Myth4-FERM Myosin Based Filopodia Initiation

MyTH4-FERM Myosin based filopodia initiation

A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY

Ashley L. Arthur

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

Margaret A. Titus, PhD ADVISOR

July 2020

I would first and foremost like to advisor my mentor, Dr. Margaret Titus, for her unassailable commitment to training, enthusiasm for science and her support of my career. Meg has been superb advisor, has made me a better scientist and communicator and I genuinely enjoyed working for her. I am grateful for the long list of positive experiences and opportunities I gained while working in the Titus lab.

I would like to thank all of my past and present lab mates. Thank you to Hilary Bauer, Sinzi Cornea and Zoe Henrot for welcoming me into the lab when I started and especially to Karl Petersen who share his imaging and analysis ex- pertise. I am so grateful for the help and from my PLA project teammate Livia Songster, you brought such great energy to the project and to the lab. Thanks Casey Eddington, Annika Schroeder for their support, encouragement and help reading and discussing many aspect of this work. Thanks to the University of MN undergraduate students who joined my on research projects over the years especially to Himanshu Jain. I would like to thank Jordan Beach at Loyola, Guillermo Marques, Mark Sanders, for their help with imaging. I thank Ashim Rai for his assistance with motor purification and motility assays. Thanks to all the members of the Houdusse Lab for their work on the PLA project and for Anne Houdusse for her critical reading of my work and her help with my manuscripts. Thanks also to Drs. Holly Goodson (Notre Dame), Robert Insall (Beatson), John Cooper (Wash- ington U), Lil Fritz-Laylin and members of the Fritz-Laylin lab (UMass Amherst) for helpful comments and discussion about the work. Thanks to Borris Demeler and Akash Bhattacharya for their help with analytical centrifugation, to Mike Fea- ley for assistance with CD. Thank you to Gaku Ashiba for many insightful conver- sations and experimental plans.

I would like to thank my thesis committee: Drs. Melissa Gardner, Sivaraj Sivaramakrishnan, Hans Othmer, Dave Thomas, with help from Chad Myers. Your support of my project, and my development as a scientist was instrumental in completing this work.

Finally thank you to my family and friends for their love and support. i

Contents ACKNOWLEDGEMENTS ...... i

List of Tables ...... iv

List of Figures ...... v

1. CHAPTER 1: Introduction ...... 1

Significance: ...... 1

Cellular interactions with the environment and filopodia function ...... 1

Filopodia characterization and diversity ...... 3

Key molecules in filopodia formation ...... 4

Filopodia formation ...... 7

Regulation of Myosin by Autoinhibition ...... 8

Myosins and binding specialized actin structures ...... 9

Dictyostelium as a model for studying filopodia formation ...... 11

Understanding MF myosin targeting for filopodia formation ...... 12

2. CHAPTER 2: Optimized filopodia formation requires myosin tail domain cooperation ...... 14

Summary ...... 17

Keywords ...... 17

Significance Statement ...... 18

Introduction ...... 19

Results and Discussion ...... 20

The proximal tail has a dominant negative effect on filopodia and promotes weak dimerization ...... 22 The distal tail of DdMyo7 is critical to promote filopodia length and number ... 23 Deletion of both N-PLA and MF2 domains abolishes activity ...... 24 Shortening the lever arm and post lever arm disrupts filopodia formation ...... 26 Disruptions in the proximal tail cause DdMyo7 accumulation in filopodia tips . 28 ii Summary and Conclusion...... 29

Materials and Methods ...... 33

Figures ...... 37

3. CHAPTER 3: VASP mediated actin dynamics activate a filopodia myosin .. 55

Introduction ...... 57

Results ...... 60

DdMyo7 targets to actin in pseudopodia ...... 60 DdMyo7 is localized to dynamic cortical actin ...... 61 Role of myosin motor function in targeting and filopodia formation...... 62 The role of VASP in DdMyo7 cortical recruitment...... 63 Myo7 - VASP cooperation is required for filopodia formation ...... 64 VASP mediated actin polymerization recruits DdMyo7...... 65 Discussion ...... 66

VASP activity promotes DdMyo7 motor domain to bind actin filaments...... 67 DdMyo7 actin binding, and release of autoinhibition ...... 69 Conserved and divergent models of filopodia myosin function ...... 69 Figures ...... 71

Materials and Methods ...... 87

4. CHAPTER 4 - DISCUSSION AND FUTURE AIMS ...... 93

...... 96

BIBLIOGRAPHY ...... 96

Reprint permission...... 115

iii List of Tables

Table 1 – Quantification of DdMyo7 mutants in myo7- cells 47 Table 2 – Molecular Biology constructs 49 Table 3 Quantification with anti-actin drugs. 84 Table 4 Quantification of filopodia number and cortical targeting. 86 Table 5 Key Resources 91

iv List of Figures

Figure 1 Characteristics of filopodia and invadopodia...... 2 Figure 2 Filopodia in diverse cell types ...... 4 Figure 3 Actin properties and modifications ...... 11 Figure 4 Filopodia initiation sequence ...... 13 Figure 5 Recruitment to the cortex and release of head-tail autoinhibition promotes filopodia formation...... 38 Figure 6 The proximal tail regulates DdMyo7 activity...... 39 Figure 7 The proximal tail domain has a dominant negative effect on filopodia and weakly dimerizes in solution...... 40 Figure 8 Filopodia formation promoted by a forced dimer of the motor ...... 41 Figure 9 Functional cooperation of the proximal tail and MF2 regions...... 42 Figure 10 Shortening the lever arm and post lever arm disrupts filopodia formation...... 43 Figure 11 Deletion of the proximal tail region causes DdMyo7 accumulation in filopodia tips...... 45 Figure 12 Model of DdMyo7 Inhibition and Activation States of DdMyo7...... 46 Figure 13 Western blots Dictyostelium cell lines ...... 53 Figure 14 Localization of various deletion mutants...... 54 Figure 15 DdMyo7 is localizes with cortical actin...... 72 Figure 16 Actin dynamics regulate DdMyo7 recruitment to the cortex...... 74 Figure 17 DdMyo7 motor activity is required to release autoinhibition...... 75 Figure 18 VASP is required for DdMyo7 cortical recruitment...... 76 Figure 19 VASP relieves DdMyo7 head-tail autoinhibition to promote targeting and filopodia formation...... 77 Figure 20 VASP mediated actin polymerization recruits DdMyo7 to the cortex to promote filopodia formation...... 78 Figure 21 Model of VASP mediated targeting of DdMyo7 during filopodia initiation...... 79 Figure 22 Concentration dependence of anti actin drugs...... 80 Figure 23 Conservation of the motor domain and motor in pseudopod targeting...... 81 Figure 24 VASP mutants in vivo ...... 82 Figure 25 Western blot analysis of DdMyo7 and VASP expression...... 83 Figure 26 Model of MF myosin and VASP cooperation in the convergent evolution model of filopodia formation...... 96

v 1. CHAPTER 1: Introduction

Significance:

Filopodia are actin based protrusions that cells use to probe their environment and aid cellular activities such as axon guidance, and cancer cell metastasis. The structure of filopodia is well characterized, but how they form is not well understood. The goal of this work is to study how two proteins: a molecular motor and an actin regulator work together to initiate the formation of these structures. Insights from this work may lead a better understanding of the conserved process of filopodia formation, which a critical in a variety of cell types and processes.

Cellular interactions with the environment and filopodia function

Cells engage with their environment to accomplish many processes including wound healing (Kopecki and Cowin, 2016), neuronal pathfinding (Davenport et al., 1993), angiogenesis (Gerhardt et al., 2003) and cancer cell metastasis (Machesky, 2008). In these cases, cells integrate cues from their environment to aid their directed migration. For example, wound healing relies on chemical cues like cytokines and chemokines signals to direct migration. Neurons integrate both attractive and repulsive cues from the environment to make a synaptic target (Davenport et al., 1993). Receptor binding of secreted signals is upstream of various processes, including gene regulation and cytoskeletal rearrangements to promote cell motility (Barrientos et al., 2008; Raman et al., 2011). In many cases, directed cell migration is aided by specialized structures called filopodia. Filopodia are actin based protrusions comprised of bundles of parallel actin (reviewed: (Mattila and Lappalainen, 2008)). Filopodia also function by promoting adhesion (Galbraith et al., 2007). A cohort of proteins are essential for filopodia formation. In general, the proteins needed to facilitate filopodia formation must integrate cues from the environment, and reorganize the actin cytoskeleton into parallel bundles of filaments that protrude from the cell’s cortex. Ena/VASP proteins have been

1 shown to be particularly important filopodia formation in neuronal growth cones (Lebrand et al., 2004). Ena/VASP proteins localize to the leading edge of cells, incorporate actin monomers and bind filaments to accelerate actin filament growth. (Dent et al., 2011; Drees and Gertler, 2008; Barzik et al., 2005; Hansen and Mullins, 2015). Ena/VASP also blocks actin-capping proteins (Bear and Gertler, 2009; Applewhite et al., 2007). VASP phosphorylation and ubiquitination both neg- atively regulate VASP activity, and in turn filopodia formation (Döppler and Storz, 2013; Menon et al., 2015). This example illustrates how regulation of a key filopodia protein can tunes filopodia formation. Cancer cell metastasis is another example where actin protrusions support cell migration and cellular interactions with the environment. Cancerous cells dis- regard cues for normal growth and proliferate. Metastatic cancer cells dedifferen- tiate, lose cell-cell adhesions and invade surrounding tissues or travel through the bloodstream. Like other migrating cells, this process is mediated through polarized assembly of the actin cytoskeleton (Machesky, 2008). Some metastatic cancer cells have specialized actin-based protrusion to aid their invasive migration: in-

Figure 1 Characteristics of filopodia and invadopodia.

Adapted from (Mukherjee et al., 2018). Filopodia are short, “fingerlike” projections whereas invadopodia are thicker projections which an actin rich core. Fi- lopodia often emerge from a lamellipodium, or the leading edge of the cell whereas invadopodia often extend out of the ventral cell surface. The actin network in filopodia is bundles of parallel filaments whereas, invadopodia utilized branched actin nucleators to form, and have a less well organized actin network. 2 vadopodia. Invadopodia are proteolytic protrusions that help to degrade the extracellular matrix (Chen, 1989) and harness the branched actin network by the Arp2/3 activator N-WASP. In addition to invadopodia, cancer cells can generate excess filopodia overexpression of Myo10 (MyoX). Myo10 overexpression correlates with cancer invasiveness in aggressive breast cancer subtypes, (Arjonen et al., 2014; Cao et al., 2014), and cervical cancer (He et al., 2020). Filopodia can be coupled to extracellular adhesion via focal adhesions and integrins and this adhesion could further power filopodia mediated invasiveness (Arjonen et al., 2011; Shibue et al., 2012, 2013). An illustration of filopodia and invadopodia is depicted in Figure 1. In addition to these examples, filopodia have roles in sensing chemical gradients (Heckman and Plummer, 2013), electrical gradients (Wang et al., 2011), and mechanical forces (Jacquemet et al., 2019). Thus, they are critical cellular protrusions in a variety of cell-environment interactions.

Filopodia characterization and diversity

Filopodia were first described in sea urchins, where these thin cell projections “filopodia” Latin for “thread protrusions” were visualized extending between cells. During gastrulation of sea urchin embryos, cells migrate to form tissue layers, and filopodia appear to provide long distance cell contacts that are critical for signaling (Miller et al., 1995). Using a combination of model systems and techniques, filopodia have been found across many different organisms. They are well studied in animals but have been observed in various Rhizaria (Cavalier-Smith and Chao, 2003), including predatory vampire amoeba (Hess et al., 2012), Discoba (Hanousková et al., 2019), Apusoza (Yabuki et al., 2013) Amoeboza and Holozoa (Sebé-Pedrós et al., 2013). Single cell relatives of metazoans including Capaspora owczaraki and Salpingoeca rosetta have actin based structure that resemble filopodia. Filopodia have been studied the amoeba Dictyostelium and may be important for adhesion and phagocytosis (Tuxworth et al., 2001). Amoeboid filopodia comprised loosely bundled actin filaments that are not perfectly parallel to each other (Medalia et al., 2007) which is in contrast to those studied in mammalian cells which are tighly bundled into parallel arrays (Svitkina et al., 2003). Recently, the novel coronavirus SARS-CoV-2 was reported to increases filopodia in host cells 3 (Bouhaddou et al., 2020). Together, these examples and others (Figure 2) highlight the structural and functional diversity of filopodia.

Key molecules in filopodia formation

Ena/VASP: Ena/VASP proteins are actin binding proteins that speed actin filament polymerization, block capping protein and bundle actin filaments (Bear et al.,

Figure 2 Filopodia in diverse cell types

A. Light microscopy showing ectodermal filopodia (black arrows) and long primary mesenchyme cell filopodia (about 80 microns) in sea urchin embryo. Scale Bar is 10 microns, Adapted from (Miller et al., 1995). B. Electron micrograph of a filopodia adapted from (Mellor, 2010). Actin filaments in the filopodia are pseudocolored blue. The filopodia tip has increased density. C. Dictyoste- lium discoideum cell imaged by SIM with actin (red) and DdMyo7 (green) SB 10 microns, Arthur and Titus, unpublished. D. Mouse CAD (neuronal cell) with actin (red) and Myo10 (green) from (Berg and Cheney, 2002). E. Choanoflag- ellates with actin (green) MTs (red) nuclei (blue) from (Laundon et al., 2019). C-E. white arrows indicate filopodia tips. 4 2002). They are critical for cell motility and overall cellular actin dynamic regulation (Krause, 2002). This family is comprised of related proteins Ena (Enabled from a Drosophila screen for suppressors of Abelson kinase; (Ahern-Djamali et al., 1998)), Mena (Mammalian Ena), and VASP (Vasodilator-stimulated phosphoprotein; identified as a phosphorylated protein in platelets, (Waldmann et al., 1987). In addition to cell migration, platelet aggregation and pathogen movement, Ena/VASP proteins are essential for filopodia formation in diverse cell types (Schirenbeck et al., 2006; Lebrand et al., 2004; Han et al., 2002). Ena/VASP proteins have three domains, and N-terminal EVH1 (Ena/VASP Homology 1) a more divergent proline rich region and a C-terminal EVH2 (Ena/VASP Homology 2) (Gertler et al., 1995; Haffner et al., 1995). EVH1 domains aid VASP in targeting itself to focal adhesion proteins zyxin and vinculin (in mammalian cells) (Reinhard et al., 1995, 1996). The EVH2 domain is sufficient for leading edge targeting to the actin (Bear et al., 2000). The EVH2 domain is tripartite itself- with a G-actin binding region, and F-actin binding region and a tetramerizating coiled-coil. The G- and F- actin cooperate to facilitate robust barbed end F-actin targeting (Hansen and Mullins, 2010), whereas filament binding and the coiled-coiled region contribute the actin bundling (Schirenbeck et al., 2006; Breitsprecher et al., 2008). Ena/VASP proteins have been well described in neuronal growth cone filopodia (Davenport et al., 1993; Geraldo and Gordon-Weeks, 2009). Ena/VASP may cooperate with formin in some cell types to mediate its activity in filopodia formation (Schirenbeck et al., 2005a; Barzik et al., 2014). A direct interaction between VASP and the parallel actin polymerase formin (specifically mDia2) could promote efficient elongation of filopodia. VASP could also be transported by the myosin motor Myo10 into filopodia tips in order to promote filopodia elongation (Tokuo and Ikebe, 2004).

Formin Formins are dimeric actin binding proteins that nucleate and elongate parallel actin filaments. Formins have formin homology (FH1, FH2) domains. FH1 domains have polyprolines tracks that interact with profilin, and the FH2 domain binds actin and promotes nucleation (Pollard, 2007). Formins promote elongation of filopodia (Schirenbeck et al., 2005b) by adding actin monomers at the barbed end of actin 5 while staying associated with the plus end (Higashida et al., 2004). The diaphanous formin (mDia2 in animals and dDia2 in amoeba) is activated by a small GTPase (Schirenbeck et al., 2005b; Pellegrin and Mellor, 2005; Peng et al., 2003). Cells with disrupted Arp2/3 actin networks can still make filopodia suggesting formin based filopodia formation is critical at least in some cell types (Steffen et al., 2006; Di Nardo et al., 2005). MyTH4-FERM Myosins A family of myosin motors have a vital role in filopodia formation that has been conserved over 600MA of evolution (Petersen et al., 2016). Filopodia formation in amoebae and animals is driven by paralogous MyTH4-FERM myosins, DdMyo7 and Myo10 (Bohil et al., 2006; Tuxworth et al., 2001). In addition to filopodia, MyTH4-FERM family myosins (MF; myosin tail homology 4, band 4.1, ezrin, radixin, moesin) organize and maintain other parallel actin-based structures such as microvilli, stereocilia (Sebé-Pedrós et al., 2013; Manor et al., 2011; Weck et al., 2017; Kollmar and Mühlhausen, 2017). Myo7, Myo15, and Myo22 have tandem MF domains in their tails whereas Myo10 has a single MF domain. Myosin 10 is restricted to a subset of metazoan lineages, and is an atypical MF myosin due to its membrane binding PH domains and antiparallel dimerization (Umeki et al., 2011; Lu et al., 2012; Ropars et al., 2016; Kollmar and Mühlhausen, 2017). Myo10 motor activity has been proposed to provides force to reorganize branched actin filaments at the cortex to converge them into parallel bundles (Tokuo et al., 2007). Myo10 is an anti-parallel dimer that can walk on bundled actin filaments (Ropars et al., 2016). It is a processive motor, which is consistent with cargo transport and/or actin filament convergence. DdMyo7 is essential for filopodia formation in amoeba, and is presumed to resemble ancestral MF myosin, from which duplication and specialized over the course of evolution allowed for cells to make specialized filopodia -like protrusions such as microvilli and stereocilia. DdMyo7 may function a distinctly from Myo10, as it is not a constitutive dimer, nor does it readily show intrafilopodia mobility. Thus, the motor properties necessary and sufficient to induce filopodia formation could have flexiblilty. For example, Myo6 is a pointed end directed myosin motor not strongly implicated in filopodia formation. Researchers found by reversing the Myo6 lever arm so that it is a barbed end directed motor, Myo6+ could ectopically induced filopodia like structures (Masters and Buss, 2017). Thus, a processive dimeric motor with a 6 cargo binding tail likely induces filopodia formation. Combining in vivo dissection of MF myosins with in vitro and other synthetic assays will be critically to understand the ways by which MF myosins indeed promote filopodia initiation.

Filopodia formation

Filopodia are slender protrusion comprised of bundles of actin, so in order to initiate, actin filaments need to be organized into a parallel configuration against the membrane. This can either be from a rearrangement of branched filaments at the cortex, or form new nucleation of parallel bundles. Thus, two models of filopodia initiation have been proposed - the convergent extension model where filopodia form from a reorganization of an Arp2/3 branched actin network (Svitkina et al., 2003; Tokuo et al., 2007) and de novo or tip nucleation model where unbranched actin filaments are bundled and elongated by formin.(Steffen et al., 2006; Faix and Rottner, 2006; Schirenbeck et al., 2006). Actin crosslinkers such as fascin are implicated in promoting and maintaining the filament organization of bundles. Rho family GTPase signaling coordinates these events (Mattila and Lappalainen, 2008; Faix et al., 2009). In the actin convergence model, filopodia emerge from the lamellipodium. Here, coalescence of a branched actin network by MF myosin dimerization followed by parallel actin bundling by fascin drives initiation (Svitkina et al., 2003; Tokuo et al., 2007). The rearrangement model considers filopodia protruding from the lamellipodium, while dorsal filopodia are ignored (Mogilner and Rubinstein, 2005). Lamellipodia nucleation is dependent actin branching by the Arp2/3 complex (Wu et al., 2012) but filopodia are not disrupted by the loss this complex (Mejillano et al., 2004). In the de novo model, actin filaments are nucle- ated to form filopodia. They are not reliant on the underlying branched actin network, and their formation is largely driven by parallel actin nucleators including formin. Here, the role of MF myosin is more focused on transport of cargo (i.e. VASP) to the filopodia tip to promote elongation (Tokuo and Ikebe, 2004). The de novo model accounts for dorsal filopodia, but cannot address low numbers of short filopodia produced in formin (dDia2) null cells (Schirenbeck et al., 2005b). Both mechanisms could be correct, and reported differences may reflect cell type vari-

7 ations (Young et al., 2015). In other words, despite structural and functional similarities of protruding filopodia across cell types, the underlying mechanism of their formation remains both varied and poorly understood.

Regulation of Myosin by Autoinhibition

Molecular motors are enzymes that utilize the cell’s energy stock to generate mechanical work. In the case of myosin motors, ATP is bound by the motor domain, and is hydrolyzed, which promotes a conformation change and therefore force along actin filaments (Sweeney and Houdusse, 2010a; Houdusse and Sweeney, 2016). Many myosins adopt an autoinhibited or catalytically inactive conformational state to preserve energy. For example, muscle myosin shows a much higher activity in vitro, compared to in vivo which was an important clue for deciphering the super-relaxed state, a low activity conformation (Stewart et al., 2010). Another well described example is Myosin V, where ionic interactions between the head and globular tail domain promote a folded conformation that allosterically inhibits ATPase activity approximately 50 fold (Trybus, 2008). Autoinhi- bition release by binding membrane lipids has been described for Myosin 10 (Umeki et al., 2009) and related modes of autoinhibition are also described for microtubule based kinesin motors (Hackney et al., 1992) illustrating that this folded conformation is a evolutionarily conserved and important property of molecular motors. In the MF class of myosins implicated in filopodia formation, head tail autoinhibition appears to regulate motor activity in this process. The activity of Myosin 7 in Drosophila is auto regulated by the FERM domain in the tail (Yang et al., 2009). Conserved lysine resides in the FERM domain likely form a salt-bridge with the motor domain to regulation autoinhibition and activity. Specially a constitutively activated DdMyo7 (lysine->alanine neutralization point mutations) stimulates filopodia formation, whereas expression levels of the molecule do not play a signification role in filopodia formation numbers (Petersen et al., 2016). Thus an outstanding question is how autoinhibition and autoactivation of DdMyo7 regulated in vivo to control motor activity during filopodia formation.

8 Myosins and binding specialized actin structures

Myosin motors bind actin, but cells are comprised of a variety of actin networks including lamellipodia, filopodia, stress fibers and cytokinesis contractile rings (Svitkina, 2018). The myosin superfamily of motors has a broad array of functions, and therefore must be able to restrict their activity to the proper actin network for a give cellular process. A variety of actin properties may direct impact motor activity differences that are dependent on their tracks (reviewed (Santos et al., 2020). Several are discussed in detail here and also are represented in Figure 3.

Polarity and Nucleotide State Actin filaments have a barbed end where monomers are readily incorporated, and a pointed end where growth is much slower. Thus, actin filaments have polarity. Actin filament polarity is not random in cells. The barded end are often adjacent to the membrane and filament elongation at the membrane is a key com- ponent of cell migration. Myosin motors also discern this filament polarity, and most processive motors walk along actin filaments toward the barbed end with Myo6 being a notable exception.

Nucleotide state is closely associated with polarity. G-actin monomers bound to ATP are incorporated mostly at the barbed or fast ending end of actin filaments. ATP actin hydrolyzes to ADP-Pi actin in the course of a few seconds, and the phosphate group released leaving ADP actin in the course of a few minutes (Pollard and Weeds, 1984; Carlier and Pantaloni, 1986). This leaves a ‘young’ actin network at the barbed end and an older actin filament toward the pointed end. This manifests in cells in a spatial distribution of young and old actin, where stable actin structures like stress fibers are comprised mostly of ADP actin, and the leading edge of cells is composed of ATP or ADP-Pi filaments. It has been shown that the nucleotide stats have different structural properties, supporting the idea that this actin again could be read out by myosin motors or other actin binding proteins (Chou and Pollard, 2019)

Isoforms

9 Actin is encoded by several genes and has six isoforms in mammalian cells. The role of isoforms is specifying differential actin networks is of interest. Beta actin is the predominant isoform in non-muscle cells, whereas alpha-cardiac and alpha-skeletal are important muscle isoforms. These isoforms differ in just a few amino acids. Myosin motors across classes are differentially impacted by actin isoform. Myosin 5 kinetic parameters are is not strongly affected by actin isoform (De La Cruz et al., 2000), whereas nonmuscle myosin II was activated by beta- actin and myosin7A by gamma-actin (Müller et al., 2013). Beta and gamma actin show distinct cellular localization in non muscle cells (Dugina et al., 2009), and more work is needed to understand the role of isoforms on myosin activity in vivo. One potential impact of isoforms is that they are have different post-translational modifications. Alpha actin in skeletal muscle is methylated, and acetylated (Dominguez and Holmes, 2011). One interesting modification is arginine, which has an impact on cell migration (Karakozova et al., 2006). This modification is restricted to the otherwise ubiquitous beta-actin isoform, and could be a possible mode by which the cells specifies specific actin tracks for particular motors and cargo.

Twist and Bending and Tension Actin is a helical polymer with a 36nm repeat. The helix of actin filaments repeats in 6 turns, with 13 monomers needed to complete the twist (Hanson and Lowy, 1963; Dominguez and Holmes, 2011). Proteins that bind actin can change its twist, such as the actin severing protein cofilin (McGough et al., 1997). The cell toxin phalloidin and in vivo fusion protein RFP-Lifeact, routinely use to visualized actin in cells, are unable to bind these twisted actin networks, demonstrating how filament twist can effect function and protein binding. Like twist, bending (inward force) or tension (outward) also compacts or extends the organization of monomers in the helix and could create a differential set of binding sites for actin interaction proteins.

Bundles A number of actin bundling proteins like fascin, fibrin and alpha-actin function in cells to organize actin filaments into bundles. These bundling proteins space adjacent actin filaments a range of distances from 6nm in the case of fascin, 10 and 35nm for alpha-actinin. This relatively wide range of bundle spacing imparts unique cellular functions of the differential actin bundles (Winkelman et al., 2016;

Figure 3 Actin properties and modifications

A. Actin filaments grow from the barbed ends by incorporating ATP actin. B. classes of actin isoform present in metazoan. C. twist, bend or tension on filaments. D. illustration of how filaments are organized into bundles by bundling proteins.

Harker et al., 2019). Myosin V step size has been correlated with the helical pitch of actin, so that the myosin can avoid spiraling around filaments as it walks (Tanaka et al., 2002). Myosin-10 step size is optimized for stepping on bundles (Ropars et al., 2016), which is likely critical for its transport role in filopodia (Berg and Cheney, 2002).

Dictyostelium as a model for studying filopodia formation

Dictyostelium discoideum is perhaps the simplest system that expresses the fun- damentally conserved players involved in filopod initiation. Efforts to study initiation in metazoan cells have not yielded clear results, possibly due to the complex regulatory systems in play, the redundancy of protein family members, and variability of filopodia and filopodia like structures across cell types. Studying this process in Dictyostelium allows efficient genetic knockout and rescue experiments, is ame- nable for a variety of live cell imaging techniques and can yield large quantities for protein expression and purification. Unlike metazoan cells, Dictyostelium filopodia

11 length is tightly regulated, which allows discrimination between growing and mature filopodia. Despite differences in the tail domains of filopodial MF myosins across kingdoms, DdMyo7 has functional conservation with Myo10, in fact, a Dicty- human chimera myosin can rescue the filopodia formation defect of myo7 null cells (Petersen et al., 2016).

Understanding MF myosin targeting for filopodia formation

MyTH4-FERM myosins such as Myosin 10 and DdMyo7 need to be recruited to the cortical actin network or the plasma membrane in order to function in filopodia formation. Characterization of filopodia initiation sites and the dynamics of key protein recruitment during their formation will provide a better understanding of initiation. Filopodia are initiated from the cell periphery, therefore, localization of key filopodial proteins to the membrane (or underlying actin cortex) is an early step in this process. Filopod initiation events monitored in cells coexpress- ing DdMyo7 and RFP-LifeAct to visualize actin filaments DdMyo7 reveal the steps of a typical initiation event (Figure 4). DdMyo7 first concentrated at the cortex. Then, a bright spot of myosin appeared, projecting from an actin-rich pseudopod. Filopodia elongated several micrometers within 7 s, with actin present along the length and DdMyo7 concentrated close to the filopod tip throughout the elongation process. These results show that filopod initiation is a highly dynamic and rapid process. The activation, recruitment and clustering of DdMyo7 as a first step of filopodia initiation was established by (Petersen et al., 2016). In my thesis work, I sought to uncover deeper details about the manner by which this myosin motor was recruited and activated so that it would function in this critical conserved cell process.

Figure 4 Filopodia initiation sequence

Top: GFP-DdMyo7 assembles into an initiation foci prior to extending into a mature filopodia. Middle: actin marked with F-actin marker RFP-LifeAct. Bot- tom, merge, time in seconds, scale bar is 5 microns. Adapted from (Petersen et al., 2016).

2. CHAPTER 2: Optimized filopodia formation requires myosin

tail domain cooperation1

Ashley L. Arthur1, Livia D. Songster1, Helena Sirkia2, Akash Bhattacharya3, Carlos Ki- kuti2, Fernanda Pires Borrega2 Anne Houdusse2* & Margaret A. Titus1*

1 Department of Genetics, Cell Biology, and Development, University of Minnesota, Min- neapolis, MN 55455 2 Structural Motility, Institut Curie, CNRS, UMR 144, F-75005 Paris, France 3 Beckman Coulter, Loveland, CO 80538

* co-corresponding authors Margaret A. Titus Department of Genetics, Cell Biology, and Development 6-160 Jackson Hall 321 Church St SE University of Minnesota Minneapolis, MN 55455

E-mail: [email protected] Phone: 612-625-8498 Fax: 612-625-4648

Anne Houdusse Institut Curie CNRS UMR144 26 rue d’Ulm,

1 Reprinted from:

Arthur, Ashley L., Livia D. Songster, Helena Sirkia, Akash Bhattacharya, Carlos Kikuti, Fernanda Pires Borrega, Anne Houdusse, and Margaret A. Titus. 2019. “Optimized Filopodia Formation Requires Myosin Tail Domain Cooperation.” Proceedings of the National Academy of Sciences of the United States of America 116 (44): 22196–204.

14 5248 Paris cedex 05 France

E-mail: [email protected] Tel : 33-(0)1-56-24-63-95 Fax: 33-(0)1-56-24-63-82

15 Detailed Author Contributions: Ashley L. Arthur – Designed experiments, created cell lines, imaged cell lines, created image analysis tools, analyzed data, assembled figures, wrote and edited the manuscript. Major contribution to Figure 4,5,6A-D,9,10.

Livia D. Songster – Created cell lines, imaged cell lines, blotted cell lines, created imaging analysis tools, analyzed data, edited figures, edited the manuscript. Major contribution to Figure 7,8.

Carlos Kikuti, - Designed experiments, purified proteins, analyzed and interpreted data.

Major contribution to Figure 6E.

Helena Sirkia – Purified proteins, major contribution to Figure 6E.

Fernanda Pires Borrega, Purified proteins, major contribution to Figure 6E.

Akash Bhattacharya – Ran analytical ultracentrifugation, analyzed and interpreted AUC data (Figure 6E).

Anne Houdusse - Designed research, designed experiments, interpreted data, wrote and edit the manuscript.

Margaret A. Titus - Designed research, designed experiments, created construcuts, interpreted data, wrote and edit the manuscript

Summary

Filopodia are actin-filled protrusions employed by cells to interact with their environment. Filopodia formation in Amoebozoa and Metazoa requires the phylogenetically diverse MyTH4-FERM (MF) myosins DdMyo7 and Myo10, respectively. While Myo10 is known to form antiparallel dimers, DdMyo7 lacks a coiled-coil domain in its proximal tail region, raising the question of how such divergent motors perform the same function. Here it is shown that the DdMyo7 lever arm plays a role in both autoinhibition and function while the proximal tail region can mediate weak dimerization, and is proposed to be working in cooperation with the C-terminal MF domain to promote partner-mediated dimerization. Additionally, a forced dimer of the DdMyo7 motor is found to weakly rescue filopodia formation, further highlighting the importance of the C-terminal MF domain. Thus, weak dimerization activity of the DdMyo7 proximal tail allows for sensitive regulation of myosin activity to prevent inappropriate activation of filopodia formation. The results reveal that the principles of MF myosin-based filopodia formation are conserved via divergent mechanisms for dimerization.

Keywords myosin | filopodia | actin | MyTH4-FERM

17 Significance Statement

Cells interact with their environment using filopodia, thin membrane protrusions supported by actin filaments. Filopodia formation in evolutionarily divergent organisms requires force generation by a MyTH4-FERM (MF) myosin that binds membrane proteins and is designed to promote cytoskeletal reorganization and filopodial transport. The amoeboid filopodial MF myosin is found to be a resilient motor in filopodia formation. Its activity and regulation appear to have emerged by multi-domain co-evolution for optimized control of filopodia formation and stability. Its properties reflect those of the ancestral MF myosin that gave rise to the metazoan MF myosins. These findings have implications for understanding the fundamental principles of how filopodia form and how MF myosins function in phylogenetically distant organisms.

18 Introduction

Filopodia are specialized cellular projections that detect extracellular cues, facilitate adhesion and aid in directed cell migration (Heckman and Plummer, 2013). They are important for processes ranging from neuronal pathfinding to cancer cell metastasis (Davenport et al., 1993; Jacquemet et al., 2015). Parallel bundles of actin support these thin membrane projections that extend from the dendritic actin network at the cell cortex. The structure and function of filopodia has been established, but the molecular mechanism of filopodia initiation is not fully understood. Specifically, it is not yet clear how the cortical actin network at the membrane is locally reorganized into the parallel actin bundles that form nascent filopodia. A core set of conserved proteins including actin bundling proteins and an unconventional myosin are required to make filopodia (Sebé-Pedrós et al., 2013). A favored model is that an activated small GTPase recruits key actin regulators such as formins and VASP at a filopodia initiation site. Actin filaments then converge into a parallel array and actin polymerization drives filopodia elongation (Svitkina et al., 2003). MyTH4- FERM myosins (myosin tail homology - band 4.1, ezrin, radixin, moesin; MF) are molecular motors found at filopodia tips and are necessary for filopodia formation across cell types (Tuxworth et al., 2001; Berg and Cheney, 2002) but the action of MF myosins in the process of filopodia initiation remains poorly understood.

The MF myosins are a group of unconventional myosins with roles in the formation and function of parallel actin-based structures such as filopodia, stereocilia and microvilli (Chen et al., 2001; Tuxworth et al., 2001; Belyantseva et al., 2005; Bohil et al., 2006; Weck et al., 2017). They are characterized by the presence of a MF domain(s) in the C-terminal tail. These domains mediate interactions with binding partners and microtubules (e.g. (Weber et al., 2004; Zhang et al., 2004; Li et al., 2017; Yu et al., 2017). The amoeboid MF myosin DdMyo7 and the phylogenetically distant metazoan MF myosin Myo10 are essential for the initiation of filopodia (Tuxworth et al., 2001; Berg and Cheney, 2002). Myo10 is proposed to organize the actin network at initiation sites and/or transport the actin regulator VASP towards the tip during filopodia formation (Tokuo and Ikebe, 2004; Tokuo et al., 2007; Kerber and Cheney, 2011). These actions of bundling and transport require a dimeric myosin. The metazoan Myo10 monomer is autoinhibited by head-tail

19 interaction, as is typical for many myosins (Umeki et al., 2011; Heissler and Sellers, 2016).

Dimerization is induced by PI(3,4,5)P3 mediated membrane binding that promotes formation of an anti-parallel dimer via a region in the proximal tail (Plantard et al., 2010; Umeki et al., 2011; Lu et al., 2012; Vavra et al., 2016; Ropars et al., 2016). This arrangement confers increased distance between motor heads, gives the molecule flexible reach and facilitates walking on bundled actin filaments in filopodia cores (Ropars et al., 2016).

Filopodia are found in a diverse array of organisms, and many proteins in the ‘filopodia toolkit’ are paneukaryotic (Sebé-Pedrós et al., 2013). MF myosins are ancient - they arose prior to the Amoebozoa/Holozoa split and are present in unicellular and multicellular Holo- zoa and Fungi, but Myo10 is notably only found in Holozoa (Sebé-Pedrós et al., 2013; Kollmar and Mühlhausen, 2017). The amoebozoan MF myosin, DdMyo7, and the metazoan Myo10 have evolved independently across ~600 Ma, yet both have essential roles in filopodia formation whereas other MF myosins evolved to have roles in the formation and maintenance of microvilli and stereocilia (Petersen et al., 2016; Weck et al., 2017). It is not known if the two distinct filopodial myosins employ unique mechanisms to generate filopodia. An understanding of the features employed by the divergent amoeboid MF myosin to form filopodia can provide insight into the conservation or divergence of the mechanism of MF myosin-based filopodia formation. In particular, while DdMyo7 appears to be regulated by head-tail autoinhibition it lacks a predicted coiled-coil dimerization region in the proximal tail, thus raising the question: did proximal dimerization evolve to tune the specialized function of metazoan Myo10, or is dimerization an ancient feature of filopodia motors? A functional dissection of the amoebozoan DdMyo7 filopodia myosin was under- taken to address this issue.

Results and Discussion

Autoinhibition regulates DdMyo7 activity DdMyo7 is essential for the formation of filopodia in Dictyostelium discoideum (Tuxworth et al., 2001). The first step in this process is the recruitment of the MF myosin

20 to the cell membrane or underlying actin cortex. Myo10 is recruited and dimerized following PH domain-mediated interaction with PI(3,4,5)P3 that promotes both activation, by releasing motor/tail autoinhibition, and dimerization, that is required for activity (Plantard et al., 2010; Umeki et al., 2011). DdMyo7 lacks both a clear lipid-binding motif and identifiable coiled-coil domain that could mediate dimerization, raising the question of how this myosin is targeted and activated.

DdMyo7 switches between an autoinhibited state and an active state that is controlled via a basic patch in the C-terminal tail that allosterically inhibits motor activity (Petersen et al., 2016). This motif is found among widely divergent Myo7s (Yang et al., 2009; Petersen et al., 2016) and autoinhibition provides spatial and temporal control of motor activity. Do- main deletion of DdMyo7 could disrupt the conformation necessary for head-tail autoinhibition and mutant motors were assessed for both their ability to rescue filopodia formation and extent of efficient targeting (that would increase in case of lack of autoinhibition). GFP-tagged full length DdMyo7 and mutants were expressed in DdMyo7 null cells (myo7- ; ), and fluorescence intensity at the cortex measured using automated image analysis (Figure 5 Recruitment to the cortex and release of head-tail autoinhibition promotes filopodia formation. Figure 5;(Petersen et al., 2016)). GFP-DdMyo7 is enriched at the cortex over the cytoplasm by a ratio of ~1.2. The motor-Pro fragment is not targeted (Figure 5A,D) while the DdMyo7 tail fragment has higher cortical intensity than the wild type DdMyo7 (a ratio of 1.61 ± 0.06 versus 1.17 ± 0.03), consistent with a lack of the head-tail interaction leading to increased cortical recruitment (Figure 5C). The link between autoinhibition and cortical targeting is supported by the finding that mutating key residues for autoinhibition (KKAA; K2333A, K2336A) results in increased cortical targeting (cortical intensity = 1.76 ± 0.08; Figure 5D). The autoinhibition mutant KKAA also shows a larger percentage of cells making at least one filopodia (DdMyo7 = 58.3%, KKAA = 78.8%) and more filopodia per cell (Table 1, Figure 5E;(Petersen et al., 2016)) consistent with autoinhibition via tail/motor interaction controlling targeting and release of autoinhibition leading to increased targeted MF myosin and more filopodia formation.

The proximal tail contributes to optimal DdMyo7 activity, but is not essential for either activity or autoinhibition.

The DdMyo7 proximal tail region, i.e. post lever arm (PLA, residues 830-1117) follows the catalytic motor domain, the Lever Arm (LA) and a single alpha helix (SAH 798-830) 21 that extends the LA (without mediating dimerization) (Knight et al., 2005; Baboolal et al., 2009; Peckham and Knight, 2009) and encompass the region up to the MyTH-FERM1 domain. The DdMyo7 PLA consists of a region possibly adopting a folded structure (N- PLA) followed by a proline-rich domain (Pro1, residues 1020-1084). Secondary structure prediction tools indeed suggested the PLA was unlikely to form a coiled-coil (Lupas et al., 1991), but its N-terminal sequence (residues 845-1000) has a hydrophobic and charged amino acid content compatible with forming a folded structural domain (Callebaut et al., 1997), that could possibly permit dimerization or self-association. Sequence alignment of amoeba Myo7s revealed such a conserved candidate dimerization domain (cDD, residues 884-952). The proline-rich (Pro1) region, on the other hand, is a candidate for adaptor binding (Williamson, 1994; Rath et al., 2005). GFP-DdMyo7 rescues filopodia formation in the myo7 null cells and the myosin is enriched at filopodia tips (Petersen et al., 2016). Deletion of the candidate dimerization domain (ΔcDD) or proline-rich region (ΔPro1) did not affect filopodia formation (meaning fraction of cells making filopodia and filopodia/cell) but the cDD has increased cortical targeting (p<0.03) (Figure 6; Table 1).

Interestingly, a DdMyo7 mutant with part of the LA deleted in addition to the proximal tail (ΔSAH-PLA) exhibits increased cortical localization, exceeding that seen for the autoinhibition (KKAA) mutant (Figure 6). Mutants are typically compared to control DdMyo7, but it is informative to also compare them to the autoinhibition mutant to evaluate the protein function relative to another uninhibited myosin. Despite this significant cortical enrichment, the ∆SAH-PLA cells do not produce an excess of filopodia suggesting that the overall activity of the ΔSAH-PLA motor to promote filopodia formation is diminished (Figure 6C,D; dashed line shows KKAA value). This reveals the role of the SAH-PLA region in optimization of filopodia formation in addition to stabilizing the autoinhibited conformation of DdMyo7. It also suggests that proximal dimerization or targeting via this domain is not absolutely required for filopodia initiation, as it has been proposed to be the case for Myo10 (Umeki et al., 2011): the motor either functions as a monomer to initiate filopodia or other regions of the DdMyo7 tail collaborate to mediate dimerization.

The proximal tail has a dominant negative effect on filopodia and promotes weak dimerization 22 If the proximal tail region promotes dimerization or self-association then filopodia formation could be inhibited by overexpression of this fragment, as has been shown for Myo10 (Bohil et al., 2006). GFP fusions of a long or short fragment of the PLA (aa 809- 1154 and aa 848-1000, respectively; Figure 7A) were expressed in wild type cells. Both the long and short PLA fragments are excluded from filopodia tips, and do not localize to the cell cortex in wild type cells and therefore do not promote targeting of the motor (Figure 7B,C). Expression of either fragment disrupted filopodia formation (actin projections >1μm) of wild type cells ( Figure 7D). These results cannot distinguish between whether the PLA associates directly with endogenous DdMyo7 in vivo to poison a functional dimeric motor, or whether it plays an inhibitory role by preventing binding and sequestering an (unknown) partner.

2Sedimentation velocity analytical ultracentrifugation (AUC) was performed on purified fragments of the post lever arm to test their ability to dimerize directly. Two N-PLA fragments were tested, PLA830-1000 and PLA830-1020. Interestingly, the shorter fragment is primarily monomeric however the longer fragment exhibits concentration dependent dimerization (Figure 7E), with a significant fraction of PLA830-1020 shifting into a higher molecular weight species at 100 - 200 µM. The failure of the shorter fragment to dimerize could suggest that the additional C-terminal sequences are required to stabilize the structural domain or the dimer. These results indicate that the PLA can self-associate, consistent with proximal dimerization of DdMyo7 that is weak and context-dependent.

The distal tail of DdMyo7 is critical to promote filopodia length and number

Models have been proposed for how dimeric and monomeric motors such as Myo10 and Myo1b would contribute to filopodia initiation and stabilization. For a dimeric motor

2 Sedimentation velocity AUC was performed and analyzed by co-author Akash Bhattacharya, with protein purified by Helena Sirkia Carlos Kikuti, Fernanda Pires Borrega and samples for AUC prepared by Ashley Arthur. 23 with proximal dimerization, the mechanism underlying filopodia initiation could be the reorganization of the actin network by the motor reaching out to different actin filaments (Tokuo et al., 2007; Ropars et al., 2016). The monomeric plus-end motor Myo1b has been proposed to function by stabilizing parallel actin bundles along the filopodia membrane (Prospéri et al., 2015). Whether dimerization of the DdMyo7 would be sufficient for function was tested by the addition of an exogenous dimerization region to the C-terminus of the DdMyo7 motor-Pro fragment (Figure 5C-E). The coiled coil sequence from mouse Myo5a followed by the leucine zipper GCN4 were added to generate a stable dimeric DdMyo7 motor (motor-Pro-CC; Figure 8A). The chimera is stably expressed and highly enriched at the cortex, exceeding the full-length DdMyo7 (Figure 8B,C; Figure 13D), and it restores filopodia formation, unlike motor-Pro (Figure 8D), showing that DdMyo7 can function as a dimer. It should be noted that while significant, the rescue is relatively weak with a smaller fraction of cells (11% versus 60% for DdMyo7) making a reduced number of filopodia (1.32 ± 0.14 per cell versus 2.26 ± 0.32 per cell for DdMyo7. Interestingly, unlike any of the other DdMyo7 deletion mutants, filopodia generated by motor-Pro-CC are shorter than wild type filopodium (Figure 8E, Table 1; 2.14 ± 0.27 µm vs 3.05 ± 0.13 µm).

The ability of the motor-Pro-CC forced dimer to promote filopodia formation is consistent with previous studies showing that initiation of filopodia occurs when a dimeric tail-less Myo10 motor presumably brings actin filament barbed ends close together adjacent the membrane (Tokuo et al., 2007). In the case of Myo10, it is not known how effectively the forced dimer promotes filopodia formation compared to full length Myo10. The weak rescue by motor-Pro1-CC shown here demonstrates that the full-length DdMyo7 requires the distal tail to efficiently initiate and fully extend filopodia.

Deletion of both N-PLA and MF2 domains abolishes activity

The MF domains and proline-rich regions of the DdMyo7 distal tail are likely cargo or partner binding sites (Williamson, 1994; Moen et al., 2011; Planelles-Herrero et al., 2016). DdMyo7 lacking either the MF1 (ΔMF1-SH3) or the MF2 region (ΔPro2-MF2) in the distal

24 tail are able to cortically target and rescue filopodia formation to wild type levels (Figure 9; (Petersen et al., 2016)). However, depletion of Pro2-MF2 in combination with the proximal tail (ΔSAH-PLA/Pro2-MF2) completely abolished targeting and filopodia formation (Figure 9B,C). In fact, removal of the candidate dimerization region in combination with Pro2-MF2 (ΔcDD/Pro2-MF2) is sufficient to abolish cortical targeting and filopodia formation, indicating either that the proximal dimerization region acts (partially) redundantly with the MF2 domain or that the two regions cooperate to promote filopodia formation. In contrast, a mutant lacking the entire proximal tail and MF1 region (ΔSAH-SH3) is targeted to wild type levels and is capable of rescuing filopodia formation, albeit less efficiently than full length DdMyo7, the proximal tail mutant (ΔSAH-PLA) or MF1 mutant (ΔMF1-SH3; Figure 9B,C; Table 1). It should be noted that the lack of cortical enrichment by ΔcDD/Pro2-MF2 indicates that the Pro1 and MF1 are not sufficient for targeting the motor to the cortex.

The ability of ∆SAH-SH3 to promote filopodia formation indicates that the presence of the Pro2-MF2 domain is sufficient for function and supports a model whereby binding of a partner to the MF2 permits function in the absence of the PLA. This could be by promoting indirect dimerization or clustering of the motor at the cortex. These data also show that the MF2 domain but not the MF1 acts redundantly in concert with the proximal tail. The PLA region could contribute to activity by either increasing targeting via the recognition of an important partner, or by stabilizing motor dimerization. The lack of targeting by PLA- long (Figure 7B) and weak dimerization by the PLA830-1020 fragment (Figure 7E) would suggest that this region has a role in promoting dimer formation. This does not entirely rule out an alternative model that this region binds to partners critical for function and further studies are needed to address this possibility.

Note also that the ΔSAH-PLA/ΔPro2-MF2 or ΔcDD/ΔPro2-MF2 mutants do not localize to the tips of filopodia in wild type cells in contrast to ΔPro2-MF2 (Figure 14B) potentially because they fail to dimerize with endogenous DdMyo7. However, it is also possible that they are unable to bind an adaptor protein that would facilitate tip targeting. Thus, although the MF1 domain contributes to cortex enrichment, it is not sufficient to allow recruitment of the motor at the tip of filopodia without the presence of either the N-PLA or MF2 domains.

The finding that the PLA showed redundancy with MF2 and not MF1 was unexpected as deletion of either of the two MF domains alone did not impact targeting or filopodia formation (Figure 8; (Petersen et al., 2016)). The observation that deletion of only MF2 in combination with the proximal tail dimerization domain results in a complete loss of filopodia formation (Figure 8D) most likely reveals a specialization of the MF2 domain in targeting and potentially inducing the dimerization or clustering of DdMyo7. In contrast, it appears that the MF1 region promotes cortical localization (likely by contributing to recognition of partners) but not dimerization or self-association of DdMyo7 (required for filopodia initiation). Overall, these results are consistent with a model whereby MF1 and MF2 domains target DdMyo7 to cortical initiation sites. The resulting increase in concentration of active monomers at initiation sites via MF2 binding partner proteins would, in turn, be stabilized by the weak self-association of the dimerization domain in the proximal tail.

Shortening the lever arm and post lever arm disrupts filopodia formation

Models of filopodia initiation and maintenance suggest that a dimeric MF myosin at the cortex could bind adjacent filaments and coerce the actin into a parallel arrangement and then deliver cargo to the tip as the filopodium grows (Tokuo et al., 2007; Kerber et al., 2009; Ropars et al., 2016). The LA is important for inter-head distance and step size, thus the LA that sets the distance between catalytic heads of MF myosin dimers could govern the efficiency of both filopodia initiation and maintenance. In the context of a monomeric myosin, the LA can also play a role in tuning the force sensing activity of myosin as previously shown for mammalian Myo1b (Laakso et al., 2010). The DdMyo7 LA is comprised of four IQ motifs and the single alpha helix (SAH). The structural features of DdMyo7 required to promote filopodia formation were explored by designing myosins with shorter lever arms (Figure 10A). Deletion of IQ domains 2-4 or IQ 2-4-SAH apparently destabilized the myosin based on the inability to obtain stable cell lines for analysis. Deletion of the two internal IQs 2-3 (Δ2IQ) resulted in increased cortical localization compared to wildtype DdMyo7 (Figure 10A, B). Interestingly, despite increased cortical targeting, Δ2IQ produced wild type numbers of filopodia. This differs from the increased numbers of filopodia

26 seen for the activated KKAA mutant (Figure 10C, KKAA: p< 0.0001). On the other hand, the Δ2IQ makes more filopodia compared to the proximal tail mutant ΔSAH-PLA (p< 0.0001) that is, like Δ2IQ, targeted to the cortex more efficiently than DdMyo7 (Table 1). These findings suggest that the Δ2IQ lever arm mutant has both dysregulated autoinhibition that increases targeting and a shorter power stroke or reach between actin filaments that decreases the efficiency of filopodia formation. These observations highlight the role of the LA and PLA regions to finely regulate the step size and proximal dimerization for DdMyo7 to have optimal activity.

Shortening of the LA in combination with deleting the PLA is predicted to disrupt motor coordination and function by both reducing the LA of the motor and impairing proximal dimerization. Deletion of 2IQ/SAH-PLA or 3IQ-PLA causes increased cortical localization, thus appears to disrupt autoinhibition (Figure 10B; Table 1). Comparison of these activated motors suggests that a proper lever arm, and subsequent motor function actually contributes to motor function. Despite the increased cortical targeting, the ∆3IQ-SAH-PLA mutant also showed a significant defect in filopodia formation compared to ∆SAH-PLA (Figure 10C; Table 1) consistent with filopodia formation requiring proper spacing and force generation between DdMyo7 heads.

The distal tail domains of Δ2IQ/ΔSAH-PLA (notably the MF1 region that now abuts IQ4) are not able to elongate the LA of this mutant, and dimerization via partner binding by the distal tail is not sufficient to promote efficient function (Figure 10), possibly by loss of coordination (gating) between heads. As stated earlier, the functionality of the ΔSAH-PLA mutants indicates that the full IQ region (4IQ motifs) provides a sufficiently long LA and that proximal dimerization is not an absolute requirement for filopodia initiation as long as the motor can cluster following proper cortical recruitment and oligomerization via the MF2 domain, as is the case with the even more dramatic deletion in ΔSAH-SH3 (Figure 9). Although filopodia can form despite the lack of proximal dimerization, the function of these activated truncated myosins is lower than for the full length myosin, as shown most clearly by the comparison with the activated autoinhibition mutant, KKAA (Figure 5). Thus, filopodia formation by DdMyo7 is resilient to changes and perturbations in the LA (Figure 10)

27 and proximal and distal tails (Figure 6,Figure 8, respectively), yet it is optimized with re- dundant functions within the tail. The minimum requirement for (near) wild type filopodia formation includes either a N-PLA or MF2 to cluster monomers, and a LA comprised of either 4IQs or 2IQ+SAH.

Disruptions in the proximal tail cause DdMyo7 accumulation in filopodia tips

The filopodia tip complex is a hub of activity that directs incorporation of actin monomers to drive filopodia growth, contains receptors that sense the environment, and adhesion receptors for binding to substrates (Heckman and Plummer, 2013). MF myosins are strikingly localized in filopodia tips, a result of either active transport by walking along actin, via restricted diffusion, or assembling in an initiation complex and ‘surfing’ out on actin as the filopodium elongates (Berg and Cheney, 2002; Tokuo et al., 2007; Baboolal et al., 2016). In fact, there is evidence that Myo10 can undergo intrafilopodia motility, accumu- lating in filopodia tips over time, suggesting Myo10 utilizes active transport to localize to tips (Berg and Cheney, 2002; Kerber et al., 2009; He et al., 2017).

The intensity of DdMyo7 in filopodia tips is consistently two-fold higher compared to the cell body (Figure 11B). The filopodia tip intensity is uniform during filopodia elongation, with little change in intensity as elongation proceeds (Figure 11A,C; (Petersen et al., 2016)). Intrafilopodial transport and accumulation of DdMyo7 in growing filopodia tips was not observed for control DdMyo7 in spite of multiple efforts to visualize this process. These data are consistent with DdMyo7 motors assembling into an initiation complex at the cortex that is pushed out as the filopod extends. While the tip intensity was the same for wild type and many of the mutants, one unexpected observation was the striking enrichment of tip intensity signal from several DdMyo7 mutants, including ΔSAH-PLA and Δ2IQ (Fig- ure 11B; Figure 14).

Quantitative analysis revealed that ΔSAH-PLA had increased signal in filopodia tips (Figure 11A,B). The autoinhibition mutant ‘KKAA’ and smaller proximal tail deletion ‘ΔcDD’ 28 also showed brighter filopodia tips to a lesser extent (Figure 11B; Table 1). Kymographs were generated from time-lapse images and filopodia tip intensity was measured during elongation. The change in tip intensity over time (slope, m) for each filopodia tip was determined (Figure 11A). In contrast to the control, ΔSAH-PLA shows a significant increase in accumulation in the tips of elongating filopodia over time (Figure 11C). Despite their overall higher filopodia tip intensity, KKAA and ΔcDD did not show significant changes in tip intensity during extension (Figure 11C). In spite of the observed accumulation, the signal in the filopodia shaft is diffuse and no moving DdMyo7ΔSAH-PLA puncta were observed. These data do not distinguish active transport of the motor along the actin bundles from dimensionally restricted diffusion of the molecule into filopodia tips but they do highlight the role of DdMyo7 autoinhibition in regulation of the filopodia tip complex.

The ΔSAH-PLA mutant has an MF2 domain that could play a critical role in targeting the mutants to the tip complex and a large change in the lever arm length that significantly impairs autoinhibition. These mutants could accumulate due to dysregulation of DdMyo7 recycling in and out of the tip complex of the filopodia, allowing their enrichment at the tip complex over time. Alternatively, the proximal tail could bind a partner that facilitates the off state or tip recycling, and deleting the domain abolishes that interaction and reduces recycling.

In both cases, control of autoinhibition may facilitate the rearward transport from the tip to the cell body by a compact DdMyo7, as the geometry of the mutant could be unlikely to favor the off state.

Summary and Conclusion

Filopodia formation requires the action of a MF myosin in Metazoa and Amoebozoa, providing the opportunity to address questions of fundamental mechanisms of action by phylogenetically distinct MF myosins. This study and others support a model whereby filopodia formation relies on a dimeric MF myosin. In the case of DdMyo7, the proximal and distal tail cooperate to dimerize (or cluster) the motor (Figure 9). The MF2 domain of DdMyo7 has a specialized role in filopodia formation likely by efficient partner binding and

29 an ability to oligomerize DdMyo7 motors. The ability of the Myo10 MF to functionally sub- stitute for the DdMyo7 MF2 domain (Petersen et al., 2016) highlights the conservation of key attributes of filopodial MF myosins. Interestingly, during the evolution of filopodia myosins, the internal MF present in the Dictyostelium DdMyo7 tail (and likely present in an ancestral MF myosin; (Kollmar and Mühlhausen, 2017) has been replaced in Myo10 by a membrane interaction domain. This reveals that filopodia formation doesn’t require a precise tail design (two MF domains or PH domains) as long as it serves to target the motor to the membrane and promote self-association. The data presented here is consistent with a model of a weak, context-dependent self-association domain of DdMyo7 being present in the proximal tail region and an MF2 domain serving to interact with a binding partner that promotes dimerization or clustering of the motor (Figure 12). Partner binding would lead to activation of the motor by preventing internal auto-inhibitory interactions and likely also lead to sufficiently high local concentrations of the motor to favor proximal dimerization. The SAH-PLA mutant indicates that this region is required to promote autoinhibition and is thus key to regulate the life time the motor stays associated in an active form. This design is likely to favor both optimized function and precise regulation of the molecule.

Myosins such as the MF myosins required for filopodia formation are regulated, so- phisticated enzymes and likely resulted from multi-domain co-evolution. The large tail domain of DdMyo7 (and others) is required to not only bind partners that regulate its activity, but also stabilize the off-state of a myosin with a long lever arm. Head-tail interaction and formation of the off-state controls both cortical targeting and filopodia formation (Figure 5; (Petersen et al., 2016)). The proximal tail of DdMyo7 is implicated in regulating the inac- tivation of DdMyo7 (Figure 6). Disruptions in the lever arm and PLA that impair the myosin’s ability to form filopodia (Figure 10) could result from compromising the ability of the motors to correctly orient actin filaments at the plasma membrane or to traffic actin regulators along the growing filopodium. The shortened lever arms mutants are sufficient for filopodial formation (Figure 10); however, more structural and reconstitution studies are required to precisely define the design of MF myosin required for reorientation of actin filaments at the membrane.

30 The SAH-PLA region regulates the levels of DdMyo7 in the filopodia tip complex as evidenced by dysregulation of tip turnover seen for ∆SAH-PLA and ∆SAH-SH3 deletion mutants (Figure 11). The increasing intensity of both mutants as the filopodium extends is consistent with this region contributing to the ability of DdMyo7 to be turned off and recy- cled back to the cytosol and reveals a previously unappreciated requirement for filopodia tip recycling. MF myosin recycling in filopodia may be required for discontinuous growth of filopodia as the elongation pauses and the tip interacts with the extracellular matrix, possibly when partners such as talin are incorporated into the filopodia tip complex via integrin binding, before resuming elongation (Jacquemet et al., 2016; He et al., 2017). Identification of DdMyo7 binding partners at the cortex and in the tip will provide needed information on how this myosin is incorporated and maintained in the initiation sites and the tip complex.

The amoebozoan filopodial myosin DdMyo7 and metazoan MF myosins are proposed to have evolved from a common ancestor that had two MF domains, with an overall domain structure similar to DdMyo7, metazoan Myo7, Myo22 and Myo15; (Kollmar and Mühlhausen, 2017). As with DdMyo7, the majority of MF myosin family members either lack a clear region of coiled-coil sequence that could promote dimerization or they only have a short stretch of coiled coil. Thus, the partner-mediated dimerization mechanism employed by DdMyo7 likely reflects the properties of its ancestral MF myosin. In the case of Myo7A, it has a small coiled-coil sequence that appears insufficient for dimer formation although binding to its partner MyRIP activates transport, consistent with partner-mediated dimerization (Sakai et al., 2011). Myo7B lacks any identifiable coiled coil sequence, similar to DdMyo7, and it also appears to be assembled into either a transport or anchoring complex through binding to partners (Li et al., 2017; Yu et al., 2017). A similar mode of partner-mediated dimerization has been shown for Myo6 (Sweeney and Houdusse, 2010b), indicating that control of myosin dimerization through partner interaction is a widely used and ancient strategy for activating and modulating myosin function.

DdMyo7 and Myo10 can both promote filopodia formation when the motor domains alone are stably dimerized (Figure 8; (Tokuo et al., 2007)). The mechanism of dimerization

31 is clearly distinct for DdMyo7 and Myo10. The metazoan filopodial MF myosin Myo10 has evolved a specialized mode for forming anti-parallel coiled-coil dimers via a short self- association region of rather high affinity (Lu et al., 2012; Ropars et al., 2016). Further studies of Myo10 are required to demonstrate whether the recycling or detachment of the motor from the tip complex controls its enrichment at the tip. In the case of DdMyo7, the results here suggest that it may function as a dimer via partner binding. Evolution has solved the interesting and complex problem of modulating motor function through co-evolution of head, lever arm, and tail to maintain stable, yet activatable off-state, and a mechanism for precisely recruiting and activating the motor when and where necessary for cellular function. The findings here not only provide a better understanding of the formation and regulation of these widely used cell sensors, but also provide insight into how other metazoan MF myosins that evolved from a common ancestor (such as those making stereocilia and microvilli) may use an array of optimized regulatory methods including partner- mediated dimerization and post lever arm regulated autoinhibition to carry out their specialized cellular functions.

32 Materials and Methods

Cells lines and Microscopy: Dictyostelium control/wildtype (AX2) or myo7 null (HTD17- 1) (Tuxworth et al., 2001) were cultured in HL5 media. Cell lines were generated and screened as described (Galdeen et al., 2007; Gaudet et al., 2007; Petersen et al., 2016). All expression plasmids were based on the cloned myoi gene (dictyBase:DDB-G0274455; (Titus, 1999)) The complete list of plasmids and oligos is provided in Supp. Table 1. Live cell imaging was done as previously described (Petersen et al., 2016). Briefly, cells are adhered to cover glass and starved for 45-75 minutes in nutrient-free buffer (SB, 16.8mM phosphate pH 6.4) then imaged at 1-4Hz on a spinning disk confocal with a 1.4 NA 63X objective (3i Marianas or Zeiss AxioObserver Z.1).

Data Analysis: Images were quantified using a custom FIJI plugin “Seven” (Schindelin et al., 2012; Petersen et al., 2016). Cells not expressing transgenic proteins were excluded from the analysis. Statistical analysis was performed in Prism (GraphPad). One-way ANOVA analysis with post hoc Tukey test or Dunnett’s multiple comparison to wildtype control was used to compare groups; student’s t-test was used when only comparing two data sets. Automated analyses data points deemed definite outliers (0.1%) by Rout method were excluded. Error bars are SEM unless noted. P values represented on graphs by p≥ 0.05 = ‘ns’; p< 0.05 = ‘*’, p< 0.01 = ‘**”, p< 0.001 = ‘***’, p< 0.0001 = ‘****’. Significant differences are in comparison to control (DdMyo7) unless noted. Filopodia formation in cells expression PLA fragments were manually scored for the presence of actin-rich protrusions. Filopodia tip accumulation was analyzed by creating kymographs of initiating filopodia in FIJI, then drawing a line along the filopodia shaft and calculating the intensity of the line.

Plasmid design: Expression constructs were generated using a combination of standard ligation cloning, PCR cloning (StrataClone, Agilent), Q5 mutagenesis (New England Bi- olabs) and Gibson Assembly (New England Biolabs) (see Supp. Table 1). DNA modification and restriction enzymes were obtained from New England Biolabs and all PCR-generated DNAs were verified by Sanger sequencing (Biomedical Genomics Center, UMN). The integrating GFP-DdMyo7 expression plasmid pDTi74, KKAA autoinhibition mutant 33 plasmid pDTi321 and ∆Pro2-MF2 mutant plasmid pDTi357 have been described (Tuxworth et al., 2001; Petersen et al., 2016). Extrachromosomal Dictyostelium expression plasmids were made with pTX-GFP (Levi et al., 2000). Fragments for expression in bacteria were cloned into either pET-23a (Novagen) or a custom modified pET-14 plasmid (Novagen) that lacks the thrombin cleavage site and instead has a TEV protease site.

Cell culture and transformations: Cells were cultured in HL5 (Formedium) at 22°C on bacteriological plastic. Media was supplemented with 60U/mL penicillin and 60µg/mL streptomycin sulfate (Sigma). Cells were transformed with expression plasmids as described (Gaudet et al., 2007) and transgenic cells were selected with either Neomycin sulfate (10 µg/mL) or Hygromycin (35 µg/mL) (Gold Biotechnology). Transformation plates were screened for fluorescence, individual colonies picked, grown to confluency, cell lysates prepared as previously described (Galdeen et al., 2007) and protein expression verified by western blotting. Blots were probed with either a mouse monoclonal anti- GFP antibody (MMS-118P clone B34; BioLegend Inc. catalog no. 902605) or a rabbit pol- yclonal antibody specific for the DdMyo7 heavy chain directed against aa 809 - 901 (UMN87 (Tuxworth et al., 2005)), and the heavy chain of MyoB (Novak et al., 1995), a class I myosin, as a loading control. Detection was performed with either Alexa Fluor 680- or 800-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Invitrogen) using an Odyssey infrared imaging system (LI-COR Biosciences).

Live Cell Imaging: Confocal microscopy was performed on an AxioObserver Z.1 stand (ZEISS) with Plan-Apochromat 63×/1.4 NA oil objective (ZEISS), a Yokogawa CSU-X1 spinning disk, and Photometrics Evolve EMCCD camera, and laser stack with two 50 mW solid-state lasers (488 nm and 561 nm). The microscope is run by SlideBook software (3i - intelligent imaging innovations). Z stack, time-lapse images were captured 1 - 2 µm from the cover glass to image the substrate adhered region with the most filopodia. Images are collected for 30 sec - 1 min to avoid photo-toxicity at a frame rate of 1 or 4 Hz.

34 Fixed cells: Cells were fixed with picric acid (Humbel and Biegelmann, 1992) and stained with Alexa647 fluorescent phalloidin and DAPI (Thermo Fisher). Slides were mounted in prolong diamond (Invitrogen) and imaged on Eclipse Ni-E microscope with a 63×/1.40 NA oil immersion objective (Nikon), a SOLA solid state white-light excitation subsystem (Lu- mencor), and a CoolSNAP ES2 CCD (Photometrics) run by Nikon Elements software.

Protein Expression and Purification: The 6xHis fusion proteins were expressed in either BL21-AI, BL21 (DE3) Rosetta (Invitrogen) or BL21-Gold (DE3) (Agilent) Escherichia coli at 20°C after induction with 0.2 mM IPTG with or without 0.2% L-arabinose, accord- ingly. Cells were collected by centrifugation, frozen in liquid N2 and stored at -80°C. Fro- zen cells were thawed and lysed using a TS cell disruptor (Celld). The soluble fraction of the lysate was applied to a Histrap FF crude column (GE Healthcare) and the His fusion proteins were eluted with 20 mM Tris, pH 7.5, 300 mM NaCl, 200 mM imidazole or 20 mM HEPES, pH 7.5, 300 mM NaCl, 1 mM DTT, 200 mM Imidazole, pH 7.5. The proteins were further purified by ion exchange chromatography on a HiScreen CaptoQ column (GE Healthcare) and gel filtration on a Superdex 200 or Superdex 75 column (GE Healthcare) in a final buffer containing 10mM Tris pH 7.5; 50 mM NaCl or 20 mM Na-Phosphate, pH 7.5, 100 mM NaCl, 1 mM DTT. The final pool was concentrated, flash-frozen in liquid N2 and stored in small aliquots at -80°C. Samples for analytical gel filtration and MALS were digested with Tobacco Etch Virus protease and the tag removed by incubation with Histrap beads prior to use.

Analytical ultracentrifugation: Analytical ultracentrifugation was performed in an Optima AUC analytical ultracentrifuge (Beckman Coulter) either through the Beckman testing pro- gram at the Center for Analytical Ultracentrifugation of Macromolecular Assemblies at the University of Texas Health Science Center at San Antonio, by Dr. Borries Demeler, Direc- tor or at the Beckman Coulter Colorado R&D Center.

The fragments were dialyzed into 10mM phosphate, 50mM sodium chloride (pH 7.4) and spun at 45,000 X g to eliminate aggregates. The fragments were diluted to a range of

35 concentrations using E280 5,120 OD mol-1 * cm-1 extinction coefficient. AUC samples were then loaded into 2-sector charcoal-epon centerpieces with quartz windows and spun at 45,000 rpm in an An-50 Ti 8 hole rotor. Sedimentation velocity scan data was acquired at both 230 nm and at 280 nm simultaneously with a linear resolution of 10 microns and at 180 second intervals. Up to 250 scans were collected. This data was fit to provide so- lutions of the Lamm equation by the software package Ultrascan III (v4, rev 2475) using a 2-dimensional gridsearch over Sedimentation Coefficient space (0.5 < S < 5) and frictional ratio (1 < f/f0 < 4). The resulting sedimentation coefficient population distributions were converted to molecular weight population.

36 Figures

Figure 5 Recruitment to the cortex and release of head-tail autoinhibition promotes filopodia formation.

A. Micrographs of D. discoideum cells expressing GFP tagged DdMyo7 constructs expressed in myo7 null cells. Note the concentration of GFP-DdMyo7 and the KKAA autoinhibition mutant in the filopodia tip and distinct cortical enrichment of the GFP-tail. Scale bar represents 10μm. B. Illustration of the quantitative image analysis pipeline (adapted from (Petersen et al., 2016). Cells expressing GFP-DdMyo7 fusions are analyzed by iden- tifying fluorescent cell bodies and filopodia tips then registering those tips to cells to cal- culate average number of filopodia per cell. The fluorescence intensity of a 0.8µm band around the cell periphery is measured and compared to the rest of the cell body to measure cortical targeting. Only cells expressing the fluorescent protein are included in analysis by thresholding. C. Schematic illustration of DdMyo7 and fragments. N-terminal motor domain (grey oval), light chain binding IQ motifs (ovals), stable α-helix (SAH), candidate dimerization domain (cDD) two proline-rich regions (Pro), MyTH4 domains (rods), FERM domains (circles) and a src-homology 3 domain (SH3). Amino acid numbers are indicated above. D. Graph of cortical band intensity. E. Violin plot of number of filopodia per cell. Cells expressing the tail or motor fragments lack filopodia and are excluded. C-D. DdMyo7 and KKAA means are represented by horizontal solid and dashed lines, respectively, for comparison. Significance indicators from Tukey’s test are in comparison to DdMyo7 control, unless otherwise indicated on the graph.; ns = not significant; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Figure 6 The proximal tail regulates DdMyo7 activity.

A. Micrographs of D. discoideum cells expressing GFP-tagged deletion constructs in myo7 null cells. Scale bar represents 10μm. B. Schematic illustration of constructs. C. Cortical band intensity of proximal tail deletion mutants. D. Violin plot of the distribution of filopodia per cell. C-D. DdMyo7 and KKAA means are represented by horizontal solid and dashed lines, respectively, for comparison. Significance indicators are in comparison to DdMyo7 control by Dunnett’s multiple comparison test, ns = not significant; * = p < 0.05; **** = p<0.0001.

Figure 7 The proximal tail domain has a dominant negative effect on filopodia and weakly dimerizes in solution.

A. Schematic of proximal tail region. Numbers represent amino acid number in the full protein. B. Representative micrographs of AX2 (control) D. discoideum cells fixed and stained with phalloidin (actin) and DAPI (nuclei). Left: control untransformed cells. White arrowheads point to filopodia. Right: GFP-PLAlong fragment or GFP-PLAshort expression in wildtype cells. C. Cortical band intensity of cells expressing either GFP-DdMyo7, GFP alone or one of two different GFP-PLA fragments. D. Graph of phalloidin-stained filopodia per cell. E. Graph of molecular weight distribution of PLA830-1000 (single concentration run) or PLA830-1000 (three increasing concentrations) by sedimentation velocity analytical centrifugation. Significance indicators, ns = not significant; ** = p < 0.01; *** = p<0.001.

Figure 8 Filopodia formation promoted by a forced dimer of the motor

A. Schematic illustration of the motor-Pro1 region or forced dimer (CC). B. Localization of the motor-Pro1 and motor-Pro1-CC motors in myo7 null cells. C. Cortical intensity of expressed motor fragments significant values. Motor-Pro-CC also enriched relative to DdMyo7 control (***, horizontal line). D. Violin plot of filopodia per cell. The horizontal line represents the mean of DdMyo7 control. E. Measurement of filopodia length of motor- Pro1-CC compared to the control DdMyo7. Significance indicators from Tukey’s test are in comparison to DdMyo7 control unless otherwise noted, ns = not significant; * = p < 0.05; **** =p<0.0001

Figure 9 Functional cooperation of the proximal tail and MF2 regions.

A. Schematic illustration of proximal tail and MyTH4-FERM double domain mutants. B. Cortical intensity of tail deletion mutants, significant values are compared to DdMyo7 control (horizontal line). C. Violin plot of filopodia per cell. The horizontal line represents the mean of DdMyo7 control. Significance indicators from Tukey’s test are in comparison to DdMyo7 control unless otherwise noted, ns = not significant; * = p < 0.05; **** =p<0.0001.

Figure 10 Shortening the lever arm and post lever arm disrupts filopodia formation.

A. Schematic illustration of deletion mutants. B. Cortical intensity of lever arm and PLA region deletion mutants, significant values are compared to DdMyo7 control (horizontal line). C. Violin plot of filopodia per cell. Solid horizontal line represents mean of DdMyo7 control, dashed line represents KKAA. Significance indicators from Tukey’s test are in comparison to DdMyo7 control unless otherwise noted, ns = not significant; *** = p< 0.001; **** = p<0.0001.

44 Figure 11 Deletion of the proximal tail region causes DdMyo7 accumulation in filopodia tips. A. Method for analysis of tip intensity during filopodia extension. Shown is a myo7 null expressing ∆SAH-Pro1. Left top: Sample micrograph dotted line drawn from the filopodia tip to cell body. Axis labels ‘x’ and ‘y’ are distance units (microns). Left bottom: Sample graph representing the fluorescence intensity of GFP-fusion protein along filopodia. The X-axis is distance along the length of a filopodia starting and the tip, and Y-axis represents GFP intensity in arbitrary units. Note the two intensity maxima at the filopodia tip and cell body/cortex. Right top: Sample kymograph of an elongating filopodium (generated from the line on the left). The line is drawn along the extending filopodia tip. The X- axis is distance (microns) and the Y- axis is time (seconds). Right bottom: Sample graph representing the intensity measurement of the filopodia tip over time from the above kymograph. X-axis is time (seconds) and Y-axis is GFP intensity (arbitrary units). B. Graph of the tip/cell body intensity ratios of GFP-DdMyo7 and mutants. All constructs are expressed in myo7 null cells except for the 2IQ/SAH-PLA mutant that does not rescue filopodia formation and was expressed in the AX2 wild type strain (noted on graph). C. Graph of filopodia tip intensity accumulation rates (change in tip intensity during elongation) of GFP-DdMyo7 and mutants. Note that a negative rate indicates the tip gets dimmer. Com- parisons are to DdMyo7 control, ns = not significant; * = p < 0.05, ** = p < 0.01; *** = p< 0.001, **** = p<0.0001.

Figure 12 Model of DdMyo7 Inhibition and Activation States of DdMyo7.

The major domains are shown as in Figure 5. (Left) The inactive state where MF2 interacts with the motor domain to autoinhibit activity. (Middle) Active DdMyo7 that is dimerized via partner binding and dimerization mediated by a dimerization region (rectangle) of weak affinity. (Right) Proposed active state for the ΔSAH-SH3 mutant that lacks the central region of the DdMyo7 encompassing the SAH, PLA, MF1 and SH3 domains. Shown here is a pair of myosin heavy chains dimerized by partner binding to the MF2 and extended motor domains that can bind two actin filaments.

46 Table 1 – Quantification of DdMyo7 mutants in myo7- cells

intensity

± SEM

/cell

accumulation

tip tip

% cells with filo- with cells % podia cell per filopodia ± SEM in- Cortex/cell ratio tensity SEM length filopod (μm) Tip ratio (cells) n (experiments) N Autoinhibition dysregulated infilopo- Activity diaformation 2.26 1.17 3.05 1.86 WT= DdMyo7 58 ± ± ± ± No 84 4 No ‘wildtype’ 0.32 0.03 0.12 0.16

More 5.32 1.76 2.69 3.05 than WT, due to KKAA 79 ± ± ± ± No 130 3 Yes increase 0.37 0.08 0.08 0.12 in active form

NONE 1.02 (lacks Motor-Pro 6 N/A ± N/A N/A N/A 206 4 Yes required 0.01 domains)

More targeted, 1.32 1.38 2.14 1.23 but less Motor-Pro- active; 11 ± ± ± ± No 181 7 Yes CC forms 0.14 0.02 0.27 0.18 shorter filopodia than WT

More in active form, but 2.52 1.49 3.0 3.15 not effi- ΔcDD 59 ± ± ± ± No 94 2 Yes cient to 0.27 0.07 0.16 0.19 lack of proximal dimerization

2.49 1.23 2.8 1.5 ΔPro1 51 ± ± ±  - 68 5 No WT 0.25 0.06 0.18 0.17

Active, Proximal tail Proximal less efficient (LA 2.29 2.16 3.1 5.08 and distal oli- ΔSAH-PLA 40 ± ± ±  Yes 78 3 Yes gomeri- 0.32 0.17 0.24 0.58 zation sufficient for activity)

47 3.20 1.33 2.38 2.03 Less active due ΔMF1-SH3 58 ± ± ±  No 203 5 Yes to less 0.27 0.04 0.11 0.13 targeting

Less efficient in filopodia for- 1.60 1.26 2.49 ΔSAH- 3.1 ± mation 25 ± ±  Yes 219 4 Yes (poor SH3 0.17 0.13 0.05 0.19 targeting + no proximal dimerization

3.11 1.38 2.7 2.99 Less ac- ΔPro2- tive due 35 ± ± ± No 82 5 Yes MF2  to less 0.47 0.08 0.23 0.35 targeting

NONE (Need proximal dimerization 0.98 when

MF Deletions MF ΔcDD/ 3 N/A ± N/A N/A N/A 84 4 Yes target- MF2 0.02 ing/ distal oli- gomeri- zation is less efficient)

NONE (Need proximal dimerization ΔSAH- 1.08 when 1 N/A ± N/A N/A N/A 165 6 Yes target- PLA/ MF2 0.02 ing/distal oli- gomeri- zation is less efficient)

More in active form, 3.18 1.43 3.8 3.31 less effi- Δ2IQ 68 ± ± ±  No 95 4 Yes cient due

0.40 0.06 0.18 0.18 to shorter lever arm

More in

Lever Arm Lever 1.17 1.70 active form, Δ3IQ-PLA 14 ± ± N/A N/A N/A 46 3 Yes less effi- 0.17 0.14 cient due to short LA and

48 proximal dimerization

NONE (short 1.51 4.32 lever Δ2IQ/SAH- arm and 0 N/A ± N/A No* 179 2 Υes PLA  no proxi- 0.03 0.26* mal di- meriza- tion) * = wildtype background

49 Table 2 – Molecular Biology constructs

Template Plasmid Expressed Protein Mutation or Primers/Ligations Backbone

full length wild type, aa pDTi741 GFP-DdMyo7 1 - 2357 pDTi3212 KKAA KK2333,2336AA cDNA clone for aa 577 pDTi20 n/a - end of the myoi gene in pBluescript precursor clone for pDTi74 gfp-myoi gene fusion add drug resistance cassette (NEO) to gen- pDTi73 n/a expression cassette driven by the Act6 erate expression plas- promoter with the mid Act15 terminator Internal PLA deletion mutant myi229 TTTATCTT- PCR ∆ clone for aa GTTGTTCTTTGAGC pDTi351 ∆DD pDTi20 855-952 myi228 AAATTT- GAATTACCACCAGG deletion of aa 712-750 ligation of pDTi351 to pDTi359 ∆DD in GFP fusion base pDTi73 pDTi73 plasmid GFP-DdMyo7 ∆ aa pDTi361 ∆DD 855-952 myi277 TCTATCTCTTTTTAGT ∆SAH-PLA PCR ∆ clone for aa AAAACTGCATTTC pDTi406 pDTi20 (SAH DD Pro1) 791-1084 myi276 TCAGCAACAGCAACA GGT deletion of aa 791- ligation of pDTi406 to pDTi410 ∆SAH-PLA 1084 in GFP fusion pDTi73 pDTi73 base plasmid GFP-DdMyo7 ∆ aa pDTi415 ∆SAH-PLA 791-1084 PLA & single MF deletion coding region for aa myi28 GAA- pDTi353 PCR clone 1188-1690 (stop TAGCAATCCATTGG before Pro2-MF2)

50 myi232 ggatccttaA- GCAACTG- GATGAACTGG GFP-DdMyo7 ∆ aa pDTi3572 ∆Pro2-MF2 1691-2357 myi277 TCTATCTCTTTTTAG- deletion of aa 791- TAAAACTGCATTTC pDTi445 ∆SAH-SH3 1684 in GFP fusion pDTi73 base plasmid myi230 CCAG- TTCATCCAGTTGC- TAC GFP-DdMyo7 ∆ aa pDTi446 ∆SAH-SH3 791-1684 aa 1-1690 in GFP ligation of pDTi353 to pDTi356 ∆Pro2-MF2 pDTi73 fusion base plasmid pDTi73 myi227 ATAAC- GTTTCTTACAACGA- deletion of aa 712-750 TAC pDTi394 ∆2IQ in GFP fusion base pDTi73 plasmid myi226 CTTAC- CTATCAAAAACAATTT AAAATC GFP-DdMyo7 ∆ aa pDTi395 ∆2IQ 712-750 myi227 ATAACGTTTCTTACAA PCR clone for ∆ aa CGATAC pDTi404 ∆3IQ-SAH pDTi20 712-880 myi274 CAACAAGATAAAAATA TTAACGAAC deletion of aa 712-880 ligation of pDTi404 to pDTi408 ∆3IQ-SAH in GFP fusion base pDTi73 pDTi73 plasmid GFP-DdMyo7 ∆ aa pDTi413 ∆3IQ-SAH 712-880 myi275 ATAACGTTTCTTACAA PCR ∆ clone for aa CGATACATTC pDTi405 ∆3IQ-PLA pDTi20 712-1084 myi276 TCAGCAACAGCAACA GGT deletion of aa 712- ligation of pDTi405 to pDTi409 ∆3IQ-PLA 1084 in GFP fusion pDTi73 pDTi73 base plasmid

51 GFP-DdMyo7 ∆ aa pDTi414 ∆3IQ-PLA 791-1084 Double domain deletions myi227 ATAAC- deletion of aa 712-750 GTTTCTTACAACGA- in GFP fusion base TAC pDTi441 ∆2IQ/∆SAH-PLA pDTi410 plasmid for ∆ 791- myi226 CTTAC- 1084 CTATCAAAAACAATTT AAAATC GFP-DdMyo7 ∆ aa pDTi442 ∆2IQ/∆SAH-PLA 712-750; 791-1084

deletion of aa 855-952 myi229 TTTATCTT- in GFP fusion base GTTGTTCTTTGAGC pDTi440 ∆DD/∆Pro2-MF2 pDTi356 plasmid for ∆ 1691- myi228 AAATTT- 2357 GAATTACCACCAGG GFP-DdMyo7 ∆ 885- pDTi443 ∆DD/∆Pro2-MF2 952; aa 1691-2357 deletion of aa 791- ∆SAH-PLA/∆Pro2- 1684 in GFP fusion ligation of pDTi353 to pDTi423 MF2 base plasmid for ∆ pDTi410 1691-2357 ∆SAH-PLA/∆Pro2- GFP-DdMyo7 ∆ aa pDTi425 MF2 791-1084; 1691-2357 Extrachromosomal expression GFP-DdMyo7 PLA- pDTi2013 PLA-long Pro1 region aa 809- pTX-GFP4 1154 myi253 ggatcccatatgGAAGAA- PCR clone of coding GAAGAATTGAAG pDTi383 PLA-short pSC-A region for aa 848-1000 myi254 ctcgag- ttaATCATCATCAGCTT CATCAAC GFP-DdMyo7 PLA pDTi385 PLA-short pTX-GFP4 region aa 848-1000

Bacterial expression

myi266 gctagcAGA- CAACTCCAAGAA- PCR clone of coding GAAC pDTi397 PLA830-1000 pSC-A region for aa 830-1000 myi267 ctcga- gATCATCATCAGCTTC ATCAAC pDTi400 PLA830-1000 (6XHis) pET23a 52 myi266 gctagcAGA- CAACTCCAAGAA- PCR clone of coding GAAC pDTi447 PLA829-1619 pSC-A region for aa 829-1619 myi150 ctcgagAG- CATTGTTTCTTAAA- TATAATG pDTi449 PLA829-1619 (6xHis) pET23a generated by Gibson assembly myi294 gcgaaaacctg- tattttcagggcGAA- pET14 pDTi426 PLA850-1000 (6xHis) aa 850-1000 GAATTGAAGAAATT- rTEV GGAAG myi295 tcgggctttgttag- cagccgTTAATCATCAT CAGCTTCATC deletion mutagenesis - pDTi426 template TEV AS pET14 GCCCTGAAAATACAG pDTi454 PLA879-1000 aa 879-1000 rTEV GTTTTC myi309 AAA- GAACAACAAGA- TAAAAATATTAACG

Figure 13 Western blots Dictyostelium cell lines

Western blots of cell lysates from cells expressing GFP-tagged DdMyo7 mutants. MyoB is used as a loading control (124kDa). A. Whole cell lysates of wildtype (Ax2) and myo7 null cells (primary Ab: αMyo7, αMyoB). B. Whole cell lysate of GFP-DdMyo7 in wildtype cells (primary Ab: αMyo7). C. Whole cell lysates of wildtype cell lines expressing LA/PLA mutants (primary Ab: αGFP, αMyoB). Upper bands represent mutants, lower band representing MyoB loading control. D. Whole cell lysates ofmyo7 null cell lines expressing motor-Pro-CC, PLA, LA/PLA, and MF/PLA mutants (primary Ab: αGFP, αMyoB). Upper bands represent mutants, lower band is the MyoB loading control.

Figure 14 Localization of various deletion mutants.

Representative images of myo7 null cells (top) or wildtype (bottom) expressing GFP tagged constructs with noted deletions, scale bar represents 10μm.

3. CHAPTER 3: VASP mediated actin dynamics activate a

filopodia myosin3

4. Ashley L. Arthur1, Anne Houdusse2 & Margaret A. Titus1* 5. 6. 7. 1 Department of Genetics, Cell Biology, and Development, University of Min- nesota, Minneapolis, MN 55455 8. 9. 2 Structural Motility, Institut Curie, CNRS, UMR 144, F-75005 Paris, France 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. * corresponding author 21. Margaret A. Titus 22. Department of Genetics, Cell Biology, and Development 23. 6-160 Jackson Hall 24. 321 Church St SE 25. University of Minnesota 26. Minneapolis, MN 55455 27. 28. E-mail: [email protected] 29. Phone: 612-625-8498 30. Fax: 612-625-4648

3 Manuscript submission under review 56

Summary

Filopodia are thin actin-based structures that cells use to interact with their environments. Filopodia initiation requires a suite of conserved proteins but the mechanism remains poorly understood. The actin polymerase VASP and a

MyTH-FERM (MF) myosin, DdMyo7 in amoeba and Myo10 in animals, are essential for initiation. DdMyo7 is localized to dynamic regions of the actin-rich cortex.

Analysis of VASP with altered activity reveals that localized actin polymerization is required for myosin recruitment and activation in Dictyostelium. Targeting of

DdMyo7 to the cortex is not sufficient for filopodia initiation; VASP activity is required as well. The actin regulator locally produces new actin filaments which activates a MF myosin. Myosin then shapes or crosslinks the actin network so parallel bundles of actin can extend during filopodia formation. This work reveals cooperativity of an actin binding protein and the actin cytoskeleton on mediating myosin activity during filopodia initiation.

Keywords

Filopodia, actin dynamics, VASP, MyTH4-FERM myosin

57 Introduction

Efficient and directed migration of cells depends in their ability to detect and respond to chemical signals and physical cues in the environment. Adherent and migrating cells utilize specialized membrane protrusions called filopodia to sense and respond to these signals. Filopodia have roles in an array of cell processes, notably neuronal growth cone guidance, cell-cell junction formation during development (reviewed by (Gomez and Letourneau, 2014; Martín-Blanco and Knust, 2001)) and metastatic invasion in some cancer cells (Arjonen et al., 2011, 2014; Cao et al., 2014; Shibue et al., 2012). Although most intensely studied in animal cells, filopodia are ubiquitous in moving cells and have been observed in various Rhizaria (Cavalier-Smith and Chao, 2003), including predatory vampire amoeba (Hess et al., 2012), Discoba (Hanousková et al., 2019), Apusoza (Yabuki et al., 2013) Amoeboza and Holozoa (Sebé-Pedrós et al., 2013).

Filopodia are thin membrane-based projections that arise from the polymerization of parallel actin bundles against the plasma membrane of a cell. A core set of conserved proteins orchestrates the formation of this actin-filled structure, including a Rho family GTPase (Rac1, Cdc42), an actin polymerase (VASP or formin) and a MyTH4-FERM Myosin (Mattila et al., 2007; Sebé-Pedrós et al., 2013; Nobes and Hall, 1995; Tuxworth et al., 2001; Faix et al., 2009). Filopodia also contain additional core proteins including actin crosslinkers (Vignjevic et al., 2006) as well as mechanosensitive and adhesion proteins (Jacquemet et al., 2019) that likely dictate the diverse repertoire of their functions. The key first step of filopodia formation is initiation and two models describing this process have been proposed - the convergent extension model where filopodia emerge via reorganization of an Arp2/3 branched actin network (Svitkina et al., 2003; Tokuo et al., 2007) and de novo or tip nucleation model where unbranched actin filaments are bundled and elongated by formin and then crosslinked by a protein such as fascin (Steffen et al., 2006; Faix and Rottner, 2006; Schirenbeck et al., 2006). It is likely that both mechanisms are used, reflecting cell type variations (Young et al., 2015). In spite of the structural and functional similarities of protruding filopodia across

58 cell types, the underlying mechanism of their formation can vary and the initial steps are still poorly understood. Filopodia formation in evolutionarily distant Amoebozoa and Metazoa is driven by paralog MyTH4-FERM myosins (MF; myosin tail homology 4, band 4.1, ezrin, radixin, moesin) - DdMyo7 and Myo10, respectively (Tuxworth et al., 2001; Berg and Cheney, 2002). The MyTH4-FERM family of myosins is ancient and members have been identified in many branches of the tree of life including Amoebozoa, Metazoa, Stramenopiles (oomycetes) and Aleveolates, (i.e. ciliates and apicom- plexans) (Odronitz and Kollmar, 2007; Kollmar and Mühlhausen, 2017). The sig- nature MF domain mediates interaction with partner proteins such as microtubules (Weber et al., 2004; Toyoshima and Nishida, 2007; Planelles-Herrero et al., 2016) and the cytoplasmic tails of adhesion and signaling receptors (Hirao et al., 1996; Hamada, 2000; Zhang et al., 2004; Zhu et al., 2007). In addition to filopodia formation, this group of myosins play a role in the formation and/or organization of parallel actin-based structures such as microvilli (Myo7) and stereocilia (Myo15) (Manor et al., 2011; Weck et al., 2017). Myo7 and Myo15 have tandem MF domains in their tails whereas Myo10 has a single MF domain. Myo10 is restricted to the Holozoan lineage that includes fungi and animals and is essential for filopodia in mammalian cells (Berg and Cheney, 2002; Bohil et al., 2006; Kollmar and Mühlhausen, 2017). It is distinguished from other MF myosins by the presence of an antiparallel dimerization domain and membrane binding PH domains that pre- cede the C-terminal MF domain (Umeki et al., 2011; Lu et al., 2012; Ropars et al., 2016). DdMyo7 is essential for filopodia formation in amoeba. It has two MF domains in it tails, similar in domain organization to mammalian Myo7 and Myo15, and resembles the presumed ancestral MF myosin (Kollmar and Mühlhausen, 2017).

MF myosins are essential for the first step of filopodia formation, initiation, in both mammalian cells and Dictyostelium (Bohil et al., 2006; Petersen et al., 2016). These myosins are found in a compact autoinhibited form in the cytosol mediated by tail binding to the motor domain (Yang et al., 2009; Umeki et al., 2009). Re- cruitment to the cortical region is accompanied by relief of autoinhibition, opening up the myosin and promoting dimerization (Umeki et al., 2011). In the case of 59 Myo10, it has been found to co-precipitate with the actin bundler/polymerase VASP and it is thought to transport VASP towards the tip of an extending filopodium to promote its growth (Tokuo and Ikebe, 2004; Kerber and Cheney, 2011). Interest- ingly, VASP is also essential for filopodia formation both animals and amoeba (Lebrand et al., 2004; Han et al., 2002). Dictyostelium vasp null mutant phenocop- ies the myo7 null (Tuxworth et al., 2001; Han et al., 2002). The functional link between the mammalian filopodial MF myosin and VASP and the shared role in filopodia formation in amoeba suggests an evolutionarily conserved cooperation between the myosin and actin regulator that both bundles and promotes actin polymerization at the cortex. MF myosins lacking a phosphoinositides binding via PH domain (including amoeboid Myo7, animal Myo7s, Myo15) are likely reliant on actin or other binding partners to mediate their recruitment and activation in vivo. This potential link between an essential actin regulator, VASP, and MF myosins was investigated by examining the role of actin dynamics and VASP in DdMyo7- based filopodia initiation.

60 Results

DdMyo7 targets to actin in pseudopodia

DdMyo7 is observed at filopodia tips and essential for filopodia formation (Figure 15A,B)(Tuxworth et al., 2001). Filopodia formation can be rescued in myo7 null cells by expressing a GFP-tagged DdMyo7 (Figure 15A, (Petersen et al., 2016)). DdMyo7 is associated to the cortex where it has been seen in growing phagocytic cups (Tuxworth et al., 2001) and at extending pseudopodia (Figure 15B and (Petersen et al., 2016)). The first step in filopodia formation is targeting DdMyo7 to initiation sites at the cell cortex but how this happens is unknown. A linescan around the cell periphery revealed there is striking overlap between the actin and GFP-DdMyo7 (Figure 15C). Quantification of actin and DdMyo7 intensity during active pseudopod extension showed a strong correlation over time (Figure 15D,E). The relative change intensity over time was quantified in multiple cells. The fluorescent intensity of GFP-DdMyo7 and LifeAct (marking F-actin, (Riedl et al., 2008) was measured in extending pseudopodia. The average normalized in- tensities of DdMyo7 were plotted relative to actin and showed a strong linear correlation (Figure 15F) revealing that DdMyo7 is highly targeted to actin-rich, actively extending pseudopodia.

Confocal images suggested a difference in localization of full length GFP- DdMyo7 (DdMyo7 hereafter) and the tail fragment. While the tail was targeted all around the cortex, full length DdMyo7 is localized asymmetrically as it is mostly in the leading edge (pseudopod) (Figure 15G, top). The actin network (and pseudopodia) also showed asymmetrical distribution typically biased toward the front of the cell, suggesting the motor domain and not the tail could be targeting this myosin to the actin pseudopod. Linescans to measure the intensity around the periphery showed that actin, DdMyo7 and a forced dimer motor fragment (motor-FD; used because a motor fragment alone shows poor cortical targeting (Arthur et al., 2019) ) were all enriched at the leading edge (Figure 15G). Cortical intensity asymmetry measurement was used as a proxy for pseudopod targeting. The radial in- tensities of a 0.8 µm wide band were measured around the cell and the variation (standard deviation, SD) indicate how uniform the localization is around the cell

61 cortex. Actin shows a high SD given its asymmetrical localization (Figure 15H). Full length DdMyo7 and a motor-FD show a similar standard deviation as actin. In contrast, the tail is uniformly localized around the perimeter of the cell and not well restricted to on part of the cell as illustrated by its lower SD (Figure 15G-H). These data show that the DdMyo7 tail can bind all along the cortex yet the motor restricts the full length myosin to regions of dynamic actin extension such as a pseudopod.

DdMyo7 is localized to dynamic cortical actin

Actin filaments incorporate new monomers and polymerize again the membrane at the leading edge of the cell (Borisy and Svitkina, 2000; Pollard et al., 2001). Altering actin dynamics using anti-actin drugs allowed us to test the role of actin polymerization on DdMyo7 recruitment (Figure 16A). The intensity ratio of a 0.8µm band of the cortex relative to the cytoplasm is 1.2, indicating that DdMyo7 is ~20% more enriched on the cortex (Figure 16B and (Arthur et al., 2019). Cyto- chalasinA (CytoA) binds to the fast-growing (plus) end of actin filaments and blocks incorporation of actin monomers, capping and stabilizing filaments. The capped actin filaments are largely considered to be ADP or old actin (Cooper, 1987) (Fig- ure 16A top). CytoA treatment of cells resulted in a total loss in cortical localization of DdMyo7 (Figure 16A,B, Figure 22, Table 3), suggesting polymerizing actin is required for recruitment of this myosin. Similarly, treatment of cells with Latruncul- inA (LatA), that sequesters monomers and prevents actin filament growth, also resulted in decreased DdMyo7 cortical recruitment (Figure 22C, Table 3). As expected, filopodia formation also is severely impaired in response to treatment with either CytoA or LatA (Figure 16C, Figure 22B,D, and Table 3). Conversely, cells treated with Jasplakinolide (Jasp), that promotes monomer nucleation and stabilizes ADP-Pi actin (Figure 16A) (Bubb et al., 2000; Pospich et al., 2020), results in increased DdMyo7 recruitment to the cell cortex (Figure 2B) and stimulates filopodia formation (Figure 16C, Figure 22E,F) likely due to the combined effect of actin polymerization and DdMyo7 targeting. Together these observations indicate that dynamic F-actin polymerization at the cortex plays a critical role in DdMyo7 recruitment to the cortex where it can then initiate filopodia formation.

Dictyostelium cells can also make actin-rich ventral waves that traverse the cell. These are generated by PIP3-mediated actin polymerization following LatA 62 treatment and washout (Gerisch et al., 2004). Interestingly, DdMyo7 does not localize to actin waves (Figure 22) suggesting that actin density and polymerization are not enough to recruit DdMyo7 and that there are differences in the dynamic actin networks found in waves and pseudopodia.

Role of myosin motor function in targeting and filopodia formation.

The DdMyo7 motor domain has a critical role in the targeting and function of this myosin during filopodia formation (Figure 15G and (Petersen et al., 2016)). The myosin motor activity could be required to generate force between the membrane and actin cytoskeleton or to act as a crosslinker to organize filaments at the cortex. Myosin motors couple ATP hydrolysis to conformational changes between the motor and lever arm to generate force (Robert-Paganin et al., 2020). A single point mutation in a highly conserved isoleucine between the relay helix and L50 uncouples force in DdMyo2 (Sasaki et al., 2003). The equivalent mutation in DdMyo7 (Figure 23; I426A, uncoupler) localizes to the cortex normally (Figure 17A,C) but it fails to efficiently rescue the filopodia formation defect of myo7 null cells (Figure 17A,D and Table 2) establishing that active force generation by MyTH4-FERM myosin is essential for function.

A second mutant that binds but cannot hydrolyze ATP was tested for functionality. Switch II is a conserved loop and connector that assists nucleotide hydrolysis (Robert-Paganin et al., 2020). Without hydrolysis, ATP-bound myosin remains in a weak actin binding state. Mutation of a highly conserved residue in Switch II of DdMyo2 results in a 106 fold decrease in the rate of hydrolysis of ATP, and at least a 10 fold decrease in actin affinity (Friedman et al., 1998). The corresponding residue in DdMyo7 (E386V,Figure 23) failed to target efficiently to the cell periphery (Figure 17B,C) or rescue the filopodia formation (Figure 17D,Table 4). The lack of cortical localization of the non-hydrolyzer indicates that E386V remains in an autoinhibited conformation and cannot switch to an active, ‘open’ state that would allow recruitment (Figure 17A). Charged residues in the distal tail mediate head- tail autoinhibition (Yang et al., 2009). Neutralization of these residues resulted in increased cortical localization and filopodia formation (Figure 17A,C,D, (Petersen et al., 2016; Arthur et al., 2019)). Introduction of the autoinhibition neutralization mutations (K2333A, K2336A, KKAA) into the tail of the E386V hydrolysis mutant 63 strikingly restores its cortical targeting defect (Figure 17A,C, Table 2) but not filopodia formation (Figure 17A,D). This shows again that motor function is required for filopodia formation although it is dispensable for its cortical targeting. This strongly suggests that transport or strong actin binding is not required to reach the place where myosin is recruited at the membrane. It is likely occurring by diffusion of an auto-inhibited form of DdMyo7 that must be activated close to the membrane. The non-hydrolyzer + KKAA double mutant does not target as efficiently to the cortex as KKAA autoactivated mutant (Figure 17C,Table 4), and is not asymmetrically localized (to the pseudopod) like KKAA (Figure 23B). Instead, the non-hydrolyzer+KKAA mutant showed more uniform targeting around the periphery, similar to the tail. The interaction between head and tail in the auto-inhibited state must be destabilized for tail recruitment. The non-hydrolyzer is unable to release autoinhibition since this likely requires hydrolysis and the mutant is thus unable to target to the cortex. However, the uniform localization of the tail or the non-hydrolyzer+KKAA mutant shows that an asymmetric location of the partners does not define where the motor is recruited (since these are not enriched in the pseudopod). While the tail domain is critical for cortical localization, actin binding by the motor domain appears to destabilize the autoinhibited conformation by promoting conformational changes in the motor domain. Thus, both the head and the tail re- fine the localization of the myosin to the actin rich pseudopod at the cell cortex.

The role of VASP in DdMyo7 cortical recruitment . Filopodia initiation and extension requires actin regulators, notably the actin bundler and polymerase VASP. The Dictyostelium vasp null mutant phenocop- ies the myo7 null mutant, it lacks of filopodia, has reduced adhesion and a smaller cell size. (Figure 18A,B, Table 4; (Han et al., 2002; Tuxworth et al., 2001)). The similarity of the phenotypes suggested functional cooperation between VASP and DdMyo7. Myo10 has been proposed to work with VASP in filopodia formation by transporting it to tips (Tokuo and Ikebe, 2004; Berg and Cheney, 2002). DdMyo7 and Dictyostelium VASP (DdVASP) are not known to interact, the role of DdVASP as a potent actin polymerase at the leading edge of cells (Breitsprecher et al., 2008). These results suggested that DdVASP activity could be required for recruitment of DdMyo7. GFP-DdMyo7 (Han et al., 2002) was expressed in wild type or

64 vasp null cells and the cortical enrichment of DdMyo7 measured. Strikingly, the cortical localization of DdMyo7 is significantly reduced in the absence of VASP (Figure 18C,D). In contrast, DdVASP localizes to the cortex regardless of the presence of DdMyo7 ((Breitsprecher et al., 2008), Figure 18C,D). These results reveal that DdVASP is required for cortical recruitment of DdMyo7.

Formins similarly play an important role in filopodia formation. Formins incorporate actin monomers on the barbed end of actin filaments, and elongate parallel actin filaments such as those found in a filopodium (Breitsprecher and Goode, 2013; Mellor, 2010). Diaphanous related formins such as dDia2 are activated by Rho family GTPase and bind DdVASP via their FH2 domain (Watanabe et al., 1997; Schirenbeck et al., 2005a). dDia2 is localized to the cortex and although not absolutely essential for filopodia formation, it is required for normal filopodia number and length (Figure 18A,B, (Schirenbeck et al., 2005b)). Interestingly, GFP- DdMyo7 localizes normally to the cortex in dDia2 null (Figure 18C,D). Together these findings support a specific role of DdVASP activity in recruiting DdMyo7 to the cell’s dynamic actin cortex.

Myo7 - VASP cooperation is required for filopodia formation

The manner by which VASP recruits DdMyo7 to the cortex could be via a VASP mediated actin network formation at the cortex or by direct binding to the DdMyo7 tail which harbors several protein interaction motifs, including tandem MyTH4-FERM and SH3 domains, and proline rich regions (Tuxworth et al., 2005; Petersen et al., 2016). The tail is robustly localized to the entire cortex in vasp null cells, (Figure 19A,C and Table 4), showing that VASP is not a binding partner of DdMyo7 and instead implicates its role in cortical actin dynamics.

The strong uniform cortical localization of the tail in vasp null cells as well as in wild type cells contrasts with the striking asymmetric targeting of full-length DdMyo7. This suggests that specific VASP recruitment at the pseudopod may play a role in regulating the localization of DdMyo7 by releasing head-tail autoinhibition. If so (and if activating DdMyo7 is its sole role), then any mutant that favors the open conformation should target normally in vasp null cells. The KKAA autoinhibition mutant was expressed in vasp null cells where it is targeted to the cortex

65 (Figure 19A,B and Table 4). Unexpectedly, the overall level of KKAA targeting in vasp null was not as robust as the targeting of KKAA in wildtype or myo7 null cells Figure 19C, Table 4) KKAA in vasp nulls is also localized more uniform compared to in control (Figure 24D). KKAA does not rescue the filopodia formation defect of vasp nulls (Figure 19D) indicating that a specific VASP activity is required for filopodia formation. The observation that the constitutively active DdMyo7 mutant can target in the absence of VASP, albeit less efficiently than expected, suggests however that VASP activity does contribute to shift the equilibrium of the autoinhibited myosin to the open state. Together with the results with non-hydrolyzer+KKAA mutant (Figure 17), this indicates that VASP’s activity is likely acting by generating a cortical actin environment that promotes optimal targeting via the motor domain binding to cortical actin polymerized by DdVASP.

In order to verify the requirement of VASP when DdMyo7 is targeted to the cortex or membrane, a prenylation sequence (CAAX, (Weeks et al., 1987)) was added to the C-terminus of DdMyo7 to promote membrane targeting. DdMyo7- CAAX was robustly localized to the cortex in myo7 null, wildtype and vasp null cells (Figure 19A,B, Table 4). DdMyo7-CAAX in myo7 nulls or wildtype cells significantly stimulated filopodia formation however, no filopodia are formed when DdMyo7- CAAX was expressed in vasp null cells (Figure 19A,D and Table 4). These results confirm that targeting DdMyo7 to the membrane alone is not sufficient for filopodia initiation and VASP presence at the cortex is also required.

VASP mediated actin polymerization recruits DdMyo7.

VASP accelerates the rate of actin polymerization, bundles actin filaments and blocks capping protein (Breitsprecher et al., 2008, 2011; Hansen and Mullins, 2010). The VASP dependent localization of DdMyo7 to dynamic actin and pseudopodia suggests the motor domain preferentially binds to newly polymerized actin filaments generated by VASP. Alternatively, myosin recruitment could be specific for VASP-generated actin bundles.

VASP mediates actin polymerization by binding G-actin and profilin-actin and incorporating monomers onto filament ends, but profilin is not essential to promote actin polymerization in Dictyostelium (Breitsprecher et al., 2008). The role of

66 VASP activity in DdMyo7 recruitment was investigated by making mutations in highly conserved regions of the VASP EVH2 domain. First, a charge switch mutation in the G actin binding domain was introduced (GAB K-E, Figure 24A,B based on (Walders-Harbeck et al., 2002; Ferron et al., 2007; Hansen and Mullins, 2010)). Surprisingly, the VASP-GAB K-E mutant potently stimulated both DdMyo7 recruitment and filopodia formation even higher than observed for the DdMyo7 constitutively active mutant with wild type VASP (Figure 24A,C and Table 4). This data reiterates that DdMyo7 targeting stimulates filopodia formation in the presence of VASP.

The requirement for VASP bundling activity was tested by co-expressing either monomeric VASP (1M) or an F-actin binding mutant (FAB K-E) with DdMyo7 in vasp null cells (Figure 20A). These mutations in conserved regions of the EVH2 region (Figure 24B) are both predicted to eliminate bundling and slow actin polymerization (Breitsprecher et al., 2008; Schirenbeck et al., 2006; Applewhite et al., 2007; Hansen and Mullins, 2010). The VASP monomer (1M) and FAB K-E mutants each partially restore DdMyo7 cortical recruitment (Figure 20B). The monomeric VASP mutant can partially rescue filopodia formation; however, the FAB K-E mutant cannot (Figure 20C). It should be noted that the residual filopodia forming activity of the VASP monomer (1M) could be attributed to the anti-capping activity that is retained by the monomer but lost in the F-actin binding mutant (Breitsprecher et al., 2008; Hansen and Mullins, 2010). The decreased recruitment of DdMyo7 to the cortex in cells lacking VASP bundling activity or anti-capping activity, supports a model that the actin polymerization activity of VASP is needed for DdMyo7 targeting.

Discussion

Filopodia formation requires both DdMyo7 and DdVASP. The cortical recruitment of DdMyo7 requires DdVASP mediated actin polymerization to promote filopodia initiation (Figure 18C,D; Figure 19D, Figure 20). VASP impacts the actin network by speeding actin elongation, bundling filaments and blocking capping proteins, however its actin polymerization activity is especially critical for DdMyo7 recruitment (Figure 20). This work shows how actin dynamics in vivo are important

67 for myosin localization, by promoting release of autoinhibition (Figure 19), and motor domain binding to actin at the leading edge (Figure 15,Figure 17).

Figure 21 shows a proposed unifying model for filopodia initiation in amoeba. In the case of Dictyostelium, filopodia initiation likely occurs via the convergent elongation mechanism, as filopodia emerge from the actin at the leading edge of these migratory cells. VASP tetramers (Haffner et al., 1995) at the cortex assemble actin into parallel filaments with a perpendicular orientation to the membrane (Bear et al., 2002; Laurent et al., 1999; Breitsprecher et al., 2008) where clustering of Vasp increases the local concentration of such filaments (ref Mullins). DdMyo7 is folded over on itself, in an inactive, autoinhibited state in the cytoplasm (Arthur et al., 2019; Petersen et al., 2016). The DdMyo7 motor domain binds young actin (i.e. ATP/ADP-Pi actin) at the cortex (Figure 15G, Figure 17C) allowing release of head-tail autoinhibition, and activation (Figure 19). DdMyo7 it then thought to dimerize (Arthur et al., 2019) and assemble into an initiation foci along with DdVASP that bundles the actin filaments (Schirenbeck et al., 2006; Breitsprecher et al., 2008). Next, DdMyo7 dimers act on the actin network to generate force (Figure 17), bending filaments into a perpendicular arrangement with respect to the membrane. VASP continues to promote actin polymerization and inhibits capping of the filaments (Figure 20) allowing actin growth against the membrane along with formin (Schirenbeck et al., 2005a), which drives filopodia elongation.

VASP activity promotes DdMyo7 motor domain to bind actin filaments.

The motor domain of DdMyo7 has an important role in targeting this myosin to the actin cortex at the front of the cell. While the tail fragment is strongly localized all around the entire cortex (Figure 15G and (Arthur et al., 2019)), the full length protein or a motor forced dimer are targeted asymmetrically, in F-actin rich pseudopodia (Figure 15G-H). This is dependent on a catalytically active motor (Figure 17) as this asymmetric targeting is lost in the non-hydrolyzer mutant (Fig- ure 16). Since localization is also lost when filaments are capped with cytochalasin 68 or polymerization is blocked by latrunculin A, an appealing model to account for the observations is that the young or new ATP or ADP-Pi actin at the cortex is specifically recognized by the motor. Indeed, there is biochemical evidence that the nucleotide and structural states of F-actin can regulate myosin motor function (Santos et al., 2020; Zimmermann et al., 2015).

VASP's polymerization and anti-capping activity, but not its ability to bundle actin, is required for building actin networks that recruit DdMyo7 (Figure 19). While the formin dDia2 also has a role in actin elongation and filopodia formation (Schirenbeck et al., 2005b), its activity is not required for DdMyo7 recruitment (Fig- ure 17,Table 4). DdVASP is a potent actin polymerase and binds the sides instead of ends of F-actin filaments (Breitsprecher et al., 2008). The opposite effect of the VASP- GAB K-E and FAB K-E (Figure 20, Figure 24) mutants strongly indicates that VASP is unlikely to control DdMyo7 release of auto-inhibition and specific recruitment by a direct interaction with the DdMyo7 tail. It is most likely via the nature of the actin network formed with VASP activity and how the activated DdMyo7 motor can subsequently exert force on this network that filopodia initiation efficiency is controlled. The finding that an increase or decrease in VASP activity en- hances or diminishes DdMyo7 targeting supports the idea that it is the actin network formed by VASP activity that regulates myosin recruitment. Interestingly, evidence for VASP-dependent changes in the cortical actin network has recently been shown in B16 melanoma cells (Damiano-Guercio et al., 2020). The normal cortical actin network is dense and with many filaments oriented perpendicular to the membrane. In the absence of VASP the network is more disperse and actin filaments are oriented in shallower angles with respect to the membrane. Thus, it remains important to consider whether actin density and geometry, additional proteins or unidentified factors are responsible for or also contribute to DdMyo7 enrichment. In either case, the motor must have a high affinity for the VASP generated network of new actin that is primarily responsible for the initial recruitment that is followed by relief of autoinhibition and activation.

DdMyo7 actin binding, and release of autoinhibition 69 Myosin head-tail autoinhibition is a widely used mechanism for controlling myosin activity (Heissler and Sellers, 2016). Autoinhibition of the motor reduces the unnecessary expenditure of ATP and maintains the myosin in a compact, more readily diffusible conformation until the motor is recruited to its site of action. The mechanism of autoinhibition relief varies across myosins; for example: the motor- proximal tail interaction in β -cardiac myosin is alleviated by sarcomere tension (Spudich, 2015; Fusi et al., 2016) , and Myo5 can be activated by Ca2+-binding to calmodulin light chains as well as by cooperative recruitment to membranes by the actin nucleator Spir and Rab11 (Krementsov et al., 2004; Pylypenko et al., 2016). The regulation of autoinhibition relief and targeting of the metazoan Myosin 7a and Myo7b is still not known but it is clear that ionic interactions between the FERM domain and head mediate autoinhibition (Yang et al., 2009), and partner complex binding to the MF domains in the tail may regulation motor function (Yu et al., 2017). It is interesting to note that these myosins must both be recruited to the actin-rich apical regions of cells and interaction with actin binding proteins or regulators may control this targeting and/or relief of head-tail autoinhibition.

Conserved and divergent models of filopodia myosin function

The role of two evolutionarily distant filopodia myosins, amoeboid DdMyo7 and metazoan Myo10, using both shared and divergent mechanisms to generate filopodia. They both dimerize upon recruitment to the actin-rich cortex (Arthur et al., 2019; Lu et al., 2012) where they likely reorganize the Arp2/3 branched actin network to orient actin filaments perpendicular to the membrane (Svitkina et al., 2003; Tuxworth et al., 2001; Berg and Cheney, 2002; Tokuo et al., 2007; Arthur et al., 2019). Interestingly, both DdMyo7 and Myo10 work in cooperation with VASP. In the case of Myo10, it transports VASP down the length of filopodia during extension (Tokuo and Ikebe, 2004; Kerber et al., 2009). In contrast, VASP activity is shown here to generate new actin ends that recruit DdMyo7. Furthermore, Dicty- ostelium are highly motile cells with very dynamic, loosely bundled (Medalia et al., 2007), short lived filopodia. Given the early origins of Amoebozoa, this simple mechanism of coupling motor activation to actin dynamics at the pseudopod could suggest amoeboid filopodia formation is driven by a minimal regulatory circuit. It is

70 interesting to speculate that VASP-dependent recruitment of an MF myosin represents an early form of cooperation between these two proteins. Perhaps as filopodia played wider roles in development and migration, a signaling-based mechanism of MF myosin recruitment emerged - PIP3 binding resulting from the replace- ment of the first MF domain by a very distinct 3 PH structural motif that can sense and activate Myo10 depending on PIP3 concentrations - and its motor activity was then used to promote VASP transport and thus filopodia extension. It is fascinating to find that the mechanism of auto-inhibition via myosin/tail stabilizing interactions has been conserved throughout the evolution of myosins involved in filopodia initiation in particular and MF myosins in general. The evolution of the functional relationship between VASP and MF myosin would be interesting to explore in organisms such as Drosophila that makes filopodia but lacks Myo10, instead it has a Myo22 (with two MF domains like DdMyo7) and in the earliest organisms that have Myo7, Myo22 and Myo10 such as the filasterean Capsaspora and choanoflagellate Salpingoeca (Kollmar and Mühlhausen, 2017). The development of genetic tools in several evolutionarily significant organisms such as Capsaspora and Salpingoeca (Parra-Acero et al., 2018; Booth et al., 2018; Booth and King, 2020), unicellular organisms at the onset of multicellularity, Spizellomyces (Medina et al., 2020), early diverging fungi, should now allow for the study of the evolution of filopodial MF myosin targeting and activation.

71 Figures

Figure 15 DdMyo7 is localizes with cortical actin.

73 A. (top) Schematic of DdMyo7 illustrating its motor domain, 4 IQ domains and tandem MyTH4-FERM domains (rods-MyTH4, circles-FERM) in the tail; (bottom) Dic- tyostelium myo7- cells expressing GFP-DdMyo7 and RFP-LifeAct (actin), or RFP- LifeAct only. Scale bar is 10µm. B. Confocal images showing localization of GFP- DdMyo7 at the cortex and in filopodia tips, and that filopodia are actin filled projections. C. (top) Tracing of cell periphery in confocal images (dotted line starting at arrow), (bottom) intensity of RFP-LifeAct or GFP-DdMyo7 of line above. D. Confo- cal image of cell extending a pseudopod, the yellow line is perpendicular to the leading edge. E. Linescan intensity profile of DdMyo7 and actin in extending pseudopod (from D). The intensity of actin and GFP-Myo7 during pseudopod extension was normalized to min and maximum intensity for each probe. F. Intensity correlation of GFP-DdMyo7 and RFP-Life-Act plotted as the average spline fit of 10 extending pseudopodia. G. (top) Micrographs of cells expressing RFP-LifeAct, GFP-DdMyo7, GFP-Tail, or GFP-Motor-Forced Dimer (FD). (bottom) Radial intensity from trace of cell periphery. H. Mean of the standard deviation of radial intensity of a cortical (0.8µm) band n>93 cells for each from 3 experiments. One-way ANOVA with multiple comparison correct compared to actin, **** p<0.001, ns not significant.

Figure 16 Actin dynamics regulate DdMyo7 recruitment to the cortex.

A. (top) Jasplakinolide promotes nucleation and stabilizes ADP-Pi actin. CytochalasinA caps actin filaments (bottom) Confocal images of cells expressing GFP-DdMyo7 and RFP-LifeAct treated with vehicle control or drug, scale bar is 10 µm. B. Quantification of the mean ratio of GFP-DdMyo7 intensity in the cortical region compared to the cytoplasm (cortical recruitment ratio). C. Quantification of the number of filopodia per cell. B-C. Circles indicate the means of individual experiments, one way ANOVA with multiple comparison correction, shown to control, p****<0.0001.

Figure 17 DdMyo7 motor activity is required to release autoinhibition.

A. Schematic of proposed effect of motor mutations on function.. B. Confocal images of myo7 null cells expressing GFP-DdMyo7 fusion proteins, scale bar is 10µm C. Quantification of cortical recruitment of DdMyo7 and mutants. KKAA mean is shown as a comparison to non-hydrolyzer+KKAA double mutant. D. Violin plot of number of filopodia per cell. B-C experimental means shown as black circles. One way ANOVA with multiple comparison correction, p***<0.001, p****<0.0001, ns not significant.

Figure 18 VASP is required for DdMyo7 cortical recruitment.

A. Confocal images of wild type or myo7 null, vasp null or dia2 null cells expressing RFP-LifeAct (actin). B. Violin plot showing distribution of filopodia per cell. C. Mi- crographs of cells expressing GFP-DdMyo7 (top) and GFP-VASP in myo7 null, vasp null or dDia2 null cells. A,C. Scale bar is 10µm D. Quantification of the cortical band (0.8µm of periphery) relative to the cytoplasmic intensity of either GFP-Myo7 or GFP-VASP. Circles are experimental means. B, D. One way ANOVA multiple comparison correction, p***<0.001, ns, not significant.

Figure 19 VASP relieves DdMyo7 head-tail autoinhibition to promote targeting and filopodia formation.

A. Micrographs of GFP-DdMyo7 fusion proteins in control and vasp null cells scale bar is 10μm B. Quantification of cortical recruitment of GFP-DdMyo7 and variants in vasp- cells. The line represents the mean GFP-DdMyo7 recruitment in wild type cells. C. Comparison of cortical targeting of activated KKAA or tail in vasp- versus control cells. D. Quantification of number of filopodia per cell in control or vasp- cells. B-D. Circles represent experimental means. One way ANOVA with multiple comparison test, ns not significant, p***<0.001, p****<0.0001, ns, not significant.

Figure 20 VASP mediated actin polymerization recruits DdMyo7 to the cortex to promote filopodia formation.

A. Schematic of wildtype, monomeric, and F-actin binding (FAB K-E) mutant VASP. B. Quantification of the cortical recruitment of DdMyo7 expressed in the vasp null along with VASP mutants. C. Quantification of filopodia per cell of vasp null cells expressing the VASP mutants. B-C. circles represent experimental means. One way ANOVA with multiple comparison correction, p****<0.0001, ns not significant.

Figure 21 Model of VASP mediated targeting of DdMyo7 during filopodia initiation.

A. players involved. ADP-PI actin (darker) and ADP actin (lighter). Tetrameric VASP, DdMyo7 shown with motor domain oval and tail is rods and circles. B. VASP polymerizes actin filaments at the membrane generating a young cortical actin network. DdMyo7 is autoinhibited in the cytoplasm and opens up to bind the young actin network. DdMyo7 forms a partner mediated dimer (Arthur et al., 2019). The activity of VASP and DdMyo7 together organize parallel actin filaments against the membrane during filopodia protrusion.

Figure 22 Concentration dependence of anti actin drugs.

A.-B. Cytochalasin A treatment, C.-D. LatrunculinA treatment, E.-F. Jas- plakinolide treatment of cells at different concentrations A,C,E. Quantification of the mean ratio of GFP-DdMyo7 intensity in the cortical region compared to the cytoplasm (cortical recruitment ratio). B,D,F. Quantification of the number of filopodia per cell with after treatment with vehicle along or actin drugs specified. A-F. One way ANOVA with multiple comparison correction, shown to control, p****<0.0001. G. Confocal images from timelapse of Latrunculin induced actin waves in cells with RFP-LifeAct (actin) and GFP-DdMyo7 (DdMyo7). H. Kymo- graph (from G.) showing DdMyo7 is not strongly associated with in actin in waves. Scale bar is 5 μm, vertical scale bar is 60 seconds.

Figure 23 Conservation of the motor domain and motor in pseudopod targeting.

A. Relay helix region of an alignment of motor domain sequences from M-coffee (Wallace et al., 2006). Boxed columns indicate the highly conserved glutamic acid in switch 2 (non-hydrolyzer, DdMyo7 E386V) and hydrophobic residue in relay loop (uncoupler, DdMyo7 I426A) ‘*’ identical, ‘:’ strongly similar, ‘.’ weakly similar. B. Bar graph of the mean of the standard deviation of radial intensity (same analysis as Figure 15H) of a cortical (0.8µm) band. **** p<0.0001, n>58 cells for each from 3 experiments.

Figure 24 VASP mutants in vivo

A. Confocal images of cells co-expressing GFP-DdMyo7 and untagged wild type and mutant VASP. Scale bar 10μm. B. Alignment of human VASP and DdVASP protein sequences with mutated residues starred above. C. Quantification of cortex to cytoplasm ratio and filopodia number in vasp null cells expressing VASP GAB K-E. D. Cortical radial asymmetry of KKAA in vasp null compared to control. C-D. Student’s t test, ****p<0.0001.

Figure 25 Western blot analysis of DdMyo7 and VASP expression.

Blots of total cell lysates were probed with anti-Myo7 or VASP antibody and MyoB antibody (loading control). The position of the molecular weight standards (Kd) is indicated. A. Western blots of total cell lysates of wild type, myo7 null or vasp null cell lines B. GFP-DdMyo7 expression in control and vasp null cells, and GFP- VASP in control and myo7 null cells. C. Expression of GFP-DdMyo7, GFP-tail, GFP-DdMyo7/KKAA or GFP-DdMyo7/CAAX in myo7 nulls. D. Expression of wild VASP and VASP mutants (not fused to a fluorescent protein).

1% Buffer Only 0.5% DMSO DMSO percent of cells with filopodia 42 66 38 filopodia number + SEM 2.01±0.11 3±0.14 1.97±0.09 cortex:cytoplasm ratio + SEM 1.18±0.01 1.2±0.02 1.2±0.02 N, n 4, 238 3, 209 3, 229 1µM Cyto- chlasinA 5µM CytochlasinA percent of cells with filopodia 0 8.5 filopodia number + SEM 0 1.3±0.04 cortex:cytoplasm ratio + SEM 1.02±0.02 1.07±0.01 N, n 3, 66 3, 234 1µM Latruncul- inA 5µM LatrunculinA percent of cells with filopodia 19 4.7 filopodia number + SEM 1.73±0.06 1.33±0.05 cortex:cytoplasm ratio + SEM 1.12±0.01 1.02±0.01 N, n 4, 387 3, 127 50nm Jas- plakinolide 100nM Jasplakinolide percent of cells with filopodia 72 48 filopodia number + SEM 3.89±0.2 2.21±0.09 cortex:cytoplasm ratio + SEM 1.34±0.03 1.24±0.02 N, n 3, 219 3, 337

Table 3 Quantification with anti-actin drugs.

Cortical recruitment ratio of DdMyo7 and filopodia per cell for GFP- DdMyo7/myo7- cells treated with actin depolymerizing or nucleation promoting drugs.

GFP- GFP- GFP- GFP- GFP- GFP- GFP- DdMyo7- GFP- DdMy DdMyo7- DdMyo7- DdMyo7- DdMyo7- KKAA E386V- VASP o7 E386V I426A CAAX Tail KKAA percent of

cells with 49 80 1 3 22 58 n.c. n.c. - filopodia filopodia myo7 2.29±0 number + 5.32±0.33 1±0 1.5±0.09 1.75±0.15 2.87±0.14 n.c. n.c. .15 SEM cortex:cytoplasm 1.14±0 1.18±0. 1.67±0.06 1±0.01 1.32±0.04 1.19±0.04 1.39±0.03 1.61±0.07 ratio + .02 29 SEM N, n 3, 158 3, 133 3, 186 3, 59 3, 37 3, 237 3, 55 3, 91

GFP- GFP- GFP- GFP- GFP- GFP- GFP- GFP- DdMyo7; DdMyo7; DdMyo7; GFP- DdMy DdMyo7; DdMyo7- DdMyo7 DdMyo7 VASP - VASP- VASP- VASP o7 VASP-1M CAAX -Tail -KKAA WT FAB-K-E GAB-K-E percent of

- cells with 7 35 69 14 75 n.c. 4 n.c. 4 filopodia vasp filopodia 1.25±0 number + 3.78±0.31 3.07±0.25 1.39±0.03 7.6±0.53 n.c. 1.2±0.03 n.c. 1±0 .07 SEM cortex:cytoplasm 1.06±0 1.35±0. 1.29±0.0 1.2±0.02 1.47±0.05 1.19±0.01 2.59±0.12 1.18±0.33 1.24±0.01 ratio + .01 02 2 SEM 86

N, n 3, 118 3, 131 3, 81 3, 322 3, 110 3, 200 4, 239 3, 213 3, 193

GFP- GFP- GFP- GFP- GFP- DdMy DdMyo7- DdMyo7- DdMyo7- VASP o7 CAAX Tail KKAA

percent of cells with 45 54 n.c. 73 n.c. filopodia filopodia 2.36±0 Ax2 control Ax2 number + 2.9±0.15 n.c. 3.65±0.2 n.c. .2 SEM cortex:cytoplasm 1.35±0 1.45±0.04 1.41±0.02 1.79±0.07 1.16±0.25 ratio + .03 SEM N, n 4, 124 5, 266 4, 351 3, 219 4, 216 Table 4 Quantification of filopodia number and cortical targeting.

Average number of filopodia per cells from cells with at least one filopodia. Cortex:cytoplasm ratio is intensity ratio of a 0.8µm band around the periphery compared to the cytoplasm. N is number of experiments, n is number of cells. SEM is standard error of the mean GFP-VASP and GFP-DdMyo7-Tail fail to efficiently target to filopodia tip and were not counted in this analysis.

87 Materials and Methods

Cell lines, cell maintenance and transformations Dictyostelium control/wild-type (AX2), myo7 null (HTD17-1) (Tuxworth et al., 2001), vasp null (Han et al., 2002) and dia2 null (Schirenbeck et al., 2005b) cells were cultured in HL5 media. For transgenic lines, cells were harvested, washed twice with ice cold H50 (20 mM HEPES, pH 7.0, 50 mM KCl, 10 mM NaCl, 1 mM MgSO4 , 5 mM NaHCO3, 1 mM NaH2PO4 and flash spun at 10,000 X g until the rotor reached speed. Cells were resuspended at 5e7 cells/mL and 100uL of cells was combined with 10µg DNA in a 0.1 cm gap cuvette. Cells were electro- porated by pulsing twice, 5 seconds apart with a Bio-Rad Gene-Pulser set to 0.85 kV, 25 µF, and 200 Ω. Cells were recovered 10 minutes on ice and plated in a 10cm dish for 24 hours before moving to selection media, either 10µg/mL G418, 35µg/mL HygromycinB or both.

Expression of the different fusions was verified by western blotting using either anti-DdMyo7 (UMN87, (Tuxworth et al., 2005)), anti-GFP (Biolegend - B34) or anti- VASP (Han et al., 2002) with anti-MyoB used as a loading control (Novak 1995) (Figure 25).

Generation of expression plasmids Cloning and expression constructs. DdMyo7 (dictyBase DDB: G0274455; (Titus, 1999) was expressed with a N-terminal GFP fusion in pDXA backbone with the actin-15 promotor and a NeoR cassette as described by (Tuxworth et al., 2001). The full length tail (aa 809 - end) (Tuxworth et al., 2001), the KKAA autoinhibition mutant (K2333A/K2336A) (Petersen et al., 2016), motor forced dimer (aa 1-1020 followed by the mouse Myo5A coiled coil region and a GCN4 leucine zipper) (Arthur et al., 2019) have been described previously. A full- length GFP-DdMyo7 with a C-terminal prenylation site (CAAX) was generated by Q5 mutagenesis (New England Biolabs) to add codons encoding the CTLL* prenylation motif from Dictyostelium RasG (UNIPROT: P15064). A motor mutant that cannot hydrolyze MgATP, the non-hydrolyzer E386V was designed based on a 88 characterized Dictyostelium Myo2 mutant (Friedman et al., 1998). The combined non-Hydrolyzer + KKAA was made by standard ligation cloning to introduce the motor domain sequence from the non-hydrolyzer mutant into KKAA full length expression plasmid by restriction enzyme digest with BsiWI and BstEII. The uncoupler mutant (I426A) was based on a characterized Dictyostelium Myo2 mutant (Sasaki et al., 2003). It cloned by Q5 mutagenesis.

GFP-VASP was a gift from Dr. Richard Firtel (UCSD) (Han et al., 2002). The VASP tetramer and FAB, 1M mutants were not fused to GFP to avoid any steric hindrance with the fluorescent protein. The full-length VASP cDNA (dicty- Base:DDB_G0289541) was cloned into the pDM344 shuttle vector (Veltman et al., 2009) and the NgoM-IV fragment from this plasmid was ligated into pDM358-mAp- ple (the mApple gene (Shaner et al., 2008) was cloned in between the act6 promoter and act15 terminator of pDM358 (Veltman et al., 2009). VASP-1M was created by introducing a SmaI site and stop codon into the vasp gene, altering the coding sequence from 334 PSLSAPL to 334 PSLSAPG* using Q5 mutagenesis. The F-actin binding mutant (FAB K-E) was based on mutating previously identified critical F-Actin binding residues (K275, R276, K278, and K280; (Schirenbeck et al., 2006) to glutamic acid (Hansen and Mullins, 2010) by Q5 mutagenesis. Oli- gonuclotides used are in the key resource table.

Microscopy and imaging experiments Live-cell imaging was done as previously described (Petersen et al., 2016). Briefly, cells are adhered to cover glass (CellVis) and starved for 45 to 75 min in nutrient- free buffer (SB, 16.8 mM phosphate, pH 6.4), and then imaged at 1 to 4 Hz on a spinning disk confocal with a 1.4 numerical aperture, 63× objective (3i Marianas or Zeiss AxioObserver Z.1). The sample is cooled to 19-21°C. Samples were illumi- nated with 50mW lasers (488nm or 561nm), and a Yokogawa CSU-X1 M1 spinning disk, and captured with an Evolve EMCCD camera. 4-6 Z sections of 0.28-0.5 microns were taken with a 50-250ms exposure with 10-40% laser power. Cells were images for 10 seconds – 10 minutes depending on experiment. Cells are plated at a density of 5x105 per mL. From each imaging dish, 10 fields of view are

89 collected with 2-20 cells per field of view. All data sets represent cells from at least three independent experiments and two independently transformed cell lines.

Drug treatment. Cells were washed free of media, adhered to glass bottom dishes and starved in nutrient-free buffer for 40 minutes. The buffer was replaced by buffer supplemented with the noted concentration of Jasplakinolide (diluted to 0.5% DMSO) , CytochalasinA, Latrunculin A or DMSO control. Jasplakinolide treatment was for 5-8 minutes, CytochalasinA or LatrunculinA for 15-20 minutes, prior to imaging for 10-15 minutes.

LatrunculinA-induced actin waves. Cells expressing GFP-Ddmyo7 and the actin reporter RFP-LimE∆coil (Gerisch et al., 2004) were induced to generate travel- ling actin waves using a modified protocol (Gerisch et al., 2004). Cells rinsed with 16mM phosphate buffer pH 6.8 were seeded on glass bottom dishes (Celvis) at 5x105 cell/mL, incubated in phosphate buffer for 30 minutes and then supplemented with 5µM LatrunculinA for 20 minutes. The solution was diluted to 0.5µM LatA and cells incubated for an additional 30 minutes, then were imaged for up to 2 hours. Images were captured in 5-10 0.3 µm Z sections by spinning disk confocal microscopy (see above) every 5 seconds for 10-30 minute movies.

Data Analysis Protein alignments. Myosin motor domains were aligned using T-coffee algorithm “expresso” (Notredame et al., 2000) and include sequences from uniprot entries: P54697, Q9U1M8, P08799,P19524, K4JEU1, Q9V3Z6, Q9HD67,Q13402, Q6PIF6. VASP mutations were made by first creating a structural alignment with DdVASP (Uniprot: Q5TJ65 ) and Human vasP (Uniprot: P50552) in T-coffee algorithm “espresso”. Next, conserved charge switch mutations in FAB and GAB domain of DdVASP and the tetramerization domain was truncated to make a monomeric VASP based on previous in vitro work (Hansen and Mullins, 2010) (Brühmann et al., 2017).

Data Analysis. Images were quantified using a custom FIJI plugin “Seven” (Petersen et al., 2016). Cells not expressing transgenic proteins were excluded

90 from the analysis. Statistical analysis was performed in Prism 8 (GraphPad). One- way ANOVA analysis with post hoc Tukey test or Dunnett’s multiple comparison to wild-type control was used to compare groups; Student’s t test was used when only comparing 2 datasets. Automated analyses data points deemed definite outliers (0.1%) by Rout method were excluded. Error bars are SEM, unless noted Significant differences are in comparison to control (DdMyo7), unless noted. Ex- tending pseudopodia and actin correlation where done in FIJI by reslicing through extending pseudopodia and making plot profiles of the edge of the cell. Intensity profiles were normalized between 0-1, and multiple cells were averaged by fitting a restricted cubic spline, and averaging the lines. Cortical asymmetry is calculated from the standard deviation of all pixels in a 0.8μm band around the cell perimeter. Tables have filopodia number as average number of filopodia in cells with at least one. SEM is standard error of the mean. Capitalized ‘N’ indicates number of experiments lowercase n is number of cell

91 Table 5 Key Resources

Reagent type Designa- Source Identifiers Additional infor- (species) or tion or refer- mation resource ence

Software Graphpad Statistical Analysis graph preparation, 8.0 statistical analysis

filopodia counting, cor- Software Seven Petersen (Petersen et al., 2016) tex:cell ratio Rabbit anti- Antibody myo7 UMN-87 1:2000 mouse anti- Bio- Antibody GFP legend B34 1:5000 Rabbit anti- Antibody vasp Han 1:500 Rabbit anti Antibody myoB 1:2000 Goat anti Antibody rabbit Licor IR680 1:10,000 Goat anti Antibody mouse Licor IR800 1:10,000 Oligonucleo- 5' -ttattaTAAAAAAATTAAAATAAAA- CAAX F pDTi346 tide TAAAATCTCGTG-3' Oligonucleo- 5' -tgtacaTTGAGAAGAATAAAATTGA- CAAX R pDTi346 tide 5'->3' TAAACTG-3' Oligonucleo- 5' -ttttgtAAATTTTAAAAAGAA- E386V F pDTi364 tide 5'->3' TAGTTTTGAACAATTTTG-3'

Oligonucleo- 5' -ccaaagATATCCAATACACCAA- E386V R pDTi364 tide 5'->3' TAAATGTTG -3'

Oligonucleo- 5' -AAAAGAAAAAgctAATTGGAGTAA- I426A F pDTi435 tide 5'->3' GATCGTATATAATG -3' Oligonucleo- 5' -TCATATTCTTCTT- I426A R pDTi435 tide 5'->3' GTTCTAATTTAAAAATATG -3' Oligonucleo- VASP339* 5' -taataaAGAG- pVASP29 tide 5'->3' F CATCTCAACATTAACTAG-3' Oligonucleo- VASP339* 5' -cccgggAGCTGA- pVASP29 tide 5'->3' R TAAGGATGGTGAAG-3' Oligonucleo- 5' -gaaatggagGCAGCAG- FAB K-E F pVASP34 tide 5'->3' CATCTCAACCAA-3' Oligonucleo- 5' -ggcttcctcGGCCA- FAB K-E R pVASP34 tide 5'->3' TAACTTCGGCCAT-3' Oligonucleo- 5' -GGGTGGACTTgaagAGACAGTTAC GAB K-E F pVASP35 tide 5'->3' -3' Oligonucleo- 5' -TTTGAGAAATTTTCAATTGAACC - GAB K-E R pVASP35 tide 5'->3' 3'

93 4. CHAPTER 4 - DISCUSSION AND FUTURE AIMS

This work provides new insights into two major topics of research:

1. MF myosin regulation by partner interactions 2. How actin tracks impact myosin function

Many unconventional myosin motors act as dimers to function in cells. The proximal tail region can harbor a coiled-coil domain that mediates dimerization directly as is the case for the processive plus end transporter Myosin 5. In the case of the pointed end trafficker, Myo6 its clear that partner binding in the distal tail mediates dimerization (Mukherjea et al., 2014; Peckham, 2012; Thirumurugan et al., 2006). Myosin-1 and -2 actin as monomers or ensembles, however we are still learning about the dimerization and regulation of other myosin family members. Myosin 10, found only in animal lineages where it is important for filopodia formation and mitotic spindle orientation, has been shown to form an antiparallel dimer via its coiled-coil domain in its proximal tail (Weber et al., 2004; Umeki et al., 2011). Myosin 7a binds a cargo MyRip which turns it into a transport motor (Sakai et al., 2011). Here I showed that DdMyo7, an ancestral MF myosin that likely emerged and then diversified into other MF myosins in Holozoa including 7- 10 and 15, forms a partner mediated dimer in vivo in order to function. In collaboration with Beckman Coulter, we found evidence for a weak (occurring at micromolar concentrations) dimer of the proximal tail region in solution. The tandem MyTH4- FERM domain in the tail bind actin and tubulin (Moen et al., 2011; Planelles- Herrero et al., 2016) and possibly other partner proteins (Arthur et al., 2019) and are likely to cluster DdMyo7 to increase the local concentration in a manner that promotes dimerization. The binding partner that might accomplish this is not yet known. Preliminary motility assays do not show strong support for a processive motor, and given that DdMyo7 in an autoinhibited state diffuses and is likely activated by a leading edge actin network, these data best support the model that DdMyo7 is a weakly processive dimeric motor. This type of myosin could be best suited to act at the cortical actin network, generate force of filaments to either promote adhesion or phagocytosis (Tuxworth et al., 2001; Bosmani et al., 2020) or

94 filopodia initiation (Arthur et al., 2019; Petersen et al., 2016). TalinA, the only cur- rently known binding partner of DdMyo7 (aside from its light chains and cytoskeletal interactions) is not critical for filopodia formation. Preliminary work using a promiscuous biotin ligase shows that a subset of putative DdMyo7 binding partners can be labeled in filopodia tips. We are optimistic that this strategy will reveal a dimerization partner that mediates DdMyo7 filopodia formation.

Another important finding from the results presented here is how actin network dynamics activate DdMyo7. DdMyo7 lacks a PH domain, thus how it as targeted to the membrane or underlying actin cortex was not known. Previous work by the Firtel group reported that filopodia formation required the actin polymerase VASP, and VASP localizes to dynamic actin networks at the cortex (Han et al., 2002). Work in mammalian cells implicated a relationship between Myosin-10 and VASP in Myo10 based cargo transport into filopodia tips (Tokuo and Ikebe, 2004). A logical experiment was to test if there was evidence for this type of direct interaction in Dictyostellium. Despite lack of evidence of such direct interaction for DdMyo7 and VASP, an interesting relationship between was uncovered. VASP mediated actin dynamics promote DdMyo7 motor domain binding at the cortex. This is not mediated through the tail region of the myosin, rather VASP promotes the release of Myosin head-tail autoinhibition and binding of the motor domain to the leading edge actin network. Capping actin filaments with cytochlasinA (Figure 16) or stimulating actin polymerization with cAMP (Schroeder, Songster in preparation) showed this principle to be true without manipulations of VASP. Thus, we favor the model that VASP is neither making such specific actin network that recruits myosin, nor directly binding DdMyo7. Rather VASP in Dictyostelium is a potent polymerize that acts in the leading edge of cells and the DdMyo7 motor domain has higher affinity of ATP-actin (made by VASP) in comparison with ADP actin, and thus binds the leading edge in a VASP dependent manner. This reveals a key insight to MF myosin regulation and possible myosin activation and regulation more broadly.

The other key actin binding protein in filopodia formation in Dictyostellium is the diaphanous related formin dDia2. Formin has a weaker role in promoting filopodia initiation, and myosin recruitment. Key next steps will be to test how activation of

95 actin polymerization might compensate for the loss of VASP. Removing the DAD domain of formin is likely to stimulate filopodia formation through increasing actin dynamics. Thus, future work will test DdMyo7 recruitment in the absence of VASP in cells expressing of activated filopodia proteins. Furthermore, results from ongo- ing promiscuous biotin ligase screening in the Titus lab screen could identify additional actin regulations (besides formin and VASP) that alter actin cytoskeleton dynamics and effect DdMyo7 recruitment during filopodia formation.

There are a few key biochemical studies that could provide mechanistic insight to DdMyo7 recruitment and function. Actin cosedimentation assays with ATP or ADP actin and myosin motor could help us determine the differential binding of the motor based on the nucleotide state of actin filaments. Similarly, in vitro motility assays with purified motors on ATP versus ADP actin tracks will reveal if nucleotide state of the filament effects on or off rates or run lengths of DdMyo7. We predict that DdMyo7 will have higher affinity or longer run lengths on new filaments, or filaments locked in an ADP-Pi like state with excess phosphate or phalloidin stabilization (Mahaffy and Pollard, 2006; Zimmermann et al., 2015). This model is supported by similar effect of run length based on actin nucleotide state for Myo5 and Myo6 (Zimmermann et al., 2015). If another property besides nucleotide state mediates DdMyo7 recruitment, those can be tested by altering the actin in the as- say in other ways (see Figure 3).

Another observation regarding DdMyo7 regulation is filopodia tip recycling, and its abrogation in the case of lever arm and proximal tail deletions (Figure 11). Desta- bilization of the off state (or possible partner binding in that proximal region of the molecule) led to an accumulation of DdMyo7 in filopodia tips. We await high resolution tomography of filopodia in Dictystelium to peer into organization of proteins in the filopodia tip complex. Work from the Gerish group pointed to a tip structure that may degrade as the filopodia retracts (Medalia et al., 2007). What does the DdMyo7 motor do in filopodia tips, and how much recycling occurs and why are still mostly unanswered questions. Current efforts to measure the turnover dynamics in filopodia tips using photoconversion and photobleaching experiments, in combination with cell-substrate adhesion mutations may begin to address these problems.

96 A unifying model for filopodia initiation in amoeba is proposed (Figure 26). In the case of Dictyostelium, filopodia initiation likely occurs via the convergent elongation mechanism, as filopodia emerge from the dense cortical actin network at the leading edge of these migratory cells. VASP tetramers (Haffner et al., 1995) localize to the at the leading edge cortex (Han et al., 2002; Cheng and Mullins, 2020) where they assemble actin into bundles perpendicular to the membrane (Bear et al., 2002; Laurent et al., 1999; Breitsprecher et al., 2008). DdMyo7 is folded over on itself, in an inactive, autoinhibited state in the cytoplasm (Arthur et al., 2019; Petersen et al., 2016). The DdMyo7 motor domain binds young actin (red/ orange, i.e. ATP/ADP-Pi actin) at the cortex allowing release of head-tail autoinhibition, and activation. DdMyo7 is then thought to dimerize (Arthur et al., 2019) and assemble into initiation foci along with VASP that bundles the actin filaments (Schirenbeck et al., 2006; Breitsprecher et al., 2008). Next, DdMyo7 dimers act on the actin network to generate force, bending filaments into a perpendicular arrangement with respect to the membrane. VASP continues to promote actin polymerization and inhibits capping of the filaments allowing actin growth against the membrane along with formin (Schirenbeck et al., 2005a), which drives filopodia elongation.

Figure 5 Model of MF myosin and VASP cooperation in the convergent evolution model of filopodia formation. VASP generates a cortical actin network that recruits DdMyo7. DdMyo7 dimerizes and together with VASP organizes filaments. Actin filament elongation drives filopodia extension.

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