text:
Published online August 31, 2021
Differential Roles of Tubby Family Proteins in Ciliary Formation and Trafficking
Julie J. Hong1,3 , Kyung Eun Kim1,3 , So Young Park1 , Jinwoong Bok2 , Jeong Taeg Seo1 , and Seok Jun Moon1, *
1Department of Oral Biology, BK 21 FOUR Project, Yonsei University College of Dentistry, Seoul 03722, Korea, 2Department of Anatomy, Yonsei University College of Medicine, Seoul 03722, Korea, 3These authors contributed equally to this work.
Correspondence to : sjmoon@yuhs.ac
Received: April 7, 2021; Revised: June 1, 2021; Accepted: June 2, 2021
Cilia are highly specialized organelles that extend from the cell membrane and function as cellular signaling hubs. Thus, cilia formation and the trafficking of signaling molecules into cilia are essential cellular processes. TULP3 and Tubby (TUB) are members of the tubby-like protein (TULP) family that regulate the ciliary trafficking of G-protein coupled receptors, but the functions of the remaining TULPs (i.e., TULP1 and TULP2) remain unclear. Herein, we explore whether these four structurally similar TULPs share a molecular function in ciliary protein trafficking. We found that TULP3 and TUB, but not TULP1 or TULP2, can rescue the defective cilia formation observed in TULP3-knockout (KO) hTERT RPE-1 cells. TULP3 and TUB also fully rescue the defective ciliary localization of ARL13B, INPP5E, and GPR161 in TULP3 KO RPE-1 cells, while TULP1 and TULP2 only mediate partial rescues. Furthermore, loss of TULP3 results in abnormal IFT140 localization, which can be fully rescued by TUB and partially rescued by TULP1 and TULP2. TUB's capacity for binding IFT-A is essential for its role in cilia formation and ciliary protein trafficking in RPE-1 cells, whereas its capacity for PIP2 binding is required for proper cilia length and IFT140 localization. Finally, chimeric TULP1 containing the IFT-A binding domain of TULP3 fully rescues ciliary protein trafficking, but not cilia formation. Together, these two TULP domains play distinct roles in ciliary protein trafficking but are insufficient for cilia formation in RPE-1 cells. In addition, TULP1 and TULP2 play other unknown molecular roles that should be addressed in the future.
Keywords cilia, cilia formation, ciliary trafficking, RPE1, TULP
Primary cilia are microtubule-based sensory organelles present on the surface of most mammalian cell types. Although long thought dispensable, cilia are now recognized to have essential roles in various cellular processes, including developmental signaling and adult homeostasis (Drummond, 2012; Fliegauf et al., 2007). Disruptions of ciliary function lead to ciliopathies, such as the developmental disorders polycystic kidney disease, retinitis pigmentosa, Bardet-Biedl syndrome, and Joubert syndrome (Ansley et al., 2003; Green et al., 1989; Hildebrandt et al., 2011; Liu et al., 2002; Pazour et al., 2000; Valente et al., 2006).
Primary cilia receive many extracellular signals, including Hedgehog, Wnt, and Notch (Ezratty et al., 2011; Huangfu et al., 2003; Oishi et al., 2006; Rohatgi et al., 2007). Therefore, to ensure proper function, the ciliary membrane houses many transmembrane signaling molecules such as platelet-derived growth factor receptors (PDGF), Smoothened (Smo), transient receptor potential (TRP) channels, and G protein-coupled receptors (GPCRs) (Christensen et al., 2007; Colbert et al., 1997; Corbit et al., 2005; Hilgendorf et al., 2016; Mukhopadhyay et al., 2013; Rohatgi et al., 2007). Together, these receptors modulate a number of critical developmental signaling pathways, but our understanding of the mechanisms by which these membrane proteins are targeted to cilia and concentrated there remains sparse.
Intraflagellar transport (IFT) is a well-characterized, conserved system that controls ciliary protein trafficking. Its specialized transport machinery comprises two discrete multi-protein complexes—IFT-A and IFT-B (Cole et al., 1998; Rosenbaum and Witman, 2002). The IFT-B complex, in cooperation with kinesin-2, mediates anterograde transport towards the ciliary tip, while the IFT-A complex, along with dynein, mediates retrograde transport back towards the ciliary base (Ishikawa and Marshall, 2011; Scholey, 2003; Taschner et al., 2012). IFT is thought to play a critical role in establishing and sustaining proper cilia formation and function (Rosenbaum and Witman, 2002; Scholey, 2003). Still, although many membrane proteins depend on IFT proteins for ciliary trafficking (Crouse et al., 2014; Keady et al., 2011; Nachury et al., 2010), it remains unclear whether the IFT complexes regulate this process directly themselves or whether other regulatory mechanisms are also required.
Mammalian TULP3 reportedly plays a key role in the ciliary trafficking of several membrane proteins, including the rhodopsin family GPCRs Sstr3, Mchr1, Npy2r, GPR161, as well as the polycystins (Badgandi et al., 2017; Mukhopadhyay et al., 2010; 2013). TULP3 captures ciliary membrane proteins in a phosphoinositide 4,5-bisphosphate (PI(4,5)P2)-dependent mechanism, presumably by binding to the PI(4,5)P2-binding domain, and then moves together with its cargo into the cilia via IFT-A binding (Badgandi et al., 2017). Tubby (TUB), the founding member of the tubby-like protein (TULP) family, is also required for the ciliary trafficking of Sstr3, Mchr1, and Npy2r (Loktev and Jackson, 2013; Sun et al., 2012). These molecular functions seem to be conserved throughout the animal kingdom, as the Drosophila tubby homolog dTULP and the C. elegans homolog tub-1 are also involved in the ciliary trafficking of membrane receptors (DiTirro et al., 2019; Park et al., 2013).
Tubby mice show maturity-onset obesity with neurosensory deficits (Kleyn et al., 1996; Noben-Trauth et al., 1996), both common ciliopathy traits. Although the particular cell types and GPCRs that cause the obesity phenotype of tubby mice have not yet been identified, the phenotype is almost certainly linked to TUB's ciliary functions. On the other hand, TULP3 was originally identified as a negative regulator of Hedgehog signaling (Norman et al., 2009), and the TULP3-dependent nature of ciliary GPCR trafficking has been clearly implicated in the underlying mechanism (Mukhopadhyay et al., 2010).
While some of the molecular functions of mammalian TULPs, especially of TULP3 and TUB, have been identified, those of TULP1 and TULP2 have not. Whereas the loss of TULP1 function leads to retinal degeneration in both humans and mice (Hagstrom et al., 1999; Ikeda et al., 2000), there are not yet any functional data available for TULP2. Because TULP1 and TULP2 each have either the conserved IFT-A-binding domain or the membrane phosphoinositide-binding domains of the TULPs, and because these domains are required for the ciliary trafficking of GPCRs, there is a good chance TULP1 and TULP2 share some of the molecular functions of TULP3 and TUB.
Here, we report the results of our investigation into the ciliary functions of the four TULPs—TULP1, TULP2, TULP3, and TUB—in hTERT RPE-1 (RPE1) cells. We found that, despite significant amino acid-level similarity, these four proteins play separate roles in primary cilia assembly and trafficking. The functions of TULP3 and TUB are largely analogous with respect to cilia formation and ciliary protein trafficking. In contrast, although TULP1 and TULP2 show limited capacity to control ciliary protein trafficking, the addition of an IFT-A binding domain increases it. Together, while TULP3 and TUB are critical ciliary membrane trafficking regulators, TULP1 and TULP2 must play different molecular roles that should be investigated in the future.
Both RPE1 wild-type (CRL-4000; American Type Cell Collection [ATCC], USA) and TULP3 knockout (KO) cells were cultured in DMEM/F-12 (11320082; Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS) (10099141; Thermo Fisher Scientific) and 1% Penicillin/Streptomycin (P/S) (15-140-122; Thermo Fisher Scientific). Cells were grown on coverslips in 12-well culture plates at a density of 0.5 × 106 cells/well and cultured in a CO2 incubator. Cells were shifted from 10% to 0.5% serum 24 h after transfection to induce ciliation and fixed 72 h post-transfection. Plasmid transfections were carried out with a NEPA21 Electroporator (NEPA GENE, Japan) at a pulse voltage of 150 V and pulse length of 5 ms.
RNA isolation and quantitative RT-PCR
Total RNA was extracted using the RNeasy mini kit (74104; Qiagen, Germany). Complementary DNA (cDNA) was synthesized from total RNA with the RevertAid Reverse Transcriptase (EP0442; Thermo Fisher Scientific) in accordance with the manufacturer's instructions. To assess gene expression, cDNAs were amplified using the SensiFast SYBR HiRox Kit (92020; Meridian Bioscience, USA). Each reaction was performed in triplicate. A comprehensive list of PCR primers appears in Supplementary Table S1. Amplification conditions were as follows: 2 min at 95°C for polymerase activation, 40 cycles at 95°C denaturation for 5 s, 60°C annealing for 10 s, and 72°C extension for 20 s. The cycle threshold (Ct) values for the target genes and GAPDH were measured and calculated with the 7500 fast system sequence detection software (Applied Biosystems, USA).
Immunoblot analysis
Cells were washed with phosphate-buffered saline (PBS), lysed on ice in RIPA buffer (BRI-9001; TIB Molbiol, Germany) containing protease inhibitors, and centrifuged at 15,000 × g for 20 min. Samples were then mixed with a loading buffer containing 2-mercaptoethanol (1610747; Bio-Rad, USA) and incubated at 95°C for 5 min. Each protein sample was then resolved via SDS-PAGE using 8% polyacrylamide gels and electrophoretically transferred to a PVDF membrane (IPVH00010; Millipore Sigma, USA). The membranes were then incubated with blocking buffer (5% skimmed milk in Tris-buffered saline [TBS] containing 0.1% Tween 20; TBS-T) for 1 h at room temperature. Primary antibodies specific to TULP3 (13637-1-AP; Proteintech, USA) at 1:1,000 and TUB (17928-1-AP; Proteintech) at 1:1,000 were diluted in blocking buffer and incubated with the membranes overnight at 4°C on a rocking platform. The following day, the membranes were washed four times at 10 min intervals with TBS-T. HRP-conjugated-anti-Rabbit IgG at 1:5,000 (A32731; Sigma-Aldrich, USA) was diluted in blocking buffer and incubated with the membranes as a secondary antibody for 1 h at room temperature on a rocking platform. The membranes were then rinsed in TBS-T four times at 10 min intervals before being visualized with an ECL detection reagent (RPN2232; GE Healthcare, USA).
Establishment of the TULP3 KO RPE1 cell line using CRISPR/Cas9
A single-guide RNA (sgRNA) sequence (5'-AGAAATGATGAAGATGCGAC-3') targeting exon 1 of the TULP3 gene was designed using the CRISPR/Cas9 design program (Sentmanat et al., 2018). Forward and reverse oligonucleotides matching the sgRNA sequence were ordered, annealed, and cloned into the pSpCas9 (BB)-2A-Puro (PX459) V2.0 vector (Addgene plasmid #62988) (Ran et al., 2013). RPE1 cells were transfected with the PX459 vector containing the sgRNA-encoding cassette by electroporation. Cells were subcultured at a ratio of 1:6 in media for 72 h and maintained until the formation of distinct colonies. Individual clones were isolated and expanded. Genomic DNA from single clones was extracted and analyzed for TULP3 mutations by sequencing. Lines with biallelic TULP3 disruption were selected, and the absence of TULP3 protein expression in these TULP3 KO RPE1 cells was confirmed with immunoblotting.
Plasmids
Mouse Tulp1, Tulp2, Tulp3, and TUB cDNAs were cloned into the pIRES2-DsRed2 vector (Clontech plasmid #632420), which expresses both the gene of interest and the dsRed gene. TUB PIP2- and IFT-A-mutant cDNAs were cloned into the pLVX-EF1α-IRES-ZsGreen vector (Clontech plasmid #631982), which expresses both the gene of interest and the ZsGreen gene. To examine the subcellular localization of TULPs in RPE1 cells, mouse Tulp1, Tulp2, Tulp3, TUB, and Tulp1IFT-A(+) cDNAs were cloned into the pCMV-Tag3B vector (Agilent Technologies plasmid #211173), which makes it possible to visualize the expression of a gene of interest using an anti-Myc antibody.
Immunofluorescence analysis
Samples were fixed with 4% paraformaldehyde in 1× PBS at room temperature for 10 min. After three washes with PBS, cells were permeabilized with 0.2% Triton X-100 (PBS-T) for 30 min and blocked with 5% goat serum in PBS for 1 h at room temperature. For TULP3 staining, cells were blocked in PBS containing 0.1% Triton X-100 and 2% donkey serum. Samples were subsequently incubated in a blocking solution containing primary antibodies at 4°C overnight. The following day, coverslips were rinsed three times with PBS, incubated with a blocking solution containing secondary antibodies at room temperature for 1 h. They were then rinsed twice with PBS, mounted in Vectashield (Vector Laboratories, USA), and examined using a Zeiss LSM700 confocal microscope (Germany) with a 60× plan-apochromat oil-immersion objective. When comparing the expression and localization of ciliary proteins between groups, all samples were prepared simultaneously, and the resulting confocal images were obtained under the same conditions. We only examined DsRed-positive RPE1 cells to exclude any non-transfected cells.
The primary antibodies used for immunofluorescence were diluted as follows: mouse anti-α-acetylated tubulin, 1:1,000 (T6793; Millipore Sigma); mouse anti-γ-acetylated tubulin, 1:800 (T6557; Sigma-Aldrich); chicken anti-mCherry, 1:700 (ab205402; Abcam, UK); rabbit anti-TULP3, 1:100 (13637-1-AP; Proteintech), 1:100; rabbit anti-ARL13B, 1:400 (17711-1-AP; Proteintech); rabbit anti-GPR161, 1:400 (13398-1-AP; Proteintech); rabbit anti-INPP5E, 1:400 (17797-1-AP; Proteintech); rabbit anti-IFT140, 1:300 (17460-1-AP; Proteintech); rabbit anti-c-Myc 1:500 (C3956; Sigma-Aldrich), 1:500; rabbit anti-IFT88, 1:300 (13967-1-AP; Proteintech). The secondary antibodies used were diluted as follows: Alexa Fluor 488, 568-conjugated anti-mouse IgG, 1:400 (A11029 and A11031; Invitrogen, USA); Alexa Fluor 568-conjugated anti-rabbit IgG, 1:400 (A11011; Invitrogen), Alexa 633-conjugated anti-rabbit IgG, 1:400 (A21070; Invitrogen); Alexa Fluor 555-conjugated anti-chicken IgY, 1:500 (A32932; Invitrogen). Control and non-transfected TULP3 KO cells were also counterstained with TO-PRO3 at 1:1,000 (1878895; Invitrogen).
All data are presented as mean ± SEM and based on results obtained from at least three independent experiments. All statistical analyses were carried out using GraphPad Prism 9 (GraphPad Software, USA). One-way ANOVAs with Tukey's post-hoc tests were used for multiple comparisons. Pearson's χ2 test was used for comparisons of ciliary IFT140 localization.
Generating a TULP-free RPE1 cell line
To investigate the role of TULPs in ciliary trafficking, we decided to utilize hTERT RPE-1 (RPE1) cells because they produce clear cilia and are commonly used to study cilia formation and ciliary protein trafficking. We first asked which TULPs are expressed in RPE1 cells. We found via quantitative RT-PCR that while TULP3 is abundant in RPE1 cells, the other TULPs are almost undetectable (Supplementary Fig. S1A). We further confirmed the presence of TULP3 and the absence of TUB in RPE1 cells via immunoblot (Supplementary Fig. S1B). In addition, we found that endogenous TULP3 is localized to cilia with a slight enrichment in the ciliary base (Supplementary Fig. S1E). Based on these preliminary results, we wanted to generate a TULP-free RPE1 cell line so we could re-introduce the TULPs and identify any shared ciliary functions. We therefore attempted to generate TULP3-knockout RPE1 (TULP3 KO RPE1) cells using the CRISPR/Cas9 system (Ran et al., 2013). We obtained one TULP3 KO RPE1 cell line by targeting the first exon of TULP3, leading to a frameshift mutation in the distal end of the second exon (Supplementary Fig. S1C). We then confirmed the successful knockout of TULP3 using immunocytochemistry and western blot analysis (Supplementary Figs. S1D and S1E).
Effect of TULPs in cilia formation in RPE1 cells
Consistent with previous results (Han et al., 2019), we found TULP3 KO RPE1 cilia were significantly disrupted compared to wild-type RPE1 cilia (Fig. 1). When we compared the cilia of serum-starved wild-type and TULP3 KO RPE1 cells, we found that while 50% of wild-type RPE1 cells assembled a cilium, only 20% of TULP3 KO RPE1 cells assembled one (Fig. 1B). In addition, the primary cilia of TULP3 KO RPE1 cells averaged only half the length of wild-type RPE1 cilia (Fig. 1C). This suggests TULP3 is critical for proper cilia formation in RPE1 cells (Fig. 1C). We then found that re-introduction of Tulp3 into TULP3 KO RPE1 cells by transfection fully rescued their cilia defects, confirming the specificity of TULP3's role in cilia formation. When we introduced other TULPs into TULP-free RPE1 cells, we found that TUB, but not Tulp1 or Tulp2, fully rescued cilia formation and length (Figs. 1B and 1C). To exclude the possibility that mislocalization of TULP1 and TULP2 in TULP3 KO RPE1 cells attributes to their inability to rescue defective cilia formation, we examined the intracellular localization of TULP1 and TULP2. TULP1 was mainly localized in the nucleus as previously reported (He et al., 2000), and TULP2 was enriched in cilia (Supplementary Fig. S2). Their subcellular localization was not dependent on TULP3, indicating that TULP1 and TULP2 did not play a role in cilia formation.
Effect of TULPs on ciliary membrane protein trafficking in RPE1 cells
TULP3 regulates the localization of several ciliary membrane-associated proteins, as well as a subset of GPCRs (Badgandi et al., 2017; Han et al., 2019; Mukhopadhyay et al., 2010; 2013). Indeed, consistent with previous reports (Han et al., 2019), we found that TULP3 KO RPE1 cells showed impaired ciliary localization of ARL13B, INPP5E, and GPR161, and that TULP3 expression rescued these trafficking defects (Fig. 2). We therefore proceeded to explore further whether the other TULPs could also rescue this impaired ciliary protein localization. While the expression of TUB induced a nearly full rescue of ciliary protein localization, Tulp1 and Tulp2 induced only a partial rescue. Specifically, compared to that facilitated by TULP3, expression of TULP1 and TULP2 in TULP3 KO RPE1 cells facilitated approximately 25% and 40%, respectively, of the ciliary localization of ARL13B, INPP5E, and GPR161 (Fig. 2). These data suggest TULP1 and TULP2 may be additional modulators of ciliary protein trafficking, though to a lesser extent than TUB and TULP3.
Effect of TULPs on IFT subunit trafficking in RPE1 cells
The core IFT-A complex is essential for the ciliary localization of TULP3, as TULP3 localization is lost upon ablation of the core IFT-A subunits IFT140, IFT122, or WDR19 (Mukhopadhyay et al., 2010; Qin et al., 2011). Because TULP3 acts as an adaptor between the core IFT-A complex and ciliary membrane cargo (Badgandi et al., 2017), we asked whether the loss of TULP3 affects the ciliary localization of the core IFT-A subunits. Specifically, we examined IFT140 localization in RPE1 cells because the phosphoinositide-dependent localization of TULP3 also affects the ciliary localization of IFT140 (Garcia-Gonzalo et al., 2015). In wild-type RPE1 cells, IFT140 is primarily localized to a singular focus at the base of the cilium, with some occasional faint staining at the ciliary tip (Fig. 3, Supplementary Fig. S3C). Upon depletion of TULP3, however, IFT140 staining was either localized in two foci, one at the ciliary base and one at the tip, or it was uniformly distributed across the entire cilium (Fig. 3). This suggests TULP3 plays an integral role in the trafficking of IFT140. Upon the expression of TUB and TULP3 in TULP3 KO RPE1 cells, approximately 70% of the cells showed a reversion of IFT140 staining to the singular focus observed in wild-type RPE1 cells (Fig. 3). Furthermore, expression of TULP1 and TULP2 also rescued IFT140 localization back to the ciliary base, though to a lesser degree than TUB and TULP3 (Fig. 3). In contrast, TULP3 depletion did not affect the ciliary distribution of IFT88, indicating that TULPs only control the trafficking of IFT-A (Supplementary Figs. S3A and S3B).
Tubby's IFT-A and PIP2-binding domains contribute differentially to ciliary protein trafficking
Previous studies have shown that TULP's N-terminal IFT-A binding domain and C-terminal PIP2 binding domain are crucial for proper regulation of ciliary membrane protein trafficking (Mukhopadhyay et al., 2010; Park et al., 2013). We decided to evaluate the contribution of each of these domains in the TULPs to cilia formation and IFT-A trafficking in RPE1 cells. When we expressed a mutant form of TUB defective in IFT-A binding (TUBIFT-A(–)) in TULP3 KO RPE1 cells, not only did it fail to rescue cilia formation, but it also induced the formation of even shorter cilia than those produced by control TULP3 KO RPE1 cells. This was reminiscent of the dominant negative effect mutant TULP3 defective in IFT-A binding has on GPCR ciliary trafficking (Mukhopadhyay et al., 2010) (Figs. 4A-4C). In contrast, a mutant form of TUB defective in PIP2 binding (TUBPIP2(–)) did partially rescue cilia formation and cilia length (Figs. 4A-4C). Furthermore, while TUBIFT-A(–) did not rescue IFT140 localization, TUBPIP2(–) permitted a partial rescue of IFT140 localization (Figs. 4D and 4E). These data indicate that while both domains of TUB are vital for both cilia formation and IFT-A trafficking, IFT-A binding is more important in RPE1 cells. Consistent with these results, TUBPIP2(–) permitted a near total rescue of the ciliary localization of ARL13B, INPP5E, and GPR161, but TUBIFT-A(–) did not (Supplementary Fig. S4). These data indicate that TUB's IFT-A binding domain is essential for the trafficking of ciliary membrane proteins.
Enhancement of TULP1 IFT-A binding improved its regulation of cilia protein trafficking
The IFT-A binding domain of the TULPs seems to be essential for cilia formation and ciliary protein trafficking. While TULP1 contains a conserved Tubby domain, it does not bind to the core IFT-A complex. It contains a far less conserved IFT-A binding domain than the other TULPs that also failed to interact with IFT-A in an in vitro protein interaction assay (Fig. 5A) (Mukhopadhyay and Jackson, 2011; Mukhopadhyay et al., 2010). We reasoned that adding IFT-A binding to TULP1 may allow it to fully rescue cilia formation and ciliary protein trafficking in TULP3 KO RPE1 cells. To test this hypothesis, we substituted the putative IFT-binding domain of TULP3 (20-59 amino acids of TULP3) for the corresponding 40-amino-acid stretch in TULP1 (22-61 amino acids of TULP1). Hereafter, we refer to this chimeric protein as TULP1IFT-A(+) (Fig. 5B). In contrast to the nuclear localization of TULP1, TULP1IFT-A(+) was mainly localized in the cilia. This implicates the IFT-A binding domain in dictating the cilia localization of TULP1IFT-A(+) (Fig. 5C). Next, we examined the effect of TULP1IFT-A(+)on cilia formation and ciliary protein trafficking. We found that expression of TULP1IFT-A(+) in TULP3 KO RPE1 cells had no effect on the formation of their cilia or on their ciliary length defects (Figs. 5D and 5E), but almost completely rescued the localization of ARL13B, INPP5E, GPR161, and IFT140 (Figs. 5F-5I). This suggests TULP1's inability to control ciliary protein trafficking was due to its inability to bind IFT-A.
Here, we have shown that TUB and TULP3, but not TULP1 or TULP2, support cilia formation and protein trafficking in RPE1 cells. The main characteristic distinguishing the TULPs with respect to this phenomenon is their capacity to bind to the IFT-A complex, as a chimeric TULP1 harboring the ITF-A binding domain of TULP3 rescued ciliary protein trafficking just as TULP3 did.
Although previous studies found that TULP3 did not affect ciliogenesis (Mukhopadhyay et al., 2010), we found complete knockout of TULP3 in RPE1 cells using CRISPR/Cas9 downregulated cilia formation. This finding is consistent with another study that used TULP3 KO RPE1 cells (Han et al., 2019). We expect that this discrepancy is due to an incomplete knockdown of TULP3 via RNA interference. It is noteworthy, however, that the cilia of Tulp3-/- mouse embryonic fibroblasts (MEFs) are comparable to those of wild-type MEFs (Norman et al., 2009). This could be due to differential requirements of TULP3 for ciliogenesis in each cell type, which would mean TULP3 is a non-universal regulator of ciliogenesis. It is also possible that TUB plays a redundant role in Tulp3–/– MEFs.
Although we found that IFT140 localization depended on TULP3 and TUB (Fig. 3), a previous study found that TULP3 knockdown did not affect IFT140 localization (Mukhopadhyay et al., 2010), perhaps due to incomplete TULP3 depletion. However, it is unclear whether the mislocalization of IFT140 is due to the inability of INPP5E to localize in cilia in TULP3 KO RPE1 cells because INPP5E affects both the ciliary localization and level of the IFT-A subunits IFT140 and IFT139 (Garcia-Gonzalo et al., 2015). Based on our findings, the interaction between IFT140 and TULP3 is more important, because we found that expression of TUB lacking its PIP2-binding domain in TULP3 KO cells still rescued INPP5E localization (Supplementary Fig. S4B), but not IFT140 localization (Figs. 4D and 4E).
Although chimeric TULP1 containing the IFT-A binding domain of TULP3 fully rescued the ciliary trafficking of IFT-A and other proteins, it did not rescue the defective cilia formation of TULP3 KO RPE1 cells. In addition, TULP2, which contains both IFT-A and PIP2-binding domains, was localized to cilia, despite lacking any ciliary rescue ability. This data suggest the IFT-A and PIP2-binding domains of TUB and TULP3 are not the only domains required for cilia formation and ciliary protein trafficking. Future studies will be necessary to determine which additional unknown domains of TULP3 and TUB contribute to proper cilia formation in RPE1 cells.
In conclusion, we have demonstrated that TULP3 and TUB have a similar capacity to regulate cilia formation and ciliary protein trafficking in RPE1 cells. It is likely that more functions of TULP3 and TUB will be revealed in the future, and it will be intriguing to explore the molecular functions of TULP1 and TULP2, both of which may not have anything to do with cilia.
This work was supported by National Research Foundation of Korea (NRF) Grants funded by the Korean Government (NRF-2016R1A5A2008630 and NRF-2018R1A2B3001668).
J.J.H. and K.E.K. conducted most of the experiments. S.Y.P. performed plasmid transfections. J.B. and J.J.H. analyzed the data. J.T.S. and S.J.M. designed and supervised the project and wrote the paper.
Fig. 1. Effects of TULPs on ciliogenesis in RPE1 cells. (A) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated TULPs. Cells were stained with an antibody specific to the axonemal marker Ac-tubulin. Transfected cells were identified with DsRed staining. Non-transfected cells were identified with the nuclear stain To-Pro3. Scale bars = 5 μm. (B and C) Quantification of ciliated cells (B) and cilia length (C). Data are presented as mean ± SEM from three independent experiments. More than 300 cells were counted for each genotype for the ciliary formation experiment and more than 100 were counted for each genotype for the ciliary length experiment. One-way ANOVAs with Tukey's post-hoc tests. ***P < 0.001.
Fig. 2. Effects of TULPs on ciliary trafficking of ARL13B, INPP5E, and GPR161 in RPE1 cells. (A-C) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated TULPs. Cells were stained with antibodies specific for the axonemal marker Ac-tubulin, as well as ARL13B (A), INPP5E (B), or GPR161 (C). Transfected cells were identified with DsRed staining. Non-transfected cells were identified with the nuclear stain To-Pro3. Enlarged images of single cilia displayed on the right. Scale bars = 5 μm. A quantification of ciliated cells expressing each ciliary protein is shown below. Data are presented as mean ± SEM from three independent experiments. More than 100 ciliated cells were counted for each condition. One-way ANOVAs with Tukey's post-hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 3. Effects of TULPs on ciliary trafficking of IFT140 in RPE1 cells. (A) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated TULPs. Cells were stained with antibodies specific for the axonemal marker Ac-tubulin and IFT140. Transfected cells were identified with DsRed staining. Non-transfected cells were identified with the nuclear stain To-Pro3. Insets, enlarged images of the cilia marked with arrowheads. Scale bars = 5 μm. (B) Quantification of IFT140 localization. Data are presented as mean ± SEM from three independent experiments. Total counted cells are 100-150 for each condition. Pearson's χ2 test. ****P < 0.0001.
Fig. 4. Differential contributions of the IFT-A and PIP2-binding domains of TUB to ciliary formation and IFT140 trafficking. (A) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated forms of TUB. TUBIFT-A(–), a mutant TUB that does not bind IFT-A; TUBPIP2(–), a mutant TUB that does not bind PIP2. Cells were stained with an antibody specific for the axonemal marker Ac-tubulin. Transfected cells were identified with either DsRed or ZsGreen. Non-transfected cells were identified with the nuclear marker To-Pro3. Scale bars = 5 μm. (B and C) Quantification of ciliated cells (B) and cilia length (C). Data are presented as mean ± SEM from three independent experiments. More than 300 cells were counted for each genotype for the ciliary formation experiment and more than 90 were counted for each genotype for the ciliary length experiment. One-way ANOVAs with Tukey's post-hoc tests. (D) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated forms of TUB. Cells were stained with antibodies specific for the axonemal marker Ac-tubulin and IFT140. Transfected cells were identified with ZsGreen. Non-transfected cells were identified with the nuclear marker To-Pro3. Insets, enlarged images of the cilia marked with arrowheads. Scale bars = 5 μm. (E) Quantification of IFT140 localization. Data are presented as mean ± SEM from three independent experiments. Total counted cells are 90-150 for each condition. Pearson's χ2 test. *P < 0.05, ***P < 0.001, ****P < 0.0001. n.s., not significant.
Fig. 5. IFT-A binding in the TULPs is essential for the proper ciliary trafficking and localization of IFT140. (A) Multiple sequence alignment of the putative IFT-A binding domain from the TULPs. (B) Schematic depicting the formation of TULP1IFT-A(+). The putative IFT-A binding domain of TULP3 (amino acids 20-59) was used to replace the corresponding 40 amino acid sequence in TULP1. (C) Immunocytochemical analysis of TULP1 and TULP1IFT-A(+) in wild-type and TULP3 KO RPE1 cells. Cells were stained with antibodies specific for the axonemal marker Ac-tubulin and c-Myc for TULP1 and TULP1IFT-A(+). Nuclei were stained with To-Pro3. TULP1IFT-A(+)-positive cilia are marked with arrowheads. Scale bars = 5 μm. (D and E) Quantification of ciliated cells (D) and cilia length (E). Data are presented as mean ± SEM from three independent experiments. More than 300 cells were counted for each genotype for the ciliary formation experiment and more than 100 were counted for each genotype for the ciliary length experiment. One-way ANOVAs with Tukey's post-hoc tests. ***P < 0.001. (F-H) Quantification of ciliated cells expressing each ciliary protein. Data are presented as mean ± SEM from three independent experiments. More than 100 cells were counted for each condition. One-way ANOVAs with Tukey's post-hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001. (I) Quantification of IFT140 localization. Data are presented as mean ± SEM from three independent experiments. Total counted cells are 90-150 for each condition. Pearson's χ2 test. *P < 0.05, **P < 0.01, ****P < 0.0001. n.s., not significant.
Ansley S.J., Badano J.L., Blacque O.E., Hill J., Hoskins B.E., Leitch C.C., Kim J.C., Ross A.J., Eichers E.R., and Teslovich T.M., et al. (2003). Basal body dysfunction is a likely cause of pleiotropic Bardet-Biedl syndrome. Nature 425, 628-633.
Badgandi H.B., Hwang S.H., Shimada I.S., Loriot E., and Mukhopadhyay S. (2017). Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins. J. Cell Biol. 216, 743-760.
Christensen S.T., Pedersen L.B., Schneider L., and Satir P. (2007). Sensory cilia and integration of signal transduction in human health and disease. Traffic 8, 97-109.
Colbert H.A., Smith T.L., and Bargmann C.I. (1997). OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans. J. Neurosci. 17, 8259-8269.
Cole D.G., Diener D.R., Himelblau A.L., Beech P.L., Fuster J.C., and Rosenbaum J.L. (1998). Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. J. Cell Biol. 141, 993-1008.
Corbit K.C., Aanstad P., Singla V., Norman A.R., Stainier D.Y., and Reiter J.F. (2005). Vertebrate Smoothened functions at the primary cilium. Nature 437, 1018-1021.
Crouse J.A., Lopes V.S., SanAgustin J.T., Keady B.T., Williams D.S., and Pazour G.J. (2014). Distinct functions for IFT140 and IFT20 in opsin transport. Cytoskeleton (Hoboken) 71, 302-310.
DiTirro D., Philbrook A., Rubino K., and Sengupta P. (2019). The Caenorhabditis elegans Tubby homolog dynamically modulates olfactory cilia membrane morphogenesis and phospholipid composition. Elife 8, e48789.
Drummond I.A. (2012). Cilia functions in development. Curr. Opin. Cell Biol. 24, 24-30.
Ezratty E.J., Stokes N., Chai S., Shah A.S., Williams S.E., and Fuchs E. (2011). A role for the primary cilium in Notch signaling and epidermal differentiation during skin development. Cell 145, 1129-1141.
Fliegauf M., Benzing T., and Omran H. (2007). When cilia go bad: cilia defects and ciliopathies. Nat. Rev. Mol. Cell Biol. 8, 880-893.
Garcia-Gonzalo F.R., Phua S.C., Roberson E.C., Garcia G. 3rd, Abedin M. 3rd, Schurmans S. 3rd, Inoue T. 3rd, and Reiter J.F. 3rd. (2015). Phosphoinositides regulate ciliary protein trafficking to modulate hedgehog signaling. Dev. Cell 34, 400-409.
Green J.S., Parfrey P.S., Harnett J.D., Farid N.R., Cramer B.C., Johnson G., Heath O., McManamon P.J., O'Leary E., and Pryse-Phillips W. (1989). The cardinal manifestations of Bardet-Biedl syndrome, a form of Laurence-Moon-Biedl syndrome. N. Engl. J. Med. 321, 1002-1009.
Hagstrom S.A., Duyao M., North M.A., and Li T. (1999). Retinal degeneration in tulp1−/− mice: vesicular accumulation in the interphotoreceptor matrix. Invest. Ophthalmol. Vis. Sci. 40, 2795-2802.
Han S., Miyoshi K., Shikada S., Amano G., Wang Y., Yoshimura T., and Katayama T. (2019). TULP3 is required for localization of membrane-associated proteins ARL13B and INPP5E to primary cilia. Biochem. Biophys. Res. Commun. 509, 227-234.
He W., Ikeda S., Bronson R.T., Yan G., Nishina P.M., North M.A., and Naggert J.K. (2000). GFP-tagged expression and immunohistochemical studies to determine the subcellular localization of the tubby gene family members. Brain Res. Mol. Brain Res. 81, 109-117.
Hildebrandt F., Benzing T., and Katsanis N. (2011). Ciliopathies. N. Engl. J. Med. 364, 1533-1543.
Hilgendorf K.I., Johnson C.T., and Jackson P.K. (2016). The primary cilium as a cellular receiver: organizing ciliary GPCR signaling. Curr. Opin. Cell Biol. 39, 84-92.
Huangfu D., Liu A., Rakeman A.S., Murcia N.S., Niswander L., and Anderson K.V. (2003). Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature 426, 83-87.
Ikeda S., Shiva N., Ikeda A., Smith R.S., Nusinowitz S., Yan G., Lin T.R., Chu S., Heckenlively J.R., and North M.A., et al. (2000). Retinal degeneration but not obesity is observed in null mutants of the tubby-like protein 1 gene. Hum. Mol. Genet. 9, 155-163.
Ishikawa H. and Marshall W.F. (2011). Ciliogenesis: building the cell's antenna. Nat. Rev. Mol. Cell Biol. 12, 222-234.
Keady B.T., Le Y.Z., and Pazour G.J. (2011). IFT20 is required for opsin trafficking and photoreceptor outer segment development. Mol. Biol. Cell 22, 921-930.
Kleyn P.W., Fan W., Kovats S.G., Lee J.J., Pulido J.C., Wu Y., Berkemeier L.R., Misumi D.J., Holmgren L., and Charlat O., et al. (1996). Identification and characterization of the mouse obesity gene tubby: a member of a novel gene family. Cell 85, 281-290.
Liu Q., Zhou J., Daiger S.P., Farber D.B., Heckenlively J.R., Smith J.E., Sullivan L.S., Zuo J., Milam A.H., and Pierce E.A. (2002). Identification and subcellular localization of the RP1 protein in human and mouse photoreceptors. Invest. Ophthalmol. Vis. Sci. 43, 22-32.
Loktev A.V. and Jackson P.K. (2013). Neuropeptide Y family receptors traffic via the Bardet-Biedl syndrome pathway to signal in neuronal primary cilia. Cell Rep. 5, 1316-1329.
Mukhopadhyay S. and Jackson P.K. (2011). The tubby family proteins. Genome Biol. 12, 225.
Mukhopadhyay S., Wen X., Chih B., Nelson C.D., Lane W.S., Scales S.J., and Jackson P.K. (2010). TULP3 bridges the IFT-A complex and membrane phosphoinositides to promote trafficking of G protein-coupled receptors into primary cilia. Genes Dev. 24, 2180-2193.
Mukhopadhyay S., Wen X., Ratti N., Loktev A., Rangell L., Scales S.J., and Jackson P.K. (2013). The ciliary G-protein-coupled receptor Gpr161 negatively regulates the Sonic hedgehog pathway via cAMP signaling. Cell 152, 210-223.
Nachury M.V., Seeley E.S., and Jin H. (2010). Trafficking to the ciliary membrane: how to get across the periciliary diffusion barrier? Annu. Rev. Cell Dev. Biol. 26, 59-87.
Noben-Trauth K., Naggert J.K., North M.A., and Nishina P.M. (1996). A candidate gene for the mouse mutation tubby. Nature 380, 534-538.
Norman R.X., Ko H.W., Huang V., Eun C.M., Abler L.L., Zhang Z., Sun X., and Eggenschwiler J.T. (2009). Tubby-like protein 3 (TULP3) regulates patterning in the mouse embryo through inhibition of Hedgehog signaling. Hum. Mol. Genet. 18, 1740-1754.
Oishi I., Kawakami Y., Raya A., Callol-Massot C., and Izpisúa Belmonte J.C. (2006). Regulation of primary cilia formation and left-right patterning in zebrafish by a noncanonical Wnt signaling mediator, duboraya. Nat. Genet. 38, 1316-1322.
Park J., Lee J., Shim J., Han W., Lee J., Bae Y.C., Chung Y.D., Kim C.H., and Moon S.J. (2013). dTULP, the Drosophila melanogaster homolog of tubby, regulates transient receptor potential channel localization in cilia. PLoS Genet. 9, e1003814.
Pazour G.J., Dickert B.L., Vucica Y., Seeley E.S., Rosenbaum J.L., Witman G.B., and Cole D.G. (2000). Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene Tg737, are required for assembly of cilia and flagella. J. Cell Biol. 151, 709-718.
Qin J., Lin Y., Norman R.X., Ko H.W., and Eggenschwiler J.T. (2011). Intraflagellar transport protein 122 antagonizes Sonic Hedgehog signaling and controls ciliary localization of pathway components. Proc. Natl. Acad. Sci. U. S. A. 108, 1456-1461.
Ran F.A., Hsu P.D., Wright J., Agarwala V., Scott D.A., and Zhang F. (2013). Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281-2308.
Rohatgi R., Milenkovic L., and Scott M.P. (2007). Patched1 regulates hedgehog signaling at the primary cilium. Science 317, 372-376.
Rosenbaum J.L. and Witman G.B. (2002). Intraflagellar transport. Nat. Rev. Mol. Cell Biol. 3, 813-825.
Scholey J.M. (2003). Intraflagellar transport. Annu. Rev. Cell Dev. Biol. 19, 423-443.
Sentmanat M.F., Peters S.T., Florian C.P., Connelly J.P., and Pruett-Miller S.M. (2018). A survey of validation strategies for CRISPR-Cas9 editing. Sci. Rep. 8, 1-8.
Sun X., Haley J., Bulgakov O.V., Cai X., McGinnis J., and Li T. (2012). Tubby is required for trafficking G protein-coupled receptors to neuronal cilia. Cilia 1, 21.
Taschner M., Bhogaraju S., and Lorentzen E. (2012). Architecture and function of IFT complex proteins in ciliogenesis. Differentiation 83, S12-S22.
Valente E.M., Silhavy J.L., Brancati F., Barrano G., Krishnaswami S.R., Castori M., Lancaster M.A., Boltshauser E., Boccone L., and Al-Gazali L., et al. (2006). Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome. Nat. Genet. 38, 623-625.
Published online August 31, 2021 https://doi.org/10.14348/molcells.2021.0082
Correspondence to:sjmoon@yuhs.ac
Keywords: cilia, cilia formation, ciliary trafficking, RPE1, TULP
Figure 1.Effects of TULPs on ciliogenesis in RPE1 cells. (A) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated TULPs. Cells were stained with an antibody specific to the axonemal marker Ac-tubulin. Transfected cells were identified with DsRed staining. Non-transfected cells were identified with the nuclear stain To-Pro3. Scale bars = 5 μm. (B and C) Quantification of ciliated cells (B) and cilia length (C). Data are presented as mean ± SEM from three independent experiments. More than 300 cells were counted for each genotype for the ciliary formation experiment and more than 100 were counted for each genotype for the ciliary length experiment. One-way ANOVAs with Tukey's post-hoc tests. ***P < 0.001.
Figure 2.Effects of TULPs on ciliary trafficking of ARL13B, INPP5E, and GPR161 in RPE1 cells. (A-C) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated TULPs. Cells were stained with antibodies specific for the axonemal marker Ac-tubulin, as well as ARL13B (A), INPP5E (B), or GPR161 (C). Transfected cells were identified with DsRed staining. Non-transfected cells were identified with the nuclear stain To-Pro3. Enlarged images of single cilia displayed on the right. Scale bars = 5 μm. A quantification of ciliated cells expressing each ciliary protein is shown below. Data are presented as mean ± SEM from three independent experiments. More than 100 ciliated cells were counted for each condition. One-way ANOVAs with Tukey's post-hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3.Effects of TULPs on ciliary trafficking of IFT140 in RPE1 cells. (A) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated TULPs. Cells were stained with antibodies specific for the axonemal marker Ac-tubulin and IFT140. Transfected cells were identified with DsRed staining. Non-transfected cells were identified with the nuclear stain To-Pro3. Insets, enlarged images of the cilia marked with arrowheads. Scale bars = 5 μm. (B) Quantification of IFT140 localization. Data are presented as mean ± SEM from three independent experiments. Total counted cells are 100-150 for each condition. Pearson's χ2 test. ****P < 0.0001.
Figure 4.Fig. 4. Differential contributions of the IFT-A and PIP2-binding domains of TUB to ciliary formation and IFT140 trafficking. (A) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated forms of TUB. TUBIFT-A(–), a mutant TUB that does not bind IFT-A; TUBPIP2(–), a mutant TUB that does not bind PIP2. Cells were stained with an antibody specific for the axonemal marker Ac-tubulin. Transfected cells were identified with either DsRed or ZsGreen. Non-transfected cells were identified with the nuclear marker To-Pro3. Scale bars = 5 μm. (B and C) Quantification of ciliated cells (B) and cilia length (C). Data are presented as mean ± SEM from three independent experiments. More than 300 cells were counted for each genotype for the ciliary formation experiment and more than 90 were counted for each genotype for the ciliary length experiment. One-way ANOVAs with Tukey's post-hoc tests. (D) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated forms of TUB. Cells were stained with antibodies specific for the axonemal marker Ac-tubulin and IFT140. Transfected cells were identified with ZsGreen. Non-transfected cells were identified with the nuclear marker To-Pro3. Insets, enlarged images of the cilia marked with arrowheads. Scale bars = 5 μm. (E) Quantification of IFT140 localization. Data are presented as mean ± SEM from three independent experiments. Total counted cells are 90-150 for each condition. Pearson's χ2 test. *P < 0.05, ***P < 0.001, ****P < 0.0001. n.s., not significant.
Figure 5.Fig. 5. IFT-A binding in the TULPs is essential for the proper ciliary trafficking and localization of IFT140. (A) Multiple sequence alignment of the putative IFT-A binding domain from the TULPs. (B) Schematic depicting the formation of TULP1IFT-A(+). The putative IFT-A binding domain of TULP3 (amino acids 20-59) was used to replace the corresponding 40 amino acid sequence in TULP1. (C) Immunocytochemical analysis of TULP1 and TULP1IFT-A(+) in wild-type and TULP3 KO RPE1 cells. Cells were stained with antibodies specific for the axonemal marker Ac-tubulin and c-Myc for TULP1 and TULP1IFT-A(+). Nuclei were stained with To-Pro3. TULP1IFT-A(+)-positive cilia are marked with arrowheads. Scale bars = 5 μm. (D and E) Quantification of ciliated cells (D) and cilia length (E). Data are presented as mean ± SEM from three independent experiments. More than 300 cells were counted for each genotype for the ciliary formation experiment and more than 100 were counted for each genotype for the ciliary length experiment. One-way ANOVAs with Tukey's post-hoc tests. ***P < 0.001. (F-H) Quantification of ciliated cells expressing each ciliary protein. Data are presented as mean ± SEM from three independent experiments. More than 100 cells were counted for each condition. One-way ANOVAs with Tukey's post-hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001. (I) Quantification of IFT140 localization. Data are presented as mean ± SEM from three independent experiments. Total counted cells are 90-150 for each condition. Pearson's χ2 test. *P < 0.05, **P < 0.01, ****P < 0.0001. n.s., not significant.
Previous Article LIST Next Article
Supplementary File
SCOPUS 1
Sub-Ciliary Segregation of Two Drosophila Transient Receptor Potential Channels Begins at the Initial Stage of Their Pre-Ciliary Trafficking
Youngtae Kwon, Jeongmi Lee, and Yun Doo Chung
Mol. Cells 2020; 43(12): 1002-1010 https://doi.org/10.14348/molcells.2020.0205
The Ciliary Transition Zone: Finding the Pieces and Assembling the Gate
Jo?o Gon?alves, and Laurence Pelletier
Figure 5.Effects of TULPs on ciliogenesis in RPE1 cells. (A) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated TULPs. Cells were stained with an antibody specific to the axonemal marker Ac-tubulin. Transfected cells were identified with DsRed staining. Non-transfected cells were identified with the nuclear stain To-Pro3. Scale bars = 5 μm. (B and C) Quantification of ciliated cells (B) and cilia length (C). Data are presented as mean ± SEM from three independent experiments. More than 300 cells were counted for each genotype for the ciliary formation experiment and more than 100 were counted for each genotype for the ciliary length experiment. One-way ANOVAs with Tukey's post-hoc tests. ***P < 0.001.|@|~(^,^)~|@|Effects of TULPs on ciliary trafficking of ARL13B, INPP5E, and GPR161 in RPE1 cells. (A-C) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated TULPs. Cells were stained with antibodies specific for the axonemal marker Ac-tubulin, as well as ARL13B (A), INPP5E (B), or GPR161 (C). Transfected cells were identified with DsRed staining. Non-transfected cells were identified with the nuclear stain To-Pro3. Enlarged images of single cilia displayed on the right. Scale bars = 5 μm. A quantification of ciliated cells expressing each ciliary protein is shown below. Data are presented as mean ± SEM from three independent experiments. More than 100 ciliated cells were counted for each condition. One-way ANOVAs with Tukey's post-hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001.|@|~(^,^)~|@|Effects of TULPs on ciliary trafficking of IFT140 in RPE1 cells. (A) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated TULPs. Cells were stained with antibodies specific for the axonemal marker Ac-tubulin and IFT140. Transfected cells were identified with DsRed staining. Non-transfected cells were identified with the nuclear stain To-Pro3. Insets, enlarged images of the cilia marked with arrowheads. Scale bars = 5 μm. (B) Quantification of IFT140 localization. Data are presented as mean ± SEM from three independent experiments. Total counted cells are 100-150 for each condition. Pearson's χ2 test. ****P < 0.0001.|@|~(^,^)~|@|Fig. 4. Differential contributions of the IFT-A and PIP2-binding domains of TUB to ciliary formation and IFT140 trafficking. (A) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated forms of TUB. TUBIFT-A(–), a mutant TUB that does not bind IFT-A; TUBPIP2(–), a mutant TUB that does not bind PIP2. Cells were stained with an antibody specific for the axonemal marker Ac-tubulin. Transfected cells were identified with either DsRed or ZsGreen. Non-transfected cells were identified with the nuclear marker To-Pro3. Scale bars = 5 μm. (B and C) Quantification of ciliated cells (B) and cilia length (C). Data are presented as mean ± SEM from three independent experiments. More than 300 cells were counted for each genotype for the ciliary formation experiment and more than 90 were counted for each genotype for the ciliary length experiment. One-way ANOVAs with Tukey's post-hoc tests. (D) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated forms of TUB. Cells were stained with antibodies specific for the axonemal marker Ac-tubulin and IFT140. Transfected cells were identified with ZsGreen. Non-transfected cells were identified with the nuclear marker To-Pro3. Insets, enlarged images of the cilia marked with arrowheads. Scale bars = 5 μm. (E) Quantification of IFT140 localization. Data are presented as mean ± SEM from three independent experiments. Total counted cells are 90-150 for each condition. Pearson's χ2 test. *P < 0.05, ***P < 0.001, ****P < 0.0001. n.s., not significant.|@|~(^,^)~|@|Fig. 5. IFT-A binding in the TULPs is essential for the proper ciliary trafficking and localization of IFT140. (A) Multiple sequence alignment of the putative IFT-A binding domain from the TULPs. (B) Schematic depicting the formation of TULP1IFT-A(+). The putative IFT-A binding domain of TULP3 (amino acids 20-59) was used to replace the corresponding 40 amino acid sequence in TULP1. (C) Immunocytochemical analysis of TULP1 and TULP1IFT-A(+) in wild-type and TULP3 KO RPE1 cells. Cells were stained with antibodies specific for the axonemal marker Ac-tubulin and c-Myc for TULP1 and TULP1IFT-A(+). Nuclei were stained with To-Pro3. TULP1IFT-A(+)-positive cilia are marked with arrowheads. Scale bars = 5 μm. (D and E) Quantification of ciliated cells (D) and cilia length (E). Data are presented as mean ± SEM from three independent experiments. More than 300 cells were counted for each genotype for the ciliary formation experiment and more than 100 were counted for each genotype for the ciliary length experiment. One-way ANOVAs with Tukey's post-hoc tests. ***P < 0.001. (F-H) Quantification of ciliated cells expressing each ciliary protein. Data are presented as mean ± SEM from three independent experiments. More than 100 cells were counted for each condition. One-way ANOVAs with Tukey's post-hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001. (I) Quantification of IFT140 localization. Data are presented as mean ± SEM from three independent experiments. Total counted cells are 90-150 for each condition. Pearson's χ2 test. *P < 0.05, **P < 0.01, ****P < 0.0001. n.s., not significant.


text:
Pieces of Me August 8, 2021
Tags: anxiety, Family, identity, Marriage, Parenting, ragtag daily prompt, rdp, Relationships, WOD Challenge, YDwordprompt
Photo by Ismael Sanchez on Pexels.com
Something I've begun to learn fairly recently is that opening up, showing vulnerability, admitting my limitations, being authentically me….it helps. It helps to talk about the not-so-shiny feelings I struggle with. But it helps even more when friends, acquaintances, even strangers reach out to me in response to some of the topics I've shared about here and elsewhere on social media. Raw stuff. Stuff that I had previously swept under the rug and tried to ignore or deny. But the more I share, the more I realize that I'm not alone. My insecurities, my fears, my anxieties; they're not unique to me.
About a week ago, I was scrolling Facebook and came across a post from a new podcaster. I'm not familiar with the creator or the podcast so this isn't a plug, necessarily. But the post I saw resonated with me so I shared it. If you've got a minute (literally, one minute) and you'd like to check it out, it's available here. But if you don't, here's a recap: The video shows a man speaking directly to the camera about his experience in therapy. He recounts telling his therapist that he feels he needs help to be great; he's only good on his own. A good employee, a good father, a good husband, a good friend. His therapist says, "Employee is someone you are for your job. Husband is someone you are for your wife. Father is someone you are for your son. Friend is someone you are for your friends. Who are you for yourself? Who are you outside of those roles?" He then admits to the camera, "I'm not sure there's much of a person at the end of all that."
BAM! Mic drop. That one minute anecdote succinctly sums up what I've been trying to find the words to say for damn near four years now.
Within the past year, my husband and I concluded a conversation we'd mooted since our daughter was first born. The big "should we have another child?" conversation. We've opened the dialog periodically over the years, in good times when parenting feels like a breeze and in bad times when I'm certain we're failing. The end result has always been a decision to table it for the future. I've found various excuses. "When she's out of diapers. Once she's out of daycare. When we're in a bigger house. After I'm settled into a new career." There's always been a reason why the time wasn't right. Until sometime mid-pandemic when I tearfully and honestly admitted to my husband, "I don't want any more children. I've lost too much of myself in motherhood and can't emotionally do it, starting over from scratch with a new baby."
I felt like a terrible mom for having even thought it, let alone breathed life into it and actually said it. I felt that it implied my daughter has somehow taken something from me or that I don't love her or appreciate the family my husband and I have created together. Let me be clear: Motherhood has been the most terrifying endeavor of my life but also my most rewarding. Watching her grow into the 8-year old she is today—from learning the basics like talking and walking to developing her own personality and opinions—I'm incredibly fortunate to have a front row seat for all of it. The prospect of ushering her into adolescence and eventually adulthood looms ahead of me, an insurmountable task. But I have faith that it'll become slightly more tractable with time and experience.
But it's true. I've lost a piece of myself when I became her mom. Just as I lost a piece of myself when I became his wife. And their friend. We—because I'm confident I'm not alone in the way I was brought up—are taught from a young age that you're supposed to hop on this sort of life escalator as soon as possible. Finish school. Find a job. Nail down a spouse. Buy a house with a picket fence. Pop out 2.5 children. Work until you die. Anything outside of that isn't normal. It isn't right. You're doing it wrong unless you do it this way. And I've bought into every last bit of it, happily cleaving off parts of Sierra and bestowing them upon others.
I've recognized for a while now that my identity is too wrapped up in others. That I'm long overdue to disentangle myself, to a degree, from the roles I fill for others. To reclaim some pieces of me, patch them back together and mod podge them into place if I have to. Part of me proffers that these weird existential ramblings going on in my brain are related to the pandemic; surely COVID has caused many of us to take a step back and examine what's important in life and what we'd like to improve upon. Another part of me wonders if it's my age; maybe all 30-somethings go through this, no matter what's going on in the world at large.
Regardless of why I'm feeling the things I'm feeling, I'm feeling them. And I've taken some steps towards reclaiming myself. But I realized this weekend that my work is far from over. I was at a party at my cousin's house, celebrating a couple of birthdays. "You two have met, right?" my cousin asked simultaneously of me and a woman across the campfire from me. We both replied at the same time, but gave different answers. Mine was, "I'm sure we have. I know I've seen her at parties here before." Hers was, "No, I don't think so." After a brief explanation from my cousin, the woman remembered me. Turns out, she hadn't recognized me because I'd walked into the party with two kids (my daughter and my nephew), neither of whom were seated with me at the time, and no partner; hubby wasn't feeling well so he had opted to stay home. Without being flanked by my daughter and my husband, I was unrecognizable as an individual. Which proves my point.
So. Friends, acquaintances, even strangers, this is a topic I'd love to hear from you about. How have you maintained (or reclaimed) your individual identity while still fulfilling the roles you've taken on in life? I'm humbly asking for guidance.
New love, forever love August 30, 2020
Tags: 30 Day Writing Challenge, dating, Family, love, Marriage, midlife crisis, Relationships
Day 19: Discuss "first love."
"I'm having a midlife crisis." The handful of people I'm closest with have had the distinct privilege of hearing me utter these words, usually amid heavy sobs or in a manic frenzy or while brooding about life in general. But am I really? Maybe, maybe not. There are so many days when I can do nothing but look around me and feel thankful for and proud of the life I'm fortunate enough to call mine. The life that I helped orchestrate through a series of careful choices and maybe a couple of reckless chances. And I'm in no way trying to detract from all the love and light and goodness around me. It's there. I see it all. But despite all that, there are some days when I've got an acute awareness that many of life's biggest decisions have already been made for/by me. Roots put down. Life cemented in place. More doors closed behind me than open in front of me.
This post has been about two years in the making. I've given it a lot of thought. Kicked the idea around in my head at all hours of the day and night. Searched for the right words that don't make me sound ungrateful. Tried to formulate a coherent string of sentences that may help identify myself to others feeling the way I am. Unfortunately, this is as close as I've come. So here goes: On those really tough days, "midlife crisis" feels like the only descriptor adequate enough to define the profound feelings of…loss? Loss of youth. Loss of choices. Loss of opportunities to experience life's big moments (and all of the feelings that are wrapped up in those experiences) just one more time.
One of those big life experiences is falling in love. And before I go on, it's important to me to interject here and say a few things, beginning with that I am happily married. There is no doubt in my mind that I married my match and will live the rest of my life loving him. But this post isn't about him. (If you want to read about him, you can do so here, where I gushed about him for our tenth wedding anniversary in 2018.) This post is about trying to find words to describe the differences between the love we have now versus "new love." Because they are undeniably different.
The most succinct way I've ever seen the differences summed up came in a novel I read last fall, What Alice Forgot by Liane Moriarty. After reading, I jotted a quote, my biggest takeaway from the story. It resonated with me then and it resonates with me even more as time goes on. Here's the quote:
"She had always thought that exquisitely happy time at the beginning of her relationship…was the ultimate, the feeling they'd always be trying to replicate, to get back, but now she realized that was wrong. That was like comparing sparkling mineral water to French champagne. Early love is exciting and exhilarating. It's light and bubbly. Anyone can love like that. But love after three children, after a separation and a near-divorce, after you've hurt each other and forgiven each other, bored each other and surprised each other, after you've seen the worst and the best–well, that sort of a love is ineffable. It deserves its own word."
—excerpt from What Alice Forgot by Liane Moriarty
There's something to be said for new love, absolutely. For me, it starts with a spark of attraction that spreads like wildfire until a kaleidoscope of butterflies is flapping wildly in my gut. There are first dates and first kisses, tentative reaching and finding a comfortable rhythm. There's giving and taking, yinning and yanging. There's talking and listening and figuring each other out. An all-encompassing, breathless wanting. An I-can't-get-enough need. My…sparkling mineral water certainly does quench a thirst, doesn't it?
But no matter how exciting new love feels, those bubbles, that fizziness, it all eventually dissipates. Tattered, broken, unshiny parts are revealed. True colors poke through as if the harsh house lights have just flicked on after last call. And if you're lucky, as I've been, you find the right tempo and the waltz truly begins. One two three, one two three. Count by count, all that feels good and right is boxed in. Fiercely protected. You lead each other through to the other side where love morphs into something more. A higher form. The French champagne.
And just as there's much to be said about new love, so, too, is there about the kind of love we've got now, over twenty years after our first date. The forever kind. Love with the depth and breadth to encompass two lifetimes in one swooping arch. That kind of love is reliable, safe, comfortable, even easy after as long as he and I have been together. But when I use these terms to describe it, he turns up his nose at me, somewhat regretful that he's no longer responsible for the butterflies and fizz. But it's important to note that you can't get to here, where we are, without having lived through the newness and beyond. Our love has gone through breakups and fights and more hard conversations than I can count. We've celebrated greatness, endured losses, faced hardships. We've created life and navigated parenting. We've supported each other in decisions that felt impossible to make, some with consequences that felt impossible to live with. And through it all, we're still each others' number one. And there's no world in which I'd ever dare to replace or dispose of that. Ever.
Instead, I'll close this with the valediction that he and I end every email, card, or letter to each other with. A phrase ripped from love letters between my paternal grandparents and inscribed on our wedding bands.
Always and all ways.
A Map to My Heart April 13, 2020
To My Husband on our 10th Wedding Anniversary… November 1, 2018
Tags: anniversary, love, Marriage, Relationships
We met a lifetime ago. We were kids, sixteen years old and clueless about all that would come. A first date, the culmination of your persistent asking. A first kiss in a parking lot on a cold January night as the snow fell around us. Prom nights. Graduations. College years spent 150 miles apart, and all the weekend road trips and all-night phone calls to stay connected. The night you proposed, and the night my car died to save our future. Every moment of our past has led us to now, almost 19 years later, celebrating our 10th wedding anniversary. And I want to thank you.
Thank you for supporting me emotionally through some of the roughest points of my past. Moving out of my parents' house at 17, my mom's brief illness followed by her passing and the emotional fallout that came after, navigating some tumultuous family relationships. When I can't catch my breath and feel like there's no way out of the hole, you're the one I turn to. You bring me logic when my mind spirals out of control. You calm me down when I need it most.
Thank you for being a constant source of encouragement. The little things you do and say have the biggest impact on me. Helping me see the bright side of a particularly rough week at work, boosting my confidence in my parenting, reminding me of my strengths when I've forgotten myself. You have helped me find my backbone countless times when I otherwise feel like a pile of mush. (And who knows? Maybe someday I'll actually write the book you've been telling me to write for years now.)
Thank you for helping me celebrate all that's good in our lives. And there's been so. much. good. It's easy to glide through life and lose sight of the positives. And sometimes I have to consciously stop and take it all in. Without our partnership in life, I don't think any of it would have been possible. From the big stuff like our home and our family to the smaller stuff like family date days…I say it all the time but it's completely sincere every time I do: "I love our life."
Thank you for being Rylin's other (often better) parent. I'll never forget how happy we were to have conceived our girl. The birthing class, setting up the nursery, and pacing the halls of Johnson Memorial Hospital with me during labor, awaiting the arrival of our future. That feeling of panic when they "let" us take her home from the hospital, unsupervised. We're not always on the same page when it comes to parenting but I wouldn't want to share the responsibility with anyone else. I am almost constantly in awe of you as a father and so thankful that she has you to teach her by example how a man should treat her.
Thank you for doing your best to help me be my best self. I've told you for 18+ years that "it's not your job to try to fix me." But for 18+ years, you've done just that. And for that, I am more appreciative than you know. Whether it's shouldering more than your fair share of household responsibilities or taking me away for a weekend getaway, you always know what my soul needs to smile brighter. And you do your best to deliver just that.
We are each wildly different people than we were when we met. And I'm absolutely proud of who we've become, both as individuals and as a couple. Our marriage isn't perfect but I'm proud that we're both willing to acknowledge its faults and find ways to bolster it. And each other. Every day, I'm proud to call you my husband and so thankful to be your wife.
I love you always and all ways and look forward to celebrating a lifetime more anniversaries with you.
Perhaps the first ever "funfetti" wedding cake (11/01/2008)
Dear Rylin April 23, 2013
Tags: daddy-to-be, Family, first time mom, Marriage, Parenting, Pregnancy, third trimester
My husband makes me smile. A lot. Today it was because of an email he sent to me. Which was intended for our daughter. Who is currently receiving mail in my uterus. It made me teary. Admittedly that's probably just due to the wonky pregnancy hormones coupled with the "is it over yet?!" third trimester crankiness. Even so, I feel the need to share….
Dear Rylin,
This is your father. I would like to tell you that I've spent the last nine months plus thinking about you. I have dreamt and imagined what it will be like to hold you, talk to you, and snuggle you. I have thought about watching you play in the yard with your mom, Bob, Gracie and me. I think about who you will be when you get older and hope that I can guide you to the best life possible. I want to teach you to be a good person. Your mother and I are so excited to finally meet you and complete this family. No matter where your life takes you we will always be there to support you through it. The big key to all of this is we need you to arrive to begin this journey together. So if you will kindly get things moving and come see us that would be great. I love you with all of my heart and soul and can't wait to take our first jaunt together with your carrier.
P.S. Tell mommy you like MaryBeth for your middle name and I'll make sure you get some extra candy on your 1st birthday.
P.P.S. Funfetti is the best cake. I'll make sure that's what you get.
Thank You August 31, 2012
Tags: Marriage, Relationships
It's been what I'd call a rough week. Chris and I have been at the center of some lively debate and it's been mentally and emotionally exhausting. I won't go into detail but I will tell you that it all began with a comment about how "marriage is like a jail sentence" and the fact that the person who made the comment "doesn't want to be handcuffed to someone for life." I'll also tell you that it's weeks like this that make me appreciate (more than usual) the man I married.
We will celebrate our 4th wedding anniversary this November. But we've been together—for the most part—for just under 13 years. I say "for the most part" because our relationship began when we were 16 and we've had our share of little teenage spats. Two of which (once during high school and once more during freshman year of college) resulted in us "taking a break." You know how it goes. You have a fight so major that there's just no getting past it. So you break up. And three weeks later you realize that you've done nothing but miss them, wonder what they're up to, and ask yourself, "What was that fight about again?" Been there, done that. And yet here we are. Almost thirteen years later. More mature. More understanding of each others' quirks and needs. And more in love than ever.
But how? We live in culture that is quick to give up on marriage, with 40+% of all marriages in the country ending in divorce. Then there's domestic violence and infidelity and all those other couples who just seem to coexist without actually being happy to be together. What makes our relationship different than those? How have we gotten to where we are today? Sure, all the usual stuff….all the stuff psychologists and marriage experts will tell you are vital to building a strong marriage. We communicate—well and frequently. We show each other respect. We have similar ideals with respect to the "big deal" topics like finances and family. We share responsibilities. We have the perfect balance between "us" time and alone time—so neither of us are defined 100% by our relationship. We know how to fight fairly. But probably most importantly, we never miss an opportunity to make each other feel special.
Every day before he leaves for work, he finds me—usually I'm still in bed, so it's not hard—and kisses my forehead. Some days he sends me a mid-day text message that just says, "Hi, beautiful." Once in a while, it's him giving me a foot massage while we curl up to watch a movie on the couch. (And, yes, I'll admit it. He's a better movie picker than I am. Just don't tell him I said that.) He has a way of making me get that "melty" feeling inside. Even after almost thirteen years.
So today, just for fun, I stopped by the commuter lot where he parks his car to catch the bus into Hartford for work. I found his car, jotted a note on the first piece of paper I found in my car (a bank envelope), and left it on his dashboard. It just took a few minutes, but I knew he'd smile when he got off the bus and found it waiting for him. And he did.
Yeah, I call my husband "Buddy." Don't judge me!
Now, I realize that marriage isn't for everyone—and you're certainly in that group if you view it as a jail sentence! And I also realize that you don't have to be married to be happy in a relationship—my parents were together for over 30 years before they got married. But I can say with certainty that marrying Chris was the best decision I've ever made because he's my best friend and I wouldn't want to experience this crazy thing we call life without him. So thank you, Chris, for being my "cell mate for life."

redpajama_set_name: RedPajamaCommonCrawl


S. Bernard water is characterized by a low content of sodium, potassium, magnesium and a reduced fixed residue (34.5 mg / L), which place it among the minimally mineralized waters. For this reason it is commonly called "light" water, precisely because of its low salt concentration. S.Bernardo Sorgente Rocciaviva is a water with high diuretic properties, because it is quickly absorbed and eliminated by the body: this favors the drainage of nitrogenous waste (urea) and prevents, thanks to the modest amount of hydrocarbonate, the formation of calculations. Its rapid assimilation and absorption, combined with low mineralization, make it suitable for feeding newborns, especially in the dilution of milk and food. The sparkling version, with a light and unmistakable gassing, is the natural complement of taste.