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8-2019

OBSCURIN MEDIATES COMPLEX FORMATION IN THE

Janani Subramaniam

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This Thesis (MS) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. OBSCURIN MEDIATES ANKYRIN COMPLEX FORMATION IN THE HEART

by

Janani Subramaniam, B.S.

APPROVED:

______Shane R. Cunha, Ph.D. Advisory Professor

______Darren F. Boehning, Ph.D.

______Carmen W. Dessauer, Ph.D.

______Jeffery A. Frost, Ph.D.

______M. Neal Waxham, Ph.D.

APPROVED:

______Dean, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences

1

OBSCURIN MEDIATES ANKYRIN COMPLEX FORMATION IN THE HEART

A

THESIS

Presented to the Faculty of

The University of Texas

MD Anderson Cancer Center UTHealth

Graduate School of Biomedical Sciences

in Partial Fulfillment

of the Requirements

for the Degree of

MASTER OF SCIENCE

by

Janani Subramaniam, B.S. Houston, Texas

August, 2019

Dedication

To my family: Mom, Dad, Utsi, and Manga, Your unconditional love and support allow me to ―burrow burrow‖ my way through life. I am so deeply grateful for you!

iii

Acknowledgements

The phrase ‗It takes a village,‘ could not be more applicable to my graduate career thus far. I am thankful for all the people I have met and the all opportunities I‘d had to grow.

I would not be here without the love, support and guidance of my family. Despite the distance you are there for me no matter what, and I am so grateful for that. Thank you for the encouragement, for celebrating my highs with me, and for being there for me during my lows. I love you more than I can express!

To all my friends from Houston, thank you for making this city feel like home for the last few years. You‘ve been there for me through it all and I‘ve had so much fun in the time we‘ve spent together. Meredith, you were my first friend in Houston and grew to be one of my best. Thank you for always believing in me and inviting me into your family with open arms! To Katie, thanks for helping me learn how to use Illustrator and answering my design questions- I enjoyed that almost as much as our wine nights. To all my grad school friends, thank you for listening to my presentations, helping me with experiments, encouraging me when I needed it, and commiserating with me when experiments/things didn‘t work out! I‘d have a lot more grey in my hair if it weren‘t for you.

To everyone in the BCB program and IBP department, thank you for helping me learn techniques, providing me with reagents and cells, and all the feedback on my projects and experiments. Special shout-out to the admin for always ensuring that things run seamlessly.

Thank you, Drs. Jeff Frost, Neal Waxham, Carmen Dessauer, and Darren Boehning, for providing a supportive yet challenging atmosphere at committee meetings. Thank you for

iv your guidance and insight along the course of my project. Dr. Carmen Dessauer, thank you for all the encouragement along the way. It always meant a lot to me as you are someone I hope to emulate one day. Dr. Darren Boehning, thank you so much teaching me techniques, letting me use your lab like I was a part of it, and answering my numerous questions.

And last but not least, thank you, Shane! You have been both a mentor and a friend these last few years and I know I wouldn‘t be here today without you. You have been patient with me when I needed it and pushed me when I needed that. Thank you for encouraging me to think critically, be an ethical and creative scientist, and deliver my ideas with clarity- all while having a good time!

v

OBSCURIN MEDIATES ANKYRIN COMPLEX FORMATION IN THE HEART

Janani Subramaniam, B.S.

Advisory Professor: Shane R. Cunha, Ph.D.

Distinctly organized domains of receptors, ion channels, transporters, signaling molecules, molecules, and contractile are crucial to cardiac function.

Interactions between adaptor proteins such as and cytoskeletal proteins such as obscurin play a pivotal role in organizing these functional domains in cardiomyocytes.

Therefore, dysfunction of both ankyrin as well as obscurin lead to a host of cardiovascular diseases such as and cardiomyopathies. of ankyrin yields numerous isoforms that interact with obscurin at various sub-cellular domains. And while some of these obscurin-ankyrin complexes have been studied, many others have not been characterized. Further, previous studies have focused on these ankyrin-obscurin complexes individually; however, how ankyrin-mediated macromolecular complexes are integrated together within the cardiomyocyte is not clear. Thus, the work in this thesis describes mechanisms by which obscurin organizes two separate ankyrin-mediated macromolecular complexes simultaneously.

Two muscle-specific ankyrin isoforms, sAnk1.5 and AnkG107/130, which do not interact with each other, were used to test if obscurin could serve to bridge these ankyrins.

Through a series of in vitro binding assays, co-precipitation assays, and FLIM-FRET analysis we demonstrate that obscurin binds both isoforms simultaneously via interactions with two ankyrin binding sites in its C-terminus. We also show that β1-, a that does not

vi directly interact with obscurin or sAnk1.5, can be recruited to a macromolecular complex via an interaction with AnkG107/130. Lastly, both ankyrin isoforms localize to the M-line of cardiomyocytes suggesting that obscurin could serve to target two separate ankyrin protein complexes to the same domain (M-line). In sum, this work provides a novel, compelling mechanism for ankyrin complex integration and coordination in cardiomyocytes.

vii

Table of Contents

Approval Page...... i

Title Page...... ii

Dedication...... iii

Acknowledgements...... iv

Abstract...... vi

Table of Contents...... viii

List of Figures...... xi

Abbreviations………………………...... xii

Chapter 1: Introduction...... 1

1.1: Spotlight on cardiovascular disease...... 2

1.2: Features of cardiomyocytes...... 2

1.3: Ankyrins...... 4

1.4: Ankyrin functional domains...... 5

1.5: Ankyrin diversity: and alternatively spliced isoforms...... 8

1.6: Ankyrins in the heart...... 11

1.7: Obscurin...... 17

1.8: Significance and scope of this thesis...... 21

viii

Chapter 2: Materials and Methods...... 23

2.1: Plasmids and antibodies...... 24

2.2: RNA isolation, reverse transcription, and PCR amplification of AnkG107/130

mRNA transcripts...... 26

2.3: In-vitro binding assays...... 26

2.4: Co-precipitation assays...... 27

2.5: FLIM-FRET analysis...... 29

2.6: Isolation and culture of neonatal rat ventricular cardiomyocytes...... 29

2.7: Fluorescence immunohistochemistry...... 30

2.8: Statistics...... 30

Chapter 3: Results...... 31

3.1: Obscurin binds AnkG107/130 and sAnk1.5 in-vitro...... 32

3.2: Obscurin ABD1 and ABD2 regulate AnkG107/130 and sAnk1.5 complex

formation...... 34

3.3: Obscurin mediates an indirect interaction between AnkG107/130 and

sAnk1.5...... 36

3.4: Obscurin regulates AnkG107/130-sAnk1.5 complex formation in cells...... 38

3.5: Virally-expressed AnkG107/130-CTD-RFP and sAnk1.5-GFP localize to the

ix

M-lines of ventricular cardiomyocytes...... 41

3.6: AnkG107/130 binds obscurin, but not -1 or -C...... 43

3.7: β1-spectrin associates with the obscurin/ankyrin complex via AnkG107/130.....47

Chapter 4: Discussion and future directions...... 49

4.1: Discussion...... 50

4.2: Future directions...... 55

4.3: Conclusion...... 57

Bibliography...... 59

Vita...... 74

x

List of Figures

Figure 1: Structure of a cardiomyocyte...... 4

Figure 2: Functional domains of canonical ankyrins...... 6

Figure 3: Diversity of ankyrin products...... 9

Figure 4: Ankyrin mediated domain organization in cardiomyocytes...... 12

Figure 5: Domain organization of obscurin isoforms...... 18

Figure 6: Diagrams of obscurin, sAnk1.5, and AnkG107/130...... 25

Figure 7: Obscurin interacts with both sAnk1.5 and AnkG107/130...... 33

Figure 8: Obscurin ABDs mediate interaction with sAnk1.5 and AnkG107/130...... 35

Figure 9: Obscurin regulates AnkG107/130 complex formation with sAnk1.5...... 37

Figure 10: FLIM-FRET analysis demonstrating obscurin regulation of ankyrin complex

formation...... 39

Figure 11: Virally-expressed sAnk1.5 and AnkG107/130 are targeted to the M-lines of

ventricular cardiomyocytes...... 42

Figure 12: AnkG107/130 preferentially interacts with obscurin via OTBD...... 44

Figure 13: AnkG107/130 does not interact with filamin-C...... 46

Figure 14: β1-spectrin associates with the obscurin/ankyrin complex via AnkG107/130...... 48

Figure 15: Summary model of obscurin mediated ankyrin complexes...... 58

xi

Abbreviations

β-DG β-

ABD ankyrin binding domain

AD Alzheimer‘s Disease

AE1 anion exchanger 1

Ank ankyrin

AnkB ankyrin-B

AnkG ankyrin-G

AnkR ankyrin-R

Ca2+ calcium

CaMKII calcium/calmodulin-dependent II

CFP Cyan Fluorescent Protein

CTD carboxy-terminal domain

DCM

DD

DGC -glycoprotein complex

DHPR dihydropyridine receptor

ECM

ER endoplasmic reticulum

ERK extracellular receptor kinase

Fluorescence Lifetime Imaging-Fluorescence Resonance Energy FLIM-FRET Transfer

xii

FNIII fibronectin type-III

GFP Green Fluorescent Protein

GST Glutathione S-

HCM hypertrophic cardiomyopathy

ICD intercalated disc

Ig immunoglobin

IP3R inositol 1,4,5 triphosphate receptors jSR junctional

KCNQ2/3 potassium voltage-gated channel subfamily Q member 2/3

KCTD6 potassium channel tetramerization domain containing 6

KO knock-out

KV3.1 voltage-gated potassium channel 3.1

L1-CAM -cell adhesion molecule

LQTS long QT syndrome lSR longitudinal sarcoplasmic reticulum

LVNC left ventricular noncompaction cardiomyopathy

MBD membrane binding domain

NaV1.5 voltage-gated 1.5

NCX Na+/Ca2+ exchanger

NKA Na+/K+ ATPase

NRVM neonatal rat ventricular cardiomyocytes

NTD amino-terminal domain obs obscurin

xiii

OTBD obscurin/ binding domain

PH pleckstrin homology

PP2A protein phosphatase 2A

RanBP9 Ran-binding protein 9

RFP Red Fluorescent Protein

Rh Rhesus

RhAG Rhesus Ammonium Transporter AG

RhoA Ras homolog gene family member A

RhoGEF Rho-guanine nucleotide exchange factor

ROCK1 Rho associated coiled-coil containing kinase 1

RyR ryanodine receptor sAnk1 small ankyrin 1

SBD spectrin binding domain

SERCA sarco/endoplasmic reticulum calcium ATPase

SH3 src homo7logy 3

SK1/2 Ser/Thr kinase domains 1 and 2

SLN sarcolipin sMyBP-Cv1 slow skeletal binding protein C variant 1

SR sarcoplasmic reticulum t-tubule transverse-tubule

T2D type 2 diabetes

TM transmembrane

Tmod1 1

xiv

Tmod3

TNF

Y2H yeast 2-hybrid

YFP Yellow Fluorescent Protein

xv

Chapter 1

Introduction

1

Spotlight on Cardiovascular Disease

Cardiovascular disease is the leading cause of death in the United States, accounting for 1 in every 4 deaths or ~31% of all deaths (CDC, 2015). Additionally, heart disease costs

~$200 billion each year in health care services, medication, and lost productivity (CDC,

2015). Therefore, it is crucial that we to continue to develop effective solutions to prevent and treat cardiovascular disease.

The production of a single heart beat is brought about by a multitude of tightly regulated electrical, chemical, and mechanical pathways brought about by ion channels, contractile machinery, transporters, receptors, signaling proteins, and cell adhesion molecules

( Bennett & Baines, 2001). Thus, the organization and coordination of these proteins within a cardiomyocyte is crucial for normal cardiac function. Adaptor proteins such as ankyrins and cytoskeletal proteins such as obscurin play pivotal roles in organizing the cardiomyocyte, and numerous studies have linked both proteins to a host of electrical and structural cardiovascular diseases (Vann Bennett and Healy 2008; Makara et al. 2014; Mohler et al.

2007; Arimura et al. 2007; Marston et al. 2015; Rowland et al. 2016). Therefore, understanding the regulation of the fundamental cellular and molecular pathways by these cytoskeletal and cytoskeletal associated proteins provides valuable information about normal cardiac function as well as dysfunction caused by disease.

Features of Cardiomyocytes

In all striated muscle, the contractile filaments, and myosin, are organized into the , the structural and functional unit of contraction, by sarcomeric cytoskeletal proteins such as α-, myomesin, titin, obscurin and (Kontrogianni-

Konstantopoulos et al. 2009). The sarcomere is made up of a dark central region known as

2 the A-band, flanked on either side by lighter regions called the I-bands, and ending in Z-discs on either side (Fig. 1). The A-bands contain filaments of the protein myosin, and the I-bands have filaments of actin, which start at the Z-disc, pass through the I-band and overlap the ends of the myosin filaments in the A-band. The part of the A-band not overlapped by actin filaments is called the H-zone, and the center of the A-band and sarcomere is known as the

M-line. The sarcolemma invaginates the myocte at the Z-line as the transverse-tubule (t- tubule), to allow rapid transmission of electrical signals into the cell interior. The sarcoplasmic reticulum (SR) extends across the entire myocyte and is a calcium (Ca2+) reservoir. It is made up of junctional SR (jSR), which contains the calcium-release channels through which calcium enters the during excitation-contraction coupling, and the longitudinal or tubular SR (lSR), that surrounds the contractile proteins for re-uptake of calcium during relaxation. Ankyrins play an essential role in coordinating these Ca2+ events and maintaining the SR membrane.

3

Figure 1: Structure of a cardiomyocyte. Diagram showing the sarcomeric structure of a cardiomyocyte, including the Z-disc, A-band, I-band, M-line, t-tubules, and sarcoplasmic reticulum (SR). Modified with permission from A.M. Katz, ―Essential Cardiology : Principles and Practice,‖ (2013). License number: 4631840358234.

Ankyrins Ankyrins (AnkR, AnkB, and AnkG) are a family of adaptor proteins that link integral membrane proteins with the underlying . The first ankyrin was discovered by Dr.

Vann Bennett in 1979, anchoring the anion exchanger to β1-spectrin in erythrocytes (V

Bennett and Stenbuck 1979). Since then, several studies have demonstrated that in addition to simply anchoring membrane bound proteins to the cytoskeleton, ankyrins regulate the expression and recruitment of membrane proteins, signaling molecules, and cytoskeletal elements to distinct domains with the cell (V Bennett and Baines 2001). Thus, these adaptor proteins are crucial for the formation and maintenance of domains, serving functions such as clustering ion channels or providing adherence between cells (Cunha and Mohler 2009; J. Q.

Davis and Bennett 1993; Hill et al. 2008).

4

Ankyrin Functional Domains

The canonical ankyrin is composed of four major domains: the membrane binding domain (MBD), spectrin binding domain (SBD), death domain (DD), and C-terminal domain

(CTD); with the DD and CTD together referred to as the regulatory domain (Fig. 2) (Rubtsov and Lopina 2000).

The MBD interacts with a diverse range of integral proteins, including channels, transporters, cell adhesion molecules, and endocytic machinery. Proteins that have been identified to bind the ankyrin MBD include: the anion exchanger (AE1) (Ding, Kobayashi, and Kopito 1996), voltage-gated sodium channels (Zhou et al. 1998), Na+/K+ ATPase

+ 2+ (NKA), Na /Ca exchanger (NCX), inositol 1,4,5 triphosphate receptors (IP3R) (Mohler et al. 2003), Rh antigen and RhAG ammonium transporter (Lopez et al. 2005), potassium voltage-gated channel subfamily Q member 2/3 (KCNQ2/3) (Rasmussen et al. 2007), and voltage-gated potassium channel 3.1 (KV3.1) (Xu et al. 2007), L1-cell adhesion molecules

(L1CAMs) (Gil et al. 2003), CD44 (Lokeshwar, Fregien, and Bourguignon 1994), N- and E- cadherin (Hoffman et al. 2007), and beta-dystroglycan (β-DG) (Ayalon et al. 2008). The canonical MBD is made up of 24 ANK repeats, which are further divided into four smaller domains (D1, D2, D3, and D4), each containing six repeats, that can bind various membrane proteins in a modular fashion (Michaely et al. 2002). Each ANK repeat is made up of 33 amino acids, which form a hairpin-like β-sheet followed by two anti-parallel α-helices, and ends in a loop which then leads into the next repeat (Michaely et al. 2002). It is one of the most common structural motifs, mediating protein-protein interactions responsible for numerous cellular processes including signal transduction, inflammatory responses, vesicular trafficking, cytoskeleton

5

Figure 2: Functional domains of canonical ankyrins. Ankyrins contain four domains: membrane binding domain (MBD), spectrin binding domain (SBD), death domain (DD), and C-terminal domain (CTD). Integral membrane proteins bind to ANK repeats in the MBD, and the SBD links the membrane-ankyrin complex to the underlying spectrin cytoskeleton. Reproduced with permission from V. Bennett & D.N. Lorenzo, ―Current Topics in Membranes,‖ (2016). License number: 4633971510344.

integrity, cell cycle regulation, and even transcriptional regulation (Li, Mahajan, and Tsai

2006). The 24 ANK repeats form a solenoid, with dynamic spring-like properties (Lee et al.

2006). Thus, the solenoid shape with grooves, nano-spring biophysical properties, and homology across ANK repeats, convey the MBD with broad and diverse binding capabilities. Without these qualities, ankyrins would lack the ability to play significant roles in a variety of physiological settings.

The ankyrin associated protein complexes are tethered to the cytoskeleton via its SBD

(Ipsaro and Mondragón 2010). The SBD contains three conserved domains, two ZU5s and a

6

UPA domain, arranged as ZU5-ZU5-UPA (Wang et al. 2012). β-spectrin repeats 14 and 15 bind ankyrin in the first ZU5 domain through both electrostatic and hydrophobic interactions, with two conserved sites (AnkB: DAR976, A1000 and AnkG: DAR999, A1024) having been identified (Mohler, Yoon, and Bennett 2004; Kizhatil et al. 2007).

The DD is part of a superfamily of domains that are known to mediate cellular functions including (Weber and Vincenz 2001). While very little is known about the function of the ankyrin DD, a yeast 2-hybrid (Y2H) screen of AnkG’s DD identified Fas and several TNF receptor family members as potential interacting partners (Del Rio et al.

2004). They further identified the site of interaction with Fas on AnkG190 (R1496), and showed that over-expression of AnkG DD promoted Fas-mediated apoptosis in MDCK cells

(Del Rio et al. 2004).

The CTD domain is highly divergent among ankyrin family members. For comparison, the MBD, SBD, and DDs of AnkG and AnkB are 74%, 68%, and 59% homologous, respectively, while their CTD domains have less than 11% amino acid sequence identity (Mohler, Gramolini, and Bennett 2002a). Circular dichroism of the AnkB CTD domain, suggests that it is a mostly flexible unstructured domain, and scanning mutagenesis and Y2H experiments identified intra-molecular interacting residues (Abdi et al. 2006).

These findings advocate for the regulatory role of the CTD, with the C-terminus forming inter-domain interactions with the MBD, which prevents MBD-integral protein interactions.

Further, studies of AnkR and AnkB have shown that fragments of the protein without their respective CTDs have a higher affinity for their binding partners compared to their full length counterparts (L. H. Davis, Davis, and Bennett 1992; Mohler, Gramolini, and Bennett 2002a).

7

In addition to restricting the interactions of the MBD, the CTD also targets ankyrin to specific regions with a cell (Chen et al. 2017).

Ankyrin Diversity: genes and alternatively spliced isoforms Three genes encode ankyrin proteins: ANK1 on 8p11 encodes ankyrin-R

(AnkR), ANK2 on chromosome 4q25-27 encodes ankyrin-B (AnkB), and ANK3 on 10q21 encodes ankyrin-G (AnkG) (V Bennett and Baines 2001). Alternative splicing of these three genes results in variants that contain specialized domains, and/or lack certain aspects of the above mentioned domains (Fig. 3) (van Oort et al. 2008). Thus, alternative splicing allows ankyrin isoforms to further diversify their binding partners and differentially localize to sub- cellular domains (Mohler, Gramolini, and Bennett 2002b).

ANK1 contains 42 that are alternatively spliced to encode various AnkR isoforms that are expressed in erythrocytes, , and striated muscle (Cunha and Mohler

2006; V Bennett and Stenbuck 1979; Vann Bennett and Lambert 1999). Two of these splice variants are 215 kDa and 186 kDa; the shorter isoform lacks a portion of the CTD, resulting in a variant with a higher binding affinity for the anion exchanger, spectrin, and (L.

H. Davis, Davis, and Bennett 1992). AnkR is also spliced into small ankyrin isoforms

(sAnk1.5, 1.6, 1.7, 1.8, and 1.9), which are the most divergent variants. sAnk1.5 integrates into the SR via a unique 21 amino acid N-terminal transmembrane domain (Porter et al.,

2015.). While it lacks the MBD and SBD, its CTD shares homology with other ankyrin proteins, such as AnkB212 (Hopitzan, Baines, and Kordeli 2006). sAnk1.5 can be localized to the M-line and to a lesser extent, at the Z-line (Porter et al. n.d.). Mutations in ANK1 are associated with hereditary (Pekrun et al. 1993), type-2

8

Figure 3: Diversity of ankyrin gene products. Figure shows domain organization, molecular weights, and tissue-specific distribution of various ankyrin isoforms. Reproduced with permission from S.R. Cunha & P.J. Mohler, ―Cardiac ankyrins: Essential components for development and maintenance of excitable membrane domains in heart,‖ (2006). License number: 4633980656875.

diabetes (T2D) (Yan et al. 2016), Alzheimer’s Disease (AD) (Higham et al. 2019), and several cancers (Hall et al. 2016).

ANK2 consists of 53 exons which are also alternatively spliced to create numerous

AnkB isoforms found in the brain, heart, , pancreas, lung, liver, and kidney (V

Bennett and Baines 2001). In fact, it gets its name, AnkB, having been characterized first in

9 the brain and also for being broadly expressed in most cell types. The largest AnkB isoform is 440 kDa and plays an important role in axogenesis in the developing brain (Kunimoto

1995). This isoform is identical to the 220 kDa, the most commonly expressed AnkB variant, except for the addition of a giant 220 kDa unstructured, brain-specific insert between the

SBD and DD (Otto et al. 1991). It was taken for granted that the heart only expressed the 220 kDa AnkB isoform; however, our lab has identified several other AnkB isoforms in the heart and further characterized two of them, AnkB188 and AnkB212 (named after their molecular weights) (Wu et al. 2015). We found that while AnkB188 is crucial for the expression and retention of the NCX at the Z-line, AnkB212 is restricted to the M-line via an interaction with obscurin (Wu et al. 2015). Mutations in ANK2 are linked to several arrhythmias such as atrial flutter, atrial fibrillation, ventricular fibrillation, sinus node dysfunction, long QT syndrome (LQTS), and ventricular tachycardia (Swayne et al. 2017; Mohler et al. 2007;

Musa et al. 2016; Cunha et al. 2011). Recently mutations in ANK2 have also been associated with both dilated cardiomyopathy (DCM) (Swayne et al. 2017) as well as hypertrophic cardiomyopathy (HCM) (Hubank et al. 2014).

Finally, ANK3 has 52 exons that account for various AnkG isoforms expressed in the brain, skeletal and , kidney and epithelial tissue (V Bennett and Baines 2001).

In the brain, two giant AnkG isoforms, AnkG270 and AnkG480, contain a serine/threonine rich, gycosylated insert that targets these isoforms to the initial segment and prevents them from diffusing to other regions of the (Zhang and Bennett 1998). The canonical

AnkG isoform AnkG194 is required for the targeting of ion channels in the heart and brain, and crucial for lateral membrane biogenesis in epithelial cells (Zhou et al. 1998; Kizhatil et al. 2007; Mohler et al. 2004). Numerous shorter AnkG isoforms have also been identified in

10 striated muscle (Yamankurt et al. 2015; Gagelin et al. 2002). AnkG107 and AnkG130 are muscle-specific isoforms that lack the MBD and have a 76 amino acid insertion in its CTD

(Gagelin et al. 2002). They contain an obscurin/titin binding domain (OTBD) and share CTD homology with ankyrin-R and ankyrin-B isoforms (Hopitzan, Baines, and Kordeli 2006).

AnkG100 and AnkG120 are two isoforms, also missing their MBD, and found in macrophages (Hoock, Peters, and Lux 1997). And AnkG119, containing 13 of 24 ANK repeats of the MBD, is found in the endoplasmic reticulum (ER) and Golgi (Devarajan et al.

1996). Mutations in ANK3 result in Brugada syndrome (Mohler et al. 2004),

(Zhu et al. 2017), epilepsy (Zhu et al. 2017), autism spectrum disorder (Kloth et al. 2017), and schizophrenia (Guo et al. 2016).

Ankyrins in the heart

Cardiomyocytes are specialized to respond to electrical activity by contracting through a phenomenon known as excitation-contraction coupling. Each heart beat is the result of an that travels down the t-tubule, depolarizes voltage-gated calcium channels, known as the dihydropyridine receptor (DHPR), and results in a calcium-induced calcium release from the ryanodine receptor (RyR) in the SR. Calcium release from the SR produces crossbridge formations between the thick and thin filaments, and causes the contraction of the cardiomyocyte. Additionally, cell-cell adhesions and electrical coupling of adjacent cells enable coordinated propagation of contraction across the ventricle to effectively eject blood. Ankyrin proteins play a pivotal role in targeting, creating and maintaining various specialized domains responsible for allowing the heart to function in this manner (Fig. 4).

11

Figure 4: Ankyrin mediated domain organization in cardiomyocytes. (A) Ankyrin-B recruits dystrophin and β-dystroglycan (DG) to the sarcolemma, and the retention of dystrophin and DG at the costamere is due to interactions with ankyrin-G. (B) Ankyrin-G interacts with components of the gap junction, desmosomal complex, and NaV1.5 at the Intercalated disc (ICD). (C) Ankyrin-B targets and retains the sodium/calcium exchanger (NCX), sodium/potassium pump (NKA), and inositol triphosphate receptor (IP3R) at T- tubules. Reproduced with permission from Yamankurt et al., ―Ankyrin-Based Trafficking and Scaffolding of Membrane Proteins: Implications for Plasma Membrane Stability, Formation, and Specialization,‖ (2013). Modified with permission under CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

12

The heart expresses various ankyrin isoforms of all three genes including sAnk1 isoforms, AnkR210, AnkB160, AnkB188, AnkB212, AnkB220, AnkG107, and AnkG190, among many other splice variants (Cunha & Mohler, 2006; Porter et al., 2015.; Yamankurt,

Nguyen, Mohler, & Cunha, 2013). While the functional role of many cardiac ankyrins is still under investigation and not entirely understood, many studies over the past decade have shed light on the various ankyrin mediated domains in the heart (Cunha and Mohler 2006).

Transverse-tubules (t-tubules) and junctional sarcoplasmic reticulum (jSR): The t- tubules are invaginations of plasma membrane, juxtaposed to the junctional sarcoplasmic reticulum, deep into the myocyte to facilitate the rapid propagation of an action potential.

The expression and regulation of several ion channels and transporters in this region is governed in part by ankyrins. Ankyrin-B binds NCX, NKA, and IP3R in this region, and has been implicated in maintaining the stability of both NCX and NKA (Cunha, Bhasin, and

Mohler 2007; Mohler, Davis, and Bennett 2005). Functionally coupling these channels and transporters are important in maintaining normal cytosolic and SR calcium levels. AnkB haploinsufficiency resulted in elevated SR calcium stores, and increased SR calcium leak, mechanisms that are speculated to contribute to arrhythmias (Camors et al. 2012). A decrease in AnkB leads to reduced NCX expression, and resulting in an elevated load on sarco/endoplasmic reticulum calcium ATPase (SERCA) for cytosolic calcium removal.

Further, Tuvia et al., showed that AnkB null cardiomyocytes displayed mislocalization of

SERCA2. Mislocalization of SERCA and NCX could cause an accumulation of Ca2+ in this region, leading to the activation of CaMKII, hyperphosphorylation of RyR, and release of

Ca2+ sparks: another proarrhythmogenic mechanism (Popescu et al. 2016). Taken together,

13 the t-tubule not only allows for RyR to be activated to release Ca2+, but also allows for proper removal of Ca2+ from the cytosol during diastole.

Longitudinal sarcoplasmic reticulum (lSR): As alluded to earlier, the SR is organized into at least two distinct functional domains, the jSR and the lSR. While the jSR are beside the t-tubules, the lSR is a system or network of tubules that wrap around the sarcomere, predominantly at the M-line and Z-line, and then connect to the jSR. This domain also contains SERCA, which functions in Ca2+ re-uptake during ventricular relaxation. Therefore, maintenance of the lSR is crucial for the uniform re-uptake of Ca2+, and loss of lSR results in loss of SR resident proteins such as sAnk1.5 at the M-line, and functionally results in prolonged twitch times (Giacomello et al. 2015). An interaction between sAnk1.5 and obscurin is necessary for the maintenance of lSR across the sarcomere as knock-out (KO) mouse models of either protein showed reduced lSR (Giacomello et al. 2015; Lange et al.

2009). The C-terminal cytoplasmic domain of sAnk1.5 binds to the CTD of obscurin at the

M-line (Giacomello et al. 2015), and to the amino-terminal domain (NTD) of titin at the Z- line (Kontrogianni-Konstantopoulos and Bloch 2003). While obscurin KO animals displayed altered lSR, and decreased expression of sAnk1.5 (Lange et al. 2009), sAnk1.5 KO animals displayed altered distribution of lSR, and older mice developed tubular aggregates accompanied by structural damage to the contractile apparatus (Giacomello et al. 2015).

These changes resulted in significant functional impairment and a reduction in ability to develop force. Further, knockdown of sAnk1.5 with siRNA also resulted in reduced and swollen lSR, destabilization of SERCA and sarcolipin (SLN) protein, and reduced release and uptake of Ca2+ from the SR (Maegen A Ackermann et al. 2011). Subsequent studies also showed the sAnk1.5 directly interacts with both SLN (Desmond et al. 2017) and SERCA

14

(Desmond et al. 2015) via its transmembrane (TM) domain, and regulates Ca2+ homeostasis.

Another interaction that maintains the lSR around the contractile apparatus is that of sAnk1.5 and tropomodulin 3 (Tmod3) (Gokhin and Fowler 2011). Loss of Tmod3 at the M-line due to loss of (Tmod1) resulted in mislocalization of sAnk1.5, reduced lSR retention, and defective Ca2+ release (Gokhin and Fowler 2011). Consequently, strong anchoring mechanisms that can withstand the stresses associated every contractile cycle are required to maintain the lSR in place. However, how this is accomplished by a small integral protein was not well understood. Previous work from our lab showed that sAnk1.5 forms dimers and oligomers. We speculate that these oligomers transverse the length of the lSR, interact with obscurin and anchor the lSR around the contractile apparatus. We also identified two sites in sAnk1.5 CTD that mediate binding but hypothesize that there may be additional sites that facilitate oligomer formation. By in large, the lSR is necessary for the uniform release and re-uptake of Ca2+ during relaxation and also for interactions between integral SR proteins and proteins of the contractile apparatus.

Intercalated discs (ICD): Adjoining cardiomyocytes are connected by intercalated discs, and these specialized domains allow for mechanical and electrical coupling of the cells. AnkG interacts with the desmosomal protein -2 gap junction protein connexin 43, and recruits NaV1.5 to this region as well; linking these proteins together into a functional domain for controlling excitability, molecular adhesion, and intercellular communication (Sato et al. 2011; Makara et al. 2014). Reduction in AnkG expression leads to reduced plakophilin-2 localization and diminished intercellular adherence (Sato et al.

2011). Conversely, knockdown of plakophilin-2 also decreases localization of AnkG at the

ICD; along with this decrease of AnkG is a loss of Nav1.5 targeting to the ICD (Makara et al.

15

2014). Loss of AnkG also resulted in reduced ICD localization of connexin 43 and consequently decreased junctional conductance (Sato et al. 2011). Finally, AnkG enriches for

Nav1.5 at the ICD and allows action potential propagation between the cells (Sato et al.

2011). As a result, this domain is vital in ensuring that myocardium contracts in sync.

Costameres: Costameres are submembranous protein complexes that facilitate the lateral transmission of contractile force to the sarcolemma, surrounding extracellular matrix

(ECM), and adjoining myocytes (Yamankurt et al. 2013). They overlie the Z-lines, and facilitate through the actions of proteins such as , , α-actinin, and β1 (Peter et al. 2011). Also at the costamere, the dystrophin- glycoprotein complex (DGC) connects the myocyte cytoskeleton through the sarcolemma to the surrounding extracellular matrix thereby providing structural integrity for the sarcolemmal membrane. While dystrophin binds to both, AnkB and AnkG, β-dystroglycan binds exclusively to AnkG (Ayalon et al. 2008). AnkB deficiency results in decreased sarcolemmal localization of dystrophin and decreased costamere-associated ; physiologically resulting in exercise induced muscular damage (Ayalon et al. 2010). Further, studies have shown that while AnkB is important for the targeting of the DGC complex,

AnkG plays an important role in its retention at the costamere (Ayalon et al. 2008).

16

Obscurin

Obscurins are a family of sarcomeric structural and signaling polypeptides encoded by the gene OBSCN on chromosome 1q42.13, spanning over 150 kb (Young, Ehler, and

Gautel 2001). Alternative splicing of its 119 exons encode various giant (800 kDa) and smaller (40-500 kDa) isoforms (Manring, Carter, and Ackermann 2017). They were discovered relatively recently, in 2001 and named for its initial difficulties in detection and characterization due to its massive size (Young, Ehler, and Gautel 2001). Two giant obscurin isoforms, obscurin-A and obscurin-B are most well characterized and expressed in striated muscle; however, recent evidence has suggested that smaller obscurin variants are found in a variety of cell types (Maegen A. Ackermann et al. 2014). Several mutations in OBSCN have been linked with the development of hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), left ventricular noncompaction cardiomyopathy (LVNC), heart failure, and skeletal myopathies (Marston et al. 2015; Arimura et al. 2007; Rowland et al.

2016).

The prototypical obscurin, obscurin A, is about 720 kDa and composed of several immunoglobulin (Ig) repeats punctuated by three fibronectin type-III (FNIII) domains, a calmodulin-binding IQ motif, a Src homology-3 domain (SH3), a Rho-guanine nucleotide exchange factor (RhoGEF) domain, a pleckstrin homology (PH) domain, two more consecutive Ig repeats, and a nonmodular CTD that contains two ankyrin binding motifs and motifs for extracellular signal regulated kinase (ERK) phosphorylation (Fig. 5) (Manring,

Carter, and Ackermann 2017). The second giant isoform, obscurin-B, differs only in its CTD, where the non-modular CTD is replaced by two sets of Ig domains and/or FNIII domains

17 followed by Ser/Thr kinase domains, known as SK1 and SK2 (Manring, Carter, and

Ackermann 2017).

Figure 5: Domain organization of obscurin isoforms. Figure shows interaction sites of various binding partners of obscurin isoforms. Domains of obscurin illustrated in the key provided. Reproduced with permission from Perry et al., ―Obscurins: Unassuming giants enter the spotlight,‖ (2013). License number: 4633990257467.

Differential localization and a plethora of binding partners impart obscurin with its diverse functionality and ability to couple spatially distinct domains. At the M-line, obscurin interacts with titin and myomesin (Kontrogianni-Konstantopoulos et al. 2004), ankyrin-B

(Cunha and Mohler 2008), Ras homolog gene family member A (RhoA) (Ford-Speelman et al. 2009), calmodulin (Carlsson, Yu, and Thornell 2008), and slow skeletal myosin binding protein C variant 1 (sMyBP-Cv1) (M. A. Ackermann et al. 2009). At the Z-line, it interacts with the NTD of titin (Young, Ehler, and Gautel 2001) and Ran-binding protein 9 (RanBP9)

(Bowman et al. 2008). Additionally, obscurin associates with sAnk1.5 at the SR (Lange et al.

2009), maintains AnkB at costameres (Randazzo et al. 2017), and with N-cadherin and NKA at the intercalated discs (Hu and Kontrogianni-Konstantopoulos 2013). Obscurin serves in both structural and signaling capacities at the M-line. Structurally, it is involved in the

18 formation and maintenance of the sarcomere by stabilizing, and providing proper spatial positioning to other myofibrillar proteins (Borisov et al. 2006). Knockdown of obscurin in adult rat ventricular cardiomyocytes using siRNA resulted in aberrant myosin and myomesin distribution, poorly formed sacromeric M-band and A-band structures, and loss of rigidity of the (Borisov et al. 2006). While the structural roles of obscurin have been explored extensively, its signaling capabilities are less familiar. An antibody to the RhoGEF domain of obscurin revealed that it localized to the M-line along with RhoA, and overexpression of the RhoGEF domain caused an increase in GTP bound RhoA and its downstream target, Rho associated coiled-coil containing kinase 1, ROCK1 (Ford-Speelman et al. 2009). Considering this signaling cascade is activated during proliferation, it possible that obscurin plays a role in injury repair by replacing injured cells with newly proliferated cells. Unchecked however, this could lead to hypertrophy, as studies have shown an upregulation in both RhoA and ROCK1 activity in cardiac hypertrophy (Molkentin and Dorn

2001). Additionally, obscurin also interacts with the CTD of AnkB to target protein phosphatase 2A (PP2A) to the M-line (Cunha and Mohler 2008). By overexpressing a dominant-negative AnkB, Cunha and Mohler, showed reduced M-line targeting of both endogenous AnkB as well PP2A, a phosphatase known to modulate Ca2+ signaling in cardiomyocytes.

While down regulation of obscurin significantly disrupted M-line formation and associated proteins, it did not affect Z-line formation or its associated proteins such as α- actinin (Borisov et al. 2004). In fact, obscurin was only observed at the Z-line at the end of sarcomeric assembly, where it interacted with titin via its CTD (Borisov et al. 2004). It is well established that obscurin localizes to the Z-line as antibodies to different regions of

19 obscurin (NTD, CTD, and SK2) map it to the Z-line, but further functions of obscurin at the

Z-line are still unknown.

As discussed previously, the interaction between sAnk1.5 and obscurin maintains the lSR around the contractile apparatus. Skeletal muscle from mice lacking obscurin displayed disruption of lSR and degradation of sAnk1.5 via the potassium channel tetramerization domain containing 6 (KCTD6)/ cullin-3 ubiquitin complex (Lange et al. 2009, 2012).

Therefore, obscurin not only provides structural support for the lSR, but also mediates Ca2+ homeostasis by stablizing sAnk1.5, which modulates SERCA mediated Ca2+ sequestration.

The importance of AnkB recruiting the DGC complex to the costamere has already been mentioned, and obscurin directs AnkB to the sarcolemma to connect the sarcomere to the sarcolemma (Randazzo et al. 2017). Skeletal muscle from mice lacking obscurin showed mislocalized AnkB and dystrophin, and altered costamere- arrangement

(Randazzo et al. 2017). Further, these mice exhibited increased muscle damage following exercise as a result of decreased sarcolemmal integrity (Randazzo et al. 2017).

Small obscurin isoforms with the kinase domains localize to the intercalated discs of cardiomyocytes, where SK-1 interacts with NKA and SK-2 interacts with N-cadherin (Hu and Kontrogianni-Konstantopoulos 2013). While the functional implications of these interactions are not fully understood, obscurin may modulate cell-cell adhesion strength with its ability to phosphorylate N-cadherin.

20

Significance and scope of this thesis

In summary, the organization of various receptors, ion channels, transporters, signaling molecules, cell adhesion molecules, and contractile proteins into distinct functional domains is paramount to normal cardiac function. Ankyrins and obscurins enable the formation of these functional domains through their intricate isoform-specific modularity and localization. Cardiac dysfunction in the form of arrhythmias and myopathy have been linked to mutations in both proteins (Vann Bennett and Healy 2008; Makara et al. 2014; Mohler et al. 2007; Arimura et al. 2007; Marston et al. 2015; Rowland et al. 2016).

As previously described, obscurin contains two ankyrin binding domains in its C- terminus, and sAnk1.5 has been shown to interact with both of them (Giacomello et al.

2015). Studies have also shown that AnkB and other ankyrin isoforms that contain the OTBD could also associate with obscurin (Hopitzan, Baines, and Kordeli 2006). Further, each of these ankyrin-obscurin interactions elicited different functions based on their differential subcellular localization. For example, obscurin-sAnk1.5 maintains the lSR around the contractile apparatus, and obscurin-AnkB allows targeting of dystrophin at the sarcolemma and PP2A at the M-line. Therefore, studying obscurin mediated targeting of ankyrin isoforms, and the functional relevance of those interactions can help us better understand both normal as well as pathological cardiac function.

We hypothesize that obscurin can interact with two different ankyrin isoforms in concert to couple functionally distinct ankyrin mediated protein complexes. The goal of my thesis was therefore to assess if obscurin could target and interact with two different ankyrin isoforms simultaneously, serving to bridge their individual complexes. Specifically, we used

21 various biochemical and imaging techniques to investigate whether obscurin could couple sAnk1.5 and AnkG107/130 together.

22

Chapter 2

Materials and Methods

23

Plasmids and antibodies

Figure 6 contains a diagram of all constructs used in experiments for this paper.

Human sAnk1.5 (AF005213, amino acids 29-155) was subcloned in frame with various carboxyl-terminal epitope tags including FLAG, HA, CFP, YFP, and GFP. Human obscurin

(NM_052843, amino acids 6148-6460) was subcloned in frame with an amino-terminal GST epitope tag or with various carboxyl-terminal epitope tags including HA, CFP, and GST. To generate ankyrin-binding domain mutants of obscurin (ΔABD1, ΔABD2, ΔABD1&2), PCR- based site-directed mutagenesis (confirmed by sequencing) was performed to substitute acidic residues with uncharged residues. AnkG107/130 (AJ428573, full-length and amino acids 656-959) was isolated by reverse-transcriptase PCR from rat ventricular mRNA and subcloned in frame with an amino-terminal GST epitope tag or with various carboxyl-terminal epitope tags including FLAG, HA, GST, YFP, GFP, and RFP. Sarcolipin

(Q6SLE7.1) was isolated by reverse-transcriptase PCR from rat ventricular mRNA and subcloned in frame with a carboxyl-terminal fusion of CFP and YFP (separated by 6 serine and glycine residues). Plectin (NM_011117, amino acids 3386-3583) and filamin-C

(NM_001081185, amino acids 669-860) were isolated by reverse-transcriptase PCR from mouse ventricular mRNA. Plectin was subcloned in frame with an amino-terminal His(X6) epitope tag. β1-spectrin (J05500.1, amino acids 1605-2129) was isolated by reverse- transcriptase PCR from human ventricular mRNA and subcloned in frame with a carboxyl- terminal YFP epitope tag.

24

Figure 6: Diagrams of obscurin, sAnk1.5, and AnkG107/130. Representations of full- length constructs and various epitope-tagged, truncated constructs used in different experiments for obscurin (A), sAnk1.5 (B), and AnkG107/130 (C). In the truncated constructs, beginning and terminal amino acids are labelled. Highlighted domains include immunoglobulin (IG), fibronectin type 3 (FN3), calmodulin-binding motif (IQ), Src homology 3 (SH3), guanine nucleotide exchange factor for Rho/Rac/Cdc42-like GTPases (RhoGEF), pleckstrin homology (PH), ankyrin-binding domains (ABD), transmembrane domain (TMD), obscurin-binding domains (OBD), zona occluden 1 and unc5-like netrin receptors (ZU5), and death domain (DD).

25

Primary antibodies used in this study include HA (26183, Thermo Scientific), FLAG

(2368, Cell Signaling Technologies), GFP (B-2, sc-9996, Santa Cruz) or (sc-8334, Santa

Cruz), RFP (AB3528, Chemico International), GST (B-14, sc138, Santa Cruz), α-actinin

(A7811, Sigma), and myomesin (Developmental Studies Hybridoma Bank, University of

Iowa). For confocal microscopy, secondary antibodies used were goat anti-rabbit conjugated to Alexa Fluor 488, goat anti-chicken conjugated to Alexa Fluor 568, and goat anti-mouse conjugated to Alexa Fluor 568 or 633 (1:500, Life Technologies).

RNA isolation, reverse transcription, and PCR amplification of AnkG107/130 mRNA transcripts

RNA was isolated from mouse with GenEluteMammalian RNA kit (Sigma

Aldrich). cDNA was synthesized using SuperScript III (Life Technologies). AnkG107/130 mRNA transcripts were amplified using primers that flanked the obscurin-binding domains

(5‘: GATATCAGGATGGCGATAGTAG, 3‘: GATTTCCTTCTTCGTCTTCACC) using

Phusion polymerase (Finnzymes). PCR products were purified, ligated into pCR2.1-TOPO vector (Life Technologies), and sequenced.

In vitro binding assays

For obscurin-binding assays, bacterially-expressed and purified GST-obscurin-CTD fusion proteins were incubated with increasing amounts of cell lysates containing over- expressed sAnk1.5-FLAG or AnkG130-FLAG in binding buffer (50 mM Tris pH 7.4, 1 mM

EDTA, 1 mM EGTA, 150 mM NaCl, 0.1% Triton X-100) overnight at 4°C. Protein complexes were pelleted with Glutathione-Sepharose beads (GE Healthcare Life Sciences),

26 washed in binding buffer supplemented with 500 mM NaCl and 1% Triton X-100 (wash buffer) three times, and resolved on a 4-12% Bis-Tris protein gel (Invitrogen). Obscurin precipitation of sAnk1.5 and/or AnkG107/130 was visualized by chemiluminescent detection of the FLAG epitope (ProSignal Femto, Genesee Scientific).

For AnkG107-binding assays, bacterially-expressed and purified GST-AnkG107 fusion proteins were incubated in binding buffer with in vitro translated, S35-labelled obscurin, plectin-1, or filamin-C (TnT T7 Coupled Reticulate Lysate System, Promega). As described above, protein complexes were pelleted, washed three times, and resolved by SDS- polyacrylamide gel electrophoresis. S35-labelled proteins precipitated with AnkG107 were measured with a phosphorimager and signal intensities are represented as relative light units

(RLU). Experiments were repeated in triplicate and statistical analysis was performed as described below. For obscurin and plectin binding assays, His-obscurin and His-plectin were bacterially-expressed and purified over a nickel column. They were then incubated with cell lysate containing over-expressed GFP alone or AnkG130-GFP in binding buffer (50 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.1% Triton X-100) overnight at 4°C.

Protein complexes were pelleted, washed in binding buffer supplemented with 500 mM NaCl and 1% Triton X-100 (wash buffer) three times, and resolved on a 4-12% Bis-Tris protein gel

(Invitrogen). Precipitation of AnkG130-GFP was visualized by chemiluminescent detection of the GFP epitope (ProSignal Femto, Genesee Scientific).

Co-precipitation assays

HEK293TN cells (System Biosciences) were transfected with DNA plasmids using

JetPRIME transfection reagent (Polyplus Transfection). After 24 hours, transfected cells

27 were subject to three freeze-thaw cycles in lysis buffer (5 mM HEPES pH 8.0, 0.5 mM

EDTA, 0.1 mM MgCl2, 1 mM Dithiothreitol, 150 mM NaCl, 1% Triton X-100, 1 mM

PMSF, 1X inhibitor cocktail) followed by a 20-min incubation at 4°C on a nutator.

Protease inhibitor cocktail (P2714, Sigma-Aldrich) contains 2 mM AEBSF, 0.3 μM

Aprotinin, 116 μM Bestatin, 14 μM E-64, 1 μM Leupeptin, and 1 mM EDTA. Lysates were then centrifuged at 13,000 g for 10 mins at 4°C, after which the supernatants were collected.

While 5% supernatant was retained for input, the remaining supernatant was incubated with

Glutathione-Sepharose beads for 1 hour at 4°C. As described above, protein complexes were pelleted, washed three times in wash buffer (binding buffer with 1% Triton X-100 and 500 mM to 1M NaCl). Pelleted protein complexes were separated by SDS-PAGE and visualized by chemiluminescence using antibodies to FLAG (1:1000), GFP (1:1000), GST (1:500), and

HA (1:500). Quantification of signal intensity was performed using NIH ImageJ software.

For quantification of ankyrin-binding to obscurin, signal intensity measurements of precipitated proteins were normalized to their corresponding inputs, then ankyrin measurements were normalized to obscurin measurements. The results are presented as % binding obscurin with AnkG107/130 and sAnk1.5 binding to wild-type obscurin set to 100%.

Calculations of AnkG107 precipitation of obscurin-HA and sAnk1.5-HA were performed in a similar manner except the measurements for obscurin-HA and sAnk1.5-HA were normalized to AnkG107-GST. The results are presented as % binding AnkG107 with wild- type obscurin binding to AnkG107-GST and sAnk1.5-HA set to 100%. Experiments were performed in triplicate.

28

FLIM-FRET analysis

Fluorescent Lifetime Imaging Microscopy-Fluorescence Resonance Energy Transfer

(FLIM-FRET) experiments were performed with a lifetime fluorescence imaging attachment

(Lambert Instruments, Leutingewolde, The Netherlands) on an inverted microscope (Nikon).

HEK293TN cells were plated on coverslips and transfected with CFP(donor)/YFP(acceptor) in a 1:2 ratio and/or CFP(donor)/YFP(acceptor)/HA(bridging) tagged constructs at a 1:2:1 ratio. After 24 hours, transfected cells were washed in 1X PBS, fixed in 2% paraformaldehyde, quenched in 50 mM NH4Cl for 10 mins, and washed in 1X PBS again.

Coverslips were mounted on slides in 10% Mowiol mounting media (Sigma-Aldrich) and dried at room temperature for 24 hours. Transfected cells were excited by using a sinusoidally modulated 3-watt 448-nm light-emitting diode at 40 MHz under epi- illumination. Fluorescein at the concentration of 1 μM was used as a lifetime reference standard (4 ns). Cells were imaged with a 40X oil-immersion objective (numerical aperture

1.3) using a CFP filter set. The phase and modulation lifetimes were determined from 12 phase settings by using the manufacturer's software (LIFA). The analysis yields the CFP lifetime of free CFP in donor only (τ1) and the CFP lifetime in donor-acceptor complexes

(τ2). FLIM data were averaged on a per cell basis and three experiments (at least 65 cells in total) were performed for each condition.

Isolation and culture of neonatal rat ventricular cardiomyocytes (NRVMs)

NRVMs were isolated from 1-2 day old Sprague-Dawley rat pups as previously described by Wu et al., and in accordance with the animal protocol approved by the Animal

Welfare Committee at University of Texas Health Science Center at Houston. Briefly,

29 neonatal rat pups were euthanized by decapitation and their hearts were excised, minced, and digested through a series of agitations in buffer containing collagenase type II (Worthington,

Collagenase type II 305U/mg) and pancreatin (Sigma). Cells from each digestion were collected and pooled, and pre-plated on Nunc plates (Thermo Fisher Scientific) to reduce fibroblasts and enrich for cardiomyocytes. Cells were then collected and plated on fibronectin-coated glass-bottom MatTek plates (MatTek Corporation). Media was changed

24-hours after plating and replaced every 2-3 days. After 4 days in culture, cardiomyocytes were transduced with lenti-viral ankyrin constructs (sAnk1.5-GFP, AnkG130-GFP or -RFP).

Fluorescent immunohistochemistry

Cardiomyocytes were washed in phosphate-buffered saline (PBS, pH 7.4) three times, blocked with 5% normal goat serum and 0.075% Triton X-100 for 30 mins at room temperature, and incubated with primary antibodies overnight at 4°C. Primary antibodies used were: α-actinin (1:1000), myomesin (1:500), GFP (sc-8334, 1:500), and RFP (1:500).

Images were obtained with a Nikon A1 confocal microscope (Nikon, Melville, NY) equipped with 40X oil, numerical aperture 1.3 objective.

Statistics

For Western blot analysis, signal intensity was quantified using ImageJ (NIH) and the results are represented as mean ±SEM with statistical significance determined by unpaired

Student‘s t-test (2-way). For FLIM-FRET analysis, the results are represented as mean

±SEM with statistical significance determined by unpaired Student‘s t-test (2-way). P value of <0.001 is represented by ***.

30

Chapter 3

Results

31

Obscurin binds AnkG107/130 and sAnk1.5 in vitro.

Previous studies have demonstrated that the C-terminal domain (CTD) of obscurin has two distinct ankyrin-binding domains and that each domain is sufficient to bind sAnk1.5

(Busby et al. 2010). To determine whether obscurin can interact with two different ankyrins simultaneously, we performed in vitro binding assays with a GST-fusion protein of obscurin-

CTD (expressed and purified from bacteria) and increasing amounts of cell lysate containing transiently-expressed sAnk1.5-FLAG, AnkG130-FLAG, or both constructs together. With this experimental set-up, we could vary the amount of sAnk1.5 or AnkG130 incubated with obscurin to investigate ankyrin competition for obscurin-binding. AnkG130 and AnkG107 are alternative isoforms in which AnkG107 lacks 196 amino acids due to use of an alternative splice acceptor site in Ank3 44. Considering both isoforms have the same obscurin-binding domains in their C-terminus, we used these constructs interchangeably in experiments and will henceforth be referred to as AnkG107/130.

We find that sAnk1.5-FLAG and AnkG107/130-FLAG individually bind to GST- obscurin-CTD (Fig. 7A&B). Specifically, obscurin precipitates more sAnk1.5-FLAG or

AnkG107/130-FLAG as the amount of ankyrin in each reaction increases. The presence of residual unbound-ankyrin suggests that sAnk1.5-FLAG or AnkG107/130-FLAG is in excess of obscurin. We also demonstrate that GST-obscurin-CTD pulls down both sAnk1.5-FLAG and AnkG107/130-FLAG (Fig. 7C). Moreover, AnkG107/130-FLAG does not precipitate with obscurin when sAnk1.5-FLAG is increased 4-fold (20 μls to 80 μls). In contrast, a 5- fold increase in AnkG107/130-FLAG (5 μls to 25 μls) does not prohibit obscurin precipitation of sAnk1.5. These findings suggest that AnkG130 competes with sAnk1.5 for

32 obscurin interaction. Also, demonstrating the presence of residual unbound-ankyrin in each reaction suggests that obscurin is the rate-limiting component.

Figure 7: Obscurin interacts with both sAnk1.5 and AnkG107/130. A bacterially- expressed GST fusion protein of obscurin-CTD incubated with increasing amounts of cell lysates precipitates increasing amounts of sAnk1.5-FLAG (A), AnkG107/130-FLAG (B), or both ankyrins together (C) as demonstrated by immunoblot to the FLAG epitope. Bound represents ankyrin precipitated by obscurin, while unbound represents ankyrin remaining in the cell lysate not bound to obscurin. Experiments performed by Brian Lauman.

33

Obscurin ABD1 and ABD2 regulate AnkG107/130 and sAnk1.5 complex formation.

To determine whether AnkG107/130 or sAnk1.5 preferentially interacts with obscurin

ABD1 or ABD2, we performed ankyrin precipitation assays with wild-type or mutant obscurin. Three mutant obscurin constructs were generated by substituting acidic residues with uncharged glutamine residues in ABD1, ABD2, or both domains (Fig. 8A).

HEK293TN cells were transfected with AnkG107/130-HA, sAnk1.5-HA, and wild-type or mutant obscurin-GST (ΔABD1, ΔABD2, ΔABD1&2). Ankyrin/obscurin protein complexes were precipitated from cell lysates with glutathione sepharose, incubated in a high-stringent wash buffer, and resolved on a protein gel by SDS-PAGE. Each condition was repeated in triplicate and the immunoblot signal was measured using ImageJ. Measurements for precipitated proteins were normalized to their corresponding inputs, then ankyrin measurements were normalized to obscurin measurements. The results are presented as % binding obscurin with AnkG130 and sAnk1.5 binding to wild-type obscurin set to 100%.

Consistent with the previous findings, both AnkG107/130-HA and sAnk1.5-HA precipitate with wild-type obscurin-GST (Fig. 8B&C). Mutating ABD1 in obscurin reduces both AnkG107/130-HA and sAnk1.5-HA binding by 26.9% and 44.6% respectively.

Interestingly, when obscurin ABD2 is mutated only sAnk1.5-HA binding is reduced by

25.7% while AnkG107/130-HA displays enhanced obscurin-binding. When both ABDs are mutated, obscurin-binding to AnkG107/130-HA and sAnk1.5-HA is reduced by over 90%, effectively demonstrating a complete loss of binding. These findings suggest that mutating

ABD1 more significantly impacts obscurin precipitation of ankyrin than mutating ABD2. In addition, obscurin precipitation of AnkG107/130-HA is only diminished by mutating ABD1.

While it’s confounding that obscurin still precipitates both AnkG107/130-HA and sAnk1.5-

34

HA even when one ABD is mutated, these findings may be explained if obscurin expression is greater than ankyrin expression. Specifically, if obscurin is in excess, AnkG107/130-HA and sAnk1.5-HA may be interacting with different populations of obscurin.

Figure 8: Obscurin ABDs mediate interaction with sAnk1.5 and AnkG107/130. (A) Alignment of obscurin amino acid sequences of wild-type (wt) and ankyrin-binding domain mutants (ΔABD1, ΔABD2, ΔABD1&2). (B) HA and GST immunoblots demonstrate expression and precipitation of AnkG107/130-HA and sAnk1.5-HA by wild-type or mutant obscurin-GST constructs. (C) Quantification of AnkG107/130-HA and sAnk1.5-HA precipitation by wild-type or mutant obscurin-GST constructs. Error bars represent standard error of the mean and statistical analysis was performed using unpaired Student’s t-test with *** representing a p-value of 0.005.

35

Obscurin mediates an indirect interaction between AnkG107/130 and sAnk1.5.

To ascertain whether obscurin can link AnkG107/130 to sAnk1.5, we first demonstrated that sAnk1.5 does not directly interact with AnkG107 (Fig. 9A). Specifically,

AnkG107-GST does not precipitate sAnk1.5-HA from cell lysate transiently expressing both ankyrins. In a similar paradigm as described above, we then investigated whether

AnkG107/130-GST precipitates sAnk1.5-HA when co-expressed with either wild-type or mutant obscurin-HA (ΔABD1, ΔABD2, ΔABD1&2). Conditions were repeated in triplicate and immunoblot signal intensity was quantified using ImageJ. After the measurements for precipitated proteins were normalized to inputs, the measurements for obscurin-HA and sAnk1.5-HA were normalized to AnkG107/130-GST. The results are presented as % binding

AnkG107/130 with wild-type obscurin binding to AnkG107/130-GST and sAnk1.5-HA set to 100%.

While AnkG107/130-GST does not directly interact with sAnk1.5-HA, it precipitates sAnk1.5-HA when co-expressed with wild-type obscurin (Fig. 9B&C). These findings demonstrate that AnkG107/130 forms a complex with sAnk1.5 via obscurin. In addition,

AnkG107/130’s interaction with obscurin and sAnk1.5 is more significantly reduced by mutating obscurin’s ABD1 than ABD2. Specifically, mutating ABD1 reduces

AnkG107/130-GST precipitation of obscurin-HA by 62.4% and sAnk1.5-HA by 89.8%. In comparison, mutating ABD2 reduces precipitation of obscurin-HA by 26.8% and sAnk1.5-

HA by 42.2%. Interestingly, the finding that mutating ABD1 most significantly disrupts ankyrin/obscurin interactions is the same irrespective of whether obscurin or AnkG107/130 precipitates the protein complexes. It is also interesting that AnkG107/130-GST precipitates sAnk1.5 even if obscurin’s ABD1 or ABD2 is mutated. Two potential explanations for this

36 finding are (1) AnkG107/130 interaction with obscurin creates another ankyrin-binding domain or (2) this obscurin construct may form a dimer, thus creating two accessible ankyrin-binding domains.

Figure 9: Obscurin regulates AnkG107/130 complex formation with sAnk1.5. (A) HA and GST immunoblots demonstrate AnkG107/130-GST alone does not precipitate sAnk1.5- HA. (B) HA and GST immunoblots demonstrate AnkG107/130-GST precipitates sAnk1.5- HA when co-expressed with wild-type but not mutant (ΔABD1&2) obscurin-HA. (C) Quantification of wild-type or mutant obscurin-HA and sAnk1.5-HA precipitation by AnkG107-GST. Error bars represent standard error of the mean and statistical analysis was performed using unpaired Student’s t-test (*** p < 0.005).

37

Obscurin regulates AnkG107130-sAnk1.5 complex formation in cells.

To validate that obscurin can link AnkG107/130 to sAnk1.5 in cells, we performed

FLIM-FRET. With this technique, the lifetime of the donor fluorophore is reduced by energy transfer between the donor (CFP) and acceptor (YFP) fluorophores when they are within 10 nanometers of each other. For these experiments, various donor and acceptor fluorophores were transiently expressed in HEK293TN cells. Using a Lambert Instruments FLIM

Attachment (LIFA) microscope, fluorescent lifetime per cell was measured and pseudocolored images represent the CFP lifetime, which ranges from 2.6 ns (red) to 2.0 ns

(blue) based on experimental results. The average lifetime (or τav) was calculated from >50 cells from three independent experiments and represented in a bar graph (Fig. 10E).

We generated a positive control for FLIM-FRET by appending CFP and YFP in succession to the small ER sarcolipin. The average CFP lifetime of this construct is 2.16 ns, which due to the energy transfer between the CFP and YFP fluorophores is shorter than the average lifetime of obscurin-CFP alone (2.58 ns) (Fig. 10A&E). In cells expressing obscurin-CFP and sAnk1.5-YFP or AnkG107-YFP, the average CFP lifetimes are reduced to 2.28 and 2.24 ns respectively (Fig. 10B&E). These findings demonstrate molecular interactions between obscurin-CFP and sAnk1.5-YFP or AnkG107-YFP, which is consistent with our biochemical results. In cells expressing sAnk1.5-CFP and AnkG107-

YFP, the average CFP lifetime is 2.51 ns (Fig. 10C&E), which is consistent with our biochemical results that AnkG107 does not directly interact with sAnk1.5. With the addition of wild-type obscurin to cells expressing sAnk1.5-CFP and AnkG107-YFP, the average CFP lifetime decreases to 2.25 ns (Fig. 10C&E), which is in agreement with our biochemical results that demonstrate obscurin connects AnkG107 to sAnk1.5. In contrast, cells

38

Figure 10: FLIM-FRET analysis demonstrating obscurin regulation of ankyrin complex formation. Pseudocolored images showing average CFP lifetime (τav) of (A) obscurin-CFP alone (control: CFP donor) or sarcolipin-CFP-YFP (positive control), (B) obscurin-CFP (donor) with acceptors sAnk1.5-YFP or AnkG107/130-YFP, and (C) sAnk1.5- CFP (donor) with acceptor AnkG107/130-YFP alone or with wild-type (WT) or mutant (ΔABD1&2) obscurin-HA. (D) HA and GFP immunoblots demonstrate similar expression of donor, acceptor, and obscurin constructs between WT and mutant (ΔABD) obscurin transfection conditions. (E) The graph shows mean fluorescence lifetime of CFP ± SEM (n ≥ 65, from three independent experiments). Statistical analysis was performed by unpaired Student’s t-test (*** p < 0.005).

39 expressing sAnk1.5-CFP, AnkG107-YFP, and mutant obscurin (ABDs mutated) display an average CFP lifetime of 2.57 ns, which indicates that sAnk1.5-CFP and AnkG107-YFP are no longer in a complex because they are not interacting with obscurin. To confirm similar expression of the fluorophores and obscurin constructs in the FLIM-FRET conditions, we performed immunoblot analysis (Fig. 10D). We found that wild-type and mutant obscurin constructs were expressed at similar levels (HA immunoblot) and that the ratio of AnkG107-

YFP to sAnk1.5-CFP was the same between conditions (GFP immunoblot). Specifically, cells were transfected with AnkG107-YFP and sAnk1.5-CFP plasmids at a 2:1 ratio, which accounts for greater expression of AnkG107-YFP compared to sAnk1.5-CFP. Taken together, our FLIM-FRET results corroborate our biochemical findings that the two ankyrin- binding domains in obscurin can facilitate AnkG107 and sAnk1.5 complex formation both in- vitro and in cells.

40

Virally-expressed AnkG107/130CTD-RFP and sAnk1.5-GFP localize to the M-lines of ventricular cardiomyocytes.

While sAnk1.5 localizes to the M- and Z-lines of skeletal muscles using an antibody to endogenous sAnk1.5 (Porter et al., 2015), there is no commercially available antibody to

AnkG107/130. Since we could not use an antibody to locate endogenous AnkG107/130, we over-expressed fluorescently-tagged AnkG107/130 lentivirus in neonatal cardiomyocytes and used confocal microscopy to assess its subcellular localization. Unfortunately, the transfection efficiency of full length AnkG107/130 in neonatal cardiomyocytes was extremely poor. The GFP-tagged CTD lentiviral construct of AnkG107/130, however, distinctly co-localized with M-line marker, myomesin, and was interdigitated by the Z-line protein, α-actinin (Fig. 11B). Similarly, virally expressed sAnk1.5-GFP also localized to the

M-line in conjunction with M-line protein myomesin, and was alternated by α-actinin (Fig.

11A). Finally, neonatal cardiomyocytes expressing both ankyrin isoforms, sAnk1.5-GFP and

AnkG107/130CTD-RFP, revealed co-localization of both protein at the M-line, along with myomesin, and flanked on either side by α-actinin (Fig. 11C).

While this data may not paint a full picture as to the subcellular localization of endogenous full-length AnkG107/130, it shows that the CTD of AnkG107/130, containing the OTBD, is recruited to the M-line most probably via its interaction with obscurin. Further, this recruitment is maintained even in the presence of sAnk1.5, which suggests that obscurin is capable of targeting two different ankyrin isoforms to the M-line simultaneously.

41

Figure 11: Virally-expressed sAnk1.5 and AnkG107/130 are targeted to the M-lines of ventricular cardiomyocytes. sAnk1.5-GFP (A) and AnkG107/130-GFP (B) co-localize with the M-line resident protein myomesin but not with the Z-line resident protein α-actinin. (C) Cardiomyocytes expressing both lenti-viral constructs display AnkG107/130-RFP and sAnk1.5-GFP co-localization with myomesin but not α-actinin. Scale bar represents 10 microns. Experiments performed by Brian Lauman.

42

AnkG107/130-CTD binds obscurin, but not plectin-1 or filamin-C.

A previous study has demonstrated by yeast 2-hybrid, in-vitro binding assays, and co- immunoprecipitation that AnkG107 also interacts with plectin-1 and filamin-C via its obscurin-binding domains (Maiweilidan, Klauza, and Kordeli 2011). Specifically, AnkG107 was shown to interact with plectin repeats 12 and 13 [amino acids 3523-3598 in human plectin-1 (Q15149) corresponding to amino acids 3411-3486 in mouse plectin-1

(NM_011117)] and filamin-C [amino acids 697-848 in human filamin-C (Q14315) corresponding to amino acids 698-849 in mouse filamin-C (NM_001081185)] (Maiweilidan,

Klauza, and Kordeli 2011). We have demonstrated that alternative isoforms of the ankyrin-B

CTD have different binding capacities for obscurin(Cunha and Mohler 2008). We hypothesized that the heart expresses different isoforms of AnkG107 and that these isoforms could display different binding capacities for obscurin, plectin-1, and filamin-C.

Using primers that flank the obscurin-binding domains (encoded by Ank3 exons 47,

48, and 49) we amplified seven PCR products from cDNA created from mouse ventricular mRNA (Fig. 12B). The different transcripts are the result of excluding Ank3 exons 46, 46 and 47, 46 - 48, and 5’-truncation of exon 44. As mentioned earlier, an alternative 3’-splice acceptor site in exon 44 causes the 5’-truncation and the in-frame exclusion of 196 amino acids, which is the principal difference between AnkG130 and AnkG107 isoforms. We subcloned the PCR products in frame with an amino-terminal GST tag and then bacterially expressed and purified these constructs for in-vitro binding assays (Fig. 12B). In-vitro translated and S35-labelled obscurin CTD (amino acids 6148-6460) was incubated with the seven GST-AnkG107/130-CTD proteins and binding was measured with a phosphorimager

(Fig. 12C). Loss of exon 47, which encodes the first obscurin-binding domain, completely

43

OTBD1 OTBD2

Figure 12: AnkG107/130 preferentially interacts with obscurin via OTBD1. (A) AnkG107 amino acids Glu892-Lys909 and Asp928-Arg945 comprise obscurin/titin-binding- related domain 1 (OTBD1) and 2 (OTBD2) respectively. (B) Coomassie Blue stain of bacterially-expressed GST fusion proteins of alternative AnkG107/130 transcripts. (C) Binding assay with in vitro translated S35-labelled obscurin (Leu6148-Asn6460) and GST- AnkG107/130 fusion proteins. Obscurin-binding was measured from three independent experiments and represented as relative light units (RLU) with error bars signifying standard error of the mean. (D) Binding assay with in vitro translated S35-labelled plectin (Tyr3386- Tyr3583) and GST-AnkG107/130 fusion proteins. Plectin-binding was measured from three independent experiments and represented as relative light units (RLU) with error bars signifying standard error of the mean. (E) Bacterially-expressed and purified obscurin (6210-6380) incubated with cell lysate precipitates AnkG107/130-GFP, but not GFP alone (GFP immunoblot, lower panel). In contrast, bacterially-expressed and purified plectin (Tyr3386-Tyr3583) does not precipitate AnkG107/130-GFP. Experiments performed by Gokay Yamankurt.

44 disrupts obscurin-binding (compare binding between AnkG107 constructs 5 and 6). We performed similar in vitro binding assays with plectin-1 (amino acids 3386-3583) and filamin-C (amino acids 669-860). While filamin-C did not bind to any of the GST-

AnkG107/130-CTD proteins (Fig. 13), plectin-1 only interacted with AnkG107 construct 4 albeit at low levels (Fig. 12D).

Considering the minimal interaction between AnkG107/130 and plectin-1, we reassessed this interaction when AnkG107/130 was precipitated by obscurin or plectin-1.

Specifically, His-tagged obscurin or plectin-1 domains containing the ankyrin-binding sites were bacterially-expressed and purified on nickel columns. The purified proteins were then incubated with cell lysates containing transiently expressed AnkG107/130-GFP or GFP alone. Precipitated complexes were washed in 500 mM NaCl, resolved by SDS-PAGE, and visualized by immunoblot detection of the GFP epitope. While GFP alone does not interact with obscurin or plectin-1, AnkG107/130-GFP only interacts with obscurin (Fig. 12E).

These findings suggest that AnkG107/130-CTD preferentially interacts with obscurin and not plectin-1 or filamin-C.

45

Figure 13: AnkG107/130 does not interact with filamin-C via OTBD1. Binding assay with in-vitro translated S35-labelled filamin-C (669-860) and GST-AnkG107/130 fusion proteins. No filamin-C binding was detected. Experiment performed by Gokay Yamankurt.

46

β1-spectrin associates with the obscurin/ankyrin complex via AnkG107/130.

Since AnkG107/130 did not associate with plectin-1 or filamen-C, we utilized BioID, a proximity dependent labeling technique (Trinkle-Mulcahy 2019), to identify physiologically relevant binding partners for AnkG107/130. Using PCR-based strategies, we subcloned the BioID tag in frame with three HA epitopes and then appended the

BioID/HA epitope to the C-terminus of full-length AnkG107/130, which was subsequently made into lenti-virus and its expression was confirmed by immunoblot detection of the HA- tag. Cultured rat neonatal ventricular cardiomyocytes were incubated with lenti-virus for 48 hours, of which the media was supplemented with 50 μM biotin for the last 18 hours.

Cardiomyocyte lysate was incubated with streptavidin-beads; then biotinylated proteins were pelleted, washed, resolved on a protein gel, and excised for mass spectrometry analysis.

Using this technique, we identified β1-spectrin as a potential protein that interacts with

AnkG107/130, which was not unexpected considering AnkG107/130 contains a SBD

(Gagelin et al. 2002).

To corroborate the interaction between AnkG107/130 and β1-spectrin, HEK293TN cells were transfected with GST-tagged AnkG107/130 and YFP-tagged β1-spectrin. A GST pulldown assay confirmed that β1-spectrin interacts with AnkG107/130 (data not shown).

Further, when HEK293TN cells were transfected with GST-obscurin, YFP-β1-spectrin, HA- sAnk1.5, and HA-AnkG107/130, YFP-β1-spectrin was pulled down as part of the complex

(Fig. 14). When HA-AnkG107/130 was not transfected into the cells, YFP-β1-spectrin was not pulled down in the complex (Fig. 14). These findings indicate that, not only can

AnkG107/130 interact with β1-spectrin but also the AnkG107/130-β1-spectrin complex is

47 recruited to and maintained in a larger macromolecular complex containing sAnk1.5 and obscurin.

Figure 14: β1-spectrin associates with the obscurin/ankyrin complex via AnkG107/130. HA, GFP, and GST immunoblots demonstrate expression (input) and precipitation (IP) of sAnk1.5-HA, AnkG107/130-HA, and β1-spectrin with obscurin-GST. Note AnkG107/130- HA co-expression is necessary for obscurin-GST precipitation of β1-spectrin-YFP.

48

Chapter 4

Discussion & Future Directions

49

Discussion

As adaptor proteins that coalesce protein complexes to distinct subcellular domains, ankyrins play critical roles in vital physiological processes such as initiating and propagating the heart beat and transmitting nerve impulses. There are many mechanisms that regulate ankyrin-mediated macromolecular complex formation, of which the principle mechanism is the generation of unique alternative ankyrin isoforms that interact with distinct cohorts of proteins and target these complexes to specific subcellular domains. Several studies have shown that alternative splicing of the three ANK genes results in various tissue-specific, modular isoforms that bind discrete sets of proteins. Many mechanisms govern how these complexes are directed to their appropriate subcellular location from interacting with cell adhesion molecules, cytoskeletal proteins, and/or large scaffolding proteins. This thesis explores the role of obscurin in linking two different ankyrin isoforms and their associated protein complexes.

Obscurin, the massive myofibrillar protein, contains two independent ankyrin-binding sites in its C-terminus (ABD1&2) (Lange et al. 2009). Various studies have shown that many muscle-specific ankyrin isoforms such as sAnk1.5, AnkB212, and AnkG107/130 contain obscurin binding motifs in their C-terminus as well (Hopitzan, Baines, and Kordeli 2006).

Obscurin serving as a regulator and scaffold of multiple ankyrin proteins simultaneously provides a novel, compelling mechanism for ankyrin complex integration and coordination.

Unpublished work from our lab has demonstrated that ankyrins can self-associate and interact with other ankyrin isoforms as well. For example, we have shown that sAnk1.5 forms dimers and large oligomers and that obscurin can associate with these complexes. We

50 have also found that sAnk1.5 interacts with the CTD of AnkB. The implications of these ankyrin-ankyrin interactions have not been fully elucidated and are currently being investigated in the Cunha lab. AnkG107/130 and sAnk1.5 are both muscle-specific ankyrin isoforms that do not interact with each other as demonstrated via a GST pulldown assay (Fig.

9). To investigate obscurin regulation of ankyrin complex formation, we used these ankyrin proteins to avoid any confounding issues with a direct interaction between ankyrins.

While it has been established that sAnk1.5 interacts with obscurin and it is presumed that this interaction maintains the lSR across the , an interaction between

AnkG107/130 and obscurin had not yet been demonstrated. Using an array of biochemical techniques and FLIM-FRET analysis, we are the first to show that AnkG107/130 interacts with obscurin (Figs. 7-10). In addition, we also demonstrate that AnkG107/130 forms a complex with sAnk1.5 only in the presence of obscurin (Fig. 9). Furthermore, mutating the ankyrin binding sites (ABD1&2) in obscurin abolished AnkG107/130 pulldown of obscurin and sAnk1.5 (Fig. 9).

Corroborating our biochemical results, FLIM-FRET analysis demonstrated that sAnk1.5-CFP and AnkG107/130-YFP do not display FRET with a CFP lifetime of 2.51 ns, which is similar to the lifetime of our negative control of obscurin-CFP alone (2.58 ns).

Addition of wild-type obscurin-HA induced complex formation between sAnk1.5-CFP and

AnkG107-YFP significantly reducing the CFP lifetime to 2.25 ns, which is comparable to the lifetime of our positive control. When wild-type obscurin was replaced with mutant obscurin, FRET no longer occurred between Ank1.5-CFP and AnkG107/130-YFP indicating that obscurin mediates ankyrin-ankyrin complex formation within cells via its two ABDs.

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As lenti-viral constructs of sAnk1.5 and AnkG107/130-CTD localize to the M-line, these data would suggest that obscurin regulates the subcellular targeting of these ankyrin constructs (Fig. 11). These results are consistent with several studies that have shown sAnk1.5 is targeted to the M-line where it interacts with obscurin to maintain the lSR across the myofibril and in close proximity to the contractile apparatus (Maegen A Ackermann et al.

2011). AnkG107 has been less extensively studied. Using an antibody generated to the C- terminus of AnkG107, Maiweilidan et al., have published data demonstrating AnkG107 localizes to the sarcolemma of rat skeletal myocytes in particular to the costameres where it co-localizes with plectin-1 and filamin-C. Unfortunately, we were unable to acquire this antibody from Dr. Kordeli’s lab and there is no commercial antibody to AnkG107.

Without a commercially available antibody to AnkG107, we could not assess the subcellular localization of endogenous AnkG107. However, fluorescently-tagged, virally- expressed AnkG107/130-CTD localized to the M-line of neonatal cardiomyocytes.

Moreover, fluorescently-tagged sAnk1.5 and AnkG107/130 both demonstrated M-line localization in neonatal cardiomyocytes as they co-localized with the M-line marker myomesin, and were flanked by the Z-line marker, α-actinin. While these data are consistent with the hypothesis that obscurin is capable of targeting two different ankyrin isoforms simultaneously to the cardiomyocyte M-line, there are other possible interpretations. First, each ankyrin isoform may be interacting with different M-line populations of obscurin.

Second, other M-line proteins like myomesin or M-protein may regulate the subcellular targeting of AnkG107/130. To determine the role of obscurin in targeting AnkG107130-

CTD, we could examine the subcellular localization of AnkG107/130-CTD with mutated obscurin-binding domains. Finally, while we demonstrated that a truncated construct of

52

AnkG107/130 (lacking the SBD) localizes to the M-line, endogenous AnkG107/130 may not demonstrate the same localization. Nevertheless, this data serves as proof of the principle that obscurin may interact and target two different ankyrin isoforms simultaneously.

To determine the function of AnkG107/130, we first investigated its interaction with plectin-1 and filamin-C. A previous study had identified these proteins as AnkG107 interacting partners via yeast 2-hybrid, in vitro binding assays, and co-immunoprecipitation

(Maiweilidan, Klauza, and Kordeli 2011). Furthermore, they demonstrated that this interaction was mediated by the obscurin-binding domains of AnkG107. Based on Dr.

Cunha’s previous work demonstrating alternative isoforms of the ankyrin-B CTD display different binding capacities for obscurin (Cunha and Mohler 2008), we hypothesized that alternative isoforms of AnkG107 CTD may display different binding capacities for obscurin, plectin-1, and filamin-C. Previous work from our lab had isolated seven splice variants of the AnkG107/130-CTD from mouse ventricular mRNA, which was subsequently subcloned in frame with an amino-terminal GST epitope for bacterial expression. While our results from the in vitro binding assay demonstrate different obscurin-binding capacities between the

AnkG107/130 CTD variants, the most salient point is that exclusion of Ank3 exon 47, which encodes the first obscurin-binding domain, significantly reduces obscurin-binding (Fig. 12).

With the same experimental paradigm, we find that AnkG107/130-CTD does not bind to filamin-C (Fig. 13) and displays minimal binding to plectin-1 (Fig. 12). Considering

AnkG107/130 displayed minimal plectin-binding, we re-assessed this interaction in a different binding assay in which AnkG107/130 is precipitated by obscurin or plectin-1.

These results demonstrate that AnkG107/130 is only precipitated by obscurin and not plectin-

1. These findings demonstrate that the obscurin-binding domains of AnkG107/130 interact

53 with obscurin and not filamin-C or plectin-1, contrary to the published study. In reviewing the experimental protocols from the published study, it becomes apparent that the binding reactions were never incubated in wash buffers with high salt concentrations to disrupt electrostatic interactions. In fact, the wash buffers consisted of PBS supplemented with 0.1%

Triton X-100 or 0.02% Tween (Maiweilidan, Klauza, and Kordeli 2011). In contrast, we incubate the precipitated binding complexes in wash buffer containing 500 mM NaCl and

0.1% Triton X-100 for five minutes three times.

Considering AnkG107/130 did not interact with plectin-1 or filamin-C, we investigated for other AnkG107/130 binding partners using the BioID technology, which would biotinylate AnkG107/130-interacting proteins. Lysate from cardiomyocytes expressing AnkG107/130-BioID yielded only two biotinylated proteins: AnkG107/130 and

β1-spectrin. This interaction is pertinent given AnkG107/130 contains a SBD and one function of ankyrin proteins is to anchor integral proteins to the underlying cytoskeleton.

Some limitations of BioID include that the large size of the biotin ligase (27 kDa) may interfere with the targeting, function, or stability of the bait protein. Also an identified candidate does not imply a direct interaction since it is simply a proximity-based labeling assay (10-15 nm) (Trinkle-Mulcahy 2019).

We used pulldown assays to corroborate that AnkG107/130 binds β1-spectrin. We also demonstrated that an AnkG107/130-β1-spectrin complex precipitates with an obscurin- sAnk1.5 complex (Fig. 14). Moreover, neither sAnk1.5 nor obscurin interacted with β1- spectrin individually or in a complex without AnkG107/130. While it is possible that

AnkG107/130 simply recruits β1-spectrin to this complex to provide cytoskeletal elements around the myofibril, we are still actively investigating the physiological role of this

54 complex. In sum, these findings show that ankyrin-associated proteins can be recruited to obscurin-nucleated protein complexes and that ankyrin-obscurin interactions do not disrupt ankyrin-protein interactions.

Future Directions

Identify additional cardiomyocyte-specific AnkG107/130 interacting proteins

We believe that AnkG107/130 has other cardiomyocyte-specific binding partners that were not revealed due to technical limitations that can be improved upon in future experiments. Firstly, lenti-viral transduction efficiency of large proteins in neonatal cardiomyocytes is low. We will improve the cardiomyocyte expression of lenti-viral

AnkG107/130-BioID with two changes: (1) use an adenovirus as opposed to a lentivirus to virally transduce cardiomyocytes, and (2) use a Percoll gradient to enrich the cardiomyocyte population and reduce the fibroblast population. Our experience reveals that it is far easier to transfect fibroblasts than cardiomyocytes; however, by increasing cardiomyocyte number and improving transfection efficiency we will enhance the expression of AnkG107/130-BioID and identify cardiomyocyte-specific protein interactions.

Measure sAnk1.5 and AnkG107/130 binding affinity for obscurin CTD

In the in vitro binding assays, we demonstrated that sAnk1.5 and AnkG107/130 could compete for obscurin-binding. Specifically, a 4-fold increase in sAnk1.5 disrupts

AnkG107/130 binding to obscurin while a 5-fold increase in AnkG107/130 did not affect sAnk1.5 binding to obscurin. Future experiments will measure the binding affinities for sAnk1.5 and AnkG107/130 to the obscurin CTD using microscale thermophoresis with a

55

NanoTemper Monolith NT.115 (in Dr. Xiaodong Cheng’s lab). In addition, using the obscurin mutants we will measure the binding affinities of sAnk1.5 and AnkG107/130 to obscurin’s first and second ankyrin-binding domains. These experiments may provide an explanation for why the ankyrin/obscurin interaction is more significantly decreased when obscurin ABD1 is mutated compared to ABD2. While the binding affinities may provide insight into the physiologic relevance of obscurin interactions with sAnk1.5 and

AnkG107/130, determining the subcellular localization of endogenous AnkG107/130 may also support the veracity of this complex.

Determine the subcellular localization of AnkG107/130 in cardiomyocytes

While the data is preliminary, AnkG107/130-BioID displays unique punctate expression in ventricular cardiomyocytes as determined by confocal microscopy. Efforts to identify the AnkG107/130-expressing organelle have been unsuccessful. Specifically, we investigated AnkG107/130-BioID co-localization with proteins in the perinucleus (IP3R type

1 antibody, provided by Darren Boehning), endoplasmic reticulum (calnexin), and the Golgi apparatus (GM130). Future experiments will focus on optimizing conditions to maximize immunodetection of the above listed organelles and lysosomes, endosomes, and vacuoles. In addition, we anticipate improved expression of AnkG107/130-BioID in cardiomyocytes will reveal additional AnkG107/130-interacting proteins, which may shed light on the identity of the organelle.

Examine obscurin dimerization by co-precipitation and FLIM-FRET

56

A perplexing observation from this study was that obscurin maintains its interaction with both ankyrin isoforms when either ABD is mutated. There are several possibilities that could explain this data. First, in the in vitro binding assays mutant obscurin may be more abundant than each ankyrin isoform and ankyrins are merely interacting with different obscurin populations. Second, it is also possible that ankyrin binding to obscurin may reveal a cryptic ankyrin . Considering various ankyrin isoforms contain an OTBD and localize to the M-line via obscurin, it is possible that protein interactions may reveal additional binding sites to facilitate macromolecular complex formation. Finally, obscurin may form dimers such that a dimer of mutant obscurins would still have two available ABDs.

Preliminary data from our lab suggests that the CTD of obscurin forms dimers. However, our construct of the CTD is a small segment of the full 800 kDa protein; therefore, whether dimerization of the full protein is relevant and/or possible is yet to be investigated and would be difficult to determine given the protein’s size. Future experiments to investigate obscurin dimerization include: performing FLIM-FRET on CFP and YFP obscurin constructs, creating larger obscurin constructs and assessing dimerization via pulldown assays, and identifying the domain that mediates dimerization.

Conclusion

In conclusion, this thesis provides the first data demonstrating obscurin can interact with two ankyrin isoforms and their associated proteins simultaneously. This observation is validated in in-vitro binding assays, co-precipitation assays, and FLIM-FRET analysis.

Moreover, we demonstrate that sAnk1.5 and AnkG107/130, which do not interact with each other, are brought together in a complex via the two independent ankyrin-binding domains in

57 the C-terminus of obscurin. We also find that AnkG107/130 recruits β1-spectrin to a complex of obscurin and sAnk1.5. Finally, we demonstrate that sAnk1.5 and AnkG107/130-

CTD are localized to the M-line presumably via obscurin. In closing, this work lays the foundation for a novel mechanism of ankyrin regulation, specifically the coalescing of two distinct ankyrin protein complexes to the same domain (i.e. the M-line) via interactions with two independent ankyrin-binding domains in the large scaffolding protein obscurin (Fig. 15).

Figure 15: Summary model of obscurin mediated ankyrin complexes. Obscurin organizes two distinct ankyrin protein complexes to the same domain (i.e. the M-line) via interactions with two independent ankyrin-binding domains.

58

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Vita

Janani Subramaniam was born on March 11, 1991, the daughter of Hema Venkatraman and

Subramaniam Iyer. After completing her work at Sophia High School, Bengaluru, India in

2009, she entered Roger Williams University in Bristol, Rhode Island. She received the degree of Bachelor of Science from Roger Williams in May 2013. For the next four years, she worked as a research assistant in the Department of Integrated Biology and

Pharmacology at the McGovern Medical School at UTHealth, Houston. In August 2017, she entered the University of Texas MD Anderson Cancer Center UTHealth Graduate School of

Biomedical Sciences.

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