Radial intercalation of ciliated cells during Xenopus skin development

Cells with motile cilia cover the skin of Xenopus tadpoles in a characteristic spacing pattern. This pattern arises during early development when cells within the inner layer of ectoderm are selected out by Notch to form ciliated cell precursors (CCPs) that then radially intercalate into the outer epithelial cell layer to form ciliated cells. When Notch is inhibited and CCPs are overproduced, radial intercalation becomes limiting and the spacing of ciliated cells is maintained. To determine why this is the case, we used confocal microscopy to image intercalating cells labeled using transplantation and a transgenic approach that labels CCPs with green fluorescent protein (GFP). Our results indicate that inner cells intercalate by first wedging between the basal surface of the outer epithelium but only insert apically at the vertices where multiple outer cells make contact. When overproduced, more CCPs are able to wedge basally, but apical insertion becomes limiting. We propose that limitations imposed by the outer layer, along with restrictions on the apical insertion of CCPs, determine their pattern of radial intercalation.     

Misshapen decreases integrin levels to promote epithelial motility and planar polarity in Drosophila

Complex organ shapes arise from the coordinate actions of individual cells. The Drosophila egg chamber is an organ-like structure that lengthens along its anterior–posterior axis as it grows. This morphogenesis depends on an unusual form of planar polarity in the organ’s outer epithelial layer, the follicle cells. Interestingly, this epithelium also undergoes a directed migration that causes the egg chamber to rotate around its anterior–posterior axis. However, the functional relationship between planar polarity and migration in this tissue is unknown. We have previously reported that mutations in the Misshapen kinase disrupt follicle cell planar polarity. Here we show that Misshapen’s primary role in this system is to promote individual cell motility. Misshapen decreases integrin levels at the basal surface, which may facilitate detachment of each cell’s trailing edge. These data provide mechanistic insight into Misshapen’s conserved role in cell migration and suggest that follicle cell planar polarity may be an emergent property of individual cell migratory behaviors within the epithelium.     

Multicellular computer simulation of morphogenesis: blastocoel roof thinning and matrix assembly in Xenopus laevis

In the blastocoel roof (BCR) of the Xenopus laevis embryo, epibolic movements are driven by the radial intercalation of deep cell layers and the coordinate spreading of the overlying superficial cell layer. Thinning of the lateral margins of the BCR by radial intercalation requires fibronectin (FN), which is produced and assembled into fibrils by the inner deep cell layer of the BCR. A cellular automata (CA) computer model was developed to analyze the spatial and temporal movements of BCR cells during epiboly. Simulation parameters were defined based on published data and independent results detailing initial tissue geometry, cell numbers, cell intercalation rates, and migration rates. Hypotheses regarding differential cell adhesion and FN assembly were also considered in setting system parameters. A 2-dimensional model simulation was developed that predicts BCR thinning time of 4.8 h, which closely approximates the time required for the completion of gastrulation in vivo. Additionally, the model predicts a temporal increase in FN matrix assembly that parallels fibrillogenesis in the embryo. The model is capable of independent predictions of cell rearrangements during epiboly, and here was used to predict successfully the lateral dispersion of a patch of cells implanted in the BCR, and increased assembly of FN matrix following inhibition of radial intercalation by N-cadherin over-expression.     

Par6b Regulates the Dynamics of Apicobasal Polarity during Development of the Stratified Xenopus Epidermis

During early vertebrate development, epithelial cells establish and maintain apicobasal polarity, failure of which can cause developmental defects or cancer metastasis. This process has been mostly studied in simple epithelia that have only one layer of cells, but is poorly understood in stratified epithelia. In this paper we address the role of the polarity protein Partitioning defective-6 homolog beta (Par6b) in the developing stratified epidermis of Xenopus laevis. At the blastula stage, animal blastomeres divide perpendicularly to the apicobasal axis to generate partially polarized superficial cells and non-polarized deep cells. Both cell populations modify their apicobasal polarity during the gastrula stage, before differentiating into the superficial and deep layers of epidermis. Early differentiation of the epidermis is normal in Par6b-depleted embryos; however, epidermal cells dissociate and detach from embryos at the tailbud stage. Par6b-depleted epidermal cells exhibit a significant reduction in basolaterally localized E-cadherin. Examination of the apical marker Crumbs homolog 3 (Crb3) and the basolateral marker Lethal giant larvae 2 (Lgl2) after Par6b depletion reveals that Par6b cell-autonomously regulates the dynamics of apicobasal polarity in both superficial and deep epidermal layers. Par6b is required to maintain the “basolateral” state in both epidermal layers, which explains the reduction of basolateral adhesion complexes and epidermal cells shedding.     

Cdc42 and Par proteins stabilize dynamic adherens junctions in the Drosophila neuroectoderm through regulation of apical endocytosis

Cell rearrangements require dynamic changes in cell–cell contacts to maintain tissue integrity. We investigated the function of Cdc42 in maintaining adherens junctions (AJs) and apical polarity in the Drosophila melanogaster neuroectodermal epithelium. About one third of cells exit the epithelium through ingression and become neuroblasts. Cdc42-compromised embryos lost AJs in the neuroectoderm during neuroblast ingression. In contrast, when neuroblast formation was suppressed, AJs were maintained despite the loss of Cdc42 function. Loss of Cdc42 function caused an increase in the endocytotic uptake of apical proteins, including apical polarity factors such as Crumbs, which are required for AJ stability. In addition, Cdc42 has a second function in regulating endocytotic trafficking, as it is required for the progression of apical cargo from the early to the late endosome. The Par complex acts as an effector for Cdc42 in controlling the endocytosis of apical proteins. This study reveals functional interactions between apical polarity proteins and endocytosis that are critical for stabilizing dynamic basolateral AJs.     

Rho GTPase controls invagination and cohesive migration of the Drosophila salivary gland through Crumbs and Rho-kinase

Coordinated cell movements shape simple epithelia into functional tissues and organs during embryogenesis. Regulators and effectors of the small GTPase Rho have been shown to be essential for epithelial morphogenesis in cell culture; however, the mechanism by which Rho GTPase and its downstream effectors control coordinated movement of epithelia in a developing tissue or organ is largely unknown. Here, we show that Rho1 GTPase activity is required for the invagination of Drosophila embryonic salivary gland epithelia and for directed migration of the internalized gland. We demonstrate that the absence of zygotic function of Rho1 results in the selective loss of the apical proteins, Crumbs (Crb), Drosophila atypical PKC and Stardust during gland invagination and that this is partially due to reduced crb RNA levels and apical localization. In parallel to regulation of crb RNA and protein, Rho1 activity also signals through Rho-kinase (Rok) to induce apical constriction and cell shape change during invagination. After invagination, Rho-Rok signaling is required again for the coordinated contraction and dorsal migration of the proximal half of the gland. We also show that Rho1 activity is required for proper development of the circular visceral mesoderm upon which the gland migrates. Our genetic and live-imaging analyses provide novel evidence that the proximal gland cells play an essential and active role in salivary gland migration that propels the entire gland to turn and migrate posteriorly.     

Eph regulates dorsoventral asymmetry of the notochord plate and convergent extension-mediated notochord formation

Background

The notochord is a signaling center required for the patterning of the vertebrate embryic midline, however, the molecular and cellular mechanisms involved in the formation of this essential embryonic tissue remain unclear. The urochordate Ciona intestinalis develops a simple notochord from 40 specific postmitotic mesodermal cells. The precursors intercalate mediolaterally and establish a single array of disk-shaped notochord cells along the midline. However, the role that notochord precursor polarization, particularly along the dorsoventral axis, plays in this morphogenetic process remains poorly understood.

Methodology/Principal Findings

Here we show that the notochord preferentially accumulates an apical cell polarity marker, aPKC, ventrally and a basement membrane marker, laminin, dorsally. This asymmetric accumulation of apicobasal cell polarity markers along the embryonic dorsoventral axis was sustained in notochord precursors during convergence and extension. Further, of several members of the Eph gene family implicated in cellular and tissue morphogenesis, only Ci-Eph4 was predominantly expressed in the notochord throughout cell intercalation. Introduction of a dominant-negative Ci-Eph4 to notochord precursors diminished asymmetric accumulation of apicobasal cell polarity markers, leading to defective intercalation. In contrast, misexpression of a dominant-negative mutant of a planar cell polarity gene Dishevelled preserved asymmetric accumulation of aPKC and laminin in notochord precursors, although their intercalation was incomplete.

Conclusions/Significance

Our data support a model in which in ascidian embryos Eph-dependent dorsoventral polarity of notochord precursors plays a crucial role in mediolateral cell intercalation and is required for proper notochord morphogenesis.     

The Rac-GAP Bcr is a novel regulator of the Par complex that controls cell polarity

Cell polarization is essential for many biological processes, including directed cell migration, and loss of polarity contributes to pathological conditions such as cancer. The Par complex (Par3, Par6, and PKCζ) controls cell polarity in part by recruiting the Rac-specific guanine nucleotide exchange factor T-lymphoma invasion and metastasis 1 (Tiam1) to specialized cellular sites, where Tiam1 promotes local Rac1 activation and cytoskeletal remodeling. However, the mechanisms that restrict Par-Tiam1 complex activity to the leading edge to maintain cell polarity during migration remain unclear. We identify the Rac-specific GTPase-activating protein (GAP) breakpoint cluster region protein (Bcr) as a novel regulator of the Par-Tiam1 complex. We show that Bcr interacts with members of the Par complex and inhibits both Rac1 and PKCζ signaling. Loss of Bcr results in faster, more random migration and striking polarity defects in astrocytes. These polarity defects are rescued by reducing PKCζ activity or by expressing full-length Bcr, but not an N-terminal deletion mutant or the homologous Rac-GAP, Abr, both of which fail to associate with the Par complex. These results demonstrate that Bcr is an integral member of the Par-Tiam1 complex that controls polarized cell migration by locally restricting both Rac1 and PKCζ function.     

Using optogenetics to link myosin patterns to contractile cell behaviors during convergent extension

Distinct patterns of actomyosin contractility are often associated with particular epithelial tissue shape changes during development. For example, a planar-polarized pattern of myosin II localization regulated by Rho1 signaling during Drosophila body axis elongation is thought to drive cell behaviors that contribute to convergent extension. However, it is not well understood how specific aspects of a myosin pattern influence the multiple cell behaviors, including cell intercalation, cell shape changes, and apical cell area fluctuations, that simultaneously occur during morphogenesis. Here, we developed two optogenetic tools, optoGEF and optoGAP, to activate or deactivate Rho1 signaling, respectively. We used these tools to manipulate myosin patterns at the apical side of the germband epithelium during Drosophila axis elongation and analyzed the effects on contractile cell behaviors. We show that uniform activation or inactivation of Rho1 signaling across the apical surface of the germband is sufficient to disrupt the planar-polarized pattern of myosin at cell junctions on the timescale of 3–5 min, leading to distinct changes in junctional and medial myosin patterns in optoGEF and optoGAP embryos. These two perturbations to Rho1 activity both disrupt axis elongation and cell intercalation but have distinct effects on cell area fluctuations and cell packings that are linked with changes in the medial and junctional myosin pools. These studies demonstrate that acute optogenetic perturbations to Rho1 activity are sufficient to rapidly override the endogenous planar-polarized myosin pattern in the germband during axis elongation. Moreover, our results reveal that the levels of Rho1 activity and the balance between medial and junctional myosin play key roles not only in organizing the cell rearrangements that are known to directly contribute to axis elongation but also in regulating cell area fluctuations and cell packings, which have been proposed to be important factors influencing the mechanics of tissue deformation and flow.     

Par1b Induces Asymmetric Inheritance of Plasma Membrane Domains via LGN-Dependent Mitotic Spindle Orientation in Proliferating Hepatocytes

The development and maintenance of polarized epithelial tissue requires a tightly controlled orientation of mitotic cell division relative to the apical polarity axis. Hepatocytes display a unique polarized architecture. We demonstrate that mitotic hepatocytes asymmetrically segregate their apical plasma membrane domain to the nascent daughter cells. The non-polarized nascent daughter cell can form a de novo apical domain with its new neighbor. This asymmetric segregation of apical domains is facilitated by a geometrically distinct “apicolateral” subdomain of the lateral surface present in hepatocytes. The polarity protein partitioning-defective 1/microtubule-affinity regulating kinase 2 (Par1b/MARK2) translates this positional landmark to cortical polarity by promoting the apicolateral accumulation of Leu-Gly-Asn repeat-enriched protein (LGN) and the capture of nuclear mitotic apparatus protein (NuMA)–positive astral microtubules to orientate the mitotic spindle. Proliferating hepatocytes thus display an asymmetric inheritance of their apical domains via a mechanism that involves Par1b and LGN, which we postulate serves the unique tissue architecture of the developing liver parenchyma.     

Mechanisms of convergence and extension by cell intercalation

The cells of many embryonic tissues actively narrow in one dimension (convergence) and lengthen in the perpendicular dimension (extension). Convergence and extension are ubiquitous and important tissue movements in metazoan morphogenesis. In vertebrates, the dorsal axial and paraxial mesodermal tissues, the notochordal and somitic mesoderm, converge and extend. In amphibians as well as a number of other organisms where these movements appear, they occur by mediolateral cell intercalation, the rearrangement of cells along the mediolateral axis to produce an array that is narrower in this axis and longer in the anteroposterior axis. In amphibians, mesodermal cell intercalation is driven by bipolar, mediolaterally directed protrusive activity, which appears to exert traction on adjacent cells and pulls the cells between one another. In addition, the notochordal-somitic boundary functions in convergence and extension by 'capturing' notochordal cells as they contact the boundary, thus elongating the boundary. The prospective neural tissue also actively converges and extends parallel with the mesoderm. In contrast to the mesoderm, cell intercalation in the neural plate normally occurs by monopolar protrusive activity directed medially, towards the midline notoplate-floor-plate region. In contrast, the notoplate-floor-plate region appears to converge and extend by adhering to and being towed by or perhaps migrating on the underlying notochord. Converging and extending mesoderm stiffens by a factor of three or four and exerts up to 0.6 microN force. Therefore, active, force-producing convergent extension, the mechanism of cell intercalation, requires a mechanism to actively pull cells between one another while maintaining a tissue stiffness sufficient to push with a substantial force. Based on the evidence thus far, a cell-cell traction model of intercalation is described. The essential elements of such a morphogenic machine appear to be (i) bipolar, mediolaterally orientated or monopolar, medially directed protrusive activity; (ii) this protrusive activity results in mediolaterally orientated or medially directed traction of cells on one another; (iii) tractive protrusions are confined to the ends of the cells; (iv) a mechanically stable cell cortex over the bulk of the cell body which serves as a movable substratum for the orientated or directed cell traction. The implications of this model for cell adhesion, regulation of cell motility and cell polarity, and cell and tissue biomechanics are discussed.     

Cell dynamics in fetal intestinal epithelium: implications for intestinal growth and morphogenesis

The cellular mechanisms that drive growth and remodeling of the early intestinal epithelium are poorly understood. Current dogma suggests that the murine fetal intestinal epithelium is stratified, that villi are formed by an epithelial remodeling process involving the de novo formation of apical surface at secondary lumina, and that radial intercalation of the stratified cells constitutes a major intestinal lengthening mechanism. Here, we investigate cell polarity, cell cycle dynamics and cell shape in the fetal murine intestine between E12.5 and E14.5. We show that, contrary to previous assumptions, this epithelium is pseudostratified. Furthermore, epithelial nuclei exhibit interkinetic nuclear migration, a process wherein nuclei move in concert with the cell cycle, from the basal side (where DNA is synthesized) to the apical surface (where mitosis takes place); such nuclear movements were previously misinterpreted as the radial intercalation of cells. We further demonstrate that growth of epithelial girth between E12.5 and E14.5 is driven by microtubule- and actinomyosin-dependent apicobasal elongation, rather than by progressive epithelial stratification as was previously thought. Finally, we show that the actin-binding protein Shroom3 is crucial for the maintenance of the single-layered pseudostratified epithelium. In mice lacking Shroom3, the epithelium is disorganized and temporarily stratified during villus emergence. These results favor an alternative model of intestinal morphogenesis in which the epithelium remains single layered and apicobasally polarized throughout early intestinal development.     

Mechanical Coupling between Endoderm Invagination and Axis Extension in Drosophila

How genetic programs generate cell-intrinsic forces to shape embryos is actively studied, but less so how tissue-scale physical forces impact morphogenesis. Here we address the role of the latter during axis extension, using Drosophila germband extension (GBE) as a model. We found previously that cells elongate in the anteroposterior (AP) axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. Here we further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, we mapped gastrulation movements temporally using light sheet microscopy to image whole Drosophila embryos. We found that both mesoderm and endoderm invaginations are synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. We next looked for evidence of this force in a simplified system without polarized cell intercalation, in acellular embryos. Using Particle Image Velocimetry, we identify posteriorwards Myosin II flows towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. We probed this posterior region using laser ablation and showed that tension is increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. We propose that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical interactions between tissues during morphogenesis.     

Numb

Epithelial to mesenchymal transition (EMT) is a critical event in embryogenesis and plays a fundamental role in cancer progression and metastasis1. Numb has been shown to play an important role in the proper functions of Par protein complex and in cell-cell junctions, both of which are associated with EMT. However, the role of Numb in EMT has not been fully elucidated. Recently, we showed that Numb is capable of binding to both Par3 and E-cadherin. Intriguingly, the interaction of Numb with E-cadherin or the Par protein complex is dynamically regulated by tyrosine phosphorylation induced by HGF or Src. Knockdown of Numb by shRNA in MDCK cells led to an lateral to apical translocation of E-cadherin and β-catenin, active F-actin polymerization, mis-localization of Par3 and aPKC, a decrease in cell-cell adhesion, and an increase in cell migration and proliferation. These data suggest a diverse role for Numb in regulating cell-cell adhesion, polarity and migration during EMT.     

Multi-cellular rosettes in the mouse visceral endoderm facilitate the ordered migration of anterior visceral endoderm cells

The visceral endoderm (VE) is a simple epithelium that forms the outer layer of the egg-cylinder stage mouse embryo. The anterior visceral endoderm (AVE), a specialised subset of VE cells, is responsible for specifying anterior pattern. AVE cells show a stereotypic migratory behaviour within the VE, which is responsible for correctly orientating the anterior-posterior axis. The epithelial integrity of the VE is maintained during the course of AVE migration, which takes place by intercalation of AVE and other VE cells. Though a continuous epithelial sheet, the VE is characterised by two regions of dramatically different behaviour, one showing robust cell movement and intercalation (in which the AVE migrates) and one that is static, with relatively little cell movement and mixing. Little is known about the cellular rearrangements that accommodate and influence the sustained directional movement of subsets of cells (such as the AVE) within epithelia like the VE. This study uses an interdisciplinary approach to further our understanding of cell movement in epithelia. Using both wild-type embryos as well as mutants in which AVE migration is abnormal or arrested, we show that AVE migration is specifically linked to changes in cell packing in the VE and an increase in multi-cellular rosette arrangements (five or more cells meeting at a point). To probe the role of rosettes during AVE migration, we develop a mathematical model of cell movement in the VE. To do this, we use a vertex-based model, implemented on an ellipsoidal surface to represent a realistic geometry for the mouse egg-cylinder. The potential for rosette formation is included, along with various junctional rearrangements. Simulations suggest that while rosettes are not essential for AVE migration, they are crucial for the orderliness of this migration observed in embryos. Our simulations are similar to results from transgenic embryos in which Planar Cell Polarity (PCP) signalling is disrupted. Such embryos have significantly reduced rosette numbers, altered epithelial packing, and show abnormalities in AVE migration. Our results show that the formation of multi-cellular rosettes in the mouse VE is dependent on normal PCP signalling. Taken together, our model and experimental observations suggest that rosettes in the VE epithelium do not form passively in response to AVE migration. Instead, they are a PCP-dependent arrangement of cells that acts to buffer the disequilibrium in cell packing generated in the VE by AVE migration, enabling AVE cells to migrate in an orderly manner.     

Dynamics of the basement membrane in invasive epithelial clusters in Drosophila

The basement membrane (BM) represents a barrier to cell migration, which has to be degraded to promote invasion. However, the role and behaviour of the BM during the development of pre-invasive cells is only poorly understood. Drosophila border cells (BCs) provide an attractive genetic model in which to study the cellular mechanisms underlying the migration of mixed cohorts of epithelial cells. BCs are made of two different epithelial cell types appearing sequentially during oogenesis: the polar cells and the outer BCs. Here, we show that the pre-invasive polar cells undergo an unusual and asymmetrical apical capping with major basement membrane proteins, including the two Drosophila Collagen IV alpha chains, Laminin A and Perlecan. Capping of polar cells proceeds through a novel, basal-to-apical transcytosis mechanism that involves the small GTPase Drab5. Apical capping is transient and is followed by rapid shedding prior to the initiation of BC migration, suggesting that the apical cap blocks migration. Consistently, non-migratory polar cells remain capped. We further show that JAK/STAT signalling and recruitment of outer BCs are required for correct shedding and migration. The dynamics of the BM represents a marker of migratory BC, revealing a novel developmentally regulated behaviour of BM coupled to epithelial cell invasiveness.     

Oscillatory behaviors and hierarchical assembly of contractile structures in intercalating cells

Fluctuations in the size of the apical cell surface have been associated with apical constriction and tissue invagination. However, it is currently not known if apical oscillatory behaviors are a unique property of constricting cells or if they constitute a universal feature of the force balance between cells in multicellular tissues. Here, we set out to determine whether oscillatory cell behaviors occur in parallel with cell intercalation during the morphogenetic process of axis elongation in the Drosophila embryo. We applied multi-color, time-lapse imaging of living embryos and SIESTA, an integrated tool for automated and semi-automated cell segmentation, tracking, and analysis of image sequences. Using SIESTA, we identified cycles of contraction and expansion of the apical surface in intercalating cells and characterized them at the molecular, cellular, and tissue scales. We demonstrate that apical oscillations are anisotropic, and this anisotropy depends on the presence of intact cell-cell junctions and spatial cues provided by the anterior-posterior patterning system. Oscillatory cell behaviors during axis elongation are associated with the hierarchical assembly and disassembly of contractile actomyosin structures at the medial cortex of the cell, with actin localization preceding myosin II and with the localization of both proteins preceding changes in cell shape. We discuss models to explain how the architecture of cytoskeletal networks regulates their contractile behavior and the mechanisms that give rise to oscillatory cell behaviors in intercalating cells.     

Morphogenetic movements driving neural tube closure in Xenopus require myosin IIB

Vertebrate neural tube formation involves two distinct morphogenetic events — convergent extension (CE) driven by mediolateral cell intercalation, and bending of the neural plate driven largely by cellular apical constriction. However, the cellular and molecular biomechanics of these processes are not understood. Here, using tissue-targeting techniques, we show that the myosin IIB motor protein complex is essential for both these processes, as well as for conferring resistance to deformation to the neural plate tissue. We show that myosin IIB is required for actin-cytoskeletal organization in both superficial and deep layers of the Xenopus neural plate. In the superficial layer, myosin IIB is needed for apical actin accumulation, which underlies constriction of the neuroepithelial cells, and that ultimately drive neural plate bending, whereas in the deep neural cells myosin IIB organizes a cortical actin cytoskeleton, which we describe for the first time, and that is necessary for both normal neural cell cortical tension and shape and for autonomous CE of the neural tissue. We also show that myosin IIB is required for resistance to deformation (“stiffness”) in the neural plate, indicating that the cytoskeleton-organizing roles of this protein translate in regulation of the biomechanical properties of the neural plate at the tissue-level.     

Par3 functions in the biogenesis of the primary cilium in polarized epithelial cells

Par3 is a PDZ protein important for the formation of junctional complexes in epithelial cells. We have identified an additional role for Par3 in membrane biogenesis. Although Par3 was not required for maintaining polarized apical or basolateral membrane domains, at the apical surface, Par3 was absolutely essential for the growth and elongation of the primary cilium. The activity reflected its ability to interact with kinesin-2, the microtubule motor responsible for anterograde transport of intraflagellar transport particles to the tip of the growing cilium. The Par3 binding partners Par6 and atypical protein kinase C interacted with the ciliary membrane component Crumbs3 and we show that the PDZ binding motif of Crumbs3 was necessary for its targeting to the ciliary membrane. Thus, the Par complex likely serves as an adaptor that couples the vectorial movement of at least a subset of membrane proteins to microtubule-dependent transport during ciliogenesis.