Multicomponent depolymerization of actin filament pointed ends by cofilin and cyclase-associated protein depends upon filament age.

Intracellular actin networks assemble through the addition of ATP-actin subunits at the growing barbed ends of actin filaments. This is followed by "aging" of the filament via ATP hydrolysis and subsequent phosphate release. Aged ADP-actin subunits thus "treadmill" through the filament before being released back into the cytoplasmic monomer pool as a result of depolymerization at filament pointed ends. The necessity for aging before filament disassembly is reinforced by preferential binding of cofilin to aged ADP-actin subunits over newly-assembled ADP-Pi actin subunits in the filament. Consequently, investigations into how cofilin influences pointed-end depolymerization have, thus far, focused exclusively on aged ADP-actin filaments. Using microfluidics-assisted Total Internal Reflection Fluorescence (mf-TIRF) microscopy, we reveal that, similar to their effects on ADP filaments, cofilin and cyclase-associated protein (CAP) also promote pointed-end depolymerization of ADP-Pi filaments. Interestingly, the maximal rates of ADP-Pi filament depolymerization by CAP and cofilin together remain approximately 20-40 times lower than for ADP filaments. Further, we find that the promotion of ADP-Pi pointed-end depolymerization is conserved for all three mammalian cofilin isoforms. Taken together, the mechanisms presented here open the possibility of newly-assembled actin filaments being directly disassembled from their pointed-ends, thus bypassing the slow step of Pi release in the aging process.

Multicomponent depolymerization of actin filament pointed ends by cofilin and cyclase-associated protein depends upon filament age.

Intracellular actin networks assemble through the addition of ATP-actin subunits at the growing barbed ends of actin filaments. This is followed by "aging" of the filament via ATP hydrolysis and subsequent phosphate release. Aged ADP-actin subunits thus "treadmill" through the filament before being released back into the cytoplasmic monomer pool as a result of depolymerization at filament pointed ends. The necessity for aging before filament disassembly is reinforced by preferential binding of cofilin to aged ADP-actin subunits over newly-assembled ADP-Pi actin subunits in the filament. Consequently, investigations into how cofilin influences pointed-end depolymerization have, thus far, focused exclusively on aged ADP-actin filaments. Using microfluidics-assisted Total Internal Reflection Fluorescence (mf-TIRF) microscopy, we reveal that, similar to their effects on ADP filaments, cofilin and cyclase-associated protein (CAP) also promote pointed-end depolymerization of ADP-Pi filaments. Interestingly, the maximal rates of ADP-Pi filament depolymerization by CAP and cofilin together remain approximately 20-40 times lower than for ADP filaments. Further, we find that the promotion of ADP-Pi pointed-end depolymerization is conserved for all three mammalian cofilin isoforms. Taken together, the mechanisms presented here open the possibility of newly-assembled actin filaments being directly disassembled from their pointed-ends, thus bypassing the slow step of Pi release in the aging process.

TaCAP1 Interacts with TaLHCB1s and Positively Regulates Wheat Resistance Against Stripe Rust.

Actin filaments and their associated actin-binding proteins play key roles in plant innate immune signaling. CAP1, or cyclase-associated protein 1, is an important regulatory factor of the actin cytoskeleton-associated signaling network and was hypothesized here to be involved in resistance against wheat stripe rust because TaCAP1 expression was upregulated in response to Puccinia striiformis f. sp. tritici (Pst). Downregulation of TaCAP1 expression led to decreased resistance against Pst, in contrast to increased resistance upon TaCAP1 overexpressing, as demonstrated by the changes of phenotypes and hyphal growth. We found increased expression of pathogenesis-responsive or relative related genes and disease grade changed in TaCAP1 overexpressing plants. Our results also showed TaCAP1-regulated host resistance to Pst by inducing the production and accumulation of reactive oxygen species and mediating the salicylic acid signaling pathway. Additionally, TaCAP1 interacted with chlorophyll a/b-binding proteins TaLHCB1.3 and TaLHCB1.4, also known as the light-harvesting chlorophyll-protein complex II subunit B, which belong to the light-harvesting complex II protein family. Silencing of two TaLHCB1 genes showed higher susceptibility to Pst, which reduced wheat resistance against Pst. Therefore, the data presented herein further illuminate our understanding that TaCAP1 interacts with TaLHCB1s and functions as a positive regulator of wheat resistance against stripe rust.

Cyclase-associated protein is a pro-formin anti-capping processive depolymerase of actin barbed and pointed ends.

Cellular actin networks display distinct assembly and disassembly dynamics resulting from multicomponent reactions occurring primarily at the two ends and the sides of actin filaments [1-3]. While barbed ends are considered the hotspot of actin assembly [4], disassembly is thought to primarily occur via reactions on filament sides and pointed ends [3, 5-11]. Cyclase-associated protein (CAP) has emerged as the main protagonist of actin disassembly and remodeling - it collaborates with cofilin to increase pointed-end depolymerization by 300-fold [6, 7], promotes filament "coalescence" in presence of Abp1 [12], and accelerates nucleotide exchange to regenerate monomers for new rounds of assembly [13-15]. CAP has also been reported to enhance cofilin-mediated severing [16, 17], but these claims have since been challenged [7]. Using microfluidics-assisted three-color single-molecule imaging, we now reveal that CAP also has important functions at filament barbed ends. We reveal that CAP is a processive barbed-end depolymerase capable of tracking both ends of the filament. Each CAP binding event leads to removal of about 5,175 and 620 subunits from the barbed and pointed ends respectively. We find that the WH2 domain is essential, and the CARP domain is dispensable for barbed-end depolymerization. We show that CAP co-localizes with barbed-end bound formin and capping protein, in the process increasing residence time of formin by 10-fold and promoting dissociation of CP by 4-fold. Our barbed-end observations combined with previously reported activities of CAP at pointed ends and sides, firmly establish CAP as a key player in actin dynamics.

Cyclase-associated protein interacts with actin filament barbed ends to promote depolymerization and formin displacement.

Cyclase-associated protein (CAP) has emerged as a central player in cellular actin turnover, but its molecular mechanisms of action are not yet fully understood. Recent studies revealed that the N terminus of CAP interacts with the pointed ends of actin filaments to accelerate depolymerization in conjunction with cofilin. Here, we use in vitro microfluidics-assisted TIRF microscopy to show that the C terminus of CAP promotes depolymerization at the opposite (barbed) ends of actin filaments. In the absence of actin monomers, full-length mouse CAP1 and C-terminal halves of CAP1 (C-CAP1) and CAP2 (C-CAP2) accelerate barbed end depolymerization. Using mutagenesis and structural modeling, we show that these activities are mediated by the WH2 and CARP domains of CAP. In addition, we observe that CAP collaborates with profilin to accelerate barbed end depolymerization and that these effects depend on their direct interaction, providing the first known example of CAP-profilin collaborative effects in regulating actin. In the presence of actin monomers, CAP1 attenuates barbed end growth and promotes formin dissociation. Overall, these findings demonstrate that CAP uses distinct domains and mechanisms to interact with opposite ends of actin filaments and drive turnover. Further, they contribute to the emerging view of actin barbed ends as sites of dynamic molecular regulation, where numerous proteins compete and cooperate with each other to tune polymer dynamics, similar to the rich complexity seen at microtubule ends.

Dynamic remodeling of actin networks by cyclase-associated protein and CAP-Abp1 complexes.

How actin filaments are spatially organized and remodeled into diverse higher-order networks in vivo is still not well understood. Here, we report an unexpected F-actin "coalescence" activity driven by cyclase-associated protein (CAP) and enhanced by its interactions with actin-binding protein 1 (Abp1). We directly observe S. cerevisiae CAP and Abp1 rapidly transforming branched or linear actin networks by bundling and sliding filaments past each other, maximizing filament overlap, and promoting compaction into bundles. This activity does not require ATP and is conserved, as similar behaviors are observed for the mammalian homologs of CAP and Abp1. Coalescence depends on the CAP oligomerization domain but not the helical folded domain (HFD) that mediates its functions in F-actin severing and depolymerization. Coalescence by CAP-Abp1 further depends on interactions between CAP and Abp1 and interactions between Abp1 and F-actin. Our results are consistent with a mechanism in which the formation of energetically favorable sliding CAP and CAP-Abp1 crosslinks drives F-actin bundle compaction. Roles for CAP and CAP-Abp1 in actin remodeling in vivo are supported by strong phenotypes arising from deletion of the CAP oligomerization domain and by genetic interactions between sac6Δ and an srv2-301 mutant that does not bind Abp1. Together, these observations identify a new actin filament remodeling function for CAP, which is further enhanced by its direct interactions with Abp1.

An association study of cyclase-associated protein 2 and frailty.

Frailty is a geriatric syndrome that results from multisystem impairment caused by age-associated accumulation of deficits. The frailty index is used to define the level of frailty. Several studies have searched for molecular biomarkers associated with frailty, to meet the needs for personalized care. Cyclase-associated protein 2 (CAP2) is a multifunctional actin-binding protein involved in various physiological and pathological processes, that might reflect frailty's intrinsic complexity. This study aimed to investigate the association between frailty index and circulating CAP2 concentration in 467 community-dwelling older adults (median age: 79; range: 65-92 years) from Milan, Italy. The selected robust regression model showed that circulating CAP2 concentration was not associated with chronological age, as well as sex and education. However, circulating CAP2 concentration was significantly and inversely associated with the frailty index: a 0.1-unit increase in frailty index leads to ~0.5-point mean decrease in CAP2 concentration. Furthermore, mean CAP2 concentration was significantly lower in frail participants (i.e., frailty index ≥0.25) than in non-frail participants. This study shows the association between serum CAP2 concentration and frailty status for the first time, highlighting the potential of CAP2 as a biomarker for age-associated accumulation of deficits.

Knockout of cyclase-associated protein CAP1 confers tolerance towards salt and osmotic stress in Arabidopsis.

As a regulator of actin filament turnover, Arabidopsis thaliana CAP1 plays an important role in plant growth and development. Here, we analyzed the phenotypes of two Arabidopsis cap1 mutants: cap1-1 (a T-DNA insertion mutant) and Cas9-CAP1 (generated by CRISPR-Cas9 gene editing). Phenotypic analysis demonstrated that loss of CAP1 results in defects in seed germination and seedling morphology, with some seedlings exhibiting one or three cotyledons. The cap1-1 mutant took longer than the wild type to complete its life cycle, but its flowering time was normal, indicating that loss of CAP1 prolongs reproductive but not vegetative growth. Moreover, loss of CAP1 severely reduces seed production in self-pollinated plants, due to disruption of pollen tube elongation. RNA-seq and qRT-PCR analyses demonstrated that CAP1 may be involved in osmotic stress responses. Indeed, the cap1-1 mutant showed increased tolerance of salt and mannitol treatment, indicating that CAP1 plays a negative role in osmotic stress tolerance in Arabidopsis. Taken together, our results demonstrate that CAP1 functions not only in plant growth and development, but also in Arabidopsis responses to osmotic stress.

CAP1 (cyclase-associated protein 1) mediates the cyclic AMP signals that activate Rap1 in stimulating matrix adhesion of colon cancer cells.

We previously reported that CAP1 (Cyclase-Associated Protein 1) regulates matrix adhesion in mammalian cells through FAK (Focal Adhesion Kinase). More recently, we discovered a phosphor-regulation mechanism for CAP1 through the Ser307/Ser309 tandem site that is of critical importance for all CAP1 functions. However, molecular mechanisms underlying the CAP1 function in adhesion and its regulation remain largely unknown. Here we report that Rap1 also facilitates the CAP1 function in adhesion, and more importantly, we identify a novel signaling pathway where CAP1 mediates the cAMP signals, through the cAMP effectors Epac (Exchange proteins directly activated by cAMP) and PKA (Protein Kinase A), to activate Rap1 in stimulating matrix adhesion in colon cancer cells. Knockdown of CAP1 led to opposite adhesion phenotypes in SW480 and HCT116 colon cancer cells, with reduced matrix adhesion and reduced FAK and Rap1 activities in SW480 cells while it stimulated matrix adhesion as well as FAK and Rap1 activities in HCT116 cells. Importantly, depletion of CAP1 abolished the stimulatory effects of the cAMP activators forskolin and isoproterenol, as well as that of Epac and PKA, on matrix adhesion in both cell types. Our results consistently support a required role for CAP1 in the cAMP activation of Rap1. Identification of the key role for CAP1 in linking the major second messenger cAMP to activation of Rap1 in stimulating adhesion, which may potentially also regulate proliferation in other cell types, not only vertically extends our knowledge on CAP biology, but also carries important translational potential for targeting CAP1 in cancer therapeutics.

Chromenopyrimidinone exhibit antitumor effects through inhibition of CAP1 (Adenylyl cyclase-associated protein 1) expression in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most malignant human cancers, with a high mortality rate worldwide. Within an HCC tumor, cancer stem cells (CSCs) are responsible for tumor maintenance and progression and may contribute to resistance to standard HCC treatments. Previously, we characterized CD133+ cells as CSCs in primary HCC and identified chromenopyrimidinone (CPO) as a novel therapeutic for the effective treatment of CD133+ HCC. However, the biological function and molecular mechanism of CD133 remain unclear. Epigenetic alterations of CSCs have impacts on tumor initiation, progression, and therapeutic response. Here, we found that pharmacological and genetic depletion of CD133 in HCC attenuated the activity of DNA methyltransferases via control of DNMT3B stabilization. Genes were ranked by degree of promoter hypo/hyper methylation and significantly differential expression to create an "epigenetically activated by CPO" ranked genes list. Through this epigenetic analysis, we found that CPO treatment altered DNA methylation-mediated oncogenic signaling in HCCs. Specifically, CPO treatment inhibited Adenylyl cyclase-associated protein 1 (CAP1) expression, thereby reducing FAK/ERK activity and EMT-related proteins in HCC. Moreover, CPO improved the efficacy of sorafenib by inhibiting CAP1 expression and FAK/ERK activation in sorafenib-resistant HCC. These novel mechanistic insights may ultimately open up avenues for strategies targeting DNA methylation in liver cancer stem cells and provides novel therapeutic function of CPO for the effective treatment of sorafenib-resistant HCC.

Characterization of a Peptide Antibody Specific to the Adenylyl Cyclase-Associated Protein of Acanthamoeba castellanii.

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.

Disease association of cyclase-associated protein (CAP): Lessons from gene-targeted mice and human genetic studies.

Cyclase-associated protein (CAP) is an actin binding protein that has been initially described as partner of the adenylyl cyclase in yeast. In all vertebrates and some invertebrate species, two orthologs, named CAP1 and CAP2, have been described. CAP1 and CAP2 are characterized by a similar multidomain structure, but different expression patterns. Several molecular studies clarified the biological function of the different CAP domains, and they shed light onto the mechanisms underlying CAP-dependent regulation of actin treadmilling. However, CAPs are crucial elements not only for the regulation of actin dynamics, but also for signal transduction pathways. During recent years, human genetic studies and the analysis of gene-targeted mice provided important novel insights into the physiological roles of CAPs and their involvement in the pathogenesis of several diseases. In the present review, we summarize and discuss recent progress in our understanding of CAPs' physiological functions, focusing on heart, skeletal muscle and central nervous system as well as their involvement in the mechanisms controlling metabolism. Remarkably, loss of CAPs or impairment of CAPs-dependent pathways can contribute to the pathogenesis of different diseases. Overall, these studies unraveled CAPs complexity highlighting their capability to orchestrate structural and signaling pathways in the cells.

A pilot study of the relative number of circulating tumor cells and leukocytes containing actin-binding proteins in head and neck cancer patients.

Circulating tumor cells (CTCs) play an important role in tumor metastases, which is positively correlated with an increased risk of death. Actin-binding proteins, including cofilin (CFL1), profilin 1 (PFN1), and adenylate cyclase-associated protein 1 (CAP1), are thought to be involved in tumor cell motility and metastasis, specifically in head and neck squamous cell carcinoma (HNSCC). However, currently, there are no published studies on CFL1, PFN1, and CAP1 in CTCs and leukocytes in HNSCC patients. We assessed serum levels of CFL1, PFN1, and CAP1 and the number of CTCs and leukocytes containing these proteins in blood from 31 HNSCC patients (T1-4N0-2M0). The analysis used flow cytometry and an enzyme-linked immunosorbent assay kit. We found that CAP1 + CTCs and CAP1 + leukocyte subpopulations were prevalent in these HNSCC patient samples, while the prevalence rates of CFL1 + and PFN1 + CTCs were relatively low. Patients with stage T2-4N1-2M0 had CFL1 + and PFN1 + CTCs with an elevated PFN1 serum level, compared with the T1-3N0M0 group. In summary, the PFN1 serum level and the relative number of PFN1 +CD326 + CTCs could be valuable prognostic markers for HNSCC metastases. The current study is the first to obtain data regarding the contents of actin-binding proteins (ABPs) in CTCs, and leukocytes in blood from HNSCC patients. This is also the first to assess the relationship between the number of CTCs subgroups and disease characteristics.

Phosphorylation of CAP1 regulates lung cancer proliferation, migration, and invasion.

PURPOSE: Cyclase-associated protein 1 (CAP1) is a ubiquitous protein which regulates actin dynamics. Previous studies have shown that S308 and S310 are the two major phosphorylated sites in human CAP1. In the present study, we aimed to investigate the role of CAP1 phosphorylation in lung cancer. METHODS: Massive bioinformatics analysis was applied to determine CAP1's role in different cancers and especially in lung cancer. Lung cancer patients' serum and tissue were collected and analyzed in consideration of clinical background. CAP1 shRNA-lentivirus and siRNA were applied to CAP1 gene knockdown, and plasmids were constructed for CAP1 phosphorylation and de-phosphorylation. Microarray analysis was used for CAP1-associated difference analysis. Both in vitro and in vivo experiments were performed to investigate the roles of CAP1 phosphorylation and de-phosphorylation in lung cancer A549 cells. RESULTS: CAP1 is a kind of cancer-related protein. Its mRNA was overexpressed in most types of cancer tissues when compared with normal tissues. CAP1 high expression correlated with poor prognosis. Our results showed that serum CAP1 protein concentrations were significantly upregulated in non-small cell lung cancer (NSCLC) patients when compared with the healthy control group, higher serum CAP1 protein concentration correlated with shorter overall survival (OS) in NSCLC patients, and higher pCAP1 and CAP1 protein level were observed in lung cancer patients' tumor tissue compared with adjacent normal tissue. Knockdown CAP1 in A549 cells can inhibit proliferation and migration, and the effect is validated in H1975 cells. It can also lead to an increase ratio of F-actin/G-actin. In addition, phosphorylated S308 and S310 in CAP1 promoted lung cancer cell proliferation, migration, and metastasis both in vitro and in vivo. When de-phosphorylated, these two sites in CAP1 showed the opposite effect. Phosphorylation of CAP1 can promote epithelial-mesenchymal transition (EMT). CONCLUSION: These findings indicated that CAP1 phosphorylation can promote lung cancer proliferation, migration, and invasion. Phosphorylation sites of CAP1 might be a novel target for lung cancer treatment.

Reduced tissue and serum resistin expression as a clinical marker for esophageal squamous cell carcinoma.

Esophageal cancer is one of the most common malignancies and leading cause of cancer-associated mortality worldwide. However, the molecular mechanisms underlying esophageal cancer progression and the development of clinical tools for effective diagnosis remain unclear. Resistin, which was originally identified as an adipose tissue-secretory factor, has been associated with obesity-related diseases, including certain types of cancer. Thus, the present study aimed to investigate the expression levels of resistin in tissue and serum specimens from patients with esophageal squamous cell carcinoma (ESCC) to determine the potential biological effects of resistin on ESCC cells. The results demonstrated that both tissue and serum resistin levels were significantly lower in patients with ESCC compared with healthy controls. In addition, resistin expression was positively associated with the body mass index of patients with ESCC. In vitro studies revealed that resistin inhibited the migratory ability of ESCC cells, while having no effect on ESCC cell proliferation. Taken together, these results suggest that resistin may have the potential to be developed into a clinical marker for ESCC. However, further studies are required to investigate resistin receptor expression and determine the potential involvement of resistin-associated biological pathways, which may provide insight for future development of targeted therapies for resistin-mediated ESCC.

Functional Redundancy of Cyclase-Associated Proteins CAP1 and CAP2 in Differentiating Neurons.

Cyclase-associated proteins (CAPs) are evolutionary-conserved actin-binding proteins with crucial functions in regulating actin dynamics, the spatiotemporally controlled assembly and disassembly of actin filaments (F-actin). Mammals possess two family members (CAP1 and CAP2) with different expression patterns. Unlike most other tissues, both CAPs are expressed in the brain and present in hippocampal neurons. We recently reported crucial roles for CAP1 in growth cone function, neuron differentiation, and neuron connectivity in the mouse brain. Instead, CAP2 controls dendritic spine morphology and synaptic plasticity, and its dysregulation contributes to Alzheimer's disease pathology. These findings are in line with a model in which CAP1 controls important aspects during neuron differentiation, while CAP2 is relevant in differentiated neurons. We here report CAP2 expression during neuron differentiation and its enrichment in growth cones. We therefore hypothesized that CAP2 is relevant not only in excitatory synapses, but also in differentiating neurons. However, CAP2 inactivation neither impaired growth cone morphology and motility nor neuron differentiation. Moreover, CAP2 mutant mice did not display any obvious changes in brain anatomy. Hence, differently from CAP1, CAP2 was dispensable for neuron differentiation and brain development. Interestingly, overexpression of CAP2 rescued not only growth cone size in CAP1-deficient neurons, but also their morphology and differentiation. Our data provide evidence for functional redundancy of CAP1 and CAP2 in differentiating neurons, and they suggest compensatory mechanisms in single mutant neurons.

Endoplasmic Reticulum Stress Induces CAP2 Expression Promoting Epithelial-Mesenchymal Transition in Liver Cancer Cells.

Cyclase-associated protein 2 (CAP2) has been addressed as a candidate biomarker in various cancer types. Previously, we have shown that CAP2 is expressed during multi-step hepatocarcinogenesis; however, its underlying mechanisms in liver cancer cells are not fully elucidated yet. Here, we demonstrated that endoplasmic reticulum (ER) stress induced CAP2 expression, and which promoted migration and invasion of liver cancer cells. We also found that the ER stress-induced CAP2 expression is mediated through activation of protein kinase C epsilon (PKCε) and the promotor binding of activating transcription factor 2 (ATF2). In addition, we further demonstrated that CAP2 expression promoted epithelial-mesenchymal transition (EMT) through activation of Rac1 and ERK. In conclusion, we suggest that ER stress induces CAP2 expression promoting EMT in liver cancers cells. Our results shed light on the novel functions of CAP2 in the metastatic process of liver cancer cells.

Mutual functional dependence of cyclase-associated protein 1 (CAP1) and cofilin1 in neuronal actin dynamics and growth cone function.

Neuron connectivity depends on growth cones that navigate axons through the developing brain. Growth cones protrude and retract actin-rich structures to sense guidance cues. These cues control local actin dynamics and steer growth cones towards attractants and away from repellents, thereby directing axon outgrowth. Hence, actin binding proteins (ABPs) moved into the focus as critical regulators of neuron connectivity. We found cyclase-associated protein 1 (CAP1), an ABP with unknown brain function, abundant in growth cones. Super-resolution microscopy and live cell imaging combined with pharmacological approaches on hippocampal neurons from gene-targeted mice revealed a crucial role for CAP1 in actin dynamics that is critical for growth cone morphology and function. Growth cone defects in CAP1 knockout (KO) neurons compromised neuron differentiation and was associated with impaired neuron connectivity in CAP1-KO brains. Mechanistically, by rescue experiments in double KO neurons lacking CAP1 and the key actin regulator cofilin1, we demonstrated that CAP1 was essential for cofilin1 function in growth cone actin dynamics and morphology and vice versa. Together, we identified CAP1 as a novel actin regulator in growth cones that was relevant for neuron connectivity, and we demonstrated functional interdependence of CAP1 and cofilin1 in neuronal actin dynamics and growth cone function.

Native cyclase-associated protein and actin from Xenopus laevis oocytes form a unique 4:4 complex with a tripartite structure.

Cyclase-associated protein (CAP) is a conserved actin-binding protein that regulates multiple aspects of actin dynamics, including polymerization, depolymerization, filament severing, and nucleotide exchange. CAP has been isolated from different cells and tissues in an equimolar complex with actin, and previous studies have shown that a CAP-actin complex contains six molecules each of CAP and actin. Intriguingly, here, we successfully isolated a complex of Xenopus cyclase-associated protein 1 (XCAP1) with actin from oocyte extracts, which contained only four molecules each of XCAP1 and actin. This XCAP1-actin complex remained stable as a single population of 340 kDa species during hydrodynamic analyses using gel filtration or analytical ultracentrifugation. Examination of the XCAP1-actin complex by high-speed atomic force microscopy revealed a tripartite structure: one middle globular domain and two globular arms. The two arms were observed in high and low states. The arms at the high state were spontaneously converted to the low state by dissociation of actin from the complex. However, when extra G-actin was added, the arms at the low state were converted to the high state. Based on the known structures of the N-terminal helical-folded domain and C-terminal CARP domain, we hypothesize that the middle globular domain corresponds to a tetramer of the N-terminal helical-folded domain of XCAP1 and that each arm in the high state corresponds to a heterotetramer containing a dimer of the C-terminal CARP domain of XCAP1 and two G-actin molecules. This novel configuration of a CAP-actin complex should help to understand how CAP promotes actin filament disassembly.

CAPt'n of Actin Dynamics: Recent Advances in the Molecular, Developmental and Physiological Functions of Cyclase-Associated Protein (CAP).

Cyclase-associated protein (CAP) has been discovered three decades ago in budding yeast as a protein that associates with the cyclic adenosine monophosphate (cAMP)-producing adenylyl cyclase and that suppresses a hyperactive RAS2 variant. Since that time, CAP has been identified in all eukaryotic species examined and it became evident that the activity in RAS-cAMP signaling is restricted to a limited number of species. Instead, its actin binding activity is conserved among eukaryotes and actin cytoskeleton regulation emerged as its primary function. However, for many years, the molecular functions as well as the developmental and physiological relevance of CAP remained unknown. In the present article, we will compile important recent progress on its molecular functions that identified CAP as a novel key regulator of actin dynamics, i.e., the spatiotemporally controlled assembly and disassembly of actin filaments (F-actin). These studies unraveled a cooperation with ADF/Cofilin and Twinfilin in F-actin disassembly, a nucleotide exchange activity on globular actin monomers (G-actin) that is required for F-actin assembly and an inhibitory function towards the F-actin assembly factor INF2. Moreover, by focusing on selected model organisms, we will review current literature on its developmental and physiological functions, and we will present studies implicating CAP in human pathologies. Together, this review article summarizes and discusses recent achievements in understanding the molecular, developmental and physiological functions of CAP, which led this protein emerge as a novel CAPt'n of actin dynamics.