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

Giardia lacks canonical actin-binding proteins. Gl-14-3-3 was identified as an actin interactor, but the significance of this interaction was unknown. Loss of Gl-14-3-3 results in ectopic short actin filaments, indicating that Gl-14-3-3 is an important regulator of the actin cytoskeleton in Giardia. Drug studies indicate that Gl-14-3-3 complex formation is in part phospho-regulated. We demonstrate that complex formation is downstream of Giardia’s sole Rho family GTPase, Gl-Rac. This result provides the first mechanistic connection between Gl-Rac and Gl-actin in Giardia. Native gels and overlay assays indicate intermediate proteins are required to support the interaction between Gl-14-3-3 and Gl-actin, suggesting that Gl-14-3-3 is regulating multiple Gl-actin complexes.

T he protein 14-3-3 belongs to a family of highly conserved eukaryotic proteins whose role is to regulate target proteins through binding of specific phosphoserine/phosphothreonine motifs. Through recognition and binding of these specific motifs, 14-3-3 functions in a variety of cellular processes, including cytoskeletal regulation. 14-3-3 can act as an adapter in order to activate/inhibit protein function, change intracellular localization of bound cargos, or mediate formation of multiprotein complexes (1)(2)(3)(4)(5)(6)(7)(8). In the current model for higher eukaryotes, 14-3-3 regulates actin through phospho-dependent sequestration of the actin-depolymerizing protein cofilin (4,9). The existence of multiple 14-3-3 isoforms in higher eukaryotes complicates the relationship between 14-3-3 and actin, leading to discrepant results about whether 14-3-3 directly interacts with actin (2). Consistent with additional interaction/regulatory mechanisms, actin has been identified as an interactor of 14-3-3 in plant and animal 14-3-3 proteomic data sets where cofilin was not found (10)(11)(12). Indeed, 14-3-3 was recently reported to be upregulated in breast cancer cells, where it forms a complex with actin and intermediate filament proteins that is utilized for cell motility during breast tumor invasion (13). The complex was also found to play a role in actin sequestration, as depletion of 14-3-3 led to an increase in filamentous actin. Whether complex formation between monomeric actin and 14-3-3 is a broadly utilized mechanism of actin regulation remains an open question.
Giardia lamblia (synonymous with G. intestinalis and G. duodenalis), is a protozoan parasite that belongs to a deep-branching group of eukaryotes known as Excavata. Giardia, in concordance with its phylogenetic position, has an evolutionarily divergent actin with only 58% average identity to other actin homologs and lacks the canonical actin-binding proteins (ABPs) once thought common to all eukaryotes (Arp2/3 complex, formin, wave, myosin, cofilin, etc.) (14)(15)(16). Other excavates, such as Trichomonas vaginalis and Spironucleus salmonicida, also lack many canonical actin-binding proteins, suggesting that the core actin regulators conserved in plants, animals, and fungi may not have solidified their cellular roles before the ancestors of these excavates branched from the eukaryotic tree (17)(18)(19)(20). The presence of ABPs in the closely related diplomonad Spironucleus, not found in Giardia (20), suggests that Giardia's minimalism is in part due to reductive evolution. Nevertheless, Giardia actin (Gl-actin) functions in conserved cellular processes, including membrane trafficking, cytokinesis, polarity, and control of cellular morphology (21). The mechanism for actin recruitment and regulation for these processes remains poorly understood. The only conserved actin regulator identified in Giardia is a Rho family GTPase, Gl-Rac, which can promote changes in actin organization without any of the actin-binding proteins known to link small G-protein signaling to the actin cytoskeleton (21). Notably, 14-3-3 has been shown to integrate G-protein signaling to the actin and tubulin cytoskeleton in Dictyostelium discoideum (7); thus, it potentially links Gl-Rac to the actin cytoskeleton in Giardia. Through actin affinity chromatography and MudPIT analysis, the single 14-3-3 homolog (Gl-14-3-3) of Giardia was identified as an actin-associated protein (19). Likewise, actin has been identified as part of the 14-3-3 interactome in Giardia (22). Here we set out to address whether Gl-14-3-3 has a role in regulating the Gl-actin cytoskeleton, characterize the nature of the interaction, and determine if Giardia's sole Rho family GTPase, Gl-Rac, is upstream of this association. critical concentration, then Giardia would require a mechanism to sequester actin. Using purified proteins as standards and custom antibodies to Gl-actin and Gl-14-3-3, we measured actin and 14-3-3 concentrations in Giardia trophozoite extracts. We found that 10 g of extract contained 102.5 Ϯ 7.4 ng of Gl-14-3-3 and 70.7 Ϯ 16.4 ng of Gl-actin or~1.8 pmol of 14-3-3 dimer and~1.7 pmol of actin (see Fig. S1 in the supplemental material). Our measurement of actin at 70 ng per 10 g of total cellular extract can be extrapolated to~4.7 M actin (where 16,927 cells ϭ 10 g and 1 cell ϭ 199.8 m 3 [23]). Compared with other eukaryotes, this actin concentration is relatively low, yet the value is at least 5ϫ higher than the concentration needed to form filaments (21), indicating that some level of actin sequestration is likely needed to prevent spontaneous filament formation. Since Gl-14-3-3 associates with monomeric actin, a portion of the total actin pool, there appears to be sufficient Gl-14-3-3 to bind and modulate actin as well as regulate the many other Gl-14-3-3 target proteins. Since 14-3-3 has a role in regulating cell division in other eukaryotes, we examined the localization of an endogenously hemagglutinin (HA) C-terminally-tagged version of Gl-14-3-3 (Gl-14-3-3-HA) (19). (See Fig. 1B for a diagram of Giardia cellular landmarks.) In interphase cells, Gl-14-3-3 was distributed throughout the cell with some enrichment at the cortex, perinuclear region, and in association with the intracytoplasmic axonemes of all flagella, but was most apparently associated with the anterior flagella ( Fig. 1C; see Fig. S2 in the supplemental material). In mitotic cells, Gl-14-3-3 disassociated with the intracytoplasmic axonemes and enrichment of 14-3-3 were observed around the spindle which may reflect association with the perinuclear membrane/ nuclear envelope (Fig. S2). Notably, we previously demonstrated a central role for actin in positioning the flagella and nuclei (21). Gl-14-3-3 was also associated with the ingressing furrow, which does not utilize a contractile ring (Fig. 1C). We recently reported that Gl-actin levels are reduced just ahead of the advancing furrow cortex, and Gl-actin is required for abscission but not furrow progression (24). Enrichment of Gl-14-3-3 just ahead of the furrow cortex may indicate a negative actin regulatory function for Gl-14-3-3 and/or a role in regulating membrane trafficking (Fig. 1C). Consistent with 14-3-3 having a role in regulating membrane trafficking (6), Gl-14-3-3-HA is associated with the nuclear envelope/endoplasmic reticulum (ER) and the bare area of the ventral disc (Fig. 1D). This void in the disc lacks microtubules and serves as a conduit for vesicle trafficking, whereas the rest of the disc is composed of a sheet of microtubules and associated proteins that would physically prevent vesicle transport (25). Although our images indicate partial colocalization between Gl-14-3-3 and Glactin, using an antibody that recognizes both monomeric and filamentous Gl-actin (F-actin), we did not observe Gl-14-3-3-HA to colocalize with F-actin structures. This result is consistent with our finding that Gl-14-3-3 complexes with monomeric actin.
To ascertain whether Gl-14-3-3 has a role in cytoskeletal regulation in Giardia, we depleted Gl-14-3-3 with an antisense translation-blocking morpholino. Knockdown (KD) of Gl-14-3-3 protein expression was monitored by immunoblotting to detect the integrated copy of Gl-14-3-3-HA. On average, a 70% reduction in Gl-14-3-3-HA levels was observed 24 h after morpholino treatment, and parasite growth was dramatically reduced in the knockdown population versus the nonspecific morpholino control, indicating a key role in cell proliferation ( Fig. 2A and B). Depletion of Gl-14-3-3 disrupted characteristic actin organization and resulted in small bright puncta distributed throughout the cell ( Fig. 2C; see Fig. S3 in the supplemental material). Detailed examination revealed that the puncta are short filaments below 1 m in length ( Fig. 2D; see Movie S1 in the supplemental material). Depletion of Gl-14-3-3 also led to polarity and cytokinesis defects, indicating that Gl-14-3-3 has a role in regulating both actin and tubulin cytoskeletal organization ( Fig. 2E; Fig. S3). The loss of cell polarity, accumulation of multinucleate cells, reduction in cell growth, and abnormal flagellar positioning associated with Gl-14-3-3 depletion are phenotypes that overlap those observed in Gl-actin-depleted Giardia (21). These results likely reflect misregulation of cytoskeletal dynamics which are exquisitely controlled in other eukaryotes. Indeed, drugs which stabilize or depolymerize cytoskeletal arrays often lead to similar defects; therefore, these results are consistent with 14-3-3 having an actin regulatory role in Giardia.
Since actin phosphorylation occurs in several eukaryotes (26-32) and 14-3-3 binding usually requires Ser/Thr target phosphorylation, the effect of kinase and phosphatase inhibitors on Gl-actin/Gl-14-3-3 complex stability was studied. The Ser/Thr phosphatase inhibitor calyculin A and the general kinase inhibitor staurosporine are both effective in Giardia (33), likely affecting the phosphorylation state of multiple proteins. Using Phos-tag phosphate-affinity electrophoresis (34), we find that a portion of actin is indeed phosphorylated in Giardia extracts (Fig. 3A), suggesting phosphorylation could be an important actin regulatory mechanism. After 45 min of treatment with either staurosporine or calyculin A, the level of Gl-actin phosphorylation increased following treatment with the phosphatase inhibitor and decreased as a result of treatment with the kinase inhibitor, respectively (Fig. 3A). Treatment of the cell extracts with lambda protein phosphatase effectively depletes the shifted band, demonstrating the specificity of our actin antibody. Remarkably, our ability to coimmunoprecipitate Gl-actin with Gl-14-3-3-HA correlated with the phosphorylation level of Gl-actin and is consistent with phospho-dependent regulation of the 14-3-3-actin interaction ( Fig. 3B and C).
Next we asked whether modulating the phosphorylation level of actin could affect the balance between F-and G-actin. Actin extraction assays were performed after treatment of parasites with staurosporine or calyculin A. Phosphatase inhibition with calyculin A increased extractable Gl-actin from 12.2% in dimethyl sulfoxide (DMSO)treated control cells to 30.4% (n ϭ 3; P Ͻ 0.05). No reduction in extractable actin was observed after treatment with the kinase inhibitor staurosporine ( Fig. 3D and E), possibly due to the limited sensitivity of this assay coupled with our observation that only a small pool of actin is free to begin with. These results do show that actin phosphorylation is correlated with a shift in the balance toward soluble, presumably, G-actin and association with Gl-14-3-3.
The increased association of Gl-14-3-3 with actin that results from calyculin A treatment could indirectly result from increased monomeric actin levels. Therefore, we sought to assess whether increased monomeric actin in the absence of increased phosphorylation could promote complex formation with Gl-14-3-3. Mutation of Arg 62, a key residue also conserved in Gl-actin, to Asp (R62D) has been shown to result in polymerization-defective ␤-actin (35). To monitor the mutant Gl-actin isoform, TS-actin The puncta in Gl-14-3-3-depleted cells are short filaments; see Movie S1 for an entire image stack. Scale bar, was mutated and transformed into a Gl-14-3-3-HA-expressing parasite line. In Gl-14-3-3-HA immunoprecipitation, both TS-actin R62D and control TS-actin coprecipitated in similar ratios compared with endogenous Gl-actin ( Fig. 3F and G). This result suggests that the association between Gl-actin and Gl-14-3-3 promoted by calyculin depends on increased Gl-actin phosphorylation rather than an increased amount of monomeric Gl-actin. To confirm that Gl-14-3-3 complexes with phosphorylated actin, pulldowns of 14-3-3-TS were performed and the phosphorylation state of the associated actin was assessed with Phos-tag gels and Western blotting. Indeed, compared with input, the phosphorylated forms of actin were enriched in 14-3-3-TS pulldown (Fig. 3H); however, most of the coimmunoprecipitated actin was not phosphorylated. The presence of both phosphorylated and unphosphorylated actin in association with Gl-14-3-3 suggests multiple modes of 14-3-3 association, which could include both direct binding and recruitment through actin-binding proteins.
Since modulation of Gl-actin phosphorylation changed the balance between F-and G-actin, we anticipated that this would be apparent as changes in cellular actin organization. To verify this hypothesis, cells were treated with calyculin A or staurosporine for 30 min and then stained for Gl-actin and Gl-14-3-3-HA or tubulin. Treatment with the phosphatase inhibitor calyculin A resulted in an apparent decrease in the robustness of actin structures and enrichment of Gl-14-3-3 along the intracytoplasmic axonemes of the anterior flagella (Fig. 4A). More severely impacted cells (27% [83/300]) lost cytoskeletal organization and became spherical (Fig. 4B). Conversely, treatment with staurosporine led to increased cortical actin and brightly labeled F-actin structures, the most apparent of which are at the anterior of the cell (Fig. 4A). Prominent actin filaments associated with the nuclei of staurosporine-treated cells were also apparent ( Fig. 4B and C). In 30% (90/300) of staurosporine-treated cells, we observed an aberrant structure containing actin and 14-3-3 in proximity to the bare area of the ventral disc, suggesting that membrane trafficking is impaired (Fig. 4A, asterisk). Nuclear size was also increased. Staurosporine treatment increase nuclear area by 89% compared to DMSO-treated control cells, and prominent nuclear actin filaments were observed to span the entire width of the nuclei (P Ͻ 0.001; n ϭ 43 for control, and n ϭ 32 for staurosporine treated). This phenotype suggests that actin may regulate nuclear size in Giardia; alternatively, staurosporine treatment caused the nuclei to swell and actin filaments grew to occupy the available space. Overall, phosphorylation of the cytoskeleton appears to be correlated with Gl-14-3-3 association and cytoskeletal disassembly, while inhibition of phosphorylation with staurosporine stabilized cytoskeletal structures.
Considering the differences in actin extractability and changes in nuclear size associated with staurosporine and calyculin A treatment, we questioned whether Gl-14-3-3 depletion might also alter actin extractability and nuclear size. In Fig. 2D, we showed that depletion of Gl-14-3-3 led to ectopic actin distribution: the small puncta appear to be short filaments that could result from changes in actin regulation such as spontaneous actin nucleation or reduced filament stability. The amount of actin extracted from the Gl-14-3-3 knockdown cells increased in comparison to the control in each of the three replicates ( Fig. 5A and B). This result confirms that Gl-14-3-3 depletion alters the state of actin. However, due to the presence of short actin filaments, it is not clear if the increased extractability is due to increased globular actin or an indication that the short filaments are simply more extractable than the larger filaments found in control cells. We examined nuclear area in 14-3-3 knockdown cells and found that like staurosporine treatment, nuclear area was increased, although to a lesser extent ( Fig. 5C and D). Intriguingly, while actin filaments in the cytoplasm appear smaller in 14-3-3 knockdown cells, actin filaments associated with nuclei are more robust and are reminiscent of those observed in staurosporine-treated cells. These results are consistent with 14-3-3 regulating both the actin cytoskeleton and nuclear size.
Next we questioned whether, Rho GTPase signaling could modulate 14-3-3-actin complex formation. Expression of an inducible N-terminally HA-tagged constitutively active Q74L Gl-Rac (HA-Rac CA ; equivalent to Q61L Rac1) was previously observed to increase overall actin fluorescence, which we suggested was due to increased filament formation (21). Indeed, detailed analysis revealed the formation of prominent Gl-actin filaments at the cell cortex (Fig. 6A). Induction of the HA-Rac CA mutant protein led to a decrease in the amount of actin that coimmunoprecipitated with vesicular stomatitis virus G glycoprotein (VSVG) tagged 14-3-3, suggesting that Gl-Rac inhibits 14-3-3-actin complex formation (Fig. 6B). This result provides the first link between Rho GTPase signaling and actin in Giardia, since all of the proteins that normally link Rho GTPases and the actin cytoskeleton are missing.
After demonstrating that 14-3-3-actin complex levels could be modulated by Rho GTPase signaling and drug treatments, we sought to determine if there are binding motifs in Gl-actin that could support direct interaction with Gl-14-3-3. The 14-3-3-Pred prediction algorithm tool identified S330 (RVRIpSSP) and S338 (RKYpSAW) as the highest-scoring predicted interaction sites (36). In agreement with this finding, the same sites were previously reported using a custom algorithm to identify binding sites in putative Giardia 14-3-3 interactors (22). These sites are similar to the canonical mode 1 site RXXp(S/T)XP, where p(S/T) are phosphorylated serine or threonine residues; while not a perfect match, many 14-3-3-interacting proteins have been found that lack canonical mode 1-3 binding motifs (3,36,37). To assess the potential involvement of these sites, shown as surface accessible (Fig. 7A), TS-actin was mutated to generate an S330A S338A double mutant. The wild-type and mutant TS-actin constructs were introduced into the endogenously tagged Gl-14-3-3-HA parasite line. Phos-tag gel analysis of the S330A S338A double mutant confirms that at least one of these two sites is phosphorylated (Fig. 7B and C). The double mutant as well as single-point mutants had reduced capacity to coprecipitate 14-3-3-HA (Fig. 7D). The mean reductions in 14-3-3 binding were similar for the S330A and S338A single mutants and the S330A S338A double mutant. The double mutant, however, showed more consistent reduction, as noted by error bar size in Fig. 7E. Incomplete disruption of the Gl-14-3-3 interaction could indicate that part of the 14-3-3 recruitment is mediated through Ctrl Rac CA *** association with ABPs. This further raises the possibility that the point mutations that reduced complex formation with 14-3-3 could cause structural changes that reduce interaction with ABPs which recruit 14-3-3. Alternatively, the incomplete disruption of 14-3-3-actin complex formation could indicate the presence of additional interaction sites. Thus, we also tested the possible involvement of T162 (VTHpTVP), a conserved residue identified by Scansite 3 as the highest-scoring 14-3-3 interaction site (38). However, mutation of T162 to alanine did not disrupt 14-3-3-actin interaction, consistent with structure homology modeling that suggested this residue was not surface accessible (see Fig. S4 in the supplemental material). These results are in line with S330 and S338 having a role in promoting 14-3-3-actin complex formation. However, our inability to completely disrupt actin complex formation indicates additional means for 14-3-3's association with actin.
To test whether Gl-14-3-3 can directly bind Gl-actin, overlay experiments were performed using a 6ϫHis-Gl-actin expressed in and purified from Giardia extracts to ensure native phosphorylation. The overlay was performed with either the recombinant wild-type glutathione S-transferase (GST)-fused Gl-14-3-3 or with the mutant GST-K53E, previously shown to be binding defective (39,40). Binding of GST-Gl-14-3-3 near the molecular mass of 6ϫHis-Gl-actin was not observed (Fig. 8A). Instead, GST-Gl-14-3-3 bound to four bands on the blot corresponding to proteins copurified with 6ϫHis-Glactin. The most prominent labeling is associated with a major copurified protein at 80 kDa also seen in silver staining. Immunoblotting with an anti-pSer antibody confirmed that a fraction of actin was phosphorylated, as well as some copurified proteins, including the 80-kDa protein band (Fig. 8A). This result confirms that some of the 14-3-3-actin interaction is due to recruitment through additional binding partners that can mediate the formation of a complex containing both Gl-14-3-3 and Gl-actin. The inability to bind directly to actin could reflect a requirement for actin to be natively folded or that the interaction site is low affinity and additional proteins are needed to stabilize binding (41).
To assess whether Giardia contains a 14-3-3 complex appropriately sized for direct interaction, we turned to native gel electrophoresis. If a 14-3-3-actin complex formed without any additional proteins, it would contain a dimer of 14-3-3 (42) and a single TS-actin monomer. Previous native gel analysis indicated that 14-3-3 dimers run at 80 kDa (42) and TS-actin is 45.2 kDa; taking into account the size of the 3ϫHA tag (3.8 kDa) the anticipated complex size for direct binding is around 130 kDa. TS-actin and associated proteins were purified, immediately run on native gels, and transferred to membrane for Western blotting. A prominent band of Gl-14-3-3 was detected running between the 66-and 140-kDa native gel markers (Fig. 8B). However, a similarly sized band is also apparent in the input, suggesting this band represents free 14-3-3 dimer and that the complexes were unstable. In an effort to stabilize actin complexes, we used the membrane-permeable cross-linker DSP to cross-link samples before lysis. Cross-linking did change the distribution of actin, and a prominent actin band running just below 146 kDa was enriched in the pulldown (Fig. 8B); however, a corresponding enrichment of 14-3-3 was not apparent. Instead, the larger-molecular-mass smear of Gl-14-3-3 became more prominent, consistent with multiple actin interactors having the capacity to bind actin. Future work will be required to identify these 14-3-3-actininteracting proteins and determine their specific roles in regulating the Gl-actin cytoskeleton. Although the relationship between Gl-14-3-3 and actin appears to be more complicated than we initially imagined, our results indicate that Gl-14-3-3 plays a decisive role in actin regulation.

DISCUSSION
Proteomic studies support complex formation between 14-3-3 and actin without the involvement of cofilin (10)(11)(12)22). A recent study of 14-3-3 function in the basal-like progression of breast cancer cells found that 14-3-3 forms a complex containing actin and two intermediate filament proteins (13). The 14-3-3 complex sequesters soluble actin in a bioavailable form that is then used for directional assembly of the cytoskeleton during cell migration. Similarly, Giardia appears to use 14-3-3 to restrict actin assembly to specific subcellular regions, as evidenced by the 14-3-3 knockdown experiments where actin filaments were dispersed throughout the cell and typical actin organization was lost.
We estimated the intracellular concentration of actin to be around 4.7 M. This relatively low concentration is nevertheless above the critical concentration for Gl-actin (21), indicating the need for actin sequestration. The emerging view of actin network homeostasis is that sequestering proteins play an important role in partitioning actin to competing F-actin networks (43,44). In other eukaryotes, the concentration of sequestering proteins exceeds the actin monomer pool (45), but Giardia lacks all known monomer sequestering proteins. Our results indicate that Gl-14-3-3 is associated with monomeric actin in complexes with other interaction partners (Fig. 7) and that complex formation has a reverse correlation with actin filament formation (Fig. 3, 4, and 6). Therefore, it seems likely that 14-3-3 functions in the partitioning of actin complexes for subfunctionalization in addition to having a role in maintenance of G/F-actin homeostasis.
Complex formation between Gl-14-3-3 and Gl-actin appears to be both dependent and independent of phosphorylation. The putative 14-3-3 phospho-binding motif centered on S338 is conserved in mammalian actin and has previously been implicated as an AKT phosphorylation site (46). The 14-3-3 binding site prediction tool 14-3-3-Pred identified S338 as the highest-scoring site for both Gl-actin and human ␤-actin. We have shown that mutation of this site as well as S330 reduces 14-3-3 recruitment, and at least one of these predicted interaction sites is phosphorylated. Our ability to perform biochemical assays with Giardia's highly divergent actin remains limited. Overlay assays and native gels did not provide support for direct interaction between Gl-14-3-3 and Gl-actin. However, it remains possible that some of the observed complexes contain Gl-14-3-3 directly bound to Gl-actin with additional interactors that work to stabilize the complex (41). Indeed, the 14-3-3 complex contains two intermediate filament proteins, yet in vitro actin polymerization assays containing only actin and 14-3-3 demonstrated that 14-3-3 could directly regulate actin dynamics (13). The need for additional proteins to stabilize the 14-3-3 interaction with S330 and S338 would reconcile our data; however, a major caveat is that these point mutants could have disrupted Gl-actin folding and therefore disrupted the binding of proteins that recruit Gl-14-3-3.
While we have yet to pursue proteins that associate with both Gl-14-3-3 and Gl-actin, independent proteomic studies aimed at identifying 14-3-3 and actin interactors point toward proteins of interest (19,22). Besides Gl-actin and Gl-14-3-3, there are 14 proteins in common between the two studies (see Table S1 in the supplemental material). Eight of these proteins belong to the highly conserved chaperonin containing TCP-1 (CCT; molecular mass, 56.3 to 64.7 kDa), which is known to have a role in actin folding (47). The TCP-1 epsilon subunit has been implicated in the regulation of actin dynamics and this subunit has two, albeit untested, canonical mode 1 14-3-3 recognition motifs (22,48). Intriguingly, the epsilon subunit was the most abundant component of the TCP-1 complex found in our actin interactome. The list also includes TIP49 (GL50803_9825 [51.4 kDa]), which we have validated as a robust actin interactor through reciprocal pulldown (19). Other proteins include an SMC domain protein (GL50803_6886 [102.8 kDa]), two dynein heavy-chain proteins (GL50803_111950 [570.3 kDa] and GL50803_42285 [834.7 kDa]), and two proteins without any conserved domains (GL50803_15251 [32.5 kDa] and GL50803_15120 [26.6 kDa]). All of these proteins have at least one canonical mode 1 14-3-3 recognition motif, but whether they are involved in recruiting 14-3-3 to actin complexes remains to be determined.
Overall, our results support a role for Gl-14-3-3 associating with monomeric Gl-actin complexes that are downstream of Gl-Rac, where phosphorylated actin and phosphorylated actin interactors are held inactive. This work illustrates the conserved role of 14-3-3 as an actin regulator, albeit through an alternative set of actin-binding proteins that remain to be identified and validated.

MATERIALS AND METHODS
Strain and culture conditions. Giardia intestinalis strain WBC6 was cultured as described in reference 49.
Morpholino and drug studies. Morpholino treatments with anti-14-3-3 CGCGTAAATGCCTCGGCCA TAGGTT and control CCTCTTACCTCAGTTACAATTTATA were performed as described in reference 52. Calyculin A and staurosporine (LC Laboratories, Woburn, MA) were diluted in DMSO and used at 1 M and 200 nM final concentrations, respectively. Cells were treated for 45 min at 37°C.
Detergent-extractable actin. The detergent-extractable actin assay compares the fraction of Triton X-100-extractable actin versus nonextractable actin within the cell. While some filaments may be extracted by detergent treatment, the detergent-extractable fraction is largely composed of monomeric actin, while actin filaments associated with larger scaffolds remain within the cell (53,54). Confluent 8-ml cultures were treated for 45 min with drugs, chilled to detach cells, and then pelleted at 700 ϫ g. The pellet was resuspended in 800 l of HBS plus protease inhibitors and moved to a microcentrifuge tube. The cells were pelleted again and then resuspended in 100 l of actin-stabilizing lysis buffer consisting of 50 mM PIPES [piperazine-N,N=-bis(2-ethanesulfonic acid); pH 6.9], 50 mM NaCl, 5 mM MgCl 2 , 5 mM EGTA, 5% (vol/vol) glycerol, 0.1% Triton X-100, 0.1% Tween 20, 0.1% 2-mercaptoethanol, 0.2 mM ATP, and HALT protease inhibitor. The samples were incubated on ice for 5 min to perforate the membrane and then pelleted at 700 ϫ g for 5 min. The supernatants were reserved, and the pellets were resuspended in 80 l of 8 M urea for 1 h on ice. Equal amounts of each sample were boiled in sample buffer, loaded for SDS PAGE, and transferred to Immobilon-P membrane (Millipore). Actin was detected with the 28PBϩ1 antibody (1:3,000) and anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (1:6,000 [BioRad]), Western Lightning Plus ECL enhanced chemiluminescence reagent, and a BioRad Chemidoc MP digital gel doc (with further details provided below).
Phos-tag gels. After lysis or immunoprecipitation, samples were loaded onto 10% SDS gel supplemented with 100 M MnCl 2 and 20 M acrylamide-pendant Phos-tag AAL-107 (NARD Institute, Ltd. [from 5 mM stock solution in 3% MeOH in distilled water, prepared according to the manufacturer's instruction]) to detect mobility shift of phosphorylated proteins. The gels were run at 100 V for approximately 2 h. Immunoblotting was performed as described below.
Native gels. TS-actin and associated proteins were purified as described above with slight modifications. After cell lysis, the complexes were bound to StrepTactin resin (IBA, Lifesciences, Germany). Unbound complexes were washed out two times, and the 14-3-3-actin complexes were eluted with wash buffer supplemented with 2 mM biotin (Sigma). The samples were dissolved in native PAGE sample buffer (4ϫ) and immediately loaded onto 4 to 16% native PAGE Novex bis-Tris minigel (Life Technologies, Inc.) according to the manufacturer's instructions. Immunoblotting was performed as described below.
Statistical analysis. For each experiment with a P value, we compared at least three biological replicates using a two-tailed t test.