How capping protein binds the barbed end of the actin filament

Cytoskeletal filaments are often capped at one end, regulating assembly and cellular location. The actin filament is a right-handed, two-strand long-pitch helix. The ends of the two protofilaments are staggered in relation to each other, suggesting that capping could result from one protein binding simultaneously to the ends of both protofilaments. Capping protein (CP), a ubiquitous alpha/beta heterodimer in eukaryotes, tightly caps (K(d) approximately 0.1-1 nM) the barbed end of the actin filament (the end favored for polymerization), preventing actin subunit addition and loss. CP is critical for actin assembly and actin-based motility in vivo and is an essential component of the dendritic nucleation model for actin polymerization at the leading edge of cells. However, the mechanism by which CP caps actin filaments is not well understood. The X-ray crystal structure of CP has inspired a model where the C termini ( approximately 30 amino acids) of the alpha and beta subunits of CP are mobile extensions ("tentacles"), and these regions are responsible for high-affinity binding to, and functional capping of, the barbed end. We tested the tentacle model in vitro with recombinant mutant CPs. Loss of both tentacles causes a complete loss of capping activity. The alpha tentacle contributes more to capping affinity and kinetics; its removal reduces capping affinity by 5000-fold and the on-rate of capping by 20-fold. In contrast, removal of the beta tentacle reduced the affinity by only 300-fold and did not affect the on-rate. These two regions are not close to each other in the three-dimensional structure, suggesting CP uses two independent actin binding tentacles to cap the barbed end. CP with either tentacle alone can cap, as can the isolated beta tentacle alone, suggesting that the individual tentacles interact with more than one actin subunit at a subunit interface at the barbed end.     

Vital role for the Plasmodium actin capping protein (CP) beta‐subunit in motility of malaria sporozoites

Successful malaria transmission from the mosquito vector to the mammalian host depends crucially on active sporozoite motility. Sporozoite locomotion and host cell invasion are driven by the parasite's own actin/myosin motor. A unique feature of this motor machinery is the presence of very short subpellicular actin filaments. Therefore, F‐actin stabilizing proteins likely play a central role in parasite locomotion. Here, we investigated the role of the Plasmodium berghei actin capping protein (PbCP), an orthologue of the heterodimeric regulator of filament barbed end growth, by reverse genetics. Parasites containing a deletion of the CP beta‐subunit developed normally during the pathogenic erythrocytic cycle. However, due to reduced ookinete motility, mutant parasites form fewer oocysts and sporozoites in the Anopheles vector. These sporozoites display a vital deficiency in forward gliding motility and fail to colonize the mosquito salivary glands, resulting in complete attenuation of life cycle progression. Together, our results show that the CP beta‐subunit exerts an essential role in the insect vector before malaria transmission to the mammalian host. The vital role is restricted to fast locomotion, as displayed by Plasmodium sporozoites.     

Capping protein binding to actin in yeast: biochemical mechanism and physiological relevance

The mechanism by which capping protein (CP) binds barbed ends of actin filaments is not understood, and the physiological significance of CP binding to actin is not defined. The CP crystal structure suggests that the COOH-terminal regions of the CP alpha and beta subunits bind to the barbed end. Using purified recombinant mutant yeast CP, we tested this model. CP lacking both COOH-terminal regions did not bind actin. The alpha COOH-terminal region was more important than that of beta. The significance of CP's actin-binding activity in vivo was tested by determining how well CP actin-binding mutants rescued null mutant phenotypes. Rescue correlated well with capping activity, as did localization of CP to actin patches, indicating that capping is a physiological function for CP. Actin filaments of patches appear to be nucleated first, then capped with CP. The binding constants of yeast CP for actin suggest that actin capping in yeast is more dynamic than in vertebrates.     

Characterization of Plasmodium phosphatidylserine decarboxylase expressed in yeast and application for inhibitor screening

Phospholipid biosynthesis is critical for the development, differentiation and pathogenesis of several eukaryotic pathogens. Genetic studies have validated the pathway for phosphatidylethanolamine synthesis from phosphatidylserine catalyzed by phosphatidylserine decarboxylase enzymes (PSD) as a suitable target for development of antimicrobials; however no inhibitors of this class of enzymes have been discovered. We show that the Plasmodium falciparum PSD can restore the essential function of the yeast gene in strains requiring PSD for growth. Genetic, biochemical and metabolic analyses demonstrate that amino acids between positions 40 and 70 of the parasite enzyme are critical for proenzyme processing and decarboxylase activity. We used the essential role of Plasmodium PSD in yeast as a tool for screening a library of anti‐malarials. One of these compounds is 7‐chloro‐N‐(4‐ethoxyphenyl)‐4‐quinolinamine, an inhibitor with potent activity against P. falciparum, and low toxicity toward mammalian cells. We synthesized an analog of this compound and showed that it inhibits PfPSD activity and eliminates Plasmodium yoelii infection in mice. These results highlight the importance of 4‐quinolinamines as a novel class of drugs targeting membrane biogenesis via inhibition of PSD activity     

Feeling at home from arrival to departure: protein export and host cell remodelling during Plasmodium liver stage and gametocyte maturation

Obligate intracellular pathogens actively remodel their host cells to boost propagation, survival, and persistence. Plasmodium falciparum, the causative agent of the most severe form of malaria, assembles a complex secretory system in erythrocytes. Export of parasite factors to the erythrocyte membrane is essential for parasite sequestration from the blood circulation and a major factor for clinical complications in falciparum malaria. Historic and recent molecular reports show that host cell remodelling is not exclusive to P. falciparum and that parasite‐induced intra‐erythrocytic membrane structures and protein export occur in several Plasmodia. Comparative analyses of P. falciparum asexual and sexual blood stages and imaging of liver stages from transgenic murine Plasmodium species show that protein export occurs in all intracellular phases from liver infection to sexual differentiation, indicating that mammalian Plasmodium species evolved efficient strategies to renovate erythrocytes and hepatocytes according to the specific needs of each life cycle phase. While the repertoireof identified exported proteins is remarkably expanded in asexual P. falciparum blood stages, the putative export machinery and known targeting signatures are shared across life cycle stages. A better understanding of the molecular mechanisms underlying Plasmodium protein export could assist in designing novel strategies to interrupt transmission between Anopheles mosquitoes and humans.     

Binding of myotrophin/V-1 to actin-capping protein: implications for how capping protein binds to the filament barbed end

The heterodimeric actin-capping protein (CP) regulates actin assembly and cell motility by binding tightly to the barbed end of the actin filament. Here we demonstrate that myotrophin/V-1 binds directly to CP in a 1:1 molar ratio with a Kd of 10-50 nm. V-1 binding inhibited the ability of CP to cap the barbed ends of actin filaments. The actin-binding COOH-terminal region, the "tentacle," of the CP beta subunit was important for binding V-1, with lesser contributions from the alpha subunit COOH-terminal region and the body of the protein. V-1 appears to be unable to bind to CP that is on the barbed end, based on the observations that V-1 had no activity in an uncapping assay and that the V-1.CP complex had no capping activity. Two loops of V-1, which extend out from the alpha-helical backbone of this ankyrin repeat protein, were necessary for V-1 to bind CP. Parallel computational studies determined a bound conformation of the beta tentacle with V-1 that is consistent with these findings, and they offered insight into experimentally observed differences between the alpha1 and alpha2 isoforms as well as the mutant lacking the alpha tentacle. These results support and extend our "wobble" model for CP binding to the actin filament, in which the two COOH-terminal regions of CP bind independently to the actin filament, and bound CP is able to wobble when attached only via its mobile beta-subunit tentacle. This model is also supported by molecular dynamics simulations of CP reported here. The existence of the wobble state may be important for actin dynamics in cells.     

Purification, characterization, and immunofluorescence localization of Saccharomyces cerevisiae capping protein

Capping protein binds the barbed ends of actin filaments and nucleates actin filament assembly in vitro. We purified capping protein from Saccharomyces cervisiae. One of the two subunits is the product of the CAP2 gene, which we previously identified as the gene encoding the beta subunit of capping protein based on its sequence similarity to capping protein beta subunits in chicken and Dictyostelium (Amatruda, J. F., J. F. Cannon, K. Tatchell, C. Hug, and J. A. Cooper. 1990. Nature (Lond.) 344:352-354). Yeast capping protein has activity in critical concentration and low-shear viscometry assays consistent with barbed- end capping activity. Like chicken capping protein, yeast capping protein is inhibited by PIP2. By immunofluorescence microscopy yeast capping protein colocalizes with cortical actin spots at the site of bud emergence and at the tips of growing buds and shmoos. In contrast, capping protein does not colocalize with actin cables or with actin rings at the site of cytokinesis.     

Dominant negative mutant of Plasmodium Rad51 causes reduced parasite burden in host by abrogating DNA double‐strand break repair

Malaria parasites survive through repairing a plethora of DNA double‐stranded breaks (DSBs) experienced during their asexual growth. In Plasmodium Rad51 mediated homologous recombination (HR) mechanism and homology‐independent alternative end‐joining mechanism have been identified. Here we address whether loss of HR activity can be compensated by other DSB repair mechanisms. Creating a transgenic Plasmodium line defective in HR function, we demonstrate that HR is the most important DSB repair pathway in malarial parasite. Using mouse malaria model we have characterized the dominant negative effect of PfRad51^K143R mutant on Plasmodium DSB repair and host–parasite interaction. Our work illustrates that Plasmodium berghei harbouring the mutant protein (PfRad51^K143R) failed to repair DSBs as evidenced by hypersensitivity to DNA‐damaging agent. Mice infected with mutant parasites lived significantly longer with markedly reduced parasite burden. To better understand the effect of mutant PfRad51^K143R on HR, we used yeast as a surrogate model and established that the presence of PfRad51^K143R completely inhibited DNA repair, gene conversion and gene targeting. Biochemical experiment confirmed that very low level of mutant protein was sufficient for complete disruption of wild‐type PfRad51 activity. Hence our work provides evidence that HR pathway of Plasmodium could be efficiently targeted to curb malaria.     

Vertebrate isoforms of actin capping protein beta have distinct functions In vivo

Actin capping protein (CP) binds barbed ends of actin filaments to regulate actin assembly. CP is an alpha/beta heterodimer. Vertebrates have conserved isoforms of each subunit. Muscle cells contain two beta isoforms. beta1 is at the Z-line; beta2 is at the intercalated disc and cell periphery in general. To investigate the functions of the isoforms, we replaced one isoform with another using expression in hearts of transgenic mice.Mice expressing beta2 had a severe phenotype with juvenile lethality. Myofibril architecture was severely disrupted. The beta2 did not localize to the Z-line. Therefore, beta1 has a distinct function that includes interactions at the Z-line. Mice expressing beta1 showed altered morphology of the intercalated disc, without the lethality or myofibril disruption of the beta2-expressing mice.The in vivo function of CP is presumed to involve binding barbed ends of actin filaments. To test this hypothesis, we expressed a beta1 mutant that poorly binds actin. These mice showed both myofibril disruption and intercalated disc remodeling, as predicted.Therefore, CPbeta1 and CPbeta2 each have a distinct function that cannot be provided by the other isoform. CPbeta1 attaches actin filaments to the Z-line, and CPbeta2 organizes the actin at the intercalated discs.     

The alpha and beta subunits of nematode actin capping protein function in yeast.

We cloned and analyzed two genes, cap-1 and cap-2, which encode the alpha and beta subunits of Caenorhabditis elegans capping protein (CP). The nematode CP subunits are 55% (cap-1) and 66% (cap-2) identical to the chicken CP subunits and 32% (cap-1) and 48% (cap-2) identical to the yeast CP subunits. Purified nematode CP made by expression of both subunits in yeast is functionally similar to chicken skeletal muscle CP in two different actin polymerization assays. The abnormal cell morphology and disorganized actin cytoskeleton of yeast CP null mutants are restored to wild-type by expression of the nematode CP subunits. Expression of the nematode CP alpha or beta subunit is sufficient to restore viability to yeast cap1 sac6 or cap2 sac6 double mutants, respectively. Therefore, despite evolution of the nematode actin cytoskeleton to a state far more complex than that of yeast, one important component can function in both organisms.     

An essential role of the basal body protein SAS‐6 in Plasmodium male gamete development and malaria transmission

Gametocytes are the sole Plasmodium parasite stages that infect mosquitoes; therefore development of functional gametes is required for malaria transmission. Flagellum assembly of the Plasmodium male gamete differs from that of most other eukaryotes in that it is intracytoplasmic but retains a key conserved feature: axonemes assemble from basal bodies. The centriole/basal body protein SAS‐6 normally regulates assembly and duplication of these organelles and its depletion causes severe flagellar/ciliary abnormalities in a diverse array of eukaryotes. Since basal body and flagellum assembly are intimately coupled to male gamete development in Plasmodium, we hypothesized that SAS‐6 disruption may cause gametogenesis defects and perturb transmission. We show that Plasmodium berghei sas6 knockouts display severely abnormal male gametogenesis presenting reduced basal body numbers, axonemal assembly defects and abnormal nuclear allocation. The defects in gametogenesis reduce fertilization and render Pbsas6 knockouts less infectious to mosquitoes. Additionally, we show that lack of Pbsas6 blocks transmission from mosquito to vertebrate host, revealing an additional yet undefined role in ookinete to sporulating oocysts transition. These findings underscore the vulnerability of the basal body/SAS‐6 to malaria transmission blocking interventions.     

A Plasmodium α/β‐hydrolase modulates the development of invasive stages

The bud emergence (BEM)46 proteins are evolutionarily conserved members of the α/β‐hydrolase superfamily, which includes enzymes with diverse functions and a wide range of substrates. Here, we identified a Plasmodium BEM46‐like protein (PBLP) and characterized it throughout the life cycle of the rodent malaria parasite Plasmodium yoelii. The Plasmodium BEM46‐like protein is shown to be closely associated with the parasite plasma membrane of asexual erythrocytic stage schizonts and exo‐erythrocytic schizonts; however, PBLP localizes to unique intracellular structures in sporozoites. Generation and analysis of P. yoelii knockout (Δpblp) parasite lines showed that PBLP has an important role in erythrocytic stage merozoite development with Δpblp parasites forming fewer merozoites during schizogony, which results in decreased parasitemia when compared with wild‐type (WT) parasites. Δpblp parasites showed no defects in gametogenesis or transmission to mosquitoes; however, because they formed fewer oocysts there was a reduction in the number of developed sporozoites in infected mosquitoes when compared with WT. Although Δpblp sporozoites showed no apparent defect in mosquito salivary gland infection, they showed decreased infectivity in hepatocytes in vitro. Similarly, mice infected with Δpblp sporozoites exhibited a delay in the onset of blood‐stage patency, which is likely caused by reduced sporozoite infectivity and a discernible delay in exo‐erythrocytic merozoite formation. These data are consistent with the model that PBLP has an important role in parasite invasive‐stage morphogenesis throughout the parasite life cycle.     

Beta-actinin is equivalent to Cap Z protein

Chicken skeletal muscle beta-actinin, previously reported to bind the slow-exchanging (pointed) ends of actin filaments was purified to homogeneity. By two dimensional gel electrophoresis, it consists of two subunits, beta I (35 kDa) and beta II (32 kDa), and each subunit has two isoforms. The amino acid sequences of V8 protease-digested peptides of beta I were nearly identical with those of portions of the muscle barbed end-blocking protein Cap Z alpha, although several amino acids were different from those deduced from cDNA sequences (Casella, J.F., Casella, S.J., Hollands, J.A., Caldwell, J.E., and Cooper, J.A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5800-5804). The amino acid sequences of two peptides from beta II were completely identical with portions of Cap Z beta deduced from cDNA sequences (Caldwell, J.E., Waddle, J.A., Cooper, J.A., Hollands, J.A., Casella, S.J., and Casella, J.F. (1989) J. Biol. Chem. 264, 12648-12652). beta-Actinin capped the barbed end of an actin filament as evidenced by actin assembly of myosin S1-decorated filaments and specifically its impairment of growth in the "barbed" direction. Thus it is concluded that highly purified beta-actinin is identical with the more recently described Cap Z, an actin barbed-end capping protein of chicken skeletal muscle.     

Oryza sativa actin‐interacting protein 1 is required for rice growth by promoting actin turnover

Rapid actin turnover is essential for numerous actin‐based processes. However, how it is precisely regulated remains poorly understood. Actin‐interacting protein 1 (AIP1) has been shown to be an important factor by acting coordinately with actin‐depolymerizing factor (ADF)/cofilin in promoting actin depolymerization, the rate‐limiting factor in actin turnover. However, the molecular mechanism by which AIP1 promotes actin turnover remains largely unknown in plants. Here, we provide a demonstration that AIP1 promotes actin turnover, which is required for optimal growth of rice plants. Specific down‐regulation of OsAIP1 increased the level of filamentous actin and reduced actin turnover, whereas over‐expression of OsAIP1 induced fragmentation and depolymerization of actin filaments and enhanced actin turnover. In vitro biochemical characterization showed that, although OsAIP1 alone does not affect actin dynamics, it enhances ADF‐mediated actin depolymerization. It also caps the filament barbed end in the presence of ADF, but the capping activity is not required for their coordinated action. Real‐time visualization of single filament dynamics showed that OsAIP1 enhanced ADF‐mediated severing and dissociation of pointed end subunits. Consistent with this, the filament severing frequency and subunit off‐rate were enhanced in OsAIP1 over‐expressors but decreased in RNAi protoplasts. Importantly, OsAIP1 acts coordinately with ADF and profilin to induce massive net actin depolymerization, indicating that AIP1 plays a major role in the turnover of actin, which is required to optimize F‐actin levels in plants.     

The Plasmodium falciparum exportome contains non‐canonical PEXEL/HT proteins

The pathogenicity of Plasmodium falciparum is partly due to parasite‐induced host cell modifications. These modifications are facilitated by exported P. falciparum proteins, collectively referred to as the exportome. Export of several hundred proteins is mediated by the PEXEL/HT, a protease cleavage site. The PEXEL/HT is usually comprised of five amino acids, of which R at position 1, L at position 3 and E, D or Q at position 5 are conserved and important for export. Non‐canonical PEXEL/HTs with K or H at position 1 and/or I at position 3 are presently considered non‐functional. Here, we show that non‐canonical PEXEL/HT proteins are overrepresented in P. falciparum and other Plasmodium species. Furthermore, we show that non‐canonical PEXEL/HTs can be cleaved and can promote export in both a REX3 and a GBP reporter, but not in a KAHRP reporter, indicating that non‐canonical PEXEL/HTs are functional in concert with a supportive sequence environment. We then selected P. falciparum proteins with a non‐canonical PEXEL/HT and show that some of these proteins are exported and that their export depends on non‐canonical PEXEL/HTs. We conclude that PEXEL/HT plasticity is higher than appreciated and that non‐canonical PEXEL/HT proteins cannot categorically be excluded from Plasmodium exportome predictions.     

Differential expression of cell surface proteins in human bone marrow mesenchymal stem cells cultured with or without basic fibroblast growth factor containing medium

Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi‐lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2‐DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin–avidin affinity column were separated by 2‐DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole‐time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin‐related proteins, F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF‐induced morphological change of MSCs.     

Mutational analysis of capping protein function in Saccharomyces cerevisiae.

To investigate physiologic functions and structural correlates for actin capping protein (CP), we analyzed site-directed mutations in CAP1 and CAP2, which encode the alpha and beta subunits of CP in Saccharomyces cerevisiae. Mutations in four different regions caused a loss of CP function in vivo despite the presence of mutant protein in the cells. Mutations in three regions caused a complete loss of all aspects of function, including the actin distribution, viability with sac6, and localization of CP to actin cortical patches. Mutation of the fourth region led to partial loss of only one function-formation of actin cables. Some mutations retained function and exhibited the complete wild-type phenotype, and some mutations led to a complete loss of protein and therefore loss of function. The simplest hypothesis that can explain these results is that a single biochemical property is necessary for all in vivo functions. This biochemical property is most likely binding to actin filaments, because the nonfunctional mutant CPs no longer co-localize with actin filaments in vivo and because direct binding of CP to actin filaments has been well established by studies with purified proteins in vitro. More complex hypotheses, involving the existence of additional biochemical properties important for function, cannot be excluded by this analysis.     

Crystal structure of CapZ: structural basis for actin filament barbed end capping

Capping protein, a heterodimeric protein composed of alpha and beta subunits, is a key cellular component regulating actin filament assembly and organization. It binds to the barbed ends of the filaments and works as a 'cap' by preventing the addition and loss of actin monomers at the end. Here we describe the crystal structure of the chicken sarcomeric capping protein CapZ at 2.1 A resolution. The structure shows a striking resemblance between the alpha and beta subunits, so that the entire molecule has a pseudo 2-fold rotational symmetry. CapZ has a pair of mobile extensions for actin binding, one of which also provides concomitant binding to another protein for the actin filament targeting. The mobile extensions probably form flexible links to the end of the actin filament with a pseudo 2(1) helical symmetry, enabling the docking of the two in a symmetry mismatch.     

Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments

We describe herein the purification of a protein from skeletal muscle that binds to ("caps") the morphologically defined barbed end of actin filaments. This actin-capping protein appeared to be a heterodimer with chemically and immunologically distinct subunits of Mr = 36,000 (alpha) and 32,000 (beta), Rs = 37 A, s20,w = 4.0 S, and a calculated native molecular weight of approximately 61,000. The protein was obtained in milligram quantities at greater than 95% purity from acetone powder of chicken skeletal muscle by extraction in 0.6 M KI, precipitation with ammonium sulfate, sequential chromatographic steps on DEAE-cellulose, hydroxylapatite, and Sephacryl S-200, followed by preparative rate zonal sucrose density gradient centrifugation. In immunoblots of myofibrillar proteins, affinity-purified antibodies selectively recognized protein bands of the same molecular weight as the subunits of the capping protein to which they were made, indicating that the isolated capping protein is a native myofibrillar protein, and not a proteolytic digestion product of a larger muscle protein. A specific interaction of the capping protein with the barbed end of actin filaments was indicated by its ability to inhibit actin filament assembly nucleated by spectrin-band 4.1-actin complex in 0.4 mM Mg2+, accelerate actin filament formation and increase the critical concentration of actin in 2-5 mM Mg2+, 75-100 mM KCl, and inhibit the addition of actin monomers to the barbed end of heavy meromyosin-decorated actin filaments as determined by electron microscopy. All of these effects occurred at nanomolar concentrations of capping protein and micromolar concentrations of actin, suggesting a high affinity interaction.