Human Costars Family Protein ABRACL Modulates Actin Dynamics and Cell Migration and Associates with Tumorigenic Growth.

Regulation of cellular actin dynamics is pivotal in driving cell motility. During cancer development, cells migrate to invade and spread; therefore, dysregulation of actin regulators is often associated with cancer progression. Here we report the role of ABRACL, a human homolog of the Dictyostelium actin regulator Costars, in migration and tumorigenic growth of cancer cells. We found a correlation between ABRACL expression and the migratory ability of cancer cells. Cell staining revealed the colocalization of ABRACL and F-actin signals at the leading edge of migrating cells. Analysis of the relative F-/G-actin contents in cells lacking or overexpressing ABRACL suggested that ABRACL promotes cellular actin distribution to the polymerized fraction. Physical interaction between ABRACL and cofilin was supported by immunofluorescence staining and proximity ligation. Additionally, ABRACL hindered cofilin-simulated pyrene F-actin fluorescence decay in vitro, indicating a functional interplay. Lastly, analysis on a colorectal cancer cohort demonstrated that high ABRACL expression was associated with distant metastasis, and further exploration showed that depletion of ABRACL expression in colon cancer cells resulted in reduced cell proliferation and tumorigenic growth. Together, results suggest that ABRACL modulates actin dynamics through its interaction with cofilin and thereby regulates cancer cell migration and participates in cancer pathogenesis.

Costars, a Dictyostelium protein similar to the C-terminal domain of STARS, regulates the actin cytoskeleton and motility.

Through analysis of a chemotaxis mutant obtained from a genetic screen in Dictyostelium discoideum, we have identified a new gene involved in regulating cell migration and have named it costars (cosA). The 82 amino acid Costars protein sequence appears highly conserved among diverse species, and significantly resembles the C-terminal region of the striated muscle activator of Rho signaling (STARS), a mammalian protein that regulates the serum response factor transcriptional activity through actin binding and Rho GTPase activation. The cosA-null (cosA(-)) cells formed smooth plaques on bacterial lawns, produced abnormally small fruiting bodies when developed on the non-nutrient agar and displayed reduced migration towards the cAMP source in chemotactic assays. Analysis of cell motion in cAMP gradients revealed decreased speed but wild-type-like directional persistence of cosA(-) cells, suggesting a defect in the cellular machinery for motility rather than for chemotactic orientation. Consistent with this notion, cosA(-) cells exhibited changes in the actin cytoskeleton, showing aberrant distribution of F-actin in fluorescence cell staining and an increased amount of cytoskeleton-associated actin. Excessive pseudopod formation was also noted in cosA(-) cells facing chemoattractant gradients. Expressing cosA or its human counterpart mCostars eliminated abnormalities of cosA(-) cells. Together, our results highlight a role for Costars in modulating actin dynamics and cell motility.

Pre-clinical assessment of chimeric antigen receptor t cell therapy targeting CD19+ B cell malignancy.

BACKGROUND: Autologous chimeric antigen receptor (CAR) T cell therapy is a promising therapeutic strategy for treating hematologic malignancies. A spectrum of serious complications caused by CAR-T cells has caught great attention. We developed a novel CAR against CD19 namely UWC19, consisting anti-CD19 single-chain variable fragment (scFv) hinged with 4-1BB and CD3z signaling domains. In this study, preclinical assessments of UWC19 were conducted to evaluate the safety and efficacy in vitro and in vivo. METHODS: To evaluate the binding activity of UWC19 cells to CD19, we measured the saturation degree of CAR with human CD19 molecules using flow cytometry in vitro. The antitumor efficacy of UWC19 cells was determined by in vitro cytotoxicity assay against CD19 positive cells and in vivo using a xenograft mouse model. Cross tissue reactivity of UWC19 cells was examined by co-culturing with cell lines from difference human tissues. Tumorigenicity was determined by subcutaneously injecting UWC19 in immunodeficient mice. Persistence was analyzed using quantitative PCR. RESULTS: We showed that UWC19 CAR T cells exerted highly specific binding affinity and cytotoxicity against CD19+ cells in vitro. In vivo, UWC19 CAR T cells are able to fully control disease progression in a Raji-xenografted immunodeficient mouse model. UWC19 exerted no obvious effects on the mean body mass and graft versus host disease were observed in surviving mice. We showed that UWC19 cells specifically recognized and eliminated CD19 positive cells, whereas CD19 negative cells were much less affected. No tumorigenicity of UWC19 in immunodeficient mice was observed. CONCLUSIONS: UWC19 treatment effectively eliminated CD19 positive tumor cells with favorable toxicity profile. The findings suggest encouraging clinical prospects for its use in patients with CD19 positive B cell malignancies. Our study presented an alternative evaluation strategy for CAR-T cell products.

Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideum.

Cell migration is involved in various physiological and pathogenic events, and the complex underlying molecular mechanisms have not been fully elucidated. The simple eukaryote Dictyostelium discoideum displays chemotactic locomotion in stages of its life cycle. By characterizing a Dictyostelium mutant defective in chemotactic responses, we identified a novel actin-binding protein serving to modulate cell migration and named it actin-binding protein G (AbpG); this 971-amino acid (aa) protein contains an N-terminal type 2 calponin homology (CH2) domain followed by two large coiled-coil regions. In chemoattractant gradients, abpG(-) cells display normal directional persistence but migrate significantly more slowly than wild-type cells; expressing Flag-AbpG in mutant cells eliminates the motility defect. AbpG is enriched in cortical/lamellipodial regions and colocalizes well with F-actin; aa 401-600 and aa 501-550 fragments of AbpG show the same distribution as full-length AbpG. The aa 501-550 region of AbpG, which is essential for AbpG to localize to lamellipodia and to rescue the phenotype of abpG(-) cells, is sufficient for binding to F-actin and represents a novel actin-binding protein domain. Compared with wild-type cells, abpG(-) cells have significantly higher F-actin levels. Collectively our results suggest that AbpG may participate in modulating actin dynamics to optimize cell locomotion.

Dictyostelium gnt15 encodes a protein with similarity to LARGE and plays an essential role in development.

LARGE is a putative glycosyltransferase found to be mutated in mice with myodystrophy or patients with congenital muscular dystrophy. By homology searches, we identified in the Dictyostelium discoideum genome four open reading frames, i.e. gnt12-15, encoding proteins with sequence similarity to LARGE. Semi-quantitative RT-PCR analysis revealed distinct temporal expression patterns of the four gnt genes throughout Dictyostelium development. To explore the gene function, we performed targeted disruptions of gnt14 and gnt15. The gnt14(-) strains showed no obvious phenotypes. However, gnt15(-) cells grew slowly, changed in morphology, and displayed a developmental phenotype arresting at early stages. Compared with the wild type, gnt15(-) cells were more adhesive and exhibited altered levels of some surface adhesion molecules. Moreover, lectin-binding analysis demonstrated that gnt15 disruption affected profiles of membrane glycoproteins. Taken together, our data suggest that Gnt15 is essential for Dictyostelium development and may have a role in modulating cell adhesion and glycosylation.

Exposure of single-stranded telomeric DNA causes G2/M cell cycle arrest in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Cdc13p is a single-stranded TG(1-3) DNA binding protein that protects telomeres and maintains telomere length. A mutant allele of CDC13, cdc13-1, causes accumulation of single-stranded TG(1-3) DNA near telomeres along with a G(2)/M cell cycle arrest at non-permissive temperatures. We report here that when the single-stranded TG(1-3) DNA is masked by its binding proteins, such as S. cerevisiae Gbp2p or Schizosaccharomyces pombe Tcg1, the growth arrest phenotype of cdc13-1 is rescued. Mutations on Gbp2p that disrupt its binding to the single-stranded TG(1-3) DNA render the protein unable to complement the defects of cdc13-1. These results indicate that the presence of a single-stranded TG(1-3) tail in cdc13-1 cells serves as the signal for the cell cycle checkpoint. Moreover, the binding activity of Gbp2p to single-stranded TG(1-3) DNA appears to be associated with its ability to restore the telomere-lengthening phenotype in cdc13-1 cells. These results indicate that Gbp2p is involved in modulating telomere length.

Design and synthesis of class-selective activity probes for protein tyrosine phosphatases.

Two mechanism-based activity probes, adopting a cassette-like design, for protein tyrosine phosphatases (PTPs) were synthesized. Both probes carry a phosphate group that serves as the recognition head for the target PTPs but differ in their reporter groups; probe LCL-1 uses a dansyl fluorophore, while LCL-2 has a biotin reporter group. LCL-1 and LCL-2 are specifically activated by phosphatase, leading to its covalent labeling, as exemplified with PTP-1B. However, they show no activation with other classes of hydrolases, including trypsin and beta-galactosidase. LCL-1 and LCL-2 thus represent the first example of class-selective probes for phosphatases.