ABSTRACT Effects of Oncomodulin on Cytoskeletal Actin Dynamics Craig

ABSTRACT

Effects of Oncomodulin on Cytoskeletal Actin Dynamics

Craig I. Heflick, M.S.

Mentor: Dwayne D. Simmons, Ph.D

Cochlear outer hair cells (OHCs) contribute to the fine sensitivity of the

mammalian auditory system by serving as a feedback mechanism in response to sound.

Regulation of filamentous and globular actin may contribute to the reversible shape

changes critical to OHC function. Previous studies have suggested that the Rho family of

GTPases play an important role in regulating intracellular actin dynamics in OHCs using

RhoA/ROCK and LIMK/cofilin mediated pathways. We hypothesize that modulation of

calcium signaling through mobile calcium buffers alters intracellular actin dynamics in

OHCs. Early studies of OHC lateral stiffness and motility suggest that calcium modifies

the actin-based cortical cytoskeleton. An EF-hand calcium buffering protein,

oncomodulin (OCM) is preferentially expressed in OHCs and may aid in sculpting calcium signals important for actin remodeling. Targeted deletion of OCM leads to early

progressive hearing loss in mice without severe OHC loss suggesting the importance of

OCM to OHC function. Effects of Oncomodulin on Cytoskeletal Actin Dynamics

Craig I. Heflick, B.S.

A Thesis

Approved by the Department of Biology

Dwayne Simmons, Ph.D., Chairperson

Submitted to the Graduate Faculty of Baylor University in Partial Fulfillment of the Requirements for the Degree of Master of Science

Approved by the Thesis Committee

Dwayne Simmons, Ph.D., Chairperson

Joseph Taube, Ph.D.

Leigh Greathouse, Ph.D.

Mary Lynn Trawick, Ph.D.

Paul Fillmore, Ph.D.

Accepted by the Graduate School August 2019

J. Larry Lyon, Ph.D., Dean

LIST OF FIGURES ...... v

LIST OF TABLES ...... vi

ACKNOWLEDGMENTS ...... vii

CHAPTER ONE ...... 1 Introduction ...... 1

CHAPTER TWO ...... 8 Material and Methods ...... 8 Animals ...... 8 Antibodies ...... 8 Cell Culture and DNA Transfection ...... 8 Gene Expression ...... 9 Relative Gene Expression ...... 10 Protein Isolation and Quantification ...... 11 Immunofluorescence ...... 12 Measurement of Area Fraction and Mean Intensity Value ...... 13 Measurement of Intracellular Calcium Levels ...... 13

CHAPTER THREE ...... 15 Results ...... 15

CHAPTER FOUR ...... 27 Discussion ...... 27

REFERENCES ...... 33

iv LIST OF FIGURES

Figure 1...... 2 Figure 2...... 4 Figure 3...... 12 Figure 4...... 15 Figure 5...... 17 Figure 6...... 19 Figure 7...... 20 Figure 8...... 21 Figure 9...... 22 Figure 10...... 23 Figure 11...... 24

v LIST OF TABLES

Table 1...... 9 Table 2...... 10 Table 3...... 26

vi ACKNOWLEDGMENTS

I would like to begin by thanking Dr. Dwayne Simmons, my advisor and thesis chair, for the continuous support of my Master study and research. I would not have been given the opportunity to pursue this degree if not for him, and for that, I am forever grateful.

My sincere thanks go to Andrew Cox and Dr. Leslie Climer for their immense amount of help and support during the past two years. Their expertise made my thesis work possible.

My thanks must also go to my lab mates, Yang Yang and Kaitlin Murtha for their continuous support of my research and sharing countless hours in lab together.

My special thanks go to my thesis committee members, Dr. Joseph Taube, Dr.

Leigh Greathouse, Dr. Mary Lynn Trawick, and Dr. Paul Fillmore, for offering their continuous support of my research

I must thank my friends Devon Plewman, Chase Smith, Kelsey Johnson,

Samantha Hodges, Robert Huff, Patrick Charapata, Yang Yang, and Kaitlin Murtha for their friendships and their eagerness to see me succeed.

Last but not least, I need to express my gratitude to my family: my parents Scott and Kristin Heflick, and my siblings Chad and Elizabeth Heflick. Because of their endless support, I am here. I can never repay or thank them enough for all they have done for me and continue to do.

vii

CHAPTER ONE

Introduction

Age-related hearing loss (ARHL) and noise-induced hearing loss (NIHL) affect

over 450 million people worldwide. The first target of hearing loss is the destruction of

cochlear OHCs. Mammalian auditory function begins as sound pressure waves travel

through the outer ear canal to the middle ear. Sound waves cause displacements on the

tympanic membrane, which causes components of the middle ear to move in mechanical

piston-like motions to produce a traveling pressure wave inside the cochlea. The cochlea

is a small snail shaped organ within the inner ear that is home to a highly specialized

sensory organ, the organ of Corti. The cochlear spiral is innervated by specialized

sensory hair cells that rest upon the basilar membrane (Figure 1). Sound waves traveling

through the cochlea cause displacements on the basilar membrane and activate the

sensory cells that allow for hearing perception1. Inner hair cells (IHCs) and OHCs are the

sensory cells within the organ of Corti (Figure 1). IHCs are primarily responsible for

neurotransmission of sound directly to vestibulocochlear nerve fibers whereas OHCs amplify and sharpen the sound induced vibrations1,2. The cochlear spiral is tonotopic,

sound waves of certain frequencies activate specific hair cells in designated locations

throughout the cochlea. High frequency waves activate hair cells at the narrow base of

the cochlea, whereas low frequency sound waves activate hair cells at the wide apex of

the cochlea3.

Figure 1. Oncomodulin expression is enriched towards the lateral wall of OHCs. Reproduced with permission from Climer et al.4 A) Schematic organ of Corti within the basilar membrane of the cochlea containing three rows of OHCs and one row of IHCs. B) A confocal image of OCM labeling (red) and phalloidin staining (green) in a cross- section of the mouse organ of Corti from the basal turn of the cochlea. OCM labeling preferentially localized to the basolateral membrane of OHCs and is not present in IHCs. Scale bar represents 10m. C) Schematic representation of OCM immunoreactivity in a cochlear OHC. Afferent terminals are shown in blue, efferent terminals are shown in red.

OHCs are unique to the mammalian cochlea and possess a cylindrical shape with

a diameter of ~9 m and a length varying between 10 and 100 m5. Components of the

OHC lateral wall include the molecular motor protein Prestin, a cytoskeletal network of actin filaments, actin crosslinking proteins and one or more layers of subsurface cisternae6,7. OHCs are able to reversibly change their shape in response to various stimuli

in a process known as “OHC motility”, which can be divided into two types: fast and

slow motility. OHC motility is responsible for the precise hearing sensitivity of the

mammalian inner ear. Prestin is the molecular motor protein in OHCs and is embedded

throughout the plasma membrane5,8. Prestin allows for extremely fast motility of OHCs.

2 Although there is discrepancy in the time scale of Prestin-dependent motility, it is most likely in the microsecond range. The term “fast-motility” is often used to describe

Prestin-dependent motility. Conversely, “slow-motility” is used to describe changes in

OHC shape that takes place in milliseconds, seconds or even minutes. Slow-motility is independent of Prestin and involves reorganization of the cellular cytoskeleton to elicit cell elongation and shortening9. It has been suggested that pathways involving members of the Rho-GTPase family are involved in regulating intracellular actin dynamics in

OHCs and thus play a role in OHC slow motility9–12 (Figure 2). In general, RhoGTPases of the Ras superfamily may also regulate OHC function.

The Ras superfamily is a protein family of small GTPases containing five main subfamilies primarily involved in cell proliferation and cell morphology. Of these subfamilies, Rho GTPases are primarily known for controlling intracellular actin dynamics. The three most studied Rho GTPases are cell division cycle 42 (Cdc42), Rac family small GTPase 1 (Rac1) and Ras homolog family member A (RhoA). Each has a different action on actin dynamics. RhoA affects stress fibers, which are contractile actin bundles found in non-muscle cells. These stress fibers are composed of actin and non- muscle myosin II (NMMII)13. Although evidence suggests Rho proteins participate in the signaling cascade that regulates OHC motility, the detailed signaling cascade has yet to be determined. Previous experiments show activation of the small G - proteins Rac1 and Cdc42 lead to OHC shortening as well as an increase in the amplitude of fast motility9. Similarly, Rac1 + RhoA activation lead to OHC elongation and decrease in fast motility14. A Rho-associated kinase (ROCK) mediated signaling cascade continuously modulates OHC fast motility by selectively targeting the cytoskeleton10,15. ROCK was

3 identified as a major effector of Rho GTPases that modulates the cytoskeleton. ROCK is a downstream effector protein of RhoA. There are two isoforms of ROCK, ROCK1 and

ROCK2. Each isoform has its own cellular localization and function despite sharing 92% homology in their kinase domains. The primary isoform of ROCK found in OHCs is

ROCK210. Different substrates can be phosphorylated by ROCK including LIM-Kinase

(LIMK), myosin light chain (MLC), and Ezrin/Radixin/Moesin (ERM) proteins10,15,16. In

OHCs, ROCK2 inhibits the depolymerization of actin filaments indirectly by

phosphorylating LIMK (a potent regulator of actin dynamics), which in turn

phosphorylates/inactivates the protein cofilin (Figure 2).

‐

Figure 2. Schematic pathway of RhoA and downstream effector proteins that regulate OHC motility as suggested by various studies. Red indicates proteins specifically identified in OHCs. Asterisks indicate the protein is phosphorylated.

Cofilin is a small protein with actin depolymerizing activity. Cells lacking cofilin

have impaired locomotion; those overexpressing cofilin are more motile11. Actin

microfilaments are constantly being remodeled in healthy OHCs. ROCK2 phosphorylates

4 the ERM protein family, which maintains healthy regulation of the actin cytoskeleton10. It has been shown that traumatic noise decreases the activity of RhoA and, consequently, decreases ROCK2 and p-ERM proteins in the cochlea10. Thus, F-actin depolymerization

may contribute to NIHL and hair cell death. As a secondary messenger, Ca2+ has been

shown to have roles in mechanoelectric transduction, cochlear amplification, and synaptic

function15,17,18. Therefore, proteins that regulate Ca2+ may also regulate OHC slow

motility.

Unique to OHCs is oncomodulin (OCM), a major Ca2+ binding protein (CaBP).

The term “oncomodulin” was acquired from the initial discovery of OCM in rat liver

hepatomas as an oncoprotein and from its similarity to other known CaBPs19,20,21. OCM

remained to be considered oncogenic due to a lack of evidence of any expression in

normal post-embryonic tissue. However, decades after its discovery, OCM was identified

as a major protein in guinea pig OHCs22–24. OCM is a small EF-hand CaBP of

approximately 12kDa and belongs to the parvalbumin family. Mammalian OCM shares

~53% sequence identity with -parvalbumin (PV), another main EF-hand CaBP25. In

addition to OCM and PV, the mammalian inner ear has other EF-hand CaBPs including

calbindins (CB-D28k), calretinin (CB-D29k), and calmodulin26. EF-hand CaBPs share a

common sequence of ~ 30 residues forming a helix-loop-helix motif which, when bound to Ca2+, undergo a conformational change that allow for activation of downstream

targets27–31. Most EF-hand CaBPs are found extensively throughout the nervous system,

but OCM is uniquely limited to OHCs and certain subtypes of immune cells32–34. EF-

hand CaBPs are difficult to study in the inner ear because their expression levels change

during maturation. For example, PV, calretinin and CB-D28k are expressed in both

IHCs and OHCs at birth, but all three are downregulated in OHCs, whereas OCM is

upregulated before the onset of hearing26,32. Using high resolution and high gain confocal microscopy in both mice and rat cochlear tissues, Simmons et al. suggested that OCM preferentially localizes to the lateral membrane, basal pole, and cuticular plate of OHCs

(Fig. 2B-C)32. In P26 rat OHCs, OCM concentrations were found to be as high as 3mM,

while other CaBPs were found to be between 15-300M35. The unique cell and tissue

distribution of OCM in addition to its overwhelming expression in OHCs in respect to

other CaBPs suggests its functional specificity. To test the importance of OCM in relation

to OHC function and hearing in general, Tong et al. engineered a conditional knockout

(KO) of OCM26. Interestingly, OCM KO mice showed little to no hearing phenotype at

one month. However, at three months OCM KO mice were completely deaf as indicated

by a distortion product otoacoustic emissions (DPOAEs) hearing assay26. DPOAEs are a

direct way to measure OHC function. An elevation in DPOAE thresholds indicate OHCs

are not present or not functional. OCM KO mice show OHC loss in one row of OHCs

only at the 60 kHzs region at the base of the cochlea. Despite the overwhelming majority

of OHCs being present, OCM KO mice display a complete lack of DPOAEs. This

suggests that although the OHCs are present, they are completely non-functional. In

addition to the hearing loss phenotype, OCM KO mice show altered cell shape in all three

rows of OHCs. A triple KO of PV, CB-D28k and calretinin resulted in no loss of

hearing17. This provides evidence that OCM is critical to hearing, while other CaBPs are not.

In summary, finely tuned changes in intracellular Ca2+ concentration modulate a

variety of cellular functions in neurons and sensory cells. Ca2+ transients are critically

6 important for actin based cellular dynamics that are mediated through G-protein

pathways. Ca2+ signals are manipulated by protein buffers of which the EF-hand family

plays a prominent role. Although many proteins regulate Ca2+ in sensory cells and

neurons, the role of the mobile EF-hand buffers is less well known. Most EF-hand protein buffers have a very wide distribution in neurons and sensory cells, with the exception of

OCM, which is found in a subset of sensory hair cells in the mammalian inner ear and certain immune cells. OHCs uniquely express OCM and directly enhance sensitivity and

frequency selectivity of the inner ear. Ca2+ signals may regulate the G-protein pathways

important to OHC actin-based motility. As a secondary messenger, Ca2+ might regulate

RhoA. Understanding the underlying mechanisms behind OHC function could provide

opportunities to reduce cellular damage seen in ARHL and NIHL. The primary goal of

this thesis project was to investigate whether or not the presence of OCM, a major Ca2+

buffering protein, leads to changes in cytoskeletal function of OHCs.

We investigated whether or not the presence of OCM would alter cell morphology

in HEK293T and Hela cells. Both HEK293T and HeLa cells do not endogenously express OCM. Additionally, intense noise has been shown to influence F:G actin ratios in the mouse cochlea, and because OCM is a protein critical to cochlear function, we measured F:G actin ratios in OCM KO mice in addition to HEK293T and HeLa cells transiently expressing OCM. Finally, because Ca2+-mediated RhoA pathways have been

shown to be involved in OHC slow motility, we tested whether or not the presence of

OCM results in gene expression changes of proteins involved in RhoA pathways.

CHAPTER TWO

Materials and Methods

Animals

Pathogen free mouse strains were bred at Baylor University and had access to water, maintained a regular diet, and were kept at 22°C before experiments. Mice were acclimated for one hour in the laboratory before being euthanized. Animals were given near lethal injections of pentobarbital (Nembutal, 100mg/kg) and euthanized by decapitation. All experimental procedures were conducted with approval and in accordance with the guidelines for Animal Research at Baylor University.

Antibodies

The antibodies used in this study were: mouse anti-OCM 1:100 (DSHB), purified mouse anti-actin 1:2000 (BD Transduction Laboratories Cat#612656), mouse anti-- tubulin 1:2000 (Sigma #T6199) and Alexa Fluor 594 phalloidin 1:500 (Invitrogen

Cat#A12381) . Secondary antibodies for western blotting were rabbit anti-mouse IgG

(HRP) (Abcam 97046) and Alexa 568 donkey anti-mouse (IgG).

Cell Culture and DNA Transfection

In order to express PV and OCM in HEK293T and HeLa, which do not endogenously express these proteins, we transfected pEGFP, PV, and OCM containing plasmids (Table 1) into each cell line using the following protocol. HEK293T and HeLa cells were maintained in DMEM (Gibco) supplemented with 10% FBS (ThermoFisher)

8 without antibiotics. The cells were plated into 10cm plates and incubated in a humidified

incubator at 37°C under 5% CO2. Cells were grown to appropriate confluency of ~80% to

be used for immunofluorescence or G:F in vivo actin assay. Plasmids were isolated from

bacteria using Qiagen MiniPrep Kits. Plasmid transfections were performed using

Lipofectamine 3000 (ThermoFisher) according to manufacturer’s instructions.

Table 1. Plasmids used to transiently transfect HEK293T and HeLa cells in this study.

Plasmid Source Citation pEGFP-N1 Flag Patrick Calsou Britton et al.36 PV in pEGFP-N1 Flag Leslie Climer Developed for this Study OCM in pEGFP-N1 Flag Leslie Climer Developed for this Study

Gene Expression

HEK293T cells were transfected with either PV-GFP or OCM-GFP plasmids for

24 hours and total RNA was isolated from HEK293T cells using Qiagen RNeasy mini kit

(Cat #74104) and Qiashredder (Cat #79654) according to the manufacturer’s instructions.

During the RNA isolation, an “on column” DNase digestion was conducted using

Qiagen’s RNase-free DNase kit as per kit instructions (Cat#79524). Total RNA was qualitatively assessed and quantified using a Nanodrop spectrophotometer and 1g of

RNA per sample was reverse transcribed using BioRad’s iScript Advanced cDNA synthesis kit (Cat #1725037). Thermo Fisher Taqman Array Human RhoA gene

expression plates (Cat #4414180) and Taqman Fast Advanced Master Mix (Cat

#4414180) were used as per manufacturer’s instructions in combination with 1l of

1g/l cDNA for gene quantification. The PV-GFP transfection served as the

9 comparison treatment for OCM-GFP and the OCM-GFP Ct values were normalized to the PV-GFP sample (see Table 2). Specifically, the 18s ribosomal subunit Ct value was used as the normalizing reference gene. Using 18S as an internal control was used because it shows less variance in expression across multiple treatments compared to other controls, such as GAPDH37,38. A Ct value of 32 was used as a cutoff value to avoid interpreting results that could be due to spurious amplification. Reactions were run on a

CFX96 Real-Time System (Biorad) and analyzed using BioRad CFX software.

Relative Gene Expression

Table 2. Relative gene expression values using the Perkin Elmer delta-delta Ct method

Treatment Gene Ct ΔΔCt ΔΔCt Relative Expression aPV-GFP 18S 17.99 Ocm-GFP 18S 18.36 aPV-GFP CHN1 22.13 4.14 Ocm-GFP CHN1 22.27 3.91 -0.23 1.172834949 aPV-GFP RHPN2 28.37 10.38 Ocm-GFP RHPN2 31.26 12.9 2.52 0.174342958 aPV-GFP KTN1 24.94 6.95 Ocm-GFP KTN1 24.5 6.14 -0.81 1.753211443 aPV-GFP EZR 28.48 10.49 Ocm-GFP EZR 27.3 8.94 -1.55 2.928171392 aPV-GFP PPAP2B 26.63 8.64 Ocm-GFP PPAP2B 27.92 9.56 0.92 0.52850902 aPV-GFP ARHGAP1 28.24 10.25 Ocm-GFP ARHGAP1 27.62 9.26 -0.99 1.986184991 aPV-GFP GNAI1 26.9 8.91 Ocm-GFP GNAI1 26.15 7.79 -1.12 2.173469725 aPV-GFP CFL2 26.97 8.98 Ocm-GFP CFL2 26.1 7.74 -1.24 2.361985323 ΔCt= (target gene-OCM-GFP) - (18S-OCM- GFP) ΔΔCt= (target gene-PV-GFP) - (18s-PV- GFP) ΔΔCt= (ΔCt-OCM-GFP) - (ΔCt-PV- GFP) Relative Expression = 2^-(ΔΔCt)

10 Relative gene expression values were obtained using the Perkin Elmer delta-delta Ct

method.39

Protein Isolation and Quantification

The G-actin/F-actin in vivo Assay Biochem Kit (Cytoskeleton Inc. #BK037) was used to evaluate F:G actin ratios from protein lysates of mouse cochlea and human cell lines. Cochlea and cultured cells were lysed and homogenized with LAS2 buffer (50 mM

KCl, 5 mM MgCl2, 5mM EGTA, 5% glycerol, 0.1% Nonidet P40, 0.1% Triton X-100,

0.1% Tween 20, 0.1% beta-mercaptoethanol, 100mM ATP, 100x Protease inhibitor

cocktail (Cytoskeleton Inc. #PIC02)). Homogenized samples were incubated at 35°C before being spun for 5 min at 350 x g to pellet cell debris. All samples were ultra- centrifuged at 100,000 g at 35C for 1 hour using an Optima XE centrifuge with type

42.2 TI rotor (Beckman Coulter) (Figure 3). Separated F-and G-actin fractions were quantified using ImageJ protein band density analysis after western blotting with mouse anti-actin antibody at a dilution of 1:2000. Both cochlea from an individual mouse were dissected in ice cold PBS containing protease inhibitors. As much bone and tissue surrounding the cochlea was removed as possible paying careful attention not to damage the cochlea. Cochlear tissues were homogenized and incubated in LAS2 buffer

(Cytoskeleton Inc.). HeLa and HEK293T cells were grown on 10cm plates and harvested

24 hours after transfection. Cells were gently scraped off and homogenized using a cell scraper and LAS2 buffer (Cytoskeleton Inc.)

11 Cochlear and cell lysates were analyzed by SDS-PAGE using Mini-PROTEAN

TGX Stain-Free Gels (BioRad #4568125) before being transferred to 0.2m PVDF membranes (BioRad #1704156). Blots were blocked in TBST/5% non-fat milk for 35 minutes before incubation overnight at 4°C in primary antibody in TBST with 0.1% non- fat milk. Blots were washed in TBST and incubated with secondary antibodies for two hours at room temperature. Clarity Western ECL Substrate (BioRad Cat# 170-5060) was used for western blot detection. Western blots were imaged using a BioRad ChemiDoc

Touch Imaging System. Densitometry for F:G ratios was determined using ImageJ.

Figure 3 G:F actin in vivo assay experimental procedure.

Immunofluorescence

HEK293T and Hela cells were grown on 12mm glass coverslips to be 75-90% confluent at collection. Cells were washed in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (32% stock solution - Electron Microscopy Sciences). Cells were permeabilized for 1 min with 0.1% Triton X-100 followed by room temperature incubation in 50mM ammonium chloride and two washes in PBS. Cells were blocked twice in 1% BSA, 0.1% saponin in PBS for 10 min before being incubated for 2 hours in primary antibody diluted in 1% fish gelatin + 0.1% saponin. Cells were washed 4 times with PBS before being incubated for 60min in secondary antibody diluted in 1% fish gelatin + 0.1% saponin. Coverslips were washed four times with PBS, rinsed with water,

and mounted on glass microscope slides using Vectasheild mounting medium with DAPI.

Confocal images were collected using a Zeiss LSM800 with 63x oil-immersion or 40x

oil-immersion.

Measurement of Area Fraction and Mean Intensity Value

Confocal images of GFP, OCM-GFP, and PV-GFP transfected HEK293T

stained with phalloidin were taken using the 40x oil-immersion using identical settings.

ImageJ basic intensity quantification was used to obtain area fraction and mean intensity

value measurements. Original images were converted to 16-bit grayscale images and duplicated to create a binary image. For mean intensity measurement, the ImageJ “auto threshold” feature was first used on GFP transfected cells to obtain a threshold, which

was then applied to both OCM-GFP and PV-GFP binary images. ImageJ drawing tools

were used to create a region of interest around the perimeter of each cell. Mean intensity

values were obtained and plotted for all transfection conditions. For area fraction measurements, the threshold was manually adjusted on the GFP image first so that only pixels indicating bright were visible. This threshold value was then applied to OCM-GFP and PV-GFP grayscale images. Using the “masks” setting in imageJ and selecting for

“area fraction” provided a single %area value for each image. This value was divided by the total number of cells in the image to obtain an area fraction/cell.

Measurement of Intracellular Calcium Levels

Transfected HEK293T cells were grown on poly-L-lysine cover glass at 37°C in a humidified CO2 atmosphere in DMEM (Gibco) supplemented with 10% FBS

(ThermoFisher) until optimal confluency was reached (~80%). Cells were then incubated

2+ for 45-60 min at 37°C at 5% CO2 with 3M Fluo-4, a Ca indicator dye. After efficient uptake of Fluo-4, cells were washed in HBSS (Hanks Balanced Salt Solution) media. 10

M ATP was added extracellularly to elevate intracellular [Ca2+] from internal stores using a controlled microinjection system. Fluo-4 intensity was monitored every 2 seconds by a real time laser confocal microscope (Zeiss LSM800) with relative intensities plotted in regard to the basal calcium level (F0).

CHAPTER THREE

Results

We hypothesize the presence of mobile CaBPs alters cytoskeletal actin dynamics.

Because Ca2+ signaling can modify Rho GTPases that have been suggested to play roles

in OHC motility, we investigated whether or not the presence of major CaBPs are

capable of modifying the actin-based cytoskeleton. To test our hypothesis, we generated

plasmids of two major CaBPs, PV and OCM tagged with GFP and transiently

transfected them in cell lines that do not naturally express these proteins. Successful

transfection with the OCM-GFP plasmid was confirmed by western blot analysis (Figure

4). Antibodies against OCM and GFP revealed bands at ~40 kDa. This was expected as

the size of OCM is 12 kDa and GFP is 27 kDa40. Our goal was to identify whether or not the presence of any CaBP alters cytoskeletal dynamics, or if the presence of OCM displays a unique phenotype.

Figure 4. Oncomodulin is efficiently tagged to GFP. Western blots labeled with mouse ∝ OCM and mouse ∝ GFP antibodies. Lanes 1 and 3 contain cell lysate of cells transfected

15 with GFP only. Lanes 2 and 4 contain cell lysate of HEK293T cells transfected with

OCM-GFP.

Although OCM is similar in structure to mobile EF hand CaBPs, there is no evidence demonstrating OCM is capable of modifying intracellular Ca2+signals. Our first aim was to test Ca2+ signaling in cells transiently transfected with the protein OCM.

Neither HEK nor HeLa cells have endogenous expression of OCM. We used adenosine triphosphate (ATP) to increase cytosolic calcium levels. Extracellular ATP activates plasma membrane P2-type purinoceptors which increases levels of cytosolic inositol

2+ 41 1,4,5-triphosphate (IP3) and subsequent release of Ca from internal stores . The duration of ATP induced Ca2+ transients were measured in OCM transfected vs. untransfected cells. Untransfected cells show a continually changing induced Ca2+ response from ATP induced IP3 signaling (Figure 5A). Although there was significant variation, the mean response (F/F0) continually increased over a 50 second window. In contrast, OCM transfected cells show a significantly decreased mean response (F/F0) in

ATP induced Ca2+ response (Figure 5B). As a matter of fact, the mean response only last one to two seconds. This is the first demonstration that OCM acts as a Ca2+ buffer.

Figure 5. Duration of ATP induced calcium transients is significantly decreased in OCM transfected cells. Fluo-4 intensity (F) was monitored by real time laser confocal microscope and plotted as relative intensity with respect to that of a basal calcium level (F0). Gray lines represent Fluo-4 intensity of individual cells. Black line represents mean Fluo-4 intensity of all cells measured. Intensity measurements were obtained with 480nm excitation, 530nm emission.

To test whether or not the presence of CaBPs results in changes to F-actin, we compared OCM-GFP expression to that of PV-GFP in two cell lines (HEK293T and

HeLa) that do not endogenously express these proteins. Confocal images of Alexa-fluor-

564 conjugated phalloidin staining was used as a readout of actin polymerization since phalloidin is known to bind preferentially to F-actin in fixed cells42. Because Ca2+ levels have been shown to influence proteins involved in cytoskeletal organization, we predicted the presence of two major CaBPs would result in structural changes in the cell due to modified levels of F-actin. Fluorescently labeled cells do not show obvious differences in cell shape amongst the three different transfection schemes (Figure 6).

Next, we looked at whether or not there are differences in the overall amount of F-actin in these cells. One way to asses total F-actin is to measure actin intensity in all three transfection schemes. There appears to be no obvious differences in mean intensity of 17

actin across all transfection conditions (Figure 7). However, it is possible OCM transfected cells display less variation in mean actin intensity.

Further investigation of the actin images revealed what appeared to be fewer areas

of concentrated phalloidin binding in the OCM-GFP cells. These locations indicate

possible stress fiber structures composed of actin and NMII. Basic intensity

quantification measurements were obtained after proper threshold adjustments were made

to each figure (Figure 8A 4-6). There appears to be possible differences in OCM-GFP

transfected cells compared to PV-GFP and GFP transfected (Figure 8A 4-6).

Particularly, there appear to be fewer amounts of these structures in OCM-GFP

transfected cells compared to PV-GFP and GFP transfected cells (Figure 8A 4-6). We

decided to quantify the structures identified Figure 8A panels 4-6 (Figure 8B). Obtaining an area fraction per cell indicates that cells transfected with OCM-GFP might contain fewer amounts of stress fibers (Figure 8B). However, more replicates need to be

performed. Overall, these experiments reveal OCM-GFP transfected cells display fewer

structures that might be stress fibers. We did not anticipate investigating these areas of

increased phalloidin staining. Therefore, we do not know for certain what these structures

are. However, based on appearance, they do appear to be stress fibers. Further

investigation and more replicates need to be performed to identify these areas of intense phalloidin labeling.

Additionally, because tubulin is a major cytoskeletal element of cells, we tested whether or not OCM-GFP would have an effect on tubulin expression in HeLa cells

(Figure 9). It has been demonstrated that microtubules become depolymerized when

cytosolic Ca2+ exceeds 0.6M43. Additionally, further investigation showed that the

CaBP protein calmodulin is the intermediary factor that stimulates microtubule depolymerization44. We predicted that the presence of OCM-GFP would result in a

decrease in the total amount of polymerized microtubules. However, there are no

significant changes in tubulin expression in OCM-GFP transfected versus untransfected

HeLa cells (Figure 9, 1 & 4).

Figure 6. Oncomodulin has no observable effect on filamentous actin. HEK293T cells transiently expressing OCM-GFP, PV-GFP, GFP,and Lipofectamine only. Asterisks indicate transfected cells. Lipofectamine without DNA served as a negative control. Actin was labelled using Alexa Fluor 594-conjugated phalloidin (ThermoFisher). The

19 actin cytoskeleton appears to be unaltered in all four experimental conditions. Confocal mages were taken on Zeiss LSM800 40x objective lens. Scale bars represent 20 m

Figure 7. Basic intensity quantification of actin labeled HEK293T cells expressing GFP, PV-GFP, OCM-GFP. Green dots indicate cells expressing GFP.

Figure 8. Area fraction measurements on potential stress fibers in GFP, PV-GFP, and OCM-GFP transfected HEK293T cells. OCM-GFP transfected cells might have decreased stress fiber formation. A.) Images used to obtain area fraction measurements before and after threshold adjustments. B.) %Area/cell for each transfection condition. n=43, n=31 and n=43 for OCM-GFP, PV-GFP, and GFP respectively.

Figure 9. Oncomodulin has no observable effect on -tubulin in HeLa cells by immunofluorescence. HeLa cells transiently transfected with OCM-GFP stained with host anti-Tubulin (Sigma Aldrich). Cells transfected with lipofectamine only with no DNA served as a negative control. Asterisks indicate cells not expressing OCM-GFP. When compared with untransfected cells, it appears that microtubule structure is not affected by expression of OCM-GFP. Scale bars represent 20 m.

Since changes in actin dynamics could be more subtle than is possible to detect by

microscopy, we performed companion analyses on the F:G actin ratios by western blot.

Transfected HEK293T and HeLa cell lysates were harvested using the G-actin/F-actin in

vivo assay kit from Cytoskeleton, Inc.to separate the F-actin polymers from the G-actin

monomers (Figure 10A-B). Densitometry of the F-actin band compared with the G-actin

band in untransfected cells gives ratios similar to those previously published for HeLa,

~0.45 (Figure 10C)45. Likewise, ratios in untransfected HEK293T cells mirrored the variability of previous reports, 0.2-0.4 (Figure 10D)46. Transfection with OCM-GFP did not significantly alter F:G ratios in either cell type (Figure 10C-D). Similar experiments were performed using cochlea from OCM WT and KO mice (Figure 11). WT cochlea

demonstrated similar ratios to those previously published by Han et al., ~0.4 (Figure

22 11B)10. F:G ratios of cochlear lysates from OCM KO mice did not differ significantly from the WT cochlea. Altogether, this data indicates that there is no impact of OCM on cytoskeletal organization in mouse cochlea, HEK293T, and HeLa cells under these conditions.

Figure 10. OCM does not impact F:G actin in HEK293T and HeLa cells by Western Blot. (A - B) Western blots of F- and G-actin measurements in HEK293T and HeLa cells transfected with OCM-GFP and GFP. Actin standards of indicated concentrations were quantified to provide a relative amount of actin in each sample. Blots were probed with host anti-Actin antibodies. (+) Lipo represents cells transfected with Lipofectamine only without DNA while (-) Lipo represents cells treated without any Lipofectamine or DNA. Cell lysates treated with a phalloidin F-actin enhancing solution (Cytoskeleton Inc.) 23 served as a positive control and are labeled “Phall – ES”. (C-D) Quantification of western blots by densitometry using ImageJ. Data represents averages of independent experiments in HEK293T cells (n=9) and HeLa cells (n=5). Error bars represent  Std deviation.

Figure 11. OCM KO mouse cochlea do not display altered F:G actin by Western Blot. (A) Western blots of F- and G-actin measurements on whole cochlear extracts from OCM KO and WT mice. Blots were probed with host anti-actin antibodies. Cochlear lysates treated with phalloidin F-actin enhancement solution (Cytoskeleton Inc.) were used as a positive control and are labeled as “Phall – ES”. (B) Quantification of western blots by densitometry using ImageJ. Data represents averages of five independent experiments. Error bars represent  Std deviation.

Actin polymerization is downstream of Ca2+-dependent RhoA signaling. Proteins

involved in RhoA signaling cascades have been shown to modify F:G actin ratios in the

mammalian cochlea and effect OHC motility. Due to the vast amount of proteins

involved in these signaling cascades, we wanted to identify protein expression that might

24 be changed in the presence of CaBPs. To more easily screen the many contributors to

RhoA signaling, we utilized the TaqMan RhoA gene expression assay from

ThermoFisher. This 96-well plate contains primer/probe pairs for 92 genes associated

with RhoA signaling pathways. Because of the similarity in structure and function of

OCM and PV, any gene expression change observed could be due to functions unique

to OCM. Genes with the biggest difference in relative gene expression are shown in

Table 3. Interestingly, Ezrin and Cofilin expression was increased. Ezrin and cofilin are downstream effectors of ROCK and both have been shown to affect F:G actin ratios in the cochlea after noise trauma10. We would expect to see a decrease in proteins associated

with ROCK because it has been suggested Ca2+ levels are a major upstream component

of ROCK activation. ARHGAP1 has functions in activating Rho GTP metabolizing

enzymes, which ultimately leads to a decrease in active GTP bound RhoA. Although

additional replicates need to be performed, results such as this align with our hypothesis

that OCM decreases levels of active Rho GTPases.

Table 3. Candidate genes for further investigation of proteins involved in RhoA signaling that might be influenced by OCM in cultured cells and mouse cochlea. Data from two separate Rho-A Taqman gene expression assays (ThermoFisher) with RNA collected from HEK293T cells transfected with Ocm-GFP and PV-GFP. Gene Ct values from the Ocm-GFP plate were normalized to those on the PV-GFP. Genes listed are based on their relative expression using the delta delta Ct method.

Gene Gene Name Relative Gene Function Symbol Expression CHN1 Chimerin 1 1.17 GTPase activating protein

KTN1 Kinectin 1 1.75 Binds to kinesin and has roles in intracellular organelle motility EZR Ezrin 2.92 Intermediate kinase between the plasma membrane and cytoskeleton

CFL2 Cofilin 2 2.36 Reversibly controls actin polymerization and depolymerization

GNAI1 G Protein Subunit Alpha subunit of an inhibitory G Alpha 1 2.17 Protein complex

ARHGAP1 Rho GTPase 1.98 Activates Rho-type GTP Activating Protein 1 metabolizing enzymes PPAP2B Phospholipid 0.52 Participate in signal transduction Phosphatase 3 mediate by phospholipase D RHPN2 Rhophilin Rho Binds both GTP and GDP bound GTPase Binding 0.17 RhoA Protein

CHAPTER FOUR

Discussion

Previous studies have suggested OHC motility is regulated by cytoskeletal changes. The Ras superfamily of small GTPases and their downstream effectors are known to modify cytoskeletal organization in multiple cell types. In particular, the small

GTPase RhoA and its associated effector proteins have been suggested to contribute to

OHC motility by modifying the actin cytoskeleton. Activation of cholinergic acetylcholine (Ach) receptors in OHCs leads to an increase in intracellular calcium levels, which have been shown to influence active levels of Rho proteins47,48. OCM is a major calcium buffer uniquely expressed in OHCs. Because OCM KO mice show OHC morphology defects and hearing loss, we hypothesized that OCM affects the actin-based cytoskeleton by manipulating intracellular calcium and affecting levels of active Rho

GTPases. We transiently transfected HEK293T and HeLa cells with OCM-GFP and labeled for cytoskeletal elements F-actin and -tubulin (Figures 6 & 9). We speculated that expressing OCM in HEK293T and HeLa cells might result in a structural change in the actin-based cytoskeleton. However, no observable cytoskeletal changes were detected in any transfection condition. A potential explanation is that these methods were not sensitive enough to detect more subtle changes to the cytoskeleton, or that we did not induce the correct conditions to elicit OCM-dependent cytoskeletal rearrangement.

HEK293T cells appear to have fewer areas of intense phalloidin binding (Figure 8).

These areas could be stress fibers composed of actin and NMII or focal adhesions. RhoA

and its effectors regulate stress fiber formation in many cell types49,50. Characterized by

their location, dorsal stress fibers, ventral stress fibers, transverse arcs, and perinuclear actin caps are all stress fibers composed of actin filament bundles51. If the areas of

concentrated phalloidin binding in our images are indeed stress fibers, they are most

likely transverse arcs due to their location on the perimeter of the cell. In addition to

actin-bundles and NMII, stress fibers often contain myosin light chain kinase, a protein

identified in OHC slow motility52. Although it would very interesting to see a difference

in stress fiber formation in OCM-GFP treated cells, it has been shown that stress fiber

formation is increased in cells growing on plastic surfaces compared to the same cells in

their natural tissue environment53,54. In HEK293T cells, extracellular ATP activates purinoceptors in the plasma membrane to induce calcium influx and release of calcium from internal stores, thereby raising intracellular calcium41,55,56. Likewise, HeLa cells can

be stimulated to release internal calcium stores in an IP3 dependent manner through

activation of stromal interaction molecule (STIM) proteins57. It is possible that simply

expressing OCM in these cells is not enough to observe a phenotype. Stimulating OCM

transfected cells with a variety of physiological stimuli such as ATP and (IP3) to release

internal calcium might elicit a change in the cytoskeleton. Another possibility is that our

experiments relied on OCM tagged to GFP (27kDa), a protein more than double the size

of OCM (12kDa). It is possible that OCM function is jeopardized while tagged to such a

large protein. Use of a smaller peptide tags such as Myc, FLAG, or Spot, to detect OCM

might be less inhibitory of OCM function. Functional analyses comparing OCM-GFP

with peptide-tagged OCM would help identify any difference in OCM efficiency. One

such method could be the use of calcium indicator dyes such as Fluo-4 to measure the

difference in ATP induced calcium transients between OCM-GFP and peptide-tagged

OCM.

To address the possibility that actin staining and microscopy were not sensitive enough to observe OCM-dependent effects on the cytoskeleton, we also investigated F:G actin ratios in transfected cells and mouse cochlea (Figures 10-11). Previous studies have shown noise induced F:G actin alterations in the mouse cochlea are regulated in a

RhoA/ROCK/LIMK/Cofilin dependent manner10,58. The mechanism by which intense

noise manipulates RhoA effector protein expression might be due to changes in calcium levels. The RhoA/ROCK pathway leads to phosphorylated cofilin, which inactivates cofilin, thus preventing actin depolymerization15. If calcium levels are responsible for

initiating RhoA/ROCK signaling, we expected to observe lower levels of F actin in cells

transfected with OCM and in the cochlea of WT mice. HeLa cells transfected with OCM-

GFP display a slightly lower F:G actin ratio compared to control cells (Figure 10C),

while HEK293T cells transfected with OCM-GFP display slightly higher F:G actin ratios compared to control and untransfected cells (Figure 10D). However, these results are not statistically significant. Adherent HeLa cells do not have developed F-actin structures and have a low surface stiffness that remains low after cell detachment59. Surface stiffness of

adherent HEK293T cells is also very low, but in contrast to HeLa cells, increases upon

cell detachment due to an increase in F-actin60. Differences in surface stiffness as

determined by levels of F-actin is a possible explanation for the variability of our HeLa

and HEK293T F:G actin ratios. Previous studies have shown that HEK293T F:G actin

ratios can be as low as 0.2 and as high as 1 without any treatment46. This is consistent

with our findings. Previous reports indicate whole cochlea homogenates possess F:G

actin ratios of 0.610. This is also consistent with our findings. Variability in our F:G actin

ratios could be due to using whole cochlea homogenates. In addition to OHCs, the

mammalian cochlea possesses many other cells who undoubtably express actin. Using

micro dissected tissue from the organ of Corti would be a more accurate representation of

any cytoskeletal change occurring in OHCs and might reduce the variability in F:G actin

measurements as seen in whole cochlea homogenates. Using carefully dissected pieces

from the organ of Corti would also allow cytoskeletal investigation of OHCs after

exposure to physiological stimuli of interest.

It is plausible that OCM might be impacting the actin polymerization pathway in

a nuanced way not discernable by the F:G actin assay. As mentioned previously, the

RhoA GTPase pathway involving actin depolymerization is large and complex and is

affected by Ca2+ dynamics in the cell. Thus, to screen if OCM impacts proteins in this

pathway a RhoA Taqman gene expression plate was implemented. Each plate is

composed of 96 assays with sequence specific primers for 92 RhoA associated proteins

known to have roles in regulating cytoskeletal dynamics. We predicted OCM might influence gene expression in known genes that have been shown to play roles in noise

induced hearing loss, such as ROCK, LIMK, ERM and cofilin12. However, the

intracellular calcium levels were not modified at all in these cells and we did not analyze

and cochlear tissue. For instance, the localization pattern of OCM in the transfected cells

differs significantly than in OHCs. In OHCs, OCM usually localizes at or near the plasma

membrane and cuticular plate. Thus, if OCM is modulating expression of RhoA pathway

proteins in transfected HEK293T cells, it may do so in a different manner in OHCs. We

would expect OCM to alter gene expression of proteins in OHCs that directly bind actin

30 near the plasma membrane such as cofilin, profilin and ERM proteins. One limitation of this experiment was the lack of negative controls. Untransfected and GFP-only transfected cells would have shown gene expression changes resulting from the transfection process and from the presence of GFP, respectively. Due to limitation of resources, the PV-GFP transfection was used as the control. This is because PV and

OCM share 53% sequence similarity, and any difference in gene expression between the two would plausibly be due to the minor differences in OCM and PV. OHCs co-opt expression of PV for OCM during development, suggesting that OCM performs a biological role PV does not. Therefore, the genes that changed in the OCM-GFP sample compared to the PV-GFP sample may indicate genes with physiological relevance.

In conclusion, OHCs are responsible for the fine frequency selectivity of mammalian hearing. Critical to OHC function is OHC slow motility, a Ca2+-dependent mechanism that involves cytoskeletal reorganization. OCM is a major CaBP that is critical for cochlear function. OCM preferentially localizes to the lateral wall of OHCs, therefore placing itself in close proximity to the actin-based cytoskeleton. OCM can potentially regulate OHC motility through Ca2+-dependent mechanisms. We hypothesized that OCM might regulate OHC slow motility in two ways. By changing internal Ca2+ concentrations, OCM might affect the number of active Rho GTPases and thus effect downstream cytoskeletal actin dynamics. Conversely, OCM might alter the actin-based cytoskeleton by directly interacting with proteins involved in cytoskeletal organization. In this work, we investigated whether or not the presence of OCM would manipulate the actin-based cytoskeleton in cells that do not naturally express OCM.

HEK293T and HeLa cells transiently transfected with OCM-GFP displayed no distinct

morphological changes under observation of confocal microscopy. Because changes in

structure might be more subtle, we investigated F:G actin ratios in OCM-GFP transfected

HEK293T and HeLa cells in addition to OCM KO and WT cochlea. There were no

distinct differences in F:G actin ratios in all samples. Lastly, we utilized RT-qPCR to

identify potential changes in gene expression due to OCM. This provided us with

information to better understand what interactions OCM might be influencing in vivo.

Although substantial work has recently been accomplished in regard to the genetic components contributing to hearing loss, little is known about its underlying mechanisms.

Further investigation of OCM and proteins involved in OHC motility is crucial to understanding cochlear function.

Based on these data, future research should focus on identifying which Rho

GTPase(s) are active in the cochlea. This information will provide a better understanding

of effector proteins that might be associated with the phenotypes observed in OCM KO

OHCs. Additionally, investigating F:G actin ratios in the organ of Corti instead of whole

cochlear homogenates might reveal significant differences in the F:G actin ratio in OCM

KO versus WT cochlea that are masked when using whole cochlear tissue. Lastly,

investigating OCMs role as a potential actin binding protein will provide evidence for its

preferential cellular localization in OHCs and give insight to the mechanisms associated

with OHC morphology changes as seen in the OCM KO cochlea.

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