Project topics and materials
Get Complete Project Material File(s) Now! »
Ena/VASP proteins in general
Ena/Vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are actin-binding proteins that were one of the main subjects of study during my PhD, so I will describe them in detail in this chapter. Ena/VASP proteins have been variously attributed to have nucleation activity, the capacity to compete with capping protein for barbed ends (called anti-capping) and barbed end elongation enhancement activity (Trichet et al., 2008) (Krause and Gautreau, 2014). These activities will be explained more fully at the end of this chapter, along with a description of the controversies surrounding the mechanisms of Ena/VASP protein action.
The first member of this family to be discovered was Drosophila Enabled (Ena), the gene for which was discovered as a dominant suppressor of lethal mutations in the tyrosine kinase gene abl, involved in axon guidance (Gertler et al., 1990). Based on this sequence, mammalian equivalents were identified, Mammalian Ena (Mena), Enabled/vasodilator-stimulated phosphoprotein-like protein (Evl) and Vasodilator-stimulated phosphoprotein (VASP), and shown to have roles in actin filament assembly (Gertler et al., 1996). VASP had been previously identified as a substrate of cyclic nucleotide-dependent kinase cAMP and cGMP in platelets (Haffner et al., 1995; Halbrügge and Walter, 1989). Ena/VASP proteins are highly conserved through evolution: in C. elegans the equivalent of Ena/VASP is UNC-34, and DdVASP in Dictyostelium (Han et al., 2002; Withee et al., 2004; Yu et al., 2002).
Role of Ena/VASP proteins in cells and in vivo
In lamellipodia and cell motility
Ena/VASP proteins are found at the leading edge of lamellipodia, at the tips of filopodia, at cell-cell contacts, in cell-substrate adhesions, and in actin stress fibers (Gertler et al., 1996; Lanier et al., 1999; Reinhard et al., 1992; Rottner et al., 1999) (Figure 2.1). In lamellipodia-based cell motility, the local level of Ena/VASP recruitment at the membrane is proportional to transient protrusion rate of that portion of membrane (Rottner et al., 1999). When Ena/VASP proteins are artificially enriched at the front of a moving cell, a network of long unbranched actin filaments form under the membrane (Figure 2.2), and although these structures protrude rapidly, they are not persistent (Bear et al., 2002; Loureiro et al., 2002), and thus increased Ena/VASP sometimes has the effect of reducing overall cell motility (Bear et al., 2000). On the other hand, when Ena/VASP is reduced at the leading edge of the cell, lamellipodia protrude more slowly than wild type, and the actin network is composed of short highly-branched filaments (Bear et al., 2002; Loureiro et al., 2002). Similarly mislocalization of Ena/VASP protein in fish keratinocytes induces altered cell shape and less efficient migration (Lacayo et al., 2007).
In vivo in Drosophila oogenesis, border cells migrate to the posterior part of the egg chamber, and Ena mutation in these cells significantly reduces their migration speed (Gates et al., 2009). In the Drosophila embryo, Ena overexpression induces an increase in the rate of haemocyte migration, while Ena depletion decreases the rate of cell migration (Tucker et al., 2011). Deletion of C. elegans VASP, UNC-34, decreases the migration speed of leader cells during ventral enclosure, a WAVE-Arp2/3 complex dependent, lamellipodia-driven event (Havrylenko et al., 2014). T-cell movement through endothelial cell layers during extravasation in mice is also reduced by Ena/VASP protein deletion (Estin et al., 2017).
Listeria bacteria hijack Ena/VASP proteins of host cells to increase bacterial motility (Chakraborty et al., 1995; Geese et al., 2002; Skoble et al., 2001; Smith et al., 1996) . Likewise in the bead/comet system, when Ena/VASP proteins are recruited to the bead surface, they increase speed of movement (Castellano et al., 2001; Havrylenko et al., 2015; Plastino et al., 2004b; Samarin et al., 2003). Altogether, studies on cells in culture and in vivo suggest that Ena/VASP proteins promote cell migration, and this is confirmed in biomimetic systems.
In filopodia
Ena/VASP proteins also play a role in the dynamics of filopodia. Ena/VASP deficient neurons have reduced filopodia formation in their growth cones (Bear et al., 2002; Lebrand et al., 2004), and Ena/VASP knockout completely suppresses filopodia formation in capping protein-deficient mouse melanoma cells and in Dictyostelium (Han et al., 2002; Mejillano et al., 2004; Schirenbeck et al., 2006) (Figure 2.3).
In cell-substrate adhesions and stress fibers
Ena/VASP proteins play an important role in stress fibers and focal adhesions. Upon mechanical stress, VASP relocalizes from focal adhesions to stress fibers, and helps in their repair, thus restoring the structural integrity and the contractility of the stress fiber (Burridge and Guilluy, 2016; Smith et al., 2010; Yoshigi et al., 2005). VASP is also involved in remodeling stress fibers through cooperation with focal adhesion protein zyxin (Hoffman et al., 2006). Furthermore, Ena/VASP proteins are an integral component of focal adhesions (Kanchanawong et al., 2010).
In cancer
In the past two decades, several studies have emerged indicating a relation between Ena/VASP protein and cancer progression. Phosphorylation of Ena/VASP, which reduces its interaction with actin, inhibits the formation of invadopodia, essential structures for cancer cell invasion and metastasis, and thus reduces colon cancer cell circulation (Zuzga et al., 2012). Fibroblasts overexpressing Ena/VASP lose contact inhibition and are considered as potential tumorigenic cells (Liu et al., 1999), and Ena/VASP overexpression in lung adenocarcinoma cells is correlated with the progress of the tumor (Dertsiz et al., 2005). Mena is overexpressed in breast cancer cell lines, and in particular one splice form of Mena is associated with increased invasion and metastasis (Philippar et al., 2008; Roussos et al., 2010). In addition to its role in invasion, Ena/VASP plays a role in the vascularization of tumors: melanoma cancer cells transplanted into Ena/VASP deficient mice do not develop well, and tumors are smaller and significantly less vascularized (Kim et al., 2011). On a global scale, Ena/VASP proteins seem to be involved at multiple levels in the coordination of the development of metastasis.
Ena/VASP domains and their functions
All Ena/VASP family members share a conserved domain structure: an amino-terminal Ena/VASP homology 1 domain (EVH1), a central proline rich region, and a carboxy-terminal Ena/VASP homology 2 (EVH2) domain, encompassing G- and F-actin binding sites and a coiled-coil motif. Ena/VASP protein interacts with many partners and performs various functions via its different domains (Figure 2.4), as described in the following sections.
EVH1 domain
The N-terminal EVH1 domain of Ena/VASP proteins is part of the pleckstrin homology (PH) domain superfamily, but unlike other members of this family, it does not bind phospholipid phosphatidyl inositol-(4,5)-bisphosphate (PIP2) (Prehoda et al., 1999; Volkman et al., 2002). The EVH1 domain binds to peptide ligands containing special poly-proline sequences with FPPPP-type sequences, such as those found in the Listeria ActA protein and in the prolinerich regions of WASP and WAVE molecules (Castellano et al., 2001; Havrylenko et al., 2015; Niebuhr et al., 1997). Through this interaction, Ena/VASP proteins are also recruited to cell-substrate adhesions and stress fibers by interaction with the focal adhesion components vinculin and zyxin, and to the leading edge of lamellipodia and filopodia via interaction with a membrane-bound protein lamellipodin (Krause et al., 2003; Krause et al., 2004). Studies on EVH1 domain in C. elegans and Drosophila revealed that EVH1 domain mutations interfere with the localization of Ena/VASP proteins, and reduce significantly their activity (Fleming et al., 2010; Gates et al., 2009; Shakir et al., 2006).
Proline rich domain
The central domain of Ena/VASP protein is a proline-rich domain that is the most diverse region in the Ena/VASP family ensuring interactions with different proteins for different regulatory mechanisms (Krause et al., 2003). The shared feature in all family members is profilin binding via this region (Reinhard et al., 1995).
EVH2 domain
The Ena/VASP homology 2 domain (EVH2) is located at the C-terminal of Ena/VASP, and is composed of three domains organized as follows: G-actin binding site, F-actin binding site, and a coiled-coil domain.
• G-actin binding domain (GAB). This domain binds G-actin, but binds profilin-complexed G-actin with an even higher affinity, unlike most GAB domains (Chereau and Dominguez, 2006). In this context, it has been proposed that profilin-bound G-actin is loaded onto the proline-rich domain and handed off to the GAB for efficient addition to the filament barbed end (Ferron et al., 2007) (Figure 2.5).
The GAB domain also has actin nucleation properties at non-physiological (low) salt conditions (Walders-Harbeck et al., 2002). In vitro, GAB domain seems to play an important role in Ena/VASP’s anti-capping activity (Barzik et al., 2005), although it doesn’t play an essential role in the capture of barbed ends (Pasic et al., 2008). In contrast, single molecule experiments show that GAB is essential for targeting Ena/VASP at the barbed end of a growing filament, and important for barbed end elongation enhancement (Hansen and Mullins, 2010). Along these same lines, a recent study shows that Ena/VASP, which is a homotetramer, uses one of its subunits to track the fast elongating barbed end, while the G-actin binding domains of the other three subunits recruit and deliver monomers to the barbed end of the filament; engineered Ena/VASP proteins with more GAB domains produce faster filament elongation (Brühmann et al., 2017). In addition to its contribution to activity, the GAB of Ena/VASP is also important for correct localization: GAB mutants localize abnormally in fibroblast filopodia (Applewhite et al., 2007; Loureiro et al., 2002).
• F-actin binding domain (FAB). FAB binds F-actin and bundles it so that Ena/VASP co-precipitates with actin filaments in both low and high speed sedimentation assays (Bachmann et al., 1999; Laurent et al., 1999). In vivo, FAB is important for localizing Ena/VASP at the leading edge of moving cells and filopodia (but not focal adhesions) (Applewhite et al., 2007; Bear et al., 2002; Loureiro et al., 2002). In Dictyostelium, the FAB domain shows actin bundling activity that is necessary for the formation of filopodia and for localization at the leading edge (Schirenbeck et al., 2006). In vitro, FAB is essential for anti-capping activity of Ena/VASP (Barzik et al., 2005), and for its localization at the barbed end and barbed end elongation enhancement (Hansen and Mullins, 2010). On the other hand, studies on DdVASP show that both FAB and GAB domains must be deleted to interfere with barbed end elongation enhancement activity, indicating a possible redundancy in functions of FAB and GAB domains (Breitsprecher et al., 2008).
• Coiled-coil domain (TET) is the tetramerization domain of Ena/VASP, found at the C-terminus of the protein (Bachmann et al., 1999; Zimmermann et al., 2002). This domain is essential for filopodia formation (Applewhite et al., 2007). In vitro, the tetramerization domain plays a role in anti-capping (Barzik et al., 2005), and in filament decoration (Hansen and Mullins, 2010). Moreover, the tetramerization domain is essential for bundle formation and barbed end elongation enhancement activity of DdVASP, although artificially clustered monomeric Ena/VASP proteins can also enhance barbed end elongation (Breitsprecher et al., 2008).
Modes of action of Ena/VASP and controversy
In keeping with its multi-domain structure, Ena/VASP has been ascribed many different modes of action as concerns actin filament dynamics, some of which are controversial.
Nucleation activity
Ena/VASP proteins nucleate the formation of actin filaments from monomers at low salt concentrations (Figure 2.6) (Hüttelmaier et al., 1999b; Schirenbeck et al., 2006). This activity depends on G-actin binding and tetramerization (Walders-Harbeck et al., 2002). This is mechanistically reminiscent of nucleators such as Spire that nucleate by clustering actin-binding sites together (Campellone and Welch, 2010), although other proteins probably participate in vivo to make this nucleation mechanism more efficient (Dominguez, 2016). In physiological salt conditions (in cells), Ena/VASP proteins do not nucleate actin polymerization (Barzik et al., 2005). The few reports of Ena/VASP nucleation at physiological salt conditions are attributable to recruitment of preformed actin filaments and barbed end elongation (Fradelizi et al., 2001; Plastino et al., 2004a; Trichet et al., 2007).
Figure 2.6 – Ena/VASP nucleation activity is dependent on salt concentration. Actin filament formation is monitored over time in the pyrene assay (see Chapter 3), in the presence of 250 nM mouse VASP and the indicated concentrations of KCl. From (Hüttelmaier et al., 1999a).
Anti-capping activity
As mentioned in a previous section (Figure 2.2), when Ena/VASP recruitment at the leading edge of moving fibroblasts in increased, long filaments are observed by electron microscopy, whereas when Ena/VASP is depleted from the leading edge, short filaments are observed (Bear et al., 2002). This led to the hypothesis that Ena/VASP proteins protect filaments from capping protein, allowing them to grow longer before being capped, not to be confused with uncapping activity. Indeed, purified Ena/VASP, coated on beads, could capture and elongate filaments, but not when the filaments were pre-capped (Figure 2.7) (Bear et al., 2002).
In keeping with this, when cells are treated with cytochalasin D (a drug that blocks barbed ends), Ena/VASP does not localize to the leading edge of protruding lamellipodia, suggesting the need of growing barbed ends for the localization of Ena/VASP (Bear et al., 2002; Krause et al., 2004). Mathematical modeling of cell shape based on actin filament dynamics also supports the anti-capping activity of Ena/VASP (Lacayo et al., 2007). Anti-capping, but not uncapping, activity of Ena/VASP is clear in in vitro studies, such as the pyrene assay where Ena/VASP inhibits the activity of capping protein and promotes filament elongation in a dose-dependent manner (Figure 2.8) (Barzik et al., 2005; Bear et al., 2002). The GAB, FAB and TET domains are required for this anti-capping activity (Barzik et al., 2005). Similar results are obtained with total internal reflection fluorescence (TIRF) microscopy experiments, where individual actin filaments are followed over time (see Chapter 3). Actin filaments grow in the presence of capping protein only when Ena/VASP is added, thus supporting the anti-capping activity of Ena/VASP (Figure 2.8) (Breitsprecher et al., 2011; Hansen and Mullins, 2010; Pasic et al., 2008). Clustering of Ena/VASP enhances its anti-capping effect: when Ena/ VASP is adsorbed on a bead surface, it induces elongation of actin filaments at high concentrations of capping protein that inhibit elongation via Ena/VASP in solution (Breitsprecher et al., 2008).
Interestingly, early data from in vitro pyrene experiments show that Ena/VASP does not rescue actin polymerization in the presence of capping activity (capping protein or gelsolin) (Boujemaa-Paterski et al., 2001a; Samarin et al., 2003). These results are difficult to reconcile with the studies mentioned in Figure 2.8, but one difference in the experimental systems in that the early pyrene assays were done in the presence of Arp2/3 complex-actin nucleation.
In conclusion on anti-capping, there is agreement that Ena/VASP proteins are not able to uncap filaments; capping proteins have a high affinity for the barbed end, and once attached, cannot be displaced by Ena/VASP (Bear et al., 2002; Schirenbeck et al., 2006). Today it is generally accepted that Ena/VASP has anti-capping activity, i.e., their interaction with the barbed end delays capping protein binding.
Effect on barbed end elongation
A controversy in the field centers on the effect of Ena/VASP on the elongation of filaments. Some studies report no effect of Ena/VASP on the elongation speed of actin filaments (Barzik et al., 2005; Bear et al., 2002; Samarin et al., 2003), while other studies show an increase in the polymerization speed in presence of Ena/VASP, similar to the barbed end elongation activity of formin (Breitsprecher et al., 2008; Hansen and Mullins, 2010). Again differences here are possibly attributable to different assays: barbed end elongation enhancement is observed in TIRF but not in pyrene assays. The role of the profilin in barbed end elongation enhancement by Ena/VASP proteins is not entirely clear; some studies report no effect of profilin on Ena/VASP-induced actin polymerization (Breitsprecher et al., 2008), while others observe an effect of profilin on polymerization speed and on enhancement of the anti-capping activity of Ena/VASP (Barzik et al., 2005; Hansen and Mullins, 2010). Structural studies described in Figure 2.5 indicate that Ena/VASP binds profilin-actin with both its proline-rich domain and its G-actin binding site, consistent with, but not proof of, a role for profilin-actin in barbed end elongation enhancement by Ena/VASP.
Effect on Arp2/3 complex branching
Ena/VASP proteins do not interact directly with Arp2/3 complex (Boujemaa-Paterski et al., 2001b), but they seem to affect Arp2/3 complex branch frequency. In general Ena/VASP protein is associated with reduced branching frequency of actin filaments by the Arp2/3 complex (Bear et al., 2002; Plastino et al., 2004b; Samarin et al., 2003; Skoble et al., 2001) although there is an exception where Ena/VASP is observed to increase branch frequency (Boujemaa-Paterski et al., 2001a) (Figure 2.9). However overall there is a consensus that Ena/VASP protein association with a network lowers the degree of branching of that network in the presence of capping protein.
In this context it is important to note that the Arp2/3 complex activators WASP and WAVE have both been observed to directly recruit Ena/VASP proteins via the interaction of WASP/WAVE proline-rich domain and the EVH1 domain of Ena/VASP (Chen et al., 2014; Havrylenko et al., 2015). This interaction could potentially place Ena/VASP proteins close to new (uncapped) barbed ends created by the Arp2/3 complex (Figure 2.10). Elongation enhancement of the barbed ends coupled with a constant on-rate for the Arp2/3 complex on the side of the growing mother filament to make a branch could explain how Ena/VASP proteins produce networks that are less highly branched.
Table of contents :
Chapter 1: General Introduction to Cell Motility and the Actin Cytoskeleton
1.1 Cell shape changes and motility
1.2 Structures of cell motility
1.2.1 Lamellipodia
1.2.2 Filopodia
1.2.3 The cell cortex
1.2.4 Stress fibers and focal adhesions
1.3 Actin polymerization and dynamics
1.3.1 Actin in general
1.3.2 From monomers to filaments
1.3.3 Assembly dynamics
1.4 Actin polymerization regulatory proteins
1.4.1 G-actin binding proteins
1.4.2 F-actin regulating proteins
1.4.3 Cross-linkers of actin networks
1.4.4 Molecular motors
1.4.5 Actin nucleating proteins
1.4.6 Activators of actin polymerization
1.4.7 Putting all the ingredients together
1.5 Biomimetic approaches to study actin dynamics and actin-based motility
1.5.1 Listeria monocytogenes motility
1.5.2 Reconstitution of actin polymerization
1.5.3 Symmetry breaking and movement generation
1.5.4 Diversity of biomimetic systems
Chapter 2: Ena/VASP Proteins
2.1 Ena/VASP proteins in general
2.3 Role of Ena/VASP proteins in cells and in vivo
2.3.1 In lamellipodia and cell motility
2.3.2 In filopodia
2.3.3 In cell-substrate adhesions and stress fibers
2.3.4 In cancer
2.2 Ena/VASP domains and their functions
2.2.1 EVH1 domain
2.2.2 Proline rich domain
2.2.3 EVH2 domain
2.4 Modes of action of Ena/VASP and controversy
2.4.1 Nucleation activity
2.4.2 Anti-capping activity
2.4.3 Effect on barbed end elongation
2.4.4 Effect on Arp2/3 complex branching
Chapter 3: Experimental Methods in vitro
3.1. Actin network reconstitution on beads
3.1.1. DNA and proteins
3.1.2. Bead preparation
3.1.3. Actin polymerization on beads
3.1.4 Two-color experiments
3.1.5 Bead observation and data processing
3.2 Actin polymerization assessment by pyrene assay
3.3 Single filament assay by TIRF microscopy
3.4 Photoswitchable Arp2/3 complex inhibitors
Chapter 4: Ena/VASP Affects Polarized Actin Network Growth and Architecture
4.1 Introduction and open questions concerning the mode of action of Ena/VASP proteins
4.2 Results
4.2.1 Mouse VASP restores polarized actin network growth in the absence of capping protein
4.2.2 Mouse VASP is a barbed end elongation enhancement protein
4.2.3 Which VASP domains are necessary for restoring polarized growth in the absence of capping protein?
4.2.4 Aggressive nucleation at the surface can compensate for the absence of capping protein
4.2.5 VASP can compensate for reduced Arp2/3 complex in the network polarity establishment.
4.2.6 Actin network density and Arp2/3 complex levels increase at the bead surface in the presence of VASP
4.3 Conclusion and perspectives
Chapter 5: Small Molecule Photoswitchable Inhibitors of the Arp2/3 Complex
5.1 Introduction to inhibition of the Arp2/3 complex
5.1.1 CK-666
5.1.2 Photoswitchable Arp2/3 complex inhibitors based on CK-666
5.2.3 Attempts to improve solubility of LU06-type compounds
5.3 Conclusions and perspectives.
Chapter 6: Exploring Actin Architecture in vivo in Nematode Embryos
6.1 Introduction
6.1.1 Goal of the study
6.1.2 Asymmetric cell division
6.1.3 Symmetry breaking
6.1.4 Polarity establishment
6.1.5 Spindle positioning
6.2 Preliminary results actin visualization
6.2.1 Actin labeling of live embryos
6.2.2 Phalloidin labeling of fixed samples
6.2.3 Conclusions actin visualization
6.3 First tests rheology of nematode embryos
6.3.1 Optical trapping of endogenous granules
6.3.2 Tests with bead injection
6.3.3 Conclusions rheology
6.4 Overall conclusion and perspectives
6.5 Procedures and solution recipes
6.5.1 Worm manipulation and embryo isolation
6.5.2 Solutions
6.5.3 Polylysine slides
General Conclusion
References
