A Thesis Entitled Identification of CALML4 As a Novel Component of the Intermicrovillar Adhesion Complex That Regulates Intestin

A Thesis

entitled

Identification of CALML4 as a Novel Component of the Intermicrovillar Adhesion

Complex that Regulates Intestinal Brush Border Assembly

Myoung Soo Choi

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Master of Science Degree in

Biology

______Dr. William Scott Crawley, Committee Chair

______Dr. Song-Tao Liu, Committee Member

______Dr. Deborah Chadee, Committee Member

______Dr. Qian Chen, Committee Member

______Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies

The University of Toledo

August 2018

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

Identification of CALML4 as a Novel Component of the Intermicrovillar Adhesion Complex that Regulates Intestinal Brush Border Assembly by

Myoung Soo Choi

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Biology

The University of Toledo

August 2018

During their terminal differentiation program, intestinal enterocytes form an apical brush border (BB): a tightly packed array of lumen-oriented microvilli that function in nutrient absorption and host-cell defense. Assembly of a highly-ordered BB is driven by a protocadherin-based adhesion complex, known as the intermicrovillar adhesion complex

(IMAC), that mediates adhesion between neighboring microvilli during BB formation. We used the innate adhesion of the IMAC protocadherins as an affinity purification step to recover endogenous IMAC, and have identified calmodulin-like protein 4 (CALML4) as a novel component of this complex. CALML4 is a small EF hand protein that has yet to be characterized in the literature. Endogenous CALML4 exhibits striking localization to the distal tip of BB microvilli, the site of IMAC function, in native intestinal tissue and CACO-

2BBE cell culture enterocytes. Overexpression of EGFP-tagged CALML4 in CACO-2BBE cells recapitulates this localization. Through a series of pull-down assays, we identified that CALML4 incorporates into the IMAC by associating with the neck region of Myo7b as a myosin light chain. Refined mapping studies revealed that CALML4 associates with the third IQ motif (IQ3) in the Myo7b neck region. Interestingly, however, binding of

iii

CALML4 to IQ3 is dependent upon the presence of neighboring IQ motifs, suggesting that

CALML4 exhibits an atypical myosin-binding mode compared to other known myosin light chains. Consistent with CALML4 functioning as light chain for Myo7b,

CALML4 fails to target to the BB in Myo7b knockdown (KD) CACO-2BBE cell lines and in a Myo7b knockout (KO) mouse. Stable KD of CALML4 in CACO-2BBE cells disrupts proper BB assembly, phenocopying the loss of Myo7b itself. This suggests that

CALML4 may be necessary for Myo7b in vivo function. Biophysical analysis of recombinant CALML4 revealed that it is monomeric and lacks the ability to bind calcium with high affinity. CALML4, therefore, is unlikely to be regulated by calcium directly within the IMAC. We present further evidence that CALML4 functions as a light chain for Myo7a in the homologous Usher syndrome adhesion complex (USAC) that functions in sensory epithelia. The USAC is primarily characterized in inner ear hair cells, where it is responsible for assembling arrays of specialized microvilli, known as stereocilia, into ‘hair bundles’ that decode sound information into neural signals.

Defects in the USAC result in Usher syndrome Type 1 (USH1), a severe form of deaf- blindness. Excitingly, CALML4 is one of 27 candidate genes for USH1H, a subtype of

USH1 whose genetic cause is currently unknown. In sum, our data supports the hypothesis that CALML4 functions as a myosin light chain for Myo7a and Myo7b in the USAC and

IMAC, respectively. CALML4 is, therefore, the second component shared genetically between these homologous adhesion complexes.

Acknowledgements

I would not have been able to complete my thesis without the guidance of my advisor, all the lab members, and committee members.

First and foremost, I would like to express my deepest appreciation and gratitude to my advisor, Dr. William Scott Crawley, for his excellent guidance, care, and patience through every step of acquiring this degree. The enormous amount of support and guidance

I received from Dr. Crawley helped me become not only a better scientist, but also a better person.

Next, my sincere gratitude goes to Dr. Tomer Avidor-Reiss for the encouragement he has given me, and most importantly believing in me.

Last but not least, I would like to thank my lab colleagues Maura Graves and

Samaneh Matoo, and the undergraduate researchers Zachary Storad and Rawnag El sheikh

Idris for the assistance and friendship. My sincere gratitude goes to Maura Graves for her emotional and physical support, as well as her daily companionship in the lab.

Contributions to the project

The identification of CALML4 as a PCDH24-associated cytoplasmic complex component and initial localization studies of CALML4 in mouse intestinal tissue and

CACO-2BBE cells were performed by Dr. William Scott Crawley. I performed the interactome analysis between CALML4 and the IMAC components, the mapping of the

IQ motif binding partners for CALML4 and Myo7a and Myo7b, the identification and validation of the atypical interaction between CALML4 and Myo7b, the localization of

CALML4 in colon mouse intestinal tissue and colocalization of CALML4 with PCDH24 in CACO-2BBE cells, the purification of CALML4 from E. coli and the biochemical characterization of this recombinant CALML4. Analytic ultracentrifugation, circular dichroism spectroscopy, and mass spectrometry analysis of the recombinant CALML4 was performed with the help of the Protein Function Discovery Core at Queen’s University, under the supervision of Kim Munro.

Table of Contents

Abstract ...... iii

Acknowledgements ...... v

Contributions to the project ...... vi

Table of Contents ...... vii

List of Figures ...... x

List of Abbreviations ...... xii

List of Symbols ...... xiv

1 Introduction...... 1

1.1 The mammalian intestinal tract ...... 1

1.2 The specialized apical domain of intestinal enterocytes ...... 4

1.3 The enterocyte cytoskeleton ...... 6

1.4 Intermicrovillar adhesion promotes BB formation ...... 7

1.5 Adhesion is used as a conserved mechanism for epithelial specialization .....10

1.6 A homologous adhesion complex functions in sensory epithelial ...... 11

Summary ...... 15

2 Materials and Methods ...... 16

2.1 Molecular biology ...... 16

2.2 Ectodomain protein production and IMAC recovery ...... 18

2.3 Recombinant protein purification from E. coli ...... 18 vii

2.4 Analytical ultracentrifugation ...... 20

2.5 Circular dichroism spectroscopy...... 20

2.6 Isothermal titration calorimetry ...... 20

2.7 Biochemical pull-down assays and nanoscale live-cell pull-downs ...... 21

2.8 Cell culture, lentivirus production, and stable line generation ...... 22

2.9 Light microscopy ...... 23

2.10 Immunohistochemistry ...... 24

2.11 Image analysis ...... 25

2.12 Statistical analysis ...... 25

3 Results...... 26

3.1 Recovery strategy for the PCDH24-associated cytoplasmic complex ...... 26

3.2 Identification of CALML4 as a PCDH24-associated cytoplasmic complex ......

component...... 32

3.3 CALML4 mRNA is found across the human intestinal tract ...... 33

3.4 CALML4 targets to the tips of BB microvilli ...... 34

3.5 CALML4 interacts with Myo7b ...... 37

3.6 CALML4 targeting to the BB requires Myo7b...... 41

3.7 CALML4 is necessary for proper BB assembly ...... 43

3.8 CALML4 is monomeric and lacks the ability to bind calcium with high ......

affinity...... 45

3.9 CALML4 is expressed in inner ear hair cells and interacts with Myo7a...... 47

3.10 CALML4 associates with IQ3 in both Myo7a and Myo7b ...... 50

4 Discussion...... 54

viii

4.1 CALML4 is a novel IMAC component ...... 54

4.2 CALML4 is the second component shared genetically between the IMAC and

USAC...... 55

4.3 CALML4 functions as a light chain for Myo7a and Myo7b ...... 55

4.4 CALML4 and Myosin Regulation ...... 59

4.5 CALML4 is an USH1H candidate gene ...... 60

4.6 Long-Term Directions: The IMAC and Implications for USH1 Research .....62

References ...... 65

A Permissions to Copyrighted Figures ...... 82

List of Figures

1 Cartoon of the architecture of the intestinal epithelium...... 2

2 The development of the BB and the IMAC molecular composition ...... 8

3 The development of hair cell stereocilia and the USAC molecular composition ..11

4 Comparison of the homologous adhesion complexes that control actin-based

protrusions in sensory and transporting epithelia ...... 12

5 PCDH24 recovery strategy ...... 28

6 CALML4 is a putative new IMAC component ...... 32

7 CALML4 short isoforms is functionally expressed and enriched at the BB...... 34

8 CALML4 localization in native intestinal tissue and CACO-2BBE monolayers ....36

9 Analysis of binding interactions between CALML4 and the IMAC components. 39

10 Nanoscale live-cell pull-downs between CALML4 and Myo7b ...... 40

11 Localization of CALML4 in HeLa cells...... 42

12 CALML4 BB targeting is dependent on Myo7b ...... 42

13 CALML4 KD disrupts proper BB assembly ...... 44

14 CALML4 is monomeric and does not bind to calcium with high affinity...... 46

15 A schematic diagram of human Myo7b and Myo7a constructs ...... 48

16 CALML4 interacts with Myo7a...... 49

17 Nanoscale live-cell pull-downs between CALML4 and Myo7a...... 50

18 CALML4 interacts with Myo7b IQ34 and Myo7a IQ3 ...... 52 x

19 The myosin neck region acts as a lever arm when bound to light chains ...... 56

20 A revised comparison between the IMAC and USAC ...... 63

List of Abbreviations

BB ...... Brush border IMAC ...... Intermicrovillar adhesion complex USAC ...... Usher syndrome adhesion complex

CACO-2BBE ...... Caucasian colon adenocarcinoma 2 brush border expressing HEK-293 ...... Human embryonic kidney cells 293 KD ...... Knockdown KO ...... Knockout WT ...... Wild-type

CALML4...... Calmodulin-like protein 4 Myo7b ...... Myosin-7b Myo7a ...... Myosin-7a ANKS4B ...... Ankyrin-repeat and sterile α-motif domain-containing 4B PCDH24 ...... Protocadherin-24 MLPCDH ...... Mucin-like protocadherin CDH23 ...... Cadherin-23 PCDH15 ...... Protocadherin-15 Sans ...... Scaffold protein containing ankyrin repeats and SAM domain CIB2 ...... Calcium and integrin binding family member 2 CIB1 ...... Calcium and integrin binding family member 1 Myo10 ...... Myosin-10

USH1...... Usher syndrome Type 1 USH1B ...... Usher syndrome Type 1B USH1C ...... Usher syndrome Type 1C USH1E ...... Usher syndrome Type 1E USH1K ...... Usher syndrome Type 1K USH1H ...... Usher syndrome Type 1H

ED ...... Ectodomain EC ...... Extracellular cadherin domain ΔEC1 ...... Deletion of the first extracellular cadherin domain ERM ...... Ezrin, radixin, moesin TM...... Transmembrane domain xii

PDZ ...... Pds-94, DlgA, ZO1 CEN...... Central domain N ...... N-domain SAM ...... Sterile alpha motif domain PBM ...... PDZ domain binding motif MF ...... MyTH4-FERM domain MyTH4 ...... Myosin tail homology 4 domain FERM ...... Four-point-one, ezrin, radixin, moesin domain SH3 ...... SRC Homology 3 Domain 3 IQ ...... IQ motif HMM...... Heavy meromyosin CC ...... Coiled-coil Fc...... Fragment crystallizable

EDTA ...... Ethylenediaminetetraacetic acid EGTA ...... Ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid

GAPDH ...... Glyceraldehyde-3-phosphate dehydrogenase EGFP ...... Enhanced green fluorescent protein GFP ...... Green fluorescent protein ITC ...... Isothermal titration calorimetry SDS-PAGE ...... Sodium dodecyl sulfate–polyacrylamide gel electrophoresis PMSF ...... Phenylmethylsulfonyl fluoride NaCl ...... Sodium chloride NaH2PO4 ...... Monosodium dihydrogen orthophosphate CaCl2 ...... Calcium chloride HEPES ...... Hydroxyethyl piperazineethanesulfonic acid His ...... Histidine ATP ...... Adenosine triphosphate RIPA ...... Radioimmunoprecipitation assay PEI ...... Polyethylenimine PBS ...... Phosphate-buffered saline FBS ...... Fetal bovine serum BSA ...... Bovine serum albumin [θ] ...... Molar ellipticity

MVID ...... Microvillous inclusion disease EPEC ...... Enteropathogenic E. coli

xiii

List of Symbols

kDa ...... kilodalton nm ...... nanometer μM ...... micromolar μm2 ...... micrometer squared

μcal/s ...... microcalorie per second μl ...... microliter mM ...... millimolar mg/ml ...... milligram per milliliter % ...... percent

[θ] ...... deg*cm2/dmol c(s)...... continuous sedimentation coefficient distribution S ...... sediment coefficient

α...... alpha

xiv

Chapter 1

Introduction

1.1 The mammalian intestinal tract

The mammalian intestinal tract is a complex organ that strives to maintain a delicate homeostatic balance as an absorptive barrier. It must make intimate contact with foodstuffs ingested from the outside world in order to extract nutrients for the organism

(Helander and Fandriks, 2014), yet it must also create a barrier against an estimated population of 1014 microbes that reside in the human intestine at any one time (Vallance et al., 2002; Wilson et al., 2001). Evolution has produced an elegant functional design for the intestine to achieve this homeostatic balance. This can be seen in the gross anatomy of the intestine, its regenerative capacity, and the functional ultrastructure of its epithelium.

The intestinal tract is a hollow tube, with the inner epithelial-lined layer facing the lumen and its contents, and an outer layer secured by mesentery to the abdominal cavity

(Rao and Wang, 2010) (Fig. 1A). In an adult human, the length of the tract is approximately 7.5 meters and can be divided into two sections: the small intestine (~6 meters) where nutrient absorption primarily occurs, and the large intestine (~1.5 meters) which is principally involved in water resorption (Hounnou et al., 2002). For the purposes

Figure 1. Cartoon of the architecture of the intestinal epithelium. (A) The small intestine is lined by finger-like protrusions known as villi. (B) A single villus is covered by a monolayer of epithelial cells. This epithelial monolayer is generated from stem cells located in the crypt domain. Stem cells receive growth signals from Paneth cells. Daughter cells derived from these stem cells differentiate into a variety of different types cell types, the most abundant being the nutrient- absorptive enterocyte. These epithelial cells migrate up the crypt–villus axis over a period of 5–6 days. At the tip of a villus, cells undergo apoptosis and are extruded from the epithelium. (C) During differentiation, enterocytes remodel their apical cytoskeleton to form a brush border composed of microvilli that are tightly packed in hexagonal arrays. Cartoon adapted from (Crawley et al., 2014a). Permission to use granted by Rockefeller University Press, ©Crawley et al., 2014. Originally published in Journal of Cell Biology. http://jcb.rupress.org/content/207/4/441.long of this document, we will focus on the small intestine, though many of its attributes are also found with the large intestine.

The mucosa of the small intestine is lined with macroscopic finger-like projections called villi (∼0.5 mm in length; ~40 villi per mm2 of intestinal mucosa; Fig. 1B) that 2

point towards the lumen (Helander and Fandriks, 2014). Each villus is comprised of a monolayer of specialized epithelial cells that rest on a basement membrane (Simons and

Clevers, 2011). The creation of villi is estimated to magnify the mucosal surface area in contact with the luminal contents of the small intestine by a factor of 6.5, which allows the intestine to mediate efficient nutrient absorption (Helander and Fandriks, 2014).

A testament to its importance, the epithelium of the intestine is constantly being renewed throughout the lifetime of an individual (van der Flier and Clevers, 2009).

New epithelial cells are born from intestinal stem cells that are found at the base of so- called ‘crypts’: moat-like invaginations of the intestinal sub-mucosa that serve as protective niches for the highly-proliferative stem cells (Fig. 1B). Also found in crypts along with these stem cells are Paneth cells. Paneth cells synthesize and secrete antimicrobial compounds to help keep the crypt domains relatively sterile in order to protect the intestinal stem cells from microbial damage (Mukherjee et al., 2008). Along with this,

Paneth cells also secrete factors to help maintain ‘stemness’ of the intestinal stem cells

(Sato et al., 2011). A single crypt feeds to and replenishes the epithelium of multiple villi, with the newly-borne epithelial cells quickly differentiating to their mature state as they undergo collective cell migration up the crypt axis to emerge as part of the villus surface

(Fig. 1B). Once part of the villus, they continue collective cell migration up the villus axis while performing their physiological functions until they reach the villus tip (Wong et al.,

2010). At the villus tip, cells undergo apoptosis and are extruded from the epithelium and become part of the fecal matter (Bertrand, 2011)(Fig. 1B). The entire surface epithelium of the intestine is completely replaced by this process every five to seven days, making the intestine the most highly regenerated organ in the human body (van der Flier and Clevers, 3

2009). Continual self-renewal is a physiological adaptation that reflects both the harsh environment that the luminal contents bestow upon the intestinal epithelium, as well as the physiological importance of the intestinal epithelium itself.

The intestinal stem cells give rise to a limited number of specialized epithelial cell types that are found on the villus surface (Chang et al., 2008; Mariadason et al., 2005).

These include goblet cells that secrete mucus to create a protective barrier for the intestinal tissue (Knoop and Newberry, 2018), enteroendocrine cells that secrete over 30 different types of hormones that play a role in digestive physiology (Gribble and Reimann, 2016), and tuft cells which are thought to survey the luminal environment and mediate immune responses (Grencis and Worthington, 2016). The dominant cell type of the intestinal epithelium, and the subject of this thesis, is the enterocyte. As we will see, enterocytes are uniquely adapted in both their ultrastructure and molecular composition to function as the sole cell-type responsible for nutrient absorption in the gut, while also playing a key role in host-cell defense.

1.2 The specialized apical domain of intestinal enterocytes

During their differentiation in the intestinal crypts, enterocytes remodel their apical domain to produce microscopic finger-like membrane protrusions called microvilli

(Fig. 1C,2C). In the mature state, each enterocyte possesses roughly ~1000 apical microvilli which exhibit striking organization, having uniform size (1 μm in length and 100 nm in diameter) and being packed into dense hexagonal arrays (Crawley et al., 2014a;

Revenu et al., 2004). Together, this dense uniform collection of microvilli forms a structure known as the intestinal brush border (BB). By creating an apical BB, enterocytes further magnify the membrane surface area exposed to the intestinal lumen by a factor of 4

13 beyond formation of the macroscopic villi (Helander and Fandriks, 2014). Indeed, the BB serves as an enormous membrane reservoir where enterocytes house a multitude of membrane-bound enzymes, transporters, and channels that are used to process and absorb the nutrients from the intestinal lumen. BB microvilli are also enriched in factors involved in host-cell defense against microbes found in the intestinal lumen (Koyama et al., 2002;

Maroux et al., 1988). Specifically, BB microvilli shed anti-microbial exosomes from their distal tips into the intestinal lumen. These exosomes can directly interact and lyse luminal microbes and are known to be highly enriched in alkaline phosphatase, an enzyme that detoxifies the lipopolysaccharide endotoxin found in the outer membrane of Gram- negative bacteria (Shifrin et al., 2012).

Genetic or acquired perturbations to BB structure pose serious health risks both in developing countries and the developed world. For example, infection with the attaching and effacing microbe Enteropathogenic E. coli (EPEC) results in BB destruction, ultimately causing severe osmotic diarrhea in patients that can be life-threatening if not treated (Vallance et al., 2002). Microvillus inclusion disease (MVID) is a rare inherited disorder characterized by the presence of microvilli-lined intracellular inclusions within the cytosol of enterocytes (Cutz et al., 1989; Khubchandani et al., 2011). The current school of thought is that the apical membrane recycling pathway of enterocytes is dysfunctional in these patients, causing near-to-complete loss of microvilli from the apical surface of enterocytes (Weis et al., 2016). MVID infant patients suffer from watery diarrhea and an inability to absorb nutrients, resulting in severe malnutrition unless total parenteral nutrition is provided (Khubchandani et al., 2011). As a final example, a recent large-scale study of Crohn’s disease patients identified significant perturbations to BB 5

structure, with patients exhibiting decreased microvillar length and reduced expression of a number of known gene sets involved in microvillar structure and function (VanDussen et al., 2018). The molecular etiology of Crohn’s disease is still unknown, so whether perturbed BB structure is a cause or a result of the disease is presently unclear. Despite the importance of the intestinal BB for human health, the underlying mechanism of its creation during terminal enterocyte differentiation is still poorly understood. Excitingly, recent proteomic studies have defined the molecular components that comprise the BB

(Lindeboom et al., 2018; McConnell et al., 2011), giving us a ‘parts list’ to begin to tease out the assembly pathway for BB construction.

1.3 The enterocyte cytoskeleton

Each microvillus has a core of ~35-40 actin filaments that are bundled together to increase their tensile strength in order to support the overlying plasma membrane (Ohta et al., 2012). Specifically, the F-actin filaments are bundled into parallel arrays by the actin- bundling proteins villin, espin, plastin-1, and EPS8 (Bartles et al., 1998; Bretscher et al.,

1979; Bretscher and Weber, 1980; Croce et al., 2004; Mooseker et al., 1980). To date, it is still unclear what actin-nucleating protein is responsible for creating the F-actin of microvilli, though proteomic analyses of isolated BBs identifies three potential candidates: cordon-bleu, diaphanous homologue 1, and the Arp2/3 complex (McConnell et al., 2011;

Revenu et al., 2012). The central F-actin core of each microvillus is anchored into a

‘terminal web’, a sub-apical mesh-like network of myosin-2, actin, plastin, and the intermediate filaments keratin and spectrin (Grimm-Gunter et al., 2009)(Fig. 1C). A number of key proteins are thought to cross-link the F-actin core to the overlying membrane in order to maintain the structure of the microvillus. These include the 6

unconventional myosins, Myosin-1a and Myosin 6, and the ERM (ezrin, radixin, moesin) family protein, Ezrin (Hegan et al., 2012; Saotome et al., 2004; Tyska et al., 2005).

Together, the proteins that generate and maintain the core F-actin along with those that couple the actin core to the microvillar membrane create the unique finger-like structure of each microvillus.

1.4 Intermicrovillar adhesion promotes BB formation

Early electron microscopy studies first visualized the striking hexagonal packing exhibited by BB microvilli (Granger and Baker, 1950), however, the molecular mechanism driving this organization remained undefined for many years. Recent studies using an enterocyte cell culture model, namely the CACO-2BBE cell line, have shed light on this subject. CACO-2BBE cells are a human colonic adenocarcinoma cell line that undergo a defined polarization program after reaching confluency (Peterson and Mooseker, 1992).

These cells construct a near tissue-like apical BB over a time-course of ~20 days post- confluency (Fig. 2A). Furthermore, these cells are amenable to Lentiviral-delivered genetic manipulation for the overexpression or suppression of genes, which allows us to easily interrogate gene function in this enterocyte cell culture model.

Using the CACO-2BBE cells as a principal model, a recent study identified an adhesion- based mechanism that plays a central role in regulating proper BB formation (Crawley et al., 2014b). Early during BB assembly, nascent microvilli created on the apical cell surface cluster together due to adhesion mediated between their distal tips (Fig. 2B). High- resolution electron microscopy revealed that neighboring microvilli are coupled together with physical thread-like links between their distal tips, pinpointing the apparent source of

Figure 2. The development of the BB and the IMAC molecular composition. (A) Time course of enterocyte polarization. Early during polarization, microvilli are small in size and sparse in number. Microvilli begin to contact each other to forms clusters. Further into differentiation, microvilli increase in size and number, and form discrete ‘tepee-like’ clusters due to intermicrovillar adhesion. This adhesion leads to the tight packing of microvilli into hexagonal arrays, eventually forming a mature BB. (B) High-magnification scanning electron microscopy images showing intact (yellow arrows) and broken (green arrowheads) intermicrovillar links from early and late polarized CACO-2BBE cells. (C) Deep-etch electron microscopy of the BB from mouse small intestine (SI) showing intermicrovillar links. (D) Super-resolution image of late CACO-2BBE cell stained for actin (red) and PCDH24 (green). (E) Cartoon of an early- polarized microvillar cluster showing intermicrovillar adhesion links formed due to trans-heterophilic adhesion between PCDH24 and MLPCDH. (F) Cartoon model summarizing the interactome of the intermicrovillar adhesion complex. Images have been adapted from (Crawley et al., 2014b). Permission to use granted by Elsevier, ©Crawley et al., 2014b. Originally published in Cell. https://www.cell.com/cell/abstract/S0092-8674(14)00215-3 Scale bars: B=100 nm, C=400 nm, D=1 μm. 8

this adhesion. As BB assembly progresses, additional microvilli incorporate into existing clusters as a result of these ‘intermicrovillar adhesion links’, causing clusters to grow in size and begin to amalgamate together (Fig. 2B). The final result is the creation of a single large-scale cluster on the apical surface, i.e., a mature BB (Fig. 2A,C).

Intermicrovillar adhesion links are comprised of a trans-heterophilic complex of two cadherin family members—protocadherin-24 (PCDH24) and mucin-like protocadherin

(MLPCDH) (Fig. 2E). Their specific localization at the distal tips of BB microvilli

(PCDH24 shown; Fig. 2D) requires interaction with several cytoplasmic binding partners: two scaffolding molecules (Harmonin and Ankyrin-repeat and sterile α-motif domain- containing 4B; ANKS4B), and a myosin motor protein (Myosin-7b; Myo7b)(Fig. 2F).

Together these proteins are known as the intermicrovillar adhesion complex (IMAC). Loss of any IMAC component disrupts BB assembly due to either the absence of intermicrovillar adhesion links or their aberrant localization (Crawley et al., 2014a; Crawley et al., 2016;

Weck et al., 2016). This can be seen with KD of IMAC components in CACO-2BBE cells and also in KO mice lacking specific IMAC components. In each case, the BB appears disheveled, with hexagonal packing not occurring. This loss of correct microvillar packing leads to a significant reduction in microvillar density. BB microvilli also exhibit non- uniform length when IMAC function is perturbed, suggesting that intermicrovillar adhesion links may also somehow communicate with the actin polymerization machinery.

Interestingly, the morphology of villi in the intestine of IMAC KO mice also appears to be perturbed (Crawley et al., 2014b). How the IMAC influences the gross anatomy of the intestine is presently unclear.

1.5 Adhesion is used as a conserved mechanism for epithelial specialization

Global proteome and gene expression analysis studies indicate that IMAC components are highly expressed in other transporting tissues that construct apical BBs, such as the kidney, liver and pancreas (Thul et al., 2017; Uhlen et al., 2015; Uhlen et al., 2017). This suggests that the IMAC might serve a conserved role in BB assembly across all transporting epithelia. Interestingly, protocadherin-based adhesion not only drives BB assembly in transporting epithelia, but is also known to control the specialized actin-based protrusions created by sensory epithelia. The most-well studied example of this in sensory epithelia are inner ear stereocilia, which are created by auditory and vestibular hair cells that function in hearing and balance, respectively (Kazmierczak et al., 2007). Hair cells use adhesion to assemble an apical hair bundle: a staircase-like collection of stereocilia with precisely graded heights (Fig. 3A). During hair bundle assembly, neighboring stereocilia are connected together by a number of adhesion links in order to control and promote the precise architecture of the hair bundle (Lagziel et al., 2005; Siemens et al.,

2004) (Fig. 3B). The staircase-like organization of the hair bundle is vital to its role in converting mechanical stimuli to neural signals. Most of the linkages used to create the hair bundle architecture are lost as hair bundle development proceeds. One prominent type of linkage that does remain in the mature hair bundle is known as the ‘tip-link’ (Tilney et al., 1992) (Fig. 3B). Tip-links connect the tips of the shorter stereocilia in the staircase to the side of its taller neighbor. Importantly, tip-links are not only critical for achieving proper hair bundle architecture, but they also play a direct role in the mechanotransduction mechanism of hearing (Assad et al., 1991; Pickles et al., 1984). Tip-links are thought to

Figure 3. The development of hair cell stereocilia and the USAC molecular composition. (A) Scanning electron micrograph of a hair bundle from an inner hair cell from a rat cochlea. The stereocilia form well-defined rows that increase in height along the excitatory axis of the bundle. Image taken from (Hackney and Furness, 2013). (B) Scanning electron micrograph showing that shorter stereocilia are connected to taller stereocilia by tip-links (arrows). Image taken from (Hackney and Furness, 2013). Permission to use granted by Elsevier, ©Hackney and Furness, 2013. Originally published in Cell. https://www.cell.com/cell/abstract/S0092-8674(14)00215-3 (C) Cartoon diagram of a tip-link and associated mechanotransduction complex. Cartoon was adapted from (Kazmierczak and Muller, 2012). Permission to use granted by Elsevier, ©Kazmierczak and Muller, 2012. Originally published in Cell. https://www.cell.com/trends/neurosciences/fulltext/S0166-2236(11)00178-0 Scale bars: A = 3 µm; B = 600 nm. gate a mechanotransduction ion channel that mediates hearing, though the precise identity of this channel (or channel complex) is still of great debate (Corey and Holt, 2016).

1.6 A homologous adhesion complex functions in sensory epithelial

The adhesion complex that comprises stereocilia tip-links is known as the Usher

Syndrome Adhesion Complex (USAC) due to the fact that mutations in any adhesion complex component results in Usher Syndrome Type 1 (USH1), the most common form of deaf-blindness in humans (Alagramam et al., 2001a; Alagramam et al., 2001b; Bitner-

Glindzicz et al., 2000; Bolz et al., 2001; Bork et al., 2001; Di Palma et al., 2001; Gibson et al., 1995; Johnson et al., 2003; Kikkawa et al., 2003; Verpy et al., 2000; Weil et al., 1995;

Weil et al., 2003) (Fig. 3C). The USAC is comprised of Cadherin-23 (CDH23) and 11

Protocadherin-15 (PCDH15), harmonin (a component that is genetically shared between the IMAC and USAC, though different splice isoforms are utilized), Sans, and Myosin-7a

(Myo7a) (Fig. 4). USH1 patients are classified into different subtypes, depending upon which USAC component is affected (Lentz and Keats, 2016). For example, defective

Myo7a results in USH1B, while mutations in harmonin underlie the pathology of USH1C.

While it was clear for many years that tip-link disruption was the underlying basis for the deafness suffered by USH1 patients, the molecular etiology of blindness suffered by these patients remained largely unexplored (Sahly et al., 2012). This is due to the fact that mouse Hair cell Photoreceptor cells hair Cone calyceal Rod bundle process outer seg

inner seg

Enterocyte USAC Function IMAC CDH23 Adhesion mol. PCDH24 PCDH15 Adhesion mol. MLPCDH Harmonin Scaffold Harmonin Sans Scaffold ANKS4B Myo7a Motor Myo7b homologs same gene

Figure 4. Comparison of the homologous adhesion complexes that control actin- based protrusions in sensory and transporting epithelia. Sensory epithelia, such as the hair cells from the inner ear and photoreceptors of the eye, use the USAC to organize their specialized actin-based protrusions into functional arrays. Hair cell image is taken from (Hudspeth, 1985). Permission to use granted by The American Association for the Advancement of Science, ©Hudspeth., 1985. Originally published in Science. http://science.sciencemag.org/content/230/4727/745. Photoreceptor cell images are from (Sahly et al., 2012). Permission to use granted by Rockefeller University Press, © Sahly et al., 2012. Originally published in Journal of Cell Biology. http://science.sciencemag.org/content/230/4727/745.Transporting epithelia, such as intestinal enterocytes, use the IMAC to organize their apical microvilli into a functional BB. Enterocyte image is taken from . Scale bars = 5 µm.

models of USH1 fail to recapitulate the blindness pathology seen in humans, indicating a fundamental difference between the rodent and human photoreceptors. Electron microscopy and other ancillary studies provided evidence for the existence of major differences between rodents and primates in the structural and molecular architecture of photoreceptors, specifically at the interface between the inner and outer segments of these specialized cells (Sahly et al., 2012; Schietroma et al., 2017). Specifically, there are actin- based microvilli-like processes (known as calyceal processes) that span the inner and outer segments of photoreceptor cells in primates which are not found in mice. Directly using primates as a model to study this phenomenon, however, is cumbersome, time-consuming, and expensive, necessitating the search for alternative animal models. It was recently discovered that photoreceptors from frogs share the same molecular architecture compared to primates (Schietroma et al., 2017). In a ground-breaking study using frogs as a substitute model, the Petit group proposed that the USAC creates ‘calyceal links’ in photoreceptor cells of the eye (Schietroma et al., 2017). Calyceal links connect the calyceal processes of the inner segment of rod and cone cells to the membrane of the outer segment, thereby stabilizing this critical interface (Fig. 4). The Petit group has proposed that loss of calyceal linkages causes a progressive degeneration of photoreceptor cell shape, leading to blindness in USH1 patients (Schietroma et al., 2017).

Despite controlling actin-based protrusions from epithelia that participate in divergent physiological functions, the IMAC and USAC exhibit striking homology in their compositions (Crawley et al., 2014a; Crawley et al., 2016; Li et al., 2016; Yu et al., 2017).

Each adhesion complex (at their core) is comprised of two protocadherins that interact in trans to form the external physical linkages, two cytoplasmic scaffolding molecules, and a 13

myosin motor protein (Fig. 4). This suggests that the two adhesions complexes may have a common ancestral origin and that the individual components have arisen due to gene duplication events followed by subsequent sequence divergence (perhaps to suit the needs of their specific tissue). Interestingly, USH1C patients with deleterious mutations in harmonin can also present with severe inflammatory enteropathy and nephropathy along with the typical USH1 deaf-blindness (Bitner-Glindzicz et al., 2000; Hussain et al., 2004).

These patients suffer from vomiting, nutrient malabsorption, intractable diarrhea, and aminoaciduria. The discovery of the IMAC many years later provided a molecular explanation for these non-sensorineural symptoms: Harmonin functions as a critical scaffold in both the USAC and IMAC. In agreement with this, Harmonin KO mice are born deaf and exhibit severe intestinal and kidney ultrastructural defects (Crawley et al.,

2014b).

Current evidence suggests the entire complement of proteins that compose both the

USAC and IMAC are still not definitively known. Three USH1 subtype genes (USH1E,

USH1K and USH1H) have not been identified, suggesting that more USAC components remain to be discovered (Ahmed et al., 2009; Chaib et al., 1997; Jaworek et al., 2012).

Indeed, structure-function studies have mapped USH1 point mutations in USAC components to protein-protein interaction domains/solvent-exposed surfaces that have no known binding partners (Cao et al., 2017; Pan and Zhang, 2012; Wu et al., 2011).

Furthermore, KO of the USAC components Sans and CDH23 in mice results in loss of stereocilia from the two shorter rows of the hair bundle, suggesting an unknown coupling may exist between the USAC and the F-actin polymerization machinery (Caberlotto et al.,

2011a; Caberlotto et al., 2011b). Similar defects are seen with IMAC-defective enterocytes: apical F-actin content and microvillar density is dramatically reduced (Crawley et al.,

2014b; Crawley et al., 2016; Weck et al., 2016). In sum, elucidating the molecular composition of both the USAC and IMAC represents a significant barrier for the field to overcome. Our study here is an effort towards this goal.

Summary

Here, we identified calmodulin-like protein 4 (CALML4) as a novel IMAC component from isolated mouse intestinal BBs. We demonstrate that CALML4 is a small calcium insensitive EF-hand protein that functions as a myosin light chain for Myo7b in the IMAC.

We also discovered that CALML4 interacts with Myo7a in a homologous manner and, therefore, may be a novel USAC component as well. Overall, this study identifies

CALML4 as a new molecular component genetically shared between the IMAC and the

USAC, and a new light chain specific for the class 7 myosins.

Chapter 2

Materials and Methods

2.1 Molecular biology

The human cDNA constructs used in this study are as follows: CALML4, GI:

110227593; ANKS4B, GI: 148664245; Harmonin, GI: 225690577; Myo7b, GI:

122937511; PCDH24, GI: 285002213; MLPCDH, GI: 285002197; Myo7a; GI:

189083797. DNA encoding the various domain constructs of CALML4, ANKS4B,

Harmonin and Myo7b were generated by PCR and TOPO-cloned into the pCR8 entry vector (Invitrogen). The resulting entry vectors were then shuttled into destination expression vectors using standard Gateway protocols. The domain boundaries for the

ANKS4B constructs used are as follows: ANKR (aa 1-252), CEN (aa 253-346), SAM (aa

348-417), ANKRCEN (aa 1-346), and CENSAM (aa 253-417). The domain boundaries for the Harmonin constructs used are as follows: NPDZ1 (aa 1-193), PDZ2 (aa 194-354),

PDZ2CC (aa 194-432), PDZ3 (aa 360-533), CCPDZ3 (aa 275-533), NPDZ12 (aa 1-354) and PDZ2CCPDZ3 (aa 194-533). The domain boundaries for the Myo7b constructs used are as follows: Myo7b Motor (aa 1-779), Myo7b Motor-IQ1 (aa 1-792), Myo7b Motor-

IQ12 (aa 1-815), Myo7b Motor-IQ123 (aa 1-836), Myo7b Motor-IQ1234 (aa 1-861),

Myo7b Motor-IQ12345 (aa 1-893), Myo7b full-length tail (aa 916-2116), MF1SH3 (aa 16

916-1542), SH3MF2 (aa 1501-2116), isolated 7BIQ12345 (aa 752-891), concatenated

IQ34 (810-861) and IQ45 (832-884). The domain boundaries for the Myo7a constructs used are as follows: Myo7a Motor (aa 1-741), Myo7a Motor-IQ1 (aa 1-769), Myo7a

Motor-IQ12 (aa 1-793), Myo7a Motor-IQ123 (aa 1-814), Myo7a Motor-IQ1234 (aa 1-

841), Myo7a Motor-IQ12345 (aa 1-876), isolated 7AIQ12345 (aa 733-876). Vectors used for expression of FLAG, FC, and V5-tagged proteins in this study have been previously described (Crawley et al., 2014b; Crawley et al., 2016). The pEGFP-C1 and pmCherry-

C1 vectors were digested with XhoI/BamHI, blunted using T4 DNA polymerase, and adapted to Gateway technology using the Gateway conversion kit (Invitrogen) to generate pEGFP-C1-GW and pmCherry-C1-GW, respectively. 6XHis-tagged CALML4 with an intervening TEV protease was created by PCR and cloned into the pET30b vector

(Novagen) for bacterial expression of CALML4. KD shRNA clones targeting CALML4 were expressed in the pLKO.1 vector, and correspond to TRC clones TRCN0000365582 and TRCN0000365583 (Sigma). A non-targeting scramble shRNA cloned into the pLKO.1 vector was used as a control (Addgene; plasmid #46896). The constructs encoding the cytoplasmic domains of PCDH24 and MLPCDH fused to GST have been previously described (Crawley et al., 2014b). The ectodomain of MLPCDH (residues 1-472) as well as the ΔEC1-MLPCDH (residues 1-472, Δ26-119) negative control were generated by PCR and TOPO-cloned into the gateway entry vector pCR8-GW (Invitrogen). The vector encoding the EGFP-tagged heavy meromyosin form of human Myo10 (pEGFP-Myo10-

HMM) was obtained from Addgene (Plasmid #87256), digested SalI/BamHI, blunted using

T4 DNA polymerase and adapted to Gateway technology using the Gateway conversion kit (Invitrogen) to generate pEGFP-Myo10-HMM-GW. 17

2.2 Ectodomain protein production and IMAC recovery

Production of recombinant MLPCDH ED and ΔEC1-MLPCDH ED was performed by transfection of HEK-293T cell cultures as previously described (Crawley et al., 2014b).

Media containing expressed protein was recovered, filtered using 0.45μm syringe filters, concentrated using MWCO 10K filter units (Millipore), then used to label magnetic

Protein-A beads (Dynabeads, Invitrogen). MLPCDH ED and ΔEC1-MLPCDH ED-coated magnetic beads were incubated with isolated enterocyte BBs (McConnell et al., 2011) in

Hanks buffered saline supplemented with 2 mM EDTA. A calcium switch was performed by spiking the mixture to a final concentration of 5 mM CaCl2. Beads were recovered using a DynaMag-2 Magnet apparatus (Invitrogen), washed twice in Hanks buffered saline supplemented with 2 mM CaCl2 and eluted by boiling in 2X SDS sample buffer. Recovered proteins were separated by SDS-PAGE, stained using Coomassie blue, excised from the gel and submitted for mass spectrometry. Eluates were also subjected to western blot analysis using goat anti-human IgG (FC specific) antibody (Sigma Cat #I2136), ANKS4B

(1:200; Sigma Cat#HPA043523), anti-PCDH24 (1:100; Sigma Cat#HPA012569), anti-

MLPCDH (1:500; Sigma Cat#HPA009081), anti-harmonin (1:250; Sigma

Cat#HPA027398), and anti-Myo7b (1:100; Sigma Cat#HPA039131).

2.3 Recombinant protein purification from E. coli

For recombinant production of CALML4, pET30b-6XHis-TEV-CALML4 was transformed into Escherichia coli strain T7 Express (New England BioLabs). Bacteria were grown in LB to an A600 of 0.6 and expression induced by addition of isopropyl-D- thiogalactopyranoside to a final concentration of 1 mM. After 8 hr at 37°C, cells were harvested, resuspended in ice-cold lysis buffer (50 mM NaH2PO4 pH 7.4, 300 mM NaCl, 18

10 mM imidazole, 1mg/ml lysozyme, 1 mM PMSF), lysed by sonication, and centrifuged at 15,000 X g for 1 hr. The resulting supernatant containing His-TEV-CALML4 was incubated with Ni-NTA Agarose resin (Qiagen) on a rocking platform for 1 hr at 4°C. The

Ni-NTA resin was then loaded into a column and subsequently washed with 20 mM imidazole, 300 mM NaCl, 50 mM NaH2PO4, pH 8.0 by gravity chromatography. His-

TEV-CALML4 was eluted from the Ni-NTA resin using 250 mM imidazole, 300 mM

NaCl, 50 mM NaH2PO4 pH 7.4. Eluate fractions were analyzed by SDS-PAGE and those fractions containing His-TEV-CALML4 were pooled and dialyzed in 20 mM Tris pH 7.4,

10 mM NaCl overnight. Further purification steps were performed using an ÄKTA Start

Protein Purification System (GE Healthcare). The dialyzed material was loaded onto a

HiTrap Q HP column (GE Healthcare) and eluted using a linear gradient from 10 mM NaCl to 1 M NaCl, in 20 mM Tris, pH 7.4. His-TEV-CALML4 typically eluted between 200 mM-300 mM NaCl. These fractions were pooled and further purified by size-exclusion chromatography using an HiPrep Sephacryl S-100HR column equilibrated in 50 mM

NaPO4, pH 7.4, 150 mM NaCl. Eluates were analyzed by SDS-PAGE and those containing

His-TEV-CALML4 were pooled, concentrated using Amicon Ultra centrifugal MWCO 3K filter units (Millipore), and dialyzed in (i) 5 mM Tris, pH 7.4, 50 mM NaCl with 2 mM

EDTA/2 mM EGTA for analytic ultracentrifugation, or (ii) 5 mM Tris, pH 7.4, 10 mM

NaCl with either 2 mM EDTA/2 mM EGTA or with 5mM CaCl2 for circular dichroism analysis, or (iii) 20 mM HEPES pH 7.4, 50 mM NaCl, 2 mM EDTA/2 mM EGTA for isothermal titration calorimetry. Purification of Dictyostelium discoideum calmodulin was performed as previously described (Crawley et al., 2011).

2.4 Analytical ultracentrifugation

Sedimentation velocity experiments were performed using a Beckman Optima XL-

1 instrument equipped with an AN 60-Ti rotor. Experiments were performed at 20°C with

His-TEV-CALML4 concentrations of 0.4, 0.8, and 1.2 mg/ml and a rotor speed of 55,000 rpm. Between 200 and 250 scans were taken for each experiment. Data were analyzed using the SEDFIT program.

2.5 Circular dichroism spectroscopy

Far- and Near-UV circular dichroism (CD) spectra were obtained using a Chirascan spectrophotometer (Applied Photophysics) and a 0.1 mm path length cuvette containing 20

μM His-TEV-CALML4. Spectra were recorded from 190 to 240 nm for Far-UV and 240 to 340 nm for Near-UV at 20°C and were averaged for six scans. Secondary structure was predicted using the program SpectralWorks (Olis Inc.) with the CONTIN/LL algorithm for

Far-UV spectra.

2.6 Isothermal titration calorimetry

Isothermal titration calorimetry (ITC) experiments were performed using a

MicroCal VP-ITC instrument. Titrations were performed at 30°C with 1.8 ml of 50 μM

His-TEV-CALML4 or D. discoideum Calmodulin in the calorimetric cell and 625 μM of peptide in the injection syringe. Typical experiments involved 29 injections of 10 μl, with a reference power of 15 μcal/s. CALML4 was titrated with a synthetic peptide corresponding to IQ4 in the neck region of human Myo7b, residues 828-855 (Ac-

QYQAMRQRTVQLQALCRGYLVRQQVQAK-NH2). As a control, D. discoideum calmodulin was titrated with a synthetic peptide corresponding to IQ1 in D. discoideum

MyoA, residues 718-746 (Ac-KRNKRLNDLATKIGSVWKMYKQRKWYLRT-NH2). 20

IQ motif peptides were synthesized by Genscript, reconstituted into 10 mM HEPES, pH

7.4, 50 mM NaCl containing 2 mM EDTA and 2 mM EGTA buffer, and dialyzed extensively against the same buffer. The time-dependent differential power signal was integrated to obtain the total heat evolved after each injection of ligand and corrected for the heat of dilution of the ligand alone. Data analysis and curve fitting were performed using the MicroCal ORIGIN software package.

2.7 Biochemical pull-down and nanoscale live-cell pull-down assays

For biochemical pull-downs, HEK293T cells were grown in T75 flasks to 90% confluency for transfection with pull-down constructs (40 μg total DNA) using 100 μg of

Polyethylenimine Linear, MW 25,000 (PEI; Polysciences). Cells were incubated with transfection media for 16 hours, after which transfection media was removed and replaced with fresh media. After 48 hours, cells were washed once in PBS, recovered by a cell scraper, and frozen for storage at -80°C. For pull-downs, cells were thawed on ice, resuspended in ice-cold CelLytic M buffer (Sigma) containing 2 mM ATP, 1X cOmplete

ULTRA protease inhibitor cocktail (Roche), and 1 mM Pefabloc SC (Roche), and lysed by several rounds of needle aspiration (27G X 11/4 needle). Cell lysates were centrifuged at

16,000 X g and the soluble material recovered and incubated with a 50 μl bed volume of either pre-equilibrated anti-FLAG M2 resin (Sigma) or glutathione resin (Sigma) with GST fused to the CDs of the IMAC protocadherins. Resins were incubated with cell lysates for

2-3 hours rocking at 4°C, pelleted by a low-speed spin, washed four times using 0.5X RIPA buffer supplemented with 2 mM ATP, 1X cOmplete ULTRA protease inhibitor cocktail

(Roche), and 1 mM Pefabloc SC (Roche), and eluted to recover bound material by boiling in 2X SDS buffer supplemented with 20 mM reduced glutathione for GST pull-downs or 21

eluted using wash buffer containing 200 μg/ml FLAG peptide for FLAG pull-downs.

Resin-bound material was detected by either staining with Coomassie blue or western analysis with the following antibody dilutions: mouse anti-FLAG M2 (1:1000; Sigma cat.

#F3165), mouse anti-V5 (1:5000; Invitrogen cat. #R960-25), or mouse anti-myc clone

9E10 (1:1000; Sigma cat. #M4439). For nanoscale live-cell pull-downs, HeLa cells were seeded onto coverslips at 50% confluency for transfection with nanoscale pull-down constructs (3.5 μg pEGFP-C1-Myo10-HMM bait construct and 3.5 μg pmCherry-C1 prey construct DNA) using 100 μg of Polyethylenimine Linear, MW 25,000 (PEI;

Polysciences). Cells were incubated with transfection media for 16 hours, after which transfection media was removed and replaced with fresh media. After 24 hours, cells were washed once in warm PBS, and processed for microscopy (see Light Microcopy section).

2.8 Cell culture, lentivirus production, and stable line generation

CACO-2BBE, HeLa, HEK293T, COS-7 and HEK293FT cells were cultured at 37°C and 5% CO2 in DMEM with high glucose and 2 mM L-glutamine. Media was supplemented with 20% fetal bovine serum (FBS) for CACO-2BBE cells and 10% FBS for

HeLa, HEK293T, COS-7 and HEK293FT cells. Lentiviral particles were generated by co- transfecting HEK293FT cells (T75 flasks at 80% confluency) with 6 μg pLKO.1 shRNA

KD plasmid, or pINDUCER20-C1-EGFP overexpression plasmid with 4 μg psPAX2 packaging plasmid and 0.8 μg pMD2.G envelope plasmid using Fugene transfection reagent (Roche) or PEI (Polysciences). Cells were incubated for 12 hours with transfection reagents, after which transfection media was removed and replaced with fresh media. Cells were subsequently incubated for two days to allow for lentiviral production. Media containing lentiviral particles was collected and filtered using 0.45μm syringe filters. 22

Lentviral particles were then concentrated using the Lenti-X concentrator according to the manufacturer’s protocol (Clontech). For lentivirus transduction, CACO-2BBE cells were grown to 90% confluency in T25 flasks. Before lentiviral infection, the medium was supplemented with 8 μg/ml polybrene. After 12 hours of incubation with lentivirus, the cells were reseeded into T75 flasks and grown for 3 days. Cells were then reseeded into

T182 flasks with media containing 50 mg/ml puromycin or 1 mg/ml G418, and grown to select for stable integration. For stable cell lines transduced with constructs cloned into the pINDUCER20-EGFP-C1 vector, 1 μg/ml doxycycline was included in the growth media at all times.

2.9 Light microscopy

For CACO-2BBE monolayers, cells were grown on coverslips for 12 days, supplemented with fresh media every 3 days. For preparation for confocal microscopy,

CACO-2BBE monolayers were washed once in warm PBS and incubated briefly with 0.02%

Saponin (Sigma) in warm PBS, then fixed for 15 min with 4% paraformaldehyde in PBS containing 0.1% Triton (Sigma). After fixation, cells were washed three times with PBS and blocked overnight in 5% BSA in PBS. Immunostaining was performed using anti-

PCDH24 (1:75; Sigma cat. #HPA012569), anti-MLPCDH (1:250; Sigma cat.

#HPA009081), anti-Harmonin (1:70; Sigma cat. #HPA027398), anti-Myo7b (1:25; Sigma cat. #HPA039131), anti-CALML4 (1:100; Proteintech cat. #15894-1-AP), or anti-GFP

(1:200; Aves Labs cat. #GFP1020) at 37°C for 2 hours. After primary antibody staining, coverslips were washed three times with PBS, and Alexa Fluor-488 donkey anti-rabbit

(1:200) secondary antibody or Alexa Fluor-488 goat anti-chicken (1:200) along with Alexa

Fluor-568 phalloidin (1:200) were applied for 1 hour at room temperature. Dual 23

immunostaining was performed using anti-PCDH24 (1:25; Sigma Cat#WH0054825M1) and anti-CALML4 (1:100; Proteintech cat. #15894-1-AP) primary antibodies at room temperature for 2 hours, after which coverslips were washed three times with PBS and

Alexa Fluor-488 donkey anti-mouse (1:200), Alexa Fluor-568 donkey anti-rabbit (1:200) secondary antibodies and Alexa Fluor-647 Phalloidin (1:200) were applied for 1 hour at room temperature. Coverslips were then washed five times in PBS and mounted with

Prolong anti-fade reagent (Invitrogen). Cells were imaged using a Leica TCS SP8 laser- scanning confocal microscope. All images shown are en face maximum projections through the full height of the BB, with the exception of x-z sections, which are single-plane confocal images. For HeLa cells images, cells were washed once in warm PBS and then fixed for 15 min with 4% paraformaldehyde in PBS, washed again in warm PBS to remove fixative and treated with 0.1% Triton (Sigma) in warm PBS for 7 mins for permeabilization. After permeabilization, cells were washed three times with PBS and blocked overnight in 5% BSA in PBS. Cell were then washed once in PBS, and incubated with Alexa Fluor-647 Phalloidin (1:200) for 1 hour at room temperature. Cells were subsequently washed five times in PBS and mounted with Prolong antifade reagent

(Invitrogen) and imaged using a Leica TCS SP8 laser-scanning confocal microscope.

2.10 Immunohistochemistry

Paraffin-embedded intestinal tissue sections were deparaffinized using Histo-Clear

II solution (Fisher), rehydrated in a descending graded ethanol series and stained using anti-CALML4 (1:100; Proteintech cat. #15894-1-AP) and villin (1:50; Santa Cruz cat.

#sc58897) followed by Alexa Fluor 488 donkey anti-rabbit (1:200; Invitrogen) and Alexa

Fluor 568 donkey anti-mouse (1:200; Invitrogen). Sections were washed three times and 24

then dehydrated in an ascending graded ethanol series, and mounted with Prolong Antifade reagent (Invitrogen). Tissue sections were imaged using a Leica TCS SP8 laser-scanning confocal microscope.

2.11 Image analysis

All image analyses were performed using ImageJ (NIH). For line-scan analysis, a line was drawn parallel to the microvillar axis using F-actin signal (visualized with phalloidin staining for cells and villin staining for tissue) as a reference, and the intensity of the CALML4 signal along that line was recorded. Intensity values were normalized by the maximum gray-scale value for an 8-bit image (i.e. 255), whereas all microvillar lengths were normalized so that the base of the microvillus was equal to 0 and the tip was equal to

1. Normalized line-scans were then plotted together (Prism v.6, GraphPad). For analysis of microvillar clustering, CACO-2BBE monolayers (12 days past confluency) were stained for F-actin using Alexa Fluor-568 Phalloidin (1:200) and either endogenous CALML4

(anti-CALML4, 1:100; Proteintech cat. #15894-1-AP) for non-transduced and pLKO.1 stable cell lines or GFP (1:200; Aves labs Cat#GFP1020) for GFP-fusion stable cell lines.

Individual cells were scored according to whether they exhibited microvillar clustering as previously described (Crawley et al., 2014b). For EGFP-fusion stable cell lines, only

EGFP-positive cells were scored.

2.12 Statistical Analysis

ImageJ(Fiji) was used for all image modifications and line-scanning data analysis.

All graphs were generated and statistical analyses performed in Prism v.6 or 7 (GraphPad).

Unpaired t-tests were used to determine statistical significance between reported values.

Chapter 3

Results

3.1 Recovery strategy for the PCDH24-associated cytoplasmic complex

The ability of PCDH24 to promote BB assembly depends on its interaction with an ill- defined cytosolic complex that is necessary for both proper targeting and function of the cadherin at the distal tips of enterocyte microvilli. Towards the goal of identifying additional components of the IMAC, we devised a protein isolation strategy to recover endogenous PCDH24 and its cytoplasmic complex from enterocyte BBs. We reasoned that we could use the innate adhesion of the microvillar protocadherins as a mechanism to affinity isolate PCDH24. Our protocol involves incubating magnetic beads, coated with the ectodomain (ED) of MLPCDH, with isolated BBs to allow for adhesion bond formation between the beads and endogenous PCDH24 found within BB microvilli (Fig. 5A).

Subsequently, PCDH24 and its associated cytoplasmic complex is recovered using typical bead-based protein pull-down methodology. The strength of our approach is three-fold: (i) it exploits the inherent specificity encoded in the trans-heterophilic adhesion mediated between the IMAC protocadherins as an affinity purification step, (ii) it utilizes the Ca2+- dependent, switch-like nature of cadherin adhesion to temporally control adhesion bond formation (first disrupting adhesion links in BB source material, then inducing adhesion 26

Figure 5. PCDH24 recovery strategy. (A) The protein isolation strategy used to recover endogenous PCDH24 and its cytoplasmic complex from enterocyte BBs (B) Image of the final sucrose gradient centrifugation step involved in the BB isolation procedure. A red arrow highlights the location of the BB fraction. (C) Confocal microscopy of an isolated BB stained for harmonin (green) and F-actin (red). Harmonin remains at the distal tips (white arrows) in isolated BB. Signal seen at the base of BB microvilli is nonspecific cross-reaction of secondary antibody. Scale bar: 1μm. Confocal microscopy of PCDH24-EGFP CACO-2BBE monolayers incubated with (D) MLPCDH-ED coated beads (white arrows) or (E) ΔEC1-MLPCDH-ED coated beads after extensive washing. Scale bar: 5 μm. (F) Quantitation of MLPCDH-ED coated beads and ΔEC1-MLPCDH-ED coated beads that remain bound to the PCDH24-EGFP CACO-2BBE cells after extensive washing. 27

between BBs and beads) , and finally (iii) the source material used for our purification, namely isolated enterocyte BBs, is highly enriched in the IMAC and is easily obtained by large-scale bucket biochemistry.

We reasoned that successful recovery of the PCDH24 cytoplasmic complex using our strategy rested on two caveats: (i) the cytoplasmic complex associated with PCDH24 remains intact during the preparation of our isolated BB source material, and (ii) our

MLPCDH ED-coated beads are able to make functional interactions with endogenous

PCDH24 found in BB microvilli. To begin to test the feasibility of our approach, we first assessed whether the IMAC remains intact in isolated BB source material. The isolation of enterocyte BBs from dissected intestinal tissue is a multi-step process that first involves using high concentrations of EDTA to disrupt the cell-cell junctions of the intestinal tissue in order to dissociate the epithelial sheet lining each villus into individual cells (which are primarily enterocytes). Enterocytes are then lysed using a hypotonic buffer in combination with mechanical disruption by a Waring blender. BBs are subsequently purified by several rounds of differential centrifugation and a final sucrose gradient centrifugation step (Fig.

5B). While previous proteomic analysis of isolated mouse enterocyte BBs revealed the presence of all known PCDH24 binding partners (McConnell et al., 2011), it is unclear whether these cytoplasmic components remain associated with PCDH24 as an intact complex after BB preparation. To assess the integrity of the PCDH24 cytoplasmic complex, native BBs were isolated from rat small intestine and stained for harmonin (Fig.

5C). The scaffold harmonin serves as an excellent marker to assess the integrity of the

PCDH24-associated cytoplasmic complex as it directly interacts with PCDH24, ANKS4B and Myo7b, and has been shown to be essential for proper distal tip localization of both 28

ANKS4B and Myo7b (Crawley et al., 2014b; Crawley et al., 2016). Confocal imaging of

BBs stained for harmonin revealed marked enrichment of the scaffold at the distal tips of microvilli, the normal site of IMAC function (Fig. 5C). We also observed signal at the base of the BB microvilli in the region containing the presumptive terminal web, though this staining was also seen in our secondary antibody controls, suggesting it is non-specific binding (data not shown). Thus, in line with previous proteomic data, our localization studies suggest that the PCDH24 cytoplasmic complex remains intact at the distal tips of microvilli from isolated BBs.

We next assessed whether MLPCDH ED-coated beads could make functional interactions with BB microvilli. To this end, we utilized a CACO-2BBE cell line stably expressing PCDH24-EGFP, which served as a marker for the IMAC. MLPCDH-ED coated beads were then added to the apical surface, and the cells were allowed to polarize an additional 6 days. As a control, beads coated with the ED of MLPCDH lacking the first extracellular cadherin (EC) domain (ΔEC1-MLPCDH ED) were also seeded onto

PCDH24-EGFP CACO-2BBE cells. It is known that adhesion is strictly dependent upon the first EC domain within the ectodomain of all cadherin family members (Sotomayor et al.,

2014). The CACO-2BBE monolayers were then washed extensively to remove unbound beads, fixed, stained for F-actin, and visualized by confocal microscopy. We observed abundant examples of BB microvilli forming intimate contacts with the MLPCDH ED- coated beads, especially from those cells in the monolayer overexpressing PCDH24-EGFP

(Fig. 5C). In most cases, BB microvilli seemed to be drawn towards and directly attached to the beads. In stark contrast, monolayers that were seeded with ΔEC1-MLPCDH ED- coated beads were essentially devoid of beads on their apical surfaces after extensive 29

washing (Fig. 5D,E). Therefore, we conclude that MLPCDH ED-coated beads are able to form functional adhesion bonds with native BB microvilli in a manner dependent upon interaction with PCDH24.

3.2 Identification of CALML4 as a PCDH24-associated cytoplasmic complex component

We proceeded by incubating BBs isolated from rat small intestine with our

MLPCDH ED- and ΔEC1-MLPCDH ED-coated beads (Fig. 5A). This incubation was first done in the absence of calcium (extraction buffer with 2 mM EDTA) to ensure that the endogenous PCDH24 in the BB microvilli was not engaged in making intermicrovillar adhesion links with endogenous MLPCDH. After a 20-minute incubation period of the beads with BBs in the absence of calcium, we performed a calcium-switch step by spiking the bead-BB mixture to a final calcium concentration of 5 mM to allow for adhesion bond formation between the beads and endogenous PCDH24 found in the BB microvilli. Beads were subsequently recovered, washed with extraction buffer, eluted with SDS sample buffer, and subjected to immunoblot analysis for the known IMAC components. This analysis confirmed that PCDH24, harmonin, Myo7b and ANKS4B were all recovered using MLPCDH ED-coated beads, consistent with the idea that these components form a stable complex within the BB (Fig. 6A). Interestingly, mass spectrometry and immunoblot analysis also detected the presence of the small EF-hand protein CALML4 in the eluate.

Importantly, beads coated with ΔEC1-MLPCDH ED failed to recover both the known

IMAC components or CALML4 (Fig. 6A). In sum, these data suggest that CALML4 is a novel component of the IMAC that is part of the cytoplasmic complex associated with

PCDH24. 30

Figure 6. CALML4 is a putative new IMAC component. (A) Identification of CALML4 as a novel IMAC component. Western blot analysis of the eluates from BB pull-downs using MLPCDH-ED and ΔEC1-MLPCDH-ED coated beads. Endogenous PCDH24 with its known cytoplasmic binding complex was recovered with MLPCDH- ED coated beads. Along with the known PCDH24 binding partners, CALML4 was also recovered with the MLPCDH-ED coated beads. ΔEC1-MLPCDH-ED coated beads failed to recover both known PCDH24 binding partners and CALML4. (B) Schematic diagram of the long and short isoforms of CALML4 from human with their respective molecular weights. Both are predicted to be comprised of two separate lobes (N-lobe and C-lobe), with each lobe having two EF-hand motifs. The long and short isoforms are identical in sequence, with the exception that the long isoform has 42-residue N- terminal extension.

3.3 CALML4 mRNA is found across the human intestinal tract

CALML4 is a novel EF-based protein that has yet to be characterized in the literature. Sequence analysis predicts that CALML4, like conventional calmodulin, is

‘dumbbell’ shaped, being comprised of two globular lobes (denoted here as N-lobe and C- lobe), connected by a linker sequence (Fig. 6B). While sequence analysis of the human

CALML4 gene predicts a number of splice isoforms, experimental evidence from

Expressed Sequence Tag data suggests that CALML4 is expressed predominantly as a

‘long’ and ‘short’ isoform (21.8 kDa and 17.6 kDa, respectively) (Fig. 6B). These isoforms are identical in sequence with the exception that the long isoform has a 42 residue N- terminal extension found before the N-lobe. Interestingly, this N-terminal extension appears to be specific to primates and is not found in lower species, such as mice. To confirm that the long isoform is indeed found as a transcript in human intestinal tissue, we screened a human gastrointestinal cDNA panel using a primer set specific towards the long isoform (Fig. 7B). We also used a primer set containing a 5’ primer that anneals to the start of the short isoform coding sequence. Although this annealing site is also found in the long isoform, we reasoned that we could gain information about the relative abundance of long vs. short isoform by comparing the amount of product formed between these two reactions. We found that both the long and the short CALML4 transcripts are present throughout the entire intestinal tract, with the short isoform transcript appearing to be more dominant (Fig. 7B). We further investigated the expression of CALML4 in intestinal tissue using whole-tissue lysates from mouse colon, as well as isolated mouse enterocyte BB lysates (Fig. 7C). In agreement with the idea that CALML4 is only found as a single

Figure 7. CALML4 short isoform is functionally expressed and enriched at the BB. (A) Amino acid sequence of the long and the short CALML4 isoforms. The coding regions amplified in the human gastrointestinal cDNA panel screen are denoted. (B) Agarose gel analysis of the expression of the long and the short CALML4 isoform transcripts throughout human intestinal tract. (C) Western blot analysis of the expression of CALML4 in intestinal tissue using a whole-tissue lysate from mouse colon, as well as lysate derived from isolated mouse enterocyte BBs. isoform in mice, we observed a single band migrating with an approximate molecular mass of ~17 kDa for both samples. We noticed that CALML4 was enriched in the BB lysate as compared to the whole-tissue sample (Fig. 7C). Taken together, these findings are consistent with CALML4 being expressed across the intestinal tract as part of the BB- enriched IMAC.

3.4 CALML4 targets to the tips of BB microvilli

The recovery of CALML4 alongside the other known IMAC components using our purification strategy suggests that CALML4 is a novel IMAC component. To further validate this, we examined CALML4 localization in mouse intestinal tissue using confocal microscopy. Consistent with our mRNA and western blot analysis, CALML4 is expressed across the full-length of the intestinal tract in mice with intense staining in the BB (Fig.

8A-D). Higher magnification imaging of mature enterocytes revealed striking enrichment of CALML4 at the distal tips of BB microvilli, the site of IMAC function. Indeed, line- scan analysis of CALML4 along the axis of microvilli revealed marked enrichment at the distal tips. This targeting was seen throughout the BB of the entire intestinal tract (Fig.

8E-H). We also noted occasional low levels of CALML4 localized to the junctional margins of enterocytes. We further examined the expression and localization of CALML4 in CACO-2BBE enterocyte monolayers. Western blot analysis revealed that CALML4 is expressed at low levels in non-polarized CACO-2BBE cells and only becomes upregulated as the cells differentiate to assembly an ordered apical BB, as is seen with other IMAC components (Fig. 8O). Confocal microscopy of polarized CACO-2BBE cells (12 days post- confluency) revealed marked enrichment of CALML4 at the distal tips of BB microvilli

(Fig. 8I). We confirmed this localization using EGFP-CALML4 as a genetically encoded probe in CACO-2BBE cells. Identical to endogenous localization, this construct exhibited robust targeting to the distal tips of BB microvilli (Fig. 8J). Line-scan analysis of endogenous and EGFP-CALML4 signal along the axis of microvilli revealed nearly identical peak signal at 0.93 and 0.92, respectively, in reference to the length along the microvillar axis (with 0= base and 1= tip; Fig. 8 K,L). Finally, if CALML4 is indeed a 34

Figure 8. CALML4 localization in native intestinal tissue and CACO-2BBE monolayers. (A-D) Confocal microscopy of mouse duodenal, jejunum, ileum and colon tissue stained for CALML4 (green) and villin (red; used as a BB marker). Boxed region denotes area in zoomed image panels. Dashed area in the zoomed images denotes individual enterocytes. Arrows highlight distal tip enrichment of CALML4 signal. (E- H) Line-scan analysis of CALML4 and villin signal along the microvillar axis in mouse intestinal tissue sections (duodenum, jejunum, ilium, and colon). Each plot shows a collection of 20 normalized intensity scans with respect to the normalized microvillar axis (where 0 = base and 1 = tip). (I) Confocal microscopy of CACO-2BBE cells (12 days post-confluency) stained for F-actin (magenta) and endogenous CALML4 (green). Boxed regions denote areas in zoomed image panels. (J) Confocal microscopy of

CACO-2BBE cells (12 days post-confluency) stained for F-actin (magenta) and EGFP tagged CALML4 (green). Boxed regions denote areas in zoomed image panels. Arrows point to examples of distal tip enrichment of CALML4 in clustering microvilli. (K) Line-scan analysis of endogenous CALML4 and F-actin signal along the microvillar axis in CACO-2BBE cells (12 days post-confluency). Each plot shows a collection of 20 normalized intensity scans with respective to the normalized microvillar axis (where 0 = base and 1 = tip). (L) Line-scan analysis of EGFP-CALML4 and F-actin signal along the microvillar axis in CACO-2BBE cells (12 days post-confluency). Each plot shows a collection of 20 normalized intensity scans with respective to the normalized microvillar axis (where 0 = base and 1 = tip). (M-N) Confocal microscopy of CACO-2BBE cells (12 days post-confluency) triple-stained for F-actin (magenta) PCDH24 (blue) and CALML4 (green). Triple-color merged image is shown in (M), and individual channels along with a merge of PCDH24 and CALML4 only is shown in (N). (O) Time course of expression of CALML4 during CACO-2BBE differentiation. Scale bars: A-D= 20μm, J, M, N= 30μm component of the IMAC, we reasoned that endogenous CALML4 should colocalize with other IMAC components. Consistent with this prediction, triple labeling of CACO-2BBE monolayers for PCDH24, CALML4 and F-actin showed colocalization of CALML4 with

PCDH24 at the tips of clustering microvilli (Fig. 8M,N). Thus, these data demonstrate that

CALML4 is positioned correctly in the enterocyte BB to be a bona fide component of the

IMAC.

3.5 CALML4 interacts with Myo7b

To begin to shed light on the functional role of CALML4, we performed pairwise protein pull-down analyses of CALML4 with the other known IMAC components to map how this novel component incorporates into the complex (Fig. 9A-F). Pull-down analyses using the recombinant cytoplasmic domains of PCDH24 and MLPCDH fused to GST incubated with COS-7 cell lysates expressing EGFP-tagged CALML4 failed to detect any interactions between these components (Fig. 9B). Similarly, no interactions were detected between Flag-tagged CALML4 and EGFP-tagged constructs of full-length, concatenated, and isolated domains of ANKS4B when they were co-expressed in COS-7 cells (Fig. 9C).

As well, no interactions were seen with Flag-tagged CALML4 and myc-tagged constructs of full-length, concatenated, and the isolated domains of harmonin using a similar pull- down strategy (Fig. 9D). Pull-down analyses did reveal, however, a robust interaction between V5-tagged CALML4 and Flag-tagged Myo7b co-expressed in COS-7 cells.

Further domain mapping studies demonstrated that CALML4 interacts specifically with the neck region of Myo7b (Fig. 9E.F), revealing that CALML4 acts as a light chain for this myosin.

To assess whether CALML4 interacts with the neck region of Myo7b in a cellular context, we employed a ‘nanoscale live-cell pull-down’ approach that has recently been developed by the Friedman lab (Bird et al., 2017). This method involves fusing a bait protein to a truncated heavy meromyosin (HMM) form of Myosin-10 (Myo10-HMM) to artificially target the chimera to the tips of filopodia of non-epithelial cells (Fig. 10A). This

Myo10-HMM-bait construct can then be used to interrogate protein-protein interactions against prey proteins in a pairwise manner, by assessing whether the Myo10-HMM-bait 37

Figure 9. Analysis of binding interactions between CALML4 and the IMAC components. (A) Cartoon domain diagrams of IMAC components. (B) Mapping binding interactions between CALML4 and cytoplasmic domain (CD) of PCDH24 and MLPCDH; GST-tagged CD of PCDH24 and MLPCDH served as bait whereas EGFP- tagged CALML4 served as prey. (C) Pull-down analysis of CALML4 and with ANKS4B. FLAG-tagged CALML4 served as bait whereas EGFP-tagged fragments of ANKS4B served as prey. (D) Mapping binding interactions between CALML4 and subdomains of harmonin; FLAG-tagged CALML4 served as bait whereas myc-tagged fragments of Harmonin served as prey. (E) Mapping binding interactions between CALML4 and the motor and neck domain of Myosin-7b; FLAG-tagged Myo7b motor and Myo7b motor neck constructs served as bait whereas V5-tagged CALML4 served as prey. (F) Mapping binding interactions between CALML4 and subdomains of Myosin-7b tail; FLAG-tagged Myo7b tail subdomains served as bait whereas V5-tagged CALML4 served as prey.

Figure 10. Nanoscale live-cell pull-downs between CALML4 and Myo7b. (A) Schematic diagram of the nanoscale live-cell pull-down procedure. Interaction- dependent colocalization is interrogated between a bait and a prey, in which the bait has been forcibly targeted to the tips of filopodia using the motor activity of Myo10. Bait is attached to the C-terminus end of EGFP-Myo10-HMM. Prey is tagged with mCherry. Myo10, Myosin10; SAH, Stable Alpha Helix; CC, Coiled-Coil. (B) HeLa cells expressing EGFP-Myo10-HMM-7BIQ12345 (green) and mCherry-CALML4 (red). Boxed region denotes area in zoomed image panels. F-actin is labeled with Alexa-647 phalloidin (magenta). (C) HeLa cells expressing EGFP-Myo10-HMM-empty (green) and mCherry-CALML4 (red). Boxed region denotes area in zoomed image panels. F- actin is labeled with Alexa-647 phalloidin (magenta). (D) Scatter plot of bait (x-axis) and prey (y-axis) fluorescence at individual filopodia tips. mCherry-CALML4 fluorescence is linearly correlated with EGFP-Myo10-HMM-7BIQ12345 fluorescence and is significantly higher than the HeLa cells expressing EGFP-Myo10-HMM-empty. Scale bars: B, C=20μm chimera forces the prey protein to localize to filopodia tips when expressed together (Fig.

10A). We fused the isolated neck region of Myo7b to the Myo10-HMM bait construct to generate EGFP-Myo10-HMM-7BIQ12345 and expressed it along with mCherry-

CALML4 in HeLa cells (Fig. 10B). Cells were then processed to visualize the fluorophores along with the F-actin cytoskeleton. As controls, mCherry-CALML4 was expressed alone and with EGFP-Myo10-HMM empty bait vector in HeLa cells (Fig. 10C). Importantly,

HeLa cells do not express endogenous CALML4 and when mCherry-CALML4 is expressed alone, it does not accumulate at the tips of filopodia (Fig. 11A,B). However, when expressed with EGFP-Myo10-HMM-7BIQ12345, we observed striking colocalization of mCherry-CALML4 to filopodia. We quantified this interaction by intensity correlation analysis. The linear correlation between EGFP-Myo10-HMM-

7BIQ12345 and mCherry-CALML4 is significantly higher (3.6 fold higher) than the empty bait control (Fig. 10D). We did note minor tip localization of mCherry-CALML4 when

Figure 11. Localization of CALML4 in HeLa cells. (A) Western blot analysis of

endogenous CALML4 in lysates from wild-type CACO-2BBE cells, scramble control

CACO-2BBE cells (20 days past-confluency) and HeLa cells. GAPDH was used as a loading control. (B) HeLa cells expressing mCherry-CALML4 (red). Boxed region denotes area in zoomed image panels. F-actin is labeled with Alexa-647 phalloidin (magenta). Scale bars: B, C=20μm expressed with empty EGFP-Myo10- HMM bait vector, but not when expressed alone in

HeLa cells (Fig. 10C,D). This suggests that CALML4 may be able to weakly interact with the one or more of the native IQ motifs found in the EGFP-Myo10-HMM construct.

However, together with our biochemical data, these results suggest that CALML4 functionally integrates into the IMAC by interacting with the neck region of the motor protein Myo7b.

3.6 CALML4 targeting to the BB requires Myo7b

Given that the sole interaction mediated by CALML4 within the IMAC is with Myo7b, we sought to investigate whether targeting of CALML4 to the BB was dependent upon this

Figure 12. CALML4 BB targeting is dependent on Myo7b. (A) Confocal images of Myo7b KO mouse duodenum tissue stained for villin as a BB marker (magenta) and CALML4 (green). Boxed regions denote the area in zoomed image panels. (B) Line- scan analysis of CALML4 and villin signal along the microvillar axis in WT and Myo7b KO duodenum mouse intestinal tissue sections. Each plot shows a collection of 20 normalized intensity scans with respective to the normalized microvillar axis (where 0 = base and 1 = tip). (C) Confocal images of CACO-2BBE cells (12 days post-confluency) stably expressing either a scramble shRNA construct or an shRNA targeting Myo7b, stained for F-actin (magenta) and CALML4 (green). Boxed regions denote the area in zoomed image panels. Dashed lines represent the location where x-z sections were taken; x-z sections are shown below each en face image. (D) Western blot analysis of endogenous Myo7B in lysates from scramble control and two independent shRNA Myo7b KD stable CACO-2BBE lines (20 days post-confluency). GAPDH was used as a loading control. Scale bars: A= 20μm, C= 30μm interaction. To this end, we assessed the targeting of endogenous CALML4 in duodenal intestinal tissue sections from a Myo7b KO mouse (Fig. 12A). Strikingly, CALML4 was completely lost from the BB in Myo7b KO mice. Indeed, line-scan analysis of the distribution of CALML4 along the microvillar axis clearly demonstrated that CALML4 fails to enrich within the BB in the absence of Myo7b (Fig. 12B). The population of

CALML4 found at the junctional margins of Myo7b KO enterocytes, albeit low, was still evident. We further assessed the dependence of CALML4-Myo7b interaction on proper

BB targeting of CALML4 using two independent stable Myo7b KD CACO-2BBE cells lines that had been previously validated (Weck et al., 2016). Identical to our Myo7b KO mouse, endogenous CALML4 fails to target to the BB in CACO-2BEE cells lacking Myo7b (Fig.

12C). Together with our protein pull-down data, these studies demonstrate that CALML4 targets to the enterocyte BB by directly interacting with the IMAC component Myo7b.

3.7 CALML4 is necessary for proper BB assembly

Loss of any of the previously identified components of the IMAC disrupts proper enterocyte BB assembly, as observed in many systems including CACO-2BBE enterocytes, intestinal tissue from USH1C patients, and a Harmonin KO mouse model (Bitner-

Glindzicz et al., 2000; Crawley et al., 2014b; Crawley et al., 2016; Hussain et al., 2004;

Weck et al., 2016). In particular, CACO-2BBE enterocytes serve as a robust model to study

IMAC function. IMAC-mediated adhesion between the distal tips of neighboring microvilli promotes the formation of ‘tepee-like’ clusters of microvilli on the apical surface of CACO-2BBE cells during BB assembly. The formation of microvillar clusters gives us a readily observable metric of the IMAC activity. To assess the role of CALML4 in proper

BB assembly, we used lentivirus-mediated transduction to create stable CACO-2BBE shRNA KD cell lines targeting CALML4. We screened 4 different shRNA constructs and identified two independent shRNAs with a KD efficiency above 90% as assessed by western blot analysis (Fig. 13B). Both of these cell lines were allowed to polarize 12 days past confluency and were then processed to visualize endogenous CALML4 and the F- actin cytoskeleton. In both cases, scoring of CACO-2BBE monolayers showed that 43

Figure 13. CALML4 KD disrupts proper BB assembly. (A) Confocal images of CACO-2BBE cells (12 days post-confluency) stably expressing either a scramble shRNA construct or an shRNA targeting CALML4, stained for F-actin (magenta) and CALML4 (green). Boxed regions denote the area in zoomed image panels. (B) Western blot analysis of endogenous CALML4 in lysates from untransduced, scramble control, and two independent shRNA CALML4 KD stable CACO-2BBE cell lines (20 days post-confluency). GAPDH was used as a loading control. (C) Quantification of microvillar clustering in scramble and CALML4 KD stable CACO-2BBE cell lines (12 days post-confluency). ****p < 0.0001, t test Scale bars: A= 30μm depletion of endogenous CALML4 resulted in a dramatic loss of microvillar clustering relative to scramble control cells (Fig. 13A). Specifically, only ~20% of the cells in

CACO-2BBE CALML4 KD monolayers exhibited microvilli clusters, whereas ~70% of WT

CACO-2BBE cells had clusters (Fig. 13C). Importantly, KD of CALML4 in CACO-2BBE

cells phenocopies the loss of Myo7b (Weck et al., 2016)(Fig, 12C). We noted that overexpression of EGFP-CALML4 in CACO-2BBE cell lines did not result in increased microvillar clustering (Fig. 8J), indicating that CALML4, by itself, is not sufficient to drive

BB assembly. This is in contrast to stable overexpression of PCDH24-EGFP in CACO-

2BBE cells, which directly promotes microvillar clustering, and drives mature BB assembly

(Crawley et al., 2014b; Crawley et al., 2016). In combination with our pull-down data, our results here suggest that CALML4 is a critical light chain for Myo7b and that loss of

CALML4 disrupts BB assembly likely through Myo7b dysfunction.

3.8 CALML4 is monomeric and lacks the ability to bind calcium with high affinity

Given that CALML4 is a novel protein that has yet to be directly characterized in the literature in any context, we undertook a basic biophysical analysis of CALML4 to assess its calcium binding ability and oligomeric properties. As a starting point, we first devised a protein purification protocol to obtain pure recombinant CALML4 from E. coli.

The short isoform of CALML4 was expressed and purified to homogeneity as a 6XHis-

TEV-tagged protein using Ni-NTA affinity resin, followed by anion exchange and size exclusion chromatography. 6XHis-TEV-CALML4 is 169 residues in length, has a calculated mass of 19.5 kDa, and electrophoresed on a 15% SDS gel with an apparent size of ∼17 kDa (Fig. 14A). Mass spectrometry of this sample revealed a mass of ~19.4 kDa

(data not shown), confirming that our recombinant product was fully intact and that the slight anomalous migration observed on SDS-PAGE is an inherent property of the full- length protein. Sedimentation velocity experiments performed in the absence of Ca2+ over a range of CALML4 concentrations (0.4–1.2 mg/ml) showed a dominant peak with a

Figure 14. CALML4 is monomeric and does not bind to calcium with high affinity. (A) SDS-PAGE of the final purified HIS-TEV-CALML4. (B) Sedimentation velocity analysis of HIS-TEV-CALML4 shows that it is a monodisperse species that is primarily a monomer. Far-UV (C) and Near-UV (D) circular dichroism analysis of HIS-TEV- CALML4 in the presence of either 5 mM CaCl2 or 2 mM EDTA, showing that CALML4 does not bind Ca2+. predicted molecular mass of 19.5 kDa (Fig. 14B), suggesting that CALML4 is primarily a monomer solution. A minor peak could also be detected that corresponded roughly to a dimer form of CALML4, however, it represents a very small percentage (~0.1%) of the total protein population.

Like conventional calmodulin, CALML4 is predicted to be comprised of two lobes that each have 2 EF-hand motifs. Conventional calmodulin and the short isoform of

CALML4 share 46% identity and ~67% similarity at the amino acid level. In contrast to

conventional calmodulin however, the EF hands of CALML4 contains substitutions that deviate from the canonical residues involved in high affinity Ca2+ binding and are predicted to not sense changes in Ca2+ levels (Mazumder et al., 2014),. To directly investigate whether CALML4 can bind Ca2+, we assessed the effect of this divalent cation on the secondary structure of CALML4 using far-UV circular dichroism (CD) spectroscopy. In the absence of Ca2+, the far-UV CD spectrum of CALML4 exhibited a large positive peak of ellipticity at 196 nm and negative peaks near 208 and 222 nm, typical of proteins that have a significant amount of α-helical secondary structure (Fig. 14C). In the presence of

Ca2+ there was no significant change in the intensities at the 196, 208, and 222 nm peaks, with the overall shape of the two spectra being nearly identical. This suggests that

CALML4 does not bind Ca2+ with appreciable affinity. In agreement with this, near-UV

CD spectra of CALML4 collected in the presence and absence of Ca2+ did not show any significant difference either (Fig. 14D). It should be noted, however, that we were able to generate large quantities of 6XHis-TEV-CALML4 (typical yield ~5mg/liter of culture) that remained soluble even when concentrated to high levels and that both the far- and near-

UV spectra show that CALML4 is well-folded, arguing against the possibility that our recombinant CALML4 is behaving aberrantly due to improper folding. Altogether, our biophysical analysis of recombinant CALML4 suggests that it functions as monomer and that it does not sense and respond to physiologically relevant changes in Ca2+ levels that occur in cells.

3.9 CALML4 is expressed in inner ear hair cells and interacts with Myo7a

To date, every known component of the IMAC has a functional equivalent operating in the USAC. Both the IMAC and USAC contain a class 7 myosin that use their 47

motor properties to mediate proper localization/function of their respective adhesion complex. The myosin found in the IMAC, Myo7b, exhibits 51% identity and 70% similarity in amino acid sequence compared to the functional equivalent, Myo7a, found the

USAC (Fig. 15). The overall domain structure of Myo7a and Myo7b is conserved; they each contain an N-terminal motor domain, followed by a neck region that functions as a lever arm, and a C-terminal tail region comprised of two MyTH4 domains with an intervening SH3 domain (Fig. 15). Two notable difference are found between these two myosins: there is a short stable α-helix (SAH) that functions to extend the lever arm of

Myo7a that is not found in Myo7b, and Myo7b possesses a short insertion in its motor domain, which may promote enhanced actin binding. The functional implications of these differences are not currently known.

Figure 15. A schematic diagram of human Myo7b and Myo7a constructs. Myo7b (1–2116 amino acids) and Myo7a (1–2215 amino acids) exhibit considerable homology, have similar domain composition with the exception of a Myo7b-specific motor insertion (not shown) and a Myo7a-specific stable alpha helix (SAH) domain with the lever arm of this myosin.

Our discovery of CALML4 as a light chain for Myo7b within the IMAC lead us to speculate that CALML4 may have a functional equivalent within the USAC. To that point, a proteomic analysis of mouse inner ear hair cells identified CALML4 as being expressed and exclusively enriched in hair bundle stereocilia (Ebrahim et al., 2016). In addition, CALML4 was recently identified as part of an immuno-enriched protein fraction

Figure 16. CALML4 interacts with Myo7a. Mapping binding interactions between CALML4 and the motor and neck domain of Myosin-7a; FLAG-tagged Myo7a motor and Myo7a motor neck constructs served as bait whereas V5-tagged CALML4 served as prey.

derived from stereocilia using a monoclonal antibody against the tail domain of Myo7a

(Morgan et al., 2016). This immuno-enriched protein fraction, however, also had other myosins found in it, making it hard to draw a firm conclusion about whether CALML4 and

Myo7a are direct binding partners using this data. In light of this, we tested whether

CALML4 could directly interact with Myo7a using pull-down analyses. We observed a robust interaction between V5-tagged CALML4 and a Flag-tagged Myo7a motor-neck construct lacking the SAH in pull-down assays from COS-7 cells (Fig. 16). Importantly, we detected no interaction between CALML4 and the motor domain alone of Myo7a, suggesting that CALML4 specifically associates with the neck region of Myo7a. We further confirmed this interaction in a cellular context using nanoscale live-cell pull-downs.

As before with Myo7b, we fused the isolated neck region of Myo7a to EGFP-Myo10-

HMM as bait and expressed it along with mCherry-CALML4 in HeLa cells (Fig. 17A).

Cells were then stained to visualize the F-actin cytoskeleton and imaged using confocal microscopy. Consistent with CALML4 associating with the neck region of Myo7a, we observed striking colocalization of mCherry-CALML4 to filopodia when expressed with

EGFP-Myo10-HMM-7AIQ12345 (Fig. 17A). The linear correlation between EGFP-

Myo10-HMM-7AIQ12345 and mCherry-CALML4 is significantly higher (8.4 fold

Figure 17. Nanoscale live-cell pull-downs between CALML4 and Myo7a. (A) HeLa cells expressing EGFP-Myo10-HMM-7AIQ12345 (green) and mCherry- CALML4 (red). Boxed region denotes area in zoomed image panels. F-actin is labeled with Alexa-647 phalloidin (magenta). (D) Scatter-plot of bait (x-axis) and prey (y-axis) fluorescence at individual filopodia tips. mCherry-CALML4 fluorescence is linearly correlated with EGFP-Myo10-HMM-7AIQ12345 fluorescence and is significantly higher than the HeLa cells expressing EGFP-Myo10-HMM-empty. Scale bars=20μm higher) than our empty bait control (Fig. 17B). Thus, we conclude that CALML4 also associates with and functions as a light chain for Myo7a.

3.10 CALML4 associates with IQ3 in both Myo7a and Myo7b

Both Myo7a and Myo7b contain five IQ motifs in their neck region that are conserved in sequence (Fig. 18A). To gain insight into which IQ motif(s) CALML4 associates with in these myosins, we created truncation constructs of Flag-tagged Myo7a and Myo7b lacking sequential IQ motifs. These truncation constructs were transfected into 50

Figure 18. CALML4 interacts with Myo7b IQ34 and Myo7a IQ3. (A-B) The lever arm of both Myo7a and Myo7b are comprised of 5 IQ motifs that exhibit considerable homology. The unusual proline residue found in IQ3 of Myo7b is highlighted in red. (C-D) Mapping binding interactions between CALML4 and IQ motifs found in Myo7a and Myo7b. Truncation beyond IQ4 results in loss of CALML4 association with the neck region of Myo7b, while truncation beyond IQ3 results in loss of CALML4 association with the neck region of Myo7a. See text for details. (E) Isothermal titration calorimetry showing that CALML4 does not bind to isolated IQ4 of Myo7b. Interaction of D. discoideum calmodulin with IQ1 from D. discoideum MyoA is used as a positive control. (F) Mapping binding interactions between CALML4 and IQ motif constructs from Myo7b reveals that the association of CALML4 with IQ3 in Myo7b is dependent upon the presence of IQ4. See text for details. 51

HEK-293T cells along with EGFP-tagged CALML4 and pull-downs performed.

Interestingly, while CALML4 binding was lost when IQ3 was truncated from Myo7a (Fig.

18D), we observed that Myo7b lacking IQ4 no longer interacted with CALML4 (Fig. 18C).

This apparent discrepancy between these two myosins lead us to examine the interaction of CALML4 with Myo7b in more detail. To further test the interaction between Myo7b

IQ4 and CALML4, we performed isothermal titration calorimetry (ITC) using a synthetic peptide based on the Myo7b IQ4 sequence (Fig. 18E). As a control, we titrated conventional calmodulin with a synthetic peptide based on the first IQ motif in the class 1 myosin, MyoA, from Dictyostelium discoideum. The neck region of D. discoideum MyoA contains two IQ motifs that both utilize calmodulin as their light chain, with IQ1 interacting with calmodulin with sub-micromolar affinity both in the presence and absence of Ca2+

(Crawley et al., 2011). We failed to detect any interaction between CALML4 and Myo7b

IQ4 (Fig. 18E), while we detected robust interaction with our control experiment. On closer inspection, we noted that IQ3 of Myo7b contains a proline residue in the center of the motif, whereas the corresponding residue in Myo7a is a lysine (Fig. 18B).

We speculated that this proline residue in Myo7b IQ3 might prevent this IQ motif from folding into an α-helix on its own, and that IQ3 of Myo7b may require one or more neighboring IQ motifs to attain its required secondary structure necessary for interaction with CALML4. To test this, we engineered constructs that fused EGFP to the full-length neck of Myo7b (EGFP-7B-IQ12345), concatenated IQ3 and IQ4 (EGFP-7B-IQ34), as well as concatenated IQ4 and IQ5 (EGFP-7B-IQ45) (Fig. 18F). These constructs were transfected into HEK-293T cells along with Flag-tagged CALML4 for pull-down experiments. We observed robust interaction of CALML4 with the full neck region of 52

Myo7b, along with a weaker, but detectable, interaction with isolated 7B-IQ34. No interaction, however, was observed between CALML4 and isolated 7B-IQ45 (Fig. 18F).

We interpret these results as meaning CALML4 interacts with IQ3 in Myo7b, but binding is dependent upon neighboring IQ4. In sum, our results suggest that CALML4 functions as a Myo7a and Myo7b light chain by associating with IQ3 in both these myosins.

Chapter 4

Discussion

4.1 CALML4 is a novel IMAC component

We devised an innovative protein isolation strategy to enrich for factors that associate with the cytoplasmic domain of PCDH24. In using this strategy, we identified

CALML4 as a novel IMAC component. In addition to the fact that CALML4 was discovered using this protocol, the evidence that CALML4 is a bona fide component of the

IMAC may be summarized as follows: (i) endogenous CALML4 is highly enriched at the tips of BB microvilli (the site of IMAC function) in intestinal tissue and CACO-2BBE cells,

(ii) EGFP-tagged CALML4 expressed in CACO-2BBE cells also exhibits robust targeting to the tips of BB microvilli, (iii) CALML4 directly interacts with the IMAC component,

Myo7b, (iv) BB targeting of CALML4 is strictly dependent upon Myo7b, as demonstrated using a Myo7b KO mouse model and shRNA-mediated KD of Myo7b in CACO-2BBE cells, and finally (v) loss of CALML4 results in BB assembly defects that phenocopy the loss of other IMAC components. We believe that these data provide compelling evidence that

CALML4 represents a novel, sixth member of the IMAC.

4.2 CALML4 is the second component shared genetically between the IMAC and

USAC

Although they have evolved to serve different tissues that have seemingly unrelated physiological functions, the IMAC and USAC bear remarkable homology in their compositions (Crawley et al., 2016). This striking homology raised the intriguing possibility that CALML4 or a ‘CALML4-like’ molecule may be a component of the

USAC. To that point, a proteomic analysis of mouse inner ear hair cells identified

CALML4 as being enriched in hair bundle stereocilia (Ebrahim et al., 2016) and CALML4 was recovered in a Myo7a pull-down assay from stereocilia lysates (Morgan et al., 2016).

We demonstrated using both biochemical and live-cell pull-down assays that CALML4 can indeed bind to Myo7a. In line with our data, we propose that CALML4 also represents a new USAC component. This discovery further reinforces the molecular parallels between the IMAC and USAC. Future studies investigating the localization and localization determinants of CALML4 in hair cells will be important to validate CALML4 as a bona fide component of the USAC.

4.3 CALML4 functions as a light chain for Myo7a and Myo7b

Myosins use the energy derived from ATP hydrolysis to generate mechanical force that can be used in the cell to do work (Weck et al., 2017). Small conformational changes in the motor domain that occur upon F-actin stimulated ATP hydrolysis result in a rotation of the neck region relative to the motor domain (Fig. 19A). The neck region acts as a rigid lever arm when stabilized by myosin light chains (Heissler and Sellers, 2014). This allows the neck to amplify the conformational changes that occur in the motor domain into a large

Figure 19. The myosin neck region acts as a lever arm when bound to light chains. (A) Small conformational changes in the myosin motor domain are translated into a large swing of the lever arm. Image adapted from (Spudich and Sivaramakrishnan, 2010). Permission to use granted by Springer Nature, ©Spudich and Sivaramakrishnan, 2010. Originally published in Nature Reviews Molecular Cell https://www.nature.com/articles/nrm2833#rightslink. (B) IQ motifs fold into α-helices that are stabilized by light chain interactions. Shown here are the first two IQ motifs of Myo5a (blue) bound to calmodulins (green and orange). Structure from (Houdusse et al., 2006); PDB code 2IX7. displacement of the cargo-binding myosin tail domain relative to the motor domain that is bound to the F-actin track. Myosin light chains belong to the calmodulin/calmodulin- related family of small EF-hand proteins and recognize their cognate myosin motors by binding to sequences in the myosin neck region known as IQ motifs (Heissler and Sellers,

2014). IQ motifs are found across a diverse array of proteins and conform to the generalized consensus sequence [I,L,V]QXXXRXXXX[R,K], where X is any amino acid with the general exception of proline (Bahler and Rhoads, 2002). Structural studies have shown that IQ motifs fold into straight α-helices, typically with no sharp bends (Houdusse et al., 2006) (Fig. 19B). Light chains interact with their cognate IQ motif(s) in an antiparallel fashion, with the C-lobe of the light chain bound to the more N-terminal region of the IQ motif, and the N-lobe associating with the C-terminal sequence of the IQ motif

(Houdusse et al., 2006) (Fig. 19B). Our interactome analysis revealed that CALML4 incorporates into the IMAC by associating with the neck region of Myo7b. Given the striking homology between IMAC and USAC, this discovery prompted us to explore whether CALML4 could also associate with the homologous myosin found in the USAC, namely Myo7a. Indeed, we discovered that CALML4 also specifically associates with the neck region of Myo7a. Our study, therefore, is the first to assign a cellular role for

CALML4: a myosin light chain.

Both Myo7a and Myo7b possess 5 IQ motifs in their neck region. These IQ motifs exhibit an overall amino acid sequence identity of 50% and sequence similarity of 70%.

Previous studies found that recombinant mouse Myo7a and Drosophila Myo7b, co- expressed with calmodulin alone in the baculovirus expression system, purify with substoichiometric amounts of calmodulin compared to the number of IQ motifs (Haithcock et al., 2011; Sakai et al., 2015; Yang et al., 2005). This indicates that one or more of the

IQ motifs in the neck of these myosins are unoccupied when the motors are purified under these conditions. Direct biochemical binding studies using purified proteins have demonstrated that calmodulin can associate with all the IQ motifs of Myo7A, with the exception of IQ3 (Sakai et al., 2015). This suggests that IQ3 in both Myo7a and Myo7b might stand alone in having a different light chain binding partner compared to the other four IQ motifs. Consistent with this, our refined mapping studies performed in COS-7 cells revealed that CALML4 associates with IQ3 in both Myo7a and Myo7b.

We did note an interesting difference between Myo7a and Myo7b in how they associate with CALML4: binding of CALML4 to IQ3 in Myo7b was dependent upon the

presence of IQ4. Myo7b IQ3 is unique due to the presence of a proline residue (P824) that is found in the center of this IQ motif. As previously mentioned, proline residues are generally not found in IQ motifs given that they act as ‘α-helix breakers’. A cursory search of the neck regions across the myosin superfamily identified only a few members that possess IQ motifs that contain proline residues. Interestingly, the Saccharomyces cerevisiae class V myosin Myo2p has a central proline residue in IQ5 within its six IQ motif-long lever arm (Pennestri et al., 2007; Stevens and Davis, 1998; Terrak et al., 2002;

Terrak et al., 2005; Terrak et al., 2003). The light chain for this IQ motif has not been identified, while calmodulin and the essential myosin light chain Mlc1p have been shown to bind to and stabilize the other IQ motifs in the lever arm (Terrak et al., 2005; Terrak et al., 2003). Whether IQ5 of Myo2p is dependent upon neighboring IQ motifs for its association with a light chain binding partner is currently unknown. The short-tailed class

1 myosin, MyoC, from Dictyostelium discoideum contains a proline residue in IQ3 of its lever arm. This particular IQ motif is the terminal IQ motif in its lever arm and was determined to be non-functional with no light chain binding identified (Crawley et al.,

2011). We speculate that the proline in Myo7b IQ3 prevents this IQ motif from forming an α-helix structure on its own that would be necessary for CALML4 binding. We propose that when the Myo7b IQ4 binds to its cognate light chain (possibly calmodulin), that this

IQ motif is stabilized into an α-helical structure which is partially propagated to IQ3. This would suggest that IQ motifs that contain proline residues may be dependent upon neighboring IQ motifs for proper folding and may require specific light chains. An alternative explanation for the atypical binding exhibited by CALML4 could be that

CALML4 and calmodulin themselves form direct interactions when bound to IQ3 and IQ4, 58

and that these interactions are necessary for CALML4 to bind to Myo7b, irrespective of the P824 residue. Given that the residue corresponding to Myo7b P824 is a lysine in

Myo7a (K805) and our binding data clearly show that association of CALML4 to Myo7a is not dependent upon Myo7a IQ4, we favor the first proposal. Going forward, exploring the effect of a K805P substitution in Myo7a would help clarify the CALML4 binding differences seen between Myo7a and Myo7b.

4.4 CALML4 and Myosin Regulation

Light chains can also serve as sites for regulation of myosin activity, either through phosphorylation or by the direct binding of Ca2+(Heissler and Sellers, 2014). Relevant to our discussion here, the motor activity of Myo7a has been shown to be regulated by Ca2+.

Prior to activation, Myo7a exists in an auto-inhibited state in which the cargo-binding tail folds back to contact and inhibit the motor-IQ region (Sakai et al., 2011; Umeki et al.,

2009; Yang et al., 2009). It is currently thought that Ca2+ binding to the light chains associated with the neck region of Myo7a or cargo binding directly to the tail domain relieves auto-inhibition of this myosin by causing the tail to disengage from the motor-IQ region (Sakai et al., 2011; Umeki et al., 2009; Yang et al., 2009). Similar mechanisms have been characterized to control the motor activity of other myosins, including Myo5a

(Siththanandan and Sellers, 2011).

It is not currently known whether Myo7b exhibits tail-mediated motor-inhibition or if its associated light chains have a role in regulating this myosin in the IMAC. It was recently discovered, however, that cargo binding to the tail of Myo7b occurs in an ordered manner, possibly due to the tail being found in an auto-inhibited state (Cao et al., 2017;

Crawley et al., 2016). The tail domain of Myo7b contains two MyTH4-FERM domains

(MF1 and MF2) with an intervening SH3 domain. MF1 and MF2 associate with the IMAC scaffolds ANKS4B and harmonin, respectively. It was discovered that harmonin has to first associate with MF2 before MF1 becomes ‘available’ to bind to ANKS4B. Whether calcium binding to the light chains associated with the neck region of Myo7b can induce changes in the fold of the tail domain is currently not known. However, our biophysical analysis of CALML4 revealed that this light chain does not bind Ca2+ with a physiologically relevant affinity, suggesting that it likely does not directly participate in any form of Ca2+-dependent regulation of either Myo7a or Myo7b. We propose that

CALML4 plays a structural role in stabilizing the neck region of Myo7a and Myo7b, allowing the neck to act as a rigid lever arm necessary for myosin force production. ln the future, it will be important to determine whether CALML4 plays a structural role in promoting the motile properties of Myo7a and Myo7b, rather than regulation of their motor activity.

4.5 CALML4 is an USH1H candidate gene

Deleterious mutations in USAC components result in USH1, the most common form of deaf-blindness in humans (affecting ~6/100,000 people in the general population;

(Lentz and Keats, 2016). While most USH1 patients only suffer from deaf-blindness, it was noted early on that some patients with mutations in the scaffold harmonin also present with severe inflammatory enteropathy and nephropathy (Bitner-Glindzicz et al., 2000;

Hussain et al., 2004). The discovery that harmonin is a shared component of the IMAC and USAC provided an explanation for the transporting epithelia dysfunction exhibited by

these patients (Crawley et al., 2014b). Our study now identifies CALML4 as the second component shared genetically between the IMAC and USAC. Excitingly, CALML4 is one of 27 candidate genes found within the genetic region mapped to contain the causative allele for USH1H, an USH1 subtype whose gene has not yet been identified (Ahmed et al.,

2009). Our discovery that CALML4 can function as a light chain for Myo7a places

CALML4 as the lead candidate gene for USH1H. If CALML4 is the USH1H gene, we would predict that USH1H patients suffer neurosensory deficits due to CALML4-mediated dysfunction of Myo7a. Consistent with this, there are known point mutations within the neck region of Myo7a that cause USH1 (Adato et al., 1997). This suggests that dysfunction of Myo7a can occur as a result of mutations that interfere with proper light chain binding for this myosin.

Another implication of CALML4 being the gene responsible for USH1H is that these patients may also suffer from undiagnosed transporting epithelia disease, as was seen with harmonin patients. Our identification of CALML4 as the likely candidate for USH1H may allow clinicians to tailor their treatments for these patients with potential enteropathy in mind. Along those lines, it is interesting to note that proteomic analysis of enterocyte

BBs detected small amounts of Myo7a in this isolated fraction (McConnell et al., 2011).

Whether this small amount of Myo7a is functionally incorporated into the IMAC, possibly acting redundant to Myo7b, or whether Myo7a in the BB plays a different role is currently unknown. Going forward, it will be imperative to determine whether or not CALML4 is the gene responsible for USH1H and whether Myo7a has a physiological role in the intestine.

4.6 Long-Term Directions: The IMAC and implications for USH1 research

To date, all known USAC components have been identified using genetic linkage analysis of USH1 patients and mutant mouse lines that exhibit hearing/balance defects.

One problem with this approach is that the discovery of new USAC components is predicated on them being essential for USAC function, but not lethal to the organism upon genetic disruption. It is more than likely that there are USAC components that are either

(i) not essential for function of the complex and, therefore, would not be discovered using this approach, or (ii) those that would be lethal to the organism when they are genetically disrupted. To circumvent these issues, a direct method to identify USAC components is necessary. A number of limitations prevent the direct identification of USAC components using protein-purification approaches from inner ear tissue, the most well-studied USAC system. Vertebrates possess only ~15,000 hair cells that are not easily isolated from the surrounding tissue (Brigande and Heller, 2009), with each hair cell expressing a relatively low amount of the USAC (Krey et al., 2017). This makes using hair cells as a purification source material non-ideal. Furthermore, a malleable cell culture model that recapitulates hair cell stereocilia formation does not exist that could be used as a substitute(Ohnishi et al., 2015). These limitations are not found with the transporting epithelia of the intestine, which use the homologous IMAC to organize their apical microvilli. For the purpose of comparison, it is estimated that there are ~130 billion microvilli/inch2 of intestinal mucosa, with each microvillus connected by ~7 intermicrovillar adhesion links (Crawley et al.,

2014b). In addition, CACO-2BBE cells provide an excellent cell culture model to

Figure 20. A revised comparison between the IMAC and USAC. A revised comparison of the IMAC and USAC, showing that CALML4 represents the second component shared genetically between these two homologous adhesion complexes. Hair cell image is taken from (Hudspeth, 1985). Permission to use granted by The American Association for the Advancement of Science, ©Hudspeth., 1985. Originally published in Science. http://science.sciencemag.org/content/230/4727/745. Scale bars = 5 µm interrogate IMAC biology. Our study provides an elegant example of how the striking homology between the USAC and IMAC introduces a new avenue to explore the pathology underlying USH1, by using the resources available for intestinal research.

Our identification of CALML4 as a putative USAC component represents the first time the homology between the USAC and IMAC has been utilized to discover a new

USAC component and, potentially, a new USH1 gene (Fig. 20). Going forward, we would like to exploit this homology further to explore the pathology of USH1. For example, mutations in calcium- and integrin- binding protein 2 (CIB2) was recently identified to cause USH1J, yet its cellular role is unclear (Jan, 2013; Riazuddin et al., 2012). We have identified the homolog CIB1 as the likely functional equivalent of CIB2 within in the

IMAC. Using cutting-edge resources available for the intestinal research, we can now recapitulate specific pathogenic mutations of CIB2 into CIB1 using CRISR-Cas9 technology in stem-cell derived intestinal enteroids to see how they affect BB assembly.

This knowledge can then be translated over to the USAC system to understand how these mutations result in deaf-blindness. We hope that these types of studies will spur the innovation of treatment therapies for people suffering from epithelia dysfunction due to mutations in either the IMAC or USAC.

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