Rab8‐Optineurin‐Myosin VI: Analysis of Interactions and Functions in the Secretory Pathway

doi:10.1016/S0076-6879(07)38002-6

Methods in Enzymology

Volume 438, 2008, Pages 11-24

https://doi.org/10.1016/S0076-6879(07)38002-6 Get rights and content

Abstract

The small GTPase Rab8 has been shown to regulate polarized membrane trafficking pathways from the TGN to the cell surface. Optineurin is an effector protein of Rab8 and a binding partner of the actin‐based motor protein myosin VI. We used various approaches to study the interactions between myosin VI and its binding partners and to analyze their role(s) in intracellular membrane trafficking pathways. In this chapter, we describe the use of the mammalian two‐hybrid assay to demonstrate protein–protein interactions and to identify binding sites. We describe a secretion assay that was used in combination with RNA interference technology to analyze the function of myosin VI, optineurin, and Rab8 in exocytic membrane trafficking pathways.

Introduction

The Rab proteins are a large family of small GTPases that participate in and regulate intracellular membrane trafficking pathways (Zerial and McBride, 2001). For example, Rab8 plays an important role in exocytic membrane traffic from the Golgi complex to the plasma membrane in polarized epithelial cells (Huber et al., 1993b), in photoreceptor cells (Moritz et al., 2001), and in polarized neurons (Huber et al., 1993a). It is localized in vesicles at/around the trans‐Golgi network, in recycling endosomes, and at the plasma membrane in membrane ruffles (Ang 2003, Peranen 1996). Rab8 binds to optineurin (Hattula and Peranen, 2000), an adapter protein associated with the Golgi complex, which is known to bind to the actin‐based motor protein myosin VI (Sahlender et al., 2005), and to huntingtin, the protein mutated in Huntington's disease (Faber et al., 1998). The characterization of Rab8 effector proteins and complexes is required to establish the precise role of Rab8 in exocytic membrane trafficking pathways. Furthermore, this approach will help us to understand the role of Rab8 and its effector proteins in a number of diseases.

Rab8 is linked by optineurin to myosin VI, which has been shown to be involved in endocytic and exocytic membrane trafficking pathways. Mutations in the myosin VI gene in mouse and humans are linked to deafness (Ahmed 2003, Avraham 1995) and to hypertrophic cardiomyopathy (Mohiddin et al., 2004), and the myosin VI knockout mouse also has neurological abnormalities (Osterweil et al., 2005). The specific intracellular functions of myosin VI are mediated by a range of interacting proteins (Buss et al., 2004). Optineurin was identified as a myosin VI–binding partner in a yeast two‐hybrid screen, and was shown to link myosin VI to the Golgi complex and to mediate its function in the secretory pathway (Au 2007, Sahlender 2005). This interaction between myosin VI and optineurin may be of medical interest, since mutations in the human optineurin gene cause primary open‐angle glaucoma (Rezaie et al., 2002). Optineurin is a 67‐kDa protein that contains two leucine zippers and one zinc finger separated by large stretches of coiled coil. This myosin VI binding partner is the cellular target of the adenoviral protein Ad E3–14.7 K, which inhibits tumor necrosis factor alpha (TNF‐A)–induced cytolysis, an important defense mechanism to protect cells against viral infection (Li et al., 1998). Optineurin also links Rab8 to huntingtin, the protein mutated in the progressive neurodegenerative disorder, Huntington's disease (Hattula and Peranen, 2000). Although a wealth of data suggests that polyglutamine‐mediated aggregation of huntingtin is a major cause of the disease, new evidence suggests that loss of normal huntingtin function may also play a role in Huntington's disease pathogenesis. Wildtype huntingtin is present at/around the TGN and on vesicles, and is suggested to play a role in endocytosis and exocytosis (Velier et al., 1998) (DiFiglia et al., 1995).

Therefore, to understand the function of Rab8, optineurin, myosin VI, and huntingtin in exocytic membrane‐trafficking pathways, it is of vital importance to establish how they interact in defined protein complexes.

This chapter describes the experimental approaches we have used to identify and verify myosin VI–binding partners and methods to test their function in the secretory pathway.

Section snippets

Identification of Myosin VI–Binding Partners

The two‐hybrid approach in yeast has been used extensively during the past decade as a tool to identify and characterize macromolecular interactions. The yeast two‐hybrid system is an in vivo system that identifies interacting proteins by the reconstitution of active transcription factor complexes (Chien 1991, Fields 1989). A protein of interest is fused to a DNA‐binding domain of a transcription factor, while a second protein is fused to an activating domain. If the proteins of interest

Verification of Yeast Two‐Hybrid Screen Results

Since one of the major problems with the yeast two‐hybrid system is the isolation of false positive interactions, we used a range of different in vitro and in vivo assays to verify “true” myosin VI interacting proteins. First, we used a mammalian two‐hybrid system to demonstrate that the interactions occur in mammalian cells, where the expressed proteins are more likely to be in their native conformation. Furthermore, since myosin VI is not expressed in yeast, the mammalian cell is more likely

Mammalian Two‐Hybrid Assay

The principles of the mammalian two‐hybrid system are the same as for the yeast two‐hybrid system, namely that the protein of interest is fused to a DNA‐binding domain and the potential interacting protein is fused to an activating domain of a transcription factor. If the proteins of interest interact, an active transcription factor is formed and expression of the luciferase reporter occurs. Figure 2.1 illustrates the components of the mammalian two‐hybrid assay. Four plasmids carrying two

Protocol for Mammalian Two‐Hybrid Assay

The mammalian two‐hybrid assay was performed in CHO (Chinese hamster ovary) cells obtained from the European Collection of Animal Cell Cultures. CHO cells were cultured in F‐12 HAM medium supplemented with 10% fetal calf serum, 2 mM L‐glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37° and 5% CO₂. For transfection, the cells were seeded out and grown to about 50 to 60% confluency in 35‐mm tissue culture dishes. The cells were transfected with highly purified plasmid DNA using

SEAP Secretion Assay

Myosin VI and optineurin are present at the Golgi complex, and both proteins have been shown to play a role in maintenance of Golgi morphology and constitutive secretion (Sahlender 2005, Warner 2003)(Figure 2.3). To measure constitutive secretion, we expressed a secreted form of alkaline phosphatase (SEAP) in tissue culture cells, and quantified the release of the enzyme into the tissue culture medium. The SEAP used is an engineered variant of the placental alkaline phosphatase (PLAP), which is

Measurement of SEAP Secretion in Fibroblasts from Myosin VI Knockout Mouse

To measure secretion in cells lacking myosin VI, fibroblasts were isolated from the myosin VI knockout mouse (Snell's waltzer mouse). A litter of newborn mice was genotyped using a PCR‐based method, as described in Self et al. (1999). Skin and muscle tissue was cut into small pieces and incubated in 0.25% trypsin for 15 to 30 min at 37°. After gentle pipetting, large tissue lumps were allowed to fall to the bottom of the tube, and cell suspension was collected and the cells plated in growth

Measurement of SEAP Secretion in siRNA Knockdown Cells

To study protein secretion in siRNA knockdown cells, stable cell lines expressing SEAP were generated. The SEAP cDNA was subcloned from pSEAP2‐control plasmid into the mammalian expression vector pIRES‐neo2 using NheI and HpaI restriction enzymes. The resulting construct was transfected into two 35‐mm culture dishes of HeLa cells using FuGENE. Two days after transfection, the cells were trypsinized and transferred to a 10‐cm culture dish. Selection commenced the next day with 500 μg/ml of the

Protocol for SEAP Assay

Growth medium from control or siRNA‐treated SEAP expressing cells was collected and heated to 65° for 30 min. In a 96‐well plate, 10 μl of 0.05% Zwittergent (Calbiochem) (0.5% stock in water, dilute 1:10 before the assay) was mixed with 20 μl of heated culture medium (or 20 μl of heated growth medium for baseline measurements) followed by 200 μl of PNPP substrate. To prepare the substrate, PNPP (para‐nitrophenyl phosphate disodium salt, Sigma 104 phosphatase substrate) was dissolved in 1 M

Acknowledgments

This work was funded by a Wellcome Trust Senior Fellowship (F.B.) and supported by the Medical Research Council. The Cambridge Institute for Medical Research is the recipient of a strategic award from the Wellcome Trust.

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The intracellular movement and positioning of organelles and vesicles is mediated by the cytoskeleton and molecular motors. Small GTPases like Rab and Arf proteins are main regulators of intracellular transport by connecting membranes to cytoskeleton motors or adaptors. However, it is becoming clear that interactions between these small GTPases and the cytoskeleton are important not only for the regulation of membrane transport. In this review, we will cover our current understanding of the mechanisms underlying the connection between Rab and Arf GTPases and the cytoskeleton, with special emphasis on the double role of these interactions, not only in membrane trafficking but also in membrane and cytoskeleton remodeling. Furthermore, we will highlight the most recent findings about the fine control mechanisms of crosstalk between different members of Rab, Arf, and Rho families of small GTPases in the regulation of cytoskeleton organization.
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Thus, we show for the first time that T cell-derived exosomes are enriched with Optn, which may constitute a mechanism by which TCR signaling is dampened. In the same vein, it has been shown that Optn binds to other proteins involved in vesicular trafficking to the plasma membrane, such as myosin VI and Rab8 (Chibalina et al., 2008, 2010). Optn has also been reported to localize to the Golgi compartment (Sahlender et al., 2005; Sippl et al., 2014).
Optineurin (Optn) is an adaptor protein with homology to NF-κB essential modulator (NEMO), the regulatory subunit of the IκB kinase (IKK) complex. Dysregulation of Optn has been linked to neurodegenerative, autoimmune and bone diseases. Optn shares a high degree of homology with NEMO, but is not part of the same high-molecular weight complex containing IKKα and IKKβ. Despite its homology with NEMO and the fact that it has been the subject of extensive study in several cell types, there are no published studies addressing the role of Optn during T cell activation. Here we demonstrate that ectopic expression of Optn down-regulates TCR-induced NF-κB activation and TNF-α production, in a manner dependent on ubiquitin-binding. Conversely, knock-down of Optn enhances NF-κB activation and the production of TNF-α. Consistent with a negative regulatory role for this protein, we observed transient loss of Optn after TCR stimulation in both cell lines and in primary murine T cells. The acute loss of Optn appears to be due to both protein degradation and exocytosis, the latter via activation-induced exosomes. This study therefore provides novel information regarding the role of Optn during TCR activation, suggesting the possible importance of Optn during inflammation and/or autoimmune diseases.
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OPTN links myosin VI to the Golgi apparatus, hence it is very likely involved in the organization of the Golgi complex and exocytosis (Park et al., 2006; Sahlender et al., 2005). OPTN interacts with huntingtin, Rab8 and transferrin receptor, and regulates endocytic trafficking and recycling of transferrin receptor, which could be interrupted by the E50K mutant (Chibalina et al., 2008; del Toro et al., 2009; Nagabhushana et al., 2010; Park et al., 2010; Vaibhava et al., 2012). The E50K mutant has been shown to induce oxidative stress leading to retinal cell death (Chalasani et al., 2007), and defects in autophagy and vesicle trafficking are also major contributors to E50K-induced cell death (Chalasani et al., 2014).
Mutations in the optineurin gene (OPTN) have been found to be associated with glaucoma and amyotrophic lateral sclerosis (ALS). However, the mechanism by which this single gene mutation leads to neurodegeneration in those two diseases remains unrevealed. To study the roles of wild-type (WT) OPTN and its pathogenic mutants in neuronal survival, here we overexpressed SH-SY5Y cells with WT OPTN or its four mutants (E50K, M98K, Q398X and E478G), and detected their effects on neuronal viability under normal or oxidative stress conditions. We found that overexpression of WT OPTN or its glaucoma-linked mutants (E50K and M98K) causes little harm in SH-SY5Y cells, while ALS-associated OPTN mutants (Q398X and E478G) leads to remarkably increased oxidative status and decreased antioxidase activity, which might result in severe mitochondrial dysfunction and neuronal injury. Further investigation suggests that overexpression of WT OPTN promotes endogenous antioxidase activation in the SH-SY5Y cells under oxidative stress and increases neuronal survival. Nevertheless, this neuroprotective effect of WT OPTN is abolished by its four mutants. Our results indicate that oxidative stress may play a central role in the pathogenesis of glaucoma and ALS caused by OPTN mutation.
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The pathophysiology for POAG remains remarkably elusive, but studying OPTN's in vivo role may lead to mechanistic clues. Disrupted protein trafficking has been proposed as a glaucoma disease mechanism (Quigley and Addicks, 1980), and OPTN itself functions as an adaptor protein for motor-based vesicular transport (Chibalina et al., 2010, 2008; Sahlender et al., 2005). Loss of OPTN in zebrafish disrupts axonal trafficking in the spinal cord (Paulus and Link, 2014), and OPTN knockdown results in loss of cultured neurons (Akizuki et al., 2013).
Primary open-angle glaucoma (POAG) is characterized by progressive neurodegeneration of retinal ganglion cells (RGCs). Why RGCs degenerate in low-pressure POAG remains poorly understood. To gain mechanistic insights, we developed a novel mouse model based on a mutation in human optineurin associated with hereditary, low-pressure POAG. This mouse improves the design and phenotype of currently available optineurin mice, which showed high global overexpression. Although both 18-month-old optineurin and nontransgenic control mice showed an age-related decrease in healthy axons and RGCs, the expression of mutant optineurin enhanced axonal degeneration and decreased RGC survival. Mouse visual function was determined using visual evoked potentials, which revealed specific visual impairment in contrast sensitivity. The E50K optineurin transgenic mouse described here exhibited clinical features of POAG and may be useful for mechanistic dissection of POAG and therapeutic development.
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Rab8‐Optineurin‐Myosin VI: Analysis of Interactions and Functions in the Secretory Pathway

Abstract

Introduction

Section snippets

Identification of Myosin VI–Binding Partners

Verification of Yeast Two‐Hybrid Screen Results

Mammalian Two‐Hybrid Assay

Protocol for Mammalian Two‐Hybrid Assay

SEAP Secretion Assay

Measurement of SEAP Secretion in Fibroblasts from Myosin VI Knockout Mouse

Measurement of SEAP Secretion in siRNA Knockdown Cells

Protocol for SEAP Assay

Acknowledgments

Am. J. Hum. Genet.

Gene

J. Biol. Chem.

Neuron

Curr. Biol.

Methods Enzymol.

Methods Enzymol.

Dev. Biol.

Exp. Neurol.

The Rab8 GTPase selectively regulates AP‐1B‐dependent basolateral transport in polarized Madin‐Darby canine kidney cells

J. Cell Biol.

Myosin VI is required for sorting of AP‐1B dependent cargo to the basolateral domain in polarised MDCK cells

J. Cell Biol.

The mouse Snell's waltzer deafness gene encodes an unconventional myosin required for structural integrity of inner ear hair cells

Nat. Genet.

Myosin VI: Cellular fuctions and motor properties

Annu. Rev. Cell Dev. Biol.

The two‐hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest

Proc. Natl. Acad. Sci. USA

COP I domains required for coatomer integrity, and novel interactions with ARF and ARF‐GAP

EMBO J.