The Pennsylvania State University

The Graduate School

Department of Physiology

A SILENT SYMPHONY: UNCONVENTIONAL MYOSIN 3A

AND HUMAN HEREDITARY HEARING LOSS

A Thesis in

Anatomy

by

Lina Jamis

© 2015, Lina Jamis

Submitted in Partial Fulfillment of the Requirements for the degree of

Master of Science

August 2015

The thesis of Lina Jamis was reviewed and *approved by the following:

Christopher Yengo Associate Professor of Cellular and Molecular Physiology Thesis Advisor

Alistair Barber Associate Professor of Ophthalmology and Cellular & Molecular Physiology

Sarah Bronson Professor and Director of Research Development and Interdisciplinary Research

Patricia McLaughlin Professor of Neural and Behavioral Sciences Chair of the Graduate Program in Anatomy

*Signatures are on file in the Graduate School. ii

A Silent Symphony: Unconventional Myosin3A and Human Hereditary Hearing Loss

Lina Jamis

(ABSTRACT)

The perception of sound is a process that converts mechanical sound waves to nerve impulses and relies heavily on actin-based protrusions in inner ear hair cells called stereocilia. Unconventional class III myosins (MYO3A and MYO3B) have been found to play a role in the maintenance of stereocilia length, which is essential for hearing. A number of proteins have been implicated in stereocilia length maintenance and many are associated with non-syndromic hereditary hearing loss. ESPIN1 is an actin bundling protein that is transported by MYO3 to the barbed-end of the stereocilia where it plays a role in cross-linking and regulating actin filament length. Recessive loss-of-function mutations in MYO3A are the cause of the inherited hearing impairment phenotype DFNB30 (Walsh et al., 2002), possibly because of the failed MYO3A-dependent ESPIN1 transport to the stereocilia tips. A mouse model of DFNB30 results in age-dependent degeneration of the stereocilia of inner ear hair cells (Walsh, 2010). An uncharacterized missense mutation (G488E) in MYO3A was recently found to be associated with human deafness (Grati et al., unpublished). This mutation is located near the switch I loop, responsible for the steric availability of myosin’s nucleotide-binding pocket. We proposed that the mutation may alter the motor ATPase cycle and motility, which were examined directly with biochemical studies. Another goal of the study was to characterize the point mutation in a cell culture model, which examines MYO3A and ESPIN1 in filopodia, actin-based protrusions with similar properties to stereocilia. We hypothesized that the mutation may prevent normal motility to the filopodial tips and proper elongation of the filopodia. MYO3A transport of ESPIN1 was tested by examining the localization of fluorescently tagged versions of MYO3A and actin bundling in COS7 cells. Since wild- type MYO3A accumulates at filopodia tips, this model provides a well-defined spatial compartment where potential interactions can be clearly visualized. Live imaging of COS7 cells transfected with WT GFP.MYO3A.ΔK and ESPIN1 showed dynamic co- localization at the filopodia tips. In contrast, GFP.MYO3A.ΔK.G488E co-expression with ESPIN1 showed a significant reduction in filopodial tip localization of both proteins. Steady-state actin-activated ATPase activity of baculovirus-expressed MYO3A. ΔK.2IQ wildtype and mutant were performed with an NADH-coupled assay. The maximum ATPase rate of the mutant was shown to be significantly reduced as compared to the wildtype, suggesting that the mutation results in the loss of MYO3A’s force- generating function. Motor activity of purified GFP-labeled MYO3A was directly examined using an in-vitro motility assay. Wild-type MYO3A was able to generate actin sliding while the mutant myosin is still is progress. The cumulative results from these experiments suggest that the mutation located on switch I and near to the conserved nucleotide binding region, disrupts this structural element that is critical for motor function. The introduction of this mutation is sufficient to produce defects in MYO3A-based ESPIN1 translocation, filopodial length, and filopodia density. The findings in the filopodia of COS7 cells may hold true for

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stereocilia in which mutant MYO3A prevents normal motility to stereocilia tips, causing height defects and degeneration of the stereocilia in the inner ear hair cells.

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TABLE OF CONTENTS

LIST OF FIGURES……………………………………………………………….viii

LIST OF TABLES…………………………………………………………………ix

LIST OF ABBREVIATIONS……………………………………………………...x

ACKNOWLEDGEMENTS………………………………………………………..xii

Chapter 1: INTRODUCTION: HEARING, STEREOCILIA, AND MYOSIN…..1

1.1 Purpose………………………………………………………………...1

1.2 Human Hereditary Deafness…………………………………………..1

1.3 The Cell Biology of Hearing…………………………………………..2

1.4 Stereocilia…………………………….………………………………..4

1.5 Cytoarchitecture and organization…………………………...... 5

1.6 ESPIN1 and maintenance of actin filaments…………………………...7

1.7 Class III Myosins………………………………………………………8

1.8 The Myosin Superfamily………………………………………………9

1.9 The Myosin Structure………………………………………………….10

1.9.1 Motor Head…………………………………………………..11

1.9.2 Nucleotide-binding Pocket………...…………………………11

1.9.3 Lever Arm……………………………………………………12

1.9.4 Tail……………………………………………………………13

1.10 Class III Unconventional Myosins…………………………………….13

1.11 Myosin 3A Kinetics……………………………………………………17

1.12 Myosin 3B Subtype……………………………………………………18

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1.13 The Myosin Nucleotide-binding Pocket………………………………19

1.14 Remaining Questions………………………………………………….21

1.15 Conclusions……………………………………………………………21

Chapter 2: RESEARCH OBJECTIVES……………………………………………22

Chapter 3: EXPERIMENTAL METHODOLOGY………………………………...23

3.1 Reagents………………………………………………………………...23

3.2 Expression Plasmids……………………………………………………23

3.2.1 Biochemistry………………………………………………….23

3.2.2 Cell biology………………………………………………...…23

3.2.3 Site-directed mutagenesis………………………………….…24

3.3 Protein Expression and Purification……………………………………24

3.4 Sequence Analysis……………………………………………………...25

3.5 Cell Culture and Transient Transfection………………………………..25

3.6 Live Cell Imaging of Cells Expressing Fluorescent Proteins…………..26

3.7 Data Analysis, Statistical Analysis, and Software Used……………….26

3.8 Steady-state ATPase Activity………….……………………………….27

3.9 In-Vitro Motility……………………….…………………………….....27

Chapter 4: RESULTS………………………………………………………………29

4.1 Sequence Analysis……………………………………………………...29

4.2 Quantification of MYO3A Tip Localization, Filopodia Length, and

Density in COS7 Cells………………………………………………….29

4.3 MYO3A Steady-state ATPase Activity………………………………...31

4.4 MYO3A In-Vitro Motility………………………………………………31

Chapter 5: DISCUSSION………………………………………………………...... 42

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Chapter 6: CONCLUSION AND FUTURE DIRECTIONS……………..…………50

WORKS CITED………………………………………………………………...... 54

APPENDIX………………………………………………………………………58

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LIST OF FIGURES

Figure 1.1 The Organ of Corti 3

Figure 1.2 Auditory transmission and the internal structure of the ear 5

Figure 1.3 The actin tread-milling mechanism 6

Figure 1.4 Cargo Transporter, Myosin V structure and important domains 10

Figure 1.5 Ribbon representation of the S1 structure of the myosin motor 11

Figure 1.6 Myosin 3A structure and important domains 15

Figure 1.7 The actomyosin ATPase cycle reaction scheme 18

Figure 1.8 Space-filling model of the myosin protein and ATP 20

Figure 4.1 Sequence alignments of amino acid residues from the Switch I 32 region of human MYO3A in comparison with human MYO3B, MYO5A, MYO7A, and Drosophila NINAC

Figure 4.2 Locations of WT and mutant MYO3A in COS7 cells 34

Figure 4.3 Impact of the G488E mutation on MYO3A filopodia elongation 35

Figure 4.4 Impact of the G488E mutation on filopodia induction in COS7 36 cells

Figure 4.5 The presence of ESPIN1 increases tip-localization of wildtype 37 MYO3A

Figure 4.6 Mutant MYO3A can function as a dominant-negative 38

Figure 4.7 Actin-activated ATPase activity of MYO3A.ΔK.2IQ and 39 MYO3A.ΔK.G488E.2IQ

Figure 4.8 In-vitro motility assay of WT MYO3A 40

Figure 5.1. WT MYO3A.ΔK is able to tip-localize on COS7 filopodia, while 41 mutant MYO3A.ΔK.G488E is not

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LIST OF TABLES

Table 1.1 Function, targeting, and regulation of unconventional myosin, 16 organized by myosin class

Table 3.1 Experimental myosin constructs 24

Table 4.1 Actin-activated ATPase activity of MYO3A.ΔK.2IQ constructs 39

Table 4.2 Sliding velocity of actin filaments on MYO3A.ΔK.2IQ constructs 40

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LIST OF ABBREVIATIONS

ADP Adenosine diphosphate

AM Acto-myosin

ATP Adenosine triphosphate

Bac Baculovirus

C° Centigrade

DNA Deoxyribonucleic acid

DTT Dithiothreitol

EGTA Ethylene glycol tetraacetic acid

Epsin1-un Unlabeled ESPIN1

GFP Green fluorescent protein

IHC Inner hair cell

ΔK Delta kinase

F-actin Filamentous-actin

Katpase Catalytic ATPase rate

Kcat Catalytic rate constant

KM Kinase motor

MD Motor dead min Minute mL Milliliter

MYO3A Myosin IIIA

MYO3B Myosin IIIB

MYO5 Myosin V

x

n Number

NADH Nicotinamide adenine dinucleotide

NBP Nucleotide binding pocket

NINAC Neither inactivation nor afterpotential C

N-terminus Amino terminus

OHC Outer hair cell

Pi Inorganic phosphate

PBS Phosphate buffered saline pFB pFastBac

S.E. Standard Error

S1 Subunit 1

THDI Tail homology domain I

THDII Tail homology domain II

TCBR Tip to cell body ratio

µg Microgram

µL Microliter

µm Micrometer

µM Micromolar

WT Wildtype

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ACKNOWLEDGEMENTS

This study was made possible by funding from the National Institute of Health. Special thanks should be given to the committee responsible for mentoring and assisting in the revisions of the experimental design. This committee included Dr. Christopher Yengo, Dr. Alistair Barber, and Dr. Sarah Bronson. Their knowledge and research experience were essential in this process.

Special thanks should be given to Dr. Yengo, my mentor, for his guidance and unending patience during my years as a master’s student at Penn State Hershey. Thank you for teaching me self-discipline for great work both inside and outside the laboratory.

I am indebted to the members of the Yengo lab: William Unrath, Darshan Trivedi, Manmeet Raval, Anja Swenson, Chris Fetrow, and Wanjian Tang, for all of their help, advice, and inspiring discussions during the course of my thesis work.

Thank you to Dr. Ira Ropson for his invaluable help in showing me how to model proteins with the available programs.

My most sincere thanks should also be given to Dr. Patricia McLaughlin, my program advisor, for opening the doors to Hershey for me. Her generosity, kindness, and insight have been invaluable during my time here.

Lastly, I would like to thank my dad for his overwhelming support and invaluable witticisms; these have been precious to me. It is your shining example that I try to emulate in all that I do.

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“Does sound have rhythm? Does it rise and fall like the ocean? Does sound come and go like wind?” ― Myron Uhlberg, Hands of My Father: A Hearing Boy, His Deaf Parents, and the Language of Love

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Chapter 1 INTRODUCTION: HEARING, STEREOCILIA, AND MYOSIN

1.1 Purpose

The precise ultra-structure of the inner ear hair cell stereocilia is essential for the

conversion of sound waves into auditory neurotransmission. Although much is known

about the impact of myosin and other cytoskeletal proteins on muscle contractility,

considerably less is known about how these proteins function to produce stability in

the inner ear. This review discusses how class III myosins (MYO3A and MYO3B) of

the myosin superfamily play a role in the organization, maintenance, and intracellular

transport, within the inner ear hair cell stereocilia, which ultimately contributes to the

reception of sound. Knowledge of the role that class III myosins play in the internal

structure and function of the ear will help in the understanding of myosin-mediated

onset of human hereditary deafness.

1.2 Human Hereditary Deafness

Genetic deafness is the most common form of sensory disorder in humans. Due to the

complex structure of the inner ear, it is believed that as many as a hundred genes might

participate in the hearing process (Redowicz, 2002). A mutation to one of these genes

can easily disturb the inner ear’s delicate balance and yield deafness. The first report

about a possible involvement of motor proteins in the hearing process was reported by

Hasson et al., (1997) who showed that MYO1C was located at the tips of stereocilia of

the hair cells. Those and subsequent studies, only some of which are supported in this

review, have led to the idea that myosin plays an essential role in maintenance of the

stereocilia ultrastructure. Several myosins have been implicated in the role of

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stereociliary growth and maintenance, including MYO15A, MYO7A, MYO6, and

MYO1C (Schwander, 2010), and of course the focus of this review, MYO3A.

MYO3A’s role in audition was first described in a study by Walsh et al., (2002), in

which the MYO3A gene was proposed to be the cause of a delayed onset, autosomal

recessive deafness condition with age-dependent penetrance called DFNB30. The

G488E missense mutation on the MYO3A gene is a novel autosomal dominant

mutation first described by our collaborators (Grati et al., personal communication)

that has been found to cause congenital progressive hearing loss. This mutation

provides an ideal model to further test the idea that MYO3A is necessary in the

process of hearing.

1.3 The Cell Biology of Hearing

The sensation of hearing is based on the detection of mechanical stimuli. Although the

proteins involved in hearing have not been well characterized, anatomical,

physiological, and biophysical studies have elucidated the fundamental processes.

Integral to these processes are hair cells, which reside in the Organ of Corti (OC) of

the inner ear. Described as “a masterpiece of cellular micro-architecture” (Betlejewski,

2008), the OC contains approximately 16,000 hair cells that are organized in one row

of inner hair cells (IHCs) and three rows of outer hair cells (OHCs) (Schwander,

2010). In no other organ in the body is it as easy to see the precise organization of the

principal cells. The IHCs and OHCs are separated by a large extracellular space called

the tunnel of Corti (Forge et al., 2002). Pictures obtained with electron microscopes

show the OC’s colonnade appearance (Figure 1.1).

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A B

Figure 1.1. The Organ of Corti. The photogenic appearance of the organ of Corti has long been appreciated by scientists and popular press. Scanning electron micrograph of the cochlear sensory epithelium featuring A) the stereocilia of a single row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs). The overlying tectorial membrane has been removed (Ryan, 2000). B) Electron micrograph of a hair bundle in the characteristic V-shape (Berg et al., 2002).

IHCs are arranged in U-shaped hair bundles that appear to form an almost continuous fence along the inner aspect of the organ of Corti. In contrast, OHCs form a characteristic ‘W’ shape. Both IHCs and OHCs contact the underside of the overlying tectorial membrane. Furthermore, the two hair cell types show very different innervation patterns. IHCs are innervated almost exclusively by afferent nerves traveling to synaptic connections at the cochlear nuclei in the brainstem (Klinke,

1986). Efferent endings to the IHC region arise ipsilaterally from the lateral superior olive in the mid-brain and contact the afferent nerves below the level of the hair cell

(Spoendlin, 1985). This means that most of the information about the acoustic world reaches the brain via the inner hair cells. The outer hair cells, meanwhile, are located near the center of the basilar membrane where vibrations will be greatest. OHCs are directly innervated by large efferents that descend mainly contralaterally in the medial portion of the superior olive. These innervation patterns alone indicate that IHCs are the primary receptor cells, while OHCs function as a cochlear amplifier that refines the

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sensitivity and frequency selectivity of the mechanical vibrations of the cochlea (Fay

et al., 2000).

1.4 Stereocilia

Each hair cell contains a hexagonal or V-shaped shaped bundle of 20-300 hair-like

projections, called stereocilia, on the apical surface of the hair cell from which they

protrude. These bundles are graded by height on top of the basilar membrane of the

Organ of Corti. Stereocilia are plasma membrane-bound projections that enclose

filaments of the cytoskeletal protein, actin. These stereocilia are ultimately responsible

for the initiation and termination of sound-based action potentials. Oscillations in air

pressure detected by the tympanic membrane are converted into fluid pressure, which

induces vibrations in the basilar membrane, where the hair cells are situated.

Movement of the basilar membrane causes the hair bundles to press up against the

tectorial membrane, causing deflection of the hair bundles in the direction of the tallest

protrusion (Figure 1.2).

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Figure 1.2. Auditory transmission and the internal structure of the ear. Agitation of the basilar membrane causes the stereocilia to bend against the tectorial membrane above. Shearing forces acting on the stereocilia cause opening of tip links, connecting a cluster of stereocilia. These deflections initiate cell depolarization and induce an action potential (Gray’s Anatomy, 2009).

These deflections of the stereocilia open the mechanically gated ion channels and

depolarize or hyperpolarize the cell, activating or inactivating, respectively, the

auditory nerve fibers that carry information into the central nervous system.

Neurotransmitter release at afferent synapses is regulated by changes in the membrane

potential of the hair cell in response to the shearing forces to the stereocilia bundle.

1.5 Cytoarchitecture and organization

Since transduction channels are gated when adjacent stereocilia slide along each other

during bundle deflections, auditory and vestibular transduction relies on the structural

integrity of stereocilia and the hair bundle. The stereocilia of each hair cell are

arranged in a precise geometry. This arrangement is asymmetrical and polarized

according to height; their staircase-like arrangement is vital to their function in

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mechano-transduction. Stereocilia with height defects have been found to degenerate and disrupt hearing function (Kiernan et al., 2002). Like other cytoskeletal structures, stereocilia are polarized with regard to height—the barbed, or ‘plus,’ ends of the actin filaments pointing towards the stereocilia tips and the ‘minus’ ends form the base.

Unlike other structures such as filopodia and microvilli, however, stereocilia are maintained at a constant length throughout life (Schwander, 2010). Although stereocilia height remains relatively constant, the actin core is dynamic. Actin monomers are polymerized at the stereocilia tips and depolymerized at the stereocilia base. Rates of actin polymerization and depolymerization must be tightly regulated to maintain stereociliary length (Figure 1.3). The actin tread-milling rate in stereocilia is

~10-fold slower than in filopodia (Rzadinksa et al., 2004), suggesting that specialized mechanisms control this process in hair cells (Lin et al., 2005).

Figure 1.3. The actin tread-milling mechanism. Actin tread-milling (right); the rate of growth at the barbed (plus) end of the filament is equal to the rate of growth at the minus end. Thus, the filaments maintain a constant length, by growing and shrinking at the same time.

Recent measurements demonstrated that actin along the length of stereocilia has a months-long half-life, whereas only the tips feature more rapid actin turnover (Zhang et al., 2012). The available evidence suggests that the core actin bundle acts as a scaffold that provides mechanical support for the stereocilium, in effect, determining

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its position, orientation, dimensions, and possibly also influencing its stiffness and

ability to pivot about its base (Zheng et al., 2000). The stereocilium tapers near its

base, so that only the central actin filaments of the core actin bundle extend a rootlet

into the apical cytoplasm of the hair cell to encounter the dense actin filament

meshwork known as the cuticular plate (Zheng et al., 2000). Immediately underlying

the lateral plasma membrane of the stereocilia is an organized cytoskeletal complex,

the cortical lattice, composed of actin filaments running helically around the cell.

Regularly arranged short pillar-like structures appear to link the lattice to the inner side

of the plasma membrane. It has been suggested that the lattice gives rigid support to

the plasma membrane preventing deformation, but also acts like a spring providing a

restorative force after length increase. Therefore, it is a functional requirement that the

actin cytoskeleton maintain its height and constant renewal of actin monomers.

1.6 ESPIN1 and maintenance of actin filaments

Actin bundles exist in a diverse set of structures from neurosensory bristles in

Drosophila to brush border microvillae (Zheng et al., 2004). The actin protrusion

structures depend on actin-bundling proteins to maintain the tight links between actin

filaments, much like scaffolds in a high-rise. The parallel actin filaments in the

stereocilia are closely packed in a semi-crystalline array and are cross-linked by the

actin-bundling protein ESPIN1 (Salles et al., 2009). In experiments in which the

ESPIN gene was modified, resulting in ‘Jerker’ mice stereocilia that lacked ESPIN,

there was a deafness phenotype and vestibular dysfunction (hence the name ‘Jerker’)

observed (Zheng, 2000; Steel, 1983). These studies revealed the importance of ESPIN

in actin bundling in hair cell stereocilia (Whitlon et al., 2004). In order to function as

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cross-linkers, ESPIN and other actin-bundling proteins must position themselves

between the actin filaments and are thought to do so after the actin monomers are

assembled at the filament tips. The question remains: how, then, does ESPIN make its

way to the newly polymerized actin monomers in order to incorporate into the bundle?

Studies have shown that MYO3A is responsible for localizing ESPIN1 to the tips of

stereocilia (Salles et al., 2009) and in doing so, increases stereocilia elongation.

1.7 Class III Myosins

The first class III myosin to be identified was Neither Inactivation Nor Afterpotential

C (NINAC) from Drosophila melanogaster, which is expressed specifically in

rhabdomeral photoreceptors (Dosé, 2008; Katti, 2009) and thought to mediate

phototransduction. Normal hearing in humans has been shown to require MYO3A, the

vertebrate-specific version of NINAC (Walsh, 2002) in which non-syndromic

progressive hearing loss is caused by loss-of-function mutations in MYO3A. An

additional finding that emphasizes the importance of class III myosins in sensory cells

is that MYO3A was recently localized to a region of cochlear and vestibular hair cells

that defines a previously unidentified compartment at the tips of the stereocilia (Salles

et al., 2009). How exactly mutations to MYO3A cause progressive hearing loss is not

entirely known, nor is it clear that this is the only myosin associated with this

particular type of human hearing loss. However, one thing is certain: studies of

deafness with mutant alleles indicate that molecular motor proteins perform

fundamental functions in the auditory system and that their function is critical to

hearing (Friedman et al., 1999).

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1.8 The Myosin Superfamily

Myosins are motor proteins that bind actin and hydrolyze adenosine triphosphate

(ATP) to produce force against fixed or mobile actin filaments. Myosin motor

domains, containing the ATPase and actin-binding activities, are connected to a range

of amino-terminal and carboxy-terminal domains corresponding to the variety of

molecular cargos that they move (Woolner et al., 2009). All myosin molecules

hydrolyze ATP via the same mechanism. Despite all having a conserved motor

domain, isoforms of the myosin superfamily have a diverse set of functions. The

sensory epithelium of the internal ear, a tissue that is particularly reliant on actin-rich

structures, exploits this diversity of myosin isoforms. Each inner ear stereocilium is

filled with hundreds of cross-linked actin filaments, where the actin concentration is

approximately 4 mM (Hasson et al., 1997). Because actin filaments in stereocilia are

uniformly polarized with their barbed end at the stereocilia tips, MYO3A can be

described as a barbed-end-directed motor and that the targeting of MYO3A to the tips

of stereocilia depends on a motor-driven translocation along the actin filament

(Rzadzinska et al., 2004). Furthermore, the myosin motor protein ‘motors’ to the

stereociliary tips towing components of actin assembly machinery to regulate the

ciliary length (Schwander et al., 2010). Of three different human MYO3A mutations

that have led to nonsyndromic hearing loss (Walsh et al., 2002), two of the mutations

truncate the MYO3A protein before the tail domain, and the third alternatively splices

the protein message leading to an unstable protein product. DFNB30 results in a lack

of functional MYO3A, which is likely degraded rapidly in the cell, thus leaving

stereocilia without a major method of material transport and maintenance. However, to

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understand the function or dysfunction of myosin, the structure of the myosin protein

must be first understood.

1.9 The Myosin structure

Myosins were originally discovered in muscle, while many non-muscle myosins have

also more recently been well-characterized. Class V myosin (Figure 1.4), functions as

a cargo transporter, much like MYO3, although structurally, it is distinct. MYO5 is a

large asymmetric molecule that has a long tail, a globular head, and is often dimerized.

MYO5 is made up of two heavy chains, with each heavy chain associated with two

light chains. The two heavy chains are wound around each other to form a helical

dimer. At one end, both chains are folded into separate globular structures to form the

two heads (that comprise the motor) of the dimer; the other end of the protein forms

the tail, where myosin binds to its cargo.

Figure 1.4. Cargo transporter, myosin V structure and important domains. Includes the motor, lever arm, and cargo-binding tail domain (Sellers et al., 2006).

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1.9.1 Motor head:

All myosins characterized thus far share a relatively conserved N-terminal motor domain. The highly conserved regions of the motor are in the nucleotide-binding region; switch I and II, and the P loop play an integral role in ATP binding and hydrolysis. The 110-kDa catalytic subunit comprising the motor is commonly referred to as subfragment 1 (S1; Figure 1.5). The S1 fragment contains three domains, the N- terminal domain, the central 50 k-Da domain and the C-terminal neck domain. The upper 50-kDa domain rotates about a region comprising switch I, such that the switch exists in different conformations during the acto-myosin cycle.

Figure 1.5. Ribbon representation of the S1 structure of the myosin motor (Geeves et al., 1999).

1.9.2 Nucleotide-Binding Pocket:

Myosin motility depends on a catalytic core in the motor that executes the nucleotide hydrolysis cycle, which consists of ATP binding, hydrolysis, and the release of the products (ADP and inorganic phosphate). Depending on their nucleotide state, myosins switch structural conformations that result in force generation and/or 11

detachment from the actin track (Sablin, 2001). A γ-phosphate-sensing mechanism in the motor domain relies upon three conserved regions, switch I, switch II, and P-loop.

These loop structures are major components of the protein’s ATPase site, and share structural and functional homology with the active sites of other ATPase and GTPases

(Sasaki et al., 1998). In the ATP state of the motor, the switch I serine and switch II glycine converge on the nucleotide to form hydrogen bonds with the γ-phosphate. The movements of key residues are thought to trigger the restructuring of the switch regions, which allows the opening and closing of the nucleotide-binding pocket. In the

ATP state, the switch regions move towards the nucleotide and close the binding pocket. In the ADP state, the absence of the bridging interactions with the γ-phosphate disengages the switch regions, causing the opening of the nucleotide-binding pocket

(Sablin, 2001) to allow the ADP to be released from this site. Mutations to either of these switch residues have been proposed to alter ATP binding, hydrolysis, and product release.

1.9.3 Lever arm:

The myosin light chains wrap around the C-terminal neck domain, which forms a long helix sometimes referred to as the lever arm. Rayment and collaborators (1993) suggested that the conformational changes in the nucleotide-binding pocket could be transmitted to the lever arm. It is proposed that small conformational changes in the nucleotide-binding region are amplified to generate larger movements of the lever arm. During specific steps in the myosin ATPase cycle, the movement of the lever arm drives the movement of myosin along actin. There are several reports that suggest that

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the length of the lever arm is proportional to the step-size of the movement (Sun et al.,

2011).

1.9.4 Tail:

The C-terminal tail domain of myosin proteins is more variable and class-specific and

is generally thought to mediate cargo binding or filament formation. While the head

domain of the myosin protein exerts force on actin, the role of a particular myosin

isoform is distinguished by its tail domain. The tail domains of myosins bind the

plasma membrane of the cell, the membranes of intracellular organelles, or other

cellular cargoes. In skeletal muscle myosin, the tail domains dimerize and form thick

filaments, which compose part of the contractile apparatus in skeletal muscle. In

contrast, unconventional myosins function as cargo transporters and walk along actin

filaments to their destination.

1.10 Class III Unconventional Myosins:

The first myosin to be characterized was the bipolar filament-forming class II myosins

of muscle and nonmuscle cells. In contrast to these filament-forming myosins, at least

16 more classes of myosins (Table 1.1), referred to as ‘unconventional,’ have been

reported from organisms ranging from Dictyostilium discoidium to humans (Dosé,

2008). Unconventional myosins comprise a class of proteins that do not fit

conventional standards of the myosin protein. These proteins can be monomeric or

dimeric but are thought not to form filaments (Bähler et al., 2000). Rather, these

myosins function as transporters for vesicles, organelles, and other cargoes along actin

filaments.

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MYO3A (Figure 1.6) also differs from other myosins since it has been shown to have a unique N-terminal kinase domain that allows autophosphorylation (Ng et al., 1996).

The kinase domain is important for phototransduction in Drosophila (Porter and

Montell, 1993) and for regulation of the motor domain in vertebrate MYO3 (Erickson,

2003). MYO3A’s actin-binding and enzymatic properties are regulated by phosphorylation of its motor, which is ultimately responsible for myosin translocation along actin filaments (Quintero, 2010 and 2013).

Deletion of MYO3A’s distinctive N-terminal kinase domain enhances the accumulation of MYO3A at the tips of filopodia in HeLa cells (Erickson, 2003) and results in elongation of the stereocilia and bulging of the stereocilia tips (Schneider,

2006; Dosé et al., 2008; Salles et al., 2009). These findings suggest that the kinase domain may regulate the myosin motor. Several studies have removed the kinase domain (MYO3ΔK) to allow examination of a constitutively active motor.

The tail domain of MYO3A is made up of two particularly important domains, 3 tail homology domain (THD) I and 3THDII. The 3THDI domain is responsible for cargo- binding and is necessary for the MYO3A:ESPIN1 interaction (Salles et al., 2009) while the 3THDII domain is responsible for the interaction with actin. The motor and tail actin-binding sites are thought to allow MYO3A to move as an inchworm along the actin filament. The two actin-binding sites allow alternating attachment and movement. The 3THDII is highly conserved in vertebrate MYO3A tails, and contains the actin-binding motif, DFRXXL. An intact DFRXXL sequence is required for

MYO3A tail:actin interactions in vitro and for localization of full-length MYO3A to filopodia tips (Manor et al., 2008). A failure of proper actin assembly at the barbed

14

end of the stereocilia results in improper length maintenance and may lead to deficits in audition.

Figure 1.6. Myosin 3A structure and important domains. Includes the kinase, motor, IQ calmodulin-binding, and 3THDI and 3THDII tail domains (adapted from Manmeet Raval, 2014).

15

Table 1.1. Function, targeting, and regulation of unconventional myosins, organized by myosin class (Sivaramakrishnan et al., 2011).

16

1.11 Myosin 3A Kinetics

The ATPase activity of myosin is coupled to a cyclic interaction with actin, which

allows the motor to do mechanical work, and with each ATP hydrolysis cycle, the

motor takes a step. The reaction pathways of the actin-activated myosin ATPase

appears to be conserved for all myosin isoforms, but the rate and equilibrium constants

that define the ATPase pathway alter significantly across the myosin superfamily

(Rayment, 1993). The kinetic differences allow myosins to carry out diverse

mechanical functions. In the absence of ATP, myosin binds tightly to actin (Figure

1.7: [1]). This conformation is known as the ‘rigor conformation.’ ATP binding to the

myosin head induces a conformational change in in the actin-binding site that weakens

its actin affinity and causes the myosin to detach from actin [2]. The binding of ATP

also causes a large conformational shift in the lever arm, bending the myosin head into

position further along the actin filament [2]. ATP is hydrolyzed to ADP and inorganic

phosphate (Pi), and the hydrolysis products dissociate slowly [3]. In the absence of

ATP, myosin rebinds to actin weakly [4] and a slight conformational change promotes

the release of Pi [5]. The release of Pi enhances the interaction between myosin and

actin and triggers the ‘power stroke,’ the key force-generating step [6]. In an actin-

bound conformation, ADP is released allowing for subsequent ATP binding and the

initiation of a new cycle (Lodish, et al., 2000). Kinetic studies of unconventional

myosins that function as cargo transporters show that: ATP promotes fast dissociation

from actin, phosphate release occurs rapidly after actin binding, and that myosin has a

high affinity for actin in the presence of ADP (De La Cruz et al., 2009). MYO3A’s

cycle time is limited by ADP release, meaning that the transition from Actomyosin-

ADP (AM.ADP) to AM is its slowest step (Dosé et al., 2006).

17

Figure 1.7. The actomyosin (AM) ATPase cycle reaction scheme (adapted from MB Info, “Myosin ATPase Activity,” last modified October 20, 2014, http://www.mechanobio.info/modules/go-0008570).

1.12 Myosin 3B subtype

Vertebrates have been shown to possess another type of class III myosin, MYO3B

(Manor et al., 2012). Since MYO3B functions similarly to MYO3A, it may compensate

for defects in MYO3A, yet little is known about its distribution and function. Support

for this theory is that patients with mutations in MYO3A exhibit hearing loss but show

no apparent defects in vision or vestibular function. MYO3B may be co-expressed with

MYO3A and there may be functional redundancies between these two proteins.

MYO3A and MYO3B have been shown to be similar in their kinase and myosin

domains and vary in their neck and tail domains due to alternative splicing (Manor et

18

al., 2012). Pairwise alignments of the amino acid sequences of mouse MYO3A and

mouse MYO3B show that the sequences are 47% identical and 60% similar (Katti et

al., 2009). Portions of the C-terminal 3A and 3B tail domain, 3THDI, show higher

levels of homology than the tail as a whole. However, MYO3B lacks the second THD

(3THDII; actin-binding) domain located at the C-terminus of MYO3A, making the

MYO3B tail shorter. Like, MYO3A, MYO3B also binds ESPIN1 and can transport it to

the tips of stereocilia, however, it utilizes ESPIN1 as its tail actin-binding domain to

walk along actin, since it lacks THDII.

1.13 The Myosin Nucleotide-Binding Pocket

The ATP molecule sits in the nucleotide-binding pocket (NBP) such that the phosphate

groups occupy the space closest to the internal structure of the motor (switch I, switch

II, P loop). To accommodate ATP in the active site, the nucleotide binding pocket must

undergo physical rearrangements (Coureux et al., 2004). Introducing a mutation to the

switch I loop (Figure 1.8), located in close proximity to the NBP, may alter the physical

rearrangement of the NBP. Conceivably, a mutation at this site could alter the affinity

of ATP binding, the ability to induce pocket closure, or the ability to facilitate ATP

hydrolysis. A study conducted by Sutoh et al. (1998) describes the introduction of a

missense mutation in Dictyostelium discoideum MYO2 at position G240A. The results

of this experiment showed the mutation resulted in a decrease in the MYO2 ATPase

activity and in-vitro motility. Cells expressing the mutation exhibited functional

phenotypes similar to that of cells lacking myosin. These results, suggest that the switch

I region may play an essential role in the integrity of the NBP and interfere with ATP

binding, hydrolysis, or product release (Sasaki et al., 1998).

19

Mutations near the NBP can result in altered interactions with ATP, which in turn can alter motor ATPase activity. The ATP binding site lies sufficiently close to the mutation such that the introduced charge of the glutamate may exert an electrostatic effect on ATP binding and thus on the ability of ATP to hydrolyze in the pocket.

Figure 1.8. Space-filling model of the myosin protein and ATP. Glycine is pictured (red) in the nucleotide binding pocket (NBP). ATP (orange) sits in the ATP binding site. The replacement of the native glycine with glutamate, specifically its negative charge at physiological pH and large size, may affect ATP-binding capabilities of the myosin protein.

For example, mutation to the switch I loop may slow a critical step in the acto-myosin

cycle, which could slow in-vitro motility. It is possible that motor defects can cause a

decrease in the ability of MYO3A to function as a transporter, which may result in

degeneration of the stereocilia of inner ear cells.

20

1.14 Remaining Questions

The auditory and vestibular systems are intimately connected and while both ultimately

function to provide different modes of perception—i.e. sound and head position,

respectively, both rely on hair cells to initiate signal transmissions to the nervous system.

Studies have shown that members of the myosin family (I, VI, VII, X, XV) also inhabit

the hair cells that reside in the vestibular sensory cells (Géléoc et al., 2003) however there

seem to be no vestibular dysfunction in individuals with both MYO3A-induced

hereditary deafness (Ahituv, 2002), which suggests that MYO3 is more essential in the

hair cells that inhabit the auditory system. Further studies are required to examine the role

of MYO3A in the vestibular system.

1.15 Conclusions

The precise role of MYO3A in the cochlea and the mechanism by which mutations in

MYO3A lead to progressive hearing impairment remains to be elucidated. However, a

role for MYO3A in transport suggests that defects in MYO3A can impair its ability to

maintain proper stereocilia ultrastructure. Thus, understanding how specific mutations

disrupt MYO3A motor function and linking these defects with the ability of MYO3A to

localize to and elongate stereocilia is a powerful approach to understanding the unique

molecular biology of the inner ear and the maintenance involved in its function.

21

Chapter 2: RESEARCH OBJECTIVES

The long-term goal of this research is to characterize the missense mutation in the switch

I region of the MYO3A motor [glycine 488 to glutamate (G488E)], which has been identified in a family of patients that show congenital deafness (Grati et al., unpublished).

The hypothesis of this research is that this mutation is sufficient to produce a defective myosin motor thereby, a) preventing MYO3A:ESPIN1 translocation to stereocilia tips, b) hindering stereociliary elongation, c) disrupting the ultrastructure of the stereocilia, and d) causing early-onset deafness.

Specific Aim 1:

To characterize the effects of the mutation on myosin motor function by assessing in- vitro motility, ATPase activity, and localization in actin-bundled based protrusions.

Specific Aim 2:

To propose a mechanism that describes how impairment to MYO3A motor properties impacts stereocilia structure and function, yielding a dominantly inherited form of deafness.

22

Chapter 3 EXPERIMENTAL METHODOLOGY

3.1 Reagents

ATP and ADP were prepared fresh from powder. Nucleotides were prepared in the

presence of equimolar amounts of MgCl2 before use. All reagents were the highest purity

available.

3.2 Expression Plasmids

Previously generated constructs of N-terminal GFP-tagged human MYO3A (Quintero et

al., 2010) were modified for both cell biology and biochemical experiments (table 1.2)

3.2.1 Biochemistry

MYO3A constructs used for biochemistry were modified containing residues 1-1143

truncated after the second IQ domain (MYO3A.2IQ). Myosin without the kinase domain

(MYO3A.2IQ.ΔK, residues 1-339) was also generated as in previous studies (Quintero,

2010). All baculovirus constructs contained a C-terminal GFP tag and C-terminal FLAG

tag for purification purposes (FLAG.MYO3A.ΔK.2IQ.GFP WT, FLAG.MYO3A.

ΔK.2IQ.GFP.G488E).

3.2.2 Cell biology

MYO3A constructs used for cell biology contained the tail domain as well as a GFP or

MCherry tag on the N-terminus (GFP.MYO3A.ΔK WT, MCherry.MYO3A.ΔK WT,

GFP.MYO3A.ΔK.G488E, MCherry.MYO3A.ΔK.G488E). Unlabeled ESPIN constructs

23

were also generated as well as a previously characterized motor dead mutation

(GFP.MYO3A.G720A.MD) (Quintero, 2010).

3.2.3 Site-directed mutagenesis:

QuikChange site-directed mutagenesis (Agilent Technologies) was used to modify

MYO3A constructs with a single point mutation, substituting glutamate in place of

glycine at residue 488 in the switch I region of the motor.

Table 3.1. Experimental Myosin Constructs. Construct Experiment pFB.N-FLAG.MYO3A.ΔK.2IQ.C-GFP.WT Biochemistry: pFB.N-FLAG.MYO3A.ΔK.2IQ.C- ~motility assay GFP.G488E ~ATPase assay pFB.N-FLAG.M3A.KM.2IQ.C-GFP.WT

pe.N-GFP.MYO3A.FL Cell Biology: pe.N-GFP.MYO3A.ΔK ~localization in COS7 filopodia pe.N-GFP.MYO3A.ΔK.G488E pe.N-GFP.MYO3B.ΔK ESPIN1 (unlabeled) pe.N-MCherry.MYO3A.ΔK pe.N-MCherry.MYO3A.ΔK.G488E pe.N-GFP.G720A.MD (Motor Dead) pe N-GFP (GFP only)

ΔK – kinase domain deletion; G488E – glutamate substitution for glycine at amino acid residue 488; GFP – green fluorescent protein; KM – kinase domain; Mchry – Mcherry fluorescent probe; MD – motor dead; MYO3A – myosin3A; MYO3B – myosin3B.

3.3 Protein Expression and Purification

Cell biology constructs (MYO3A 2IQ: containing 2IQ domains, ΔK: deleted kinase

domain, and C-GFP: containing a C-terminal GFP) were expressed via recombinant

baculoviruses generated with the FastBac system (Invitrogen). MYO3A.2IQ.C-GFP wild

type (WT), ΔK, and G488E were co-expressed in insect Sf9 cells. Expression in Sf9 cells

24

and purification with anti-FLAG affinity chromatography were performed as described

(Dosé, 2007). Actin was purified from rabbit skeletal muscle using an acetone powder

method (Pardee and Spudich, 1982).

3.4 Sequence Analysis

MYO3A WT and mutant nucleotide sequences were verified via Sanger Sequencing

performed at GENE WIZ. The ExPASy and NCBI Protein BLAST programs were used

to compare the sequence to that in the database.

3.5 Cell Culture and Transient Transfection

COS7 cells were obtained from ATCC (Mannassas, VA) and grown in Dulbecco’s

modified Eagle’s medium (GIBCO) containing 4.5 g/L glucose, 50 mL 5% fetal bovine

serum, and 5 mL penicillin-streptomycin. Cells were passaged using 0.05% trypsin-

EDTA (Invitrogen). Prior to transfection, the cells were plated onto acid-washed 22-mm-

square, coverslips at 35,000-45,000 COS7 cells per coverslip (one coverslip per well of a

6-well dish) and allowed to adhere overnight. Cells were transiently transfected using

Fugene transfection agent HD (400 µg of MYO3 plasmid DNA or 30 µg of ESPIN1

plasmid DNA was diluted into 100 µl of Opti-MEM media (Invitrogen) and mixed with 3

µl Fugene). The transfection mixes were allowed to incubate at room temperature for 15

minutes (min) and then added drop-wise to the well containing the cells to be transfected

(3 mL of medium per well). Transfection efficiency was assessed by examining the

fluorescence intensity of the cells during the fluorescence microscopy imaging.

25

3.6 Live Cell Imaging of Cells Expressing Fluorescent Proteins

Transfected cells were imaged at ~24 hours following transfection. The coverslips were

assembled into Rose chambers containing imaging medium (Opti-MEM without phenol

red supplemented with 5% fetal bovine serum and 100 units of penicillin-streptomycin).

Single images and time-lapse image sequences were collected using a Nikon TE2000-

PFS fluorescence microscope with a 60 x 1.4 N.A. phase objective. Images were obtained

using a CoolSnap HQ2 cooled charge-coupled device digital camera (Photometrics) and

NIS-Elements AR software (Nikon). For single images, exposure times were held

constant at 400 milliseconds (ms) for all fluorescence channels (GFP and mCherry) and

60 ms for the bright field channel (phase contrast). All imaging settings were kept

constant between groups.

3.7 Data Analysis, Statistical Analysis, and Software Used

Both ImageJ and NIS-Element AR (Nikon) software were used for image analysis. To

calculate the ratio of tip intensity to cell body intensity (rt/c), integrated intensity was

measured for a 5 x 5-pixel region of the background (bb), the filopodial tip (Bbt), and the

cell body (bc) and calculated using the following equation:

!" ! !! ��/� = (Eq. 1) !"!!!

26

Filopodial lengths were measured using the straight-line tool in ImageJ, and filopodial

density was calculated by dividing the number of protrusions extending from a cell

margin by the length of that cell margin (greater than 1 µm). Regions of the cell

contacting other cells were not considered for quantification. Data are expressed as

means ± S.E. of the mean.

3.8 Steady-state ATPase Activity

Steady-state ATP hydrolysis by MYO3A.ΔK.2IQ.C-GFP in the presence of actin was

examined by using the NADH-linked assay. The ATPase assay was performed in KMg50

buffer (10 mM imidazole-Cl, pH 7.0,50 mM KCl, 1 mM EGTA, 1 mM MgCl2, 1 mM

DTT) at 25° C. The NADH-coupled assay was performed in an Applied Photophysics

stopped-flow spectrofluorometer in which the NADH absorbance at 340 nm was

monitored continuously for 200 seconds. The ATPase rate at each actin concentration

was determined, and the Michaelis-Menten equation was used to calculate the kcat and

KATPase (V0 + ((kcat [actin])/(KATPase + [actin]))), where V0 is the ATPase rate in the

absence of actin; kcat is the maximal ATPase rate, and KATPase is the actin concentration at

which the ATPase activity is one-half maximal.

3.9 In-vitro Motility

The actin-filament sliding assay was performed on WT and G488E M3A.ΔK.2IQ.C-GFP.

Myosin was adhered to a 1% nitrocellulose coated cover slip directly. The surface was

blocked with BSA at 1 mg·ml-1 before the addition of actin labeled with either rhodamine

phalloidin (TRITC filter cube; excitation/emission: 545/620 nm) or ALEXA (FITC filter

cube; excitation/emission: 500/535 nm). An activation buffer was added containing 27

KMg50 buffer and the following: 0.35% methylcellulose, 2.5 mM phosphenolpyruvate,

20 units·ml-1 pyruvate kinase, 0.1 mg·ml-1 glucose oxidase, 5 mg·ml-1 glucose, 0.018 mg·ml-1 catalase, and 1 mM ATP. The slide was promptly viewed using a NIKON

TE2000 microscope equipped with a 60 x 1.4NA phase objective and a Perfect Focus

System. Images were acquired at 5 s intervals using a shutter controlled Coolsnap HQ2 cooled CCD digital camera (Photometrics) binned 2 x 2. Temperature was maintained at

26 ± 1°C and monitored using a thermocouple meter (Stable Systems International).

Image stacks were transferred to Image J for analysis via MTrackJ (Trivedi et al., 2013).

28

Chapter 4: RESULTS

4.1 Sequence Analysis

To identify well-conserved regions within the switch I loop, we compared the switch I

domain of human MYO3A to other myosin sequences (Figure 4.1). Alignment revealed

conserved residues of the MYO3 switch I region and the other myosin family members.

The glycine at position 488 in human MYO3A was conserved across all queried human

myosin sequences. The corresponding residue in Drosophila NINAC and human

MYO3A were not identical (hMYO3A: glutamate, dNINAC: valine), however the switch

I sequences showed 30% identity. Taken together, these data suggest the glycine at

position 488 plays a conserved role in the mechanism of ATPase activity across the

vertebrate class III myosins.

4.2 Quantification of MYO3A Tip Localization, Filopodia Length and

Density in COS7 Cells

MYO3A is not naturally expressed at detectable levels in COS7 cells. However, MYO3A

has been shown to induce filopodial actin protrusions and localize to their tips in cultured

cells particularly well when its kinase domain has been removed (MYO3A.ΔK) (Salles et

al., 2009). COS7 cells transfected with GFP-tagged MYO3A.ΔK and G488E-containing

MYO3A.ΔK were examined for their ability to tip localize as well as induce and elongate

filopodia. Figure 4.2 demonstrates the average filopodia tip to cell body ratio (TCBR) for

the MYO3A constructs. We find that the wildtype MYO3A.ΔK average TCBR was

greater than that of the mutant GFP.MYO3A.ΔK.G488E, GFP.MYO3A.MD, and GFP-

29

alone (GFP.MYO3A.ΔK: 1.96 ± 0.14, GFP.MYO3A.ΔK.G488E: 0.3 ± 0.08,

GFP.MYO3A.MD: 0.22 ± 0.01, GFP-alone: 0.23 ± 0.02). Filopodia lengths (Figure 4.3) were nearly 2-fold longer in GFP.MYO3A.ΔK-expressing cells (2.7 ± 0.13) compared to

GFP.MYO3A.ΔK.G488E-expressing cells (1.4 ± 0.05). GFP.MYO3A.MD (2.1 ± 0.1), and GFP-only (2.07 ± 0.1) expressing cells showed similar filopodia lengths as compared to GFP.MYO3A.G488E. Filopodia density (Figure 4.4) along the cell periphery was 0.13

± 0.01 for GFP.MYO3A.ΔK and 0.04 ± 0.01 for GFP.MYO3A.ΔK.G488E-expressing cells, showing a three-fold reduction in the mutant. The filopodia density of the cells expressing GFP.MYO3A.MD and GFP-only (0.05 ± 0.01 and 0.04 ± 0.01, respectively) were similar to GFP.MYO3A.ΔK.G488E. When ESPIN1 was co-transfected with myosin

(Figure 4.5), its presence was found to increase tip-localization of wildtype myosin

(Mchr.MYO3A.ΔK + ESPIN1: 3.19 ± 0.65) but not mutant (GFP.MYO3A.ΔK.G488E +

ESPIN1: 0.14 ± 0.02) or GFP-only (GFP-only + ESPIN1: 0.1 ± 0.01) myosin on COS7 cell filopodia (GFP.MYO3A.ΔK.G720A + ESPIN1 was not performed). Mutant MYO3A co-transfected with WT MYO3A in the presence of ESPIN1 (Figure 4.6) shows reduced

TCBR compared to WT MYO3A only and WT MYO3A and ESPIN1, suggesting a dominant-negative function of the G488E mutation (GFP.MYO3A.ΔK: 1.97 ± 0.14;

GFP.MYO3A.ΔK + ESPIN1: 2.17 ± 0.31; Mch.MYO3A.ΔK.G488E +

GFP.MYO3A.ΔK + ESPIN1: 0.16 ± 0.02; Mch.MYO3A.ΔK.G488E +

GFP.MYO3A.ΔK + ESPIN1: 1.17 ± 0.18). In other words, the presence of the WT not sufficient to rescue the mutant phenotype; rather, the mutant MYO3A is severe enough such that it reduces the function of WT MYO3A in co-transfected conditions.

30

4.3 MYO3A Steady-state ATPase Activity

To examine the impact of the switch I G488E mutation on MYO3A motor activity, we

performed actin-activated motor ATPase activity assays with the MYO3A.ΔK.2IQ and

MYO3A.ΔK.G488E.2IQ constructs. We determined the ATPase activity in the presence

of varying concentrations of actin (0-70 µM) using the NADH-coupled assay at 25 °C.

The actin concentration dependence of the ATPase activity allowed us to fit the data to a

hyperbolic relationship to determine the maximum ATPase activity (kcat) and actin

concentration at which the ATPase activity is one-half maximal (KATPase) (Figure 4.7).

There was a 2-fold reduction in kcat and a 10-fold increase in KATPase in

-1 MYO3A.ΔK.G488E.2IQ. The catalytic efficiency (µM/sec ) examined by kcat/KATPase

was reduced by 10-fold as a result of the switch I mutation (table 4.1).

4.4 MYO3A In-vitro Motility

Sliding velocity of actin filaments was measured using the in vitro motility assay (Table

4.2). Up to n >50 filaments were analyzed per condition and the mean velocity and

standard error of the mean was calculated for each actin concentration (MYO3A.ΔK.2IQ:

76.4 ± 0.7). The sliding velocity of actin filaments detected in buffer containing

MYO3A.ΔK.G488E.2IQ is in progress (Figure 4.8).

31

Consensus switch I % Identity

hMYO3A 474----GNACTIINDNSSRFGKYLEM----493 100%

hMYO3B 487----GNSCTAINDNSSRFGKYLEM----506 90%

hMYO5A 207----GNAKTIRNDNSSRFGKYIEI----226 80% hMYO7A 200----GNAKTIRNDNSSRFGKYIDI----219 80%

dNINAC 467----VNAGTPVNNDSTRCVLQYCL----487 30%

Figure 4.1. Sequence alignments of amino acid residues from the switch I region of human MYO3A in comparison with human MYO3B, MYO5A, MYO7A, and Drosophila NINAC. hMYO3A shows 90% identity with hMYO3B, 80% identity with hMYO5A and hMYO7A, and 30% identity with dNINAC.

32

A)

B)

Figure 4.2. Localization of WT and mutant MYO3A in COS7 cells. A) Live cell imaging shows localization of GFP.MYO3A.ΔK but not mutant MYO3A in transfected COS7 cell tips. White arrows indicate fluorescence at filopodia tip. Scale bar: 5 µm. B) The G488E mutation reduces tip localization of MYO3A as indicated by the ratio of tip to cell body fluorescence. (GFP.MYO3A.ΔK: 1.96 ± 0.14, GFP.MYO3A.ΔK.G488E: 0.3 ± 0.08, GFP.MYO3A.MD: 0.22 ± 0.01, GFP-alone: 0.23 ± 0.02). n > 10 filopodial tips from 10 or more cells per condition. *indicates significant difference (p < 0.001, Tukey test). ΔK 33

– kinase domain deletion; G488E – glutamate substitution for glycine at amino acid residue 488; GFP – green fluorescent protein; Mchry – Mcherry fluorescent probe; MD – motor dead; MYO3A – myosin3A.

34

Figure 4.3. Impact of the G488E mutation on MYO3A filopodia elongation. The G488E mutation reduces the length of MYO3A (GFP.MYO3A.ΔK.G488E: 1.40 ± 0.05) compared to WT (GFP.MYO3A.ΔK: 2.71 ± 0.13), motor dead (GFP.MYO3A.MD: 2.10 ± 0.1), and GFP-only (GFP-only: 2.07 ± 0.1)). n > 10 filopodial tips from 10 or more cells per condition. *indicates significant difference (p < 0.001, Tukey test).

35

Figure 4.4. Impact of the G488E mutation on filopodia induction in COS7 cells. The G488E mutation reduces filopodial density on mutant-expressing MYO3A (GFP.MYO3A.ΔK.G488E: 0.05 ± 0.01) compared to WT (GFP.MYO3A.ΔK: 0.13 ± 0.01) along the COS7 cell periphery. n > 10 filopodial tips from 10 or more cells per condition. *indicates significant difference (p < 0.001, Tukey test).

36

A)

B)

Figure 4.5. The presence of ESPIN1 increases tip-localization of wildtype MYO3A but not mutant or GFP-only myosin on COS7 cell filopodia. A) Live cell imaging shows localization of GFP:MYO3A and ESPIN1 in co-transfected COS7 cells. White arrows indicate fluorescence at the filopodia tip. Scale bar: 5 µm. B) The presence of ESPIN1 increases tip-localization of wildtype MYO3A (GFP.MYO3A.ΔK + ESPIN1: 2.17 ± 0.31) but not mutant (GFP.MYO3A.ΔK.G488E + ESPIN1: 0.14 ± 0.02) or GFP-only (GFP-only + ESPIN1: 0.1 ± 0.01) myosin on COS7 cell filopodia. n > 10 filopodial tips from 10 or more cells per condition. *indicates significant difference (p < 0.001, Tukey test). 37

A)

B)

Figure 4.6. Mutant MYO3A can function as a dominant-negative. WT MYO3A, when co-expressed with mutant MYO3A and ESPIN1 shows adequate tip localization, while mutant MYO3A co-expressed under the same conditions, does not. A) Live cell imaging shows localization of GFP:MYO3A and ESPIN1 co-transfected COS7 cells. White arrows indicate fluorescence at filopodia tip. Scale bar: 5 µm. B) Mutant MYO3A co- transfected with WT MYO3A in the presence of espin shows reduced TCBR compared to WT MYO3A only and WT MYO3A and ESPIN1, suggesting a dominant-negative phenotype of the G488E mutation (GFP.MYO3A.ΔK: 1.97 ± 0.14; GFP.MYO3A.ΔK + ESPIN1: 2.17 ± 0.31; Mch.MYO3A.ΔK.G488E + GFP.MYO3A.ΔK + ESPIN1: 0.16 ± 0.02; Mch.MYO3A.ΔK.G488E + GFP.MYO3A.ΔK + ESPIN1: 1.17 ± 0.18;) n > 10

38

filopodial tips from 10 or more cells per condition. *indicates significant difference (p < 0.001, Tukey test).

39

1.6

WT 1.4 G488E -1 1.2

1.0

0.8

0.6

0.4 ATPase Activity (sec) 0.2

0 0 10 20 30 40 50 60 70

[Actin] µM

Figure 4.7. Actin-activated ATPase activity of MYO3A.ΔK.2IQ and MYO3A.ΔK G488E.2IQ. (error bars represent mean ± standard deviation). The results are summarized in Table 4.1. ATPase activity was determined in the presence of varying concentrations of actin (0-70 µM) using the NADH-coupled assay at 25 °C. The actin concentration dependence of the ATPase activity allowed us to fit the data to a hyperbolic relationship to determine the maximum ATPase activity (kcat) and actin concentration at which the ATPase activity is one-half maximal (KATPase). There was a 2-fold reduction in kcat and 10-fold increase in KATPase in MYO3A.ΔK.G488E.2IQ. V0 = ATPase rate in the absence of actin; Kcat/KATPase = catalytic efficiency.

Table 4.1. Actin-activated ATPase activity of MYO3A.2IQ constructs.

Construct V0 kcat KATPase kcat/KATPase s-1 s-1 µM µM−1·s−1 MYO3A.ΔK.2IQ 0.07 ± 0.02 1.05 ± 0.03 1.55 ± 0.23 0.68 ± 0.08 MYO3A.ΔK.G488E.2IQ 0.02 ± 0.02 0.70 ± 0.04 20.24 ± 0.03 0.03 ± 0.01

40

100

MYO3A ΔK 2IQ cGFP 80

60

40

Number of Filaments 20

0 40 50 60 70 80 90 100 110 Velocity (nm/sec)

Figure 4.8. In-vitro motility assay of WT MYO3A. The motility was determined as a function of number of filaments and sliding velocity (nm/sec). MYO3A.ΔK.2IQ shows maximal sliding velocity of actin filaments at 76.4 nm/sec. MYO3A.ΔK.G488E.2IQ motility is in progress. Refer to table 4.2 for velocities.

Table 4.2. Sliding velocity of actin filaments on MYO3A.ΔK.2IQ constructs.

Construct Velocity (nm/sec) WT MYO3A.ΔK.2IQ.C-GFP 76.4 ± 0.7 MYO3A.ΔK.G488E.2IQ.C-GFP In progress

41

Chapter 5: DISCUSSION

5.1. Class III myosins transport ESPIN1 to the tips of stereocilia

Stereocilia, the primary mechanosensory detectors in the auditory and vestibular systems are made up of parallel actin bundles aligned at their core. To produce a bundle of unidirectional actin filaments with ~12-13 nm interfilament spacing, stereocilia require specialized actin-bundling proteins to establish the dimensions of the filaments and influence their functional properties (Sekerková et al., 2011). Class III myosins function as transporters of the myosin-binding protein and actin-bundler, ESPIN1, to filopodia tips in stereocilia. Actin bundling proteins such as the ESPIN class constitute an important function in the growth and maintenance of actin filaments and thus, the ultrastructure of actin-based protrusions (Zheng et al., 2014; Salles et al., 2009).

5.2. ESPIN1 stabilizes, increases filopodia length in COS7 cells

Previous studies have shown that when co-expressed, MYO3A enhances localization of

ESPIN1 at COS7 filopodia tips (Salles et al., 2009). The presence of ESPIN1 on these tips promotes enhanced elongation of the filopodia, because ESPIN cross-links actin filaments thereby stabilizing them and allowing continued polymerization at the tip of the actin protrusion. ESPIN1 binds to myosin through the interaction of the ankyrin repeat domain (ARD) of Espin1 and the tail homology domain I (THD1) of MYO3A. Through this interaction, ESPIN1 has been proposed to act as cargo for MYO3A, allowing the

ESPIN to perform its stabilizing function as it ascends the actin. This activity is physiologically relevant, since stereocilia undergo an approximate two-fold increase in diameter during morphogenesis and general maintenance during its lifetime. This

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widening likely involves assembling additional layers of actin filaments at the periphery of a pre-existing bundle, and therefore requires ESPIN. When absent in stereocilia, mutant hair cells degenerate soon after birth, resulting in mice lacking in auditory and vestibular function (Avraham, 1995; Sekerková et al., 2011).

5.3. The G488E mutation negatively impacts motor function in MYO3A

In the current work, we determined the functional consequences of the introduction of a missense mutation at amino acid residue 488 of the switch I loop of MYO3A by examining the impact of the mutation on MYO3A tip localization, and filopodia density and length in COS7 cells. We also directly examined the impact of the mutation on motor

ATPase activity. It is known that the motor domain of myosins is highly conserved across different classes (Cheney et al., 1993). Our own sequence analysis results suggest the switch I loop, located in the myosin nucleotide-binding domain, is classically conserved amongst the class III myosins and extends to Drosophila class III myosin called NINAC.

Our results support the hypothesis that switch I plays an important role in facilitating key steps in the ATP hydrolysis cycle. ATP hydrolysis in myosins occurs by cleavage of the phosphoanhydride bond between the β- and γ-phosphates and requires a conserved nucleotide-binding region. After ATP hydrolysis, switch I can move to an open position, which breaks the salt bridge between Arg238 and Glu459 and accelerates phosphate (Pi) release through a newly formed ‘backdoor’ (Reubold et al., 2003). Loss of Pi disrupts the hydrogen bond between switch II and the γ-phosphate, allowing switch II to open, leading to ADP release, lever arm swing, and force generation. Steric hindrance due to the presence of a larger charged amino acid may disrupt one or more of these key conformational changes. The glycine 488 residue is located near the γ-phosphate of ATP

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and the substitution of the glutamic acid may prevent switch I from properly interacting with ATP in the active site. Therefore, the G488E mutation may alter the kinetics of ATP binding, hydrolysis, or other key conformational changes in the active site, resulting in functional defects of the MYO3A cycle. The negatively charged glutamic acid may repel the negatively charged γ-phosphate of ATP. Even though glycine at position 488 does not directly play a role in ATP binding and hydrolysis, it likely is important for allowing flexibility in the switch I region.

5.4. The G488E mutation reduces MYO3A catalytic efficiency

Our observation that G488E MYO3A (MYO3A.ΔK.G488E.2IQ) decreases catalytic efficiency compared to the WT (MYO3A.ΔK.2IQ) is consistent with our previous conjecture that the mutation reduces MYO3A’s enzymatic activity. The mutation demonstrated reduced enzymatic properties including: Kcat, and V0, as well as elevated

KATPase. The maximal ATPase rate (Kcat) of MYO3A, a measure that provides valuable information on the motor’s turnover rate (number of ATP molecules hydrolyzed per second), was significantly reduced in the mutant, suggesting overall reduced enzymatic function (MYO3A.ΔK.2IQ: Kcat = 1.05 ± 0.03, MYO3A.ΔK.G488E.2IQ: Kcat = 0.70 ±

0.04). The rate of ATPase, or the actin concentration at which the ATPase activity is half maximal, was elevated in the mutant (MYO3A.ΔK.2IQ: KATPase = 1.55 ± 0.23,

MYO3A.ΔK.G488E.2IQ: KATPase = 20.24 ± 0.03). This measurement provided insight into the relative actin affinity for both constructs. The actin necessary to achieve half- maximal ATPase activity was nearly 10-fold greater in the mutant as compared to the

WT. Another measure used to analyze MYO3A’s functionality was the basal ATPase rate, V0, or the ATPase rate in the absence of actin (MYO3A.ΔK.2IQ: V0 = 0.07 ± 0.02,

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MYO3A.ΔK.G488E.2IQ: V0 = 0.02 ± 0.02). Thus, the intrinsic ATPase rate is also altered. By measuring MYO3A’s maximal ATPase rate (Kcat) and KATPase, we calculated the protein’s catalytic efficiency (Kcat/KATPase). WT MYO3A yielded a catalytic efficiency that was nearly 20-fold greater than mutant MYO3A (MYO3A.ΔK.2IQ:

Kcat/KATPase = 0.68; MYO3A.ΔK.G488E.2IQ Kcat/KATPase = 0.03). These studies provide a glimpse into the effects of the mutation on MYO3A’s enzymatic activity, and we have speculated on how the mutation may impact the structure of the active site. Future studies may allow examination of how the mutation alters key conformational changes of the active site and specific kinetic steps.

5.4.1. MYO3A kinetics revisited: The G488E mutation may slow ADP-release during the ATPase cycle

MYO3A is a high-duty ratio myosin motor, meaning that the myosin head spends a significant amount of its cycle time attached to the actin filament. This makes sense in the context of myosin translocation along the actin filament during cargo transport. During the acto-myosin (AM) ATPase cycle, the myosin motor head remains bound to the actin until ADP leaves and a new ATP binds to the head, releasing it from the actin filament.

Kinetic studies of MYO3A show that it has a high affinity for actin in the presence of

ADP and a rate-limiting ADP-release step, which results in the AM-ADP state being the predominant intermediate (Dosé, 2007). However, if the mutation sterically hinders the process of ADP release, then this step will be even more slowed than usual, resulting in a longer actin-binding time. The observed reduction in maximal ATPase activity fits in with this observation, and would reduce the rate of movement to the stereocilia tips.

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5.4.2. The G488E mutation may slow ATP binding during the ATPase cycle

It is also possible that ATP binding could also be disrupted due to the presence of the large positively charged glutamic acid, which would further slow the rate of myosin detachment from actin. If this step were slowed sufficiently, the detachment of myosin from actin would be the new rate-limiting step and as a result, the myosin would remain bound to actin in its rigor conformation for longer. In depth molecular analysis is still necessary to complete our understanding of how specifically, the G488E mutation affects the ATPase kinetics and impacts force generation. It is possible that it may alter one critical step, or many, but what is certain is that the specific steps in the ATPase cycle altered by the mutation will dictate if the mutant MYO3A can produce force or move along actin.

5.5. The G488E mutation may disrupt processive motility

Despite showing signs of ATPase activity, albeit reduced compared to WT MYO3A, mutant MYO3A did not exhibit proper cellular localization. The motility assays and further detailed biochemical analysis may shed light on this question. However, the traditional motility assay we utilized for our studies has limitations; this assay demonstrates movement by multiple myosins working together to produce actin sliding as is found in muscle contraction. As fluorescent actin is flowed over myosin bound to the coverslip, conventional myosins will move the actin in a concerted effort as many myosins interact with a single actin filament. This synchronized movement is ultimately what causes the shortening of muscle fibers and gives skeletal muscles the ability to contract rapidly in response to a stimulus. In contrast, MYO3A and other unconventional myosins move as processive motors—meaning that they can travel long distances along

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the actin filament without falling off. However, MYO3A is proposed to walk along actin bundles as an ‘inchworm.’ Its tail and motor actin-binding sites allow MYO3A to alternate between motor and tail actin binding, which prevents it from falling off the actin. The slow ADP-release step allows MYO3A to have a higher duty ratio, while also making it a slow-moving motor. Furthermore, the introduction of the mutation may disrupt motor and tail domain coordination required for processive motility, resulting in no tip localization in filopodia. A single molecule motility assay may be required to assess the impact of the mutation on MYO3A processive movement.

5.6. The G488E mutation abolishes MYO3A filopodia tip localization activity in COS7 cells

The impact of the G488E mutation on MYO3A cellular function is demonstrated by the

COS7 cell analysis. The mutant myosin expression in COS7 cells showed virtually no tip-localization (GFP.MYO3A.ΔK.G488E TTCB ratio: 0.3 ± 0.08) versus the WT (1.97 ±

0.14), despite showing residual ATPase activity in-vitro. Possible reasons for this discrepancy are that the mutation was severe enough to prevent myosin motility although it still has ATPase activity (Figure 5.1). Further studies on motility are necessary to determine this possibility.

Our experiments corroborate the idea that WT MYO3A.ΔK is able to transport itself efficiently along the actin filaments to COS7 filopodia tips. The tail-less (ΔK) MYO3A is able to facilitate motility at 76.4 ± 0.7 nm/sec. WT MYO3A also shows efficient tip localization at TTCB ratio of 1.97 ± 0.14. Co-expression of MYO3A.ΔK and ESPIN1 resulted in enhanced tip localization of MYO3A (GFP.MYO3A.ΔK + ESPIN1: 2.17 ±

0.31). The increase in TTCB ratio is due to the MYO3A-ESPIN1 interactions via the

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MYO3A 3THDI and ESPIN1 ARD domains (Quintero et al., 2013), which stabilize the complex at the filopodia tips. Further experiments demonstrated that the presence of

ESPIN1 could not rescue tip localization of mutant MYO3A (MYO3A.ΔK.G488E 0.14 ±

0.02). Instead, mutant MYO3A and ESPIN1 were found to be co-expressed inside of the cell, suggesting a failure to translocate. The known ‘motor-dead’ mutation

(MYO3A.ΔK.G720A) showed a comparable tip-localization ratio (0.22 ± 0.01), suggesting that the G488E mutation, like the motor-dead phenotype, also prevents translocation to the filopodia tips. The pervasiveness of the mutation was evidenced in experiments in which MYO3A.ΔK and MYO3A.ΔK.G488E were co-expressed in the presence of ESPIN1, yielding greater tip localization of MYO3A.ΔK

(Mchr.MYO3A.ΔK.G488E + GFP.MYO3A.ΔK + ESPIN1: 1.17 ± 0.18; GFP channel), but not the mutant (Mchr.MYO3A.ΔK.G488E + GFP.MYO3A.ΔK + ESPIN1: 0.16 ±

0.02; Mchr channel). Interestingly, in the presence of mutant MYO3A, WT MYO3A showed decreased tip localization even in the presence of ESPIN1. We believe that the expression of mutant MYO3A was sufficient to suppress WT MYO3A translocation and expression on COS7 filopodia tips, suggesting that the mutation acts as a dominant- negative. This might be due to the fact that ESPIN1, competitively bound to the mutant myosin, is unable to bind to WT myosin, thereby resulting in decreased ESPIN1 translocation. Furthermore, the WT MYO3A may be prevented from entering the protrusion because the mutant MYO3A:ESPIN1 complex is at the base of the protrusion, effectively blocking its pathway.

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5.7. The G488E mutation prevents ESPIN translocation to COS7 filopodia tips

Our studies revealed that mutant myosin was unable to translocate to filopodia tips, resulting in the abolishment of ESPIN1 expression on the tips. Our biochemistry results suggest that 1) actin binding is not abolished and 2) ATPase activity is reduced. Together with the observation that mutant myosin and ESPIN1 fail to tip-localize in COS7 cells, and instead co-localize inside the cell, suggest that ESPIN1 binding is also not abolished.

These results allow us to propose a model to explain the distribution of mutant and WT

MYO3A within the cell and how this is related to the impact of MYO3A mutation in stereocilia. In this model, mutant myosin binds actin and ESPIN1, but stays trapped within the cell due to the G488E-induced impairment in motility (Figure 5.1).

Figure 5.1. WT MYO3A.ΔK is able to tip-localize on COS7 filopodia, while mutant MYO3A.ΔK.G488E is not. Despite intact interactions with actin and ESPIN1, the mutation prevents its movement out of the cell soma. ΔK – kinase domain deletion; G488E – glutamate substitution for glycine at amino acid residue 488; GFP – green 49

fluorescent protein; Mchry – Mcherry fluorescent probe; MD – motor dead; MYO3A – myosin3A.

Ultimately, the binding of mutant myosin to ESPIN1 plays an important proposed role in the etiology of human hereditary deafness; because mutant myosin is still able to bind to

ESPIN1, it sequesters the ESPIN from WT myosin, preventing its translocation to stereocilia tips. With ESPIN1 trapped inside the cell, the stereocilia suffer from a lack of sufficient ESPIN-based maintenance to the actin filaments, and as a result, they begin to degrade.

5.8. MYO3B WT may compensate for mutant MYO3A in DFNB30 but not G488E

Our results agree with previously published reports on the impaired function of expressed

MYO3A mutation and late-onset hearing loss (Walsh, et al., 2010), which suggest that while other less severe MYO3A mutations occur gradually and later on in life, more severe mutations may affect the inner ear physiology during development, causing congenital deafness. We hypothesize that the novel G488E mutation is of this type: immediate with severe phenotypic impact to the stereocilia.

Since DFNB30 results in a lack of functional MYO3A, which is likely degraded rapidly in the cell, MYO3B may compensate and prevent an early onset and severe deafness.

MYO3B is a slower motor and it lacks 3THDII (Manor et al., 2012) thus, it may be less efficient at transporting ESPIN1 to the stereocilia tips. But its compensatory effect may be sufficient in DFNB30 where there is no functional MYO3A that can interfere with espin-binding. DFNB30 has been shown to be caused by recessive mutations (Walsh, et al., 2010), thus, two copies of the mutation to MYO3A are necessary to cause the

DFNB30 phenotype. In contrast, the novel G488E mutation has been proposed to be a dominant one (Grati, et al., unpublished), which may explain the severity of the 50

phenotype upon onset at birth. Only one copy of the mutation is necessary to elicit the dominant-negative effect, on the WT MYO3A by sequestering ESPIN1 inside the cell.

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Chapter 6: CONCLUSION AND FUTURE DIRECTIONS

Inherited hearing loss is genetically heterogeneous, caused by mutations in genes controlling molecular motors, hair-cell structure, neuronal innervation, signal transduction, and a variety of other processes (Walsh et al., 2002). Recessive hearing loss with congenital or very young onset can be caused by mutation of MYO7A, OR MYO15.

Any dominant hearing loss that progresses with age can be caused by mutation of

MYO7A, MYO6, or myosin heavy chain 9 (MYH9). The hearing loss described by the

G488E mutation is unique in that it is both dominantly inherited and congenital, unlike many known DFNB30 mutations, which are characterized as recessively inherited, yet progressive. The effects of the G488E mutation on MYO3A are two-fold: it hinders the transport function of MYO3A, but still allows ESPIN1 to bind MYO3A. It is likely that is, ESPIN1 binds to mutant myosin with the same affinity as WT MYO3A, sequestering the ESPIN inside the cell, and preventing its translocation to the filopodia tips, and ultimately acting as a dominant-negative. The lack of ESPIN to these tips contributes greatly to their degeneration of the actin skeleton that forms these protrusions.

Furthermore, it is extremely likely that this same process occurs in the stereocilia of inner ear hair cells, causing hearing loss. Other MYO3A mutations may not be as severe because there is no functional MYO3A expression, therefore it cannot act as a dominant negative. Instead, MYO3B may be able to compensate for the loss of MYO3A. It is also necessary to consider the likelihood of other MYO3-based mutations. The existence of three different MYO3A mutations also suggests that other mutations in this gene may exist.

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Future directions for this research include additional biochemical experiments to further elucidate the processive walking of MYO3A on the actin filament and how the mutation may alter this intrinsic property. Further experiments also include the study of the G488E mutation in-vivo and the resulting deafness phenotype as well as the study of stereociliary ultrastructure in G488E-expressing organisms.

The etiological study of biophysical players and their roles in disease states remains an important endeavor in the ever-growing field of sensory physiology. Human deafness, particularly sensorineural deafness, is no exception. Current therapies aim to manage hearing loss via cochlear implants, but not to prevent or even more radically, reverse it.

The rate of discovery and design for reversing hearing loss has remained minimal over the last decades in comparison to other biological pursuits, such as cancer and HIV treatment; however, there is much potential for new therapies, particularly hair cell regeneration using stem cell and gene therapy.

The study presented here is certainly not the first to suggest that precise motor activity is essential for proper localization of MYO3A in stereocilia, however, our data provides a unique example of how a substitution of a single amino acid residue can uncouple enzymatic and mechanical activity of the myosin motor domain. Ultimately, the G488E mutation, while novel, remains to be further studied and will pave the way for characterizing many more novel mutations in the study of human hereditary deafness.

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Appendix

Table 5.1. Average tip to cell body ratio of COS7 cell filopodia-expressing myosin constructs and standard error.

Tip to Cell Body Ratio Construct Average ± S.E. Wildtype G488E Mutant GFP.MYO3A.ΔK 1.97 ± 0.14 GFP.MYO3A.ΔK.G488E 0.26 ± 0.02 GFP-only 0.23 ± 0.02 GFP.MYO3A.G720A (MD) 0.22 ± 0.01 GFP.MYO3A.ΔK + ESPIN1 2.17 ± 0.31 GFP.MYO3A.ΔK.G488E + 0.14 ± 0.02 ESPIN1 GFP-only + ESPIN1 0.1 ± 0.01 Mchr.MYO3A.ΔK.G488E + 1.17 ± 0.18 0.16 ± 0.02 GFP. MYO3A.ΔK + ESPIN1

Table 5.2. Average lengths of COS7 cell filopodia-expressing myosin constructs and standard error.

Filopodia Length Construct Average Standard Error GFP.MYO3A.ΔK 2.71 ± 0.13 GFP.MYO3A.ΔK.G488E 1.40 ± 0.05 GFP-only 2.07 ± 0.10 GFP.MYO3A.G720A (MD) 2.10 ± 0.10 GFP.MYO3A.ΔK + ESPIN1 6.36 ± 0.62 GFP.MYO3A.ΔK.G488E + 3.33 ± 0.16 ESPIN1 GFP-only + ESPIN1 3.07 ± 0.25

Table 5.3. Average densities of COS7 cell filopodia-expressing myosin constructs and standard error.

Filopodia Density Construct Average Standard Error GFP.MYO3A.ΔK 0.13 ± 0.01 GFP.MYO3A.ΔK.G488E 0.05 ± 0.01 GFP-only 0.04 ± 0.01 GFP.MYO3A.G720A (MD) 0.05 ± 0.01 GFP.MYO3A.ΔK + ESPIN1 0.06 ± 0.02 GFP.MYO3A.ΔK.G488E + 0.03 ± 0.003 ESPIN1 GFP-only + ESPIN1 0.02 ± 0.003

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