 (LZTFL1::YFP plus the endogenous LZTFL1) in cilia at WT level (SI Appendix, Fig. S5B). Furthermore, LZTFL1 (LZTFL1::YFP plus the endogenous LZTFL1), BBS3, BBS1, and BBS5 enriched at the basal body at WT levels in LZTFL1::YFP cells (SI Appendix, Fig. S5C). These results excluded the extracellular LZTFL1 from promoting more LZTFL1 targeting to the basal body for ciliary entry, from affecting cellular BBS3 and BBSome abundance, and from affecting the abundance of LZTFL1, BBS3, and the BBSome at the basal body and in cilia, suggesting that only a minimal amount of cellular LZTFL1 is required for normal BBS3 and BBSome basal body recruitment, and LZTFL1 does not mediate ciliary entry of BBS3 and the BBSome directly.

LZTFL1 Stabilizes IFT25/27.

IFT25 and IFT27 bind to form the heterodimer IFT25/27 (8, 44, 46). IFT25/27 integrates into the rest of IFT-B1 for promoting the BBSome to remove out of cilia via IFT (8, 27, 29, 44). Underlying this process, mammalian LZTFL1 associates with the BBSome and thus bridges the BBSome to retrograde IFT trains through interacting with IFT25/27 at the ciliary tip (27, 38). This is not the case in Chlamydomonas cells as LZTFL1 does not interact with IFT-B1 in cilia (Fig. 2). Supportive of this notion, IFT25/27 did not bind LZTFL1 (SI Appendix, Fig. S6A) nor did it cosedimented with LZTFL1 in sucrose density gradient of cell body extracts in the presence or absence of the BBSome (Fig. 5A). In C. reinhardtii, the partial depletion of LZTFL1 did not affect cellular and ciliary contents of the IFT-A subunits IFT43 and IFT139; the IFT-B1 subunits IFT22, IFT46, and IFT70; and the IFT-B2 subunits IFT38 and IFT57 (Fig. 5 B and C), consistent with the fact that LZTFL1 does not affect IFT dynamics (Fig. 3 G–J and Movies S9 and S10 and Table 1). Remarkably, IFT25/27 was dramatically reduced in cilia, as reflected by its reduced content in the whole-cell sample of LZTFL1^miRNA cells (Fig. 5 B and C). After the cellular content of IFT25/27 was restored to WT level by rescuing LZTFL1 expression, its ciliary content was also rescued to be normal, as shown in LZTFL1^Res cells (Fig. 5 B and C). Therefore, LZTFL1 stabilizes IFT25/27.

Fig. 5.

LZTFL1 stabilizes IFT25/27. (A) Immunoblots of sucrose density gradient of cell body extracts (CBE) of CC-125 and bbs1-1 cells probed for BBS4, BBS5, IFT25, IFT27, and LZTFL1. (B and C) Immunoblots of whole-cell extracts (WCE) (B) and ciliary extracts (CE) (C) of CC-125, LZTFL1^miRNA, and LZTFL1^Res cells probed for the IFT-B1 subunits IFT25, IFT27, IFT22, IFT46, and IFT70; the IFT-B2 subunits IFT38 and IFT57; and the IFT-A subunits IFT43 and IFT139. For panels B and C, α-tubulin or acetylated α-tubulin (Ac-tubulin) was used as a loading control.

Our previous study and others have shown that IFT25 binds IFT27 for stabilizing the latter (29, 44). This is not the case for LZTFL1 to stabilize IFT25/27, as they do not interact with one another (Fig. 5A and SI Appendix, Fig. S6A). We next isolated the nuclear and cytoplasmic extracts from CC-125 and LZTFL1::YFP cells and found that both the native LZTFL1 and LZTFL1::YFP are present in the cytoplasmic but not the nuclear extracts, even when 50 times more nuclear extracts than whole-cell extracts were loaded, opposing the assumption that LZTFL1 could translocate from the cytoplasm to the nucleus for inhibiting IFT25/27 transcription (SI Appendix, Fig. S6B). We next compared the expression profiles of global messenger RNAs (mRNAs) between CC-125 and LZTFL1^miRNA cells by performing RNA-seq, with stringent, filtering criteria of fold-change value 2 as a cutoff, and found that 234 and 422 genes raise and drop their expressions, respectively, in LZTFL1^miRNA cells (SI Appendix, Fig. S6C and Dataset 1). However, the 22 IFT protein mRNAs remained at WT levels in LZTFL1^miRNA cells (SI Appendix, Fig. S6D). After mRNAs of IFT25, IFT27, and several IFT proteins, including IFT22, IFT46, IFT70, IFT38, IFT57, IFT43, and IFT139, were confirmed to be at WT levels in LZTFL1^miRNA cells by qPCR assay (SI Appendix, Fig. S6E), we conclude that LZTFL1 does not affect the mRNA abundance of IFT proteins and the protein content of IFT proteins other than IFT25/27. Instead, LZTFL1 is required specifically for stabilizing IFT25/27.

IFT25/27 Promotes BBSome Reassembly at the Ciliary Tip.

IFT25 binds IFT27 for stabilizing the latter, and the loss of IFT25, therefore, reduces the global abundance of IFT25/27 (29, 44). In C. reinhardtii and murine cells, this eventually causes the BBSome to accumulate in cilia, revealing that IFT25/27 promotes BBSome removal from cilia (8, 27, 29, 44). To investigate the mechanism of how LZTFL1 and IFT25/27 coordinate to function in this event, we used vector-based miRNA to knock IFT27 down to ∼10% of WT level (resulting strain IFT27^miRNA). IFT27 knockdown, similar to IFT25 depletion, did not alter cell morphology, flagellar length, cell growth, and cell swimming behavior, excluding IFT27 from assembling cilia (SI Appendix, Fig. S7 A–C and Movies S11–S13) (47). Remarkably, IFT27 knockdown did not affect cellular and ciliary contents of LZTFL1 and IFT proteins but reduced IFT25 and IFT27 to ∼9% of WT level in cilia (Fig. 6 A and B and SI Appendix, Fig. S7 D and E). Of note, IFT27 knockdown did not affect the cellular content of BBS1 and BBS5 but caused their buildup in cilia (Fig. 6 A and B) (44). This notion was confirmed as BBS1 and BBS5 were restored to WT levels in the IFT27-rescuing IFT27^Res-WT cells, revealing that IFT25/27 negatively maintains BBSome content in cilia (Fig. 6 A and B) (44).

Fig. 6.

IFT25/27 promotes BBSome reassembly at the ciliary tip. (A and B) Immunoblots of whole-cell extracts (WCE) (A) and ciliary extracts (CE) (B) of CC-125, the IFT27-knockdown IFT27^miRNA, and the IFT27-rescuing IFT27^Res-WT (IFT27::HA::GFP-expressing IFT27^miRNA cells) cells probed with α-IFT25, α-IFT27, α-BBS1, α-BBS5, and α-LZTFL1. (C and D) Immunoblots of WCE (C) and CE (D) of CC-125, BBS5::YFP, and BBS5::YFP^27-miRNA (BBS5::YFP-expressing IFT27^miRNA cells) cells probed with α-BBS5. Protein levels of BBS5 and BBS5::YFP were quantified and presented as fold change, relative to CC-125 protein (D). (E) TIRF images and corresponding kymograms of BBS5::YFP and BBS5::YFP^27-miRNA cells (Movies S14 and S15, 15 frames per second). (F) Speeds and frequencies of BBS5::YFP molecules to traffic inside cilia of the listed cells. Ant. and Ret. stand for anterograde and retrograde, respectively. Error bar indicates SD. n: number of the fluorescent particles analyzed. n.s.: nonsignificance, *: significance at P < 0.005. The actual P value was listed. (G) CC-125, IFT27^miRNA, and IFT27^Res-WT cells stained with α-BBS1 (red) and α-BBS5 (red). The inset shows the ciliary tip staining. The fluorescence intensity of the stained proteins at the ciliary tip was quantified and shown on the right. Error bar indicates SD. n: number of cells analyzed. **: significance at P < 0.001. The actual P value was listed. (Scale bars, 10 µm.) (H) Immunoblots of sucrose density gradient of CE of CC-125, IFT27^miRNA, and IFT27^Res-WT cells probed for BBS1 and BBS5. For panels A–D, MW stands for molecular weight. α-tubulin or acetylated α-tubulin (Ac-tubulin) was used as a loading control.

To check how the BBSome accumulates in cilia of IFT27^miRNA cells, we expressed BBS5::YFP in IFT27^miRNA (resulting strain BBS5::YFP^27-miRNA) to the same level as in the BBS5::YFP-expressing CC-125 cells (BBS5::YFP) (Fig. 6C) (34). For both strains, BBS5::YFP entered cilia (Fig. 6D). BBS5::YFP cilia contained BBS5 (BBS5::YFP plus the endogenous BBS5) at WT level (Fig. 6D). As expected, BBS5::YFP^27-miRNA cilia accumulated BBS5 (BBS5::YFP plus the endogenous BBS5) and BBS5::YFP alone ∼3- and ∼4.5-folds higher than BBS5::YFP cilia, respectively (Fig. 6D). TIRF imaging of living cells identified that BBS5::YFP undergoes IFT in both strains at similar anterograde and retrograde speeds and anterograde frequency as Chlamydomonas BBSome subunits (Fig. 6 E and F and Movies S14 and S15 and Table 1) (12). However, BBS5::YFP exited BBS5::YFP^27-miRNA cilia at a reduced frequency, identifying that IFT27 promotes the ciliary removal of the BBSome (Fig. 6 E and F and Movies S14 and S15 and Table 1).

According to the BBS5::YFP kymogram, BBS5::YFP rarely drops from IFT in cilia of both CC-125 and IFT27^miRNA cells, indicating that the BBSome binds IFT tightly when trafficking between the ciliary base and tip (Fig. 6E). IFT/BBS remodels before undergoing turnaround to exit cilia at the ciliary tip (30–32). BBS1 and BBS5 accumulated at the ciliary tip of IFT27^miRNA cells rather than CC-125 and IFT27^Res-WT cells, revealing that IFT27 is required for the BBSome to load onto retrograde IFT trains for exiting cilia (Fig. 6G) (27). However, BBS1 and BBS5 cosedimented right in BBSome fractions in cilia of CC-125 and IFT27^Res-WT cells, while they remained to be separated and peaked at much lower fractions than the BBSome in cilia of IFT27^miRNA cells, revealing that the BBSome, unlike in the cell body (SI Appendix, Fig. S8A), cannot be reassembled into an intact complex after remodeling at the ciliary tip in the absence of IFT25/27 (Fig. 6H and SI Appendix, Fig. S8B). These data together demonstrate that IFT25/27 promotes the BBSome reassembly for coupling with IFT-B1 during the BBSome turnaround at the ciliary tip.

LZTFL1 Is a Positive Regulator of Phototaxis.

C. reinhardtii bbs mutants are nonphototactic (7, 12). As determined by both population and single-cell assays determining the cell’s locomotive reaction in response to light, the BBS1-null mutant bbs1-1 was nonphototactic (Fig. 7 A and B). The BBS1-rescuing strain bbs1-1^Res, like CC-125 cells, became normal in phototaxis, confirming BBSome loss in cilia to be sufficient to cause nonphototactic phenotype (Fig. 7 A and B). It remains unknown which biochemical defects of bbs cilia causes the nonphototactic phenotype, while maintenance of BBSome dynamics in cilia appears to be critical for C. reinhardtii to respond to light. Supportive of this notion, LZTFL1^miRNA cells contained reduced BBSome content in cilia, dramatically reducing the capacity for performing phototaxis (Fig. 7 C and D). Therefore, LZTFL1 is a positive regulator of phototaxis in C. reinhardtii. To test whether abnormal BBSome accumulation in cilia disrupts the phototaxis of C. reinhardtii cells as well, we performed the same assays on CC-125, IFT27^miRNA, and IFT27^Res-WT cells. Of note, IFT27^miRNA cells were resistant to light stimulation by exhibiting severely disrupted phototaxis, as compared to CC-125 and IFT27^Res-WT cells (Fig. 7 E and F). Therefore, disrupted ciliary BBSome dynamics by either abnormal buildup or the loss of the BBSome in cilia prevents C. reinhardtii cells from performing phototaxis.

Fig. 7.

LZTFL1 is a positive regulator of phototaxis. (A and B) Population phototaxis assay (A) and single-cell motion assay (B) of CC-125, bbs1-1, and the BBS1-rescuing bbs1-1^Res cells. (C and D) Population phototaxis assay (C) and single-cell motion assay (D) of CC-125, LZTFL1^miRNA, and LZTFL1^Res cells. (E and F) Population phototaxis assay (E) and single-cell motion assay (F) of CC-125, IFT27^miRNA, and IFT27^Res-WT cells. For panels A, C, and E, the direction of light is indicated (white arrows). For panels B, D, and F, the direction of light is indicated (green arrows). The radial histograms show the percentage of cells moving in a particular direction relative to the light (six bins of 60° each). Composite micrographs show the tracks of single cells. Each of the five merged frames was assigned a different color (blue is frame 1 and red is frame 5, corresponding to a travel time of 1.5 s). (Scale bar, 50 μm.)

Discussion

By using C. reinhardtii as a model organism, we observed that LZTFL1 recruits the BBSome to the basal body by facilitating BBS3 for basal body targeting, therefore deciding the BBSome amount available for loading onto anterograde IFT trains for entering cilia. At the ciliary tip, the BBSome disassembles to separate from anterograde IFT trains, followed by reassembling into intact complexes. IFT25/27 promotes BBSome reassembly for loading onto retrograde IFT trains for exiting cilia. LZTFL1 mediates BBSome exit from cilia via stabilizing IFT25/27 in the cell body. Our data show that LZTFL1 maintains BBSome dynamics in cilia through such a dual and independent process, thus closing a gap in our understanding of how LZTFL1 affects cell behavior (e.g., phototaxis) of C. reinhardtii.

How Does LZTFL1 Maintain BBSome Dynamics in Cilia?

LZTFL1 bridges the BBSome to retrograde IFT trains at the ciliary tip in murine cells, thus promoting the BBSome to exit cilia via IFT (27). Therefore, LZTFL1-null mutant prevents the BBSome from loading onto retrograde IFT trains at the ciliary tip, disrupting BBSome dynamics by causing BBSome accumulation in cilia (27, 38, 39). This does not apply to C. reinhardtii, as LZTFL1 does not interact with IFT in cilia (Fig. 2). Instead, Chlamydomonas LZTFL1, by residing in the cell body, promotes the BBSome to target to the basal body. In such a way, cells control the BBSome amount available for pickup by anterograde IFT trains at the basal body for entering cilia and, in turn, could control the presence and amount of BBSomes in cilia, which signaling molecules rely on for ciliary export and import via IFT (34, 48). Our previous study identified that RABL5/IFT22 binds and stabilizes BBS3 in the cell body (34). IFT22/BBS3 binds the BBSome and recruits it to the basal body when both are in a GTP-bound state (Fig. 8) (10, 34). We identified that LZTFL1 does not bind IFT22/BBS3 (SI Appendix, Fig. S9 A–C) nor affects the stability of IFT22 and BBS3 (Figs. 4H and 5B) but promotes BBS3 targeting to the basal body (Fig. 4J). It remains unknown how this process initiates and proceeds, while LZTFL1 controls BBSome ciliary content by mediating BBSome targeting to the basal body via BBS3, therefore playing a critical role in maintaining BBSome dynamics in cilia.

Fig. 8.

Hypothetical model of LZTFL1-mediated BBSome dynamics maintenance in cilia of C. reinhardtii. In the cell body, LZTFL1 stabilizes IFT25/27 and, by such a way, controls the amount of IFT25/27 available for trafficking to the basal body for entering cilia by integrating into IFT-B1 through direct interaction between IFT25/27 and IFT74/81 (23, 57) so as to control the ciliary abundance of IFT25/27. Partial IFT25/27 dissociates with IFT-B1 at the ciliary tip (35) and promotes the IFT-B1–separated BBSome subunits to reassemble into BBSome entities at the ciliary tip, eventually making BBSomes available for loading to retrograde IFT trains for removal out of cilia. LZTFL1 also promotes BBS3 to target to the basal body, which, in the aid of IFT22 by binding and stabilizing BBS3 (34), determines the amount of the BBSome available for being recruited to the basal body for integrating into anterograde IFT trains for entering cilia. By such a dual-mode system, LZTFL1 maintains BBSome dynamics in cilia of C. reinhardtii. LZTFL1 itself traffics to the basal body and diffuses into cilia and can bind the reassembled, but IFT-B1 separated, BBSome at the ciliary tip with unknown function. The kinesin-II anterograde motor and the cytoplasmic dynein-1b retrograde motor were also shown (20, 58). After turnaround at the ciliary tip, kinesin-II diffuses back to the ciliary base (59, 60). Please see our Discussion for more details.

In contrast to murine LZTFL1 that does not affect IFT25/IFT27 content (27), LZTFL1 is required for maintaining IFT25/27 stability in C. reinhardtii. As reflected in mammalian cilia, IFT25/27 provides the BBSome a docking site in the IFT-B1 component of retrograde IFT trains (27) or acts as a BBS3-specific GEF (8). For the latter, IFT-B1–separated IFT25/27, through mediating BBS3 GTPase cycling, controls the loading of the BBSome to retrograde IFT trains at the ciliary tip (8). Our data and others have shown that IFT25 binds and stabilizes IFT27 but not vice versa (Fig. 6A) (27, 29, 44). Therefore, no matter which above hypothesis is correct, their common outcome for the depletion of either IFT25 or IFT27 is to accumulate BBSomes in cilia (Fig. 6B) (27, 29, 44). Our study showed that the depletion of LZTFL1 reduces global IFT25/27 abundance, depriving IFT25/27 of being present in cilia (Fig. 5 B and C). Since knockdown of IFT27 or IFT25 alone causes BBSome accumulation at the ciliary tip by abolishing its ciliary removal through IFT, LZTFL1 is thus able to control BBSome export out of cilia via the IFT25/27 pathway (Fig. 6) (44). Taking all the evidence together, we conclude that LZTFL1 maintains BBSome dynamics in cilia by such a dual effect.

The Possible Role of the Ciliary Positioned LZTFL1.

LZTFL1 has been previously reported to reside in cilia of certain murine cell types but not human cells (38, 39). Although contradictory results about its ciliary localization, even in the same murine IMCD3 cell line, were recorded in the literature (38), our data and others together identified that LZTFL1 resides in the matrix fraction of cilia but at low abundance in C. reinhardtii (40). Upon entering cilia, Chlamydomonas LZTFL1 does not undergo IFT but diffuses between the ciliary base and tip (Fig. 2H). Underlying this process, LZTFL1 does not bind the BBSome nor interacts with IFT, even if they reside spatiotemporally in the same ciliary compartment (Fig. 2 A–F). LZTFL1 turns out to be likely to bind the BBSome at the ciliary tip (Fig. 2), supporting the observation that LZTFL1 binds the BBSome in hTERT-RPE1 cells (38). However, we barely found even a weak interaction between LZTFL1 and IFT/BBS in the cell body of C. reinhardtii cells (Fig. 4), questioning if human LZTFL1 does not enter cilia, especially when biochemical assays for identifying the interaction between LZTFL1 and the BBSome was performed on whole-cell rather than cell body extracts of hTERT-RPE1 cells (38).

Our study visualized the BBSome in a disassembled state in cilia, providing biochemical evidence to support the notion that the BBSome remodels upon arriving at the ciliary tip (30–32). This process initiates with BBSome disassembly for separating from anterograde IFT trains, followed by BBSome reassembly for reloading onto retrograde IFT trains (Fig. 8). The underlying mechanism of how BBSome remodeling initiates and proceeds at the ciliary tip remains unknown, while LZTFL1 likely binds the IFT-separated BBSome at the ciliary tip (Fig. 2), indicating that LZTFL1 could participate in promoting BBSome reassembly or BBSome reloading onto retrograde IFT trains or both. Afterward, LZTFL1 is assumed to dissociate with the BBSome, which thereafter exits cilia via IFT.

The Role of IFT25/27 in BBSome Remodeling at the Ciliary Tip.

Depletion of IFT25 or IFT27 disrupts the formation of IFT25/27 and causes BBSome accumulation in cilia (8, 27, 29, 44). BBSome retention in cilia is caused by defects in its removal out of cilia, assuming that IFT25/27 provides the BBSome a docking site in the IFT-B1 complex component of retrograde IFT trains in murine cells (27). Our previous study has shown that, in contrast to its mammalian counterpart (8), BBS3 is not required for the BBSome to load onto retrograde IFT trains in C. reinhardtii (35), thus opposing the above mammalian BBS3/IFT27 model to apply in C. reinhardtii (8). Interestingly, IFT25/27 absence in cilia of C. reinhardtii causes the BBSome to retain at a disassembled state, suggesting that IFT25/27 promotes BBSome reassembly at the ciliary tip (Fig. 8). This finding does not necessarily disapprove the mammalian model that IFT25/27 provides the BBSome a docking site in IFT-B1 (27). However, whether IFT25/27 promotes BBSome loading onto retrograde IFT trains at the ciliary tip remains to be determined in C. reinhardtii.

Do LZTFL1 and IFT25/27 Play Species- and Tissue/Cell-Specific Roles in Regulating Ciliogenesis?

LZTFL1 does not affect the ciliation of mammalian somatic cells other than the retinas but mediates BBSome ciliary trafficking for regulating ciliary signaling (27, 38, 39). In contrast, knockout of LZTFL1 reduces mouse fertility by disrupting the ciliogenesis of sperm flagella (49). Consistent with our finding, LZTFL1 knockout causes a significant decrease of the testicular IFT27 protein but maintains other IFT proteins (checked with IFT20, IFT81, IFT88, and IFT140) at WT levels (49). Of note, rodent IFT25 and IFT27 knockouts also maintain normal ciliation in somatic cells (27, 29). Sperm flagellum formation instead relies on IFT25/27 in the mouse (50, 51). Other than this example, knockdown of IFT27 causes defects in the ciliary assembly by disassembling IFT-B in Trypanosoma brucei (52). These findings together assume species- and tissue/cell-specific differences in the role of LZTFL1 and IFT25/27 in ciliation. Chlamydomonas IFT27, when depleted by RNA interference knockdown, was reported to disrupt ciliogenesis by destabilizing both IFT-A and IFT-B (47). Since the authors did not perform a functional rescue experiment to verify the phenotype observed, the conclusion was suspicious. Our studies did not find any defects in maintaining the stability of IFT-A and IFT-B and ciliation as well by knocking down IFT25, IFT27, or LZTFL1 alone by miRNA interference in C. reinhardtii (SI Appendix, Figs. S3 and S7) (44). Given that three Chlamydomonas proteins were not depleted completely by knockdown, their possible role in ciliogenesis could be determined eventually only when null mutants for these proteins become available.

How Does LZTFL1 Regulate Phototaxis?

In C. reinhardtii, mutations in BBSome subunits cause the ciliary deprivation of the BBSome, thus impairing phototaxis due to biochemical defects of the ciliary membrane (7, 12, 28). These defects may arise from abnormal buildup of receptors in cilia due to their disrupted removal out of cilia in the absence of the BBSome or from BBSome accumulation in cilia, as this prevents receptors from exiting cilia as well. Among these receptors, the photoreceptors channelrhodopsin 1 (ChR1) and ChR2 initiates phototaxis by inducing light-induced cilium beating in C. reinhardtii (53–55). In response to light stimulation, ChR1 transports from eyespots to cilia in an IFT-dependent manner, suggesting that ciliary positioning might be a prerequisite for channelrhodopsins to mediate phototaxis (56). It is most likely that disrupted BBSome dynamics in cilia causes the ciliary accumulation of channelrhodopsins, which eventually impairs the phototaxis of C. reinhardtii cells. Therefore, LZTFL1 could regulate phototaxis by mediating the ciliary dynamics of channelrhodopsins via the BBSome.

Materials and Methods

Chlamydomonas Strains and Culture Conditions.

Chlamydomonas strain CC-125 was used throughout this study and was purchased from the Chlamydomonas Genetic Center at the University of Minnesota (http://www.chlamycollection.org/). The BBS1-null mutant bbs1-1, the BBS5::YFP-expressing strain BBS5::YFP, the IFT46::YFP-expressing strain IFT46::YFP, the BBS3::YFP-expressing strain BBS3::YFP, the BBS3 knockdown strain BBS3^miRNA, and the BBS3-rescuing strain BBS3^Res-WT have been reported previously (12, 34, 43). All the strains used in this study were listed in SI Appendix, Table S1. All the strains were cultured at room temperature in Tris acetic acid phosphate medium in a continuous light with constant aeration. Depending on a specific strain, cells were cultured with or without the addition of 20 µg/mL paromomycin (Sigma-Aldrich), 15 µg/mL bleomycin (Invitrogen), or both antibiotics with 10 µg/mL paromomycin and 5 µg/mL bleomycin.

Various experimental protocols were applied in this study, and most of the experiments were briefly introduced in the text to make the content easier to be understood. The details of each protocol are available in SI Appendix.

Data Availability

All study data are included in the article and/or supporting information.