Alessio Fragasso

All Publications

• Diameter dependence of transport through nuclear pore complex mimics studied using optical nanopores. eLife Klughammer, N., Barth, A., Dekker, M., Fragasso, A., Onck, P. R., Dekker, C. 2024; 12

  Abstract

  The nuclear pore complex (NPC) regulates the selective transport of large biomolecules through the nuclear envelope. As a model system for nuclear transport, we construct NPC mimics by functionalizing the pore walls of freestanding palladium zero-mode waveguides with the FG-nucleoporin Nsp1. This approach enables the measurement of single-molecule translocations through individual pores using optical detection. We probe the selectivity of Nsp1-coated pores by quantitatively comparing the translocation rates of the nuclear transport receptor Kap95 to the inert probe BSA over a wide range of pore sizes from 35 nm to 160 nm. Pores below 55 ± 5 nm show significant selectivity that gradually decreases for larger pores. This finding is corroborated by coarse-grained molecular dynamics simulations of the Nsp1 mesh within the pore, which suggest that leakage of BSA occurs by diffusion through transient openings within the dynamic mesh. Furthermore, we experimentally observe a modulation of the BSA permeation when varying the concentration of Kap95. The results demonstrate the potential of single-molecule fluorescence measurements on biomimetic NPCs to elucidate the principles of nuclear transport.

  View details for DOI 10.7554/eLife.87174

  View details for PubMedID 38376900

• Dynamin A as a one-component division machinery for synthetic cells. Nature nanotechnology De Franceschi, N., Barth, R., Meindlhumer, S., Fragasso, A., Dekker, C. 2024; 19 (1): 70-76

  Abstract

  Membrane abscission, the final cut of the last connection between emerging daughter cells, is an indispensable event in the last stage of cell division and in other cellular processes such as endocytosis, virus release or bacterial sporulation. However, its mechanism remains poorly understood, impeding its application as a cell-division machinery for synthetic cells. Here we use fluorescence microscopy and fluorescence recovery after photobleaching measurements to study the in vitro reconstitution of the bacterial protein dynamin A inside liposomes. Upon external reshaping of the liposomes into dumbbells, dynamin A self-assembles at the membrane neck, resulting in membrane hemi-scission and even full scission. Dynamin A proteins constitute a simple one-component division machinery capable of splitting dumbbell-shaped liposomes, marking an important step towards building a synthetic cell.

  View details for DOI 10.1038/s41565-023-01510-3

  View details for PubMedID 37798563

• Synthetic Membrane Shaper for Controlled Liposome Deformation ACS NANO De Franceschi, N., Pezeshkian, W., Fragasso, A., Bruininks, B. H., Tsai, S., Marrink, S. J., Dekker, C. 2022: 966-78

  Abstract

  Shape defines the structure and function of cellular membranes. In cell division, the cell membrane deforms into a "dumbbell" shape, while organelles such as the autophagosome exhibit "stomatocyte" shapes. Bottom-up in vitro reconstitution of protein machineries that stabilize or resolve the membrane necks in such deformed liposome structures is of considerable interest to characterize their function. Here we develop a DNA-nanotechnology-based approach that we call the synthetic membrane shaper (SMS), where cholesterol-linked DNA structures attach to the liposome membrane to reproducibly generate high yields of stomatocytes and dumbbells. In silico simulations confirm the shape-stabilizing role of the SMS. We show that the SMS is fully compatible with protein reconstitution by assembling bacterial divisome proteins (DynaminA, FtsZ:ZipA) at the catenoidal neck of these membrane structures. The SMS approach provides a general tool for studying protein binding to complex membrane geometries that will greatly benefit synthetic cell research.

  View details for DOI 10.1021/acsnano.2c06125

  View details for Web of Science ID 000891678100001

  View details for PubMedID 36441529

  View details for PubMedCentralID PMC9878720

• Transport receptor occupancy in nuclear pore complex mimics NANO RESEARCH Fragasso, A., de Vries, H. W., Andersson, J., van der Sluis, E. O., van der Giessen, E., Onck, P. R., Dekker, C. 2022; 15 (11): 9689-9703

  View details for DOI 10.1007/s12274-022-4647-1

  View details for Web of Science ID 000819726800010