Centriole distal appendages promote membrane docking, leading to cilia initiation.

The distal appendages (DAPs) of centrioles have been proposed to anchor cilia to the plasma membrane, but their molecular composition, assembly, and exact function in ciliogenesis remain poorly understood. Using quantitative centrosome proteomics and superresolution microscopy, we identified five DAP components, including one previously described (CEP164), one partially characterized (CEP89 [ccdc123]), and three novel (CEP83 [ccdc41], SCLT1, and FBF1) DAP proteins. Analyses of DAP assembly revealed a hierarchy. CEP83 recruits both SCLT1 and CEP89 to centrioles. Subsequent recruitment of FBF1 and CEP164 is independent of CEP89 but mediated by SCLT1. All five DAP components are essential for ciliogenesis; loss of CEP83 specifically blocks centriole-to-membrane docking. Undocked centrioles fail to recruit TTBK2 or release CP110, the two earliest modifications found on centrioles prior to cilia assembly, revealing centriole-to-membrane docking as a temporal and spatial cue promoting cilia initiation.

IQGAP1 controls tight junction formation through differential regulation of claudin recruitment.

IQGAP1 is a scaffolding protein previously implicated in adherens junction formation. However, its role in the establishment or maintenance of tight junctions (TJs) has not been explored. We hypothesized that IQGAP1 could regulate TJ formation by modulating the expression and/or localization of junctional proteins, and we systematically tested this hypothesis in the model Madin-Darby canine kidney (MDCK) cell line. We find that IQGAP1 silencing enhances a transient increase in transepithelial electrical resistance (TER) observed during the early stages of TJ formation (Cereijido et al., 1978). Quantitative microscopy and biochemical experiments suggest that this effect of IQGAP1 on TJ assembly is accounted for by reduced expression and TJ recruitment of claudin 2, and increased TJ recruitment of claudin 4. Furthermore, we show that IQGAP1 also regulates TJ formation through its interactor CDC42, because IQGAP1 knockdown increases the activity of the CDC42 effector JNK and dominant-negative CDC42 prevents the increase in TER caused by IQGAP1 silencing. Hence, we provide evidence that IQGAP1 modulates TJ formation by a twofold mechanism: (1) controlling the expression and recruitment of claudin 2 and recruitment of claudin 4 to the TJ, and (2) transient inhibition of the CDC42-JNK pathway.

Quantitative Determination of Primary Cilia Protein Distribution Using Immunofluorescence Staining and MATLAB Analysis.

Primary cilia are microtubule-based sensory organelles surrounded by membrane. They can detect mechanical and chemical stimuli. The last few years have uncovered cilia as unique signaling hubs that host a number of receptors and effector molecules. Thus, defining how specific proteins localize and are distributed along the cilium is critical to understanding its function. Quantitative immunofluorescence can be used to accurately assess the localization of receptors and signaling molecules within the primary cilia. However, image analysis can be time consuming, and there are limited programs that can accurately determine staining intensity along the cilia. To overcome these issues, we developed a series of MATLAB scripts to accurately measure staining intensity along the length of the cilia, in both a semi-automated and automated fashion. Here, we describe the scripts and include a protocol for image analysis for each. With these scripts, the protocols can be used to analyze the distribution of any ciliary protein using immunofluorescence images.

Super-resolution microscopy reveals coupling between mammalian centriole subdistal appendages and distal appendages.

Subdistal appendages (sDAPs) are centriolar elements that are observed proximal to the distal appendages (DAPs) in vertebrates. Despite the obvious presence of sDAPs, structural and functional understanding of them remains elusive. Here, by combining super-resolved localization analysis and CRISPR-Cas9 genetic perturbation, we find that although DAPs and sDAPs are primarily responsible for distinct functions in ciliogenesis and microtubule anchoring, respectively, the presence of one element actually affects the positioning of the other. Specifically, we find dual layers of both ODF2 and CEP89, where their localizations are differentially regulated by DAP and sDAP integrity. DAP depletion relaxes longitudinal occupancy of sDAP protein ninein to cover the DAP region, implying a role of DAPs in sDAP positioning. Removing sDAPs alter the distal border of centrosomal γ-tubulins, illustrating a new role of sDAPs. Together, our results provide an architectural framework for sDAPs that sheds light on functional understanding, surprisingly revealing coupling between DAPs and sDAPs.

Phosphorylation of CEP83 by TTBK2 is necessary for cilia initiation.

Primary cilia are microtubule-based organelles that play important roles in development and tissue homeostasis. Tau-tubulin kinase-2 (TTBK2) is genetically linked to spinocerebellar ataxia type 11, and its kinase activity is crucial for ciliogenesis. Although it has been shown that TTBK2 is recruited to the centriole by distal appendage protein CEP164, little is known about TTBK2 substrates associated with its role in ciliogenesis. Here, we perform superresolution microscopy and discover that serum starvation results in TTBK2 redistribution from the periphery toward the root of distal appendages. Our biochemical analyses uncover CEP83 as a bona fide TTBK2 substrate with four phosphorylation sites characterized. We also demonstrate that CEP164-dependent TTBK2 recruitment to distal appendages is required for subsequent CEP83 phosphorylation. Specifically, TTBK2-dependent CEP83 phosphorylation is important for early ciliogenesis steps, including ciliary vesicle docking and CP110 removal. In summary, our results reveal a molecular mechanism of kinase regulation in ciliogenesis and identify CEP83 as a key substrate of TTBK2 during cilia initiation.

Abi-1 forms an epidermal growth factor-inducible complex with Cbl: role in receptor endocytosis.

The Abl-interactor (Abi) proteins are involved in the regulation of actin polymerization and have recently been shown to modulate epidermal growth factor receptor (EGFR) endocytosis. Here we describe the identification of a novel complex between Abi-1 and the Cbl ubiquitin ligase that is induced by stimulation with EGF. Notably, an Abi-1 mutant lacking the SH3 domain (DeltaSH3) fails to interact with Cbl and inhibits EGFR internalization. We show that expression of the Abi-1DeltaSH3 mutant inhibits Cbl accumulation at the plasma membrane after EGF treatment. We have previously shown that the oncogenic Abl tyrosine kinase inhibits EGFR internalization. Here we report that the oncogenic Abl kinase disrupts the EGF-inducible Abi-1/Cbl complex, highlighting the importance of Abl kinases and downstream effectors in the regulation of EGFR internalization. Thus, our work reveals a new role for oncogenic Abl tyrosine kinases in the regulation of the Abi-1/Cbl protein complex and uncovers a role for the Abi-1/Cbl complex in the regulation of EGFR endocytosis.

An emerging role for IQGAP1 in tight junction control.

IQGAP1 is a scaffold protein involved in the assembly of adherens junctions. Our work has recently revealed a novel role for IQGAP1 in the regulation of tight junctions (TJ) through differential recruitment of claudins to the nascent TJ. Here, we discuss the potential mechanisms of this regulation, including IQGAP1 effects on CDC42, and IQGAP1 interactions with sorting/trafficking molecules (e.g. Exo70). Given the many roles of IQGAP1 and the large number of interacting partners, we focus our discussion of these functions in the context of junction formation, trafficking, growth factor signaling and cancer. We also propose a potential role for IQGAP1 in regulating epithelial integrity and compartmentalized signaling in epithelia.

Coupling bimolecular PARylation biosensors with genetic screens to identify PARylation targets.

Poly (ADP-ribose)ylation is a dynamic protein modification that regulates multiple cellular processes. Here, we describe a system for identifying and characterizing PARylation events that exploits the ability of a PBZ (PAR-binding zinc finger) protein domain to bind PAR with high-affinity. By linking PBZ domains to bimolecular fluorescent complementation biosensors, we developed fluorescent PAR biosensors that allow the detection of temporal and spatial PARylation events in live cells. Exploiting transposon-mediated recombination, we integrate the PAR biosensor en masse into thousands of protein coding genes in living cells. Using these PAR-biosensor "tagged" cells in a genetic screen we carry out a large-scale identification of PARylation targets. This identifies CTIF (CBP80/CBP20-dependent translation initiation factor) as a novel PARylation target of the tankyrase enzymes in the centrosomal region of cells, which plays a role in the distribution of the centrosomal satellites.

Super-resolution architecture of mammalian centriole distal appendages reveals distinct blade and matrix functional components.

Distal appendages (DAPs) are nanoscale, pinwheel-like structures protruding from the distal end of the centriole that mediate membrane docking during ciliogenesis, marking the cilia base around the ciliary gate. Here we determine a super-resolved multiplex of 16 centriole-distal-end components. Surprisingly, rather than pinwheels, intact DAPs exhibit a cone-shaped architecture with components filling the space between each pinwheel blade, a new structural element we term the distal appendage matrix (DAM). Specifically, CEP83, CEP89, SCLT1, and CEP164 form the backbone of pinwheel blades, with CEP83 confined at the root and CEP164 extending to the tip near the membrane-docking site. By contrast, FBF1 marks the distal end of the DAM near the ciliary membrane. Strikingly, unlike CEP164, which is essential for ciliogenesis, FBF1 is required for ciliary gating of transmembrane proteins, revealing DAPs as an essential component of the ciliary gate. Our findings redefine both the structure and function of DAPs.

Primary Cilia Mediate Diverse Kinase Inhibitor Resistance Mechanisms in Cancer.

Primary cilia are microtubule-based organelles that detect mechanical and chemical stimuli. Although cilia house a number of oncogenic molecules (including Smoothened, KRAS, EGFR, and PDGFR), their precise role in cancer remains unclear. We have interrogated the role of cilia in acquired and de novo resistance to a variety of kinase inhibitors, and found that, in several examples, resistant cells are distinctly characterized by an increase in the number and/or length of cilia with altered structural features. Changes in ciliation seem to be linked to differences in the molecular composition of cilia and result in enhanced Hedgehog pathway activation. Notably, manipulating cilia length via Kif7 knockdown is sufficient to confer drug resistance in drug-sensitive cells. Conversely, targeting of cilia length or integrity through genetic and pharmacological approaches overcomes kinase inhibitor resistance. Our work establishes a role for ciliogenesis and cilia length in promoting cancer drug resistance and has significant translational implications.

Dual Targeting of PDGFRα and FGFR1 Displays Synergistic Efficacy in Malignant Rhabdoid Tumors.

Subunits of the SWI/SNF chromatin remodeling complex are mutated in a significant proportion of human cancers. Malignant rhabdoid tumors (MRTs) are lethal pediatric cancers characterized by a deficiency in the SWI/SNF subunit SMARCB1. Here, we employ an integrated molecular profiling and chemical biology approach to demonstrate that the receptor tyrosine kinases (RTKs) PDGFRα and FGFR1 are coactivated in MRT cells and that dual blockade of these receptors has synergistic efficacy. Inhibitor combinations targeting both receptors and the dual inhibitor ponatinib suppress the AKT and ERK1/2 pathways leading to apoptosis. MRT cells that have acquired resistance to the PDGFRα inhibitor pazopanib are susceptible to FGFR inhibitors. We show that PDGFRα levels are regulated by SMARCB1 expression, and assessment of clinical specimens documents the expression of both PDGFRα and FGFR1 in rhabdoid tumor patients. Our findings support a therapeutic approach in cancers with SWI/SNF deficiencies by exploiting RTK coactivation dependencies.

A kinase-independent function of AKT promotes cancer cell survival.

The serine-threonine kinase AKT regulates proliferation and survival by phosphorylating a network of protein substrates. In this study, we describe a kinase-independent function of AKT. In cancer cells harboring gain-of-function alterations in MET, HER2, or Phosphatidyl-Inositol-3-Kinase (PI3K), catalytically inactive AKT (K179M) protected from drug induced cell death in a PH-domain dependent manner. An AKT kinase domain mutant found in human melanoma (G161V) lacked enzymatic activity in vitro and in AKT1/AKT2 double knockout cells, but promoted growth factor independent survival of primary human melanocytes. ATP-competitive AKT inhibitors failed to block the kinase-independent function of AKT, a liability that limits their effectiveness compared to allosteric AKT inhibitors. Our results broaden the current view of AKT function and have important implications for the development of AKT inhibitors for cancer.

Abl tyrosine kinase regulates endocytosis of the epidermal growth factor receptor.

Signal attenuation from ligand-activated epidermal growth factor receptor (EGFR) is mediated in part by receptor endocytosis and trafficking to the lysosomal degradative compartment. Uncoupling the activated EGFR from endocytosis and degradation has emerged as a mechanism for oncogenic activation of the EGFR. The Abl nonreceptor tyrosine kinase is activated by ligand-stimulated EGFR, but the role of Abl in EGFR signaling has not been defined. Here we uncovered a novel role for the activated Abl kinase in the regulation of EGFR endocytosis. We show that activated Abl impairs EGFR internalization. Moreover, we show that activated Abl phosphorylates the EGFR primarily on tyrosine 1173, and that mutation of this site to phenylalanine restores ligand-dependent endocytosis of the EGFR in the presence of activated Abl. Furthermore, we show that activated Abl allows the ligand-activated EGFR to escape Cbl-dependent down-regulation by inhibiting the accumulation of Cbl at the plasma membrane in response to epidermal growth factor stimulation and disrupting the formation of the EGFR.Cbl complex without affecting Cbl protein stability. These findings reveal a novel role for Abl in promoting increased cell-surface expression of the EGFR and suggest that Abl/EGFR signaling may cooperate in human tumors.