The Role of Calcium Flux in the Regulation of Filopodia Dynamics

THE ROLE OF CALCIUM FLUX IN THE REGULATION OF FILOPODIA DYNAMICS

Omolade Ademuyiwa

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

August 2019

Committee:

Carol Heckman, Advisor

Michael Geusz

David Giovannucci

Omolade Ademuyiwa

Carol Heckman, Advisor

Filopodia are finger-like projections on cells that allow the cells to sense and explore their external microenvironment. They are required for cell migration, neurogenesis and several other physiological processes. Filopodia dynamics have been widely studied in nerve cells. Here, I investigated the role of increased cytoplasmic calcium levels in the regulation of filopodia through activation of store-operated calcium entry in epithelial cells. The depletion of the endoplasmic reticulum store caused a transient increase in filopodia. The increase in fraction of cells with filopodia and percentage coverage of filopodia was seen at 10 min followed by a decrease at 30 min of treatment with cyclopiazonic acid (CPA). Filopodia dynamics are more sensitive to and regulated by Transient receptor potential cation (TRPC) channels. The downstream effectors of calcium such as calpain and calmodulin are negative regulators of filopodia. The possible mechanisms by which calcium regulates filopodia through these downstream effectors were reported here. The effect of calcium-like protein 2 (CALP2) in filopodia dynamics was first described in this study.

To my parents, Chief A.S.A. Ademuyiwa and Mrs. Adenike Adetoye-Ademuyiwa v ACKNOWLEDGMENTS

My sincere gratitude goes to my advisor, Dr. Carol Heckman. Thank you for standing by me every step of the way towards this achievement. This would not have been possible without you. I appreciate your guidance and for giving me the opportunity to work in your lab. You are like a mother to me and I pray for you this day that God will reward you and create a special place for you in Heaven because you are Heaven sent. I want to thank Dr. Cayer for showing me the ropes in the lab. I have learned so much in the past 2 years and I have you to thank for it. I will also like to thank my committee, Dr Michael Geusz and Dr. David Giovannucci for your guidance and for always ready to render your assistance when needed. In addition, Katie, Blair,

Robyn and Nicole, thank you for all your help with filopodia counts. I will like to thank

Bowling Green State University for my assistantship and general support.

Lastly, I will like to thank my parents. Daddy thank you for my upbringing. I hope you are proud of me in heaven. May you continue to rest in the bosom of the Lord. Mummy, thank you for your infinite prayers and for always encouraging me to be the best I can be.

TABLE OF CONTENTS

Page

CHAPTER ONE. INTRODUCTION ...... 1

1.1 Filopodia structure and formation ...... 1

1.2 Structural components of filopodia ...... 5

1.2.1 Actin filaments ...... 5

1.2.2 Ena/VASP and Mena ...... 7

1.2.3 Small GTPases and their effectors ...... 8

1.2.4 Wiskott-Aldrich syndrome protein (WASP) ...... 9

1.2.5 Fascin ...... 9

1.2.6 Myosin-X ...... 9

1.3 Filopodia regulation ...... 12

1.3.1 Integrins...... 12

1.3.2 Protein kinase C (PKC) ...... 13

1.4 Calcium signaling effect on filopodia formation ...... 14

1.4.1 Calcium in the body ...... 14

1.4.2 Calcium concentration needed for physiological processes ...... 14

1.4.3 Calcium signaling ...... 14

1.4.4 Intracellular store depletion ...... 15

1.4.5 Downstream effectors of calcium ...... 17

1.4.5.1 Calmodulin (CaM) ...... 17

1.4.5.2 Calmodulin kinase (CaMK) ...... 18

1.4.5.3 Calpain ...... 20 vii

1.4.5.4 Calcineurin ...... 20

1.5 Calcium-filopodia relationship ...... 20

1.5.1 Calcium channel effects on filopodia formation ...... 23

1.6 Aims and objectives ...... 23

1.7 Hypothesis ...... 24

1.8 Significance of the study ...... 24

CHAPTER TWO. MATERIALS AND METHODS ...... 25

2.1 Cell culture...... 25

2.1.1 Germanium (Ge) substrates for cell culture ...... 25

2.2 Calcium store depletion and replenishment ...... 25

2.3 Chemical treatment and fixation ...... 26

2.3.1 Fixation ...... 26

2.4 Filopodia determination...... 27

2.5 Relative changes in calcium concentration ...... 27

2.6 Statistical analysis ...... 28

CHAPTER THREE. RESULTS...... 29

3.1 Effect of ER depletion on filopodia formation ...... 29

3.1.2 Effect of ER stress causes an increase in calcium ...... 31

3.2 Effect of calcium influx after store depletion on filopodia ...... 35

3.2.1 Role of calcium channels in the regulation of filopodia ...... 35

3.2.1.1 TRPC ...... 35

3.2.1.2 Orai ...... 35

3.2.1.3 VGCC ...... 36 viii

3.3 Effect of downstream effectors of Calcium on filopodia regulation...... 40

3.3.1 Calpain ...... 40

3.3.2 Calcineurin ...... 40

3.3.3 CaMKII ...... 40

3.3.4 CaM...... 41

CHAPTER FOUR. DISCUSSION ...... 44

4.1 Increased cytoplasmic calcium regulates filopodia...... 44

4.2 Calcium influx through TRPC channel may be important in filopodia

regulation ...... 46

4.3 The role of downstream effectors of calcium in the regulation of filopodia ...... 49

REFERENCES ...... 52

LIST OF FIGURES

Figure Page

1 Architectural arrangement of filopodia in neurons ...... 2

2 GFP-actin time-lapse images showing stages of filopodia dynamics during dorsal

closure at two-minute intervals ...... 4

3 Actin filament assembly and disassembly in filopodia ...... 6

4 Filopodia formation and elongation stages during cell migration ...... 10

5 Structure of integrin and its subunits ...... 12

6 Main paths of calcium flux ...... 16

7 ER depletion effect on filopodia dynamics ...... 30

8 Hypothetical interpretation of time course ...... 32

9 Increase in Ca2+ concentration measured by calcium orange intensity...... 34

10 Role of calcium channels in filopodia regulation ...... 37

11 Inhibitors of proteins that may be downstream effectors during ER stress ...... 42

12 Effect of calpain, calcineurin and CaMKII in filopodia dynamics ...... 43

13 Representation of the observed time course during ER depletion ...... 45

14 Calcium-calmodulin inactivation of VGCC ...... 48

LIST OF TABLES

Table Page

1 Summary of CaMK and their downstream targets ...... 19

2 Experiments carried out on nerve cells with conflicting results ...... 22

3 Post hoc test of means for fraction of cells with filopodia upon

treatment with calcium channel effectors ...... 38

4 Post hoc test of means for percentage coverage of filopodia upon treatment

with calcium channel effectors ...... 39

CHAPTER ONE. INTRODUCTION

1.1 Filopodia structure and formation

Filopodia are actin-rich, spinous projections on cell membranes that allow cells to sense and explore their microenvironment. Their capability of extension and retraction is dependent on several physiological processes. The filopodium was first discovered in the 1960s in the context of migration of mesenchyme during invagination of sea urchin endoderm. At that time, they were called pseudopods and were structures about 0.5 µM in diameter seen during the migration. They were studied using time lapse cinemicrography to monitor the larvae during development

[Gustafson et.al, 1999]. Studying the structure and formation of filopodia in mammalian cell lines with fluorescence microscopy, Ahmed and coworkers observed that filopodia protrude from the cell individually and never in clumps. The filopodia were between 0.6-1.2 µM in diameter and not more than 15 µM long [Ahmed et.al, 2010]. They are present in various cells like the neurons, endothelial cells, and epithelial cells (Fig. 1).

The involvement of filopodia in migration of growth cones was studied by observing the mode of axon migration in embryonic limbs [O'Connor et.al, 1990]. Axonal guidance, also called axonal pathfinding, leads growing axons to specific target sites. The axonal growth cone is probably the system most often used to study filopodia.

Fig. 1. Architectural arrangement of filopodia in neurons (A), migrating cells (B), and epithelial cells (C). https://www.mechanobio.info/cytoskeleton-dynamics/what-are-filopodia/

Filopodia have also been discovered during embryonic dorsal closure in Drosophila.

Jacinto and coworkers monitored the cytoskeletal activity during the fusion of the opposing epithelial sheets [Jacinto et.al, 2000]. Through time lapse confocal analysis with green fluorescent protein (GFP)–actin fusion protein, they observed filopodia protrusion guiding the leading edge to its target site (Fig. 2). There are also a few studies done with the endothelial tip cell, where filopodia mark the direction of growth of the endothelial vessel [ Sawamiphak et.al, 2010].

Fig. 2. GFP-actin time-lapse images showing stages of filopodia dynamics during dorsal closure at two-minute intervals. In the early stage of dorsal closure, filopodia went through three phases.

These include extension, sensory movement and retraction (b, c and d respectively). Similarly, in the zippering stage of dorsal closure, you can see filopodia extending to the opposite leading edge, then they made contact and fused (led to retraction) with the opposite epithelial sheet (f, g and h).

Images from reference [Jacinto et.al, 2000] used by permission of Elsevier.

1.2 Structural components of filopodia

1.2.1 Actin filaments

Actin filaments in filopodia are arranged in parallel arrays by actin-bundling proteins such as fascin. Fascin was localized throughout the length of filopodia and its knockdown of resulted in filopodia loss in melanoma cells of mouse [Vignjevic et.al, 2006]. They are very important for maintenance of cell shape, adhesion, and migration. The filopodia are best understood for the growth cone of the axon. There are three regions in the growth cone known as the central (C), transitional (T) and the peripheral (P) domains. At the peripheral domain, extension and retraction of filopodia helps the growth cone to survey its environment to aid axonal guidance (see [Suter et.al, 2000] for review). Each filopodium contains a bundle of about 10-30 actin filaments. In growth cones, these filaments build up at the peripheral domain and their turnover in this domain is regulated by both depolymerization and actin bundle severing [Medeiros et.al, 2006] as illustrated in Fig. 3. 6

Fig. 3. Actin filament assembly and disassembly in the filopodia and cytoplasmic domains in a growth cone [Athamneh et.al, 2015]. Used under the Creative Commons.

Further investigation of filopodia has revealed that apart from actin filaments, there are some other proteins that contribute to its structure and functions.

1.2.2 Ena/VASP and Mena

Enabled/vasodilator-stimulated phosphoproteins (Ena/VASP), are regulatory proteins highly expressed in developing nervous system. They are found at the leading edge of lamellipodia and at the tip of the filopodia where they form a tetrameric complex [ Breitsprecher et.al, 2011;

Tokuo et.al, 2004]. Inhibition of Ena/VASP consequently resulted in defect of the central nervous system (CNS) and peripheral nervous system (PNS) axonal structure of a fly (see for review

[Krause et.al, 2003]). Their ability to bind to profilin, which is required for actin polymerization, indicates that they are important for regulation of actin architecture [Hüttelmaier et.al, 1999;

Ahern-Djamali et.al, 1999]. The proteins in the Ena/VASP family are characterized by an amino terminal domain, proline-rich central region and a carboxyl terminal domain. Further investigation of their role in filopodia formation was conducted and it was reported that they are not required for cell migration. However, cortical neurons from Ena/VASP knockout mice cannot form neurites [Dent et.al, 2007]. This finding is not surprising as filopodia production is the first step in neurite formation (see [Sainath et.al, 2015] for review). These proteins are important for elongation of the actin filament [Krause et.al, 2003]. They prevent capping of the actin filament at the barbed end and therefore can be thought of as anti-capping proteins [Bear et.al, 2002]. A capping protein protects the plus end of the filament and prevents addition of new actin subunits.

Ena/VASP proteins may also have inhibitory functions in integrin regulation, cell motility and axon guidance (see [Reinhard et.al 2001] for review).

1.2.3 Small GTPases and their effectors

Small GTPases such as Cdc42 and Rho in filopodia (RIF) promote filopodia formation by binding to protein effectors. Many dynamic processes in cells rely on the effect of these small

GTPase interactions (Fig. 4). Filopodia produced by RIF appeared to be longer than those induced by Cdc42 although both interact with Dia2 [Pellegrin et.al, 2005; Campellone et.al, 2010]. Cdc42- induced filopodia projected from the cell periphery while those of RIF emerged from the apical region of the cell. [Pellegrin et.al, 2005]. Cdc42 interacts with Wiskott-Aldrich syndrome protein

(WASP) to activate Arp2/3 complex which is required for actin filament nucleation (see [Suetsugu et.al, 2010] for review). Another important interaction is when a GTPase effector, the insulin- receptor substrate p53 (IRSp53), binds Cdc42 and Rac through its Cdc42/Rac interacting binding

(CRIB) motif. IRSp53 has an I-BAR region containing the MIM domain (missing-in-metastasis- domain), and when overexpressed in cells forms a bridge between the small GTPase and verprolin- homologous protein (WAVE) or Ena/VASP thereby promoting filopodia formation [Aspenstrom et.al, 2014 , Scita et.al, 2008]. WAVE3 has been localized at the tip of the filopodia [Nozumi et.al, 2003]. IRSp53 independently induces filopodia formation by initiating membrane deformity that allows filopodia protrusion [Millard et.al, 2005; Lee et.al, 2007].

Diaphanous-related formin-2 (Dia2), on the other hand, induces filopodia formation from lamellipodia. This was reported from an experiment carried out by Yang and coworkers [Yang et.al, 2007]. They showed that the inhibition of Dia2 disrupted filopodia formation and cell migration. Dia2 has also been localized at the tip of filopodia [Breitsprecher et.al, 2011].

1.2.4 Wiskott-Aldrich syndrome protein (WASP)

The WASP gene was discovered in 1994 as a mutant gene related to Wiskott-Aldrich syndrome hence the name, Wiskott-Aldrich syndrome protein (WASP). The central domain in its structure binds and activates the Arp2/3 complex for actin filament nucleation.

1.2.5 Fascin

It has been shown that the actin bundling filament protein, fascin, is also required for filopodia formation. It guides the actin filaments to extend to the tip of the filopodia thereby acting as an anchor. Phosphorylation of serine 39 in the N-terminal actin-binding domain regulates its activity [Vignjevic et.al, 2006]. Overexpression of fascin molecule has been indicated in certain types of cancer [Kureishy et.al, 2002].

1.2.6 Myosin-X

Myosin-X is essential for initiation of filopodia extension. Through its motor activity, myosin-X transports molecules to the tip of the filopodia [Nagy et.al, 2008]. An experiment carried out to determine the effect of myosin-X on the dorsal surface of Hela cells showed that it induces elongation of filopodia [Bohil et.al, 2006].

Sigal and coworkers reported that lipid phosphatase related protein 1 (LPR1) promotes formation of filopodia at the periphery and dorsal surfaces of cells [Sigal et.al, 2007]. Activity of

LPR1 is independent of other protein influences on filopodia (proteins such as Cdc42, Ena/VASP and RIF) even though the filopodia induced by LPR1 appear identical to those initiated by RIF.

Myosin-X was reported to be localized at the tip of filopodia in HeLa cells that express LPR1 suggesting their interaction [Sigal et.al, 2007]. 10

Fig. 4. Filopodia formation and elongation stages during cell migration [Jacquemet et.al, 2015].

(a) filopodia form adhesion complexes by interacting with extracellular matrix (ECM) during migration, (b) filopodia explore the ECM then form adhesion complexes at the base, shaft, and tip,

(c) filopodia formation is facilitated by proteins such as IRSp53 (or other inverse (I)-BAR domain- 11 containing proteins) which deform and/or tubulate the plasma membrane, and by the motor activity of myosin-X which triggers actin filament convergence at the cell periphery. Within a filopodium, actin crosslinking protein such as fascin or α-actinin bundle actin filaments together, and formins including Dia2 (and/or actin regulators such as Ena/VASP) promote actin filament elongation. Myosin-X also transports various proteins to filopodia tips, including adhesion receptors such as integrins. Copy permitted for use under Creative Common Attribution license.

1.3 Filopodia regulation

Since filopodia are responsible for several physiological processes, it is important to understand how they are regulated. Mallavarapu and Mitchison reported that extension and retraction of filopodia can be regulated by actin filament assembly rate in growth cones of neuroblastoma cells [Mallavarapu et.al, 1999]. Their data suggested that actin filaments assemble at the tip of the filopodia then flow retrogradely as a single unit.

1.3.1 Integrins

Several other proteins have been reported to regulate filopodia but the molecular mechanism is still unknown. The transmembrane protein required for cell to adhere to its extracellular matrix, integrin, is a major component of filopodia regulation. A functional integrin complex has two glycoprotein α and β subunits making a heterodimer (Fig. 5). In mammals, there are 24 α and 9 β subunits.

Fig. 5. Structure of integrin and its subunits forming a cell adhesion complex with cytoplasmic proteins such as vinculin and talin. Figure 19-45 from reference [Alberts et.al, 2015].

Integrin forms cell adhesive contacts with intracellular proteins, vinculin, paxillin, talin, and alpha-actinin, upon activation. Filopodia suppression as a result of integrin signaling was observed with live imaging and quantitative image analysis in developing Drosophila. Working on attachment of tendon to muscle during development, Richier and corkers reported that the loss of αPS2 and βPS integrin subunits caused an increase in filopodia at a stage where they are normally suppressed. The expression of the integrin subunit inhibits filopodia production as soon as filopodia guide the muscle to the site of attachment [Richier et.al, 2018].

A similar observation was reported by Weitkunat and coworkers. Their report indicated that filopodia were needed for initiation of muscle-tendon attachment. βPS integrin accumulated at the myotendinous junction but it wasn’t activated at the initiation stage. It was essential for maturation of the junctional interface [Weitkunat et.al, 2014]. These findings are a major indication of integrin’s role in filopodia regulation.

Myosin-X binds to integrin and transports it to the tip of the filopodia. Myosin regulates filopodia independent of its 4.1/ezrin/radixin/moesin (FERM) domain attachment to integrin

[Bohil et.al, 2016].

1.3.2 Protein kinase C (PKC)

The PKCs are the largest family of serine-threonine protein kinases, consisting of 11 isoforms. These kinases regulate other proteins by their ability to phosphorylate them. PKCs have been shown to regulate filopodia dynamics in cultured epithelial cells [Heckman et.al, 2017]. 14

Consistent with this is a report suggesting that PKC reduces the length of filopodia in the growth cone. PKC was activated with phorbol-myristate-13-acetate (PMA). The filopodia appeared longer when PKC was inhibited [Bonsall et.al, 1999; Cheng et.al, 2000]. The regulatory effect of

PKCα on cell migration is a downstream effect of phosphorylating the actin-binding protein, fascin. Because fascin is required for filopodia formation, this leads to loss of filopodia [Adams et.al, 1999]. Inhibition of PKC/fascin interaction resulted in increased cell migration and fascin- activated protrusion [Anilkumar et.al 2003].

1.4 Calcium signaling effect on filopodia formation

1.4.1 Calcium in the body

Calcium ions (Ca2+) are the most abundant mineral in the body. They are responsible for various physiological processes, such as signal transduction that leads to neurotransmitter release, muscle contraction, vasodilation, contraction, etc. As abundant as calcium is in the body, only about 1% is needed for these physiological roles while 99% is stored in bones.

1.4.2 Calcium concentration needed for physiological processes

At rest, intracellular calcium concentration is less than about 100 nM, but can increase to micromolar concentrations during various cellular functions which are regulated by feedback mechanisms to maintain calcium homeostasis.

1.4.3 Calcium signaling

Intracellular calcium is mostly stored in the endoplasmic reticulum (ER) and mitochondria.

Calcium signaling is driven by calcium influx, release from internal stores or both. The pathway of interest in the context of my work is the inositol triphosphate (IP3)-mediated calcium release initiated by the phospholipase C (PLC) – IP3 signaling pathway as shown in Fig. 6. When an extracellular signal molecule binds with the G-protein coupled receptor (GPCR) on the cell, it 15 activates PLC, an enzyme located on the plasma membrane. The lipase hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to form the products, inositol 1,4,5-trisphosphate

(IP3) and diacylglycerol (DAG). This can also occur with activation of tyrosine kinase receptors or ligand-gated channels. IP3 binds with the IP3 receptor (IP3R) in the membrane of the endoplasmic reticulum. The IP3R is a Ca2+ channel, and its opening leads to calcium influx from the extracellular compartment through various calcium channels on the plasma membrane of a cell

(Fig. 6). This process stimulates the opening of calcium channels on the plasma membrane to allow entry of store-operated calcium (SOC) depending on the strength of the stimulus.

1.4.4 Intracellular store depletion

Release of calcium from the intracellular stores such as the ER activates stromal interaction molecule (STIM) which interacts with store-operated calcium channels such as TRPC and ORAI to induce calcium influx from the extracellular compartment (Fig. 6). 16

Fig. 6. Main paths of calcium flux. Calcium influx can be from Voltage-Gated Calcium Channel

(VGCC) which is controlled by the electrochemical gradient. Calcium can also come in through the transient receptor potential canonical channel (TRPC), which is activated by DAG downstream of receptors. Plasma membrane calcium ATPase (PMCA) pumps calcium out of the cell with the consumption of ATP. Sodium calcium exchanger (NCE) lets in 2 sodium ions and exports 3 calcium ions. The sarcoplasmic or endoplasmic reticulum calcium ATPase (SERCA) is a pump that brings calcium into the ER with the consumption of ATP. The IP3R resides in the membrane of the ER and is also a calcium channel. IP3 binds to its receptor on the ER inducing calcium release. Orai forms the pore of another calcium release-activated channel. 17

Classical experiments on the physiological regulation of voluntary muscles provided another basic model for calcium signaling. For muscle contraction to occur, the cell membrane is depolarized to activate calcium influx through voltage-gated calcium channels. Cytosolic calcium ions bind to ryanodine receptors on the ER to activate calcium efflux from the ER stores (calcium- induced calcium release (CICR)). In the process of muscle relaxation, calcium has to be pumped back into the ER by the SERCA transporter on the ER.

1.4.5 Downstream effectors of calcium

1.4.5.1 Calmodulin (CaM)

Calmodulin is a calcium-dependent protein required for a number of signal transduction pathways in immune response, inflammation, and apoptosis amongst others. It contains 146 amino acids in its structure and has 2 domains known as the N- and C- domains. Each domain contains

EF hand motifs, and hence, it’s able to bind to four calcium ions at once. It has been reported that when the N- and C- domains of calmodulin bind to calcium, they have opposing effects on P/Q - type voltage gated calcium channel also known as Cav2.1 [Zhou et.al, 2005]. The C-domain promotes channel opening while the N-domain activates channel closure. Calmodulin is widely distributed in various types of cells. It has been localized in the cytoplasm as well as in the organelles (see [Mulder et.al, 2007] for review). It has been shown to co-localize with myosin III at the tip of the filopodia in HeLa cells [Erickson et.al, 2003; Rogers et.el, 2001]. Also, a calmodulin-like protein (CLP) has also been found at the tip of the filopodia, like myosin-X

[Bennett et.al, 2007]. This suggests that calmodulin may be a cargo of myosin during filopodia formation.

1.4.5.2 Calmodulin kinase (CaMK)

CaMK are a Ca2+/calmodulin-dependent protein kinase class of enzymes that transfer phosphates from ATP to defined serine or threonine residues in other proteins. They are activated by increased intracellular calcium concentration and are expressed in many cell types. When activated, they phosphorylate transcription factors, consequently, regulate gene expression. The members of these family can be grouped into two based on their downstream targets (Table 1). Calcium/calmodulin- dependent protein kinase II (CaMKII) has been reported to trigger filopodia growth as a mechanism of inducing long term potentiation in hippocampal neurons [Jourdain et al., 2003]. It has also been reported to be activated by calcium oscillations [Koninck et al., 1998]. Myosin light chain kinase (MLCK), a substrate specific CaMK, phosphorylates the regulatory light chain of myosin needed for muscle contraction [Kamm et al., 1985].

Table 1. Summary of CaMK and their downstream targets [Swulius et al., 2008].

Subunit Mechanism of Physiological Kinase Type Composition Activation Targets Role CaMKK Multi- Monomer Ca2+/CaM CaMKI, Gene functional CaMKIV Transcription, Apoptosis CaMKI Multi- Monomer Ca2+/CaM and Synapsin 1, Gene functional CaMKK CREB Transcription, Vesicle Mobilization CaMKII Multi- Dodecamer Ca2+/CaM CaMKII, Synaptic functional Autophosphorylation AMPA/NMDA Plasticity, receptors, L- Regulation of type Ca2+ Ion Channels, channels Gene Transcription CaMKIV Multi- Monomer Ca2+/CaM,CaMKK CaMKIV, Gene functional and CREB, CBP, Transcription Autophosphorylation SRF, HDAC4, Oncoprotein 18 CaMKIII Substrate Monomer Ca2+/CaM Elongation Facilitate Specific Factor 2 Protein Translation MLCK Substrate Monomer Ca2+/CaM RLC of Myosin Muscle Specific Contraction, Intracellular Transport Phosphorylase Substrate Tetramer of Ca2+/CaM PKA Glycogen Glycogen Kinase Specific Tetramers Phosphorylase Metabolism

1.4.5.3 Calpain

Calpain belongs to a family of proteins called the cysteine proteases in mammals as well as other organisms. This protein is calcium dependent for activation. It has been indicated in several physiological processes such as cell apoptosis, long term potentiation in neurons etc. Marthiasen and coworkers showed that calpain activation induced cell death in breast cancer cells after treatment with vitamin D which was used to increase intracellular calcium from the ER

[Marthiasen et al. 2002]. Inhibition of calpain activity in integrin-mediated cell migration has been shown to increase cell adhesion consequently, reducing cell migration. This suggests that calpain is important for cell migration [Huttenlocher et al. 1997; Palecek et al., 1998]. In contrast, Robles and coworkers reported that calpain activity reduces filopodial motility [Robles et al., 2003]. The role of calpain in filopodia protrusion and retraction is not well understood.

1.4.5.4 Calcineurin

Calcineurin is a calcium-calmodulin dependent serine/threonine phosphatase required for activation of T cells of the immune system. The role of this enzyme in filopodia dynamics is not well established although in neurons, it has been reported to have a role in the regulation of growth cone filopodia [Chang et.al., 1995]. Further investigation needs to be carried out to determine its role and possible mechanism in filopodia regulation.

1.5 Calcium-filopodia relationship

Elevated levels of calcium have been reported to induce filopodia formation and elongate existing filopodia in neuronal growth cones [Rehder et.al, 1992; Mattson et.al, 1987; Lau et.al,

1999]. Longer-term elevations of calcium had the opposite effect to short-term elevation, however. Rehder and Kater showed that membrane depolarization with KCl increased cytosolic 21 calcium but the filopodia only remained for 15 min before reversing to the pre-treated state. The treatment with KCl caused a phase of elongation of pre-existing filopodia for about 10 min, then in a second phase the filopodia were lost over 45 min-2 hours [ Rehder et.al, 1992]. The evidence suggested that elevated calcium regulates filopodia in a cyclic fashion that includes the initiation of a phase lasting for about 10 mins followed by an adaptation phase, and then a resting phase.

More recently, Lohmann and coworkers found that high frequency transients elevated calcium and caused the filopodia to cease elongating [Lohmann et.al, 2005]. A similar tendency to stabilize filopodia was noted by Gomez and coworkers, using Xenopus spinal neuron [Gomez et.al, 2001]. Calcium signaling had no role in regulating filopodia in neurons from the dorsal root ganglion [ Bonsall et.al, 1999]. These signaling mechanisms are important, but the results of the previous studies vary depending on the system studied and the endpoints analyzed, as shown in

Table 2.

Table 2. Experiments carried out on nerve cells with conflicting results.

Treatment Ca2+ Filopodia Duration Result Reference

increase initiated

initiated

Electrical 2 secs 5 min 20-30 min linear correlation with Davenport

potential length and number et.al, 1992

Caged Ca2+ immediate 2 min 15 min increased Ca2+ induces Cheng et.al,

elongation 2002

KCl immediate 2 min 15 min but biphasic, stops elongating Rehder et.al,

(17 mM) (length) transient at 10 min 1992

Caged Ca2+ immediate 1-5 min 15 min local Ca2+ transient Lau et.al,

induces new filopodia 1999

branching

BAY 3-fold 1 <1 min > 1 min L-type Ca2+ channels Jacquemet

K8644 min gone regulate filopodia stability et. al, 2015

Caged Ca2+ 5-11sec 2 min 4 min low Ca2+ transient signals Lohmann

facilitate filopodia et.al, 2005

outgrowth

Calpain 15 min >30 min N.A. Calpain inhibitor reverses Robles et.al,

inhibitors Ca2+-induced stabilization 2003

Caged Ca2+ immediate >2 min N.A. rapid Ca2+ transients Gomez et.al,

stabilize filopodia 2001 23

1.5.1 Calcium channel effects on filopodia formation

Several calcium channels have been discovered as shown in the illustration of Fig. 6. Their mechanisms are known, but their effect on filopodia formation is rarely understood.

VGCCs are plasma membrane proteins with selective permeability for calcium. Membrane voltage depolarization induces calcium influx through VGCC in excitable and non-excitable cells.

They are classified into two types based on their voltage dependency and are known as high and low voltage-gated calcium channels. The high voltage-gated channel that has been studied in relation to filopodia is the L-type present in epithelial cells. Ivaska and coworkers blocked the channels with VGCC blockers such as amlodipine, felodipine and so on, and showed that L-type

VGCCs could be a major regulator of filopodia formation [Jacquemet et.al, 2015]. There is evidence that TRPC channels are involved from genetic ablation of the TRPC gene in zebra fish, which prevents the formation of endothelial tip cell filopodia [Yu et.al, 2010]. The imidazole compound SKF96365, which inhibits TRPC channels, is an effective blocker of receptor-mediated calcium entry at concentrations that do not affect Ca2+ release from internal stores. SKF96365 treatment caused reduced cell growth in neuroblastoma through the reverse operation of Na+/Ca2+ exchanger. Na+/Ca2+ exchanger normally exports calcium out of the intracellular store [Song et.al,

2014]. Thus, the data suggested that SKF96365 inhibits the exchanger from taking out calcium from internal stores. However, the above reports only worked on one type of calcium channel at a time.

1.6 Aims and objectives

The goal of this study is to determine the following: 24

1. To make a controlled system in which the filopodia dynamics can be studied in relation to the calcium channels by releasing calcium from intracellular stores followed by calcium replenishment from extracellular compartments.

2. To understand the mechanism by which calcium regulates filopodia formation by studying the relationship between calcium and its downstream effectors such as calpain, calmodulin, calcineurin and CaMKII in filopodia dynamics.

1.7 Hypothesis

I hypothesize that long term elevation of cytoplasmic calcium regulates filopodia by activating a negative regulatory effect on filopodia through its downstream effectors such as calmodulin,

CaMKII, calcineurin and calpain.

1.8 Significance of the study

Filopodia regulation is more widely studied in neurons than other type of cells. This research is focused on filopodia regulation in epithelial cells. Filopodia has been indicated in cancer metastasis. The outcome of this study will be very useful to researchers and pharmaceutical companies in the fight to eradicate cancer progression and metastasis by developing therapeutic drugs needed to hopefully inhibit cancer cell migration.

CHAPTER TWO. MATERIALS AND METHODS

2.1 Cell culture

The 1000 W line was generated from rat tracheal epithelium after in vivo exposure to 7, 12- dimethylbenz(a)anthracene. The cells were maintained in WIHC, a modified Waymouth's medium (Sigma-Aldrich Technologies, St. Louis, MO) containing penicillin, streptomycin, 10%

FBS (fetal bovine serum, Hyclone, UT or Atlanta Biologicals, GA) supplemented with 0.1 µg/ml insulin and 0.1 µg/ml hydrocortisone. The cells were incubated in 37° C and 5% CO2 in air. The cells were subcultured by detachment with a solution of trypsin (Invitrogen, Grand Island, NY) and EDTA made up in a calcium- and magnesium-free Hanks balanced salt solution (CMF-HBSS).

2.1.1 Germanium (Ge) substrates for cell culture

Round glass coverslips of 25mm diameter and thickness #1 (Electron Microscopy Sciences,

Hatfield, PA) were coated with germanium after being rendered molecularly clean by soaking in

1N HCL overnight followed by repeated rinsing with deionized water and 70% ethanol. To make the substrates adhesive for cultured cells, a thin film of Ge (0.02 g, Structure Probe, Inc., West

Chester, PA) was applied in a Denton BTT-IV high vacuum evaporator. The Ge was placed in a tungsten basket (low voltage B) and evaporated with a resistive current of 9 amps as previously described [Heckman et.al., 2017]. Coated coverslips were sterilized by ultraviolet irradiation and placed in 35mm Petri dishes. For experiments, 2–3x105 cells were plated in each 35-mm culture dish and left overnight to become attached.

2.2 Calcium store depletion and replenishment

To deplete the calcium stores, the medium was replaced with CMF-HBSS containing 5 µM cyclopiazonic acid (CPA) or 1.5 µM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'- tetraacetic acid (EGTA). For calcium store replenishment, the cells were first depleted as 26 described above for 30 min followed by 5 min rinse in CMF-HBSS to rinse off CPA from the cells.

Then I transferred the cells into a Ca2+-replete Hanks balanced salt solution (Ca2+-HBSS) for 7.5 min to replenish the cells with calcium.

2.3 Chemical treatment and fixation

Cells were treated with 5 µM cyclopiazonic acid (CPA) to deplete the ER store. To inhibit Orai and TRPC channels respectively, 3,5-bis(trifluoromethyl)pyrazole (BTP2, also called YM-58483) and SKF96365 were used. Nifedipine was used to inhibit voltage-gated calcium channels

(VGCC). The channel blocking reagents were not completely specific for one type of channel.

BTP2 and nifedipine were active against CRAC channels and VGCC channels, respectively at concentrations below 1 µM [Zitt et.al, 2004; Wang et.al, 2018; Franckowiak et.al, 1985]. Because the compounds had no effect at similar concentrations in preliminary experiments, they were used at concentrations several-fold higher than the IC50. SKF96365 and the compounds used against calcium signaling effectors (see below) were used at concentrations around the IC50 for the respective activities [Merritt et.al, 1990; Schrøder et.al, 2008].

ALLN, a cell-permeable, peptide aldehyde inhibitor of the cysteine proteases, calpain, was obtained from Focus Biomolecules (Plymouth, PA). To inhibit the effect of CaM, calcineurin and CaMKII, respectively, calcium-like peptide 2 (CALP 2), calcineurin autoinhibitory peptide

(CAP) EMD Millipore Corp, Germany), and Autocamtide-2-related inhibitory peptide (AIP) were used.

2.3.1 Fixation

Cells were fixed with warm, buffered 3% formaldehyde (pH 7.4) made fresh from paraformaldehyde in cytoskeletal buffer. The samples were rinsed with phosphate-buffered saline and stored in buffer at 4° C until observations were made. 27

2.4 Filopodia determination

Samples were mounted on slides and assigned code numbers before being examined. For each sample, single cells were analyzed by phase contrast microscopy on a Zeiss Axiophot. Each cell a score reflecting the proportion of the perimeter covered with filopodia, as previously described

[Heckman et.al, 2017]. Values obtained from three independent observers, without knowledge of the sample treatment, were averaged for data analysis. The coverage of the cell perimeter could vary considerably in repeated experiments. Consequently, the counts in treated samples were presented as a ratio to counts in the sham-treated control.

2.5 Relative changes in calcium concentration

The level of cytoplasmic calcium was estimated in cells subjected to ER stress. This was done by exposing cells to calcium orange AM (Invitrogen Eugene, OR) for 20 min under normal culture conditions, followed by collecting fluorescence images once per minute in time-lapse mode.

Calcium orange was made up in dimethylsulfoxide (DMSO) and used at a final concentration of

5µg/ml. Fluorescence images were acquired using a rhodamine filter set with wavelength specifications of 546/10 nm excitation and 585/40 emission, on a confocal Leica DMI3000B inverted microscope (Leica Microsystems, Buffalo Grove, IL) equipped with a Lumen Dynamics light engine, Spectra X LED source (Lumencore, Beaverton, OR), X-Light spinning-disk confocal unit (CrestOptics, Rome, Italy) and a Rolera Thunder cooled CCD camera with back-thinned, back-illuminated, electron-multiplying sensor (QImaging, Surrey, British Columbia, Canada), running under MetaMorph software (Molecular Devices, Sunnyvale, CA). Images taken were processed by background subtraction, and the intensity values of the region of interest (ROI) per cell were averaged for each image frame. The time-dependent change in fluorescence was evaluated as: 28

F/F0

Where F, is the measured fluorescence intensity and F0, is the base fluorescence.

2.6 Statistical analysis

Averages and standard deviations were calculated with Microsoft Excel. The analysis of variance was performed with ANOVA statistics which was followed by Scheffe post hoc test. A P value of

<0.05 was considered significant.

CHAPTER THREE. RESULTS

3.1 Effect of ER depletion on filopodia formation

Intracellular calcium is mostly stored in the ER and mitochondria. To test if intracellular calcium induces filopodia loss as reported by Rehder and Kater [Rehder et.al, 1992], I depleted the intracellular stores with CPA. CPA is known to cause net release of calcium from the ER by blocking the SERCA ATPase needed to pump calcium back into the store at the same time. This calcium pump inhibition would consequently increase calcium concentration in the cytoplasm.

The ER stores of cells in four dishes were depleted with CPA for 0, 10, 20, 30 min respectively

(Fig. 7). Then the cells were fixed and filopodia were counted. At 10 min of treatment, the fraction of cells with filopodia and coverage of cells with filopodia increase by 2-fold compared with control but at 30 min of treatment, filopodia are back to the starting point. Although Rehder and

Kater depolarized the plasma membrane with extracellular K+ to increase calcium concentration in the cytoplasm which is different from our approach, the results are similar. They showed a transient increase in filopodia length at 15 min followed by a steep decline in filopodia to the level of control at 35mins.

Fig. 7. ER depletion effect on filopodia dynamics. Fraction of cells with filopodia (Top) and the perimeter covered by filopodia (Bottom). There was a significant difference in the perimeter covered by filopodia between 10 min CPA treatment and control (F=8.333, P=0.003). 30 min CPA treatment showed a significant difference compared to the 10 min treatment (F=8.154, P=0.003). 31

3.1.2 ER stress causes an increase in calcium

If cytoplasmic calcium increases filopodia, then the result (Fig. 7) suggests that calcium concentration increases for 10 min then it is pumped out of the cell by plasma membrane

Ca2+ATPase pump (PMCA) and Na+/Ca2+ exchanger (NCE) (Fig. 8). For further confirmation of this interpretation, I did a time-lapse recording with a confocal microscope to study the increase in calcium concentration with a calcium indicator, calcium orange. Cells were loaded with calcium orange for 20 min and steady state calcium was recorded for about 25 min followed by 30 min recording during CPA treatment to deplete the ER (Fig. 9).

Fig. 8. Hypothetical interpretation of time course. (A) Steady state at time 0 allows Ca2+ to enter and exit the cytoplasm and ER. (B) Net efflux occurs, because there is net Ca2+ release from the

ER causing Ca2+ to be extruded from the cytoplasm. This Ca2+ is taken out of the cell by PMCA and NCE. (C) There is less efflux by 30 min, because the ER is depleted and the driving force for calcium release from the ER has declined.

The result showed significant increase in calcium at 10 min of treatment with CPA and continuously increased afterwards. This suggests that filopodia sprouting occurs during initial increase of calcium but continuous increase may trigger negative regulatory effect on filopodia.

When compared with Fig. 7, the data indicates that filopodia formation is dependent on local calcium transients.

To duplicate my findings, I tried using 2 µM fura-2-AM and 5 µM Fluo-4 but did not get any signal from the cells with various treatments (data not shown). The dye also made them detach from the cover slips and hence interfered with imaging.

3 0 1.5 F/F

0 5 10 15 20 25 30 Time

Fig. 9. Increase in Ca2+ concentration measured by calcium orange intensity with confocal microscopy. At 30 min of CPA treatment, calcium level is higher when compared to time zero.

(P=0.002).

3.2 Effect of calcium influx after store depletion on filopodia

Calcium influx occurs through several calcium channels such as the TRPC, Orai and VGCC. To determine the role of calcium influx in the regulation of filopodia. I replenished the cells with calcium as described in materials and methods. The results show that calcium influx after ER stress increased filopodia when compared with starting point (Fig. 10).

3.2.1 Role of calcium channels in the regulation of filopodia

To determine the role of calcium channels in filopodia dynamics, the calcium channels, VGCC,

TRPC and Orai were blocked with nifedipine, SKF 96365 and BTP2 respectively during the calcium replenishment phase.

3.2.1.1 TRPC

To determine the role of TRPC activation by store operated calcium entry, SKF 96365, a potent inhibitor of TRPC was added during calcium replenishment. Filopodia were reduced in comparison with control (Fig. 10) indicating that TRPC is a positive regulator of filopodia.

3.2.1.2 Orai

Orai, a calcium release-activated channel, has been reported to open allowing calcium influx into the cell when store operated calcium entry is activated. BTP2 is a cell-permeable, potent blocker of STIM and Orai. Treatment with 15 µM BTP2 had no effect on filopodia (Fig. 10) suggesting that calcium influx through Orai did not have an effect on filopodia formation during calcium replenishment phase at that concentration.

3.2.1.3 VGCC

VGCC is widely expressed in non-excitable cells. Calcium influx through L-type VGCC has been reported to increase filopodia in breast cancer cells [Jacquemet et.al, 2016]. They inhibited the channel with calcium channel blockers and their result suggested that L-type VGCC has a positive regulatory effect on filopodia. To test the role of VGCC after store depletion in 1000W cells, I depleted the ER for 30 min with CPA, followed by the Ca2+ replenishment treatment with 10 µM nifedipine in Ca2+ replete HBSS. Inhibition with nifedipine had no significant effect on filopodia

(Fig. 10). These results suggest that it is either not activated or it is rapidly negatively regulated by Ca2+ replenishment. To get supportive evidence on the role of VGCC in the regulation of filopodia, cells were treated with Ca2+ replete KCl HBSS to depolarize the plasma membrane after

ER stress. Filopodia were reduced to half of control (Fig. 10).

Fig. 10. Role of calcium channels in filopodia regulation. (Top) Fraction of cells with filopodia.

There was a significant difference between calcium alone and SKF 96365(F=2.88, P=0.043)

Comparison of calcium alone and 25mM KCl showed a significant difference as well (F= 2.77,

P= 0.04). (Bottom) % coverage compared to starting point. There was a significant difference in the perimeter covered with filopodia between calcium alone and KCl treatment (F=4.26, P=0.04). 38

Table 3. Post hoc test of means for fraction of cells with filopodia upon treatment with calcium channel effectors.

Fraction with filopodia F value P value

BTP vs Ca2+ alone 0.04 0.99

NIF vs Ca2+ alone 0.57 0.68

NIF vs BTP2 0.19 0.94

SKF vs Ca2+ alone 2.88 0.04

SKF vs BTP2 1.69 0.18

SKF vs NIF 1.12 0.36

KCL vs Ca2+ alone 2.77 0.04

KCL vs BTP2 1.61 0.2

KCL vs NIF 1.04 0.4

KCl vs SKF 0.01 1

Table 4. Post hoc test of means for percentage coverage of filopodia upon treatment with calcium channel effectors

% Coverage F Value P Value

BTP vs Ca2+ alone 0.77 0.98

NIF vs Ca2+ alone 1.8 0.7

NIF vs BTP2 0.77 0.98

SKF vs Ca2+ alone 3.43 0.14

SKF vs BTP2 2.29 0.49

SKF vs NIF 1.87 0.67

KCL vs Ca2+ alone 4.26 0.04

KCL vs BTP2 3.03 0.23

KCL vs NIF 2.75 0.32

KCl vs SKF 0.82 0.97

3.3 Effect of downstream effectors of calcium on filopodia regulation

Increased calcium concentration in the cytoplasm activates a host of proteins needed for physiological processes. Downstream effectors of calcium such as calpain, calcineurin, CaMKII and CaM are activated by increased calcium concentration. To determine their effect on filopodia regulation, I treated the cells with the respective inhibitors during Ca2+ depletion and replenishment phases.

3.3.1 Calpain

Calpain’s role as a proteolytic enzyme is well known but its role in cell motility and filopodia protrusion has not been established. During the depletion phase, cells were treated with 150nM and 250nM calpain inhibitor 1 (ALLN). The fraction of cells with filopodia and percentage coverage did not show significant effect during ER stress (Fig. 11). A similar result was observed during the calcium replenishment phase (Fig. 12).

3.3.2 Calcineurin

Cells were treated with a cell permeable calcineurin-autoinhibitory peptide II (CAP). During Ca2+ depletion, and calcium replenishment phases, 20 µM CAP did not have an effect on filopodia (Fig.

11, 12).

3.3.3 CaMKII

Cells were treated with autocamtide-2- related inhibitory peptide (AIP) to inhibit CaMKII activity.

The results (Fig. 11 and 12) showed that 10 µM AIP did not have any effect on filopodia regulation during both phases.

3.3.4 CaM

To determine the role of CaM in filopodia regulation, I inhibited its activity with 20 µM CALP2.

During depletion and replenishment phases, the fraction of cells with filopodia increased significantly suggesting that CaM might have an inhibitory effect on filopodia that might be due to calcium channel inactivation by accumulation of cytoplasmic calcium concentration as suggested by Peterson and coworkers (1999).

Fig. 11. Inhibitors of proteins that may be downstream effectors during ER stress. (Top) Fraction of cells with filopodia (Bottom) percentage coverage compared to starting point. The fraction of cells with filopodia and percentage coverage showed a significant increase with CALP2 treatment during ER depletion compared to ER depletion alone (F=6.46, P=0.001; F=3.01, P= 0.034 respectively). 43

Fig. 12. Effect of calpain, calcineurin and CaMKII inhibitors on filopodia dynamics during the calcium replenishment phase. (Top) Fraction of cells with filopodia. (Bottom) Percentage coverage compared to starting point. The perimeter covered by filopodia showed a significant difference between calcium replenishment alone and CALP2 treatment (F=5.29, P=0.04). 44

CHAPTER FOUR. DISCUSSION

4.1 Increased cytoplasmic calcium regulates filopodia

Intracellular calcium increase has been extensively studied in neurons as reviewed in the

Introduction section. In this present study, I investigated the role of calcium in filopodia using a tracheal epithelial cell line. The fraction of cells with filopodia and the perimeter covered with filopodia were increased transiently. At 30 min, filopodia were back to starting point, and a schematic summary of the dynamic changes are given in Fig. 13. The different outcome of increased calcium concentration on filopodia as described in the Introduction section, collectively suggests the idea that calcium causes a transient change in filopodia. The initiation phase, lasting for about 10 min, is followed by an adaptation phase [Rehder and Kater, 1992].

Fig. 13. Representation of the observed time course during ER depletion. At 30 min of ER depletion, high intracellular calcium level was observed.

Although the calcium was high at 30 min, as shown by calcium imaging, reduced filopodia were observed at this point. I assumed that the intracellular calcium would increase further during replenishment phase after 30 min of ER depletion. I expected to observe less filopodia due to high cytoplasmic calcium levels. It was therefore unexpected to find that filopodia increased again during 7.5 min of calcium re-entry. This further supports the idea that filopodia formation is dependent of calcium transients. Calcium efflux or influx has an effect on filopodia. The main source of calcium during depletion is the ER, and so net traffic of the calcium is efflux from the

ER. The source of calcium during calcium replenishment is the extracellular media, and so the predominating pattern is influx in the cytoplasm and efflux from the cytoplasm into the ER.

4.2 Calcium influx through the TRPC channel may be important in filopodia regulation

Since calcium influx has been indicated to have a role in filopodia, I decided to test for the calcium channel that can be a regulator of filopodia. I blocked Orai, TRPC and VGCC with their respective channel blockers (Fig. 10) during the calcium replenishment phase. These blockers are relatively non-specific, so I selected the final concentrations for treatment on the IC50s for the desired channels. In hippocampal neurons, Orai1 has been shown to be essential for filopodia during synapse formation [Korkotian et. al., 2016]. The BTP2 that was used to block the Orai channel did not have any effect on filopodia at the concentration used (15µM). The inhibition of TRPC channel with SKF caused a significant decrease in the fraction and perimeter covered by filopodia

(Fig. 10). This indicates that TRPC channel may be important in filopodia dynamics and can be targeted for therapeutic purposes to regulate filopodia.

Jacquemet and coworkers (2016) showed that inhibition of L-type VGCC decreased filopodia in breast cancer cell lines. In the tracheal epithelial cells, treatment with 10 µM nifedipine had no significant effect when compared with control during replenishment. This difference in our results could be because of different approach and inhibitors used. Jacquemet and coworkers inhibited the VGCC for 1 hour while I did it for 7.5 min.

To understand the role of VGCC in filopodia dynamics, I depolarized the membrane with

25 mM KCl after ER stress. I expected the channel to open and allow calcium influx but this caused a significant decrease in filopodia prevalence after 7.5 min (Fig. 10). If depolarization caused a high amplitude calcium current, as expected, this may lead to inactivation of VGCC by calcium- dependent inactivation [Peterson et.al., 1999]. This conveys the idea that at 7.5 min of membrane depolarization, the channel may have closed which resulted in reduced filopodia prevalence (Fig.

14).

Fig. 14. Calcium-calmodulin inactivation of VGCC is a possible mechanism by which calcium regulates filopodia. When activated by calcium, calmodulin binds to the IQ motif of the VGCC

[Peterson et.al, 1999]. This leads to inactivation of the channel and may result in less filopodia formation.

This idea made me to inhibit calmodulin during VGCC activation through depolarization (data not shown). This would keep the channel open longer. In preliminary results the percentages observed, as a ratio to the coverage on controls, were 3.19 and 1.75 for depolarization in the presence of CALP2 compared to depolarization alone. Values obtained in the experiment suggested that the filopodia almost returned to the level of Ca2+-replete cells, providing some evidence that calcium-dependent inactivation may be a regulatory pathway relevant to filopodia.

4.3 The role of downstream effectors of calcium in the regulation of filopodia

Calmodulin has been reported to compete with the IP3 receptor for a binding site on the TRPC.

IP3 receptor deactivates calmodulin during activation of TRPC and vice versa to maintain calcium homeostasis in the cell. Accumulation of cytoplasmic calcium has been reported to inactivate several calcium channels by binding to calmodulin in the cytoplasmic region of the channels

[Peterson et. al., 1999; Levitan 1999, Halling et.al, 2005]. I inhibited calmodulin during the ER depletion and replenishment phases using CALP2, a cell-permeable calmodulin antagonist that increases intracellular calcium concentration through calcium flux by inhibiting the mechanism for closing calcium channels. 20 µM CALP2 increased the fraction of cells and perimeter covered with filopodia by 2-fold during ER depletion (Fig. 11). During the calcium replenishment phase, these was a significant increase in the perimeter covered by filopodia with CALP2 treatment (Fig.

12). These results support the ideas by Peterson et. al., 1999; Levitan 1999, Halling et.al, 2005.

Where they suggested that calmodulin might have an inhibitory effect on calcium channels.

Since calmodulin co-localizes with a class of myosin at the filopodia tip of HeLa cells [Erickson et.al, 2003], calmodulin could be regulating filopodia by interacting with calcium channels like 50 the TRPC at the tip of filopodia. The role of CALP2 in filopodia dynamics was described for the first time in this study.

Calcineurin inhibition caused retraction of growth cone filopodia in neurons suggesting it has a role in filopodia dynamics [Chang et.al., 1995]. The inhibition of calcineurin with 20 µM

CAP had no significant effect on filopodia during calcium depletion and replenishment phases

(Fig. 11, 12). This could be due to the non-specificity of the inhibitor.

Overexpression of CaMKII was seen to increase neurite outgrowth and growth cone motility in neuronal cells [Goshima et. al., 1992]. Blockade of CAMKII was also reported to have an effect on filopodia growth of hippocampal neurons [Jourdain et.al, 2003]. To elucidate the role of CaMKII in filopodia, I inhibited the enzyme with a cell-permeable analogue of AIP. No significant effect was observed in the 1000W cells with this inhibitor at 10 µM concentration,

CAMKII might be acting on filopodia of neurons, and more studies should be done to determine its effect on filopodia in epithelial cells.

Several isoforms of calpain have been expressed in the trachea. Three isoforms have been localized in the ER [Hood et.al, 2004; Sakai et.al, 1989] and two on the plasma membrane

[Gil-Parrado et.al, 2003; Lane et.al, 1992]. Calcium release from ER has been shown to activate calpain in breast cancer cells that led to cell death [Mathiasen et. al., 2002]. The mechanism of calpain’s role in filopodia is not well accounted for. The inhibition of calpain by calpastatin in

NIH-3T3 fibroblasts [Potter et.al., 1998] and in neutrophils [Lokuta et.al, 2003] resulted in increased filopodia, suggesting it has an inhibitory role but in growth cones, calpain stabilizes filopodia [Robles et.al, 2003]. I inhibited calpain activity with 150nM and 250nM ALLN during both phases to target calpains on the ER and plasma membrane. The results show that ALLN did 51 not have any significant effect at these concentrations (Fig. 11, 12). Different levels of intracellular calcium have been reported to activate calpain [Melloni et al., 1992, Matsumura et.al., 2001]. The intracellular concentration of calcium at the time of the experiments might not be high enough to activate calpain. Further studies should be done to determine the role of calpain in filopodia dynamics.

In summary, as hypothesized that increased cytoplasmic calcium level activates inhibitory effect on filopodia, calmodulin may be a downstream regulator of calcium in filopodia formation of epithelial cells. Calcium influx through TRPC may be important for filopodia formation. The role of CALP2 in filopodia regulation was first described in this study. More studies should be done in identifying the possible mechanisms by which calcium regulates filopodia.

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