Macrophages remodel their mechanics during differentiation toward different subtypes and drastically adapt their shapes during phagocytosis or entry to inflamed tissues. Although these functions... Show moreMacrophages remodel their mechanics during differentiation toward different subtypes and drastically adapt their shapes during phagocytosis or entry to inflamed tissues. Although these functions depend on cell mechanical properties, the mechanical behavior of macrophages is still poorly understood and accurate physiologically relevant data on basic mechanical properties of different macrophage subtypes are lacking almost entirely. By combining several complementary single-cell force spectroscopy techniques, whole cell mechanics of M1 (differentiated by granulocyte macrophage colony-stimulating factor [GM-CSF]) and M2 (differentiated by macrophage colony-stimulating factor [M-CSF]) macrophages is systematically analyzed, and it is revealed that M2 macrophages exhibit solid-like behavior, whereas M1 macrophages behave more fluid-like. In addition, the findings indicate that M2 macrophages exhibit increased dynamic motility as compared to M1 macrophages, consistent with their mechanical phenotypes. The technology presented herein can be used to distinguish macrophage subtypes based on their mechanical phenotype, and suggests that mechanical properties of macrophages are linked to their immune function. Show less
DNA polymerase catalyzes the accurate transfer of genetic information from one generation to the next, and thus it is vitally important for replication to be faithful. DNA polymerase fulfills the... Show moreDNA polymerase catalyzes the accurate transfer of genetic information from one generation to the next, and thus it is vitally important for replication to be faithful. DNA polymerase fulfills the strict requirements for fidelity by a combination of mechanisms: 1) high selectivity for correct nucleotide incorporation, 2) a slowing down of the replication rate after misincorporation, and 3) proofreading by excision of misincorporated bases. To elucidate the kinetic interplay between replication and proofreading, we used high-resolution optical tweezers to probe how DNA-duplex stability affects replication by bacteriophage T7 DNA polymerase. Our data show highly irregular replication dynamics, with frequent pauses and direction reversals as the polymerase cycles through the states that govern the mechanochemistry behind high-fidelity T7 DNA replication. We constructed a kinetic model that incorporates both existing biochemical data and the, to our knowledge, novel states we observed. We fit the model directly to the acquired pause-time and run-time distributions. Our findings indicate that the main pathway for error correction is DNA polymerase dissociation-mediated DNA transfer, followed by biased binding into the exonuclease active site. The number of bases removed by this proofreading mechanism is much larger than the number of erroneous bases that would be expected to be incorporated, ensuring a high-fidelity replication of the bacteriophage T7 genome. Show less
