Lotte B. Pedersen

The primary cilium at a glance

The primary cilium, which was first so named by Sergei Sorokin ([Sorokin, 1968][1]), is a solitary organelle that emanates from the cell surface of most mammalian cell types during growth arrest. Increasing evidence suggests that primary cilia are key coordinators of signaling pathways during

Sensory Cilia and Integration of Signal Transduction in Human Health and Disease

The primary cilium is a hallmark of mammalian tissue cells. Recent research has shown that these organelles display unique sets of selected signal transduction modules including receptors, ion channels, effector proteins and transcription factors that relay chemical and physical stimuli from the extracellular environment in order to control basic cellular processes during embryonic and postnatal development, as well as in tissue homeostasis in adulthood. Consequently, defects in building of the cilium or in transport or function of ciliary signal proteins are associated with a series of pathologies, including developmental disorders and cancer. In this review, we highlight recent examples of the mechanisms by which signal components are selectively targeted and transported to the ciliary membrane and we present an overview of the signal transduction pathways associated with primary and motile cilia in vertebrate cells, including platelet‐derived growth factor receptor‐alpha (PDGFRα), hedgehog and Wnt signaling pathways. Finally, we discuss the functions of these cilia‐associated signal transduction pathways and their role in human health and development.

Primary cilia and signaling pathways in mammalian development, health and disease.

Although first described as early as 1898 and long considered a vestigial organelle of little functional importance, the primary cilium has become one of the hottest research topics in modern cell biology and physiology. Primary cilia are nonmotile sensory organelles present in a single copy on the surface of most growth-arrested or differentiated mammalian cells, and defects in their assembly or function are tightly coupled to many developmental defects, diseases and disorders. In normal tissues, the primary cilium coordinates a series of signal transduction pathways, including Hedgehog, Wnt, PDGFRα and integrin signaling. In the kidney, the primary cilium may function as a mechano-, chemo- and osmosensing unit that probes the extracellular environment and transmits signals to the cell via, e.g., polycystins, which depend on ciliary localization for appropriate function. Indeed, hypomorphic mutations in the mouse ift88 (previously called Tg737) gene, which encodes a ciliogenic intraflagellar transport protein, result in malformation of primary cilia, and in the collecting ducts of kidney tubules this is accompanied by development of autosomal recessive polycystic kidney disease (PKD). While PKD was one of the first diseases to be linked to dysfunctional primary cilia, defects in this organelle have subsequently been associated with many other phenotypes, including cancer, obesity, diabetes as well as a number of developmental defects. Collectively, these disorders of the cilium are now referred to as the ciliopathies. In this review, we provide a brief overview of the structure and function of primary cilia and some of their roles in coordinating signal transduction pathways in mammalian development, health and disease.

Assembly of Primary Cilia

Primary cilia are microtubule‐based, hair‐like sensory organelles present on the surface of most growth‐arrested cells in our body. Recent research has demonstrated a crucial role for primary cilia in regulating vertebrate developmental pathways and tissue homeostasis, and defects in genes involved in primary cilia assembly or function have been associated with a panoply of disorders and diseases, including polycystic kidney disease, left‐right asymmetry defects, hydrocephalus, and Bardet Biedl Syndrome. Here we provide an up‐to‐date review focused on the molecular mechanisms involved in the assembly of primary cilia in vertebrate cells. We present an overview of the early stages of the cilia assembly process, as well as a description of the intraflagellar transport (IFT) system. IFT is a highly conserved process required for assembly of almost all eukaryotic cilia and flagella, and much of our current knowledge about IFT is based on studies performed in Chlamydomonas and Caenorhabditis elegans. Therefore, our review of the IFT literature includes studies performed in these two model organisms. The role of several non‐IFT proteins (e.g., centrosomal proteins) in the ciliary assembly process is also discussed. Developmental Dynamics 237:1993–2006, 2008.

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.

TGF-β Signaling Is Associated with Endocytosis at the Pocket Region of the Primary Cilium

Transforming growth factor β (TGF-β) signaling is regulated by clathrin-dependent endocytosis (CDE) for the control of cellular processes during development and in tissue homeostasis. The primary cilium coordinates several signaling pathways, and the pocket surrounding the base and proximal part of the cilium is a site for CDE. We report here that TGF-β receptors localize to the ciliary tip and endocytic vesicles at the ciliary base in fibroblasts and that TGF-β stimulation increases receptor localization and activation of SMAD2/3 and ERK1/2 at the ciliary base. Inhibition of CDE reduced TGF-β-mediated signaling at the cilium, and TGF-β signaling and CDE activity are reduced at stunted primary cilia in Tg737orpk fibroblasts. Similarly, TGF-β signaling during cardiomyogenesis correlated with accumulation of TGF-β receptors and activation of SMAD2/3 at the ciliary base. Our results indicate that the primary cilium regulates TGF-β signaling and that the ciliary pocket is a compartment for CDE-dependent regulation of signal transduction.

A Proteome-wide Screen for Mammalian SxIP Motif-Containing Microtubule Plus-End Tracking Proteins

Microtubule plus-end tracking proteins (+TIPs) are structurally and functionally diverse factors that accumulate at the growing microtubule plus-ends, connect them to various cellular structures, and control microtubule dynamics [1, 2]. EB1 and its homologs are +TIPs that can autonomously recognize growing microtubule ends and recruit to them a variety of other proteins. Numerous +TIPs bind to end binding (EB) proteins through natively unstructured basic and serine-rich polypeptide regions containing a core SxIP motif (serine-any amino acid-isoleucine-proline) [3]. The SxIP consensus sequence is short, and the surrounding sequences show high variability, raising the possibility that undiscovered SxIP containing +TIPs are encoded in mammalian genomes. Here, we performed a proteome-wide search for mammalian SxIP-containing +TIPs by combining biochemical and bioinformatics approaches. We have identified a set of previously uncharacterized EB partners that have the capacity to accumulate at the growing microtubule ends, including protein kinases, a small GTPase, centriole-, membrane-, and actin-associated proteins. We show that one of the newly identified +TIPs, CEP104, interacts with CP110 and CEP97 at the centriole and is required for ciliogenesis. Our study reveals the complexity of the mammalian +TIP interactome and provides a basis for investigating the molecular crosstalk between microtubule ends and other cellular structures.

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. Copyright

Chlamydomonas FAP133 is a dynein intermediate chain associated with the retrograde intraflagellar transport motor

Intraflagellar transport (IFT) is the bi-directional movement of particles along the length of axonemal outer doublet microtubules and is needed for the assembly and maintenance of eukaryotic cilia and flagella. Retrograde IFT requires cytoplasmic dynein 1b, a motor complex whose organization, structural composition and regulation is poorly understood. We have characterized the product of the Chlamydomonas FAP133 gene that encodes a new WD-repeat protein similar to dynein intermediate chains and homologous to the uncharacterized vertebrate protein WD34. FAP133 is located at the peri-basal body region as well as in punctate structures along the flagella. This protein is associated with the IFT machinery because it is specifically depleted from the flagella of cells with defects in anterograde IFT. Fractionation of flagellar matrix proteins indicates that FAP133 associates with both the LC8 dynein light chain and the IFT dynein heavy chain and light intermediate chain (DHC1b-D1bLIC) motor complex. In the absence of DHC1b or D1bLIC, FAP133 fails to localize at the peri-basal body region but, rather, is concentrated in a region of the cytoplasm near the cell center. Furthermore, we found that FAP133, LC8, DHC1b, D1bLIC, the FLA10 kinesin-2 necessary for anterograde IFT and other IFT scaffold components associate to form a large macromolecular assembly.

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

We previously demonstrated that the primary cilium coordinates platelet-derived growth factor (PDGF) receptor (PDGFR) α–mediated migration in growth-arrested fibroblasts. In this study, we investigate the functional relationship between ciliary PDGFR-α 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 Tg737orpk MEFs, which have no or very short primary cilia. In growth-arrested NIH3T3 cells, NHE1 is activated by the specific PDGFR-α 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 Tg737orpk MEFs. PDGF-AA failed to stimulate migration in NHE1-null fibroblasts. In conclusion, stimulation of directional migration in response to ciliary PDGFR-α signals is specifically dependent on NHE1 activity, indicating that NHE1 activation is a critical event in the physiological response to PDGFR-α stimulation.