Rabin8 phosphorylated by NDR2/STK38L, the canine early retinal degeneration (erd) gene product, directs rhodopsin Golgi-to-cilia trafficking.

The Rab11-Rabin8-Rab8 ciliogenesis complex regulates the expansion of cilia-derived light-sensing organelles, the rod outer segments, via post-Golgi rhodopsin transport carriers (RTCs). Rabin8, an effector of Rab11 and a nucleotide exchange factor (GEF) for Rab8, is phosphorylated at S272 by NDR2 kinase (aka STK38L), a canine erd gene product linked to the human ciliopathy Leber congenital amaurosis (LCA). Here, we define the step at which NDR2 phosphorylated Rabin8 regulates Rab11-Rab8 succession in X.

The ins and outs of the Arf4-based ciliary membrane-targeting complex.

The small GTPase Arf4-based ciliary membrane-targeting complex recognizes specific targeting signals within sensory receptors and regulates their directed movement to primary cilia. Activated Arf4 directly binds the VxPx ciliary-targeting signal (CTS) of the light-sensing receptor rhodopsin. Recent findings revealed that at the -Golgi, marked by the small GTPase Rab6, activated Arf4 forms a functional complex with rhodopsin and the Arf guanine nucleotide exchange factor (GEF) GBF1, providing positive feedback that drives further Arf4 activation in ciliary trafficking.

An interaction network between the SNARE VAMP7 and Rab GTPases within a ciliary membrane-targeting complex.

The Arf4-rhodopsin complex (mediated by the VxPx motif in rhodopsin) initiates expansion of vertebrate rod photoreceptor cilia-derived light-sensing organelles through stepwise assembly of a conserved trafficking network. Here, we examine its role in the sorting of VAMP7 (also known as TI-VAMP) - an R-SNARE possessing a regulatory longin domain (LD) - into rhodopsin transport carriers (RTCs). During RTC formation and trafficking, VAMP7 colocalizes with the ciliary cargo rhodopsin and interacts with the Rab11-Rabin8-Rab8 trafficking module.

The Arf GEF GBF1 and Arf4 synergize with the sensory receptor cargo, rhodopsin, to regulate ciliary membrane trafficking.

The small GTPase Arf4 and the Arf GTPase-activating protein (GAP) ASAP1 cooperatively sequester sensory receptor cargo into transport carriers targeted to primary cilia, but the input that drives Arf4 activation in this process remains unknown. Here, we show, by using frog retinas and recombinant human proteins, that during the carrier biogenesis from the photoreceptor Golgi/-Golgi network (TGN) a functional complex is formed between Arf4, the Arf guanine nucleotide exchange factor (GEF) GBF1 and the light-sensing receptor, rhodopsin. Rhodopsin and Arf4 bind the regulatory N-terminal dimerization and cyclophillin-binding (DCB)-homology upstream of Sec7 (HUS) domain of GBF1.

Novel topography of the Rab11-effector interaction network within a ciliary membrane targeting complex.

Small GTPases function as universal molecular switches due to the nucleotide dependent conformational changes of their switch regions that allow interacting proteins to discriminate between the active GTP-bound and the inactive GDP-bound states. Guanine nucleotide exchange factors (GEFs) recognize the inactive GDP-bound conformation whereas GTPase activating proteins (GAPs), and the GTPase effectors recognize the active GTP-bound state. Small GTPases are linked to each other through regulatory and effector proteins into functional networks that regulate intracellular membrane traffic through diverse mechanisms that include GEF and GAP cascades, GEF-effector interactions, common effectors and positive feedback loops linking interacting proteins.

The Arf and Rab11 effector FIP3 acts synergistically with ASAP1 to direct Rabin8 in ciliary receptor targeting.

Primary cilia have gained considerable importance in biology and disease now that their involvement in a wide range of human ciliopathies has been abundantly documented. However, detailed molecular mechanisms for specific targeting of sensory receptors to primary cilia are still unknown. Here, we show that the Arf and Rab11 effector FIP3 (also known as RAB11FIP3) promotes the activity of Rab11a and the Arf GTPase-activating protein (GAP) ASAP1 in the Arf4-dependent ciliary transport of the sensory receptor rhodopsin.

Molecular complexes that direct rhodopsin transport to primary cilia.

Rhodopsin is a key molecular constituent of photoreceptor cells, yet understanding of how it regulates photoreceptor membrane trafficking and biogenesis of light-sensing organelles, the rod outer segments (ROS) is only beginning to emerge. Recently identified sequence of well-orchestrated molecular interactions of rhodopsin with the functional networks of Arf and Rab GTPases at multiple stages of intracellular targeting fits well into the complex framework of the biogenesis and maintenance of primary cilia, of which the ROS is one example. This review will discuss the latest progress in dissecting the molecular complexes that coordinate rhodopsin incorporation into ciliary-targeted carriers with the recruitment and activation of membrane tethering complexes and regulators of fusion with the periciliary plasma membrane.

Crosstalk of Arf and Rab GTPases en route to cilia.

Small GTPases are versatile temporal and spatial regulators of virtually all cellular processes including signal transduction, cytoskeleton dynamics and membrane trafficking. They function as molecular switches, aided by a multitude of regulatory and effector proteins that link them into functional networks. A picture is beginning to emerge whereupon scaffold proteins with many functional domains perform the regulatory and effector functions, thus allowing the ordered recruitment and activation of small GTPases.

The Arf GAP ASAP1 provides a platform to regulate Arf4- and Rab11-Rab8-mediated ciliary receptor targeting.

Dysfunctional trafficking to primary cilia is a frequent cause of human diseases known as ciliopathies, yet molecular mechanisms for specific targeting of sensory receptors to cilia are largely unknown. Here, we show that the targeting of ciliary cargo, represented by rhodopsin, is mediated by a specialized system, the principal component of which is the Arf GAP ASAP1. Ablation of ASAP1 abolishes ciliary targeting and causes formation of actin-rich periciliary membrane projections that accumulate mislocalized rhodopsin.

Molecular assemblies that control rhodopsin transport to the cilia.

This review will focus on the conserved molecular mechanisms for the specific targeting of rhodopsin and rhodopsin-like sensory receptors to the primary cilia. We will discuss the molecular assemblies that control the movement of rhodopsin from the central sorting station of the cell, the trans-Golgi network (TGN), into membrane-enclosed rhodopsin transport carriers (RTCs), and their delivery to the primary cilia and the cilia-derived sensory organelle, the rod outer segment (ROS). Recent studies reveal that these processes are initiated by the synergistic interaction of rhodopsin with the active form of the G-protein Arf4 and the Arf GTPase activating protein (GAP) ASAP1.

Investigation of the hyper-reflective inner/outer segment band in optical coherence tomography of living frog retina.

This study is to test anatomic correlates, including connecting cilium (CC) and inner segment (IS) ellipsoid, to the hyper-reflective band visualized by optical coherence tomography (OCT) and commonly attributed to the photoreceptor inner/outer segment (IS/OS) junction. A line-scan OCT (LS-OCT) was constructed to achieve sub-cellular resolution (lateral: ≈ 2 μm; axial: ≈ 4 μm) of excised living frog retinas. An electro-optic phase modulator was employed for rapid and vibration-free phase modulation.

A conserved signal and GTPase complex are required for the ciliary transport of polycystin-1.

Primary cilia regulate epithelial differentiation and organ function. Failure of mutant polycystins to localize to cilia abolishes flow-stimulated calcium signaling and causes autosomal dominant polycystic kidney disease. We identify a conserved amino acid sequence, KVHPSST, in the C-terminus of polycystin-1 (PC1) that serves as a ciliary-targeting signal.

Syntaxin 3 and SNAP-25 pairing, regulated by omega-3 docosahexaenoic acid, controls the delivery of rhodopsin for the biogenesis of cilia-derived sensory organelles, the rod outer segments.

The biogenesis of cilia-derived sensory organelles, the photoreceptor rod outer segments (ROS), is mediated by rhodopsin transport carriers (RTCs). The small GTPase Rab8 regulates ciliary targeting of RTCs, but their specific fusion sites have not been characterized. Here, we report that the Sec6/8 complex, or exocyst, is a candidate effector for Rab8.

Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4.

Dysfunctions of primary cilia and cilia-derived sensory organelles underlie a multitude of human disorders, including retinal degeneration, yet membrane targeting to the cilium remains poorly understood. Here, we show that the newly identified ciliary targeting VxPx motif present in rhodopsin binds the small GTPase Arf4 and regulates its association with the trans-Golgi network (TGN), which is the site of assembly and function of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GTPase-activating protein ASAP1.

Assay for in vitro budding of ciliary-targeted rhodopsin transport carriers.

Primary cilia and cilia-derived sensory organelles are cell's antennas that contain sensory receptors and signal transduction modules. Defects in the expression and targeting of ciliary proteins to this specialized cellular compartment lead to human disorders collectively known as ciliopathies. To examine the molecular basis for the ciliary targeting of the light receptor rhodopsin, we have developed a cell-free assay that reconstitutes its packaging into the specific post-Golgi rhodopsin transport carriers (RTCs).

A role for rhodopsin in a signal transduction cascade that regulates membrane trafficking and photoreceptor polarity.

This review summarizes the most recent progress in the understanding of the role of rhodopsin C-terminal domain in the regulation of intracellular trafficking and photoreceptor morphogenesis. A proposed cascade of molecular interactions, initiated by the rhodopsin C-terminal sequence VXPX-COOH during trafficking from the Golgi/TGN in retinal photoreceptors, is relayed by the small GTPase ARF4 to the downstream effectors. One of the candidates for an ARF4 effector is the ARF-GAP ASAP1, which may function as a subunit of, or form a novel protein coat involved in trafficking from the TGN and in cytoskeletal remodeling, whose assembly is regulated by the binding of ARF4 to rhodopsin, and whose function is essential for the polarized trafficking toward the ROS.

Rhodopsin C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4).

The maintenance of photoreceptor cell polarity is compromised by the rhodopsin mutations causing the human disease autosomal dominant retinitis pigmentosa. The severe form mutations occur in the C-terminal sorting signal of rhodopsin, VXPX-COOH. Here, we report that this sorting motif binds specifically to the small GTPase ARF4, a member of the ARF family of membrane budding and protein sorting regulators.

Phosphoinositides, ezrin/moesin, and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors.

The post-Golgi trafficking of rhodopsin in photoreceptor cells is mediated by rhodopsin-bearing transport carriers (RTCs) and regulated by the small GTPase rab8. In this work, we took a combined pharmacological-proteomic approach to uncover new regulators of RTC trafficking toward the specialized light-sensitive organelle, the rod outer segment (ROS). We perturbed phospholipid synthesis by activating phospholipase D with sphingosine 1-phosphate (S1P) or inhibiting phosphatidic acid phosphohydrolase by propranolol (Ppl).

Retinal cAMP levels during the progression of retinal degeneration in rhodopsin P23H and S334ter transgenic rats.

Purpose: To test whether high levels of cAMP promote apoptosis and shorten the life of retinal rod photoreceptors, the changes in cAMP levels during retinal degeneration were analyzed in two transgenic rat models that express rhodopsin P23H and S334ter mutations.

Methods: Dark- and light-adapted heterozygous P23H (lines 1 and 3; P23H-1 and -3), S334ter line 4 (S334ter-4), and Sprague-Dawley (control) rats were studied at 4 to 8 weeks by cAMP enzyme competitive immunoassay and by cAMP immunocytochemistry.

Results: In control animals retinal cAMP content reached a steady state level at 30 days of age.

Mutant rab8 Impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus rods.

Rab8 is a GTPase involved in membrane trafficking. In photoreceptor cells, rab8 is proposed to participate in the late stages of delivery of rhodopsin-containing post-Golgi membranes to the plasma membrane near the base of the connecting cilium. To test the function of rab8 in vivo, we generated transgenic Xenopus laevis expressing wild-type, constitutively active (Q67L), and dominant negative (T22N) forms of canine rab8 in their rod photoreceptors as green fluorescent protein (GFP) fusion proteins.

Towards the proteome of the rhodopsin-bearing post-Golgi compartment of retinal photoreceptor cells.

Polarized sorting of rhodopsin in retinal rod photoreceptor cells is mediated by post-Golgi carrier membranes that bud from the trans-Golgi network and fuse with the specialized domain of the plasma membrane in the rod inner segment. The identity of the majority of the resident proteins of this organelle still remains elusive, despite multifaceted approaches to study this compartment. In the present study we have taken a proteomic approach to the analysis of the post-Golgi carriers.

Evectins: vesicular proteins that carry a pleckstrin homology domain and localize to post-Golgi membranes.

We have identified two vesicular proteins, designated evectin (evt)-1 and -2. These proteins are approximately 25 kDa in molecular mass, lack a cleaved N-terminal signal sequence, and appear to be inserted into membranes through a C-terminal hydrophobic anchor. They also carry a pleckstrin homology domain at their N termini, which potentially couples them to signal transduction pathways that result in the production of lipid second messengers.

Post-Golgi trafficking of rhodopsin in retinal photoreceptors.

Rod outer segment renewal in retinal rod photoreceptors is mediated by polarised sorting of rhodopsin, and its associated proteins and lipids, on post-Golgi vesicles that bud from the trans-Golgi network and fuse with the specialised domain of the plasma membrane in the rod inner segment. This domain surrounds the cilium that connects the inner segment and the rod outer segment to which mature rhodopsin is delivered. The intracellular sorting machinery that regulates budding, targeting and fusion of rhodopsin carrier vesicles has been studied using multiple means including a newly developed cell-free assay that reconstitutes vesicle budding.