Coordination of actin plus-end dynamics by IQGAP1, formin, and capping protein.

Cell processes require precise regulation of actin polymerization that is mediated by plus-end regulatory proteins. Detailed mechanisms that explain plus-end dynamics involve regulators with opposing roles, including factors that enhance assembly, e.g., the formin mDia1, and others that stop growth (capping protein, CP). We explore IQGAP1's roles in regulating actin filament plus-ends and the consequences of perturbing its activity in cells. We confirm that IQGAP1 pauses elongation and interacts with plus ends through two residues (C756 and C781). We directly visualize the dynamic interplay between IQGAP1 and mDia1, revealing that IQGAP1 displaces the formin to influence actin assembly. Using four-color TIRF, we show that IQGAP1's displacement activity extends to formin-CP "decision complexes," promoting end-binding protein turnover at plus-ends. Loss of IQGAP1 or its plus-end activities disrupts morphology and migration, emphasizing its essential role. These results reveal a new role for IQGAP1 in promoting protein turnover on filament ends and provide new insights into how plus-end actin assembly is regulated in cells.

Capping protein regulators of actin assembly in budding yeast.

Cellular functions of actin capping protein (CP) regulators are poorly understood. Di Pietro and colleagues (https://doi.org/10.1083/jcb.202306154) shed unprecedented light on this topic using budding yeast. Two proteins with CPI (capping protein interacting) motifs recruit CP to sites of actin assembly, while a third contributes to CP turnover.

Transcriptional Profiling of the Candida albicans Response to the DNA Damage Agent Methyl Methanesulfonate.

The infection of a mammalian host by the pathogenic fungus Candida albicans involves fungal resistance to reactive oxygen species (ROS)-induced DNA damage stress generated by the defending macrophages or neutrophils. Thus, the DNA damage response in C. albicans may contribute to its pathogenicity. Uncovering the transcriptional changes triggered by the DNA damage-inducing agent MMS in many model organisms has enhanced the understanding of their DNA damage response processes. However, the transcriptional regulation triggered by MMS remains unclear in C. albicans. Here, we explored the global transcription profile in response to MMS in C. albicans and identified 306 defined genes whose transcription was significantly affected by MMS. Only a few MMS-responsive genes, such as MGT1, DDR48, MAG1, and RAD7, showed potential roles in DNA repair. GO term analysis revealed that a large number of induced genes were involved in antioxidation responses, and some downregulated genes were involved in nucleosome packing and IMP biosynthesis. Nevertheless, phenotypic assays revealed that MMS-induced antioxidation gene CAP1 and glutathione metabolism genes GST2 and GST3 showed no direct roles in MMS resistance. Furthermore, the altered transcription of several MMS-responsive genes exhibited RAD53-related regulation. Intriguingly, the transcription profile in response to MMS in C. albicans shared a limited similarity with the pattern in S. cerevisiae, including COX17, PRI2, and MGT1. Overall, C. albicans cells exhibit global transcriptional changes to the DNA damage agent MMS; these findings improve our understanding of this pathogen's DNA damage response pathways.

Gene co-expression network analysis reveals the positive impact of endocytosis and mitochondria-related genes over nitrogen metabolism in Saccharomyces cerevisiae.

Nitrogen metabolism is essential for most cellular activities. Therefore, a deep understanding of its regulatory mechanisms is necessary for the efficient utilization of nitrogen sources for Saccharomyces cerevisiae. In this study, a gene co-expression network was constructed for S. cerevisiae S288C with different nitrogen sources. From this, a key gene co-expression module related to nitrogen source preference utilization was obtained, and 10 hub genes centrally located in the co-expression network were identified. Functional studies verified that the endocytosis-related genes CAP1 and END3 significantly increased the utilization of multiple non-preferred amino acids and reduced the accumulation of the harmful nitrogen metabolite precursor urea by regulating amino acid transporters and TOR pathway. The mitochondria-related gene ATP12, MRPL22, MRP1 and NAM9 significantly increased the utilization of multiple non-preferred amino acids and reduced accumulation of the urea by coordinately regulating nitrogen catabolism repression, Ssy1p-Ptr3p-Ssy5p signaling sensor system, amino acid transporters, TOR pathway and urea metabolism-related pathways. Furthermore, these data revealed the potential positive effects of endocytosis and mitochondrial ribosomes protein translation on nitrogen source preference. This study provides new analytical perspectives for complex regulatory networks involving nitrogen metabolism in S. cerevisiae.

A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks.

Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP's C-terminal "tentacle" extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the ß tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes-capping and nucleation-in branched actin network assembly.

In vivo proximity proteomics of nascent synapses reveals a novel regulator of cytoskeleton-mediated synaptic maturation.

Excitatory synapse formation during development involves the complex orchestration of both structural and functional alterations at the postsynapse. However, the molecular mechanisms that underlie excitatory synaptogenesis are only partially resolved, in part because the internal machinery of developing synapses is largely unknown. To address this, we apply a chemicogenetic approach, in vivo biotin identification (iBioID), to discover aspects of the proteome of nascent synapses. This approach uncovered sixty proteins, including a previously uncharacterized protein, CARMIL3, which interacts in vivo with the synaptic cytoskeletal regulator proteins SrGAP3 (or WRP) and actin capping protein. Using new CRISPR-based approaches, we validate that endogenous CARMIL3 is localized to developing synapses where it facilitates the recruitment of capping protein and is required for spine structural maturation and AMPAR recruitment associated with synapse unsilencing. Together these proteomic and functional studies reveal a previously unknown mechanism important for excitatory synapse development in the developing perinatal brain.

Capping Actin Protein Overexpression in Human Colorectal Carcinoma and Its Contributed Tumor Migration.

OBJECTIVE: 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. STUDY DESIGN: To investigate the expression levels of CapG in human tissue array by immunohistochemistry (IHC) staining. Moreover, the mRNA and protein levels were also confirmed in four CRC cell lines and determined using real-time RT-PCR and Western blotting. Finally, a Matrigel transwell invasion assay was used to evaluate the invasion ability in CapG high or low expression cells. RESULTS: We demonstrated that CapG could be determined in the normal colon tissue and human CRC specimens. However, CapG was significantly overexpressed in the mCRC specimens compared with that in CRC specimens and normal cases. It was also detectable in the four CRC cell lines including mRNA and protein levels. We also found that knockdown of the expression of CapG reduced tumor migration. CONCLUSIONS: In this study, we suggested that CapG could be used as a biomarker for metastatic CRC in the clinical specimens. Moreover, our in vitro study demonstrated that CapG might contribute on tumor metastasis in human CRCs.

Structural basis of actin monomer re-charging by cyclase-associated protein.

Actin polymerization powers key cellular processes, including motility, morphogenesis, and endocytosis. The actin turnover cycle depends critically on "re-charging" of ADP-actin monomers with ATP, but whether this reaction requires dedicated proteins in cells, and the underlying mechanism, have remained elusive. Here we report that nucleotide exchange catalyzed by the ubiquitous cytoskeletal regulator cyclase-associated protein (CAP) is critical for actin-based processes in vivo. We determine the structure of the CAP-actin complex, which reveals that nucleotide exchange occurs in a compact, sandwich-like complex formed between the dimeric actin-binding domain of CAP and two ADP-actin monomers. In the crystal structure, the C-terminal tail of CAP associates with the nucleotide-sensing region of actin, and this interaction is required for rapid re-charging of actin by both yeast and mammalian CAPs. These data uncover the conserved structural basis and biological role of protein-catalyzed re-charging of actin monomers.

Regulation of IL-1 signaling through control of focal adhesion assembly.

IL-1 signaling is adhesion-restricted in many cell types, but the mechanism that drives it is not defined. We screened for proteins recruited to nascent adhesions in IL-1-treated human fibroblasts with tandem mass tag-mass spectrometry. We used fibronectin bead preparations to enrich 10 actin-associated proteins. There was a 1.2 times log 2-fold enrichment of actin capping protein (ACP) at 30 min after IL-1 stimulation. Knockdown (KD) of ACP by siRNA reduced IL-1-induced ERK activation(by 56%, matrix metalloproteinase-3 (MMP-3) expression by 48%, and MMP-9 expression by 62% (in all reductions, P < 0.01). Confocal or structured illumination microscopy showed that ACP was diffused throughout the cytosol but strongly accumulated at the ruffled border of spreading cells. ACP colocalized with nascent paxillin- and vinculin-containing adhesions at the ruffled border, but not with mature adhesions in the center. ACP KD promoted the formation of large, stable adhesions. Immunoprecipitation and proximity ligation analysis showed that ACP was associated with the IL-1 signal transduction proteins myeloid differentiation factor 88 (MyD88) and IL-1 receptor-associated kinase (IRAK) at the ruffled border of the leading edge. IL-1-induced phospho-ERK and MyD88 or IRAK colocalized at the leading edge. We concluded that ACP is required for recruitment and function of IL-1 signaling complexes in nascent adhesions at the leading edge of the cell.-Wang, Q., Delcorde, J., Tang, T., Downey, G. P., McCulloch, C. A. Regulation of IL-1 signaling through control of focal adhesion assembly.

Yeast Aim21/Tda2 both regulates free actin by reducing barbed end assembly and forms a complex with Cap1/Cap2 to balance actin assembly between patches and cables.

Yeast Aim21 is recruited by the SH3-containing proteins Bbc1 and Abp1 to patches and, with Tda2, reduces barbed end assembly to balance the distribution of actin between patches and cables. Aim21/Tda2 also interacts with Cap1/Cap2, revealing a complex interplay between actin assembly regulators.

MoCAP proteins regulated by MoArk1-mediated phosphorylation coordinate endocytosis and actin dynamics to govern development and virulence of Magnaporthe oryzae.

Actin organization is a conserved cellular process that regulates the growth and development of eukaryotic cells. It also governs the virulence process of pathogenic fungi, such as the rice blast fungus Magnaporthe oryzae, with mechanisms not yet fully understood. In a previous study, we found that actin-regulating kinase MoArk1 displays conserved functions important in endocytosis and actin organization, and MoArk1 is required for maintaining the growth and full virulence of M. oryzae. To understand how MoArk1 might function, we identified capping protein homologs from M. oryzae (MoCAP) that interact with MoArk1 in vivo. MoCAP is heterodimer consisting of α and ß subunits MoCapA and MoCapB. Single and double deletions of MoCAP subunits resulted in abnormal mycelial growth and conidia formation. The ΔMocap mutants also exhibited reduced appressorium penetration and invasive hyphal growth within host cells. Furthermore, the ΔMocap mutants exhibited delayed endocytosis and abnormal cytoskeleton assembly. Consistent with above findings, MoCAP proteins interacted with MoAct1, co-localized with actin during mycelial development, and participated in appressorial actin ring formation. Further analysis revealed that the S85 residue of MoCapA and the S285 residue of MoCapB were subject to phosphorylation by MoArk1 that negatively regulates MoCAP functions. Finally, the addition of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) failed to modulate actin ring formation in ΔMocap mutants, in contrast to the wild-type strain, suggesting that MoCAP may also mediate phospholipid signaling in the regulation of the actin organization. These results together demonstrate that MoCAP proteins whose functions are regulated by MoArk1 and PIP2 are important for endocytosis and actin dynamics that are directly linked to growth, conidiation and pathogenicity of M. oryzae.

Vaccination with toxofilin DNA in combination with an alum-monophosphoryl lipid A mixed adjuvant induces significant protective immunity against Toxoplasma gondii.

BACKGROUND: A widely prevalent disease, toxoplasmosis poses serious health threats to both humans and animals; therefore, development of an ideal DNA vaccine against Toxoplasma gondii is needed eagerly. The purpose of the present study is to assess the protective efficacy of a DNA vaccine encoding the T. gondii toxofilin gene (pEGFP-toxofilin). In addition, toxofilin DNA vaccine combined with the individual adjuvants, alum or monophosphoryl lipid A (MPLA), or a mixture of alum-MPLA adjuvant were screened for their ability to enhance antibody responses. METHODS: Using bioinformatics, we analyzed the gene and amino acid sequences of the toxofilin protein, recognizing and identifying several potential linear B and T helper (Th)-1 cell epitopes. BALB/c mice were immunized three times with either toxofilin DNA vaccine alone or in combination with the adjuvants such as alum, MPLA or an alum-MPLA mixture. The systemic immune response was evaluated by cytokine, the percentage of CD4 (+) and CD8 (+) T cells and specific antibody measurement. Two weeks after the last immunization, protective efficacy was evaluated by challenging intraperitoneally with 1 × 104 tachyzoites of T. gondii or intragastrically with 20 cysts of T. gondii PRU strain. RESULTS: All experimentally immunized mice developed strong humoral and cellular immune responses compared with the control groups. Moreover, by comparison with the non-adjuvant toxofilin DNA vaccine group, adding alum adjuvant to toxofilin DNA vaccine resulted in an increase in humoral response and a skewed Th2 response. However, the MPLA adjuvant with toxofilin DNA vaccine induced significantly enhanced humoral and Th1-biased immune responses. Importantly, the co-administration of alum-MPLA adjuvant in combination with the toxofilin DNA vaccine shifted the Th2 immune response to a Th1 response compared with the alum-toxofilin group, and elicited the strongest humoral and Th1 responses among all the groups. At the same time, a longer survival time and less cyst amounts against T. gondii infection were also observed in the alum-MPLA-toxofilin group in comparison with single or no adjuvant groups. CONCLUSIONS: Toxoplasma gondii toxofilin is a promising vaccine candidate that warrants further development. Co-administration of the alum-MPLA adjuvant mixture with DNA vaccine could effectively enhance immunogenicity and strongly skew the cellular immune response towards a Th1 phenotype.

V-1 regulates capping protein activity in vivo.

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."

The F-actin capping protein is required for hyphal growth and full virulence but is dispensable for septum formation in Botrytis cinerea.

Filamentous (F-) actin is an integral part of the cytoskeleton allowing for cell growth, intracellular motility, and cytokinesis of eukaryotic cells. Its assembly from G-actin monomers and its disassembly are tightly regulated processes involving a number of actin-binding proteins (ABPs) such as F-actin nucleators and cross-linking proteins. F-actin capping protein (CP) is an alpha/beta heterodimer known from yeast and higher eukaryotes to bind to the fast growing ends of the actin filaments stabilizing them. In this study, we identified the orthologs of the two CP subunits, named BcCPA1 and BcCPB1, in the plant pathogenic fungus Botrytis cinerea and showed that the two proteins physically interact in a yeast two-hybrid approach. GFP-BcCPA1 fusion proteins were functional and localized to the assumed sites of F-actin accumulation, i.e. to the hyphal tips and the sites of actin ring formation. Deletion of bccpa1 had a profound effect on hyphal growth, morphogenesis, and virulence indicating the importance of F-actin capping for an intact actin cytoskeleton. As polarized growth - unlike septum formation - is impaired in the mutants, it can be concluded that the organization and/or localization of actin patches and cables are disturbed rather than the functionality of the actin rings.

TOR complex 2 localises to the cytokinetic actomyosin ring and controls the fidelity of cytokinesis.

The timing of cell division is controlled by the coupled regulation of growth and division. The target of rapamycin (TOR) signalling network synchronises these processes with the environmental setting. Here, we describe a novel interaction of the fission yeast TOR complex 2 (TORC2) with the cytokinetic actomyosin ring (CAR), and a novel role for TORC2 in regulating the timing and fidelity of cytokinesis. Disruption of TORC2 or its localisation results in defects in CAR morphology and constriction. We provide evidence that the myosin II protein Myp2 and the myosin V protein Myo51 play roles in recruiting TORC2 to the CAR. We show that Myp2 and TORC2 are co-dependent upon each other for their normal localisation to the cytokinetic machinery. We go on to show that TORC2-dependent phosphorylation of actin-capping protein 1 (Acp1, a known regulator of cytokinesis) controls CAR stability, modulates Acp1-Acp2 (the equivalent of the mammalian CAPZA-CAPZB) heterodimer formation and is essential for survival upon stress. Thus, TORC2 localisation to the CAR, and TORC2-dependent Acp1 phosphorylation contributes to timely control and the fidelity of cytokinesis and cell division.

CARMIL2 is a novel molecular connection between vimentin and actin essential for cell migration and invadopodia formation.

Cancer cell migration requires the regulation of actin networks at protrusions associated with invadopodia and other leading edges. Carcinomas become invasive after undergoing an epithelial-mesenchymal transition characterized by the appearance of vimentin filaments. While vimentin expression correlates with cell migration, the molecular connections between vimentin- and actin-based membrane protrusions are not understood. We report here that CARMIL2 (capping protein, Arp2/3, myosin-I linker 2) provides such a molecular link. CARMIL2 localizes to vimentin, regulates actin capping protein (CP), and binds to membranes. CARMIL2 is necessary for invadopodia formation, as well as cell polarity, lamellipodial assembly, membrane ruffling, macropinocytosis, and collective cell migration. Using point mutants and chimeras with defined biochemical and cellular properties, we discovered that localization to vimentin and CP binding are both essential for the function of CARMIL2 in cells. On the basis of these results, we propose a model in which dynamic vimentin filaments target CARMIL2 to critical membrane-associated locations, where CARMIL2 regulates CP, and thus actin assembly, to create cell protrusions.

CPI motif interaction is necessary for capping protein function in cells.

Capping protein (CP) has critical roles in actin assembly in vivo and in vitro. CP binds with high affinity to the barbed end of actin filaments, blocking the addition and loss of actin subunits. Heretofore, models for actin assembly in cells generally assumed that CP is constitutively active, diffusing freely to find and cap barbed ends. However, CP can be regulated by binding of the 'capping protein interaction' (CPI) motif, found in a diverse and otherwise unrelated set of proteins that decreases, but does not abolish, the actin-capping activity of CP and promotes uncapping in biochemical experiments. Here, we report that CP localization and the ability of CP to function in cells requires interaction with a CPI-motif-containing protein. Our discovery shows that cells target and/or modulate the capping activity of CP via CPI motif interactions in order for CP to localize and function in cells.

The effect of toxofilin on the structure and dynamics of monomeric actin.

The effects of toxofilin (an actin binding protein of Toxoplasma gondii) on G-actin was studied with spectroscopy techniques. Fluorescence anisotropy measurements proved that G-actin and toxofilin interact with 2:1 stoichiometry. The affinity of toxofilin to actin was also determined with a fluorescence anisotropy assay. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of toxofilin. The results can be explained by the shift of the nucleotide binding cleft into a closed conformational state. Differential scanning calorimetry measurements revealed that actin monomers become thermodynamically more stable due to the binding of toxofilin.

Actin-capping proteins play essential roles in the asymmetric division of maturing mouse oocytes.

Actin polymerization is essential for various stages of mammalian oocyte maturation, including spindle migration, actin cap formation, polar body extrusion and cytokinesis. The heterodimeric actin-capping protein is an essential element of the actin cytoskeleton. It binds to the fast-growing (barbed) ends of actin filaments and plays essential roles in various actin-mediated cellular processes. However, the roles of capping protein in mammalian oocyte maturation are poorly understood. We investigated the roles of capping protein in mouse oocytes and found that it is essential for correct asymmetric spindle migration and polar body extrusion. Capping protein mainly localized in the cytoplasm during maturation. By knocking down or ectopically overexpressing this protein, we revealed that it is crucial for efficient spindle migration and maintenance of the cytoplasmic actin mesh density. Expression of the capping-protein-binding region of CARMIL (also known as LRRC16A) impaired spindle migration and polar body extrusion during oocyte maturation and decreased the density of the cytoplasmic actin mesh. Taken together, these findings show that capping protein is an essential component of the actin cytoskeleton machinery that plays crucial roles in oocyte maturation, presumably by controlling the cytoplasmic actin mesh density.