Actin and Actin-Binding Proteins.
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ABSTRACT:
SUBMITTER: Pollard TD
PROVIDER: S-EPMC4968159 | biostudies-literature | 2016 Aug
REPOSITORIES: biostudies-literature
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Project description:We modified the actin gene in budding yeast and analyzed the cellular phenotypes of this modification. One important control is to verify the expression of actin binding proteins in these strains.
2021-11-25 | GSE189312 | GEO
Project description:BackgroundFilamin (FLN) and non-muscle alpha-actinin are members of a family of F-actin cross-linking proteins that utilize Calponin Homology domains (CH-domain) for actin binding. Although these two proteins have been extensively characterized, little is known about what regulates their binding to F-actin filaments in the cell.ResultsWe have constructed fusion proteins consisting of green fluorescent protein (GFP) with either the entire cross-linking protein or its actin-binding domain (ABD) and examined the localization of these fluorescent proteins in living cells under a variety of conditions. The full-length fusion proteins, but not the ABD's complemented the defects of cells lacking both endogenous proteins indicating that they are functional. The localization patterns of filamin (GFP-FLN) and alpha-actinin (GFP-alphaA) were overlapping but distinct. GFP-FLN localized to the peripheral cell cortex as well as to new pseudopods of unpolarized cells, but was observed to localize to the rear of polarized cells during cAMP and folate chemotaxis. GFP-alphaA was enriched in new pseudopods and at the front of polarized cells, but in all cases was absent from the peripheral cortex. Although both proteins appear to be involved in macropinocytosis, the association time of the GFP-probes with the internalized macropinosome differed. Surprisingly, the localization of the GFP-actin-binding domain fusion proteins precisely reflected that of their respective full length constructs, indicating that the localization of the protein was determined by the actin-binding domain alone. When expressed in a cell line lacking both filamin and alpha-actinin, the probes maintain their distinct localization patterns suggesting that they are not functionally redundant.ConclusionThese observations strongly suggest that the regulation of the binding of these proteins to actin filaments is built into the actin-binding domains. We suggest that different actin binding domains have different affinities for F-actin filaments in functionally distinct regions of the cytoskeleton.
| S-EPMC2275727 | biostudies-literature
Project description:The actin cytoskeleton plays a crucial role in many cellular processes while its reorganization is important in maintaining cell homeostasis. However, in the case of cancer cells, actin and ABPs (actin-binding proteins) are involved in all stages of carcinogenesis. Literature has reported that ABPs such as SATB1 (special AT-rich binding protein 1), WASP (Wiskott-Aldrich syndrome protein), nesprin, and villin take part in the initial step of carcinogenesis by regulating oncogene expression. Additionally, changes in actin localization promote cell proliferation by inhibiting apoptosis (SATB1). In turn, migration and invasion of cancer cells are based on the formation of actin-rich protrusions (Arp2/3 complex, filamin A, fascin, ?-actinin, and cofilin). Importantly, more and more scientists suggest that microfilaments together with the associated proteins mediate tumor vascularization. Hence, the presented article aims to summarize literature reports in the context of the potential role of actin and ABPs in all steps of carcinogenesis.
| S-EPMC7600575 | biostudies-literature
Project description:The assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1-CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells. We demonstrate that the binding kinetics of CH1-CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs and other binding proteins. These findings suggest that conformational changes of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop.
| S-EPMC7688639 | biostudies-literature
Project description:Actin binding proteins expression levels when modifying the actin sequence
| PRJNA782536 | ENA
Project description:Actin-binding proteins (ABPs) regulate the assembly of actin filaments (F-actin) into networks and bundles that provide the structural integrity of the cell. Two of these ABPs, filamin and alpha-actinin, have been extensively used to model the mechanical properties of actin networks grown in vitro; however, there is a lack in the understanding of how the molecular interactions between ABPs and F-actin regulate the dynamic properties of the cytoskeleton. Here, we present a native-like assay geometry to test the rupture force of a complex formed by an ABP linking two quasiparallel actin filaments. We readily demonstrate the adaptability of this assay by testing it with two different ABPs: filamin and alpha-actinin. For filamin/actin and alpha-actinin/actin, we measured similar rupture forces of 40-80 pN for loading rates between 4 and 50 pN/s. Both ABP unfolding and conformational transition events were observed, demonstrating that both are important and may be a significant mechanism for the temporal regulation of the mechanical properties of the actin cytoskeleton. With this modular, single-molecule assay, a wide range of ABP/actin interactions can be studied to better understand cytoskeletal and cell dynamics.
| S-EPMC2453742 | biostudies-literature
Project description:Understanding how numerous actin-binding proteins (ABPs) work in concert to control the assembly, organization, and turnover of the actin cytoskeleton requires quantitative information about the levels of each component. Here, we measured the cellular concentrations of actin and the majority of the conserved ABPs in Saccharomyces cerevisiae, as well as the free (cytosolic) fractions of each ABP. The cellular concentration of actin is estimated to be 13.2 µM, with approximately two-thirds in the F-actin form and one-third in the G-actin form. Cellular concentrations of ABPs range from 12.4 to 0.85 µM (Tpm1> Pfy1> Cof1> Abp1> Srv2> Abp140> Tpm2> Aip1> Cap1/2> Crn1> Sac6> Twf1> Arp2/3> Scp1). The cytosolic fractions of all ABPs are unexpectedly high (0.6-0.9) and remain so throughout the cell cycle. Based on these numbers, we speculate that F-actin binding sites are limited in vivo, which leads to high cytosolic levels of ABPs, and in turn helps drive the rapid assembly and turnover of cellular F-actin structures.
| S-EPMC10558290 | biostudies-literature
Project description:Actin is the major protein constituent of the cytoskeleton that performs wide range of cellular functions. It exists in monomeric and filamentous forms, dynamics of which is regulated by a large repertoire of actin binding proteins. However, not much was known about existence of these proteins in trypanosomatids, till the genome sequence data of three important organisms of this class, viz. Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, became available. Here, we have reviewed most of the findings reported to date on the intracellular distribution, structure and functions of these proteins and based on them, we have hypothesized some of their functions. The major findings are as follows: (1) All the three organisms encode at least a set of ten actin binding proteins (profilin, twinfilin, ADF/cofilin, CAP/srv2, CAPz, coronin, two myosins, two formins) and one isoform of actin, except that T. cruzi encodes for three formins and several myosins along with four actins. (2) Actin 1 and a few actin binding proteins (ADF/cofilin, profilin, twinfilin, coronin and myosin13 in L. donovani; ADF/cofilin, profilin and myosin1 in T. brucei; profilin and myosin-F in T.cruzi) have been identified and characterized. (3) In all the three organisms, actin cytoskeleton has been shown to regulate endocytosis and intracellular trafficking. (4) Leishmania actin1 has been the most characterized protein among trypanosomatid actins. (5) This protein is localized to the cytoplasm as well as in the flagellum, nucleus and kinetoplast, and in vitro, it binds to DNA and displays scDNA relaxing and kDNA nicking activities. (6) The pure protein prefers to form bundles instead of thin filaments, and does not bind DNase1 or phalloidin. (7) Myosin13, myosin1 and myosin-F regulate endocytosis and intracellular trafficking, respectively, in Leishmania, T. brucei and T. cruzi. (8) Actin-dependent myosin13 motor is involved in dynamics and assembly of Leishmania flagellum. (9) Leishmania twinfilin localizes mostly to the nucleolus and coordinates karyokinesis by effecting splindle elongation and DNA synthesis. (10) Leishmania coronin binds and promotes actin filament formation and exists in tetrameric form rather than trimeric form, like other coronins. (11) Trypanosomatid profilins are essential for survival of all the three parasites.
| S-EPMC7581878 | biostudies-literature
Project description:Cell migration plays a vital role in both health and disease. It is driven by reorganization of the actin cytoskeleton, which is regulated by actin-binding proteins cofilin and profilin. Stress-inducible phosphoprotein 1 (STIP1) is a well-described co-chaperone of the Hsp90 chaperone system, and our findings identify a potential regulatory role of STIP1 in actin dynamics. We show that STIP1 can be isolated in complex with actin and Hsp90 from HEK293T cells and directly interacts with actin in vitro via the C-terminal TPR2AB-DP2 domain of STIP1, potentially due to a region spanning two putative actin-binding motifs. We found that STIP1 could stimulate the in vitro ATPase activity of actin, suggesting a potential role in the modulation of F-actin formation. Interestingly, while STIP1 depletion in HEK293T cells had no major effect on total actin levels, it led to increased nuclear accumulation of actin, disorganization of F-actin structures, and an increase and decrease in cofilin and profilin levels, respectively. This study suggests that STIP1 regulates the cytoskeleton by interacting with actin, or via regulating the ratio of proteins known to affect actin dynamics.
| S-EPMC7246624 | biostudies-literature
Project description:BackgroundNeural crest cells are multipotent cells that migrate extensively throughout vertebrate embryos to form diverse lineages. Cell migration requires polarized, organized actin networks that provide the driving force for motility. Actin-binding proteins that regulate neural crest cell migration are just beginning to be defined.ResultsWe recently identified a number of actin-associated factors through proteomic profiling of methylated proteins in migratory neural crest cells. Here, we report the previously undocumented expression pattern of three of these proteins in chick early neural crest development: doublecortin (DCX), tropomyosin-1 (TPM-1), and actin depolymerizing factor (ADF). All three genes are expressed with varying degrees of specificity and intensity in premigratory and migratory neural crest cells, and their resulting proteins exhibit distinct subcellular localization in migratory neural crest cells. Morpholino knock down of ADF reveals it is required for Sox10 gene expression, but minimally important during neural crest migration.ConclusionsNeural crest cells express DCX, TPM-1, and ADF. ADF is necessary during neural crest specification, but largely dispensable for migration.
| S-EPMC4048876 | biostudies-literature