Dual-color superresolution microscopy reveals nanoscale organization of mechanosensory podosomes

Podosomes are multimolecular mechanosensory assemblies that coordinate mesenchymal migration of tissue-resident dendritic cells. They have a protrusive actin core and an adhesive ring of integrins and adaptor proteins, such as talin and vinculin. We recently demonstrated that core actin oscillations correlate with intensity fluctuations of vinculin but not talin, suggesting different molecular rearrangements for these components. Detailed information on the mutual localization of core and ring components at the nanoscale is lacking. By dual-color direct stochastic optical reconstruction microscopy, we for the first time determined the nanoscale organization of individual podosomes and their spatial arrangement within large clusters formed at the cell–substrate interface. Superresolution imaging of three ring components with respect to actin revealed that the cores are interconnected and linked to the ventral membrane by radiating actin filaments. In core-free areas, αMβ2 integrin and talin islets are homogeneously distributed, whereas vinculin preferentially localizes proximal to the core and along the radiating actin filaments. Podosome clusters appear as self-organized contact areas, where mechanical cues might be efficiently transduced and redistributed. Our findings call for a reevaluation of the current “core–ring” model and provide a novel structural framework for further understanding the collective behavior of podosome clusters.     

CP-46,665-1: A novel lipoidal amine with antimetastatic and immunomodulatory properties

Summary CP-46-665-1 [4-aminomethyl-1(2,3-(di-n-decyloxy)-n-propyl)-4-phenylpiperidine dihydrochloride] is a novel, low-molecular-weight, synthetic compound with antimetastatic activity in rodent models. In the B-16 melanoma/C57Bl/6 mouse system, in which the hind limb bearing a primary foot tumor is amputated 21 days after tumor implantation, intravenous infusion of CP-46,665-1 after surgery at doses ranging from 0.15–2.5 mg/kg increases the fraction of metastasis-free animals at 50 days to as high as 56% (significantly different from the 19% seen in surgery-only controls, P<0.001). In more preliminary experiments with a mammary adenocarcinoma (13762)/Fischer 344 rat system, there are suggestions that the combination of CP-46,665-1 administration and surgical removal of the primary tumor results in increased numbers of long-term survivors that are free of metastatic disease.The mechanisms underlying the antimetastatic activity of CP-46,665-1 are not fully evident, but certain lines of evidence suggest that the compound may act by affecting the reticuloendothelial system (RES). Peritoneal macrophages from animals dosed with CP-46,665-1 display elevated cytolytic activity against tumor target cells. Further, radiolabeled CP-46,665-1 and/or its metabolites preferentially accumulate in organs of the RES, and lymph nodes, spleens, and livers from animals dosed chronically with CP-46,665-1 contain scattered foci of histiocytic cells with foamy vacuoles containing material staining as complex lipid. CP-46,665-1 augments the vaccine potency of irradiated L-1210 leukemia cells, but does not induce interferon in rodents.CP-46,665-1 appeared to be well tolerated in rats and dogs when infused at doses of up to 25 mg/kg at 4-day intervals for 92 days. In addition to the changes in the lymphoreticular system described above, the principal findings in dogs that received high doses by injection were erythrocyte lysis accompanied by the expected serum chemistry and hematological correlates; microcytic anemia and injection site irritation were found in rats receiving high doses. Neither species displayed notable toxic reactions when chronically dosed at the 1 mg/kg level, a level well within the antimetastatic activity dose range. Cats and dogs with a variety of malignancies tolerated weekly doses of the agent as high as 5 mg/kg with minimal side-effects for periods up to 30 weeks. We believe that this combination of good chronic toleration and potent antimetastatic activity commend CP-46,665-1 well for more extensive study as an experimental drug for the treatment of cancer.