Application of Laser Feedback Interference Microscopy to Localize the Ciliary Vector

Anchorage-independent myelomonocytic cells acquire adherence within minutes of differentiation stimuli, such as the proteolytically inactive N-terminal fragment of urokinase binding to its cognate glycosylphosphatidylinositol (GPI)-anchored receptor. Here, we report that urokinase-treated differentiating U937 monocyte-like cells exhibit a rapid and transient inhibition of p56/59(hck) and p55(fgr) whereas no changes in the activity of other Src family kinases, such as p53/56(lyn) and p59(fyn) were observed. U937 transfectants expressing a kinase-defective (Lys267 to Met) p56/59(hck) variant exhibit enhanced adhesiveness and a marked F-actin redistribution in thin protruding structures. Conversely, urokinase as well as expression of wild-type or constitutively active (Tyr499 to Phe) p56/59(hck) stimulates the directional migration of uninduced U937 cells. Accordingly, expression of constitutively active or kinase inactive p56/59(hck) selectively prevents urokinase receptor-dependent induction of either adhesion or motility, indicating that a specific activation state of p56/59(hck) is required for each cell response. In conclusion, modulation of the intracellular p56/59(hck) tyrosine kinase activity switches cell motility towards adherence, providing a mutually exclusive mechanism to regulate these properties during monocyte/macrophage differentiation in vivo.

The interplay between specific integrin-mediated matrix adhesion and directional persistence in cell migration is not well understood. Here, we characterized fibroblast adhesion and migration on the extracellular matrix glycoproteins fibronectin and vitronectin, focusing on the role of α5β1 and αvβ3 integrins. Fibroblasts manifested high directional persistence in migration on fibronectin-, but not vitronectin-coated substrates, in a ligand density-dependent manner. Fibronectin stimulated α5β1-dependent organization of the actin cytoskeleton into oriented, ventral stress fibers, and assembly of dynamic, polarized protrusions, characterized as regions free of stress fibers and rich in nascent adhesions at their edge. Such protrusions correlated with persistent, local leading edge advancement, but were not sufficient, nor necessary for directional migration over longer times. Selective blocking of αvβ3 or α5β1 integrins using small molecule integrin antagonists reduced directional persistence on fibronectin, indicating integrin cooperativity in maintaining directionality. On the other hand, patterned substrates, designed to selectively engage either integrin, or their combination, were not sufficient to establish directional migration. Overall, our study demonstrates adhesive coating-dependent regulation of directional persistence in fibroblast migration and challenges the generality of the previously suggested role of β1 and β3 integrins in directional migration.

Primary cilia are evolutionarily conserved cellular organelles with a distinctive rod-like structure that protrudes from the cell surface. Primary cilia are non-motile and near-ubiquitous in animal body. Studies using model organisms have demonstrated that primary cilia are involved in signal transduction pathways that control embryonic development and adult homeostasis. Moreover, defects in the formation or function of primary cilia have been shown to underlie various genetic disorders in human. The biogenesis of cilia requires both the assembly of a microtubule-based core structure, called the axoneme, and the establishment of the ciliary membrane. Emerging evidence suggests that specialized vesicle trafficking is critical for the formation and maintenance of the ciliary membrane, whose composition is distinct from that of the surrounding plasma membrane. The signaling functions of primary cilia also depend on selective delivery of ciliary membrane proteins via vesicle trafficking routes to primary cilia. The list of genes that are thought to be required for ciliary membrane biogenesis increases at a steady pace. However, the sorting and trafficking mechanisms of cilium-bound transport vesicles remain elusive. Here, we review current findings on several key molecules that may regulate vesicle trafficking during and after ciliogenesis.

Primary cilia are unique sensory organelles that coordinate cellular signaling networks in vertebrates. Inevitably, defects in the formation or function of primary cilia lead to imbalanced regulation of cellular processes that causes multisystemic disorders and diseases, commonly known as ciliopathies. Mounting evidence has demonstrated that primary cilia coordinate multiple activities that are required for cell migration, which, when they are aberrantly regulated, lead to defects in organogenesis and tissue repair, as well as metastasis of tumors. Here, we present an overview on how primary cilia may contribute to the regulation of the cellular signaling pathways that control cyclic processes in directional cell migration.

Defects in primary cilia lead to a variety of human diseases. One of these, polycystic kidney disease, can be caused by defects in a Ca²⁺-gated ion channel (TRPP2) found on the cilium. Other ciliary functions also contribute to cystogenesis, and defects in apical Ca²⁺ homeostasis have been implicated. By recording directly from the native cilia of mIMCD-3 cells, a murine cell line of renal epithelial origin, we have identified a second Ca²⁺-gated channel in the ciliary membrane: the transient receptor potential cation channel, subfamily M, member 4 (TRPM4). In excised primary cilia, TRPM4 was found to have a low sensitivity to Ca²⁺, with an EC₅₀ of 646 μM at +100 mV. It was inhibited by MgATP and by 9-phenanthrol. The channel was not permeable to Ca²⁺ or Cl⁻ and had a permeability ratio PK/PNa of 1.42. Reducing the expression of Trpm4 mRNA with short hairpin (sh) RNA reduced the TRPM4 current by 87% and shortened primary cilia by 43%. When phospholipase C was inhibited, the sensitivity to cytoplasmic Ca²⁺ greatly increased (EC₅₀ = 26 μM at +100 mV), which is consistent with previous reports that phosphatidylinositol 4,5-bisphosphate (PIP2) modulates the channel. MgATP did not restore the channel to a preinactivation state, suggesting that the enzyme or substrate necessary for making PIP2 is not abundant in primary cilia of mIMCD-3 cells. The function of TRPM4 in renal primary cilia is not yet known, but it is likely to influence the apical Ca²⁺ dynamics of the cell, perhaps in tandem with TRPP2.

Odontoblast differentiation is regulated by an interplay between primary cilia and the canonical Wnt pathway.

Cilia develop long-lasting contacts, with other cilia

The primary cilium is a critical sensory organelle that is built of axonemal microtubules ensheathed by a ciliary membrane. In polarized epithelial cells, primary cilia reside on the apical surface and must extend these microtubules directly into the extracellular space and remain a stable structure. However, the factors regulating cross-talk between ciliation and cell polarization, as well as, axonemal microtubule growth and stabilization in polarized epithelia are not fully understood. In this study, we find TTLL12, a previously uncharacterized member of the Tubulin Tyrosine Ligase-Like (TTLL) family, localizes to the base of primary cilia and is required for cilia formation in polarized renal epithelial cells. We also show that TTLL12 directly binds to the α/β-tubulin heterodimer in vitro and regulates microtubule dynamics, stability, and post-translational modifications (PTMs). While all other TTLLs catalyze the addition of glutamate or glycine to microtubule C-terminal tails, TTLL12 uniquely affects tubulin PTMs by promoting both microtubule lysine acetylation and arginine methylation. Together, this work identifies a novel microtubule regulator and provides insight into the requirements for apical extracellular axoneme formation.

A Smoothened-Evc2 Complex Transduces the Hedgehog Signal at Primary Cilia

The primary cilium (PC) is a microtubule-based tiny sensory organelle emanating from the centrosome and protruding from the surface of most eukaryotic cells, including neurons. The extremely severe phenotypes of ciliopathies have suggested their paramount importance for multiple developmental events, including brain formation. Neuronal migration is an essential step of neural development, with all neurons traveling from their site of birth to their site of integration. Neurons perform a unique type of cellular migration called cyclic saltatory migration, where their soma periodically jumps along with the stereotyped movement of their centrosome. We will review here how the role of the PC on cell motility was first described in non-neuronal cells as a guide pointing to the direction of migration. We will see then how these findings are extended to neuronal migration. In neurons, the PC appears to regulate the rhythm of cyclic saltatory neuronal migration in multiple systems. Finally, we will review recent findings starting to elucidate how extracellular cues sensed by the PC could be intracellularly transduced to regulate the machinery of neuronal migration. The PC of migrating neurons was unexpectedly discovered to display a rhythmic extracellular emergence during each cycle of migration, with this transient exposure to the external environment associated with periodic transduction of cyclic adenosine monophosphate (cAMP) signaling at the centrosome. The PC in migrating neurons thus uniquely appears as a beat maker, regulating the tempo of cyclic saltatory migration.

Broad-Minded Links Cell Cycle-Related Kinase to Cilia Assembly and Hedgehog Signal Transduction

Disclosure: N. Mojahed: None. S.W. Athul: None. B.S. Ellsworth: None. Pituitary gland deficiency can cause infertility, metabolic dysfunction, and growth insufficiency. Many gene mutations that cause pituitary gland deficiency also cause severe developmental brain defects. During development, sonic hedgehog (SHH)signaling is crucial for the specification and differentiation of pituitary cells, with primary cilia acting as hubs for processing developmental signals. While organellescalled primary cilia play an essential role in SHH signaling, their specific influence on pituitary development remain unknown. Over the last two decades, ciliopathies have been identified as a novel category of pleiotropic diseases, characterized by overlapping phenotypes caused by dysfunction in primary cilia. Using ARL13B, a small GTPase involved in ciliary membrane dynamics and signaling, and pericentrin, a marker of centrioles, as markers of primary cilia, we explored the spatial and temporal patterns of primary cilia during pituitary gland development. Immunohistochemistry results revealed the presence of primary cilia as early as embryonic day (e)10.5. To determine if primary cilia are expressed in distinct anterior pituitarycell types, we performed co-immunohistochemistry. These studies revealed the presence of ARL13B in a subset of somatotropes, lactotropes, thyrotropes, corticotropes, and gonadotropes. These findingshighlight the potential role of primary cilia in facilitating sensory functions that may influence hormonesecretion. To investigate the role of the ciliopathy gene, Mks1, in pituitary gland development, we used the Mks1 gene trap mouse model. Mutations in MKS1 causes ciliopathies such as Bardet-Biedl, Joubert, and Meckel syndromes. The MKS1 gene is located on chromosome 17q22 in humans and encodes the MKS1 protein, which is a key factor in the transition zone of primary cilia. We examined e12.5 and e14.5 wild-type and Mks1-null mouse embryos and found that in null embryos, the pituitary gland was extremely hypoplastic and the ventral diencephalon was dysmorphic. Interestingly expression of critical transcription factors, including LHX3, PITX2, and POU1F1, which are essential for cell lineage specification and pituitary differentiation, was markedly absent in null embryos, indicating a failure of oral ectoderm to adapt apituitary fate. These findings highlight the essential role of MKS1 during pituitary morphogenesis and development. The severe morphological, molecular and cellular defects observed in Mks1-null embryos demonstrate the critical dependence of pituitary development on functional primary cilia. Our studyprovides insights into how ciliopathies, such as Bardet-Biedl and Meckel syndromes, may disrupt endocrine organogenesis through impaired ciliary function. Presentation: Saturday, July 12, 2025

Recent studies in Cdc42 knockout mouse embryonic stem (ES) cells and ES-derived fibroblastoid cell lines raise concern on a body of literature derived by dominant mutant expression approach in a variety of cell lines implicating mammalian Cdc42 as a key regulator of filopodia induction, directional migration and cell cycle progression. To resolve the physiological function of mammalian Cdc42, we have characterized the Cdc42(-/-) and Cdc42GAP(-/-) primary mouse embryonic fibroblasts (MEFs) produced by gene targeting as the Cdc42 loss- or gain-of-activity cell model. The Cdc42(-/-) cells were defective in filopodia formation stimulated by bradykinin and in dorsal membrane ruffling stimulated by PDGF, whereas the Cdc42GAP(-/-) cells displayed spontaneous filopodia. The Cdc42 loss- or gain-of-activity cells were defective in adhesion to fibronectin, wound-healing, polarity establishment, and migration toward a serum gradient. These defects were associated with deficiencies of PAK1, GSK3beta, myosin light chain, and FAK phosphorylation. Furthermore, Cdc42(-/-) cells were defective in G1/S-phase transition and survival, correlating with deficient NF-kappaB transcription and defective JNK, p70 S6K, and ERK1/2 activation. These results demonstrate a different requirement of Cdc42 activity in primary MEFs from ES or ES-derived clonal fibroblastoid cells and suggest that Cdc42 plays cell-type-specific signaling roles.

cAMP is a universal second messenger that relies on precise spatio-temporal regulation to control varied, and often opposing, cellular functions. This is achieved via selective activation of effectors embedded in multiprotein complexes, or signalosomes, that reside at distinct subcellular locations. cAMP is also one of many pathways known to operate within the primary cilium. Dysfunction of ciliary signaling leads to a class of diseases known as ciliopathies. In Autosomal Dominant Polycystic Kidney Disease (ADPKD), a ciliopathy characterized by the formation of fluid-filled kidney cysts, upregulation of cAMP signaling is known to drive cystogenesis. For decades it has been debated whether the primary cilium is an independent cAMP sub-compartment, or whether it shares a diffusible pool of cAMP with the cell body. Recent studies now suggest it is a specific pool of cAMP generated in the cilium that propels cyst formation in ADPKD, supporting the notion that this antenna-like organelle is a compartment within which cAMP signaling occurs independently from cAMP signaling in the bulk cytosol. Here we present examples of cAMP function in the cilium which suggest this mysterious organelle is home to more than one cAMP signalosome. We review evidence that ciliary membrane localization of G-Protein Coupled Receptors (GPCRs) determines their downstream function and discuss how optogenetic tools have contributed to establish that cAMP generated in the primary cilium can drive cystogenesis.

Specific proteins are concentrated within primary cilia, whereas others remain excluded. To understand the mechanistic basis of entry into cilia, we developed an in vitro assay using cells in which the plasma membrane was permeabilized, but the ciliary membrane was left intact. Using a diffusion-to-capture system and quantitative analysis, we find that proteins >9 nm in diameter (∼100 kD) are restricted from entering cilia, and we confirm these findings in vivo. Interference with the nuclear pore complex (NPC) or the actin cytoskeleton in permeabilized cells demonstrated that the ciliary diffusion barrier is mechanistically distinct from those of the NPC or the axon initial segment. Moreover, applying a mass transport model to this system revealed diffusion coefficients for soluble and membrane proteins within cilia that are compatible with rapid exploration of the ciliary space in the absence of active transport. Our results indicate that large proteins require active transport for entry into cilia but not necessarily for movement inside cilia.