Polyclonal and monoclonal antibodies against purified 32/34-kDa F-actin-capping protein from Dictyostelium discoideum were generated and used to isolate clones coding for both subunits from a lambda gt11 expression library. In addition genomic clones were isolated for the 34-kDa subunit. The sequences of the corresponding inserts were determined and the amino acid sequences of the proteins deduced. The amino acid sequences have been confirmed by sequencing tryptic peptides of both subunits. As judged from immunological data and sequence comparison, the subunits are completely different. Each of them is encoded by a single copy gene. The mRNAs are present throughout all stages of development. Severing proteins, which exert also capping activity, show no sequence similarity with the cap 32/34-kDa protein. However, a pronounced homology between the 32-kDa subunit and a hypothetical yeast protein was found indicating the presence of a protein homologous to the 32-kDa subunit of the D. discoideum capping protein in yeast.

Actin capping proteins belong to the core set of proteins minimally required for actin-based motility and are present in virtually all eukaryotic cells. They bind to the fast-growing barbed end of an actin filament, preventing addition and loss of monomers, thus restricting growth to the slow-growing pointed end. Actin capping proteins are usually heterodimers of two subunits. The Plasmodium orthologs are an exception, as their α subunits are able to form homodimers. We show here that, while the β subunit alone is unstable, the α subunit of the Plasmodium actin capping protein forms functional homo- and heterodimers. This implies independent functions for the αα homo- and αβ heterodimers in certain stages of the parasite life cycle. Structurally, the homodimers resemble canonical αβ heterodimers, although certain rearrangements at the interface must be required. Both homo- and heterodimers bind to actin filaments in a roughly equimolar ratio, indicating they may also bind other sites than barbed ends.

The PIM family kinases promote cancer cell survival and motility as well as metastatic growth in various types of cancer. We have previously identified several PIM substrates, which support cancer cell migration and invasiveness. However, none of them are known to regulate cellular movements by directly interacting with the actin cytoskeleton. Here we have studied the phosphorylation-dependent effects of PIM1 on actin capping proteins, which bind as heterodimers to the fast-growing actin filament ends and stabilize them.

Actin polymerization in cells occurs via filament elongation at the barbed end. Proteins that cap the barbed end terminate this elongation. Heterodimeric capping protein (CP) is an abundant and ubiquitous protein that caps the barbed end. We find that the mouse homolog of the adaptor protein CARMIL (mCARMIL) binds CP with high affinity and decreases its affinity for the barbed end. Addition of mCARMIL to cell extracts increases the rate and extent of Arp2/3 or spectrin-actin seed-induced polymerization. In cells, GFP-mCARMIL concentrates in lamellipodia and increases the fraction of cells with large lamellipodia. Decreasing mCARMIL levels by siRNA transfection lowers the F-actin level and slows cell migration through a mechanism that includes decreased lamellipodia protrusion. This phenotype is reversed by full-length mCARMIL but not mCARMIL lacking the domain that binds CP. Thus, mCARMIL is a key regulator of CP and has profound effects on cell behavior.

Actin arginylation regulates lamella formation in motile fibroblasts, but the underlying molecular mechanisms are unknown. To understand how arginylation affects the actin cytoskeleton, we investigated the biochemical properties and the structural organization of actin filaments in wild-type and arginyltransferase (Ate1) knockout cells. We found that Ate1 knockout results in a dramatic reduction of the actin polymer levels in vivo accompanied by a corresponding increase in the monomer level. Purified nonarginylated actin has altered polymerization properties, and actin filaments from Ate1 knockout cells show altered interactions with several associated proteins. Ate1 knockout cells have severe impairment of cytoskeletal organization throughout the cell. Thus, arginylation regulates the ability of actin to form filaments in the whole cell rather than preventing the collapse of preformed actin networks at the cell leading edge as proposed in our previous model. This regulation is achieved through interconnected mechanisms that involve actin polymerization per se and through binding of actin-associated proteins.

Coactosin, a 16 kDa protein associated with the actin cytoskeleton from Dictyostelium discoideum, was purified by an improved method, in which other components of the cytoskeleton were removed. The highly purified coactosin had no effect on the time course of actin polymerization, but when added to actin in presence of capping proteins, coactosin counteracted the capping activity of these proteins. The capping proteins cap32/34 and severin domain 1 retarded actin polymerization, on addition of coactosin to samples containing one of these capping proteins the time course of actin polymerization became close to controls without capping proteins.

Arp2/3 complex-mediated actin assembly at cell membranes drives the formation of protrusions or endocytic vesicles. To identify the mechanism by which different membrane deformations can be achieved, we reconstitute the basic membrane deformation modes of inward and outward bending in a confined geometry by encapsulating a minimal set of cytoskeletal proteins into giant unilamellar vesicles. Formation of membrane protrusions is favoured at low capping protein (CP) concentrations, whereas the formation of negatively bent domains is promoted at high CP concentrations. Addition of non-muscle myosin II results in full fission events in the vesicle system. The different deformation modes are rationalized by simulations of the underlying transient nature of the reaction kinetics. The relevance of the regulatory mechanism is supported by CP overexpression in mouse melanoma B16-F1 cells and therefore demonstrates the importance of the quantitative understanding of microscopic kinetic balances to address the diverse functionality of the cytoskeleton.

Radiotherapy (RT), is a fundamental treatment for malignant tumors and is used in over half of cancer patients. As radiation can promote anti-tumor immune effects, a promising therapeutic strategy is to combine radiation with immune checkpoint inhibitors (ICIs). However, the genetic determinants that impact therapeutic response in the context of combination therapy with radiation and ICI have not been systematically investigated. To unbiasedly identify the tumor intrinsic genetic factors governing such responses, we perform a set of genome-scale CRISPR screens in melanoma cells for cancer survival in response to low-dose genotoxic radiation treatment, in the context of CD8 T cell co-culture and with anti-PD1 checkpoint blockade antibody. Two actin capping proteins, Capza3 and Capg, emerge as top hits that upon inactivation promote the survival of melanoma cells in such settings. Capza3 and Capg knockouts (KOs) in mouse and human cancer cells display persistent DNA damage due to impaired homology directed repair (HDR); along with increased radiation, chemotherapy, and DNA repair inhibitor sensitivity. However, when cancer cells with these genes inactivated were exposed to sublethal radiation, inactivation of such actin capping protein promotes activation of the STING pathway, induction of inhibitory CEACAM1 ligand expression and resistance to CD8 T cell killing. Patient cancer genomics analysis reveals an increased mutational burden in patients with inactivating mutations in CAPG and/or CAPZA3, at levels comparable to other HDR associated genes. There is also a positive correlation between CAPG expression and activation of immune related pathways and CD8 T cell tumor infiltration. Our results unveil the critical roles of actin binding proteins for efficient HDR within cancer cells and demonstrate a previously unrecognized regulatory mechanism of therapeutic response to radiation and immunotherapy.

How actin structures of distinct identities and functions coexist within the same environment is a critical self-organization question. Fission yeast cells have a simple actin cytoskeleton made of four structures: Arp2/3 assembles actin patches around endocytic pits, and the formins For3, Cdc12, and Fus1 assemble actin cables, the cytokinetic ring during division, and the fusion focus during sexual reproduction, respectively. The focus concentrates the delivery of hydrolases by myosin V to digest the cell wall for cell fusion. We discovered that cells lacking capping protein (CP), a heterodimer that blocks barbed-end dynamics and associates with actin patches, exhibit a delay in fusion. Consistent with CP-formin competition for barbed-end binding, Fus1, F-actin, and the linear filament marker tropomyosin hyper-accumulate at the fusion focus in cells lacking CP. CP deletion also rescues the fusion defect of a mutation in the Fus1 knob region. However, myosin V and exocytic cargoes are reduced at the fusion focus and diverted to ectopic foci, which underlies the fusion defect. Remarkably, the ectopic foci coincide with Arp2/3-assembled actin patches, which now contain low levels of Fus1. We further show that CP localization to actin patches is required to prevent the formation of ectopic foci and promote efficient cell fusion. During mitotic growth, actin patches lacking CP similarly display a dual identity, as they accumulate the formins For3 and Cdc12, normally absent from patches, and are co-decorated by the linear filament-binding protein tropomyosin and the patch marker fimbrin. Thus, CP serves to protect Arp2/3-nucleated structures from formin activity.

Dendritic spines are small actin-rich protrusions essential for the formation of functional circuits in the mammalian brain. During development, spines begin as dynamic filopodia-like protrusions that are then replaced by relatively stable spines containing an expanded head. Remodeling of the actin cytoskeleton plays a key role in the formation and modification of spine morphology, however many of the underlying regulatory mechanisms remain unclear. Capping protein (CP) is a major actin regulating protein that caps the barbed ends of actin filaments, and promotes the formation of dense branched actin networks. Knockdown of CP impairs the formation of mature spines, leading to an increase in the number of filopodia-like protrusions and defects in synaptic transmission. Here, we show that CP promotes the stabilization of dendritic protrusions, leading to the formation of stable mature spines. However, the localization and function of CP in dendritic spines requires interactions with proteins containing a capping protein interaction (CPI) motif. We found that the CPI motif-containing protein Twinfilin-1 (Twf1) also localizes to spines where it plays a role in CP spine enrichment. The knockdown of Twf1 leads to an increase in the density of filopodia-like protrusions and a decrease in the stability of dendritic protrusions, similar to CP knockdown. Finally, we show that CP directly interacts with Shank and regulates its spine accumulation. These results suggest that spatiotemporal regulation of CP in spines not only controls the actin dynamics underlying the formation of stable postsynaptic spine structures, but also plays an important role in the assembly of the postsynaptic apparatus underlying synaptic function.

Cell processes require precise regulation of actin polymerization at filament plus ends to execute normal functions. The detailed mechanisms used to control filament assembly at plus ends in the presence of diverse and often opposing regulators is not clear. Here we explore and identify residues important for the plus-end related activities of IQGAP1. In multi-wavelength TIRF assays, we directly visualize dimers of IQGAP1, mDia1, and CP on filament ends alone and as a multicomponent end binding complex. IQGAP1 promotes the turnover of end-binding proteins reducing the dwell times of CP, mDia1, or mDia1-CP 'decision complexes' by 8-18-fold. Loss of these activities in cells disrupts actin filament arrays, morphology, and migration. Together, our results reveal a role for IQGAP1 in promoting protein turnover on filament ends and provide new insights into how actin assembly is regulated in cells.

Alpha-adducin (Add1) is a critical component of the actin-spectrin network in erythrocytes, acting to cap the fast-growing, barbed ends of actin filaments, and recruiting spectrin to these junctions. Add1 is highly expressed in T cells, but its role in T-cell activation has not been examined. Using a conditional knockout model, we show that Add1 is necessary for complete activation of CD4+ T cells in response to low levels of antigen but is dispensable for CD8+ T cell activation and response to infection. Surprisingly, costimulatory signals through CD28 were completely abrogated in the absence of Add1. This study is the first to examine the role of actin-capping in T cells, and it reveals a previously unappreciated role for the actin cytoskeleton in regulating costimulation.

The actin capping protein (CP) tightly binds to the barbed end of actin filaments, thus playing a key role in actin-based lamellipodial dynamics. V-1 and CARMIL proteins directly bind to CP and inhibit the filament capping activity of CP. V-1 completely inhibits CP from interacting with the barbed end, whereas CARMIL proteins act on the barbed end-bound CP and facilitate its dissociation from the filament (called uncapping activity). Previous studies have revealed the striking functional differences between the two regulators. However, the molecular mechanisms describing how these proteins inhibit CP remains poorly understood. Here we present the crystal structures of CP complexed with V-1 and with peptides derived from the CP-binding motif of CARMIL proteins (CARMIL, CD2AP, and CKIP-1). V-1 directly interacts with the primary actin binding surface of CP, the C-terminal region of the alpha-subunit. Unexpectedly, the structures clearly revealed the conformational flexibility of CP, which can be attributed to a twisting movement between the two domains. CARMIL peptides in an extended conformation interact simultaneously with the two CP domains. In contrast to V-1, the peptides do not directly compete with the barbed end for the binding surface on CP. Biochemical assays revealed that the peptides suppress the interaction between CP and V-1, despite the two inhibitors not competing for the same binding site on CP. Furthermore, a computational analysis using the elastic network model indicates that the interaction of the peptides alters the intrinsic fluctuations of CP. Our results demonstrate that V-1 completely sequesters CP from the barbed end by simple steric hindrance. By contrast, CARMIL proteins allosterically inhibit CP, which appears to be a prerequisite for the uncapping activity. Our data suggest that CARMIL proteins down-regulate CP by affecting its conformational dynamics. This conceptually new mechanism of CP inhibition provides a structural basis for the regulation of the barbed end elongation in cells.

Cell locomotion requires rapid growth of cortical actin filaments whose barbed ends are capped in the resting cell. Phosphatidylinositol-4,5-bisphosphate (PIP2) may play a critical role as an intracellular messenger in cytoskeletal rearrangement after stimulation. We have examined the effects of PIP2 micelles on the Ca2+-independent actin filament capping activity in high speed supernatants of neutrophil lysates which we had previously demonstrated to be almost entirely due to capping protein-beta2, a homologue of cap Z. High concentrations of PIP2 totally prevented the capping of exogenous spectrin-F-actin seeds by dilute supernatants of neutrophil extracts. Capping could also be inhibited, albeit less effectively, by PIP and PI, but not by other phospholipids. When incubated with filaments in the absence of supernatant, PIP2 increased the number of growing ends. PIP2 also uncapped previously capped actin filaments, as demonstrated by incubating supernatant-capped and uncapped seeds with and without PIP2 and then comparing the initial elongation rates after addition of pyrenyl-G-actin. Incubation of capped seeds with high concentrations of PIP2 increased the number of free barbed ends to a level comparable to that of the uncapped seeds exposed to PIP2. PIP2 caused uncapping to occur too quickly to be explained simply by the off-rate of capping protein-beta2, implying that PIP2 interacted directly with capping protein on the filament ends. In fact, PIP2 transiently uncapped capped seeds in the presence of excess free capping protein. From our data, we estimate that millimolar concentrations of PIP2 (almost 100-fold higher than the amount predicted from the effective concentration in purified systems) would be required to inhibit all the capping protein-beta2 in the cytosol. This discrepancy probably results, in large part, from sequestration of PIP2 by other PIP2-binding proteins in the cytoplasm. If PIP2 mediates differential cytoskeletal growth after chemoattractant stimulation in vivo, very high concentrations may be required subjacent to the plasma membrane for regional severing and uncapping of actin filaments to occur quickly near the perturbed membrane.

Actin-based remodelling underlies spine structural changes occurring during synaptic plasticity, the process that constantly reshapes the circuitry of the adult brain in response to external stimuli, leading to learning and memory formation. A positive correlation exists between spine shape and synaptic strength and, consistently, abnormalities in spine number and morphology have been described in a number of neurological disorders. In the present study, we demonstrate that the actin-regulating protein, Eps8, is recruited to the spine head during chemically induced long-term potentiation in culture and that inhibition of its actin-capping activity impairs spine enlargement and plasticity. Accordingly, mice lacking Eps8 display immature spines, which are unable to undergo potentiation, and are impaired in cognitive functions. Additionally, we found that reduction in the levels of Eps8 occurs in brains of patients affected by autism compared to controls. Our data reveal the key role of Eps8 actin-capping activity in spine morphogenesis and plasticity and indicate that reductions in actin-capping proteins may characterize forms of intellectual disabilities associated with spine defects.

The interaction of capping protein (CP) with actin filaments is an essential element of actin assembly and actin-based motility in nearly all eukaryotes. The dendritic nucleation model for Arp2/3-based lamellipodial assembly features capping of barbed ends by CP, and the formation of filopodia is proposed to involve inhibition of capping by formins and other proteins. To understand the molecular basis for how CP binds the barbed end of the actin filament, we have used a combination of computational and experimental approaches, primarily involving molecular docking and site-directed mutagenesis. We arrive at a model that supports all of our biochemical data and agrees very well with a cryo-electron microscopy structure of the capped filament. CP interacts with both actin protomers at the barbed end of the filament, and the amphipathic helix at the C-terminus of the β-subunit binds to the hydrophobic cleft on actin, in a manner similar to that of WH2 domains. These studies provide us with new molecular insight into how CP binds to the actin filament.

Actin works in concert with myosin I to regulate the transcription of ribosomal genes in the nucleolus. Recently, nucleolar actin has been shown to be active in its polymeric form raising the question how actin dynamics is regulated in the nucleolus. Here, we show that the actin capping protein CapG localizes in the nucleolus of cultured cells. CapG transport to the nucleolus is an active and ATP-dependent process. Association of CapG with the nucleolus requires active RNA Polymerase I transcription. In addition, we show that activated Ran GTPase, an interaction partner of CapG, is also transported to the nucleolus. A constitutively active Ran mutant promotes CapG accumulation in the nucleolus indicating that CapG transport to the nucleolus can be supported by Ran. Our results suggest that filamentous actin in the nucleolus might be regulated by actin binding proteins such as CapG.

Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s(-1), respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays.

Human colorectal cancer (CRC) is the third most common cancer; patients with metastatic colorectal cancer (mCRC) show poor prognosis than those with CRC cases. There are no reliable molecular biomarkers for the diagnosis of CRC prognosis except with pathological features. Therefore, it is urgent to develop a biomarker for diagnosis and/or prediction of human CRC. In addition, capping actin protein (CapG) belongs to the gelsolin family and has been reported to contribute on tumor invasion/metastasis in multiple human cancers. Here, we are the first to evaluate the expression of CapG in human CRCs.

The actin-Capping Protein heterodimer, composed of the α and β subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and β subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth.