14-3-3 Negatively Regulates Actin Filament Formation in the Deep Branching Eukaryote Giardia lamblia

The phosphoserine/phosphothreonine-binding protein 14-3-3 is known to regulate actin, this function has been previously attributed to sequestration of phosphorylated cofilin. The deep branching eukaryote Giardia lamblia lacks cofilin and all other canonical actin-binding proteins (ABPs), and 14-3-3 was identified as an actin-associated protein in Giardia, yet its role in actin regulation was unknown. Gl14-3-3 depletion resulted in an overall disruption of actin organization characterized by ectopically distributed short actin filaments. Using phosphatase and kinase inhibitors, we demonstrated that actin phosphorylation correlated with destabilization of the actin network and increased complex formation with 14-3-3, while blocking actin phosphorylation stabilized actin filaments and attenuated complex formation. Giardia's sole Rho family GTPase, GlRac, modulates Gl14-3-3's association with actin, providing the first connection between GlRac and the actin cytoskeleton in Giardia. Giardia actin contains two putative 14-3-3 binding motifs, one of which (S330) is conserved in mammalian actin. Mutation of these sites reduced, but did not completely disrupt, the association with 14-3-3. Native gels and overlay assays indicate that intermediate proteins are required to support complex formation between 14-3-3 and actin. Overall, our results support a role for 14-3-3 as a negative regulator of actin filament formation. Importance Giardia lacks canonical actin binding proteins. 14-3-3 was identified as an actin interactor but the significance of this interaction was unknown. Loss of 14-3-3 results in ectopic short actin filaments, indicating that 14-3-3 is an important regulator of the actin cytoskeleton in Giardia. Drug studies indicate that 14-3-3 complex formation is in part phospho-regulated. We demonstrate that complex formation is downstream of Giardia’s sole Rho family GTPase, GlRac, this result provides the first mechanistic connection between GlRac and actin in Giardia. Native gels and overlay assays indicate intermediate proteins are required to support the interaction between 14-3-3 and actin suggesting that 14-3-3 is regulating multiple actin complexes. Overall, we find that 14-3-3 is a negative regulator of actin filament formation in Giardia.

Introduction 43 14-3-3 belongs to a family of highly conserved eukaryotic proteins whose role is to regulate 44 target proteins through binding of specific phosphoserine/phosphothreonine motifs. Through 45 recognition and binding of these specific motifs, 14-3-3 functions in a variety of cellular 46 processes, including cytoskeletal regulation and function as an adapter protein that can 47 activate/inhibit protein function, change intracellular localization of bound cargos, or mediate 48 formation of multi-protein complexes (1)(2)(3)(4)(5)(6)(7)(8). In the current model for higher eukaryotes, 14-3-3 49 regulates actin through phospho-dependent sequestration of the actin-depolymerizing protein, 50 cofilin (4,9). The existence of multiple 14-3-3 isoforms in higher eukaryotes complicates the 51 relationship between 14-3-3 and actin, leading to discrepant results about whether 14-3-3 52 directly interacts with actin (2). Consistent with additional interaction/regulatory mechanisms, 53 actin has been identified as an interactor of 14-3-3 in plant and animal 14-3-3 proteomic 54 datasets where cofilin was not found (10-12). Indeed, 14-3-3σ was recently reported to be 55 upregulated in breast cancer cells, where it forms a complex with actin and intermediate 56 filament proteins that is utilized for cell motility during breast tumor invasion (13). The complex 57 was also found to play a role in actin sequestration, as depletion of 14-3-3σ led to an increase in 58 filamentous actin. Whether complex formation between monomeric actin and 14-3-3 is broadly 59 utilized mechanism of actin regulation remains an open question. Giardia lamblia (synonymous with G. intestinalis and G. duodenalis), is a protozoan parasite 62 that belongs to a deep-branching group of eukaryotes known as Excavata. Giardia, in 63 concordance with its phylogenetic position, has an evolutionarily divergent actin with only 58% 64 average identity to other actin homologs and lacks the canonical actin-binding proteins once 65 thought common to all eukaryotes (Arp2/3 complex, formin, wave, myosin, cofilin, etc.) (14-16). 66 Other excavates, such as Trichomonas vaginalis and Spironucleus salmonicida, also lack many 67 canonical actin-binding proteins suggesting that the core actin regulators conserved in plants, 68 animals, and fungi may not have solidified their cellular roles before the ancestors of these 69 excavates branched from the eukaryotic tree (17)(18)(19)(20). Nevertheless, Giardia actin (GlActin) 70 functions in conserved cellular processes including membrane trafficking, cytokinesis, polarity, 71 and control of cellular morphology (21). The mechanism for actin recruitment and regulation 72 for these processes remains poorly understood. The only conserved actin regulator identified in 73 Giardia is a Rho family GTPase, GlRac, which can promote changes in actin organization without 74 any of the actin-binding proteins known to link small G-protein signaling to the actin 75 cytoskeleton (21). Notably, 14-3-3 has been shown to integrate G-protein signaling to the actin 76 and tubulin cytoskeleton in Dictyostelium discoideum (7); thus, it potentially links GlRac to the 77 actin cytoskeleton in Giardia. Through actin affinity chromatography and MudPIT analysis, the 78 single 14-3-3 homolog (Gl14-3-3) of Giardia was identified as an actin-associated protein (19). 79 Likewise, actin has been identified as part of the 14-3-3 interactome in Giardia (22). Here we 80 set out to address whether 14-3-3 has a role in regulating the actin cytoskeleton, characterize 81 the nature of the interaction, and determine if Giardia's sole Rho family GTPase, GlRac, is 82 upstream of this association.

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Results 85 We previously reported that 14-3-3 complexes with actin and the biochemical conditions used 86 to isolate interactors suggested the interaction was likely with monomeric G-actin (19). 87 Pulldown of TwinStrep-tagged GlActin (TS-actin) supports this assertion. Cleared lysates ( Figure   88 1A) contain both endogenous GlActin and TS-actin. If 14-3-3 bound to F-actin or complexes 89 containing dimeric actin, then the filaments would contain a mixture of native and tagged actin. 90 After pulldown with StrepTactin resin in buffers not expected to support filament formation, 91 only TS-actin was detected ( Figure 1A). This finding is consistent with the 14-3-3 complex 92 containing monomeric actin, but does not exclude interaction with F-actin. 93 94 Actin levels in Giardia have yet to be measured; assignment of the intracellular concentration 95 relative to the critical concentration has important regulatory implications. If the concentration 96 of actin is above the critical concentration, then Giardia would require a mechanism to 97 sequester actin. We questioned whether there would be sufficient 14-3-3 to bind and modulate 98 actin function. Using purified proteins as standards and custom antibodies to GlActin and Gl14-99 3-3, we measured actin and 14-3-3 concentrations in Giardia trophozoite extracts. We found 100 that 10 µg of extract contained 102.5±7.4 ng of Gl14-3-3 and 70.7± 16.4 ng of GlActin or ~1.8 101 picomoles of 14-3-3 dimer and ~1.7 picomoles of actin ( Figure S1). Our measurement of actin at 102 70 ng per 10 µg of total cellular extract can be extrapolated to ~4.7 µM actin (16,927 cells=10 103 µg; 1cell=199.8µm 3 (23)). Compared with other eukaryotes, this actin concentration is relatively 104 low; yet, the value is at least 5x higher than the concentration needed to form filaments (21), 105 indicating that some level of actin sequestration is likely needed to properly regulate filament 106 formation. 107 108 Since 14-3-3 has a role in regulating cell division in other eukaryotes, we examined the 109 localization of an endogenously C-terminally tagged version of Gl14-3-3 (Gl14-3-3-HA)(19); see 110 Figure 1B for a diagram of Giardia cellular landmarks. In interphase cells, 14-3-3 is distributed 111 throughout the cell with some enrichment at the cortex, nuclear envelope, and in association 112 with the intracytoplasmic axonemes of the anterior flagella. In mitotic cells, 14-3-3 113 disassociated with the intracytoplasmic axonemes but maintained association with the nuclear 114 envelopes/spindles ( Figure 1C). Notably, we previously demonstrated a central role for actin in 115 positioning the flagella and nuclei (21). Gl14-3-3 was also associated with the ingressing furrow, 116 which does not utilize a contractile ring ( Figure 1C). We recently reported that actin levels are 117 reduced just ahead of the advancing furrow cortex, and actin is required for abscission but not 118 furrow progression (Hardin et al, in review). Enrichment of Gl14-3-3 just ahead of the furrow 119 cortex may indicate a negative actin regulatory function for Gl14-3-3 and/or a role in regulating 120 membrane trafficking ( Figure 1C). Consistent with 14-3-3 having a role in regulating membrane 121 trafficking (6), Gl14-3-3-HA is associated with the nuclear envelope/ER and the bare area of the 122 ventral disc ( Figure 1D). This void in the disc lacks microtubules and serves as a conduit for 123 vesicle trafficking whereas the rest of the disc is composed of a sheet of microtubules and 124 associated proteins that would physically prevent vesicle transport (24). The Gl14-3-3-HA fusion 125 protein did not appear to co-localize with filamentous actin (F-actin) structures and is 126 consistent with our finding that Gl14-3-3 complexes with monomeric actin.

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To ascertain whether Gl14-3-3 has a role in cytoskeletal regulation in Giardia, we depleted 129 Gl14-3-3 with an antisense translation-blocking morpholino. Knockdown of Gl14-3-3 protein 130 expression was monitored by immunoblotting to detect the integrated copy of Gl14-3-3-HA. On 131 average, a 70% reduction in Gl14-3-3-HA levels was observed 24 hours after morpholino 132 treatment and parasite growth was dramatically reduced in the knockdown population versus 133 the non-specific morpholino control, indicating a key role in cell proliferation (Figure 2A  The increased association of Gl14-3-3 with actin that results from calyculin A treatment could 169 indirectly result from increased monomeric actin levels. Therefore, we sought to assess Since modulating actin phosphorylation changed the balance between F and G-actin, we 187 anticipated that this would be apparent as changes in cellular actin organization. To verify this 188 hypothesis, cells were treated with calyculin A or staurosporine for 30 minutes and then stained 189 for GlActin and Gl14-3-3-HA or tubulin. Treatment with the phosphatase inhibitor calyculin A 190 resulted in an apparent decrease in the robustness of actin structures and enrichment of  3-3 along the intracytoplasmic axonemes of the anterior flagella ( Figure 4A). More severely 192 impacted cells (27%, 83/300) lost cytoskeletal organization and became spherical ( Figure 4B). 193 Conversely, treatment with staurosporine led to increased cortical actin and brightly labeled F-194 actin structures, the most apparent of which are at the anterior of the cell ( Figure 4A). 195 Prominent actin filaments associated with the nuclei of staurosporine-treated cells were also 196 apparent ( Figure 4B and C). In 30% (90/300) of staurosporine treated cells, we observed an 197 aberrant structure containing actin and 14-3-3 in proximity to the ventral disc, suggesting that 198 membrane trafficking is also impaired ( Figure 4C, asterisk). Nuclear size was also increased. 199 Staurosporine treatment led to prominent nuclear actin filaments and a corresponding 89% After demonstrating that 14-3-3-actin complex levels could be modulated, we sought to 221 determine if there are binding motifs in GlActin that could support direct interaction with Gl14-222 3-3. The 14-3-3-Pred prediction algorithm tool identified S330 (RVRIpSSP) and S338 (RKYpSAW) 223 as the highest scoring predicted interaction sites (35). In agreement, the same sites were 224 previously reported using a custom algorithm to identify binding sites in putative Giardia 14-3-3 225 interactors (22). These sites are similar to the canonical mode 1 site RXXp(S/T)XP [where p(S/T) 226 are phosphorylated serine or threonine residues]; while not a perfect match, many 14-3-3 227 interacting proteins have been found that lack canonical mode 1-3 binding motifs (3, 35, 36). To 228 assess the potential involvement of these sites, TS-actin was mutated to generate a 229 S330A/S338A double mutant. The wild type and mutant TS-Actin constructs were introduced 230 into the endogenously tagged Gl14-3-3-HA parasite line. Phos-tag gel analysis of the 231 S330A/S338A double mutant, confirms that at least one of these two sites is phosphorylated 232 ( Figure 6B, C). The double mutant as well as single point mutants had reduced capacity to co-233 precipitate 14-3-3-HA ( Figure 6D). The mean reduction in 14-3-3 binding was similar for S330A, 234 S338A, and S330A/S338A double mutant. The double mutant, however, showed more 235 consistent reduction as noted by error bar size in Figure 6E. Incomplete disruption of the 14-3-3 236 interaction could indicate that part of the 14-3-3 recruitment is mediated by association with 237 actin binding proteins, but could also indicate the presence of additional interaction sites. Thus 238 we also tested the possible involvement of T162 (VTHpTVP), a conserved residue identified by 239 Scansite 3 as the highest scoring 14-3-3 interaction site (37). However, mutation of T162 to 240 alanine did not disrupt 14-3-3-actin interaction, consistent with structure homology modeling 241 that suggested this residue was not surface accessible ( Figure S3). These results are in line with 242 S330 and S338 having a role in promoting 14-3-3 and monomeric actin complex formation. 243 However, our inability to completely disrupt actin complex formation indicates additional 244 means for 14-3-3 association with actin.

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To test whether Gl14-3-3 can directly bind GlActin, overlay experiments were performed using 247 a 6XHis-GlActin expressed in and purified from Giardia extracts to ensure native 248 phosphorylation. The overlay was performed with either the recombinant wild type GST-fused 249 Gl14-3-3 or with the mutant GST-K53E, previously shown to be binding defective (38, 39). 250 Binding of GST-Gl14-3-3 near the molecular weight of 6XHis-GlActin was not observed ( Figure   251 7A). Instead, GST-Gl14-3-3 bound to four bands on the blot corresponding to proteins co-252 purified with 6XHis-GlActin. The most prominent labeling is associated with a major co-purified 253 protein at 80 kD also seen in silver staining. Immunoblotting with an anti-pSer antibody 254 confirmed that a fraction of actin was phosphorylated, as well as some co-purified proteins, 255 including the 80 kD protein band ( Figure 7A). This result confirms that some of the 14-3-3-actin 256 interaction is due to recruitment through additional binding partners that can mediate the 257 formation of a complex containing both Gl14-3-3 and GlActin. The inability to bind directly to 258 actin could reflect a requirement for actin to be natively folded or that the interaction site is 259 low affinity and additional proteins are needed to stabilize binding (40). To assess whether Giardia contains a 14-3-3 complex appropriately sized for direct interaction, 262 we turned to native gel electrophoresis. If a 14-3-3-actin complex formed without any 263 additional proteins, it would contain a dimer of 14-3-3 (41) and a single TS-actin monomer. 264 Previous native gel analysis indicates that 14-3-3 dimers run at ~80 kD (41) and TS-actin is 45.2 265 kD; taking into account the size of the 3XHA tag (3.8KD) the anticipated complex size for direct 266 binding is around 130 kD. TS-actin and associated proteins were purified and immediately run 267 on native gels and transferred to membrane for western blotting. A prominent band of 14-3-3 268 was detected running between the 66 and 140 kD native gel markers ( Figure 7B). However, a 269 similarly sized band is also apparent in the input, suggesting this band represents free 14-3-3 270 dimer and that the complexes were unstable. In an effort to stabilize actin complexes, we used 271 the membrane permeable crosslinker DSP to crosslink samples before lysis. Crosslinking did 272 change the distribution of actin and a prominent actin band running just below 146 kD was 273 enriched in the pulldown ( Figure 7B); however, a corresponding enrichment of 14-3-3 was not 274 apparent. Instead, the higher molecular weight smear of 14-3-3 became more prominent, 275 consistent with multiple actin interactors having the capacity to bind actin. Future work will be 276 required to identify these 14-3-3-actin interacting proteins and determine their specific roles in 277 regulating the actin cytoskeleton. Although the relationship between 14-3-3 and actin appears 278 to be more complicated than we initially imagined, our results indicate that 14-3-3 plays a 279 decisive role in actin regulation.

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Proteomic studies support complex formation between 14-3-3 and actin without the Complex formation between 14-3-3 and actin appears to be both phosphodependent and 304 independent. The putative 14-3-3 phospho-binding motif centered on S338 is conserved in 305 mammalian actin and has previously been implicated as an AKT phosphorylation site (45). The 306 14-3-3 binding site prediction tool 14-3-3PRED identified S338 as the highest scoring site for 307 both GlActin and human β-actin. We have shown this site as well as S330, contribute to 14-3-3 308 recruitment and at least one of these predicted interaction sites is phosphorylated as shown by 309 a change in mobility on Phos-tag gels. Our ability to perform biochemical assays with Giardia's 310 highly divergent actin remains limited. Overlay assays and native gels did not provide support 311 for direct interaction between 14-3-3 and actin. However, it remains possible that some of the 312 observed complexes contain 14-3-3 directly bound to actin with additional interactors that 313 work to stabilize the complex (40). Indeed the 14-3-3σ complex contains two intermediate 314 filament proteins, yet in vitro actin polymerization assays containing only actin and 14-3-3 315 demonstrated that 14-3-3 could directly regulate actin dynamics (13). The need for additional 316 proteins to stabilize the 14-3-3 interaction with S330 and S338 would reconcile our data; 317 however, a caveat is that these point mutants could have disrupted actin folding and therefore 318 disrupted the binding of proteins that recruit 14-3-3. While we have yet to pursue proteins that 319 associate with both 14-3-3 and actin, independent proteomic studies aimed at identifying 14-3-320 3 and actin interactors point towards proteins of interest (19,22). Besides actin and 14-3-3 321 there are 14 proteins in common between the two studies (Table S1) and two proteins without any conserved domains (GL50803_15251, 32.5kD; GL50803_15120, 331 26.6 kD). All of these proteins have at least one canonical mode 1 14-3-3 recognition motif but 332 whether they are involved in recruiting 14-3-3 to actin complexes remains to be determined.     . 14-3-3-Actin complex formation is phosphodependent. (A) Immunoblot after Phos-tag phosphate-a nity electrophoresis. Cells were pre-treated with DMSO or inhibitors and then HALT phosphatase inhibitor (HALT PI) was added at lysis to preserve the phosphorylation state. Calyculin A treatment increased phosphorylated-actin levels (P-actin) and the kinase inhibitor staurosporine reduced P-actin. Phosphoisoforms were removed after lambda protein phosphatase treatment. (B) Immunoprecipitation of Gl14-3-3-HA after calyculin A treatment led to increased actin interaction while staurosporine treatment reduced the association of actin with Gl14-3-3-HA.