Mechanism of transport of IFT particles in C. elegans cilia by the concerted action of kinesin-II and OSM-3 motors

The assembly and function of cilia on Caenorhabditis elegans neurons depends on the action of two kinesin-2 motors, heterotrimeric kinesin-II and homodimeric OSM-3-kinesin, which cooperate to move the same intraflagellar transport (IFT) particles along microtubule (MT) doublets. Using competitive in vitro MT gliding assays, we show that purified kinesin-II and OSM-3 cooperate to generate movement similar to that seen along the cilium in the absence of any additional regulatory factors. Quantitative modeling suggests that this could reflect an alternating action mechanism, in which the motors take turns to move along MTs, or a mechanical competition, in which the motors function in a concerted fashion to move along MTs with the slow motor exerting drag on the fast motor and vice versa. In vivo transport assays performed in Bardet-Biedl syndrome (BBS) protein and IFT motor mutants favor a mechanical competition model for motor coordination in which the IFT motors exert a BBS protein-dependent tension on IFT particles, which controls the IFT pathway that builds the cilium foundation.     

Immunoaffinity-based mass spectrometric characterization of the FMRFamide-related peptide family in the pericardial organ of Cancer borealis

The tetrapeptide, FMRFamide, was first discovered in 1977 in the molluscan nervous system and was found to affect the contractile force of molluscan cardiac muscle and other muscles [1]. Since then, numerous FMRFamide-related peptides (FaRPs) have been reported in both invertebrate and vertebrate species [2], [3], [4], [5], [6], [7], [8] and [9]. We have previously reported the detection and identification of numerous FaRPs in Cancer borealis pericardial organs (POs), one of the major neurosecretory structures in the crustaceans [2] and [3]. Here, we have developed two immunoaffinity-based methods, immunoprecipitation (IP) and immuno-dot blot screening assay, for the enrichment of FaRPs in C. borealis POs. A combined mass spectrometry (MS)-based approach involving both matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) and nanoscale liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-QTOF MS/MS) is used for a more comprehensive characterization of the FaRP family by utilizing high mass accuracy measurement and efficient peptide sequencing. Overall, 17 FMRFamide-related peptides were identified using these two complementary immuno-based approaches. Among them, three novel peptides were reported for the first time in this study.     

Tyrosines in the Kinesin-5 Head Domain Are Necessary for Phosphorylation by Wee1 and for Mitotic Spindle Integrity

Mitotic spindle assembly and maintenance relies on kinesin-5 motors that act as bipolar homotetramers to crosslink microtubules [1], [2], [3], [4] and [5]. Kinesin-5 motors have been the subject of extensive structure-function analysis [5], but the regulation of their activity in the context of mitotic progression remains less well understood [2]. We report here that Drosophila kinesin-5 (KLP61F) is regulated by Drosophila Wee1 (dWee1). Wee1 tyrosine kinases are known to regulate mitotic entry via inhibitory phosphorylation of Cdk1 [6], [7], [8], [9] and [10]. Recently, we showed that dWee1 also plays a role in mitotic spindle positioning through γ-tubulin and spindle fidelity through an unknown mechanism [11]. Here, we investigated whether a KLP61F-dWee1 interaction could explain the latter role of dWee1. We found that dWee1 phosphorylates KLP61F in vitro on three tyrosines within the head domain, the catalytic region that mediates movement along microtubules. In vivo, KLP61F with tyrosine→phenylalanine mutations fails to complement a klp61f mutant and dominantly induces spindle defects similar to ones seen in dwee1 mutants. We propose that phosphorylation of the KLP61F catalytic domain by dWee1 is important for the motor's function. This study identifies a second substrate for a Wee1 kinase and provides evidence for phosphoregulation of a kinesin in the head domain.     

Deceptively simple but simply deceptive - Caenorhabditis elegans lifespan studies: Considerations for aging and antioxidant effects

The nematode worm Caenorhabditis elegans (C. elegans) is increasingly popular as a model organism for aging studies as well as for testing antioxidants and other compounds for effects on longevity. However, results in the literature are sometimes confusing and contradictory [1], [2], [3] and [4]. This review introduces C. elegans as a model organism, discusses aspects that make it attractive for aging and antioxidant research, and addresses some problems and potential artifacts.     

Arp2/3 phosphorylation kickstarts cells

The kinesin-13 motor protein family members drive the removal of tubulin from microtubules (MTs) to promote MT turnover. A point mutation of the kinesin-13 family member mitotic centromere-associated kinesin/Kif2C (E491A) isolates the tubulin-removal conformation of the motor, and appears distinct from all previously described kinesin-13 conformations derived from nucleotide analogues. The E491A mutant removes tubulin dimers from stabilized MTs stoichiometrically in adenosine triphosphate (ATP) but is unable to efficiently release from detached tubulin dimers to recycle catalytically. Only in adenosine diphosphate (ADP) can the mutant catalytically remove tubulin dimers from stabilized MTs because the affinity of the mutant for detached tubulin dimers in ADP is low relative to lattice-bound tubulin. Thus, the motor can regenerate for further cycles of disassembly. Using the mutant, we show that release of tubulin by kinesin-13 motors occurs at the transition state for ATP hydrolysis, which illustrates a significant divergence in their coupling to ATP turnover relative to motile kinesins.     

In vivo differential effects of fasting, re-feeding, insulin and insulin stimulation time course on insulin signaling pathway components in peripheral tissues

Background: Components of the insulin receptor signaling pathway are probably some of the best studied ones. Even though methods for studying these components are well established, the in vivo effects of different fasting regimens, and the time course of insulin receptor phosphorylation and that of its downstream components in insulin-sensitive peripheral tissues have not been analyzed in detail. Rationale: When assessing insulin signaling, it may be beneficial to drive insulin levels as low as possible by performing an overnight fast before injecting a supra-physiological dose of insulin. Recent studies have shown however that 5 or 6 h fast in mice is sufficient to assess physiological responses to insulin and/or glucose in glucose tolerance tests [1], insulin tolerance tests [2], [3] and [4] and euglycemic hyperinsulinemic clamp studies [5] and [6]. Moreover, mice are nocturnal feeders, with ∼70% of their daily caloric intake occurring during the dark cycle [5], and their metabolic rate is much higher than humans. Therefore, an overnight fast in mice is closer to starvation than just food withdrawal. Thus our aim was to assess insulin signaling components from the insulin receptor to downstream targets IRS1, Akt/PKB, GSK3, Erk1/2 and ribosomal protein S6 in muscle, liver and adipose tissue in 5 h versus 16 h (overnight) fasted mice, and the time course (0-30 min) of these phosphorylation events. We also assessed whether re-feeding under 5 h and 16 h fasting conditions was a more robust stimulus than insulin alone. Conclusions: Our study determines that a short food withdrawal from mice, for a period of 5 h, results in a similar insulin-stimulated response in phosphorylation events as the long overnight fast, presenting a more physiological experimental set up. We also demonstrate that in vivo, insulin-stimulated phosphorylation of its signaling components is different between different peripheral tissues, and depending on the tissue(s) and protein(s) of interest, an appropriate time course should be chosen.     

A Highlights from MBoC Selection: Regulation of microtubule-based transport by MAP4

Microtubule (MT)-based transport of organelles driven by the opposing MT motors kinesins and dynein is tightly regulated in cells, but the underlying molecular mechanisms remain largely unknown. Here we tested the regulation of MT transport by the ubiquitous protein MAP4 using Xenopus melanophores as an experimental system. In these cells, pigment granules (melanosomes) move along MTs to the cell center (aggregation) or to the periphery (dispersion) by means of cytoplasmic dynein and kinesin-2, respectively. We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs. Overexpression of XMAP4 inhibited pigment aggregation by shortening dynein-dependent MT runs of melanosomes, whereas removal of XMAP4 from MTs reduced the length of kinesin-2–dependent runs and suppressed pigment dispersion. We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2–based movement. Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2–dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect.     

Impact-Free Measurement of Microtubule Rotations on Kinesin and Cytoplasmic-Dynein Coated Surfaces

Knowledge about the three-dimensional stepping of motor proteins on the surface of microtubules (MTs) as well as the torsional components in their power strokes can be inferred from longitudinal MT rotations in gliding motility assays. In previous studies, optical detection of these rotations relied on the tracking of rather large optical probes present on the outer MT surface. However, these probes may act as obstacles for motor stepping and may prevent the unhindered rotation of the gliding MTs. To overcome these limitations, we devised a novel, impact-free method to detect MT rotations based on fluorescent speckles within the MT structure in combination with fluorescence-interference contrast microscopy. We (i) confirmed the rotational pitches of MTs gliding on surfaces coated by kinesin-1 and kinesin-8 motors, (ii) demonstrated the superiority of our method over previous approaches on kinesin-8 coated surfaces at low ATP concentration, and (iii) identified MT rotations driven by mammalian cytoplasmic dynein, indicating that during collective motion cytoplasmic dynein side-steps with a bias in one direction. Our novel method is easy to implement on any state-of-the-art fluorescence microscope and allows for high-throughput experiments.     

Dissecting the molecular mechanisms of intraflagellar transport in chlamydomonas

BACKGROUND: The assembly and maintenance of eukaryotic cilia and flagella are mediated by intraflagellar transport (IFT), a bidirectional microtubule (MT)-based transport system. The IFT system consists of anterograde (kinesin-2) and retrograde (cDynein1b) motor complexes and IFT particles comprising two complexes, A and B. In the current model for IFT, kinesin-2 carries cDynein1b, IFT particles, and axonemal precursors from the flagellar base to the tip, and cDynein1b transports kinesin-2, IFT particles, and axonemal turnover products from the tip back to the base. Most of the components of the IFT system have been identified and characterized, but the mechanisms by which these different components are coordinated and regulated at the flagellar base and tip are unclear. RESULTS: Using a variety of Chlamydomonas mutants, we confirm that cDynein1b requires kinesin-2 for transport toward the tip and show that during retrograde IFT, kinesin-2 can exit the flagella independent of the cDynein1b light intermediate chain (LIC) and IFT particles. Furthermore, using biochemical approaches, we find that IFT complex B can associate with cDynein1b independent of complex A and cDynein1b LIC. Finally, using electron microscopy, we show that the IFT tip turnaround point most likely is localized distal to the plus end of the outer-doublet B MTs. CONCLUSION: Our results support a model for IFT in which tip turnaround involves (1) dissociation of IFT complexes A and B and release of inactive cDynein1b from complex B, (2) binding of complex A to active cDynein1b, and (3) reassociation of complex B with A prior to retrograde IFT.     

Mechanism for Anaphase B: Evaluation of “Slide-and-Cluster” versus “Slide-and-Flux-or-Elongate” Models

Elongation of the mitotic spindle during anaphase B contributes to chromosome segregation in many cells. Here, we quantitatively test the ability of two models for spindle length control to describe the dynamics of anaphase B spindle elongation using experimental data from Drosophila embryos. In the slide-and-flux-or-elongate (SAFE) model, kinesin-5 motors persistently slide apart antiparallel interpolar microtubules (ipMTs). During pre-anaphase B, this outward sliding of ipMTs is balanced by depolymerization of their minus ends at the poles, producing poleward flux, while the spindle maintains a constant length. Following cyclin B degradation, ipMT depolymerization ceases so the sliding ipMTs can push the poles apart. The competing slide-and-cluster (SAC) model proposes that MTs nucleated at the equator are slid outward by the cooperative actions of the bipolar kinesin-5 and a minus-end-directed motor, which then pulls the sliding MTs inward and clusters them at the poles. In assessing both models, we assume that kinesin-5 preferentially cross-links and slides apart antiparallel MTs while the MT plus ends exhibit dynamic instability. However, in the SAC model, minus-end-directed motors bind the minus ends of MTs as cargo and transport them poleward along adjacent, parallel MT tracks, whereas in the SAFE model, all MT minus ends that reach the pole are depolymerized by kinesin-13. Remarkably, the results show that within a narrow range of MT dynamic instability parameters, both models can reproduce the steady-state length and dynamics of pre-anaphase B spindles and the rate of anaphase B spindle elongation. However, only the SAFE model reproduces the change in MT dynamics observed experimentally at anaphase B onset. Thus, although both models explain many features of anaphase B in this system, our quantitative evaluation of experimental data regarding several different aspects of spindle dynamics suggests that the SAFE model provides a better fit.     

Kif18A uses a microtubule binding site in the tail for plus-end localization and spindle length regulation

The mitotic spindle is a macromolecular structure utilized to properly align and segregate sister chromatids to two daughter cells. During mitosis, the spindle maintains a constant length, even though the spindle microtubules (MTs) are constantly undergoing polymerization and depolymerization [1]. Members of the kinesin-8 family are important for the regulation of spindle length and for chromosome positioning [2-9]. Kinesin-8 proteins are length-specific, plus-end-directed motors that are proposed to be either MT depolymerases [3, 4, 8, 10, 11] or MT capping proteins [12]. How Kif18A uses its destabilization activity to control spindle morphology is not known. We found that Kif18A controls spindle length independently of its role in chromosome positioning. The ability of Kif18A to control spindle length is mediated by an ATP-independent MT binding site at the C-terminal end of the Kif18A tail that has a strong affinity for MTs in?vitro and in cells. We used computational modeling to ask how modulating the motility or binding properties of Kif18A would affect its activity. Our modeling predicts that both fast motility and a low off rate from the MT end are important for Kif18A function. In addition, our studies provide new insight into how depolymerizing and capping enzymes can lead to MT destabilization.     

Deletion of the Tail Domain of the Kinesin-5 Cin8 Affects Its Directionality

The bipolar kinesin-5 motors are one of the major players that govern mitotic spindle dynamics. Their bipolar structure enables them to cross-link and slide apart antiparallel microtubules (MTs) emanating from the opposing spindle poles. The budding yeast kinesin-5 Cin8 was shown to switch from fast minus-end- to slow plus-end-directed motility upon binding between antiparallel MTs. This unexpected finding revealed a new dimension of cellular control of transport, the mechanism of which is unknown. Here we have examined the role of the C-terminal tail domain of Cin8 in regulating directionality. We first constructed a stable dimeric Cin8/kinesin-1 chimera (Cin8Kin), consisting of head and neck linker of Cin8 fused to the stalk of kinesin-1. As a single dimeric motor, Cin8Kin switched frequently between plus and minus directionality along single MTs, demonstrating that the Cin8 head domains are inherently bidirectional, but control over directionality was lost. We next examined the activity of a tetrameric Cin8 lacking only the tail domains (Cin8Δtail). In contrast to wild-type Cin8, the motility of single molecules of Cin8Δtail in high ionic strength was slow and bidirectional, with almost no directionality switches. Cin8Δtail showed only a weak ability to cross-link MTs in vitro. In vivo, Cin8Δtail exhibited bias toward the plus-end of the MTs and was unable to support viability of cells as the sole kinesin-5 motor. We conclude that the tail of Cin8 is not necessary for bidirectional processive motion, but is controlling the switch between plus- and minus-end-directed motility.     

IFT54 directly interacts with kinesin‐II and IFT dynein to regulate anterograde intraflagellar transport

The intraflagellar transport (IFT) machinery consists of the anterograde motor kinesin‐II, the retrograde motor IFT dynein, and the IFT‐A and ‐B complexes. However, the interaction among IFT motors and IFT complexes during IFT remains elusive. Here, we show that the IFT‐B protein IFT54 interacts with both kinesin‐II and IFT dynein and regulates anterograde IFT. Deletion of residues 342–356 of Chlamydomonas IFT54 resulted in diminished anterograde traffic of IFT and accumulation of IFT motors and complexes in the proximal region of cilia. IFT54 directly interacted with kinesin‐II and this interaction was strengthened for the IFT54^Δ342–356 mutant in vitro and in vivo. The deletion of residues 261–275 of IFT54 reduced ciliary entry and anterograde traffic of IFT dynein with accumulation of IFT complexes near the ciliary tip. IFT54 directly interacted with IFT dynein subunit D1bLIC, and deletion of residues 261–275 reduced this interaction. The interactions between IFT54 and the IFT motors were also observed in mammalian cells. Our data indicate a central role for IFT54 in binding the IFT motors during anterograde IFT.     

Microtubule-associated proteins and motors required for ectopic microtubule array formation in Saccharomyces cerevisiae

The mitotic spindle is resilient to perturbation due to the concerted, and sometimes redundant, action of motors and microtubule-associated proteins. Here, we utilize an inducible ectopic microtubule nucleation site in the nucleus of Saccharomyces cerevisiae to study three necessary steps in the formation of a bipolar array: the recruitment of the γ-tubulin complex, nucleation and elongation of microtubules (MTs), and the organization of MTs relative to each other. This novel tool, an Spc110 chimera, reveals previously unreported roles of the microtubule-associated proteins Stu2, Bim1, and Bik1, and the motors Vik1 and Kip3. We report that Stu2 and Bim1 are required for nucleation and that Bik1 and Kip3 promote nucleation at the ectopic site. Stu2, Bim1, and Kip3 join their homologs XMAP215, EB1 and kinesin-8 as promoters of microtubule nucleation, while Bik1 promotes MT nucleation indirectly via its role in SPB positioning. Furthermore, we find that the nucleation activity of Stu2 in vivo correlates with its polymerase activity in vitro. Finally, we provide the first evidence that Vik1, a subunit of Kar3/Vik1 kinesin-14, promotes microtubule minus end focusing at the ectopic site.     

Kinesin-3 KLP-6 regulates intraflagellar transport in male-specific cilia of Caenorhabditis elegans

Cilia are cellular sensory organelles whose integrity of structure and function are important to human health. All cilia are assembled and maintained by kinesin-2 motors in a process termed intraflagellar transport (IFT), but they exhibit great variety of morphology and function. This diversity is proposed to be conferred by cell-specific modulation of the core IFT by additional factors, but examples of such IFT modulators are limited. Here we demonstrate that the cell-specific kinesin-3 KLP-6 acts as a modulator of both IFT dynamics and length in the cephalic male (CEM) cilia of Caenorhabditis elegans. Live imaging of GFP-tagged kinesins in CEM cilia shows partial uncoupling of the IFT motors of the kinesin-2 family, kinesin-II and OSM-3/KIF17, with a portion of OSM-3 moving independently of the IFT complex. KLP-6 moves independently of the kinesin-2 motors and acts to reduce the velocity of OSM-3 and IFT. Additionally, kinesin-II mutants display a novel CEM cilia elongation phenotype that is partially dependent on OSM-3 and KLP-6. Our observations illustrate modulation of the general kinesin-2-driven IFT process by a cell-specific kinesin-3 in cilia of C. elegans male neurons.     

Crosslinkers and motors organize dynamic microtubules to form stable bipolar arrays in fission yeast

Microtubule (MT) nucleation not only occurs from centrosomes, but also in large part from dispersed nucleation sites. The subsequent sorting of short MTs into networks like the mitotic spindle requires molecular motors that laterally slide overlapping MTs and bundling proteins that statically connect MTs. How bundling proteins interfere with MT sliding is unclear. In bipolar MT bundles in fission yeast, we found that the bundler ase1p localized all along the length of antiparallel MTs, whereas the motor klp2p (kinesin-14) accumulated only at MT plus ends. Consequently, sliding forces could only overcome resistant bundling forces for short, newly nucleated MTs, which were transported to their correct position within bundles. Ase1p thus regulated sliding forces based on polarity and overlap length, and computer simulations showed these mechanisms to be sufficient to generate stable bipolar bundles. By combining motor and bundling proteins, cells can thus dynamically organize stable regions of overlap between cytoskeletal filaments.     

The microtubule cross-linker Feo controls the midzone stability,motor composition,and elongation of the anaphase B spindle in Drosophila embryos

Chromosome segregation during anaphase depends on chromosome-to-pole motility and pole-to-pole separation. We propose that in Drosophila embryos, the latter process (anaphase B) depends on a persistent kinesin-5–generated interpolar (ip) microtubule (MT) sliding filament mechanism that “engages” to push apart the spindle poles when poleward flux is turned off. Here we investigated the contribution of the midzonal, antiparallel MT-cross-linking nonmotor MAP, Feo, to this “slide-and-flux-or-elongate” mechanism. Whereas Feo homologues in other systems enhance the midzone localization of the MT-MT cross-linking motors kinesin-4, -5 and -6, the midzone localization of these motors is respectively enhanced, reduced, and unaffected by Feo. Strikingly, kinesin-5 localizes all along ipMTs of the anaphase B spindle in the presence of Feo, including at the midzone, but the antibody-induced dissociation of Feo increases kinesin-5 association with the midzone, which becomes abnormally narrow, leading to impaired anaphase B and incomplete chromosome segregation. Thus, although Feo and kinesin-5 both preferentially cross-link MTs into antiparallel polarity patterns, kinesin-5 cannot substitute for loss of Feo function. We propose that Feo controls the organization, stability, and motor composition of antiparallel ipMTs at the midzone, thereby facilitating the kinesin-5–driven sliding filament mechanism underlying proper anaphase B spindle elongation and chromosome segregation.     

Microtubule Depolymerization by the Kinesin-8 Motor Kip3p: A Mathematical Model

Proteins from the kinesin-8 family promote microtubule (MT) depolymerization, a process thought to be important for the control of microtubule length in living cells. In addition to this MT shortening activity, kinesin 8s are motors that show plus-end directed motility on MTs. Here we describe a simple model that incorporates directional motion and destabilization of the MT plus-end by kinesin 8. Our model quantitatively reproduces the key features of length-versus-time traces for stabilized MTs in the presence of purified kinesin 8, including length-dependent depolymerization. Comparison of model predictions with experiments suggests that kinesin 8 depolymerizes processively, i.e., one motor can remove multiple tubulin dimers from a stabilized MT. Fluctuations in MT length as a function of time are related to depolymerization processivity. We have also determined the parameter regime in which the rate of MT depolymerization is length dependent: length-dependent depolymerization occurs only when MTs are sufficiently short; this crossover is sensitive to the bulk motor concentration.     

A non-motor microtubule binding site is essential for the high processivity and mitotic function of kinesin-8 Kif18A

Background

Members of the kinesin-8 subfamily are plus end-directed molecular motors that accumulate at the plus-ends of kinetochore-microtubules (kt-MTs) where they regulate MT dynamics. Loss of vertebrate kinesin-8 function induces hyperstable MTs and elongated mitotic spindles accompanied by severe chromosome congression defects. It has been reported that the motility of human kinesin-8, Kif18A, is required for its accumulation at the plus tips of kt-MTs.

Methodology/Findings

Here, we investigate how Kif18A localizes to the plus-ends of kt-MTs. We find that Kif18A lacking its C-terminus does not accumulate on the tips of kt-MTs and fails to fulfill its mitotic function. In vitro studies reveal that Kif18A possesses a non-motor MT binding site located within its C-proximal 121 residues. Using single molecule measurements we find that Kif18A is a highly processive motor and, furthermore, that the C-terminal tail is essential for the high processivity of Kif18A.

Conclusion/Significance

These results show that Kif18A like its yeast orthologue is a highly processive motor. The ability of Kif18A to walk on MTs for a long distance without dissociating depends on a non-motor MT binding site located at the C-terminus of Kif18A. This C-proximal tail of Kif18A is essential for its plus-end accumulation and mitotic function. These findings advance our understanding of how Kif18A accumulates at the tips of kt-MTs to fulfill its function in mitosis.     

Primary Cilia Are Not Required for Normal Canonical Wnt Signaling in the Mouse Embryo

Background

Sonic hedgehog (Shh) signaling in the mouse requires the microtubule-based organelle, the primary cilium. The primary cilium is assembled and maintained through the process of intraflagellar transport (IFT) and the response to Shh is blocked in mouse mutants that lack proteins required for IFT. Although the phenotypes of mouse IFT mutants do not overlap with phenotypes of known Wnt pathway mutants, recent studies report data suggesting that the primary cilium modulates responses to Wnt signals.

Methodology/Principal Findings

We therefore carried out a systematic analysis of canonical Wnt signaling in mutant embryos and cells that lack primary cilia because of loss of the anterograde IFT kinesin-II motor (Kif3a) or IFT complex B proteins (Ift172 or Ift88). We also analyzed mutant embryos with abnormal primary cilia due to defects in retrograde IFT (Dync2h1). The mouse IFT mutants express the canonical Wnt target Axin2 and activate a transgenic canonical Wnt reporter, BAT-gal, in the normal spatial pattern and to the same quantitative level as wild type littermates. Similarly, mouse embryonic fibroblasts (MEFs) derived from IFT mutants respond normally to added Wnt3a. The switch from canonical to non-canonical Wnt also appears normal in IFT mutant MEFs, as both wild-type and mutant cells do not activate the canonical Wnt reporter in the presence of both Wnt3a and Wnt5a.

Conclusions

We conclude that loss of primary cilia or defects in retrograde IFT do not affect the response of the midgestation embryo or embryo-derived fibroblasts to Wnt ligands.