Caenorhabditis elegans embryogenesis, epidermal cell shape changes transform ovoid embryos into vermiform larvae. This process is divided into two phases: early and late elongation. Early elongation involves the contraction of filamentous actin bundles by phosphorylated non-muscle myosin in a subset of epidermal  cells. The genes controlling early elongation are associated with two parallel pathways. The first one involves the rho-1/RHOA-specific effector let-502/Rho-kinase and mel-11/myosin phosphatase regulatory subunit. The second pathway involves the CDC42/RAC-specific effector pak-1. Late elongation is driven by mechanotransduction in ventral and dorsal hypodermal cells in response to body-wall muscle contractions, and involves the CDC42/RAC-specific Guanine-nucleotide Exchange Factor (GEF) pix-1, the GTPase ced-10/RAC and pak-1.Cell shape changes are crucial for metazoan development. During pix-1 is shown to control early elongation in parallel with let-502/mel-11, as previously shown for pak-1. We show that pix-1, pak-1 and let-502 control the rate of elongation, and the antero-posterior morphology of the embryos. In particular, pix-1 and pak-1 are shown to control head, but not tail width, while let-502 controls both head and tail width. This suggests that let-502 function is required throughout the antero-posterior axis of the embryo during early elongation, while pix-1/pak-1 function may be mostly required in the anterior part of the embryo. Supporting this hypothesis we show that low pix-1 expression level in the dorsal-posterior hypodermal cells is required to ensure high elongation rate during early elongation.In this study,  Drosophila melanogaster and Caenorhabditis elegansC. elegans appears as an excellent model to better understand the function of α/β-PIX and PAKs during complex morphogenic events in living organisms.In mammals, the CDC42/RAC-specific Guanine-nucleotide exchange factor (GEF) α/β-PIX and the CDC42/RAC-specific effector kinase PAKs were shown to control cell migration, cell polarity, cytoskeleton remodeling and focal adhesion complex assembly/disassembly dynamics C. elegans, embryonic elongation involves the extension of the embryo along its longitudinal axis and a reduction of its transverse diameter, resulting in a 4-fold increase in length. This morphogenetic event involves dramatic changes in the shape of the epidermal  cells. Elongation is divided into an early and a late phase. The early phase, from comma to 1.75-fold stage – corresponding to embryos that are 1.75-fold in length compared to non-elongated embryos –, occurs through contraction of filamentous actin bundles (FBs) in hypodermal cells In the nematode The late phase of elongation involves mechanotransduction signaling from the body-wall muscles to the dorsal and ventral hypodermal cells rho-1/RHOA, ced-10/RAC and mig-2/RHOG) have been implicated in pathways controlling elongation, only three of their regulators (GAPs and GEFs) have been shown to be involved in this process The signal transduction pathways that regulate early and late elongation have been extensively investigated over the last 15 years. Interestingly, many genes controlling morphological changes of the hypodermis during elongation are effectors or regulators of Rho GTPases C. elegans ortholog of the CDC42-effector myotonic dystrophy kinase MRCK-1/MRCK and the CDC42/RAC-effector kinase PAK-1/PAK1. These kinases act antagonistically with the MEL-11/PP-1M and are organized in two parallel pathways: The let-502/mel-11 pathway including mrck-1 and a second pathway involving pak-1In hypodermal cells, contraction of the FBs during early elongation depends on the regulation of myosin-light-chain (MLC-4/MLC) phosphorylation by three serine-threonine kinases, the RHO-1/RHOA-effector kinase LET-502/ROCK, the let-502/mel-11 pathway and an essential regulator of elongation mlc-4 loss-of-function-associated elongation defects, while expression of MLC-4/MLC in ventral and dorsal cells cannot LET-502/ROCK is an essential component of the The second pathway involves the CDC42/RAC-effector PAK-1, the PP2C phosphatase FEM-2/POPX2 and a RHO/RAC-specific GTP-nucleotide exchange factor (GEF) UNC-73/TRIO. The function of these two later proteins in the regulation of MLC-4 phosphorylation and/or PAK-1 function remains unknown pak-1 pathway during early elongation remain unknown. Moreover, the biological significance of the functional redundancy of the mel-11/let-502 and pak-1 pathways is not clear. This redundancy is intriguing since it does not appear to add robustness to the elongation system: a single perturbation in any component of the mel-11/let-502 pathway induces a high proportion of embryonic lethality mel-11/let-502 and pak-1 pathways have unique functions during elongation that remain to be identified.To date, the genes controlling the C. elegans, pix-1 codes for a protein homologous to the mammalian β-PIX  during gonad morphogenesis in larvae The CDC42/RAC-specific GEF, PIX is a well-known activator of PAKs in several organisms an β-PIX . It contan β-PIX . These cpix-1 controls early elongation in parallel with mel-11/let-502. Our data suggest that pix-1 is a novel component of the pak-1 pathway while retaining some function during early elongation independent from pak-1. We show that the pix-1/pak-1 pathway controls the antero-posterior morphology of the embryo during early elongation through regulating head width, while let-502/ROCK controls both the head and tail width. Our study proposes a novel model for early elongation where the mel-11/let-502 and pix-1/pak-1 pathways have redundant and complementary functions to shape the antero-posterior axis of the embryo.In this study, we demonstrate that pix-1 during early elongation, we examined the phenotypes of pix-1(gk416) and pix-1(ok982) embryos. As controls, we characterized the elongation phenotypes of pak-1(ok448) and let-502(sb118ts) mutant embryos, which display early elongation defects pix-1(gk416) and pak-1(ok448) are null alleles pix-1(ok982) contains a 1002 bp deletion spanning exons 8 to 11 and may code for a protein that retains the N-terminal SH3 and RhoGEF/DH domains of PIX-1 (let-502(sb118ts) is a thermosensitive allele coding for the RHO-1/RHOA-effector kinase LET-502/ROCK. This allele shows no obvious phenotypes at 20°C, but displays strong elongation defects and L1-larval arrest characteristic of strong hypomorphic and null let-502 alleles at 25.5°C To investigate the role of of PIX-1 . let-502pix-1(gk416), pix-1(ok982) and pak-1(ok448) alleles revealed low penetrance embryonic lethality (Emb) and early larval arrest (Lva) confirming previous findings (pix-1(gk416), pix-1(ok982), pak-1(ok448) and let-502(sb118ts) arrested larvae showed that they were significantly shorter than synchronized wt L1 larvae at 25.5°C   (n = 10), pix-1(gk416) (n = 25) and pak-1(ok448) (n = 20) alleles developed significantly slower than wt animals (n = 17) during early elongation  . To betttracting . Animalsongation . We alsoongation , and fou embryos . These dpix-1 and pak-1 arrested larvae had very similar morphologies and behavior. For example, they were thin and clear when compared to let-502 arrested larvae , pix-1(ok982) and pak-1(ok448) had significantly higher ratios than wt and let-502 larvae , pak-1(ok448) and let-502(sb118ts) embryos at the beginning, 1.2-fold stage  and tail (T) width during early elongation. To do so, we compared the width of the head and the width of the tail of the embryos at the beginning and the end of early elongation  embryos were significantly lower than wt embryos and not significantly different than 1, suggesting that in let-502 mutant animals neither the head, nor the tail width of the animal reduce during early elongation  genetically interacts with mel-11(it26) and let-502(sb118ts) temperature sensitive mutants, as reported for pak-1mel-11(it26) mutant embryos rupture during elongation as previously described (mel-11(it26); pak-1(ok448) double mutant embryos display phenotypes similar to mel-11(it26) at both 18°C and 25.5°C (mel-11(it26)-associated embryonic rupturing is completely suppressed by pix-1(gk416) at 18°C and 25.5°C (mel-11(it26); pix-1(gk416) hatched larvae arrest at the L1 stage with characteristic pix-1 phenotypes, and a penetrance similar to pix-1(gk416) at 18°C (mel-11(it26); pix-1(gk416) embryos arrest between 1.2- and 2-fold stages without rupturing and 40.3% of them display late elongation defects and stop developing without hatching (pix-1(gk416) suppresses both the expressivity – the embryos arresting at 1.2–2 fold stages without rupturing – and the penetrance of mel-11 early elongation defects at 25.5°C. Considering mel-11(it26) to be null at 25.5°C pix-1 functions in parallel with mel-11 during early elongation. The aggravation of pix-1 associated late elongation defects in a mel-11(it26) background is somewhat intriguing and provides the first evidence suggesting an involvement of mel-11 in late elongation, in parallel with pix-1.escribed , line 3 d 25.5°C  line 10 d 25.5°C , line 4.hatching , line 4.pix-1(gk416) and pak-1(ok448) interacts with let-502(sb118ts) at 25.5°C. As previously shown, few let-502(sb118ts) embryos arrest between the 1.2- and 2-fold stage at restrictive temperature and the vast majority of the animals hatch as non-elongated larvae (let-502(sb118ts) embryos arrest between 1.2- and 2-fold stages in pix-1(gk416) and pak-1(ok448) backgrounds, respectively at 25.5°C (let-502(sb118ts); pix-1(gk416) hatched larvae arrested with more severe elongation defects than let-502(sb118ts) animals (wt as observed for let-502(sb118ts) arrested larvae  appears to be weaker than that observed for pak-1(ok448), suggesting that pix-1 is redundant with a yet unidentified gene in parallel with let-502.We then assessed whether d larvae  [10]. 89 animals , while td larvae . These rpix-1 functions in parallel with the let-502/mel-11 pathway, we generated mel-11(it26); let-502(sb118ts); pix-1(gk416) triple mutants. At 18°C, mel-11(it26); let-502(sb118ts) display similar elongation defects to mel-11(it26) single mutant embryos due to the thermosensitive nature of let-502(sb118ts) ; let-502(sb118ts); pix-1(gk416) animals arrest during early elongation when compared to mel-11(it26); let-502(sb118ts) (mel-11(it26) early elongation defects by pix-1(gk416) (mel-11(it26); let-502(sb118ts) are viable and embryos display 24% early elongation arrest (mel-11(it26); let-502(sb118ts); pix-1(gk416) triple mutants in comparison to mel-11(it26); let-502(sb118ts) double mutants (mel-11(it26); let-502(sb118ts)), and 31% of animals hatched as non-elongated larvae presenting pix-1 mutant-associated morphology (compared to 0% in mel-11(it26); let-502(sb118ts); mel-11(it26); let-502(sb118ts); pix-1(gk416) arrested larvae present a H/T width ratio significantly higher than wt and similar to that observed in pix-1(gk416) arrested larvae (mel-11(it26) background, loss of let-502 function is not anymore able to suppress pix-1-induced H/T ratio defect.To assess if 1(gk416)  line 4. n arrest  line 7. d larvae . This sumel-11(it26); let-502(sb118ts); pak-1(ok448) embryos failed to hatch and displayed early elongation arrest  at the cell periphery and at the most apical part of the lateral hypodermal cells pix-1 endogenous promoter in pix-1(gk416) animals immuno-stained with MH27 antibodies , dorsal-posterior (DP), ventral-anterior (VA), ventral-posterior (VP), lateral-anterior (LA) and lateral-posterior (LP) hypodermal cells ;sajEx1) and one stable transgenic line carrying an integrated array (unc-119(ed3);pix-1(gk416);sajIs1 see Methods). We also found that all measured ratios were constant throughout early elongation  animals. We found that the transgene significantly rescued the larval arrest phenotype (Lva) of pix-1(gk416) from 8.5% (N = 1216) to 2.0% (N = 917) . Using time-lapse microscopy, we also measured the elongation rate of wild-type (wt), pix-1(gk416) and pix-1(gk416);sajEx1 embryos and found that the transgene did not significantly rescue the elongation rate of mutant animals ;sajEx1), and a stable transgenic line carrying an integrated array (unc-119(ed3);pix-1(gk416);sajIs1). These data suggest that transgenic animals expressing PIX-1::GFP homogeneously in dorsal-anterior, lateral and ventral hypodermal cells, but two times less in dorsal-posterior cells, elongate at a wt-rate during early elongation. However, animals expressing the transgene in dorsal-posterior cells at a similar/higher level when compared to other cells elongated significantly slower.We then observed the elongation rate of embryos at the single animal level and attempted to see if there was any correlation between a given expression pattern of the transgene and the ability of the transgene to efficiently rescue the mutant elongation rate defect. We found that transgenic embryos with similar elongation rates to  animals . Overalllin-26p. This promoter is thought to drive the expression of coding sequences only in hypodermal cells and in a homogenous manner lin26p::vab-10(ABD)::GFP and lin-26p::vab-10(ABD)::mCherry transgenic animals carrying integrated arrays expressing actin-binding fluorescent probes in all hypodermal cells under the control of lin-26pPIX-1::GFP is expressed in cells other than the hypodermis, and we cannot exclude the possibility that differential expression of PIX-1::GFP in other cells may affect the rescuing ability of the transgene. To test this possibility, we generated transgenic animals expressing PIX-1::GFP under the control of the hypodermal specific promoter, pix-1p::pix-1::GFP expressing animals, transgenic animals carrying an integrated array expressing PIX-1::GFP under the control of lin-26p segregated into two populations. The embryos of the first population displayed an elongation rate similar to wt  animals. They also suggest that expression of the transgene in dorsal-posterior cells above a threshold corresponding to half the expression in the other hypodermal cells decreases the efficiency of early elongation. This suggests that pix-1 may be submitted to a tight control of its expression level in hypodermal cells, particularly to decrease its level in the dorsal-posterior cells.Altogether, these data suggest that homogenous expression of PIX-1::GFP in dorsal-anterior, lateral and ventral hypodermal cells, and decreased expression in dorsal-posterior cells significantly rescued the elongation defects observed in the Embryonic elongation transforms the ovoid embryo into the long, thin vermiform nematode. This morphogenetic process occurs in two phases. The early elongation is driven by the contraction of Filamentous actin Bundles (FBs) in hypodermal cells. The late elongation is driven by a mechanotransduction pathway in the dorsal and ventral hypodermis resulting from contraction of the underlying muscle cells.Two parallel pathways control early elongation. One pathway involves two kinases: the RHO-1/RHOA effector LET-502/ROCK and the ortholog of the CDC-42-effector human myotonic dystrophy kinase MRCK-1/MRCK pix-1 controls early elongation in parallel with the mel-11/let-502 pathway. Our data suggest that pix-1 controls this process as part of the pak-1 pathway in parallel with a gene (or a group of genes) that remains to be identified. pix-1 may also function independently from pak-1. Our data suggest that PIX-1/PAK-1 and LET-502/ROCK have different function along the antero-posterior axis of the embryo during early elongation: PIX-1, PAK-1 and LET-502/ROCK appear to control the constriction of the head while only LET-502/ROCK controls the constriction of the tail of the elongating embryo. Our study also revealed that PIX-1::GFP fusion protein is homogeneously distributed in the cytoplasm and at the cell periphery of hypodermis during early elongation. We showed that this fusion protein rescues the reduced elongation rate observed pix-1(gk416) in a subset of transgenic animals expressing the transgene homogenously in dorsal-anterior, lateral and ventral cells and at a lower level in dorsal-posterior cells. Our results also suggest that PIX-1 expression above a certain threshold in dorsal-posterior hypodermal cells is detrimental for early elongation.In this study, we show that the CDC42/RAC-GEF pix-1 and pak-1 mutants have similar elongation phenotypes and both aggravate the elongation defects observed in mel-11; let-502 double mutant suggesting that these two genes control together a subset of developmental mechanisms in parallel with mel-11; let-502 during early elongation. However, pix-1 mutant aggravates elongation defects associated with mel-11; let-502 to a lesser extent than the pak-1 mutant. This suggests that pix-1 functions with other genes that act in parallel with mel-11/let-502 pathway  or GTPase-independent  manner pix-1(gk416) than in wt embryos, suggesting that PIX-1 regulates CED-10/RAC activity during embryonic development unc-73/TRIO was also shown to control early elongation in parallel to mel-11/let-502rh40, consists of a missense mutation that eliminates its exchange activity towards CED-10/RAC, RAC-2/RAC and MIG-2/RHOG without affecting its activity towards RHO-1/RHOA In both mammals and pix-1 and pak-1 may be part of the same pathway in parallel with mel-11/let-502, mel-11(it26)-inducing embryo rupturing is suppressed by pix-1(gk416) but not by pak-1(ok448)  . This infem-2 null allele induces weak larval arrest with non-elongated larvae similar to that observed in pix-1 and pak-1 mutants fem-2 mutant can also aggravate mel-11; let-502 double mutants and suppress the mel-11 rupturing phenotype. pix-1, and fem-2/POPX2 function together to control early elongation in parallel with mel-11/let-502. Interestingly, the PP2C-like serine/threonine phosphatases, POPX2/Protein Phosphatase 1F, the closest homolog of FEM-2 in mammals, was shown to interact with β-Pix (ortholog of PIX-1) and to dephosphorylate and inactivate PAK1 (ortholog of PAK-1) kinase activity in mammalian cells C. elegans and mammals, we hypothesize that PIX-1, FEM-2/POPX2 and PAK-1 may have functional relationships similar to their homologs in mammals. Following this hypothesis, PIX-1 and FEM-2/POPX2 may control the activation/inactivation turnover dynamics of PAK-1 during early elongation. Considering that pix-1 and fem-2 mutant have similar interactions with mel-11 and let-502 during early elongation, this hypothesis suggests that a reduced or a sustained activation of PAK-1 alters similarly early elongation process. While this hypothesis would explain the genetic data obtained with pix-1, fem-2 and pak-1 mutants during elongation, it still remains to be confirmed through a careful analysis of a possible relationship existing between PAK-1 activation/inactivation dynamics and the regulation of myosin contraction in C. elegans hypodermal cells during early elongation.It was shown that the mel-11(it26) allele pix-1 may function together with pak-1 in parallel with mel-11 and may also function upstream of mel-11 independently of pak-1. This would constitute an alternative explanation for the suppression of mel-11(it26)-inducing rupturing by pix-1 but not by pak-1 allele. Interestingly, the kinase MRCK-1/MRCK, whose mammalian ortholog, is an effector of CDC-42 mel-11mel-11/let-502  specifically involved in the control of contractions in the posterior part of the embryo.Observation of the head to tail morphology of the elongating embryo showed that the anterior part of the embryo at 1.2-fold stage is much wider than the posterior part. This suggests that contraction forces applied on the anterior part of the embryo is higher than in the posterior part at that stage. We showed that the mechanism leading to a faster reduction of the head width during early elongation is dependent on wt. Higher expression of PIX-1::GFP in the dorsal-posterior cells is also shown to be detrimental for the elongation rate of embryos during early elongation. This suggests that the function of pix-1 is required at a low level in the dorsal-posterior hypodermal cells to ensure optimal elongation rate. This supports the hypothesis that the pix-1/pak-1 pathway may induce more contractions in the anterior part of the embryo than in the posterior part. However, this hypothesis will have to be tested through careful measurement of contraction forces induced by the let-502/mel-11 and pix-1/pak-1 pathways in individual sets of hypodermal cells during early elongation.Interestingly, we found that animal expressing PIX-1::GFP at a reduced level in the dorsal posterior cells present an elongation rate similar to let-502 function suppresses the increased head/tail width ratio observed in pix-1 arrested larvae when compared to wt and that this suppression occurs only in embryos expressing an active mel-11  mutants. These data confirm that MEL-11 function antagonizes both contraction forces induced by LET-502 and those induced by the PIX-1/PAK-1 pathway  X, pix-1(ok982) X, pak-1(ok448) X, mcIs40 [lin-26p::ABDvab-10::mcherry + myo-2p::gfp] and mcIs50 [lin-26p::ABDvab-10::gfp + myo-2p::gfp], were obtained from the Caenorhabditis Genetic Center (CGC). Mutant strains were backcrossed at least 3 times against wild-type (wt) animals prior to analysis. Strains carrying let-502(sb118) I, mel-11(it26) unc-4(e120)/mnC1 II and mel-11(it26) unc-4(e120) II; let-502(sb118) I, were kindly provided by Dr Paul Mains . mel-11(it26) unc-4(e120) II; let-502(sb118) I were maintained at 25.5°C. mel-11(it26) unc-4(e120) II; let-502(sb118) I; pix-1(gk416) X and mel-11(it26) unc-4(e120) II; let-502(sb118) I; pak-1(ok448) X were generated after crossing mel-11(it26) unc-4(e120) II; let-502(sb118) I hermaphrodites with pix-1(gk416) X or pak-1(ok448) X males. Genotyping of F2 progeny was done through isolation of Unc F2 (mel-11(it26) unc-4 (e120) II homozygotes) at 15°C. Mutations in pix-1 and pak-1 genes were identified using Polymerase chain Reaction (PCR) and let-502(sb118) I homozygotes were identified through scoring of embryonic lethality (Emb) and larval arrest (Lva) phenotypes of populations grown at 18°C and 25.5°C.Control N2 and other animals were maintained in standard conditions at 20°C pix(gk416); sajEx1 animals were generated by injection. Translational PIX-1::GFP fusion construct (pix-1p::pix-1::gfp) was obtained from Dr Chen HJ's laboratory  rol-6(su1006) at 50 ng/μl) in pix(gk416) animals. Rol transgenic animals were isolated and expression of PIX-1::GFP was assessed by fluorescent microscopy. R]unc-119(ed3); pix-1(gk416); sajIs1 and unc-119(ed3); pix-1(gk416); sajIs3[lin-26p::pix-1::GFP] were independent, stable transgenic lines generated also using biolostic bombardment. It contains 5kb of the lin-26 promoter (lin-26p), the pix-1 coding sequence including the intron between exon 1 and 2, and the GFP coding sequence in pMB14 vector. This construct was also integrated by biolistic bombardment in unc-119(ed3); pix-1(gk416) strain, using a PDS-1000/He system with the Hepta adaptor (Bio-Rad).pix(gk416); sajEx1, unc-119(ed3); pix-1(gk416); sajIs1, unc-119(ed3); pix-1(gk416); sajIs2 and unc-119(ed3); pix-1(gk416); sajIs3 hermaphrodites with mcIs40[lin-26p::ABDvab-10::mcherry+myo-2p::gfp] males. Stable lines expressing GFP, mCherry transgenes and carrying pix-1(gk416) allele were isolated from the F2 progeny.Transgenic animals expressing PIX-1::GFP together VAB-10(ABD)::mCherry were obtained through crossing wt) L1 arrested by starvation after hypochlorite treatment. The head/tail ratio was calculated as the ratio of the head width with the tail width in μm per animal. The head (and tail) width reduction ratio was calculated as the ratio of the head width (or tail width) at 1.2-fold stage over the head width (or tail width) at the end of early elongation. Statistical significance was calculated using unpaired Student's T-test.To score Emb and Lva phenotypes, worms were synchronized by hypochlorite treatment. After synchronisation 10–20 worms were deposited on NGM agar with OP50 as a source of food Worms were allowed to lay eggs at 18°C or 25.5°C for 4 to 5 hours and were washed off the plate with M9 medium. After 24 and 48 hours dead eggs and arrested L1 larvae were counted and observed at high magnification. The stage of embryonic arrest was confirmed in mutant animals using four-dimension microscopy. Embryos dissected from adult hermaphrodites were mounted on 3% agarose pads in M9 buffer and coverslips were sealed with drawing gum (pébéo). Elongation was recorded using 4-dimensional microscopy (3D and time), which recorded a Z-stack every 2 minutes during 10 hours at 23–24°C using a Leica DM5500 microscope equipped with a 63X oil immersion objective upon differential interference contrast illumination (DIC). Images were captured using the Leica LAS AF imaging software. These recording were used to measure the duration of early elongation for at least 20 eggs from 1.2- to the end of early elongation – identified as the moment when body-wall muscles start contracting, the length of the embryos at the end of early elongation, the width of the head , the width of the tail  at 1.2-fold and at the end of early elongation in the different mutant animals. These measurements were done using Leica LAS AF6000 imaging analysis tools. The reproducibility of these measurements was tested as detailed in the supplementary 2HPO4, 1.47 mM KH2PO4 Adjust to a final pH of 7.4.), embryos were incubated 10 minutes in cold methanol, and extensively washed with PBS. Fixed embryos were incubated overnight at 4°C with appropriate dilutions of primary antibodies in culture media . Mouse anti-MH-27 antibodies  were used at 1∶10 dilution, rabbit anti-GFP antibodies were used at 1∶500 (Invitrogen). After three washes with culture media , embryos were incubated at room temperature for 1 h with 1∶200 dilution of either anti-mouse IgG conjugated to TRITC or anti-rabbit IgG conjugated to FITC (Jackson ImmunoResearch) in 1X-PBS. Embryos were washed three times with culture media and incubated with 100 ng/ml of DAPI for 1 minute at room temperature, washed with water, resuspended in 40 µl of Mowiol (sigma Aldrich) and mounted on slides.For indirect immunofluorescence, embryos were fixed using 3% paraformaldehyde at room temperature for 10 minutes. Following washes with PBS  and a Z-step of 0.15 µm. Images were captures using NIS-element software (Nikon). Deconvolution was done using Autoquant 3X, 3D deconvolution software. Orthogonal views, and fluorescence quantifications were generated using ImageJ software. For fluorescence quantification, individual Z-steps were extracted from Z-stacks recording of elongating embryos. DP, DA, LP, LA, VP, VA hypodermal cells were selected using ImageJ ‘polygon selections’ tool. Nuclei of cells in these areas were also selected using the same tool. The Raw intensities of selected areas were measured using area and gray value function. Raw Intensities of nuclei were subtracted to raw intensities of selected hypodermal sections. The pixel mean value was then calculated dividing this adjusted raw intensity by the area size selected excluding also size of nuclei. Intensity ratios and raw intensities were calculated and used using the resulting value. Comparison of raw intensities between different animals was done on image captured within a week, to limit the impact of laser fluctuation. This fluctuation was controlled through comparison of intensities obtained for the same sample at different days. Fluorescence intensity was compared on images captured using the same laser power and adjusted photomultiplier tube (PMT). Statistical significance of quantification was assessed using the unpaired student T-test. Spearman correlation coefficients and statistical tests for significance of correlation between two quantitative variables were calculated using spearman cor.test function in R (Bioconductor).Figure S1Schematic representation of human (Hs) and C. elegans (Ce) PIX and PAKs. Modular structure has been identified using the SMART tool (www.smart.org) or by alignment of consensus sequence using CLUSTALW. Binding-domains for protein partners reported in the literature are indicated. Proteins coded by C. elegans mutant alleles are indicated. * indicate the location of translation arrest. CH: calponin homology domain; SH3: src homology domain; DH: dbl homology domain; PH: Pleckstrin homology domain; PR: Proline Rich sequence, GBD: GIT-binding domain, CC: coil-coiled domain, ZB: PDZ binding domain, PBD: GTPase binding domain; Ser/Thr kinase: serine threonine kinase domain.(TIF)Click here for additional data file.Figure S2pix-1(ok982) and pak-1(ok448) arrested larvae present severe pharynx pumping defects. 48 hours after egg-laying, pharynx pumping rates were counted on arrested L1 animals and escaper L3 animals moving freely on a bacterial lawn. At least 10 animals per genotype were examined during 15-sec periods. N = 3. ** T-test p (mutant/N2)<0.001(TIF)Click here for additional data file.Figure S3Establishment of embryo width measurement as a robust metrics to characterize embryonic elongation. A) We tested the robustness and reproducibility of head width measurement of embryos. To do so, head width was measured five times on a given population of wt embryos at 1.2-fold stage (n = 12 embryos). Means and standard deviation were calculated and Brown-Forsythe test  was used to test for homogeneity of variances among the five different groups of measurement. This test revealed no significant variance difference amongst the measurements . B) The repeatability and batch effect of our measurements were assessed through measurement of the head width of wt embryos at 1.2-fold stage from 4D-recording done at three different days (n = 12 embryos). Means and standard deviation were calculated and Brown-Forsythe test was used to test for homogeneity of variances among the three different groups of measurements. This test revealed no significant variance difference amongst the measurements . Similar results were obtained for tail width measurements and for measurement done at different stages of early elongation (data not shown). These data indicate that the significant differences observed between genotypes using head-width, tail-width and head/tail width ratio measurements are not due to measurement variability and batch effect.(TIF)Click here for additional data file.Figure S4PIX-1 is homogeneously distributed in the cytoplasm and at the cell periphery of hypodermal cells during early elongation. Confocal microscopy analysis of pix-1(gk416) embryos carrying sajEx1; mcIs40[lin-26p::ABDvab-10::mCherry + myo-2p::gfp]. PIX-1::GFP is observed in B and E  and ABD::mCherryVAB-10 in A and D . Embryos are oriented anterior to the left and dorsal up. Enlarged views (lower panels) show areas indicated by white rectangles in upper panels. Apical and basolateral membrane are indicated by arrow and arrowhead, respectively . Scale bars upper panels: 10 µm; lower panels 5 µm.(TIF)Click here for additional data file.Figure S5PIX-1::GFP intensity ratio are constant throughout early elongation. Dorsal-posterior/dorsal-anterior (DP/DA), lateral-posterior/lateral-anterior (LP/LA), ventral-posterior/ventral-anterior (VP/VA), dorsal-posterior/ventral (DP/V), dorsal-anterior/ventral (DA/V) and dorsal-anterior/lateral (DA/L) fluorescence intensity ratio were measured as detailed in methods and in R]pix-1(gk416); unc-119; sajIs2 embryos (n = 20). The spearman correlation (R2) between the elongation rate and the PIX-1::GFP intensity ratio are indicated, as well as the p-values rejecting the null hypothesis being that the two values are not significantly correlated. Similar results were obtained from two independent transgenic lines.(TIF)Click here for additional data file.Figure S7High expression of PIX-1::GFP in dorsal-posterior hypodermis is detrimental for elongation rate of embryos. Dorsal-posterior/dorsal-anterior (DP/DA), lateral/ventral (L/V), dorsal-anterior/lateral (DA/L) and dorsal-posterior/lateral (DP/L) fluorescence intensity ratio were measured as detailed in methods and in R]pix-1(gk416); unc-119; sajIs2[lin-26p::pix-1::GFP;unc-119 embryos elongating at a wt-rate (elongation≥288 nm/min) or elongating slower (elongation<288 nm/min) during early elongation. Bar correspond to the mean and error bars to the standard deviation. ** T-test p-value<0.01.(TIF)Click here for additional data file.


Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several acquired immunodeficiency syndrome-related malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and a subset of multicentric Castleman's disease. Regulation of viral lytic replication is critical to the initiation and progression of KS. Recently, we reported that herpes simplex virus type 1 (HSV-1) was an important cofactor that activated lytic cycle replication of KSHV. Here, we further investigated the possible signal pathways involved in HSV-1-induced reactivation of KSHV.By transfecting a series of dominant negative mutants and protein expressing constructs and using pharmacologic inhibitors, we found that either Janus kinase 1 (JAK1)/signal transducer and activator of transcription 3 (STAT3) or JAK1/STAT6 signaling failed to regulate HSV-1-induced KSHV replication. However, HSV-1 infection of BCBL-1 cells activated phosphatidylinositol 3-kinase (PI3K)/protein kinase B  pathway and inactivated phosphatase and tensin homologue deleted on chromosome ten (PTEN) and glycogen synthase kinase-3β (GSK-3β). PTEN/PI3K/AKT/GSK-3β pathway was found to be involved in HSV-1-induced KSHV reactivation. Additionally, extracellular signal-regulated protein kinase (ERK) mitogen-activated protein kinase (MAPK) pathway also partially contributed to HSV-1-induced KSHV replication.HSV-1 infection stimulated PI3K/AKT and ERK MAPK signaling pathways that in turn contributed to KSHV reactivation, which provided further insights into the molecular mechanism controlling KSHV lytic replication, particularly in the context of HSV-1 and KSHV co-infection. Kaposi's sarcoma (KS) is a multifocal angioproliferative disease that often occurs in human immunodeficiency virus (HIV)-infected patients . Now thePreviously, we demonstrated that both interleukin-4 (IL-4)/signal transducer and activator of transcription 6 (STAT6) and IL-6/Janus kinase 2 (JAK2)/STAT3 signal pathways modulated HIV-1 transactivative transcription protein (Tat)-induced KSHV replication . RecentlTo verify this hypothesis, in this study, we focused on the major pathways activated by IL-10/IL-10 receptor (R) and IL-4/IL-4R to evaluate their functions in HSV-1-induced KSHV lytic cycle replication. By transfecting a series of dominant negative mutants and protein expressing constructs and using pharmacologic inhibitors, we found that either IL-10/JAK1/STAT3 or IL-4/JAK1/STAT6 signaling was not involved in HSV-1-induced KSHV replication. However, activation of both phosphatidylinositol 3-kinase (PI3K)/protein kinase B  and extracellular signal-regulated protein kinase (ERK) mitogen-activated protein kinase (MAPK) signal pathways contributed to HSV-1-induced KSHV replication. These novel findings are believed to be the first report on the mechanisms of KSHV activation by HSV-1 and shed light on the pathogenesis of KSHV-induced malignancies.2 atmosphere. HSV-1 (McKrae strain) was propagated and viral titers were determined in Vero cells as described previously [BCBL-1 cells (KSHV-positive and EBV-negative PEL cell lines) were obtained through acquired immunodeficiency syndrome (AIDS) Research and Reference Reagent Program, National Institutes of Health. Vero cells (African green monkey kidney fibroblasts) were obtained from American Type Culture Collection (ATCC). BCBL-1 and Vero cells were maintained in RPMI-1640 and Dulbecco's modified Eagle's medium (DMEM) respectively, both of which contained 10% fetal bovine serum (FBS), 2 mmol/l L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified, 5% COAnti-phospho-STAT3 (Tyr705) rabbit monoclonal antibody (mAb), anti-phospho-PI3K p85 (Tyr458)/p55 (Tyr199) rabbit polyclonal antibody (pAb), anti-phospho-AKT (Ser473) mouse mAb, anti-phospho-GSK-3β  rabbit pAb, anti-phospho-c-Raf (Ser338) rabbit pAb, anti-phospho-MEK1/2  rabbit pAb, anti-phospho-ERK1/2 (Thr202/Tyr204) rabbit mAb, anti-STAT3 rabbit pAb, anti-PI3K p85 rabbit pAb, anti-GSK-3β rabbit mAb, anti-c-Raf rabbit pAb, anti-MEK1/2 rabbit pAb, anti-Flag M2 mouse mAb, anti-hemagglutinin (HA) rabbit mAb and LY294002 (PI3K inhibitor) were purchased from Cell Signaling Technologies . Anti-PTEN (PTEN: phosphatase and tensin homologue deleted on chromosome ten) mouse mAb, anti-β-actin mouse mAb, anti-α-Tubulin mouse mAb, anti-GAPDH mouse mAb and horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit IgG were obtained from Santa Cruz Biotechnologies . Anti-AKT rabbit pAb were obtained from BioVision . Anti-ERK1/2 rabbit pAb were obtained from Shanghai Kangchen Biotechnologies . Piceatannol (JAK1 inhibitor) was purchased from BIOMOL Research Laboratories Inc. . Both anti-phospho-STAT6 (Tyr641) mouse mAb and Peptide II (ERK inhibitor) were obtained from Calbiochem . Anti-STAT6 rabbit pAb was purchased from Bethyl Laboratories Inc. . Anti-KSHV ORF59 mAb and viral IL-6 (vIL-6) rabbit pAb were obtained from Advanced Biotechnologies Inc. . Anti-KSHV Rta (replication and transcription activator) antibody was generated by immunization of rabbits with ORF50 peptide (amino acids 667-691) [After infection, cells were harvested and lysed in RIPA buffer containing protease and phosphatase inhibitors. 60-80 μg of proteins were loaded onto sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with diluted primary Abs for overnight at 4°C, and then incubated with HRP-conjugated species-specific second Abs for 1 h at 37°C. Proteins were visualized by enhanced chemiluminescence (ECL) reagents  according to the manufacture's instructions.Total RNA was isolated from cells by using Trizol reagent . RT-qPCR was performed in a GeneAmp 7300 sequence detection machine  as described previously . The seqhttp://www.addgene.org). The PTEN cDNA plasmid (there was a tag of Flag) was constructed in our lab. BCBL-1 cells were electroporated at 250 V and 960 μF using a Gene Pulser  as described elsewhere [The dominant negative STAT3 construct  was kindly provided by D. Link  . The domlsewhere .After BCBL-1 cells were infected with HSV-1 for 48 h, supernatant from cell cultures was harvested and filtered through a 0.45-μm-pore-size filter. The filtered supernatant was centrifugated for 30 min at a speed of 15 000 rpm at 4°C and the precipitation contained KSHV progeny virions. The virions were resuspended in PBS and viral DNA was extracted using the high pure viral nucleic acid kit  as per the manufacturer's instructions. Purified viral DNA was used for real-time DNA-PCR analysis. The KSHV ORF26 gene cloned in the pcDNA3.1  was used to generate the standard curve.IFA was performed as described elsewhere . BrieflyWe have previously demonstrated that the production of IL-10 and IL-4 from HSV-1-infected BCBL-1 cells partially contributed to HSV-1-induced KSHV replication . CommonlBesides signal pathways from JAK1/STAT3 by IL-10 and JAK1/STAT6 by IL-4, both IL-10 and IL-4 can also induce activation of PI3K/AKT pathway -20. To eTo examine whether PI3K/AKT pathway was involved in KSHV lytic cycle replication by HSV-1, PI3K-specific inhibitor LY294002 was first used. RT-qPCR demonstrated that ORF26 mRNA in HSV-1-infected BCBL-1 cells pretreated with LY294002 was decreased 3.27-fold at 12 h, 3.64-fold at 24 h, and 2.81-fold at 48 h post infection of HSV-1, respectively, compared to HSV-1-infected BCBL-1 cells pretreated with DMSO Figure . To confFrom Figure Because HSV-1 infection of BCBL-1 cells increased phosphorylated GSK-3β Figure  and tranTaken together, these data suggest that PTEN/PI3K/AKT/GSK-3β pathway may play an important role in HSV-1-induced KSHV reactivation.Because IL-4 can also induce activation of ERK MAPK pathway ,22, we r2O , nuclear factor kappa B (NF-κB) and Notch have been shown to participate in KSHV infection, replication and angiogenesis ,23-29. Iet al. just only used chemical inhibitors. Since chemical inhibitors are known to have pleiotropic effects, the use of dominant negative protein expression plasmids is of value. In addition, authors of these two studies detected only the effects of inhibition of PI3K or AKT on the reactivation of KSHV in PEL cell lines, but the upstream and downstream effectors were not shown.PI3K/AKT signaling pathway plays an important role in cell growth and survival. PI3K is a heterodimer composed of a catalytic subunit p110 and an adaptor/regulatory subunit p85 . PI3K acet al. demonstrated that inhibiting B-Raf/MEK/ERK signaling by using MEK-specific inhibitors or siRNA construct targeting B-Raf restrained 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced KSHV lytic replication [et al. also showed an essential role of ERK signaling in TPA-induced reactivation of KSHV by using MEK-specific inhibitors [et al. revealed that Raf/MEK/ERK pathway mediated Ras-induced KSHV reactivation and the same pathway also mediated TPA-induced KSHV reactivation and spontaneous reactivation in PEL cells, by screening expression of a mammalian cDNA library [MAPK cascades are key signaling pathways involved in the regulation of cell proliferation, survival and differentiation. It is not surprising that many viruses including KSHV target MAPK pathways as a means to manipulate cellular function and to control viral infection and replication. Studies from Gao's group demonstrated that ERK, c-Jun N-terminal kinase (JNK) and p38 multiple MAPK pathways had general roles in regulating the life cycle of KSHV by mediating both viral infection and switch from viral latency to lytic replication ,40. Amonlication . Cohen ehibitors . Yu et a library . A more  library . Here, wIn summary, we have showed that not JAK1/STAT3 or JAK1/STAT6 but PTEN/PI3K/AKT/GSK-3β and ERK MAPK signal pathways partially contributed to HSV-1-induced KSHV replication. These findings provided further insights into the molecular mechanism controlling KSHV lytic replication and shed light on the pathogenesis of KSHV-induced malignancies.DQ, NF and WF carried out the experiments. DQ drafted the manuscript. XM, QY and ZL participated in Western blot and IFA. YZ and JZ participated in discussion in preparing the manuscript. CL designed the study and revised the manuscript. All authors read and approved the final manuscript.


Caenorhabditis elegans and mammalian cells, we investigated the transport mechanisms underlying compartmentalization of JS-associated ARL13B/ARL-13, which we previously found is restricted at proximal ciliary membranes. We now show evolutionary conservation of ARL13B/ARL-13 localisation to an Inv-like subciliary membrane compartment, excluding the TZ, in many C. elegans ciliated neurons and in a subset of mammalian ciliary subtypes. Compartmentalisation of C. elegans ARL-13 requires a C-terminal RVVP motif and membrane anchoring to prevent distal cilium and nuclear targeting, respectively. Quantitative imaging in more than 20 mutants revealed differential contributions for IFT and ciliopathy modules in defining the ARL-13 compartment; IFT-A/B, IFT-dynein and BBS genes prevent ARL-13 accumulation at periciliary membranes, whereas MKS/NPHP modules additionally inhibit ARL-13 association with TZ membranes. Furthermore, in vivo FRAP analyses revealed distinct roles for IFT and MKS/NPHP genes in regulating a TZ barrier to ARL-13 diffusion, and intraciliary ARL-13 diffusion. Finally, C. elegans ARL-13 undergoes IFT-like motility and quantitative protein complex analysis of human ARL13B identified functional associations with IFT-B complexes, mapped to IFT46 and IFT74 interactions. Together, these findings reveal distinct requirements for sequence motifs, IFT and ciliopathy modules in defining an ARL-13 subciliary membrane compartment. We conclude that MKS/NPHP modules comprise a TZ barrier to ARL-13 diffusion, whereas IFT genes predominantly facilitate ARL-13 ciliary entry and/or retention via active transport mechanisms.Cilia are microtubule-based cell appendages, serving motility, chemo-/mechano-/photo- sensation, and developmental signaling functions. Cilia are comprised of distinct structural and functional subregions including the basal body, transition zone (TZ) and inversin (Inv) compartments, and defects in this organelle are associated with an expanding spectrum of inherited disorders including Bardet-Biedl syndrome (BBS), Meckel-Gruber Syndrome (MKS), Joubert Syndrome (JS) and Nephronophthisis (NPHP). Despite major advances in understanding ciliary trafficking pathways such as intraflagellar transport (IFT), how proteins are transported to subciliary membranes remains poorly understood. Using  C. elegans nematodes and mammalian cell experimental systems, we uncovered differential requirements for sequence motifs, IFT and ciliopathy modules in regulating ARL-13 ciliary restriction, mobility and compartment length. Also, we provide essential insight into how IFT and ciliopathy-associated protein complexes and modules influence ciliary membrane protein transport, diffusion across the TZ, the integrity of the ciliary membrane, and subciliary protein composition.Protruding from most cells surfaces is a hair-like extension called the primary cilium. This organelle functions as a cellular antenna, receiving physical and chemical signals such as light, odorants, and molecules that coordinate cell growth, differentiation and migration. Underscoring their importance, cilium defects underlie an expanding spectrum of diseases termed ciliopathies, characterised by wide-ranging symptoms such as cystic kidneys, blindness and bone abnormalities. A key question is how ciliary proteins are targeted to and retained within cilia. The best understood system is intraflagellar transport (IFT), thought to ferry proteins between the ciliary base and tip. Also, ciliopathy protein modules organise protein diffusion barriers at the ciliary base transition zone (TZ). Despite major advances, it remains poorly understood how proteins are targeted to cilia, and ciliary membrane subdomains in particular. Here, we investigated how Joubert syndrome-associated ARL13B/ARL-13 is compartmentalized at subciliary membranes. Using  Primary cilia are organized into specific subcompartments, defined by distinct ultrastructure, protein and lipid compositions, and include the basal body (BB), the adjacent transition zone (TZ), and axonemal regions consisting of doublet and singlet microtubules C. elegans and flagellar export of signaling proteins in ChlamydomonasTargeting of proteins to cilia depends on intracellular transport mechanisms. The best studied is intraflagellar transport (IFT), an evolutionarily conserved motor protein-driven bidirectional motility of macromolecular assemblies along ciliary axonemes, essential for cilium formation and function  TZs, and bipartite axonemal structures consisting of doublet microtubules (middle segment) or singlet microtubules  C. elegans cilia are environmentally exposed, relaying chemosensory, thermosensory and osmosensory signals. Many ciliary transport and ciliopathy genes are conserved in worms, and loss-of-function alleles are available for most of them. Importantly, unlike other systems, some resemblance of cilium structure remains in most IFT and ciliopathy gene mutants, thus allowing ciliary protein targeting to be investigated.We investigate ciliary protein transport in C. elegans, the ARL-13 orthologue is further refined to a proximal ciliary subdomain Despite major progress, the targeting and retention mechanisms regulating trafficking of cytosolic and membrane ciliary proteins are not well understood. For example, although IFT is an assumed driver of ciliary transport, only a handful of IFT cargos have been identified and there is evidence that a number of membrane proteins  still localize to cilia in IFT disrupted cells, although ciliary abundance levels may be elevated C. elegans. We also show distinct roles for IFT, BBS and TZ modules  in regulating ARL-13 compartmentalization and diffusion across ciliary membranes. Finally, we determined the composition of human ARL13B complexes and uncovered robust biochemical associations with IFT complex B via IFT46 and IFT74 interactions. Overall, this study represents a comprehensive analysis of the transport mechanisms organizing the ARL13B/ARL-13 ciliary signaling subdomain, and provides important insight into how IFT and ciliopathy-associated protein complexes and modules influence ciliary transport and diffusion, the integrity of the ciliary membrane, and subciliary protein composition.Here we employed genetics, quantitative imaging, fluorescence recovery after photobleaching (FRAP) and affinity proteomics in nematode and cell culture models to investigate the mechanisms of ARL13B/ARL-13 transport and retention within a subciliary membrane domain. We show evolutionary conservation of ARL13B/ARL-13 localisation to an Inversin-like compartment and the requirement of RVVP and palmitoylation modification sequence motifs to prevent distal cilium and nuclear targeting in C. elegans ARL-13 localises to the proximal ciliary region of amphid (head) and phasmid (tail) channel cilia C. elegans ARL-13 proximal ciliary domain in amphid and phasmid channel cilia corresponds to the middle segment (MS) and does not include the transition zone (TZ); specifically, ARL-13 is juxtaposed to MKSR-1/B9D1 at the TZ, and OSM-6/IFT52 basal body signals are separated from ARL-13 signals by an ∼1 µm ‘gap’, which corresponds to the TZ length . At L2 stage, distal ciliary signals are dramatically reduced and by L3 stage, most worms show ARL-13 restriction to the proximal cilium (∼3 µm), which elongates slightly during development to adulthood . This developmental pattern was not observed for the overlapping heterotrimeric kinesin-II compartment Figure S1A). Also, ARL-13 is excluded from the phasmid TZs of all larval stages and the TZ ‘gap’ between ciliary ARL-13 and basal body CHE-13/IFT57 appears shorter in L1 larvae versus adults, suggesting TZ elongation as the worm ages , concomitant with a reduction of the non-bleached ciliary signals . Recovery is wave-like, emanating from the non-bleached pool, and re-establishes pre-bleach uniform distribution of ARL-13 across the MS . Thus, ARL-13 continuously exchanges at the MS membrane, but not between ciliary and dendritic membranes. These results are consistent with Arl13b FRAP dynamics in cultured cells Using a fluorescence recovery after photobleaching (FRAP) approach, we investigated if ; Movie S1). Although motility was more prominent in the distal cilium, movement was also detectable in proximal ciliary regions. For various technical reasons , it was difficult to obtain many usable video microscopy-derived kymographs to measure motility rates. Nonetheless, for the particles we could measure, an anterograde speed of 0.65±0.09 µm.s−1 (n = 16) in phasmid cilia was determined, which is similar to reported MS anterograde IFT rates Although it was previously reported by us and others that ARL-13 does not undergo IFT in adult worms Figure S2A).Previously we and others found that an N-terminal palmitoylation  modification motif and the disordered C-terminal tail restrict ARL-13 at ciliary membranes  transgene rescued the cilium integrity defect (measured by a dye-filling assay) of an arl-13(tm2322) mutant, indicating that GDP-locked ARL-13 is functional . In contrast, Pal motif-disrupted arl-13 constructs were previously reported by us to induce a mild dominant negative dye-filling (Dyf) phenotype, indicating disrupted function arl-13(Δ285–370) and arl-13(ΔRVVP) transgenes, which induce a fully penetrant dominant negative Dyf defect . To further investigate this defect, a CHE-13/IFT57 marker was used to stain cilia, which revealed that ARL-13(ΔRVVP)-expressing amphid cilia were abnormally dispersed and mis-positioned; furthermore, axonemal CHE-13 signals were reduced or absent, indicating an IFT defect . These phenotypes were specific for arl-13(ΔRVVP), and not found in arl-13 and arl-13(T38N) expressing worms . Since arl-13(tm2322) worms also possess misplaced amphid cilia and reduced ciliary signals for IFT proteins Finally we investigated whether altered ARL-13 localisations disrupt its function. Consistent with normal localisations, an These data show that a C-terminal RVVP motif restricts ARL-13 compartment size by preventing leakage into distal segments and suggest that palmitoylation regulates shuttling of ARL-13 between the ciliary membrane and the nucleus. Furthermore, our findings indicate that these motifs are required for the ciliogenic and IFT-related functions of ARL-13.C. elegans anterograde IFT assemblies, the kinesin-II motor is physically more closely connected with IFT-A versus IFT-B C. elegans MKS and NPHP modules play redundant roles in establishing the TZ unc-61 (class 1B) and unc-59  Next, we questioned if ARL-13 ciliary targeting and restriction requires IFT-A/B, kinesin-2, IFT-dynein, BBS, MKS, NPHP or septin genes, all of which are associated with distinct aspects of ciliary protein transport. In simplified models, protein entry into cilia is facilitated by kinesin-2/IFT-B-driven anterograde IFT, whereas protein recycling from the ciliary tip involves IFT-dynein/IFT-A-driven retrograde IFT (S8). Quantification of signal intensities revealed that IFT-B gene mutants (except dyf-6/IFT46 and dyf-13/TTC26) possessed the highest levels of PCM accumulation , whereas IFT-A and IFT-dynein mutants displayed reduced PCM accumulation levels, with levels reduced further in BBS mutants (Table S1). Although there was a trend between cilium length and accumulation levels , this correlation was not absolute. For example, osm-3/KIF17 cilia are shorter than bbs mutant cilia mks-5/RPGRIP1L, mksr-1/B9D2, mksr-2/B9D1) or NPHP (nphp-4) genes showed relatively weak ARL-13 PCM accumulations . However, moderate levels of PCM staining was found in mks-2/TMEM16;nphp-4 and mksr-1;nphp-4 double mutants, indicating redundant functions for MKS and NPHP genes in regulating ARL-13 PCM exclusion . We also observed punctate cell body spots of ARL-13 in mks-2;nphp-4 and mksr-1;nphp-4 worms, and to a lesser extent in mks-5 worms, suggesting an additional role for TZ genes in an early ARL-13 sorting event . Finally, ARL-13 localisation was unaffected in septin (unc-59/61) and kinesin-2 (osm-3/KIF17 and klp-11/KIF3B) worms . Whilst the above phenotypes are reported for phasmid neurons, similar observations were made in amphid neurons.In WT worms, ARL-13 is found exclusively in cilia. However, in IFT-B, IFT-A, IFT-dynein and BBS gene mutants, ARL-13 was specifically mislocalised at the periciliary membrane (PCM) region and not in other regions of the sensory neuron S8. QuantS8. QuantS8. QuantS8. QuantS8. Quantdyf-13/TTC26, klp-11/KIF3B, bbs-7 and bbs-8), the ARL-13 compartment was modestly elongated, suggesting a role for IFT in restricting ARL-13 subdomain length . Conversely, ARL-13 was frequently observed at the TZ region of most mks-5 single mutants, as well as mks-2;nphp-4 and mksr-1;nphp-4 double mutants, demonstrating that MKS/NPHP modules regulate the composition of the TZ membrane . This TZ staining in MKS/NPHP mutants results in an elongated ARL-13 compartment, which in some worms appears to extend into distal ciliary regions also  back to pre-bleach ratios . However, for the nphp-4 mutant, PCM/ciliary exchange was relatively slow  and recovery plateaued well below pre-bleach ratios in cilium quenching experiments . In these experiments, signals recovered from non-bleached pools (cilium or PCM) and not from other parts of the cell because no significant recovery was observed when both ciliary and periciliary ARL-13 signals were quenched ; also, as bleached pools recovered, ARL-13 signal intensity at the non-quenched pools diminished . Thus, in mks-5 and mks-2;nphp-4 animals, but not nphp-4 worms, the ciliary and PCM pools of ARL-13 are rapidly exchanging. In contrast, ARL-13 FRAP rates and recovery plateaus were much lower in most examined IFT mutants , indicating these worms possess slow and low level diffusion between ciliary and PCM pools . The one exception was a dyf-13/TTC26 (IFT-B) mutant, which displayed moderately fast FRAP rates  and high recovery levels . Interestingly, dyf-13;nphp-4 double mutants possessed faster bidirectional recovery kinetics than single mutants , possibly indicating partially redundant functions for these genes in regulating ARL-13 ciliary/PCM diffusion . From these data we conclude that the TZ barrier to ARL-13 diffusion is disrupted in mks-5, mks-2;nphp-4 and dyf-13; nphp-4 mutants; thus the TZ and PCM accumulations in these worms are due to ARL-13 leakage out of its compartment. However, in most IFT and nphp-4 mutants, the TZ diffusion barrier appears mostly intact, suggesting that any observed ARL-13 PCM accumulations are caused by defects in active transport driving ciliary entry and/or retention.One explanation for ARL-13 association with mutant periciliary and TZ membranes is that the TZ barrier is compromised, which causes ARL-13 to leak out of its compartment. An alternative non-mutually exclusive explanation is that active processes facilitating ARL-13 ciliary entry or retention might be defective. To test these hypotheses, we used our che-11/IFT140  and dyf-6/IFT46  worms, compared to wild-type worms (t1/2 = 124 sec) . Qualitatively similar results were observed when distal ARL-13 MS signals were bleached . In contrast, wild-type and mks-5 worms  possessed similar recovery rates . In all experiments the area, length and signal intensities of the photobleached MS region was comparable . We conclude that IFT-A and IFT-B proteins , but not TZ-associated MKS-5, retard ARL-13 exchange kinetics at the MS membrane.We also investigated if IFT and TZ modules regulate ARL-13 diffusion at the ciliary  membrane. Using the same FRAP assay described in Figure S6). We first performed stringent two-step (tandem) affinity purifications (TAP), followed by mass-spectrometric identification of the co-precipitated proteins. Specific interactors were identified by comparing SF-tagged ARL13B precipitate profiles with control precipitates from cells expressing the SF tag alone. Two experiments were conducted for N-SF-ARL13B and one for C-SF-ARL13B. We also performed one experiment on cells expressing GDP-locked (T35N) ARL13B. Following removal of non-specific and obvious false positive proteins routinely found in TAP experiments (see methods section), these four experiments produced a final dataset of 47 proteins co-purifying with ARL13B . Highly represented are components of the IFT complex B  and one putative IFT-B protein (TTC26/DYF-13) . Most of these proteins are suggested in Chlamydomonas to form a ∼500 kDa IFT-B core Table S2), indicating that ARL13B associates with IFT-B independent of GDP-GTP exchange.To shed further light on ARL-13/ARL13B transport and compartmentalisation mechanisms, we employed affinity proteomics to identify the composition of human ARL13B complexes. ARL13B was fused with a Strep-Flag (SF) tandem affinity purification tag (TAP) ; ; Table S3). Like the TAP data, ciliopathy proteins were not enriched in SILAC datasets (Table S3). Thus, the SILAC data confirmed the IFT-B association found by TAP and increased the depth of our analysis.To validate the ARL13B complex components identified by TAP and to increase sensitivity in order to detect additional, more labile and transient ARL13B module components, we employed stable isotope labeling of amino acids in cell culture (SILAC), in combination with single step affinity purification and quantitative mass spectrometry. This allowed a quantitative comparison of SF-tagged ARL13B and the SF-TAP alone, or mutated SF-tagged ARL13B. After purification, eluates were combined and non-specific contaminants subtracted to detect potential interaction partners by their specific enrichment compared to the control We further validated the IFT-B associations in HEK293 kidney epithelial cells using co-immunoprecipitations followed by western blotting and showed that transiently transfected ARL13B-Flag immunoprecipitated IFT88 and IFT52 . Mechanistically, MKS and NPHP genes, as well as DYF-13/TTC26, appear to regulate a TZ barrier to ARL-13 diffusion, whereas most examined IFT proteins regulate ARL-13 ciliary entry and/or retention via active transport processes. Consistent with this conclusion, human ARL13B interacts biochemically with the IFT-B complex via IFT46 and IFT74 interactions, and C. elegans ARL-13 can be observed to undergo IFT-like motility.To investigate mechanisms underpinning protein sequestration to ciliary membranes, we assessed how Joubert syndrome-associated ARL13B/ARL-13 is targeted to and restricted at ciliary membranes. We show that ARL13B/ARL-13 is compartmentalised within an evolutionarily conserved Inversin-like ciliary membrane subdomain and requires palymitoylation modification and RVVP motifs to prevent inappropriate targeting of C. elegans ARL-13 C. elegans ARL-13 extends to the ciliary tips of young larval cilia, before restricting to a proximal domain. Thus, the ARL13B/ARL-13 domain is differentially defined in different cell types and at different developmental stages, reflecting age and cell subtype-specific requirements for this G-protein. Another interesting age distinction is that we have only observed IFT-like motility for ARL-13 in young larval worms and not in later larvae or adults. Although there may be technical considerations that prevent us seeing ARL-13 processive movement in older worms , our data indicates that as the cilium ages, the proportion of ARL-13 undergoing active transport may reduce compared to the fraction undergoing diffusion. Thus, for ciliary membrane proteins considered as potential IFT cargo, it may be fruitful to perform experiments on developing or newly formed cilia.Our localisation studies in oviduct and tracheal epithelial cells show that mammalian ARL13B joins a group of other ciliopathy proteins  that localise to proximal ciliary compartments, excluding the TZ C. elegans TZ. Using subcellular localisation and in vivo FRAP assays we were then able to show that this barrier is bidirectional and dependent on MKS and NPHP genes, but not most IFT genes. These observations are consistent with and extend published findings implicating a membrane diffusion barrier at the ciliary base, including a previous report by us and others showing that plasma membrane-associated RPI-2 (retinitis pigmentosa gene 2 orthologue) and transmembrane TRAM-1 (Sec61 ER translocon component) abnormally leak into the ciliary axonemes of TZ gene-disrupted worms in vivo FRAP assay to measure barrier integrity and ciliary/periciliary exchange kinetics will help further dissection of ciliary ‘gating’ at the TZ.Our work showing that ARL-13 readily diffuses at the middle segment membrane but fails to enter the adjacent TZ membrane subdomain clearly demonstrates an ARL-13 diffusion barrier at the nphp-4 single mutants, despite previous findings that non-ciliary plasma transmembrane and membrane-associated proteins  abnormally leak into the cilia of these worms dyf-13/TTC26, which is genetically and biochemically associated with the IFT-B complex dyf-13 single mutants, and even further compromised in dyf-13;nphp-4 double mutants, suggesting a synthetic functional relationship between these genes. In C. elegans, DYF-13 has been placed in a distinct OSM-3/KIF17 accessory motor module with DYF-1/IFT70, on the basis that it is required for building at least part of the ciliary distal segment dyf-13 for IFT and the integrity of the TZ in dyf-13 and dyf-13;nphp-4 mutants should be revealing.Not all MKS, NPHP and IFT genes neatly fit our model, however. For example, the ARL-13 barrier appears mostly intact in TZ-associated How MKS, NPHP and DYF-13/TTC26 define the ARL-13 membrane diffusion barrier at the TZ is unknown. The absence of TZ proteins from our biochemically defined ARL13B complexes makes it unlikely that the barrier involves direct inhibitory interactions between ARL13B and these proteins; however, we cannot discount weak or transient interactions, nor technical limitations with standard TAP in identifying interactions with TZ-associated proteins, most of which are membrane proteins. More likely is that MKS and NPHP modules regulate TZ membrane lipid compositions or steric properties. Indeed, the latter is at the heart of the ‘picket fence’ membrane diffusion barrier model, where cytoskeletal-anchored membrane proteins form an obstacle barrier to molecular diffusion C. elegans ARL-13 entry into distal segment membranes. Nonetheless, there is evidence in our data that this barrier may be partially dependent on MKS, NPHP and dyf-13 genes, as well as BBS genes, because in the corresponding mutants we found a significant number of worms with weak ARL-13 signals in more distal ciliary regions . Also, our finding of a correlation between distal segment localisation of an ARL-13(ΔRVVP) variant together with reduced ciliary levels of CHE-13/IFT57 is consistent with a possible role for IFT in restricting ARL-13 distal segment entry. The availability of hypomorphic IFT mutants retaining distal segments would help to further address this issue.However, TZ ultrastructural features are not found elsewhere in the axoneme and cannot explain the diffusion barrier preventing dyf-13, discussed above). Third, human ARL13B interacts directly with IFT-B complexes via IFT46/74 interactions. The very specific accumulation of ARL-13 at the PCM of IFT mutants and not elsewhere in the cell suggests IFT facilitates ARL-13 trafficking from the PCM into cilia and/or prevents ARL-13 from exiting cilia and accumulating at the PCM. Although we have been unable to distinguish between these two non-mutually exclusive possibilities, our biochemical and IFT motility data suggests that the mechanism involves ARL-13 directly interacting with IFT trains as cargo. In the first scenario, PCM-localised ARL-13, derived from upstream transport or leakage out of the cilium, would be captured by IFT-B complexes at the basal body and then moved back into the cilium, across the TZ, by anterograde IFT. In the alternative scenario, ARL13B could be held in cilia by dynamic association with moving IFT trains, perhaps with rapid on/off rates. In both cases, IFT disruption would lead to PCM accumulation of ARL-13. Thus, as shown in our model  algae C. elegans IFT mutants . Thus, IFT appears to differentially regulate the ciliary transport and mobility of PKD2 and ARL-13. Future efforts using FRAP or photoconversion techniques will help to further tease out the role of IFT in regulating protein dynamics at the ciliary membrane.Since the ARL-13 TZ diffusion barrier does not depend on IFT genes, we were surprised that ARL-13 diffusion rates were increased at the middle segment membrane of IFT mutants, suggesting that IFT restricts ARL-13 mobility in cilia. One possible explanation is that ARL-13 diffusion rates are retarded due to frequent interactions of ARL-13 with IFT trains as they move through the ARL-13 compartment. Alternatively, through its role in targeting proteins to cilia, IFT may regulate steric hindrance to free diffusion at the ciliary membrane. In ChlamydomonasUsing affinity proteomics, we identified reproducible high ranking associations between ARL13B and most of the proteins that form a ∼500 kDa IFT-B core in We have performed a thorough analysis of transport mechanisms organising the ARL-13/ARL13B ciliary signalling subdomain. Our findings reveal differential requirements for sequence motifs, IFT-related complexes and TZ-associated ciliopathy modules in defining an ARL-13 subciliary membrane domain. We find that MKS and NPHP modules, as well as DYF-13/TTC26, regulate a TZ barrier to ARL-13 diffusion, whereas most IFT proteins facilitate ARL-13 ciliary entry and/or retention predominantly via active transport mechanisms. Furthermore, our protein complex and protein-protein interaction data represents a good starting point to further address mechanisms of ARL-13 ciliary protein transport and function, and provides new research avenues for investigating the pathomechanisms of Joubert Syndrome-related disorders.N2 (Bristol), N2;oqEx58[Parl-13::ARL-13::GFP+pRF4], N2;oqEx[Parl-13::ARL-13::tdTomato+pRF4], N2;mnIs17[Posm-6::OSM-6::gfp], N2;oqEx51[Parl-13::ARL-13::GFP+pRF4], N2;oqEx[Parl-13::ARL-13(Δ285–370)::GFP+pRF4], N2;oqEx[Parl-13::ARL-13(T38N)::GFP+pRF4], N2;oqEx[Parl-13::SYN-16::DsRed], N2;myEx[Pche-12::CHE-13::mCherry+Punc-122::GFP], N2;ejEx[Pkap-1::KAP-1::GFP+pRF4], MX349: dpy-5(e907); nxEx[Pmksr-1::MKSR-1::GFP+dpy-5 (+)], che-2(e1033), dyf-3(m185), che-13(e1805), dyf-6(m175), dyf-13(mn396), che-11(e1810), dyf-2(m160), xbx-1(ok279), che-3(e1124), klp-11(tm324), osm-3(p802), mks-5(tm3100), mksr-1(ok2092), mksr-2(tm2452), mks-2(nx111), nphp-4(tm925), bbs-8(nx77), bbs-7(n1606), bbs-1(ok1111), unc-59(e261), unc-61(n3169).C. elegans worms were maintained and cultured at 20°C using standard techniques nx77, tm324, ok2092, nx111, tm925 and ok990 mutations. All other mutations were followed using a dye-filling assay or an Unc phenotype (unc-59/61).Standard genetic crossing techniques were used to make double mutants and to introduce transgenes into genetic backgrounds. PCR using primers flanking deletions were used to follow arl-13p::arl-13::tdTomato and arl-13p::arl-13(Δ285–370)::gfp, plasmid amplified gfp (from pPD95.77) or tdTomato  was fused in frame with genomic DNA fragments containing 300 bp of arl-13 5′ untranslated region, and either the entire exonic/intronic arl-13 sequence  for the tdTomato construct or a truncated sequence (1–2998 nucleotides) for the amino acid 285–370 deletion construct. For the arl-13p::arl-13(T38N)::gfp variant, a PCR fragment was genomically amplified to contain the arl-13 promoter (214 bp) plus the first two exons of arl-13 to nucleotide position 1001, with nucleotides 999–1001 (threonine codon) altered by primer design to create an asparagine codon. This fragment was then fused to a PCR fragment containing the remainder of arl-13 sequence (minus the stop codon), and the resultant amplicon was fused in frame with gfp. All constructs were coinjected at 1–10 ng/µl with 50 ng/µl pRF4 into N2 worms to generate roller transgenic animals harboring extrachromosomal arrays.All constructs were generated by fusion PCR as previously described xbx-1 , dyf-6 , che-2, dyf-13;nphp-4 , and mks-5 . These exceptions correlate with experiments where FRAP recovery kinetics and levels were low, which makes it more difficult to get a good fit.Early adult worms were immobilised with 0.1 µm polystyrene microspheres (Polysciences) on a 10% agarose pad and covered with a coverslip. Experiments were performed on a Nikon Eclipse Ti microscope fitted with a 100×1.4NA Plan APO VC objective (Nikon), a 50 mW 488 nm laser, and CSU-X1 spinning disk unit (Yokogawa). Samples were excited using the 488 nm laser at 50% and images were recorded using a charge-coupled device camera  controlled by Andor Technology iQ 2.6 software. Samples were imaged pre-bleach, and then bleached using a single pulse of the 488 nm laser at 100% with a dwell time of 100 µs. Images were recorded immediately post-bleach, at 15 s, 30 s, 60 s, 120 s, 180 s, 240 s, 360 s, 480 s, and 600 s for intraciliary FRAP experiments, and post-bleach at 15 s, 30 s, 60 s, 120 s, 180 s, 240 s, 300 s, 600 s, 900 s, and 1200 s for periciliary membrane (PCM) and cilium compartment FRAP experiments. For intraciliary FRAP experiments EM gain was set to 6; for compartment FRAP experiments the EM gain was set to 20, with an exposure time of 50 ms in all experiments. Images were imported into ImageJ and converted into a stack. Photobleached and non-photobleached regions of the cilium were selected and intensity measured at each timepoint. After background subtraction, ratios of bleached∶non-bleached regions were calculated. Ratios were normalised to pre-bleach ratio. Curves were fitted and half-time recovery calculated using GraphPad Prism 5.0 software. Most FRAP curves (saturation plots) returned high goodness-of-fit statistics (R-squared>0.75), except for 12C6, 14N2 lysine and 12C6, 14N4 arginine. Heavy SILAC medium was supplemented with either 13C6 lysine and 13C6, 15N4 arginine or 13C6, 15N2 lysine and 13C6, 15N4 arginine. 0.5 mM proline was added to all SILAC media to prevent arginine to proline conversion HEK293T cells were cultured as described previously Protein complex detection and comparison was done essentially as described before LC-MS/MS analysis was performed on an Ultimate3000 RSLCnano HPLC system (Thermo Fisher Scientific) coupled to a LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) by a nano spray ion source. Tryptic peptide mixtures were automatically injected and loaded at flow rate of 6 µl/min in 0.5% trifluoroacetic acid in HPLC grade water onto a nano trap column . After 5 minutes, peptides were eluted and separated on the analytical column  by a linear gradient from 5% to 35% of buffer B  in buffer A  at a flow rate of 300 nl/min over 80 minutes. Remaining peptides were eluted by a short gradient from 40% to 100% buffer B in 5 minutes. Eluted peptides were analyzed by the LTQ Orbitrap Velos mass spectrometer. From the high resolution MS pre-scan with a mass range of 300 to 1500, the ten most intense peptide ions were selected for fragment analysis in the linear ion trap if they exceeded an intensity of at least 250 counts and if they were at least doubly charged. The normalized collision energy for CID was set to a value of 35 and the resulting fragments were detected with normal resolution in the linear ion trap. The lock mass option was activated, the background signal with a mass of 445.12002 was used as lock mass For non-quantitative experiments, the raw data were analyzed using Mascot (Version 2.4) and Scaffold (Proteome Software) against the human subset of the SwissProt Database  as earlier described The direct interaction between ARL13B and other ciliary proteins was tested using a GAL4-based yeast two-hybrid system . The DNA binding domain (GAL4-BD) fused to full length ARL13B was used as a bait to test the interaction with previously described ciliopathy and cilium-associated proteins fused to an activation domain (GAL4-AD). Constructs encoding GAL4-BD and GAL4-AD fusion proteins were co-transformed in yeast strain PJ69-4A. The direct interaction between baits and preys induced the activation of the reporter genes, resulting in the growth of yeast colonies on selective media (deficient of histidine and adenine) and induction of α-galactosidase and β-galactosidase colorimetric reactions Following perfusion with 4% paraformaldehyde (PFA)/PBS, mouse tissues were dissected out and incubated in 4% PFA/PBS at 4°C overnight. Fixed tissues were transferred to 30% sucrose/PBS solution, incubated for a few days, and embedded in OCT compound and stored at −80°C before use. Frozen tissue sections immunostained using anti-ARL13B HEK293T cells were transfected with a C-terminal Flag-tag expression vector with or without (as “Mock”) human full-length Arl13b cDNA. Two days after transfection, the cell lysates were prepared using extraction buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% NP-40, 5 mM MgCl2, 1 mM DTT, 0.1 mM PMSF, 2 µg/ml aprotinin, 2 µg/ml leupeptin) and subjected to immunoprecipitation using anti-Flag M2 beads (Sigma). Immunocomplexes were then subjected to Western blot analysis using anti-IFT88 antibody (Proteintech), anti-IFT52 antibody (Proteintech) and anti-INVS antibody (a generous gift from D. Shiba and T. Yokoyama), and anti-Flag antibody (Sigma).Figure S1A) Phasmid cilia from wild-type worms at different larval and adult stages expressing the indicated transgene-encoded protein. Arrowhead; basal body. Bar; 1 µm. (B) Phasmid cilia of worms expressing ARL-13::GFP showing FRAP recovery after photobleaching ∼40% of proximal ciliary signal. Bar; 1 µm.Developmental timecourse of ARL-13 compartment formation and mobility of ARL-13 at the ciliary membrane, linked to (PDF)Click here for additional data file.Figure S2A) Amphid neuronal cell bodies from wild-type worms expressing the TGN marker SYN-16::dsRed with either ARL-13(Δ203–370)::GFP or ARL-13(ΔRVVP)::GFP. n; nucleus. Bar; 1 µm.  Dye-fill images of arl-13(tm2322) and wild-type worms expressing the indicating ARL-13 sequence variant. Bars; 5 µm. (D) Amphid images of worms expressing CHE-13/IFT57::mCherry and the indicated ARL-13 sequence variant. BB; basal body regions. ax; axonemes. Bar; 2 µm.Analysis of ARL-13 sequence variant localisation and function, linked to (PDF)Click here for additional data file.Figure S3A) Percentage worms exhibiting ARL-13::GFP accumulation at the periciliary membrane of phasmid neurons in the indicated mutant genotype. (B) Percentage worms of the indicated genotype displaying punctate ARL-13::GFP signals in the cell bodies of phasmid neurons. Images are of phasmid neurons showing ARL-13::GFP signals in the cell bodies of mks-2;nphp-4 mutants. TZ; transition zone. PCM; periciliary membrane. Bars; 2 µm. (C) Assessment of ARL-13::GFP localisation at the TZ of phasmid neurons. TZ localisations typically identifiable as a narrowing of ARL-13 signal between the ciliary axonemal and wider periciliary membrane compartment.Quantification of ARL-13 signals at the periciliary membrane, cell body and transition zone in ciliopathy and ciliogenic gene mutants, linked to (PDF)Click here for additional data file.Figure S4A) Each graph shows the PCM and ciliary FRAP curves. All curves are subtracted for background photobleaching and any very low level recovery from PCM+ ciliary bleaches (shown in B) Representative images from phasmid cilia photobleaching experiments. Brackets denote bleached regions. All images identically imaged and scaled. Bars; 2 µm.Additional ARL-13 FRAP curves (bleaches of PCM or cilium) and images, linked to shown in . Data sh(PDF)Click here for additional data file.Figure S5A) Representative FRAP images after photobleaching entire PCM+ciliary ARL-13 signals in phasmid neurons. Bar; 1 µm. (B) Representative FRAP images after photobleaching ∼40% of proximal-most ARL-13 signals in phasmid cilia. Bars; 1 µm. (C) Box and whisker (min to max) distribution plots showing the % area, length and intensity of photobleached ARL-13::GFP in the partial ciliary FRAP experiments shown in Additional ARL-13 FRAP curves (bleaches of PCM+cilium) and quantitative analysis of bleached region in partial ciliary FRAP experiments, linked to (PDF)Click here for additional data file.Figure S6Strep/Flag (SF)-tagged ARL13B localises to hTERT-RPE1 primary cilia, linked to (PDF)Click here for additional data file.Figure S7Investigation of ARL13B complexes using SILAC-based quantitative affinity proteomics, linked to (PDF)Click here for additional data file.Movie S1Time-lapse video recording  of ARL-13::GFP processive movement along an amphid channel cilium of a wild-type worm (L1 stage) expressing ARL-13::GFP. Video captured at 3 frames per second.(AVI)Click here for additional data file.Movie S2che-3 images, ARL-13::GFP signals it the PQR cilium are also evident. TZ; transition zone. Asterisk; periciliary membrane accumulation. Bars; 2 µm.Rotating Z-stacked and volume rendered images of PHA/B cilia in adult worms of the indicated genotype expressing ARL-13::GFP. In N2 and (AVI)Click here for additional data file.Movie S3Rotating Z-stacked image of entire PHA/B neurons showing ARL-13::GFP localisation in wild-type (N2) worms. Bar; 10 µm.(AVI)Click here for additional data file.Movie S4che-2/IFT80 mutant. Bar; 10 µm.Rotating Z-stacked image of entire PHA/B neurons showing ARL-13::GFP localisation in a (AVI)Click here for additional data file.Movie S5che-11/IFT140 mutant. Bar; 10 µm.Rotating Z-stacked image of entire PHA/B neurons showing ARL-13::GFP localisation in a (AVI)Click here for additional data file.Movie S6che-3/DHC mutant. Bar; 10 µm.Rotating Z-stacked image of entire PHA/B neurons showing ARL-13::GFP localisation in a (AVI)Click here for additional data file.Movie S7osm-12/BBS7 mutant. Bar; 10 µm.Rotating Z-stacked image of entire PHA/B neurons showing ARL-13::GFP localisation in an (AVI)Click here for additional data file.Movie S8mks-5/RPGRIP1L mutant. Bar; 10 µm.Rotating Z-stacked image of entire PHA/B neurons showing ARL-13::GFP localisation in an (AVI)Click here for additional data file.Movie S9FRAP movies following bleaching of periciliary signals (denoted by brackets) in worms of the indicated genotype expressing ARL-13::GFP. All movies taken from phasmid cilia. Bars; 2 µm.(AVI)Click here for additional data file.Movie S10FRAP movies following bleaching of ciliary signals (denoted by brackets) in worms of the indicated genotype expressing ARL-13::GFP. All movies taken from phasmid cilia. Bars; 2 µm.(AVI)Click here for additional data file.Movie S11FRAP movies following bleaching of periciliary + ciliary signals in worms of the indicated genotype expressing ARL-13::GFP. All movies taken from phasmid cilia. Bars; 2 µm.(AVI)Click here for additional data file.Movie S12dyf-6/IFT46 mutants vs the WT worms. Bars; 1.5 µm.FRAP movies following bleaching of proximal or distal regions of ARL-13::GFP middle segment signals. Note the faster recovery in (AVI)Click here for additional data file.Table S1Statistical analysis of periciliary/ciliary localisation data, linked to (XLSX)Click here for additional data file.Table S2Human ARL13B complex proteins identified using tandem affinity proteomics, linked to (XLSX)Click here for additional data file.Table S3Columns D, E; four experiment averages showing protein enrichment ratios and p-values from Strep/Flag (SF)-tagged ARL13B(WT) purifications quantified against purifications of the SF tag alone. Columns F–I; peptide enrichment scores from the 4 individual experiments. n.def. (not defined).Human ARL13B complex proteins identified using SILAC-based quantitative affinity proteomics, linked to (XLSX)Click here for additional data file.