The conserved ancestral signaling pathway from cilium to nucleus.

Many signaling molecules are localized to both the primary cilium and nucleus. Localization of specific transmembrane receptors and their signaling scaffold molecules in the cilium is necessary for correct physiological function. After a specific signaling event, signaling molecules leave the cilium, usually in the form of an endocytic vesicle scaffold, and move to the nucleus, where they dissociate from the scaffold and enter the nucleus to affect gene expression. This ancient pathway probably arose very early in eukaryotic evolution as the nucleus and cilium co-evolved. Because there are similarities in molecular composition of the nuclear and ciliary pores the entry and exit of proteins in both organelles rely on similar mechanisms. In this Hypothesis, we propose that the pathway is a dynamic universal cilia-based signaling pathway with some variations from protists to man. Everywhere the cilium functions as an important organelle for molecular storage of certain key receptors and selection and concentration of their associated signaling molecules that move from cilium to nucleus. This could also have important implications for human diseases such as Huntington disease.

CILIA: before and after.

This is a history of cilia research before and after the discovery of intraflagellar transport (IFT) and the link between primary cilia ciliogenesis and polycystic kidney disease (PKD). Before IFT, ca. the beginning of the new millennium, although sensory and primary cilia were well described, research was largely focused on motile cilia, their structure, movement, and biogenesis. After IFT and the link to PKD, although work on motile cilia has continued to progress, research on primary cilia has exploded, leading to new insights into the role of cilia in cell signaling and development. Genomics, proteomics, and new imaging techniques have unified the field and pointed out the critical role of cilia as a restricted cell organellar compartment, functionally integrated with other cell organelles including the autophagosome and the nucleus.

Chirality of the cytoskeleton in the origins of cellular asymmetry.

Self-assembly of two important components of the cytoskeleton of eukaryotic cells, actin microfilaments and microtubules (MTs) results in polar filaments of one chirality. As is true for bacterial flagella, in actin microfilaments, screw direction is important for assembly processes and motility. For MTs, polar orientation within the cell is paramount. The alignment of these elements in the cell cytoplasm gives rise to emergent properties, including the potential for cell differentiation and specialization. Complex MTs with a characteristic chirality are found in basal bodies and centrioles; this chirality is preserved in cilia. In motile cilia, it is reflected in the direction of the effective stroke. The positioning of the basal body or cilia on the cell surface depends on polarity proteins. In evolution, survival depends on global polarity information relayed to the cell in part by orientation of the MT and actin filament cytoskeletons and the chirality of the basal body to determine left and right coordinates within a defined anterior-posterior cell and tissue axis.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.

CLEM Methods for Studying Primary Cilia.

CLEM (correlated light and electron microscope) imaging is a highly useful technique for examining primary cilia. With CLEM, it is possible to determine the distribution of tagged proteins along the ciliary membrane and axoneme with high precision. Scanning electron microscopy (SEM) permits measurement of ciliary length and orientation in relation to nearby cellular structures in a 3D image; in optimal cases, this can be combined with superresolution microscopy of selected ciliary components as they enter or leave the cilium. This chapter discusses CLEM methods. In the method described in detail, samples are completely processed for sequential fluorescence and SEM observation. This method is ideal for robust antibody localization and minimizes image manipulation in correlating the fluorescent and SEM images. Alternative methods prepare samples for fluorescence imaging followed by processing for SEM then observation in the SEM. This method is ideal for optimal fluorescence imaging, particularly live cell imaging.

Evolutionary implications of localization of the signaling scaffold protein parafusin to both cilia and the nucleus.

Parafusin (PFUS), a 63 kDa protein first discovered in the eukaryote Paramecium and known for its role in apicomplexan exocytosis, provides a model for the common origin of cellular systems employing scaffold proteins for targeting and signaling. PFUS is closely related to eubacterial rather than archeal phosphoglucomutases (PGM) - as we proved by comparison of their 88 sequences - but has no PGM activity. Immunofluorescence microscopy analysis with a PFUS-specific peptide antibody showed presence of this protein around the base region of primary cilia in a variety of mammalian cell types, including mouse embryonic (MEFs) and human foreskin fibroblasts (hFFs), human carcinoma stem cells (NT-2 cells), and human retinal pigment epithelial (RPE) cells. Further, PFUS localized to the nucleus of fibroblasts, and prominently to nucleoli of MEFs. Localization studies were confirmed by Western blot analysis, showing that the PFUS antibody specifically recognizes a single protein of ca. 63 kDa in both cytoplasmic and nuclear fractions. Finally, immunofluorescence microscopy analysis showed that PFUS localized to nuclei and cilia in Paramecium. These results support the suggestion that PFUS plays a role in signaling between nucleus and cilia, and that the cilium and the nucleus both evolved around the time of eukaryotic emergence. We hypothesize that near the beginnings of eukaryotic cell evolution, scaffold proteins such as PFUS arose as peripheral membrane protein identifiers for cytoplasmic membrane trafficking and were employed similarly during the subsequent evolution of exocytic, nuclear transport, and ciliogenic mechanisms.

Onward from the cradle.

This essay records a voyage of discovery from the "cradle of cell biology" to the present, focused on the biology of the oldest known cell organelle, the cilium. In the "romper room" of cilia and microtubule (MT) biology, the sliding MT hypothesis of ciliary motility was born. From the "summer of love," students and colleagues joined the journey to test switch-point mechanisms of motility. In the new century, interest in nonmotile (primary) cilia, never lost from the cradle, was rekindled, leading to discoveries relating ciliogenesis to autophagy and hypotheses of how molecules cross ciliary necklace barriers for cell signaling.

A Structural Basis for How Motile Cilia Beat.

The motile cilium is a mechanical wonder, a cellular nanomachine that produces a high-speed beat based on a cycle of bends that move along an axoneme made of 9+2 microtubules. The molecular motors, dyneins, power the ciliary beat. The dyneins are compacted into inner and outer dynein arms, whose activity is highly regulated to produce microtubule sliding and axonemal bending. The switch point hypothesis was developed long ago to account for how sliding in the presence of axonemal radial spoke-central pair interactions causes the ciliary beat. Since then, a new genetic, biochemical, and structural complexity has been discovered, in part, with Chlamydomonas mutants, with high-speed, high-resolution analysis of movement and with cryoelectron tomography. We stand poised on the brink of new discoveries relating to the molecular control of motility that extend and refine our understanding of the basic events underlying the switching of arm activity and of bend formation and propagation.

Functional interaction between autophagy and ciliogenesis.

Nutrient deprivation is a stimulus shared by both autophagy and the formation of primary cilia. The recently discovered role of primary cilia in nutrient sensing and signalling motivated us to explore the possible functional interactions between this signalling hub and autophagy. Here we show that part of the molecular machinery involved in ciliogenesis also participates in the early steps of the autophagic process. Signalling from the cilia, such as that from the Hedgehog pathway, induces autophagy by acting directly on essential autophagy-related proteins strategically located in the base of the cilium by ciliary trafficking proteins. Whereas abrogation of ciliogenesis partially inhibits autophagy, blockage of autophagy enhances primary cilia growth and cilia-associated signalling during normal nutritional conditions. We propose that basal autophagy regulates ciliary growth through the degradation of proteins required for intraflagellar transport. Compromised ability to activate the autophagic response may underlie some common ciliopathies.

Analysis of primary cilia in directional cell migration in fibroblasts.

Early studies of migrating fibroblasts showed that primary cilia orient in front of the nucleus and point toward the leading edge. Recent work has shown that primary cilia coordinate a series of signaling pathways critical to fibroblast cell migration during development and in wound healing. In particular, platelet-derived growth factor receptor alpha (PDGFRα) is compartmentalized to the primary cilium to activate signaling pathways that regulate reorganization of the cytoskeleton required for lamellipodium formation and directional migration in the presence of a specific ligand gradient. We summarize selected methods in analyzing ciliary function in directional cell migration, including immunofluorescence microscopy, scratch assay, and chemotaxis assay by micropipette addition of PDGFRα ligands to cultures of fibroblasts. These methods should be useful not only in studying cell migration but also more generally in delineating response pathways in cells with primary cilia.

PDGFRα signaling in the primary cilium regulates NHE1-dependent fibroblast migration via coordinated differential activity of MEK1/2-ERK1/2-p90RSK and AKT signaling pathways.

In fibroblasts, platelet-derived growth factor receptor alpha (PDGFRα) is upregulated during growth arrest and compartmentalized to the primary cilium. PDGF-AA mediated activation of the dimerized ciliary receptor produces a phosphorylation cascade through the PI3K-AKT and MEK1/2-ERK1/2 pathways leading to the activation of the Na(+)/H(+) exchanger, NHE1, cytoplasmic alkalinization and actin nucleation at the lamellipodium that supports directional cell migration. We here show that AKT and MEK1/2-ERK1/2-p90(RSK) inhibition reduced PDGF-AA-induced cell migration by distinct mechanisms: AKT inhibition reduced NHE1 activity by blocking the translocation of NHE1 to the cell membrane. MEK1/2 inhibition did not affect NHE1 activity but influenced NHE1 localization, causing NHE1 to localize discontinuously in patches along the plasma membrane, rather than preferentially at the lamellipodium. We also provide direct evidence of NHE1 translocation through the cytoplasm to the leading edge. In conclusion, signals initiated at the primary cilium through the PDGFRαα cascade reorganize the cytoskeleton to regulate cell migration differentially through the AKT and the MEK1/2-ERK1/2-p90(RSK) pathways. The AKT pathway is necessary for initiation of NHE1 translocation, presumably in vesicles, to the leading edge and for its activation. In contrast, the MEK1/2-ERK1/2-p90(RSK) pathway controls the spatial organization of NHE1 translocation and incorporation, and therefore specifies the direction of the leading edge formation.

The IFT-A complex regulates Shh signaling through cilia structure and membrane protein trafficking.

Two intraflagellar transport (IFT) complexes, IFT-A and IFT-B, build and maintain primary cilia and are required for activity of the Sonic hedgehog (Shh) pathway. A weak allele of the IFT-A gene, Ift144, caused subtle defects in cilia structure and ectopic activation of the Shh pathway. In contrast, strong loss of IFT-A, caused by either absence of Ift144 or mutations in two IFT-A genes, blocked normal ciliogenesis and decreased Shh signaling. In strong IFT-A mutants, the Shh pathway proteins Gli2, Sufu, and Kif7 localized correctly to cilia tips, suggesting that these pathway components were trafficked by IFT-B. In contrast, the membrane proteins Arl13b, ACIII, and Smo failed to localize to primary cilia in the absence of IFT-A. We propose that the increased Shh activity seen in partial loss-of-function IFT-A mutants may be a result of decreased ciliary ACIII and that the loss of Shh activity in the absence of IFT-A is a result of severe disruptions of cilia structure and membrane protein trafficking.

The ciliary cytoskeleton.

Cilia and flagella are surface-exposed, finger-like organelles whose core consists of a microtubule (MT)-based axoneme that grows from a modified centriole, the basal body. Cilia are found on the surface of many eukaryotic cells and play important roles in cell motility and in coordinating a variety of signaling pathways during growth, development, and tissue homeostasis. Defective cilia have been linked to a number of developmental disorders and diseases, collectively called ciliopathies. Cilia are dynamic organelles that assemble and disassemble in tight coordination with the cell cycle. In most cells, cilia are assembled during growth arrest in a multistep process involving interaction of vesicles with appendages present on the distal end of mature centrioles, and addition of tubulin and other building blocks to the distal tip of the basal body and growing axoneme; these building blocks are sorted through a region at the cilium base known as the ciliary necklace, and then transported via intraflagellar transport (IFT) along the axoneme toward the tip for assembly. After assembly, the cilium frequently continues to turn over and incorporate tubulin at its distal end in an IFT-dependent manner. Prior to cell division, the cilia are usually resorbed to liberate centrosomes for mitotic spindle pole formation. Here, we present an overview of the main cytoskeletal structures associated with cilia and centrioles with emphasis on the MT-associated appendages, fibers, and filaments at the cilium base and tip. The composition and possible functions of these structures are discussed in relation to cilia assembly, disassembly, and length regulation.

Primary cilia and coordination of receptor tyrosine kinase (RTK) signalling.

Primary cilia are microtubule-based sensory organelles that coordinate signalling pathways in cell-cycle control, migration, differentiation and other cellular processes critical during development and for tissue homeostasis. Accordingly, defects in assembly or function of primary cilia lead to a plethora of developmental disorders and pathological conditions now known as ciliopathies. In this review, we summarize the current status of the role of primary cilia in coordinating receptor tyrosine kinase (RTK) signalling pathways. Further, we present potential mechanisms of signalling crosstalk and networking in the primary cilium and discuss how defects in ciliary RTK signalling are linked to human diseases and disorders.

Controlling the direction of division.

Quyn and colleagues report that gut stem cells have a biased spindle orientation and asymmetric retention of label-retaining DNA. These features are lost in mouse and human tissues when the microtubule binding protein Apc is mutated. In the developing kidney, Apc acts downstream from primary cilium signaling to influence spindle orientation when noncanonical Wnt signaling predominates. Do gut stem cells also have primary cilia?

Directional cell migration and chemotaxis in wound healing response to PDGF-AA are coordinated by the primary cilium in fibroblasts.

Cell motility and migration play pivotal roles in numerous physiological and pathophysiological processes including development and tissue repair. Cell migration is regulated through external stimuli such as platelet-derived growth factor-AA (PDGF-AA), a key regulator in directional cell migration during embryonic development and a chemoattractant during postnatal migratory responses including wound healing. We previously showed that PDGFRalpha signaling is coordinated by the primary cilium in quiescent cells. However, little is known about the function of the primary cilium in cell migration. Here we used micropipette analysis to show that a normal chemosensory response to PDGF-AA in fibroblasts requires the primary cilium. In vitro and in vivo wound healing assays revealed that in ORPK mouse (IFT88(Tg737Rpw)) fibroblasts, where ciliary assembly is defective, chemotaxis towards PDGF-AA is absent, leading to unregulated high speed and uncontrolled directional cell displacement during wound closure, with subsequent defects in wound healing. These data suggest that in coordination with cytoskeletal reorganization, the fibroblast primary cilium functions via ciliary PDGFRalpha signaling to monitor directional movement during wound healing.

"Smart dust" biosensors powered by biomolecular motors.

The concept of a microfabricated biosensor for environmental and biomedical monitoring applications which is composed of environmentally benign components is presented. With a built-in power source (the biological fuel ATP) and driven by biological motors (kinesin), sensing in the microdevice can be remotely activated and the presence of a target molecule or toxin remotely detected. The multifaceted progress towards the realization of such a device is described.

The Na+/H+ exchanger NHE1 is required for directional migration stimulated via PDGFR-alpha in the primary cilium.

We previously demonstrated that the primary cilium coordinates platelet-derived growth factor (PDGF) receptor (PDGFR) alpha-mediated migration in growth-arrested fibroblasts. In this study, we investigate the functional relationship between ciliary PDGFR-alpha and the Na(+)/H(+) exchanger NHE1 in directional cell migration. NHE1 messenger RNA and protein levels are up-regulated in NIH3T3 cells and mouse embryonic fibroblasts (MEFs) during growth arrest, which is concomitant with cilium formation. NHE1 up-regulation is unaffected in Tg737(orpk) MEFs, which have no or very short primary cilia. In growth-arrested NIH3T3 cells, NHE1 is activated by the specific PDGFR-alpha ligand PDGF-AA. In wound-healing assays on growth-arrested NIH3T3 cells and wild-type MEFs, NHE1 inhibition by 5'-(N-ethyl-N-isopropyl) amiloride potently reduces PDGF-AA-mediated directional migration. These effects are strongly attenuated in interphase NIH3T3 cells, which are devoid of primary cilia, and in Tg737(orpk) MEFs. PDGF-AA failed to stimulate migration in NHE1-null fibroblasts. In conclusion, stimulation of directional migration in response to ciliary PDGFR-alpha signals is specifically dependent on NHE1 activity, indicating that NHE1 activation is a critical event in the physiological response to PDGFR-alpha stimulation.