Mechanism and Regulation of Centriole and Cilium Biogenesis.

The centriole is an ancient microtubule-based organelle with a conserved nine-fold symmetry. Centrioles form the core of centrosomes, which organize the interphase microtubule cytoskeleton of most animal cells and form the poles of the mitotic spindle. Centrioles can also be modified to form basal bodies, which template the formation of cilia and play central roles in cellular signaling, fluid movement, and locomotion. In this review, we discuss developments in our understanding of the biogenesis of centrioles and cilia and the regulatory controls that govern their structure and number. We also discuss how defects in these processes contribute to a spectrum of human diseases and how new technologies have expanded our understanding of centriole and cilium biology, revealing exciting avenues for future exploration.

Pathogenic RAB34 variants impair primary cilium assembly and cause a novel oral-facial-digital syndrome.

Oral-facial-digital syndromes (OFDS) are a group of clinically and genetically heterogeneous disorders characterized by defects in the development of the face and oral cavity along with digit anomalies. Pathogenic variants in over 20 genes encoding ciliary proteins have been found to cause OFDS through deleterious structural or functional impacts on primary cilia. We identified by exome sequencing bi-allelic missense variants in a novel disease-causing ciliary gene RAB34 in four individuals from three unrelated families. Affected individuals presented a novel form of OFDS (OFDS-RAB34) accompanied by cardiac, cerebral, skeletal and anorectal defects. RAB34 encodes a member of the Rab GTPase superfamily and was recently identified as a key mediator of ciliary membrane formation. Unlike many genes required for cilium assembly, RAB34 acts selectively in cell types that use the intracellular ciliogenesis pathway, in which nascent cilia begin to form in the cytoplasm. We find that the protein products of these pathogenic variants, which are clustered near the RAB34 C-terminus, exhibit a strong loss of function. Although some variants retain the ability to be recruited to the mother centriole, cells expressing mutant RAB34 exhibit a significant defect in cilium assembly. While many Rab proteins have been previously linked to ciliogenesis, our studies establish RAB34 as the first small GTPase involved in OFDS and reveal the distinct clinical manifestations caused by impairment of intracellular ciliogenesis.

Membranes in balance: mechanisms of sphingolipid homeostasis.

Sphingolipids and their metabolites play key cellular roles both as structural components of membranes and as signaling molecules that mediate responses to physiologic cues and stresses. Despite progress during the last two decades in defining the enzymatic machinery responsible for synthesizing and degrading sphingolipids, comparatively little is known about how these enzymes are regulated to ensure sphingolipid homeostasis. Here, we review new insights into how cells sense and control sphingolipid biosynthesis and transport. We also discuss emerging evidence that sphingolipid metabolism is closely coordinated with that of sterols and glycerolipids and with other processes that occur in the secretory pathway. An improved understanding of sphingolipid homeostasis promises to shed light on basic processes in cell biology and disease, including how cells establish and maintain the complex membrane composition and architecture that is a defining feature of eukaryotic cell biology.

Orm family proteins mediate sphingolipid homeostasis.

Despite the essential roles of sphingolipids both as structural components of membranes and critical signalling molecules, we have a limited understanding of how cells sense and regulate their levels. Here we reveal the function in sphingolipid metabolism of the ORM genes (known as ORMDL genes in humans)-a conserved gene family that includes ORMDL3, which has recently been identified as a potential risk factor for childhood asthma. Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production. We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted. Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism. Our work identifies the Orm proteins as critical mediators of sphingolipid homeostasis and raises the possibility that sphingolipid misregulation contributes to the development of childhood asthma.

Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae.

The Orm family proteins are conserved integral membrane proteins of the endoplasmic reticulum that are key homeostatic regulators of sphingolipid biosynthesis. Orm proteins bind to and inhibit serine:palmitoyl-coenzyme A transferase, the first enzyme in sphingolipid biosynthesis. In Saccharomyces cerevisiae, Orm1 and Orm2 are inactivated by phosphorylation in response to compromised sphingolipid synthesis (e.g., upon addition of inhibitor myriocin), thereby restoring sphingolipid production. We show here that protein kinase Ypk1, one of an essential pair of protein kinases, is responsible for this regulatory modification. Myriocin-induced hyperphosphorylation of Orm1 and Orm2 does not occur in ypk1 cells, and immunopurified Ypk1 phosphorylates Orm1 and Orm2 robustly in vitro exclusively on three residues that are known myriocin-induced sites. Furthermore, the temperature-sensitive growth of ypk1(ts) ypk2 cells is substantially ameliorated by deletion of ORM genes, confirming that a primary physiological role of Ypk1-mediated phosphorylation is to negatively regulate Orm function. Ypk1 immunoprecipitated from myriocin-treated cells displays a higher specific activity for Orm phosphorylation than Ypk1 from untreated cells. To identify the mechanism underlying Ypk1 activation, we systematically tested several candidate factors and found that the target of rapamycin complex 2 (TORC2) kinase plays a key role. In agreement with prior evidence that a TORC2-dependent site in Ypk1(T662) is necessary for cells to exhibit a wild-type level of myriocin resistance, a Ypk1(T662A) mutant displays only weak Orm phosphorylation in vivo and only weak activation in vitro in response to sphingolipid depletion. Additionally, sphingolipid depletion increases phosphorylation of Ypk1 at T662. Thus, Ypk1 is both a sensor and effector of sphingolipid level, and reduction in sphingolipids stimulates Ypk1, at least in part, via TORC2-dependent phosphorylation.

A novel protein LZTFL1 regulates ciliary trafficking of the BBSome and Smoothened.

Many signaling proteins including G protein-coupled receptors localize to primary cilia, regulating cellular processes including differentiation, proliferation, organogenesis, and tumorigenesis. Bardet-Biedl Syndrome (BBS) proteins are involved in maintaining ciliary function by mediating protein trafficking to the cilia. However, the mechanisms governing ciliary trafficking by BBS proteins are not well understood. Here, we show that a novel protein, Leucine-zipper transcription factor-like 1 (LZTFL1), interacts with a BBS protein complex known as the BBSome and regulates ciliary trafficking of this complex. We also show that all BBSome subunits and BBS3 (also known as ARL6) are required for BBSome ciliary entry and that reduction of LZTFL1 restores BBSome trafficking to cilia in BBS3 and BBS5 depleted cells. Finally, we found that BBS proteins and LZTFL1 regulate ciliary trafficking of hedgehog signal transducer, Smoothened. Our findings suggest that LZTFL1 is an important regulator of BBSome ciliary trafficking and hedgehog signaling.

A disease-associated PPP2R3C-MAP3K1 phospho-regulatory module controls centrosome function.

Centrosomes have critical roles in microtubule organization and in cell signaling.1-8 However, the mechanisms that regulate centrosome function are not fully defined, and thus how defects in centrosomal regulation contribute to disease is incompletely understood. From functional genomic analyses, we find here that PPP2R3C, a PP2A phosphatase subunit, is a distal centriole protein and functional partner of centriolar proteins CEP350 and FOP. We further show that a key function of PPP2R3C is to counteract the kinase activity of MAP3K1. In support of this model, MAP3K1 knockout suppresses growth defects caused by PPP2R3C inactivation, and MAP3K1 and PPP2R3C have opposing effects on basal and microtubule stress-induced JNK signaling. Illustrating the importance of balanced MAP3K1 and PPP2R3C activities, acute overexpression of MAP3K1 severely inhibits centrosome function and triggers rapid centriole disintegration. Additionally, inactivating PPP2R3C mutations and activating MAP3K1 mutations both cause congenital syndromes characterized by gonadal dysgenesis.9-15 As a syndromic PPP2R3C variant is defective in centriolar localization and binding to centriolar protein FOP, we propose that imbalanced activity of this centrosomal kinase-phosphatase pair is the shared cause of these disorders. Thus, our findings reveal a new centrosomal phospho-regulatory module, shed light on disorders of gonadal development, and illustrate the power of systems genetics to identify previously unrecognized gene functions.

Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise.

A major goal of biology is to provide a quantitative description of cellular behaviour. This task, however, has been hampered by the difficulty in measuring protein abundances and their variation. Here we present a strategy that pairs high-throughput flow cytometry and a library of GFP-tagged yeast strains to monitor rapidly and precisely protein levels at single-cell resolution. Bulk protein abundance measurements of >2,500 proteins in rich and minimal media provide a detailed view of the cellular response to these conditions, and capture many changes not observed by DNA microarray analyses. Our single-cell data argue that noise in protein expression is dominated by the stochastic production/destruction of messenger RNAs. Beyond this global trend, there are dramatic protein-specific differences in noise that are strongly correlated with a protein's mode of transcription and its function. For example, proteins that respond to environmental changes are noisy whereas those involved in protein synthesis are quiet. Thus, these studies reveal a remarkable structure to biological noise and suggest that protein noise levels have been selected to reflect the costs and potential benefits of this variation.

A comprehensive strategy enabling high-resolution functional analysis of the yeast genome.

Functional genomic studies in Saccharomyces cerevisiae have contributed enormously to our understanding of cellular processes. Their full potential, however, has been hampered by the limited availability of reagents to systematically study essential genes and the inability to quantify the small effects of most gene deletions on growth. Here we describe the construction of a library of hypomorphic alleles of essential genes and a high-throughput growth competition assay to measure fitness with unprecedented sensitivity. These tools dramatically increase the breadth and precision with which quantitative genetic analysis can be performed in yeast. We illustrate the value of these approaches by using genetic interactions to reveal new relationships between chromatin-modifying factors and to create a functional map of the proteasome. Finally, by measuring the fitness of strains in the yeast deletion library, we addressed an enigma regarding the apparent prevalence of gene dispensability and found that most genes do contribute to growth.

Rab34 GTPase mediates ciliary membrane formation in the intracellular ciliogenesis pathway.

The primary cilium is an essential organizing center for signal transduction, and ciliary defects cause congenital disorders known collectively as ciliopathies.1-3 Primary cilia form by two pathways that are employed in a cell-type- and tissue-specific manner: an extracellular pathway in which the cilium grows out from the cell surface and an intracellular pathway in which the nascent cilium first forms inside the cell.4-8 After exposure to the external environment, cilia formed via the intracellular pathway may have distinct functional properties, as they often remain recessed within a ciliary pocket.9,10 However, the precise mechanism of intracellular ciliogenesis and its relatedness to extracellular ciliogenesis remain poorly understood. Here we show that Rab34, a poorly characterized GTPase recently linked to cilia,11-13 is a selective mediator of intracellular ciliogenesis. We find that Rab34 is required for formation of the ciliary vesicle at the mother centriole and that Rab34 marks the ciliary sheath, a unique sub-domain of assembling intracellular cilia. Rab34 activity is modulated by divergent residues within its GTPase domain, and ciliogenesis requires GTP binding and turnover by Rab34. Because Rab34 is found on assembly intermediates that are unique to intracellular ciliogenesis, we tested its role in the extracellular pathway used by polarized MDCK cells. Consistent with Rab34 acting specifically in the intracellular pathway, MDCK cells ciliate independently of Rab34 and its paralog Rab36. Together, these findings establish that different modes of ciliogenesis have distinct molecular requirements and reveal Rab34 as a new GTPase mediator of ciliary membrane biogenesis.

Pericentrin Knocks Down Cilia in Trisomy 21.

Down syndrome is a developmental disorder caused by chromosome 21 trisomy, whereas ciliopathies result from defective primary cilia. In this issue of Developmental Cell, Galati et al. (2018) establish a link between these diseases, finding that cilium function is compromised in Down syndrome as a result of increased Pericentrin expression.

A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies.

Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.

Analysis of soluble protein entry into primary cilia using semipermeabilized cells.

The primary cilium is a protrusion from the cell surface that serves as a specialized compartment for signal transduction. Many signaling factors are known to be dynamically concentrated within cilia and to require cilia for their function. Yet protein entry into primary cilia remains poorly understood. To enable a mechanistic analysis of soluble protein entry into cilia, we developed a method for semipermeabilization of mammalian cells in which the plasma membrane is permeabilized while the ciliary membrane remains intact. Using semipermeabilized cells as the basis for an in vitro diffusion-to-capture assay, we uncovered a size-dependent diffusion barrier that restricts soluble protein exchange between the cytosol and the cilium. The manipulability of this in vitro system enabled an extensive characterization of the ciliary diffusion barrier and led us to show that the barrier is mechanistically distinct from those at the axon initial segment and the nuclear pore complex. Because semipermeabilized cells enable a range of experimental perturbations that would not be easily feasible in intact cells, we believe this methodology will provide a unique resource for investigating primary cilium function in development and disease.

The intraflagellar transport protein IFT27 promotes BBSome exit from cilia through the GTPase ARL6/BBS3.

The sorting of signaling receptors into and out of cilia relies on the BBSome, a complex of Bardet-Biedl syndrome (BBS) proteins, and on the intraflagellar transport (IFT) machinery. GTP loading onto the Arf-like GTPase ARL6/BBS3 drives assembly of a membrane-apposed BBSome coat that promotes cargo entry into cilia, yet how and where ARL6 is activated remains elusive. Here, we show that the Rab-like GTPase IFT27/RABL4, a known component of IFT complex B, promotes the exit of BBSome and associated cargoes from cilia. Unbiased proteomics and biochemical reconstitution assays show that, upon disengagement from the rest of IFT-B, IFT27 directly interacts with the nucleotide-free form of ARL6. Furthermore, IFT27 prevents aggregation of nucleotide-free ARL6 in solution. Thus, we propose that IFT27 separates from IFT-B inside cilia to promote ARL6 activation, BBSome coat assembly, and subsequent ciliary exit, mirroring the process by which BBSome mediates cargo entry into cilia.

Sphingolipid homeostasis in the endoplasmic reticulum and beyond.

Sphingolipids are a diverse group of lipids that have essential cellular roles as structural components of membranes and as potent signaling molecules. In recent years, a detailed picture has emerged of the basic biochemistry of sphingolipids-from their initial synthesis in the endoplasmic reticulum (ER), to their elaboration into complex glycosphingolipids, to their turnover and degradation. However, our understanding of how sphingolipid metabolism is regulated in response to metabolic demand and physiologic cues remains incomplete. Here I discuss new insights into the mechanisms that ensure sphingolipid homeostasis, with an emphasis on the ER as a critical regulatory site in sphingolipid metabolism. In particular, Orm family proteins have recently emerged as key ER-localized mediators of sphingolipid homeostasis. A detailed understanding of how cells sense and control sphingolipid production promises to provide key insights into membrane function in health and disease.

An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier.

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.

Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors.

The dynamic organization of signaling cascades inside primary cilia is key to signal propagation. Yet little is known about the dynamics of ciliary membrane proteins besides a possible role for motor-driven Intraflagellar Transport (IFT). To characterize these dynamics, we imaged single molecules of Somatostatin Receptor 3 (SSTR3, a GPCR) and Smoothened (Smo, a Hedgehog signal transducer) in the ciliary membrane. While IFT trains moved processively from one end of the cilium to the other, single SSTR3 and Smo underwent mostly diffusive behavior interspersed with short periods of directional movements. Statistical subtraction of instant velocities revealed that SSTR3 and Smo spent less than a third of their time undergoing active transport. Finally, SSTR3 and IFT movements could be uncoupled by perturbing either membrane protein diffusion or active transport. Thus ciliary membrane proteins move predominantly by diffusion, and attachment to IFT trains is transient and stochastic rather than processive or spatially determined. DOI:http://dx.doi.org/10.7554/eLife.00654.001.

Primary cilia: how to keep the riff-raff in the plasma membrane.

A recent report suggests that plasma membrane proteins are excluded from primary cilia via anchoring to the cortical actin cytoskeleton. These findings challenge the existence of a diffusion barrier at the base of the cilium.