CLIC5 maintains lifelong structural integrity of sensory by promoting phosphorylation in hair cells of the inner

A thesis presented to The Honors Tutorial College, Ohio University

In partial fulfillment of the requirements for graduation from the Honors Tutorial College for the degree of Bachelor of Science in Biological Sciences

Benjamin Waddell Advisor: Dr. Mark Berryman Director of Studies: Dr. Soichi Tanda May 2016

ABSTRACT

Chloride intracellular channel 5 (CLIC5) and Radixin (RDX) are deafness-linked pro- teins required for in both and mice. Both are necessary for proper development and maintenance of delicate sensory stereocilia of inner-ear hair cells. Although the biochemical role of CLIC5 is poorly understood, recent research indicates that it functions in a multiprotein complex containing RDX, which is known to crosslink filaments to the plasma membrane. This study found that CLIC5 and

RDX are expressed in stereocilia of older adult animals, suggesting that the proteins are important for long-term maintenance of stereocilia. Additionally, a phosphorylated form of RDX, essential for regulating its membrane-cytoskeletal crosslinking activity was found in stereocilia of older adults. In CLIC5-deficient jitterbug mice (jbg), there was a significant reduction in phosphorylated RDX, suggesting that CLIC5 is a positive reg- ulator of RDX function in hair cells. A biochemical assay demonstrated direct binding of CLIC5 to the membrane binding domain of RDX. We propose that the interaction between CLIC5 and RDX is required for maintaining the structural integrity of stereo- cilia throughout life, which points to new therapeutic targets that may mitigate age- related . Experiments with cultured cells indicate that two highly conserved amino acids of CLIC5 (cysteine 32 and phenylalanine 227) are critical for the localiza- tion of CLIC5 to stereocilia-like structures as well as the creation of these critical sensory organelles.

______2 Waddell ACKNOWLEDGEMENTS

I cannot express how grateful I am to Dr. Mark Berryman for the tremendous amount of help he has given me over the past two years. He has done everything from teaching me how to transfect cells to taking me into his home for Thanksgiving dinner when all my friends were out of town. He and his family are some of the nicest people I’ve had the pleasure of meeting. Dr. Soichi Tanda given me direction not only through my thesis research, but also through my college career. He has always met with me, often on mo- ment’s notice, to give me guidance on how to succeed in my coursework, to point me in the right direction when I am getting off track, and to help me plan for my future after finishing my undergrad. I hope that I will someday be able to convey ideas as clearly as Dr. Tanda when explaining something as complex as fine dissection of a mouse . I am con- stantly amazed by the amount of time and effort that Dr. Berryman and Dr. Tanda dedicate to helping their students.

I would like to thank the Provost Undergraduate Research Fund (PURF) for sup- porting this research. A big thank you to Jeff Thuma for training me on the confocal microscope. Though I never met either of you, thank you to Liz Mathias and Katie Plona: aspects of this research would not have been possible without the data you collected. Thank you to Leona Gagnon and Ken Johnson of the Jackson Labs, both for the mice and for your contributions mentioned in this research. Thank you to OU Laboratory Animal Resources and to the Kopchick Laboratory for providing us with the mice needed for this research.

Finally, I would like to thank the Honors Tutorial College for providing me with amazing opportunities throughout my undergraduate year, including the opportunity to con- duct this research.

3 CONTENTS

Index of Tables and Figures ...... 5 Introduction

I. The importance of hearing loss ...... 6 II. Cell ...... 8 III. Actin and actin-based surface structures ...... 11 IV. Stereocilia of inner-ear hair cells ...... 15 V. ERM family: , Radixin, and ...... 20 VI. CLIC5 ...... 24

Experimental Design

I. Overview ...... 28 II. Model systems ...... 28 III. Research questions and hypotheses ...... 29

Materials and Methods ...... 34 Results ...... 46 Discussion ...... 63 References ...... 70 Appendix ...... 75

______4 Waddell INDEX OF TABLES AND FIGURES

Figure 1: Portion of the population with hearing loss dramatically increases with age...... 6 Figure 2: Variations in cell morphology between different cell types...... 8 Figure 3: The four basic tissue types of the body...... 8 Figure 4: Arrangement of the three types of cytoskeletal protein filaments in the cell...... 10 Figure 5: Illustration of a cross section of a sheet of epithelial cells with microvilli projections lining the apical surface...... 12 Figure 6: Microvilli of the brush border of the small intestine...... 13 Figure 7: Microvilli of the brush border of a kidney proximal tubule...... 13 Figure 8: The anatomy of the ear...... 15 Figure 9: Diagram presenting a cross section of the cochlea...... 16 Figure 10: Cross section of the ...... 17 Figure 11: Bundle of stereocilia protruding from a ...... 18 Figure 12: Structure and function of mechanosensory hair bundles...... 19 Figure 13: The three domains of Radixin...... 21 Figure 14: Model of stepwise activation and conformational regulation of ERM function...... 22 Figure 15: CLIC5 localizes to the base of the stereocilia...... 26 Figure 16: Using ImageJ to make an intensity profile of pERM staining in a single stereocilium...... 39 Figure 17: CLIC5 is expressed throughout adulthood in wild-type mice...... 47 Figure 18: RDX is expressed throughout adulthood in wild-type mice...... 47 Figure 19: CLIC5 mutant mice have lower levels of pERM in IHCs and OHCs during postnatal develop- ment...... 49 Figure 20: CLIC5 mutant mice have dramatically lower levels of pERM in the IHCs and OHCs during adult life...... 50 Figure 21: CLIC5 mutant mice have lower levels of pERM in stereocilia of vestibular hair cells ...... 51 Figure 22: pERM levels in mutant mice declined with age...... 52 Figure 23: Percentage of CLIC5 -/- mutant IHC with normal morphology rapidly declines with age, pERM levels correlate with normal morphology of the IHCs...... 53 Figure 24: Diffuse localization of pERM along the shaft of IHC stereocilia of 1-year old C57/B6J Jbg2J CLIC5 (+/-) heterozygous mice than 1-year old Balb/c CLIC5 +/+ animals...... 54 Figure 25: CLIC5 binds directly to RDX FERM domain...... 56 Figure 26: GFP-CLIC5F227S mislocalizes to the whereas GFP-CLIC5WT staining is stronger in the microvilli...... 59 Figure 27: GFP-CLIC5 staining in COS-7 cells transfected with various GFP-CLIC5 constructs...... 61 Figure 28: Quantification of COS-7 cells transfected with wild-type and various mutant GFP-CLIC5 con- structs...... 62 Figure 29: DNA gel showing genotyping of CLIC5 +/- and CLIC5 -/- Jbg2J mouse ...... 76

Table 1: DNA Constructs Used in this Study ...... 45 Table 2: CLIC5 -/- mutant mice had significantly lower pERM levels in IHC stereocilia than CLIC5 +/- heterozygous control animals at all ages ...... 51

5 INTRODUCTION

I. The importance of hearing

Hearing loss is underreported and often overlooked, but it is difficult to ignore its prev- alence in the aging population. A 2012 study from the Centers for Disease Control and

Prevention reported that almost a third of the population experience a noticeable degree of hearing loss by the age of 65, and that figure increases to nearly half of the population beyond age 75 (Fig. 1) (Blackwell et al., 2014).

Figure 1. Percent of the population with hearing loss dramatically increases with age. Data from Centers for Disease Control National Health Interview Survey, 2012 (Blackwell et al., 2014).

Hearing loss has a profound impact on cognitive and physical function, inde- pendence, and general quality of life. The detriment to quality of life affects family members and loved ones, as hearing loss strains communication within these relation- ships (Bainbridge and Wallhagen, 2014). The economic impact of hearing loss must also be taken into consideration, as it affects not only individuals, but society as a whole.

Studies have shown that people with communication disorders, particularly deafness, are more likely to be unemployed or earn lower wages than unaffected indi- viduals (Ruben, 2000). Altogether, communication disorders are estimated to cost the

______6 Waddell United States 2.5% to 3% of its Gross Domestic Product each year; in 2013, that would mean a loss of around $420 billion to $500 billion (Ruben, 2000; The World Bank,

2015). By 2030, the World Health Organization predicts that hearing loss will be one of the top ten costliest health conditions in developed countries due to the prevalence of hearing loss increasing with increased life expectancy, as well as prevalence of hearing loss in the younger population increasing due in part to noise exposure, particularly from in-ear headphones (World Health Organization, 2004).

Approximately 360 million people, over 5% of the world’s population, live with disabling hearing loss (World Health Organization, 2015). Disabling hearing loss is de- fined by the World Health Organization as a loss of greater than 40 decibels (dB) in the better-hearing ear. This is the difference between quiet conversation, which is about 50 dB, and a loud food blender, which is about 90 dB.

In my research with Dr. Mark Berryman and Dr. Soichi Tanda, we investigated the role of a CLIC5 (Chloride Intracellular Channel protein 5) in the inner-ear hair cells, which are essential for hearing. CLIC5 plays a key role in development and maintenance of stereocilia, tiny subcellular structures that are key to the function of hair cells (Gag- non et al., 2006; Salles et al., 2014). The aim of this research was to gain a better understanding of the workings of the at the molecular level with the goal of identifying potential therapeutic targets for strengthening important inner ear sensory structures.

7 II. Cell cytoskeleton

The human body is composed of over 200 different types of cells (Fig. 2), each of which has a specialized function, such as digesting food, moving the body, or integrating sen- sory information from the outside world (Cooper and Hausman, 2013). This vast array of cell types can be grouped into four tissue types: muscle tissue, connective tissue, nervous tissue, and epithelial tissue (Fig. 3).

Figure 2. Variations in cell morphology between different cell types. Source: Human Anatomy (7th edition) Martini et al., 2011.

Figure 3. The four basic tissue types of the human body. Source: U.S. National Library of Medicine (2013), nlm.nih.gov/medlineplus/ency/imagepages/8682.htm

______8 Waddell Cell morphology reflects a cell’s role in the body. For example, muscle cells are elongated and able to contract, thereby creating motion. Nerve cells, called neurons, have branching projections to other nerve cells, forming a chain of cell-to-cell commu- nication for transmitting electrical signals throughout the body. Many connective tissue cells are organized in a manner to support and protect the body, as in bone and cartilage.

Generally, epithelial cells group together to form a sheet covering surfaces in or on the body. This characteristic serves different purposes in different types of epithelial cells: for protection—often with sheet-like cells like those of the skin, for secretion—often with globular cells like those of mucous membranes, and for absorption—often with columnar cells that display elaborate structures that increase the surface area for absorp- tion, as in the intestine and kidney (Cooper and Hausman, 2013). A key morphological characteristic of epithelial cells involved in absorption is their finger-like projections, called microvilli, which protrude from the apical surface of the cell. Microvilli increase absorption efficiency by increasing the surface area for absorption. For example, micro- villi in the brush-border of cells in the small intestine facilitate in enzymatic breakdown, transport, and absorption of nutrients (Crawley et al., 2014).

The differences in the shape and mechanical functions of various cell types of the body are controlled in large part by differences in the architecture of the cytoskeleton

(Fletcher and Mullins, 2010). The cytoskeleton is a network of protein filaments that determine the internal structural framework of the cell. Just as girders provide stability and determine the general shape of a building, the cytoskeleton provides the cell with both its stability and its shape. The cytoskeleton is composed of three types of protein

9 filaments: , intermediate filaments, and actin filaments (also called micro- filaments, or F-actin). The protein filaments themselves are polymers composed of smaller protein subunits. In F-actin and microtubules, each monomeric protein subunit possesses intrinsic structural polarity, such that when aligned in a head-to-tail fashion to form a linear polymer, the filaments are structurally and functionally polarized, with distinct plus end and minus ends (Fletcher and Mullins, 2010). Conversely, protein sub- units in intermediate filaments organize in a symmetrical, tetramer pattern. This symmetry results in no distinct plus or minus ends, resulting in the complete polymeric filament being non-polar. Each type of cytoskeletal protein is composed of different monomers and has a unique pattern of organization within the cell (Fig. 4). The bio- physical properties, subcellular organization, and dynamic activity (assembly, disassembly, turnover rate) of cytoskeletal filaments are modulated by a cadre of spe- cific regulatory proteins unique to each type of filament.

Figure 4. Arrangement of the three types of cytoskeletal protein filaments in the cell. Source: The Open University (2016), www.open.edu/openlearn/ocw/mod/oucontent/view.php?id=13785&printable=1

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Microtubules are the largest of the filament types, being about 25 nm in diameter

(O’Connor and Adams, 2010). They are rigid, hollow rods composed of the proteins alpha and beta . Microtubules separate the during cell division, serve as tracks for transport of membrane vesicles around the cell, and form motile cilia, which line the surface of certain epithelial cells to facilitate movement of extracellular substances. Intermediate filaments are about 10 nm in diameter and play a role in cell- to-cell adhesion (interactions with ) and cell-to-matrix adhesion (interac- tions with ), and form the nuclear lamina, which provides support for the nuclear envelope during cell division (O’Connor and Adams, 2010). They are com- posed of several families of proteins including , desmins, and . Finally, actin filaments are about 6 nm in diameter, earning the name (Cooper and Hausman, 2013). The proteins of interest in the research presented in this thesis are known to associate with the actin cytoskeleton.

III. Actin and actin-based surface structures

Actin is the most abundant structural protein of most cell types and is the building block for actin filaments. Of the three types of cytoskeletal filaments, actin filaments are most abundant beneath the plasma membrane of the cell. Together, the plasma membrane and the actin filaments just beneath this membrane form the cell cortex. This is especially true in epithelial cells, where actin filaments provide structural support for the plasma membrane, dictate the shape of cell surfaces, and control movements associated with

11 the membrane (Cooper and Hausman, 2013). Some specialized functions—such as nu- trient absorption by epithelial cells lining the small intestine and the detection of sound by sensory cells in the inner ear—rely on elaborate arrays of actin-based projections that line cell surfaces.

As shown in Fig. 5, actin-based surface structures project from the apical surface of the cell, which is the surface of the cell that is exposed to inside of a body tube or cavity. For example, in the small intestine, the microvilli project toward the gut lumen where the inner intestinal contents are digested and absorbed (Cooper and Hausman,

2013). At the opposite end of the cell is the basal surface, which anchors the cell to the basement membrane. The basement membrane connects the cell to other tissues that comprise the organ in which the cell functions.

Figure 5. Illustration of a cross section of a sheet of epithelial cells with microvilli projections lin- ing the apical surface. Source: Dorland’s Illustrated Medical Dictionary, 32nd edition (2010)

Actin protein monomers (G-actin) are polymerized into filaments (F-actin).

Each G-actin monomer is polar, and G-actin monomers are oriented in a uniform direc- tion when forming F-actin filaments; therefore, these filaments are polarized with a plus

______12 Waddell and minus end (Fletcher and Mullins, 2010). In most of these filaments, the plus end, or fastest growing end, of actin filaments is the end that associates with the plasma mem- brane. Actin filaments are organized into one of two main types of arrangements: actin networks and actin bundles. In actin networks, filaments are cross-linked into a criss- crossed, branching, three-dimensional meshwork of filaments, functioning to support the surface of the plasma membrane. Filaments of actin bundles are cross-linked into a tight, parallel form with all of the filaments within a bundle having the same polarity.

Parallel actin bundles support cell projections, such as the microvilli of intestine and kidney epithelial cells and stereocilia of sensory hair cells of the inner ear (Crawley et al., 2014; Drummond et al., 2012).

Figure 6. Microvilli of the brush border of Figure 7. Microvilli of the brush border of a the small intestine. Closely packed microvilli kidney proximal tubule. Again, microvilli line of uniform length and diameter make up the the lumen of the proximal tubule, greatly in- brush border that lines the of the creasing the surface area to facilitate efficient small intestine. The microvilli increase the sur- reabsorption of fluid and other substances in the face area 30-fold; thereby increasing capacity filtrate. for absorption of nutrients (Crawley et al., 2014). Source: King, 2013. Source: McDarby, 2008.

13 Microvilli create a prominent brush border in epithelial cells of the small intes- tine (Fig. 6) and in the proximal convoluted tubule of the kidney (Fig. 7). In cases where they create a brush border on the apical surface of epithelial cells, microvilli have a characteristic size, shape, and packing density with remarkable uniformity within and between cells (Crawley et al., 2014). Stereocilia are closely related to microvilli and can be considered structurally modified versions of microvilli. In fact, stereocilia begin their development as precursor microvilli. Though stereocilia are typically longer with a greater diameter, both stereocilia and microvilli are apical membrane protrusions sup- ported by a core bundle of parallel actin filaments (Zheng, 2010).

In actin-based structures like microvilli and stereocilia, there is a constant turn- over of G-actin within F-actin filaments, a process called actin-treadmilling (O’Connor and Adams, 2010; Cooper and Hausman, 2013). This process consists of a constant polymerization at the plus end and depolymerization at the minus end. Treadmilling is required for assembly and maintenance of actin filaments. If the rate of monomer addi- tion at the plus end is faster than the depolymerization at the minus end, the filament will grow. If the rate at each end is equal, the filament will maintain its current length.

If the rate of depolymerization is faster than the rate of polymerization, the filament will decrease in length. A plethora of proteins control the rate of treadmilling, some of which will be discussed in the following sections.

______14 Waddell IV. Stereocilia of inner-ear hair cells

To better understand the function of sensory stereocilia, we must first look at the over- arching anatomical mechanism of hearing (Fig. 8). First, sound waves are directed by the external ear (pinna) into the external auditory meatus, or the . At the end of the canal is the tympanic membrane, colloquially called the . This membrane is vibrated by sound waves and transmits these vibrations to the air-filled , which is composed of three bony, auditory : the , the , and the sta- pes. The transmits vibrations to the of the cochlea, which turns these vibrations into pressure waves within the fluid-filled cochlea of the inner ear.

Figure 8. The anatomy of the ear. The pathway by which sound waves in air are conducted through the , converted to vibrations in the middle ear, converted into pressure waves in fluid of the inner ear, and fi- nally converted to an electric stimulus transferred by the auditory nerve to the brain. Source: Krames, 2008.

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Figure 9. Diagram presenting a cross section of the coch- lea. Three compartments: the scala vestibuli, scala media, and scala tympani. Source: Hudspeth, 2013.

The inner ear contains two mechanosensory systems: the , which serves to give the sensation of equilibrium (balance), and the cochlear system, which serves to give the sensation of sound. The vestibular and cochlear systems are both housed within the , which, as its name suggests, is a complex, wind- ing bone. The vestibular portion contains a group of sensory organs: three at three different angles, which detect angular acceleration with different rota- tions of the head and help balance the body, and the and , which play roles in detecting linear accelerations. The sensory organ of the cochlear system, the organ of

Corti, is enclosed in a fluid-filled, snail-shell-shaped portion of the bony labyrinth called the cochlea.

Inside the cochlea are three membrane-bound tubes coiled in parallel: an upper, middle, and lower compartment (Fig. 9). The upper compartment—the scala vestibuli— is separated from the middle ear by the membrane of the oval window, as previously mentioned, and the lower compartment—the scala tympani—is separated from the mid- dle ear by the . The middle compartment is called the scala media, and

______16 Waddell the floor is formed by the . The basilar membrane houses the sensory organ of Corti (Fig. 10), which is lined with approximately 16,000 hair cells. These cells are organized in four parallel rows, which can be divided into three rows of outer hair cells and one row of inner hair cells, with stereocilia projecting from each cell’s surface

(Schwander et al., 2010). The hair cells are surrounded by non-sensory support cells such as pillar cells.

Stereocilia are highly specialized actin-containing structures of the inner ear, formed from several hundred parallel actin filaments, which are bundled together in a uniformly polarized manner by special actin-bundling proteins. Stereocilia are found on the apical surface of hair cells, both in the cochlear and the vestibular system. Each hair cell has a cluster of stereocilia projecting from its apical surface, forming a functional unit called a hair bundle (Fig. 11). The bundles on each hair cell are organized in a staircase-like shape with sequentially taller rows of stereocilia (Holt and Corey, 2000).

Bundles are planar polarized; that is, their spatial orientation on each cell is tightly con- trolled to orient the shortest row toward the middle of the cochlea and the tallest row laterally (Hudspeth, 2013).

Figure 10. Cross section of the organ of Corti. The organ of Corti contains three rows of outer hair cells (OHC) and one row of inner hair cells (IHC). Pressure waves in the basilar membrane cause de- flections in the IHCs, the opening of channels. Afferent nerves transfer signals from the IHCs to the brain. Source: Lu and Sipe, 2015. ______17

Figure 11: Bundle of stereocilia protruding from a hair cell. A scanning electron micrograph of a turtle hair bundle. Note the staircase-like shape and tightly organized arrangement of the rod-shaped stereocilia. Arrow pointing too tallest row. Source: Fettiplace and Kim, 2014.

As vibrations are transmitted from the stapes of the middle ear to the oval win- dow, the fluid () in the scala vestibuli is transformed into fluid waves which propagates toward the apex of the cochlear spiral. These fluid pressure waves result in the vibration of the basilar membrane, and this stimulus is transmitted through the organ of Corti, causing the hair bundles to deflect toward the tallest row of stereocilia, physi- cally opening mechanically gated ion channels (Hudspeth, 2013). Each stereocilia is tapered at the base, allowing for a widened range of deflection in response to stimulation

(Holt and Corey, 2000). Upon deflection and opening of ion channels, the cells depo- larize, which causes the release of from the basal end of the cell. These neurotransmitters bind postsynaptic (afferent) neurons, generating impulses in the audi- tory cortex of the brain, which are interpreted as sound (Hudspeth, 2013). Inner hair cells are responsible for converting the energy created by this stimulus to an electrical impulse to the brain via the ; these impulses are interpreted by the brain as sound. The tips of the tallest row of stereocilia in outer hair cells are embedded in the , a specialized extracellular matrix (Fig. 10). The role of the tectorial

______18 Waddell membrane is not fully understood, but mathematical models have indicated that its role may be longitudinal propagation of energy, enabling a higher basilar membrane sensi- tivity (Meaud and Grosh, 2010). Outer hair cells send little information to the brain but, instead, are involved in a process called cochlear amplification, which magnifies the effects of basilar membrane vibrations via electromotility, increasing the strength of signals reaching inner-ear hair cells (Schwander et al., 2010). Unlike many other cells in the body, mammalian hair cells are unable to regenerate, so damage caused by loud noises or chemical toxins (e.g., certain antibiotics and chemotherapy drugs) accumulates throughout life and is irreparable with current medical interventions (Hawkins and

Lovett, 2004).

Figure 12. Structure and function of mecha- nosensory hair bundles. (A) Scanning electron micrograph at 2,500x magnification of two hair bundles in the inner ear of a bull frog. These bun- dles are about 8 µm tall and contain 50-60 stereocilia. (B) Transmission electron micro- graph at 50,000x magnification of a stereocilium and its adjacent neighbor in the “staircase” of the hair bundle. Note the faint filamentous tip-link from the top of the shorter stereocilia to the side of its neighbor. (C) Diagram of the hair bundle during deflection, causing a positive stimulus and opening of ion channels via (D) mechanically gated ion channels. This is called the gating spring model of transduction. In (A) and (C) note the tapered base of the stereocilia and in (C) note how this tapered base allows the stereocilia to pivot rather than bend. Source: Holt and Corey, 2000.

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Many hair-bundle proteins play important roles in defining the structure of ste- reocilia. For example, two proteins called Protocadherin-15 and -23 bind together to form the tip-links of stereocilia (Fig. 12B and D), which interconnect the individual stereocilia within a bundle. These tip-links are hypothesized to be the site of mechanotransduction; that is, they are thought to open spring-gated ion channels upon deflection of the stereocilia (Fig. 12) (Holt and Corey, 2000). Other proteins in the ste- reocilia—including , TRIOBP, and Fimbrin—are among a large group of proteins known to regulate and model the organization of the F-actin within the stereocilia. For example, TRIOBP has been implicated in maintaining the rigidity of the stereocilia by tightly organizing actin filaments in the rootlet of stereocilia (Kitajiri et al., 2010). Espin and Fimbrin help maintain this organization by crosslinking adjacent actin filaments along the length of the shaft of stereocilia (Volkmann et al., 2001; Zheng et al., 2014).

Another group of proteins localize at the tapered base of the stereocilia: the ERM protein

Radixin (RDX), the -motor protein Myosin VI (MYO6), Taperin (TPRN), pro- tein tyrosine phosphatase receptor type Q (PTPRQ) and CLIC5 (Salles et al., 2014).

Research published in 2014 by Salles et al. suggested that these proteins may interact in a complex at the base of the stereocilia to link F-actin to the membrane, thereby playing a crucial role in the development and maintenance of hair cell stereocilia.

V. ERM protein family: Ezrin, Radixin, and Moesin

The ERM (Ezrin/Radixin/Moesin) protein family consists of three closely related pro- teins implicated in connecting actin filaments to the plasma membrane. ERM proteins have three domains: a 300 amino acid residue FERM domain (F for 4.1 protein, ERM

______20 Waddell for Ezrin/Radixin/Moesin) at the N-terminus; a 100-residue C-ERMAD domain (stand- ing for C-terminus ERM Association Domain) at the C-terminus; and a 200-residue, α- helical domain connecting FERM and C-ERMAD (Fig. 13) (McClatchey, 2014).

Figure 13. The three domains of Radixin. The N-terminal FERM domain (purple), the C-ERMAD region (orange), and the α-helical domain (white) connecting the two. Binding site in gray is for EBP50, also called NHERF1, a scaffolding protein. Also at the FERM domain is the site for PIP2 binding. The F- actin binding site at the C-ERMAD is where the molecule binds filamentous actin. Curtosey Dr. Mark Berryman and Dr. Soichi Tanda

ERM proteins exist in the cell in at least two distinct conformational states: (1) inactive, where the FERM domain is intramolecularly bound to the C-ERMAD domain; and (2) active, where the two domains are not directly bound, and the ERM protein is able to perform its membrane-actin linking function (Gary and Bretscher 1995;

McClatchey, 2014). In the inactive state, ERM proteins are free in the , interact- ing with no protein other than themselves and a small group of ligands such as SCYL3 and FHOD1 (Viswanatha et al., 2013). Inactive ERM proteins become active by the binding of the N-terminus FERM domain to the phospholipid phosphatidylinositol 4,5- bisphosphate (PI(4,5)P2 or PIP2) and by phosphorylation at a conserved threonine resi- due within the C-ERMAD (T564 in RDX, see Fig. 13) by Rho kinase or a host of other protein kinases (Yu et al., 2011; Jayasundar et al., 2012). Activation of ERM proteins relieves the intramolecular association between the FERM domain and C-ERMAD, thereby allowing these two domains to crosslink cytoskeletal F-actin to the membrane ______21 by direct association with transmembrane proteins, such as CD43, CD33, ICAM-1 and

ICAM-2 (Bretscher et al., 1997; Yu et al., 2011). A model for the activation of ERM protein RDX is shown in Fig. 14.

Figure 14. Model of stepwise activation and conformational regulation of ERM function. In the cytosol, ERM protein (RDX in this model) is in an inactive state whereby the membrane binding domain is bound to the F-actin binding domain, preventing the molecule from binding F-actin. Step 1: The mem- brane binding domain of inactive RDX binds membrane lipid PIP2. Step 2: A kinase phosphorylates RDX at T564, inducing a conformational change that activates RDX, exposing the F-actin binding site. Active RDX then links F-actin to membrane proteins. Step 3: RDX can be dephosphorylated by a phosphatase and return to its inactive state. Model adapted from Bretscher et al., 2000.

There are two modes of membrane-ERM linkage: one where the FERM domain interacts with membrane proteins directly, and one where the FERM domain interacts via scaffolding proteins. ERM proteins bound via the FERM domain to the membrane are able to bind scaffolding proteins NHERF1 (also known as EBP50) and NHERF2

(Bretscher et al., 1997; Yu et al., 2011). As their name suggests, scaffolding proteins

______22 Waddell are like scaffolds on a building under construction, connecting multiple parts of a com- plex. NHERF1 was identified by affinity chromatography to find binding partners of the N-terminal domain of Ezrin (Reczek et al., 1997). NHERF1 and NHERF2 bind ERM and, in turn, directly associate with several types of membrane proteins (Yu et al., 2011).

ERM proteins bind to the C-terminus of NHERF proteins.

To conclude, ERM are conformationally regulated by PIP2 membrane lipid bind- ing and phosphorylation at a conserved threonine residue. The active form of ERM binds F-actin at the exposed C-ERMAD domain and the N-terminal FERM domain can bind PIP2, membrane proteins, or scaffolding proteins NHERF1/2, which, in turn, asso- ciate with membrane proteins.

Zhu et al. (2007) have proposed a model of ‘phosphocycling,’ where ERM ac- tivity is regulated by multiple rounds of phosphorylation and dephosphorylation at the conserved threonine within the C-ERMAD domain (Thr567 in Ezrin, Thr564 in RDX,

Thr558 in Moesin). This model also suggests that ERM proteins closely associate with kinase and phosphatase enzymes that facilitate the phosphocycling of ERM, thus main- taining the dynamic relationship between ERM activity and membrane-actin binding

(Zhu et al., 2007). Goodyear et al. (2014) demonstrated that use of a broad spectrum kinase inhibitor in mouse cochlear cultures reduced the levels of phosphorylated ERM protein in the cells, which coincided with a loss of regular hair bundle morphology, suggesting that the integrity of the hair bundle may depend on the phosphorylation status of ERM proteins (Goodyear et al., 2014).

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This research focuses on the ERM protein RDX because it is a known deafness- linked in both mice and humans and has been suggested to interact as part of a protein complex with my protein of interest, CLIC5 (Kitajiri et al., 2004; Khan et al.,

2007; Salles et al., 2014). Interestingly, there may be some functional redundancy of the

ERM proteins: Kitajiri et al. (2004) found that in RDX-deficient knockout mice, stere- ocilia still developed with upregulation of Ezrin protein expression, but progressively degenerated after the end of development (Kitajiri et al., 2004).

VI. CLIC5

CLIC5 (Chloride Intracellular Channel 5) is a protein first isolated from extracts of pla- cental microvilli and is encoded by the CLIC5 gene (Berryman and Bretscher, 2000).

Using an immobilized, truncated C-terminus of the Ezrin molecule as a probe, this pre- viously undiscovered protein was isolated as part of a complex containing the C- terminus of Ezrin and a multitude of F-actin-associated proteins, including IQGAP1, α- , , and actin itself. This newly discovered protein had high amino-acid- to p64, a bovine protein. The novel protein was

60-70% identical to proteins CLIC1, 2, 3, and 4, and, therefore, was named the newest member of the CLIC family of proteins: CLIC5. The CLIC family of proteins is now known to have six forms in vertebrates (CLIC1-6). The role of CLIC proteins seems to be regulating membrane-actin linkage. The term “chloride intracellular channel protein” may be a misnomer; the current understanding of CLIC proteins is that they function not as ion channels but perhaps as channel modulators (Littler et al., 2010).

______24 Waddell CLIC5 is crucial for proper hearing and balance. CLIC5 associates with the actin cytoskeleton located just underneath the and is required for the proper development and maintenance of the stereocilia of the inner ear. It has been shown that decreasing CLIC5 expression leads to progressive hearing loss in both humans and mice

(Gagnon et al., 2006; Seco et al., 2015). Mice homozygous for a recessive

(named jitterbug, jbg) in the CLIC5 gene, show loss of both hearing and balance, which indicates that all sensory hair cells in the inner ear are affected (Gagnon et al., 2006).

This mutation is called jitterbug because it is associated with behavioral attributes com- monly observed in mice with vestibular dysfunction, including hyperactivity, head bobbing, inability to swim, and frequent bidirectional circling.

CLIC5 has been implicated in acting in conjunction with ERM proteins to con- trol processes at the cell membrane, including the development of actin-based surface structures such as microvilli and stereocilia (Gagnon et al., 2006; Jiang et al., 2014;

Salles et al., 2014). It was previously reported that phosphorylated ERM levels are sig- nificantly lower in the renal glomeruli of CLIC5-deficient jitterbug mutant mice than in wild-type mice (Wegner et al., 2010; Al-Momany et al., 2014). Also, overexpressing

CLIC5 has been shown to promote the formation of actin-rich surface structures con- taining phosphorylated ERM (pERM) (Al-Momany et al., 2014). These results suggest that CLIC5 may function to positively regulate the phosphorylation status of ERM pro- teins, allowing ERM proteins to exist in the active conformation and to bind actin to the membrane to develop or maintain stereocilia.

______25

Figure 15. CLIC5 localizes to the base of the stereocilia. Fluorescence confocal micrographs show CLIC5 concentrates at the base of stereocilia. Photographs were taken in an adult mouse organ of Corti. Here, F-actin is stained red using phalloidin and CLIC5 is stained green using a specific antibody. In both the IHCs (A) and OHCs (B), CLIC5 is concentrated at the base of each stereocilia (arrows). Both scale bars: 10 µm. Source: Salles et al., 2014.

Research by Gagnon et al. (2006) and Salles et al. (2014) comparing mice with Fig. 3. CLIC5 concentrates at the base of the hair bundle. (A, B) Fluorescence confocal micrographs of adult rat whole mount organ of Corti preparations stained with specific antibody (APB56) against CLIC5 (green); actin filaments were counterstained with phal- loidin (red). Specimens were gently squashed to facilitate visualization of staining along the proximal-distal axis of stereocilia; note functionalthat requisite copies use of heat-inducedof the CLIC5 antigen retrieval gene compromised to CLIC5 the morphological-deficient preservation jitterbug of bundles mice as well asdemonstrated the quality of F-actin staining. In both inner (A) and outer (B) hair cells, CLIC5 is concentrated at the base of stereocilia (arrows). (C–E) Expres- sion of GFP-CLIC5 fusion protein in vestibular hair cell. The transfected cell (C) exhibits accumulation of CLIC5 towards the prox- that CLIC5imal end oflocalizes the stereocilia. at (D) the Actin stereociliary filaments were counterstained base in with both phalloidin developing (red). (E) Merged and images mature of C and D.hair Scale cells bars: 5 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] (Fig. 15). The stereociliary base can be defined as a sub-compartment of the stereocil- P21, TPRN localization was confined to the base of control mal in P5 mutant cells (data not shown). The altered distri- stereocilia (Figs. 6M and 6O, arrows); however, in mutant butions of PTPRQ and RDX before completion of hair cell ium thatcells includes TPRN staining the wastapered punctuated region and distributed at proximalmaturation end inclosest the mutant to were the confirmed cell body by quantitative as well as throughout both fused (Figs. 6P and 6R, arrows) and analyses of pixel intensity profiles taken from the base to unfused (Figs. 6P and 6R, arrowheads) stereocilia. These the tip of stereocilia at P10 (Fig. 8). Together, these findings data suggest that CLIC5 may play a role in maintaining the indicate that CLIC5 influences the proper localization of the membranelocalization of and PTPRQ, closely RDX, andassociated TPRN at the actin base of filamentsPTPRQ and between RDX to the individual base of stereocilia stereocilia during post-. Sal- stereocilia after hair cells mature (P17). natal maturation of hair bundles. Prior to hair cell maturation, at P10, PTPRQ was les et restrictedal. also to demonstrated the base of stereocilia that (Figs. that 7A CLIC5 and 7C, is required for proper localization of RDX arrows); however, this localization was altered in the jbg CLIC5 Interacts Biochemically with ERM mutant even in cells where individual stereocilia were dis- Proteins and TPRN and PTPRQtinguishable at (Figs.the 7Dbase and of 7F, the arrow). stereocilia Localization (Salles of Previous et al., studies 2014). have shown that CLIC5 interacts with PTPRQ, although still stronger at the base, was not multiple proteins in distinct tissues and subcellular com- restricted to this compartment, but also was seen along the partments. CLIC5 associates with actin, ezrin, a-actinin, shaftResearchers of stereocilia (Figs. in 7Dthe and field 7F, arrow). of activity Similarly, at-basedgelsolin, protein and IQGAP1 profiling in placental have microvilli screen [Berrymaned native P7 RDX was more dispersed along the length of mutant and Bretscher, 2000; Berryman et al., 2004], with ezrin and (Figs. 7J and 7L, arrow) than control stereocilia (Figs. 7G podocalyxin in kidney podocyte processes [Pierchala et al., proteomesand 7I, arrows).using Inhighly contrast, TPRNreactive localization chemical at the base probes2010; Wegnerto identify et al., 2010], reactive and with amino AKAP350, acids a large sites. of stereocilia appeared similar between control (Figs. 7M scaffolding protein that concentrates at the centrosome and and 7O, arrows) and mutant (Figs. 7P and 7R, arrows) the Golgi apparatus [Shanks et al., 2002a,b]. Based on these One suchinner hairstudy cells; likewise,determined TPRN localization that a appeared conserved nor- findings, cysteine we hypothesized residue that in CLIC5 CLIC1, might beCLIC4, part of a and

CLIC5CYTOSKELETON (amino acid position 32 in CLIC5, hereafterCLIC5 termed Links Membrane-Actin C32) is Core highly in Stereocilia reactive,65 ᭿ sug- gesting that this amino acid plays an important role in the biochemical function of

CLICs (Weerapana et al., 2008). In a separate study, Ponsioen et al. (2009) found that a cysteine at the corresponding position in CLIC4 is required for its rapid translocation

______26 Waddell from the cytosol to the plasma membrane following stimulation of RhoA through G12- coupled membrane receptors. Given that this cysteine residue is conserved in all 6 ver- tebrate CLIC proteins and resides in a region of the protein that could have redox activity, it was proposed to constitute an enzymatic-reactive site (Ponsioen et al., 2009); however, no specific substrates have yet been identified.

In yet another study, unpublished research by Kathleen Plona revealed that mu- tations of the C32 residue to an alanine (designated C32A) significantly reduced the localization of CLIC5 to microvilli of LLC-PK1 epithelial cells, supporting the notion that this amino acid residue plays a role in CLIC5’s localization at the cell cortex.

In unpublished research, Dr. Kenneth Johnson (The Jackson Laboratory) found that an N-ethyl-N-nitrosourea-induced missense mutation in C57BL/6J mice (desig- nated nmf318) changed a phenylalanine residue at amino acid position 227 of CLIC5 to a serine (designated F227S) leading to profound deafness. Presumably, these do not affect the level of CLIC5 protein expression, rather they may affect CLIC5’s stability, conformation, or ability to interact with other proteins.

______27

EXPERIMENTAL DESIGN

I. Overview

My research concerns the stereocilia of inner-ear hair cells: the mechanosensory orga- nelle, which is the site at which mechanical stimuli from sound waves are transformed into electrical signals sent to the brain. The focus of my research is on how CLIC5 interacts with ERM proteins and what role this interaction plays in the development and maintenance of the stereocilia of inner-ear hair cells. As discussed previously, CLIC5 is essential for hearing, but how it functions at the molecular level is still not well un- derstood.

II. Model systems

Mice were used as a tractable model system: their genome is relatively easy to manipu- late and analyze and they are easy to breed. Unlike other common model systems such as worms and flies (which are excellent models for developmental processes), mice are , making them far better models for complex physiological processes shared with humans. For research purposes, thousands of mouse mutant strains have been bred and used to study disorders caused by certain . In fact, a current initiative called the Knockout Mouse Project aims to generate a comprehensive and public resource con- sisting of mouse embryonic stem cells containing null mutations in each individual gene in the mouse genome. The mouse is an ideal model for human deafness because the cochlea of mice is similar to that of humans and can be easily dissected. In addition, the

______28 Waddell morphology and molecular composition of hair cells can be readily observed under scanning electron microscopy and confocal microscopy, respectively.

The other model system used in this research consisted of cultured mammalian cells which can be propagated continuously using defined growth media. COS-7 fibro- blast-like cells are derived from monkey kidney tissue and transformed by Simian virus

40. In general, these cells do not have prominent actin-based cell surface structures, such as microvilli and membrane ruffles. Hence, COS-7 cells can be used in transient transfection experiments aiming to induce actin-based surface structures, as in the case of overexpression of certain actin regulatory genes (Viswanatha et al., 2013). LLC-PK1 pig kidney epithelial cells display abundant actin-based cell-surface microvilli, which are the precursor to stereocilia. Thus, LLC-PK1 cells have been used as a model system to study the effects of actin regulatory genes on stereocilia development (Zheng et al.,

2010).

III. Research questions and hypotheses

Our overarching research question was this: Does CLIC5 interact with ERM proteins in the stereocilia throughout life? Based on the research publications mentioned above and on our preliminary results, we hypothesize that CLIC5 promotes or stabilizes the phos- phorylated, conformationally active form of ERM proteins. We performed various experiments, approaching our hypothesis from different angles in order to form a larger picture of the workings of the CLIC5-ERM interaction. The experiments performed fol- lowed three lines of questioning.

______29

1. Does CLIC5 positively regulate the phosphorylation status of ERM protein in

inner-ear hair cells?

Hypothesis: CLIC5 regulates the conformation and activity of ERM by promoting nor- mal, steady-state levels of phosphorylated ERM (pERM), and this role is critical to maintaining membrane-cytoskeletal attachments at the base of the stereocilia and the overall structural integrity of stereocilia throughout adult life.

Rationale: CLIC5 promotes phosphorylation of Ezrin in podocytes and COS-7 cells

(Al-Momany et al., 2014). In the podocytes of CLIC5-deficient jitterbug mice, pERM levels are lower than they are in wild-type (Wegner et al., 2010).

Experimental design: Two experiments in mouse-model system.

1.1 Verify expression of CLIC5, RDX, and pERM in inner-ear hair cells of older

adult mice. If these proteins are not present in older adult mice with healthy

hearing, this would indicate to us that these proteins were nonessential to the

maintenance of the stereocilia, and our hypothesis would be rejected. Using a

protocol similar to Salles et al. (2014) and previously characterized antibodies,

we fixed and stained the organ of Corti of wild-type mice with functional CLIC5

and RDX genes. Tissue samples were double-stained using antibodies or phal-

loidin to label actin, which allows us to visualize the stereocilia and antibodies

______30 Waddell against either (1) CLIC5, (2) RDX, or (3) pERM—which would recognize phos-

phorylated RDX. We used dual-color immunofluorescence such that actin was

labeled red and antibodies against CLIC5, RDX, and pERM were labeled green.

Confocal microscopy was used to record images of protein localization and ex-

pression in older adult mice.

1.2 Test if CLIC5 has a positive regulatory effect on pERM levels. Using CLIC5-

deficient jitterbug mice as a model, homozygote mutant mice were compared to

heterozygote littermate controls by double-staining the organ of Corti for actin

and pERM using commercial antibodies. We used immunofluorescence and

confocal microscopy to record protein localization and to quantify levels of

pERM for mice of different ages, from development and young adult to older

adult.

2. Does CLIC5 directly bind ERM protein?

Hypothesis: CLIC5 binds directly to ERM protein.

Rationale: Previous research has shown an interaction between CLIC5 and Ezrin via pull-down assay: however, this research did not distinguish between direct binding of

CLIC5 to Ezrin or indirect binding mediated by another protein (Berryman and

Bretscher, 2000). Unpublished results by Elizabeth Mathias have suggested that CLIC5 may bind directly to the N-terminal FERM domain of ERM. The experiment showed that the binding of the FERM domain to a known FERM ligand (NHERF1) was blocked

______31 by pre-incubation of the FERM with an excess amount of CLIC5. These results suggest that CLIC5 may compete with FERM for binding to NHERF1. The goal of this experi- ment was to test for CLIC5-ERM binding with a more direct approach.

Experimental design: Affinity pull-down experiment to test the ability of CLIC5 to bind directly to RDX and Ezrin FERM domains in vitro. GST-CLIC5 bound to glutathione beads was incubated with the RDX or Ezrin FERM domain then analyzed via SDS-

PAGE to directly test the ability of CLIC5 to bind the FERM domain.

3. What amino acid residues on CLIC5 facilitate the CLIC5-ERM interaction?

Hypothesis: Amino acids cysteine at position 32 (C32) and phenylalanine at position

227 (F227) of CLIC5 are essential for CLIC5 to interact with ERM protein.

Rationale: C32 and F227 are highly conserved among CLIC1-6 and from humans to flies. Additionally, C32 is a highly reactive nucleophile in living cells (Weerapana et al.,

2008); C32 is required for proper localization of CLIC5 to microvilli in LLC-PK1 epi- thelial cells (Kathleen Plona, unpublished); a non-conservative amino acid substitution changing F227 to a serine residue (F227S) causes deafness in mice (Kenneth John- son/The Jackson Laboratory, unpublished).

______32 Waddell Experimental design: Using cultured mammalian cells, we will test the effects of over- expressing wild-type and mutant versions of CLIC5 in terms of their ability to localize to or induce the formation of ERM/actin-based surface structures.

3.1 Determine the localization of wild-type CLIC5 versus CLIC5 with mutations in

F227 (changes to either serine or alanine) to microvilli in LLC-PK1. Wild-type

CLIC5 should interact with ERM proteins in the cell and localize to the ERM-

containing microvilli. If CLIC5 with a mutation in F227 is unable to bind ERM

proteins, we expect it to localize less to the microvilli and more to the cytoplasm

of the cell.

3.2 Assess the requirement of C32 and F227 for biological function of CLIC5.

COS-7 cells naturally create very few ERM-containing surface structures such

as microvilli and membrane ruffles, however, Al-Momany et al. (2014) demon-

strated that overexpressing wild-type CLIC5 dramatically increased the

percentage of COS-7 cells with ERM containing surface structures. Determine

the percentage of cells with ERM-rich surface structures with overexpression of

wild-type CLIC5 versus C32 and F227 mutant versions of CLIC5. If CLIC5

with mutations in C32 or F227 has a reduced ability to bind ERM, we would

expect the percentage of cells with ERM-containing surface structures would be

lower in cells overexpressing these mutant proteins as compared to wild-type.

______33

MATERIALS AND METHODS

Animals

CD-1 outbred mice (from OU Lab Animal Resources) and mice of mixed Balb/c and

129Ola genetic background (from the Kopchick Laboratory, GHR/BP +/+, Coschigano et al., 2000) were used in experiments to test for expression of CLIC5 and RDX in hair cells of older adult mice: both strains were CLIC5 +/+.

To assess the relationship between CLIC5 and pERM levels, Jbg2J CLIC5- deficient mice (deletion of CLIC5 exon 3) in the C57B6J genetic background and Jit- terbug CLIC5-deficient mice (97 deletion that causes skipping of exon 5, creating frame shift and premature stop codon; Gagnon et al., 2006) in the C3H/HeJ genetic background (C3H/HeJ-Clic5jbg/J, Stock Number: 004965), both from The Jack- son Laboratory were used. These experiments compared control heterozygote (CLIC5

+/-) and experimental homozygous mutant (CLIC5 -/-) animals.

Genotypes of all animals were verified by PCR (see appendix).

Fixation and dissection of mouse cochlear and vestibular systems

Mice were euthanized via carbon dioxide euthanasia. Inner were dissected in PBS.

A hole was made in the oval and round window using forceps and the was gently perfused through the round window with fixative by one-way injection of the fixative using a fine pipette tip. Fixatives used were ice cold 10% trichloroacetic acid

(TCA) in deionized water for anti-pERM and anti-CLIC5 antibody staining and room

______34 Waddell temperature 4% electron microscopy grade formaldehyde in PBS for anti-RDX anti- body staining. See the following sections for how fixation and staining conditions varied for each antibody. The apical turn of the organ of Corti was dissected from the cochlea.

In some animals the was also dissected from the vestibular system. For each age examined, control and experimental tissues were fixed and stained in parallel.

pERM antibody staining

Tissue was fixed for 1 hour in 10% TCA on ice and processed according to the method of Hayashi et al., 1999. After fixation, tissue samples were then dissected in 30 mM glycine-PBS (G-PBS), permeabilized for 30 minutes in 0.5% Triton X-100/G-PBS at room temperature, then washed in G-PBS. Tissue samples were placed in G-PBS con- taining 4% BSA that had been filtered through a 0.4µm disk filter for 16 hours at 4oC to block nonspecific protein binding during antibody staining. Tissue was incubated for 2 hours at room temperature in a primary antibody mixture of 1:50 rabbit anti-pERM (Cell

Signaling Technologies, Inc., cat #3419) and 5 µg/mL mouse monoclonal antibody against Beta-actin (Cytoskeleton, Inc., cat #AAN02) diluted in 4% BSA/G-PBS. After washing in G-PBS, tissue was incubated for 1 hour at room temperature in a secondary antibody mixture of 1:600 goat anti-rabbit Alexa 488 (Invitrogen, cat # A11034) and

1:600 goat anti-mouse Alexa 546 (Invitrogen, cat # A11036) diluted in G-PBS. Tissue was washed in G-PBS, then rinsed in PBS before being mounted on slides in ProLong

Gold Antifade mountant. The organ of Corti and cristae ampullaris were mounted on the slide with the stereocilia-side facing upward. Coverslips (#1.5 thickness) on slides

______35 containing organ of Corti samples were gently pressed with forceps to splay the stereo- cilia, allowing for better viewing during confocal microscopy.

CLIC5 antibody staining

Anti-CLIC5 antibody staining followed the same TCA fixation methods as pERM anti- body staining. A 1:10 dilution of rabbit APXB5/6 anti-CLIC5 antibody (Salles et al.,

2014) was used in place of the anti-pERM primary antibody.

RDX antibody staining

Anti-RDX antibody staining followed the same procedure as pERM/CLIC5 antibody staining with the following differences: tissue was fixed for 20 minutes in 4% electron- microscope grade formaldehyde rather than TCA, PBS was used in the place of G-PBS, and the primary antibody was 2.25 µg/mL rabbit anti-Radixin (Sigma-Aldrich, cat

#R3653) diluted in 4% BSA/PBS. Secondary antibody was 1:600 goat anti-rabbit Alexa

488; a 1:200 dilution of phalloidin Alexa 546 was used to stain actin filaments.

Confocal microscopy

Images were acquired using a Zeiss LSM 510 confocal laser scanning microscope with an alpha-Plan-FLUAR 100x/1.45 numerical aperture objective lens. Alexa 488-stained fluorescent proteins (green) were detected using a 488 nm Argon/2 laser. Alexa 546- stained fluorescent proteins (red) were detected using a 543 nm HeNe1 laser.

______36 Waddell In experiments looking at the effect of CLIC5 on pERM levels, imaging varia- bles such as laser transmission power, digital offset, pinhole diameter, and master gain were kept constant between genotypes at each age. Settings were first carefully adjusted to avoid pixel saturation in the area of interest for pERM staining in the CLIC5 +/- heterozygous control tissue, then identical conditions were used to collect data from the

CLIC5 -/- mutant tissue samples. The master gain and laser transmission percentage for the red channel, which was used to take pictures of Beta-actin counterstain was adjusted between samples to maintain threshold of pixel saturation. Actin staining was used to determine the distal and proximal focal planes required to capture images of all stereo- cilia when creating a Z-stack. The IHCs and OHCs within the apical turn of the organ of Corti and hair cells of the cristae ampullaris were photographed.

Quantification of pERM intensities of IHC stereocilia

For CLIC5 heterozygote (+/-) versus mutant (-/-) animals, pERM levels in the proximal third (base) of IHC stereocilia were quantified and compared. For each Z-stack of con- focal images in Zeiss Zen 2.1, the 0.8 µm thick optical section with the strongest pERM staining was chosen and exported to ImageJ. Channels were split into red (Beta-actin) and green (pERM). Note that pillar cells, a type of support cell surrounding the IHCs, showed pERM staining, so care was taken not to analyze stereocilia in which stereocil- iary pERM staining overlapped the support cell pERM staining. Criteria for choosing

IHC stereocilia were as follows: only the tallest row of IHC stereocilia were chosen, no overlapping stereocilia, and minimal overlap of the stereocilia with support cell pERM

______37 staining. The length of individual stereocilia was measured using the line tool, then the proximal third of the total length was selected as a rectangular selection the width of the stereocilia. Average green channel pixel intensity in the selected region was recorded for 40 stereocilia chosen from 20 or more individual cells for each experimental condi- tion. Statistical significance between heterozygote versus mutant groups of the same age was calculated using a two-tailed Student’s t-test.

Quantification of IHC morphology

For CLIC5 +/- heterozygous control and -/- mutant mice of each age, 30 or more IHCs were categorized as having either normal or abnormal morphology based on Beta-actin staining. Characteristics of abnormal morphology were: reduced numbers of stereocilia

(ranging from few to none) and/or elongated or fused stereocilia characteristic of the membrane lifting phenotype as described in Salles et al., 2014. CLIC5 heterozygote +/- and mutant -/- animals were compared and statistical significance was evaluated using a two-tailed Student’s t-test.

Intensity profiles of pERM protein localization

The procedure for quantification of pERM localization along the long axis of stereocilia from IHCs was adapted from similar procedures used in Zhao et al., 2012 and Salles et al., 2014. Confocal images from Zen 2.1 were exported to ImageJ. Channels were split into red (Beta-actin) and green (pERM). A rectangular box was drawn around individual stereocilia using the area selection tool. The same exclusion criteria were used as in

______38 Waddell “Quantification of pERM intensities of IHC stereocilia”. The “plot profile” function was used on the selected stereocilia to make an intensity profile of the green channel pixel intensity from the base to tip of each individual stereocilia (Fig. 16). Ten stereocilia were analyzed for each experimental condition. Intensity profiles were exported to Mi- crosoft Excel, where the mean percentage of maximum pixel intensity in each row of pixels along the stereocilia was found for the stereocilium sampled. Statistical signifi- cance between the groups was calculated for the mean at each point along the stereocilia using a two-tailed Student’s t-test.

Figure 16. Using ImageJ to make an intensity profile of pERM staining in a single stereo- . A single stereocilium is selected in the green, pERM stain channel with the area select tool (yellow rectangle). Image from Balb/c CLIC5 +/+ animal. Note how pixel intensity is slightly stronger closer to the base then fades in the half of the stereocilium closer to the tip.

Purification of GST fusion proteins

GST-CLIC5 (Berryman et al., 2004), GST-Ezrin (amino acids 555-585) F-actin binding domain (Gary and Bretscher, 1995), and GST alone (pGEX-2T; Pharmacia Biotech) proteins were purified under native conditions. Cultures of host BL21 strain E. coli were grown to express the GST fusion proteins. Upon reaching an optical density of 0.5 at

______39

595 nm, production of the GST fusion proteins was induced by adding 0.2 mM Isopro- pyl β-D-1-thiogalactopyranoside (IPTG). Cultures were allowed to incubate for another

90 minutes, then were chilled for 10 minutes on ice before being centrifuged at 4,000 rpm for 10 minutes. Bacterial pellets were frozen then resuspended in PBS with 0.1 mg/mL phenylmethane sulfonyl fluoride and 0.15 mg/mL benzamidine. The cells were lysed by sonication for 15-20 seconds followed by the addition of Triton X-100 to give the solution 1% final detergent concentration. The lysate was then centrifuged at 20,000 rpm for 10 minutes to pellet unbroken cells and insoluble material.

Supernatants were added to 15 mL culture tubes containing 0.2 mL of glutathi- one agarose resin and incubated on a rotor at 4°C for one hour. Glutathione agarose beads with the bound GST fusion proteins were washed five times in PBS with 0.1%

Triton X-100, and then eluted from the beads in 10 mM reduced glutathione and 50 mM

Tris, pH 8.0. The eluted proteins were dialyzed against 2 L of PBS overnight followed by an additional round of dialysis. A BCA protein assay was performed to determine the final concentration of each purified protein.

Preparation of soluble RDX and Ezrin FERM domains

Untagged RDX FERM and Ezrin FERM domains were grown in cultures of host M15 strain E. coli. Upon reaching an optical density of 0.5 at 595 nm, production of the

FERM proteins was induced by adding 0.2 mM IPTG. Cultures were incubated for an additional 3 hours, then the same procedure for chilling, centrifugation at 4000 rpm,

______40 Waddell sonication, and subsequent centrifugation at 20,000 rpm was followed as in “Purifica- tion of GST fusion proteins”. After the final centrifugation, the supernatant was removed, transferred to a fresh tube, and kept on ice. This supernatant included the in- duced RDX/Ezrin FERM domain as the predominant protein in a bacterial extract of other soluble proteins from the M15 host strain.

Affinity pull-down assay

15 µL of glutathione beads containing 1 µg/µL of GST fusion protein were incubated for 60 minutes at 4°C on a rotor with 50 µg RDX/Ezrin FERM domain in a bacterial extract or PBS alone as a control. Beads were then washed three times in PBS with 0.1%

Triton X-100. PBS was removed and 18 µL of 2x Laemmli sample buffer containing

2% 2-mercaptoethanol was added to the beads. The mixture was boiled for 5 minutes to denature and elute the GST fusion proteins and any FERM domain that was bound to them. 8 µL of this protein sample was separated on an 11.5% SDS-PAGE gel and then stained with Coomassie blue for 1 hour. Gels were destained and then photographed using a NIKON D90 camera (AF Nikkor 50mm f/1.4D lens).

Cell culture and transfection

LLC-PK1 and COS-7 cells were routinely maintained in 100 mm culture dishes with

Eagle’s Minimum Essential Medium (MEM) containing 10% fetal bovine serum with penicillin, streptomycin, and L-glutamine. Cells were kept in a humidified incubator at

37°C under 5% CO2. LLC-PK1 cells are derived from pig kidney epithelial cells (Tyska

______41 and Mooseker, 2000). COS-7 cells are fibroblast-like cells derived from CV-1 cells, which were in turn derived from monkey kidney epithelial cells (Gluzman, 1981). Cells were routinely split to new plates using 0.125% trypsin, 1 mM EDTA.

For cell transfection in morphological/protein localization experiments, LLC-

PK1 and COS-7 cells were split to 35 mm culture dishes containing 12 mm circle glass coverslips and grown to 30-40% confluence. Serum-containing media was replaced with serum-free MEM and a mixture of 1.7 µg of plasmid DNA and 5-7 µg of polyeth- yleneimine (PEI) was added to the cells. After 4 hours, the DNA-containing media was removed, cells were washed twice with PBS, and serum containing media was added back to the cells. Cells were grown for an additional 10 hours before fixation and stain- ing.

Fixation and antibody staining of LLC-PK1 and COS-7 cells

Transfected LLC-PK1 cells on coverslips were fixed in 4% electron microscopy grade formaldehyde in PBS for 10 minutes at room temperature. Cells were washed with PBS then permeabilized with 0.1% Triton X-100/PBS for 60 seconds. Cells were incubated for 30 minutes at room temperature in a mixture of 2 µg/mL CPTC-Ezrin-1 monoclonal antibody and 2 µg/mL MSN-1 monoclonal antibody (Developmental Studies Hybrid- oma Bank, University of Iowa) in PBS. Only the anti-Ezrin antibody was used for staining LLC-PK1 cells. Cells were rinsed briefly 20 times in PBS and incubated in PBS for 10 minutes at room temperature, followed by incubation in a secondary antibody

______42 Waddell mixture containing a 1:600 dilution of goat anti-mouse IgG Alexa-546 (Life Technolo- gies/Molecular Probes) and DAPI (Life Technologies/Molecular Probes) in PBS. Cells were again rinsed and incubated for 10 minutes at room temperature in PBS. Coverslips were mounted in 6 µL Prolong Gold antifade, placed in the dark overnight, and then sealed with clear nail polish.

Immunofluorescence microscopy of transfected cells

Cells were evaluated using a Nikon Eclipse E600 epi-fluorescent microscope at 1000x magnification. The UV-2E/C blue channel was set for excitation from 330-380 nm and emission from 435-485 nm, the FITC-HYQ green channel was set for excitation from

460-500 nm and emission from 600-660 nm, and the Texas Red channel was set for excitation from 540-580 nm and emission from 600-660. Images were taken with a

SPOT camera using SPOT Advanced software version 4.7.

Quantification of GFP-CLIC5 localization in transfected LLC-PK1 cells

The percentage of LLC-PK1 cells with GFP-enriched microvilli staining stronger than cytoplasmic green fluorescent protein (GFP) staining was evaluated in cells transfected with GFP-CLIC5WT, GFP-CLIC5F227S, and GFP-CLIC5F227A plasmid DNA. Cells with- out a nucleus or in mitosis were excluded, as evaluated in the blue channel detecting

DAPI staining. The experiment was repeated five times; each time at least 200 cells were counted in each group for a total sample size of 1000 or greater. Statistical signif- icance between groups was calculated using a two-tailed Student’s t-test.

______43

Quantification of GFP-CLIC5 induced ERM rich surface structures in COS-7 cells

The percentage of GFP-transfected cells with ERM-containing surface structures was evaluated in COS-7 cells transfected with various GFP-CLIC5 DNA constructs. Cells were transfected with GFP-CLIC5WT, GFP-CLIC5 with the following mutations:

CLIC5F227S, CLIC5F227A, CLIC5C32A, CLIC5S35A, CLIC5C32A/S35A, and GFP alone as a negative control. In addition to these constructs with GFP tags at the N-terminus, con- structs with C-terminal GFP tags were also tested for each wild-type and mutant CLIC5 construct (denoted CLIC5F227A-GFP, for example). Cells were categorized by first find- ing GFP-transfected cells in the green channel, then evaluating ERM-containing surface structures in the red channel. GFP-transfected cells with ERM-containing surface struc- tures were counted as a positive and GFP-transfected cells with no visible ERM- containing surface structures were counted as a negative. Cells without a nucleus or in mitosis were excluded, as evaluated in the blue channel detecting DAPI of DNA. The experiments were repeated four times; each time at least 200 cells were counted in each group for a sample size of 800 or greater. Statistical significance between groups was calculated using a two-tailed Student’s t-test.

______44 Waddell Table 1. DNA Constructs Used in this Study Construct* Vector Resistance** Host Reference GFP-CLIC5WT pEGFP-N3 Kan DH5alpha Salles et al. 2014 GFP-CLIC5F227S pEGFP-N3 Kan DH5alpha S. Tanda & H. Tanda GFP-CLIC5F227A pEGFP-N3 Kan DH5alpha S. Tanda & H. Tanda GFP-CLIC5C32A pEGFP-N3 Kan DH5alpha S. Tanda & K. Plona GFP-CLIC5S35A pEGFP-N3 Kan DH5alpha S. Tanda & K. Plona GFP-CLIC5C32A/S35A pEGFP-N3 Kan DH5alpha S. Tanda & K. Plona CLIC5WT-GFP pEGFP-C3 Kan DH5alpha Salles et la., 2014 CLIC5F227S-GFP pEGFP-C3 Kan DH5alpha S. Tanda & H. Tanda CLIC5F227A-GFP pEGFP-C3 Kan DH5alpha S. Tanda & H. Tanda CLIC5C32A-GFP pEGFP-C3 Kan DH5alpha S. Tanda & K. Plona CLIC5S35A-GFP pEGFP-C3 Kan DH5alpha S. Tanda & K. Plona CLIC5C32A/S35A-GFP pEGFP-C3 Amp DH5alpha S. Tanda & K. Plona GST-CLIC5WT pGEX-2T Amp BL21 M. Berryman RDX 1-296 (FERM domain) pQE70 Amp & Kan M15 M. Berryman Ezrin 1-296 (FERM domain) pQE16 Amp & Kan M15 Reczek et al., 1997 GST-Ezrin 555-585 pGEX-3X Amp BL21 Gary & Bretscher, 1995 (F-actin-binding domain) GFP alone pEGFP-C3 Kan DH5alpha CloneTech Inc. GFP alone pEGFP-N3 Kan DH5alpha CloneTech Inc. GST alone pGEX-2T Amp BL21 Pharmacia Biotech *All proteins are the human form. All CLIC5 fusion proteins contain full length CLIC5 amino acid sequence (1-253). **Kanamycin (Kan), Ampicillin (Amp)

______45

RESULTS

CLIC5 and RDX are expressed throughout adulthood in wild-type mice.

We first examined if CLIC5 and RDX were expressed in the stereocilia of older adult wild-type mice using whole mount immunofluorescence microscopy. Our hypothesis was that these two proteins play a role not only in development of the stereocilia, but in lifelong maintenance of the stereocilia, and therefore we predicted that CLIC5 and RDX would still be expressed in older adult animals. CLIC5 (Fig. 17) and RDX (Fig. 18) were readily detected in both IHC and OHC stereocilia of 2-month old CD-1 (CLIC5

+/+) mice, ~1-year old Balb/c (CLIC5 +/+) mice, and ~1-year old C57/B6J Jbg2J het- erozygote (CLIC5 +/-) mice. Note that the formaldehyde fixative used with the RDX antibody staining preserves the morphology of the stereocilia significantly better than the TCA fixation required for CLIC5 antibody staining.

CLIC5 promotes ERM phosphorylation in the stereocilia during all stages of life.

Previous research by Wegner et al. (2010) showed that in the podocyte of Jitterbug

CLIC5 -/- mutant mice, levels of pERM were lower than in control animals. Similarly, research by Al-Momany et al. (2014) demonstrated that CLIC5 promoted the phosphor- ylation of Ezrin in podocytes and in COS-7 cells. The aim of our experiment was to test if the levels of pERM in the stereocilia decreased in CLIC5 -/- mutant mice versus

CLIC5 +/- heterozygous controls.

______46 Waddell

Figure 17. CLIC5 is expressed throughout adulthood in wild-type mice. Confocal images of whole mount organ of Corti fixed with TCA and stained with antibodies against CLIC5 (green) and Beta-actin (red). 2-month old mouse was CD-1 strain. ~1-year old mice were Balb/c (IHC) and C57/B6J Jbg2J heterozygote (OHC). Scale bar: 10 µm.

Figure 18. RDX is expressed throughout adulthood in wild-type mice. Whole mount of organ of Corti fixed with formaldehyde and stained with antibody against RDX (green) and phalloidin (red) to label F- actin. 2-month old mouse was CD-1 strain. ~1-year old mouse was Balb/c. Scale bar: 10 µm.

______47

Under identical fixation, staining, and microscope imaging conditions, lower levels of pERM were detected in IHC and OHC stereocilia during both development

(Fig. 19, Table 2) and adulthood (Fig. 20, Table 2) inCLIC5 mutant (-/-) mice com- pared to heterozygote littermate controls. Similarly, the vestibular hair cells of CLIC5 mutant animals had lower levels of pERM than their heterozygote littermate controls

(Fig. 21). In the CLIC5 mutant mice, pERM levels rapidly declined with age relative to heterozygote pERM levels (Fig. 22). In the P10 animal there was a 39.8% reduction

(p<0.001) in pERM levels in the mutant animal versus control. At P17, there was a

37.6% reduction (p<0.001) in pERM levels in mutant animal versus control. In adult- hood, the difference between mutant and control was even larger; at P26 there was a

57.3% reduction (p<0.001) in pERM levels in the mutant relative to the control animal and at P65 there was a 69.6% reduction (p<0.001) in pERM levels. Postnatal day 5 mice were could not be quantified by this method due to the short length of the immature stereocilia and 1-year old mice were not quantified because individual stereocilia were virtually absent in mutant mice of this age.

______48 Waddell

Figure 19. CLIC5 mutant mice have lower levels of pERM in IHCs and OHCs during postnatal development. Organ of Corti of CLIC5 +/- heterozygous littermate controls and CLIC5 -/- mutant mice were fixed with TCA and processed in parallel for immunostaining with antibodies against pERM (green) and Beta-actin (red). Confocal images of pERM were taken from similar regions of the cochlea (mid- apical turn) using identical scanning conditions. Relative to controls, CLIC5 mutants showed reduced pERM levels in both IHCs (single arrowhead) and OHCs (double arrowheads) at all ages. Scale bar: 10 µm. ______49

Figure 20. CLIC5 mutant mice have dramatically lower levels of pERM in the IHCs and OHCs during adult life. Organ of Corti of CLIC5 +/- heterozygous littermate controls and CLIC5 -/- mutant mice. Immunostaining with antibodies against pERM (green) and Beta-actin (red). Relative to controls, CLIC5 mutants showed greatly reduced pERM levels in both IHCs (single arrowhead) and OHCs (double arrowheads) at all ages. Additionally, pERM levels in CLIC5 mutant mice declined with age. There is little to no detectable pERM in mutant mice stereocilia at 2 months (in either IHCs or OHCs). At 12 months, virtually all mutant stereocilia have an abnormal morphology. Scale bar: 10 µm.

______50 Waddell

Figure 21. CLIC5 mutant mice have lower levels of pERM in stereocilia of vestibular hair cells. The cristae ampullaris of CLIC5 +/- heterozygous littermate control and CLIC5 -/- mutant mice were fixed, stained, and imaged under identical conditions. Immunostaining with antibodies against pERM (green) and Beta-actin (red). The CLIC5 mutants showed greatly reduced pERM at the base of stereocilia at both 2 months and 12 months. Similar to the cochlear system, the vestibular hair bundles of the mutant animal were sparse and morphologically abnormal in the mutant adult animals. Scale bar: 10 µm.

Table 2. CLIC5 -/- mutant mice had significantly lower pERM levels in IHC stereocilia than CLIC5 +/- heterozygous control animals at all ages (n=40 stereocilia from 20>IHCs/group) Mean ± SD P value Age (days) % CLIC5 (-/-) relative to CLIC5 (+/-) CLIC5 (+/-) CLIC5 (-/-) (2-tailed t-test) 10 63.5 ± 11.9 38.3 ± 8.52 p < .001 60.2% 17 36.0 ± 6.86 22.5 ± 8.24 p < .001 62.4% 26 47.2 ± 12.9 20.2 ± 5.25 p < .001 42.7% 65 34.8 ± 9.11 10.6 ± 2.84 p < .001 30.4%

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Figure 22. pERM levels in mutant mice declined with age. The relative level of pERM in CLIC5 -/- mutant animals rapidly declined after age of hearing onset compared to CLIC5 +/- heterozygous control mice.

Percentage of IHCs with morphologically normal hair bundles dramatically de- creases in aging CLIC5 mutant mice

As animals aged, the percentage of IHCs with morphologically normal hair bundles rapidly declined in the CLIC5 mutant mice relative to the CLIC5 heterozygote controls

(Fig. 23A). In the CLIC5 heterozygotes, the percentage of IHC hair bundles with normal morphology stayed at or above 90% from development (P5) to at least 1 year old adults.

Although there was no significant difference between CLIC5 mutants and heterozygotes at P5 (p=0.32) or P10 (p=0.27), there was a dramatic decline in the percentage of CLIC5 mutant IHC with irregular morphology after the onset of hearing.

At P17 only 48.4% of CLIC5 mutant IHC bundles (n=40) had a normal mor- phology. This was significantly lower than the heterozygote control (95.7%, p<0.001).

The other 51.6% of hair bundles were either absent or exhibited abnormal morphology.

At P17, the most common type of abnormality present in the mutant animals was fusions of small groups of stereocilia, which can be seen in figure 20. At P26 the percentage of

CLIC5 mutant IHC bundles (n=44) with normal morphology further decreased to 36.6%,

______52 Waddell again significantly lower than the heterozygote control (93.55%, p<0.001). At P65 the percentage of CLIC5 mutant IHC bundles (n=47) declined to 12.8% and by P365, 0.0% of IHC bundles (n=37) had normal morphology. At both P65 and P365, there was a significant difference between the mutants and the heterozygote controls (p<0.001). At

P365, a vast majority of CLIC5 mutant IHCs were missing or did not have any stereo- cilia. The stereocilia that remained were abnormally long and fused together. Regression analysis revealed a positive correlation (R2=0.74) between pERM level and normal hair bundle morphology (Fig. 23B).

Figure 23. Percentage of CLIC5 -/- mutant IHC bundles with normal morphology rapidly declines with age, pERM levels correlate with normal morphology of the IHCs. (A) At all ages past the age of onset of hearing (P17, P26, P65, & P365) there is a significantly lower (p<0.001*) percentage of IHC bundles in the mutant animal with normal morphology versus the heterozygote control. By P365, there were virtually no hair bundles remaining in the mutant animal that were characterized as having a normal morphology. (B) A correlation was found between the pERM levels in the IHCs and the percentage of IHC bundles with normal morphology (R2=0.74). The P10 mutant animal (**) was an outlier. If this data point is omitted, the correlation has an R2=0.96.

pERM is less tightly localized to the base of CLIC5 +/- adult stereocilia versus CLIC5 +/+ mice.

In CLIC5 +/+ Balb/c and CD-1 mice, pERM localized strongest to the base of the ste- reocilia. In contrast, after the onset of hearing in CLIC5 +/- heterozygote animals, pERM was less prominent at the stereociliary base and more diffuse along the length of ______53 the stereociliary shaft (Fig. 24). In comparing a year old Balb/c CLIC5 +/+ mouse and a Jbg2J CLIC5 +/- mouse using intensity plot profiles of individual stereocilia, we found a significant (p<0.05) increase in pERM within the distal half of CLIC5 +/- stereocilia versus the CLIC5 +/+ animal.

A

B

Figure 24. Diffuse localization of pERM along the shaft of IHC stereocilia of 1-year old C57/B6J Jbg2J CLIC5 (+/-) heterozygous mouse compared with a1-year old Balb/c CLIC5 +/+ animal. (A) Organ of Corti of Jbg2J CLIC5 +/- and Balb/c CLIC5 +/+ mice fixed with TCA and processed in parallel for immunostaining with antibodies against pERM (green) and Beta-actin (red). Confocal images of pERM were taken from similar regions of the cochlea (mid-apical turn) using identical scanning condi- tions. Scale bar: 10 µm. (B) Quantification of pERM localization by intensity plot showed that pERM was predominantly localized to the lower 25% of Balb/c CLIC5 +/+ IHC stereocilia. In contrast, Jbg2J CLIC5 +/- stereocilia displayed pERM intensity that was significantly higher (p<0.05) in the upper 50% of the stereocilia than in the Balb/c CLIC5 +/+ mouse.

______54 Waddell GST-CLIC5 binds directly to RDX and Ezrin FERM domains

Berryman and Bretscher (2000) discovered CLIC5 as a binding partner of Ezrin in an affinity pull-down assay, though this research did not distinguish between direct binding of CLIC5 to Ezrin versus indirect binding mediated by another protein. Unpublished research by Elizabeth Mathias demonstrated that the binding of Ezrin FERM domain to a known FERM ligand (NHERF1) was blocked by pre-incubation of FERM with an excess amount of CLIC5, suggesting that CLIC5 may bind directly to the N-terminal

FERM domain of ERM. Our goal was to do a simpler test for ERM-CLIC5 interaction, specifically testing if CLIC5 binds directly to the RDX and Ezrin FERM domains.

Purified GST-CLIC5 was shown to bind RDX FERM (Fig. 25) and Ezrin FERM

(not shown) in affinity pull-down assays. Both experiments were run with a positive control—a GST fusion of the Ezrin F-actin-binding domain (amino acids 555-585)— which strongly bound to FERM. GST alone was run as a negative control and showed no detectable binding to FERM. Relative to the positive control, the FERM appeared to bind weakly to CLIC5 under the in vitro conditions used here. The majority of the pro- tein interaction network in human cells is dominated by weak, sub-stoichiometric interactions, many of which have previously gone undetected (Hein et al., 2015).

______55

Figure 25. CLIC5 binds directly to RDX FERM domain. Immobilized GST fusion proteins were in- cubated with buffer alone or RDX FERM domain (*). Note that the RDX FERM Input lane was loaded with 2% of the quantity of protein used in the incubation with GST fusion proteins. Bound proteins were eluted and separated by SDS-PAGE. GST-Ezrin F-actin binding domain was positive control (Pos), GST- CLIC5 (C5) was experimental, GST was negative control (Neg). White box: RDX FERM bound to both the positive control and to GST-CLIC5 (arrow), whereas no binding was detected in the negative control. Note that the faint bands in the 40-50 kDa range and a stronger band at 30 kDa for GST-CLIC5 and to a lesser extent around the 28 kDa range for GST-Ezrin F-actin binding domain are likely degradation prod- ucts of the GST fusion proteins. DF = dye front.

______56 Waddell CLIC5 F227S deafness-associated mutation disrupts CLIC5 localization of CLIC5 to cell surface microvilli

In unpublished research, Dr. Kenneth Johnson (The Jackson Laboratory) identified a chemically induced point mutation in the CLIC5 gene that caused a missense mutation in which a phenylalanine residue at amino acid position 227 is changed to a serine res- idue (hereafter referred to as CLIC5F227S); this mutation results in profound deafness in mice. Our goal in this experiment was to determine how mutations in CLIC5F227 affect localization of CLIC5 in LLC-PK1 cells. The abundant microvilli naturally present in

LLC-PK1 cells have been used as a model for the stereocilia (Zheng et al. 2010). Salles et al. (2014) transfected LLC-PK1 cells with GFP-CLIC5 and found that wild-type

CLIC5 localizes to the microvilli as well as the nucleus of these cells. LLC-PK1 cells naturally express little to no endogenous CLIC5 (data not shown).

In contrast to wild-type GFP-CLIC5, which concentrates more in surface micro- villi than the cytoplasm, CLIC5F227S, containing a nonconservative F227S (phenyl- alanine to a serine residue) deafness-associated mutation, localized stronger to the cy- toplasm than the microvilli (Fig. 26). The percentage of GFP-CLIC5 transfected LLC-

PK1 cells with stronger CLIC5 localization in the microvilli was 99.6% ± 0.36% in cells transfected with wild-type CLIC5 but was only 29.1% ± 5.73% in cells transfected with

CLIC5F227S mutation (p<0.001). A conservative amino acid substitution, CLIC5F227A

(phenylalanine to an alanine residue), on the other hand, had no significant effect on the localization of GFP-CLIC5 relative to cells transfected with CLIC5WT (p=0.99).

______57

GFP-CLIC5 Ezrin Merge

WT CLIC5 - GFP

F227S CLIC5 - GFP

F227A CLIC5 - GFP

Figure 26. GFP-CLIC5F227S mislocalizes to the cytoplasm whereas GFP-CLIC5WT staining is stronger in the microvilli. Fluorescence for GFP (green) and immunostaining with antibody against endogenous Ezrin (red). In LLC-PK1 cells transfected with GFP-CLIC5WT or GFP-CLIC5F227A, GFP- CLIC5 staining was stronger in the microvilli than in the cytoplasm. In cells transfected with GFP- CLIC5F227S, GFP-CLIC5 staining was stronger in the cytoplasm Scale bar: 10 µm.

______58 Waddell Conserved amino acids at cysteine position 32 or phenylalanine position 227 are critical for CLIC5’s ability to induce formation of actin rich surface structures

In an unbiased proteome screening, Weerapana et al. (2008) determined that a conserved cysteine residue in CLIC proteins (C32 in CLIC5) was a highly reactive nucleophile

(Weerapana et al., 2008). This finding suggests that this amino acid plays an important role in the biological function of CLIC5. Unpublished research by Kathleen Plona demonstrated that mutating the C32 residue to alanine (CLIC5C32A) significantly re- duced the localization of CLIC5 to microvilli of LLC-PK1 cells. In addition to C32, we tested the importance of the serine residue at position 35 (S35) residue because it is part of a conserved motif of amino acids neighboring the C32 residue in the CLIC family of proteins. This motif, either CxxC or CxxS, could serve as a redox-reactive center in

CLIC proteins. The reason for our interest in the deafness-associated F227 residue is described in the previous results section.

COS-7 fibroblast-like cells naturally have a low level of ERM-containing actin- based surface structures. Viswanatha et al. (2013) demonstrated that overexpressing the active form of ERM proteins or ERM binding partners such as CLIC4 in these cells increased the percentage of COS-7 cells containing ERM-containing surface structures.

We performed a similar assay, overexpressing CLIC5WT and CLIC5 with mutations at

C32, S35, or F227 to test the necessity of these amino acids in CLIC5’s biological ac- tivity in the context of its ability to stimulate de novo assembly of actin-based surface structures.

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COS-7 cells transfected with CLIC5WT DNA displayed a significantly higher

(p<0.05) percentage of cells with ERM-containing surface structures than cells trans- fected with CLIC5 DNA harboring mutations in either C32 or F227 (Figs. 27 and 28).

The mutations that significantly reduced the biological activity of CLIC5 in creating

ERM-contain surface structures included C32A (p<0.001), a double mutant

C32A/S35A (p<0.001), F227S (p<0.001) and F227A (p<0.05). Cells transfected with

CLIC5 S35A mutant plasmid DNA, on the other hand, showed no significant difference from cells transfected with CLIC5WT DNA, suggesting that S35 does not play a critical role in CLIC5 function in terms of regulating assembly of cortical actin-based structures.

Placement of the GFP tag at the N- or C-terminus of CLIC5 fusion proteins had no significant effects with regard to the percentage of cells displaying ERM-containing surface structures for any of the GFP-CLIC5 constructs except for when comparing the

GFP-CLIC5F227A and CLIC5F227A-GFP constructs (p<0.05).

______60 Waddell GFP-CLIC5 ERM Merge

WT CLIC5 - GFP

C32A CLIC5 - GFP

F227S CLIC5 - GFP

F227A CLIC5 - GFP

GFP alone GFP

Figure 27. GFP-CLIC5 staining in COS-7 cells transfected with various GFP-CLIC5 constructs. Fluorescence for GFP (green) and immunostaining with antibodies against ERM (red). GFP-CLIC5WT transfected cells produced abundant ERM-containing surface structures; see the hair like protrusions (ar- row) and membrane ruffles (double headed arrow). In these surface structures, GFP-CLIC5 WT is colocalized with the ERM protein in the surface structures. Cells shown here transfected with GFP-CLIC5 with C32A, F227S or F227 mutant DNA produced few or no surface structures, giving them a relatively smooth appearance. The morphology of the C32A and F227S/F227A transfected cells were similar to cells transfected with GFP alone. Scale bar: 50 µm.

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Figure 28. Quantification of COS-7 cells transfected with wild-type and various mutant GFP-CLIC5 con- structs. Mutations in CLIC5 residue C32 or F227 produce significantly fewer ERM-containing surface structures than cells transfected with wild-type CLIC5.

______62 Waddell DISCUSSION

The expression of both CLIC5 and RDX in stereocilia during development and through- out adult life in mice supports our hypothesis that these proteins are required for lifelong maintenance of inner-ear hair cell structure and function in humans. Furthermore, our demonstration that phosphorylation of RDX at threonine position 564 occurs throughout adult life suggests that this particular post-translational modification of RDX is likewise critical in lifelong maintenance of stereocilia. Studying the jitterbug deafness model of

CLIC5 knockout mice revealed a critical role for CLIC5 in positively regulating the phosphorylation status of RDX in inner-ear hair cells. Presumably, this positive regula- tory role of CLIC5 on the phosphorylation status of ERM protein is also necessary for both development and lifelong maintenance of the stereocilia. We demonstrated that

CLIC5 directly binds the FERM domain of ERM proteins, and thus it is highly likely that this binding mediates the regulatory activity of CLIC5 on ERM protein function.

Finally, our results demonstrate that the CLIC5-ERM interaction is dependent on at least two highly conserved amino acids in the CLIC protein family from humans to flies, cysteine 32 and phenylalanine 227.

CLIC5 promotes ERM phosphorylation in the stereocilia

We demonstrated that CLIC5 and RDX are still expressed in the stereocilia of elderly adult mice (Fig. 17 and 18). This finding supports our hypothesis that these proteins are required for maintenance of the stereocilia into old age. Our experiments with CLIC5 knockout mice revealed reduced pERM levels in stereocilia of cochlear IHCs and OHCs

______63 at all stages of development and adulthood (Figs. 19 and 20, Table 2). Similarly, the vestibular hair cells of the cristae ampullaris in adult CLIC5 knockout mice displayed dramatically reduced levels of pERM compared to age-matched CLIC5 heterozygote control mice (Fig. 21). Experiments were preformed under carefully controlled condi- tions from fixation and staining of the tissue to microscope conditions and data acquisition. IHCs were quantified as they are the most amenable hair cells for analysis with our methodology. Together, these findings in hair cell stereocilia are consistent with results from Wegner et al. (2010) and Al-Momany et al. (2014), which showed reductions in podocyte pERM levels in CLIC5-deficient mice. Thus, the results in this study strongly support the proposed role for CLIC5 as a positive regulator of the phos- phorylated form of ERM protein in the hair cells of the inner-ear.

There is a positive correlation between pERM levels and the percentage of IHCs with normal morphology (R2=0.73) (Fig. 23B). We found that pERM levels rapidly decreased with age in the CLIC5 knockout animals (Fig. 22), and this decrease in pERM level was accompanied by a decrease in the percentage of hair cells with normal mor- phology (Fig. 23 A). There is an outlier in the data (P10, CLIC5 mutant, Fig. 23B **) that when removed gives an R2=0.96. This outlier may give us important information regarding the accuracy of the quantification of IHCs with normal morphology. It fits our hypothesis that declining pERM levels lead to weaker membrane actin linkages at the base of the stereocilia, which leads to the membrane lifting phenotype described in

Salles et al. (2014). However, we cannot exclude the possibility that the decrease in pERM levels over time in the CLIC5 mutant animals may be a secondary to the loss of

______64 Waddell normal stereocilia morphology. Based on our hypothetical model (Fig. 14), we would expect decreased pERM level to precede a decline in abnormal stereocilia morphology.

However, the P10 CLIC5 mutant outlier shows just the opposite: the percentage of hair cells scored as having normal morphology was 92.5% (not a significant difference from

CLIC5 heterozygote control) yet the pERM level was 40% lower than the control animal

(p<0.001). This anomaly may be explained in at least three ways: (1) P10 is still before the age of hearing onset and abnormal morphology simply does not occur before hair cell maturation, (2) the demand for pERM is greater after hearing onset, when cells become functional, or (3) characterizing abnormality in P10 mice is more difficult than in older adult hair cells where the differences are readily apparent, resulting in mischar- acterization of hair cells with irregular morphology as normal. Additionally, the use of

TCA fixation, which denatures tissue proteins rather than crosslinking them together, is not ideal for studying subtle morphological differences. Nonetheless, our data do not distinguish whether the reduced pERM causes the loss of normal hair cell morphology or whether the loss of normal morphology causes the decline of pERM levels, and there- fore further investigation is warranted. Another note is that the role of pERM may differ during stereocilia formation, maturation, and long-term maintenance, and we have no knowledge of the rates of phosphorylation/dephosphorylation cycles, or RDX protein turnover via degradation and biosynthesis. Additional studies will be required to distin- guish between these possibilities.

Based on the data collected in this study, CLIC5 appears to be a dosage-depend- ent gene. Adult mice with two copies of CLIC5 (CLIC5 +/+; 1 year Balb/c, 2 month

______65

CD-1 mice) displayed tight localization of pERM to the base, or proximal end of stere- ocilia, whereas adult mice with only one copy of CLIC5 (CLIC5 +/-; Jbg2J mice) exhibited more diffuse pERM localization along the shaft of the stereocilia (Fig. 24).

This finding has two major implications: (1) our findings on the reduction of pERM levels at the base of stereocilia in CLIC5 knockout would have been even more pro- nounced if we had used CLIC5 wild-type rather than CLIC5 heterozygote mice as a control, and (2) increasing levels of CLIC5 in vivo could help pERM correctly localize and and increase the biological activity of ERM proteins in heterozygote animals carry- ing a dysfunctional CLIC5 allele. These findings warrant further investigation into the effects of CLIC5 gene dose (+/+, +/-, -/-) not only on levels of pERM, but also on the localization of pERM along the proximal-distal axis of the stereociliary shaft. Accord- ingly, more detailed studies on hair cell morphology and accompanying physiological measurements of hearing will be required to assess the true role of CLIC5 gene dose during aging.

CLIC5 directly binds the FERM domain of ERM protein

Our affinity pull-down assay demonstrated that CLIC5 binds directly to the FERM do- main of both RDX (Fig. 25) as well as Ezrin. The interaction between CLIC5 and RDX is consistent with the hypothesis put forward by Salles et al. (2014) that the two proteins are part of a multiprotein complex at the base of the stereocilia. Although CLIC5 may bind to activated phosphorylated RDX and stabilize it, perhaps preventing dephosphor- ylation, there are several alternative possibilities to explain how CLIC5 promotes steady

______66 Waddell state phosphorylation of RDX. For example, CLIC5 may promote kinase activity, or inhibit phosphatase activity. Salles et al. (2014) suggested that CLIC5 and RDX are in a multiprotein complex localized at the base of stereocilia, including the membrane pro- tein PTPRQ, Taperin, and the minus-end directed F-actin motor protein Myosin VI. Our findings showing that CLIC5 positively regulates pRDX levels imply the existence of additional components to this membrane-cytoskeletal linking “machine”, such as a ki- nase and phosphatase that may facilitate phosphorylation/dephosphorylation cycles of

RDX, as proposed by Zhu et al. (2007) in gastric parietal cells.

The binding between CLIC5 and the ERM FERM domain appears to be rela- tively weak, especially when compared to the binding of the ERM FERM to the ERM

F-actin binding domain. Hines et al. (2015) demonstrated that a vast majority of the human interactome is dominated by weak protein interactions that often have transient interactions rather than forming more permanent, stable complexes. A weak binding between CLIC5 and ERM proteins fits the model put forward by Zhu et al. (2007) in which phosphocycling of ERM is required for maintaining a dynamic relationship be- tween F-actin and the membrane in gastric parietal cells (Zhu et al., 2007). Thus, a low affinity interaction between CLIC5 and RDX may promote activity of functional RDX molecules, allowing for plasticity of membrane-cytoskeleton attachments that may ex- hibit different dynamics during stereocilia development versus long-term maintenance.

Our data indicate that amino acid residues cysteine 32 (C32) and and phenylal- anine 227 (F227) of CLIC5 are critical to its function in terms of localization to stereocilia-like structures such as microvilli and the assembly of these structures on the

______67 surface of cultured mammalian cells. We demonstrated that amino acid F227 is critical for the localization of CLIC5 to ERM-containing stereocilia-like surface structures in LLC-PK1 cells (Fig. 26). Additionally, we showed that amino acids C32 and F227 are critical to the biological activity of CLIC5 protein in its interaction with

ERM proteins to create actin-based surface structures, such as stereocilia-like microvilli and membrane ruffles in COS-7 cells (Fig. 27 and 28). This finding may explain the deafness phenotype in mice with the CLIC5 F227S mutation (Kenneth Johnson, un- published data). We found that the serine residue at position 35 was nonessential for the

CLIC5-ERM interaction in terms of either localization or biological activity of CLIC5 in cultured cells.

Future research must be done to paint a more complete picture of the CLIC5-

ERM protein interaction in inner-ear hair cells. We showed that CLIC5 is able to bind the FERM domain of ERM proteins, which suggests that CLIC5 can bind ERM proteins in their phosphorylated, functionally active state. However, further research is necessary to determine the role and mode of CLIC5-ERM interaction. Although we demonstrated that CLIC5 can bind directly to the FERM domain, it will be important to determine whether CLIC5 binds only to the phosphorylated form of ERM or whether it can also bind to dephosphorylated forms. We demonstrated that the CLIC5 C32 or F227 residues are crucial for the biological activity and localization of CLIC5, but more direct ap- proaches, such as in vitro binding assays, must be taken to determine if mutations in these amino acids reduce the binding affinity of CLIC5 for ERM protein.

______68 Waddell The findings of this research raise the intriguing possibility that CLIC5 and RDX may serve as important pharmaceutical targets in the future for strengthening mem- brane-F-actin linkages in the stereocilia. Additionally, the kinase and phosphatase potentially involved in RDX phosphocycling may also be targets to increase the amount of phosphorylated RDX active in the stereocilia. Overexpressing CLIC5 or RDX to in- crease pRDX may lessen the accumulation of damage in inner-ear hair cells and ultimately combat age-related hearing loss. Deafness-linked genes have been the target of gene therapy and in one recent study gene therapy was able to restore auditory func- tion in TMC (Transmembrane channel-like) deficient mice (Askew et al., 2015), showing us that a similar approach taken with CLIC5 and RDX may be achievable. In addition to overexpression of CLIC5 and RDX to combat the natural decline of hearing with age, gene therapy by methods similar to those in Askew et al. (2015) may be used as a curative measure in CLIC5 -/-, RDX -/-, or haploinsufficient individuals at an early age before the morphology of the hair cells becomes abnormal.

______69

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______74 Waddell APPENDIX

Breeding

Homozygous CLIC5 mutant -/- male mice (jbg/jbg or jbg2J/jbg2J), and heterozygous

CLIC5 +/- female siblings were breed to give both heterozygote and mutant offspring.

Genotyping

A small 1-2 mm piece of the tail tip of each mouse was taken to confirm the mouse’s genotype. Genomic DNA was extracted from tail tips using the Hot Shot procedure

(Truett et al., 2000). This procedure began by placing the tissue in 75 µL of alkaline lysis solution (25 mM sodium hydroxide, 0.2 mM disodium EDTA, pH 12) and then heating the solution to 95°C for 30 min. After the DNA extraction, the solution was cooled to 4°C and 75 µL of neutralization agent was added (40 mM Tris-HCl, pH 5) to reduce the pH. This solution was centrifuged at 14,000 rpm for 2 minutes.

Gnomic DNA was used to prepare solutions for a polymerase chain reaction

(PCR) to amplify the target gene sequences. The following primers were used in this

PCR reaction: jbg2J MF (forward primer) GATTTGCCTGCATGTGTGTC Primer stock# 2M1F jbg2J CF (forward primer) GTCATCCCATGGGTAATGCT Primer stock# 2JC1F jbg2J commonR (common reverse) AGGCCTTTTCTCACCATCCT Primer stock# 2JC1R

PCR reactions contained 2 µL of template DNA, 2 µL of mutant forward primer,

2 µL of control forward primer, 2 µL of common reverse primer, 2 µL of distilled water, and 10 µL of 2x Taq PCR Mastermix (Promega) for a total reaction volume of 20 µL.

Tubes were placed in a PTC-1050 MiniCycler (MJ Research) and the following program was performed to amplify the target sequences: ______75

(1) 94°C for 2 min (2) 58°C for 30 sec (3) 72°C for 2 min (4) 94°C for 30 sec (5) Repeat steps 2-4 (40x) (6) 72°C for 2 min

5 µL of 5x glycerol loading dye was added to each 20 µL PCR product. 10 µL of each complete solution was run on a 1.2% agarose gel. Bands were run against a ladder with bands at 1.7Kbp, 864bp, 621bp, 450bp, and 340bp. These markers were used to tell the relative positions of the Jbg2J mutant allele band at 192bp and the wild- type (control) allele band at 306bp. After electrophoresis, the gels were soaked in an ethidium bromide-containing solution on a rocker for 15 minutes. Images of the bands were taken using Bio-Rad ChemiDoc XRS imaging system and analyzed in Bio-Rad

Image Lab 3.0.1.

1.7 kb

864 bp 621 bp 450 bp 340 bp WT allele Mutant allele

Figure 29: DNA gel showing genotyping of CLIC5 +/- and CLIC5 -/- Jbg2J mouse. Center lane: CLIC5 +/- mouse with both the WT allele (306 bp) and the mutant allele (192 bp). Right lane: CLIC5 -/- mouse with only the mutant allele. Left land: DNA ladder.

______76 Waddell