Biology subjects

De Franceschi, N.

Publications and source records attributed to De Franceschi, N..

10 recordsLinked to original sources

bioRxiv2026

Hydration-dehydration cycles drive compartment dynamics in minimal protocells

Compartmentalization is a defining feature of cellular systems, yet how early compartments could undergo repeated cycles of growth, division, and content organization without complex chemistry remains unresolved. Here we study a minimal membrane-based system subjected to periodic hydration- dehydration cycles, mimicking fluctuating physical environments on the early Earth. We show that cyclic environmental conditions alone drive a sequence of reproducible compartment dynamics, including macromolecule encapsulation, membrane growth, division, and the generation of a highly crowded interior. These processes emerge from biophysical transformations of a single-component membrane and do not require any chemical reactions or metabolic activity. Importantly, compartments retain their structural integrity across multiple cycles, enabling repeated encapsulation without loss of individuality. Our results demonstrate that fluctuating physical conditions can be transduced by membrane biophysics into sustained, cell-like cycles, challenging the view that primordial cellular dynamics necessarily required chemically driven growth and division.

synthetic biology↗

bioRxiv2025

Detergent-free reconstitution of transmembrane proteins in giant liposomes of complex curvature by the Synthetic Membrane Transfer

Transmembrane proteins perform many crucial functions in the cell, including transfer of matter and information across the membrane. Giant Unilamellar Vesicles (GUVs) are the ideal tool for studying these processes; yet functional transmembrane protein reconstitution in GUVs still represents a bottleneck in bottom-up synthetic biology. Here, we developed a novel approach that we call Synthetic Membrane Transfer (SMT), where transmembrane proteins reconstituted into styrene-maleic acid particles in a detergent-free environment can be transferred to the GUV membrane. The SMT approach is a one-step, facile and general method that works for structurally diverse proteins, and it does not require any specific lipid or buffer composition. Moreover, the SMT is fully compatible with the Synthetic Membrane Shaper (SMS) technology, which allows to deform GUVs in a controlled fashion, obtaining dumbbell-shaped GUVs exhibiting a catenoid-like geometry. The combination of SMT and SMS results in functional reconstitution of transmembrane proteins in catenoid membrane necks. Using this approach, we demonstrate that Mic10, a component of the MICOS complex, directly senses membrane curvature and localizes at the neck of dumbbells GUVs, a geometry that recapitulates the shape of mitochondria cristae junctions. This paves the way for bottom-up reconstitution of the MICOS complex on a physiological membrane geometry.

synthetic biology↗

bioRxiv2024

Interaction hierarchy among Cdv proteins drives recruitment to membrane necks

Cell division in the crenarchaea is accomplished by the Cdv system. In Sulfolobus cells, it was observed that an initial non-contractile ring of CdvA and CdvB forms at the mid location of the cell, which is followed by a second ring of CdvB1 and CdvB2 that appear to drive the constriction of the cell membrane. Here, we use an in vitro reconstituted system to explore how protein interactions among these Cdv proteins govern their recruitment to the membrane. We show that CdvA can bind to lipid membranes, but does so more efficiently when it is in complex with CdvB. We find that CdvB2 can polymerize if its self-inhibitory domain is removed, and that by itself is exhibits poor binding to the membrane. However, CdvB2 can be efficiently recruited to the membrane by both CdvB1 and CdvB. Furthermore, the CdvB1:CdvB2 co-polymer can be recruited to the membrane by CdvA:CdvB. By reconstituting these proteins in dumbbell-shaped liposomes, we show that Cdv proteins have a strong preference to localize at membrane necks of high curvature. Our findings clarify many of the mutual protein interactions of the Cdv system and their interaction with the membrane, thus helping to build a mechanistic understanding of cell division in archaeal cells.

biochemistry↗

bioRxiv2022

Dynamin A as a one-component division machinery for synthetic cells

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

synthetic biology↗

bioRxiv2022

Engineering ssRNA tile filaments for (dis)assembly and membrane binding

Cytoskeletal protein filaments such as actin and microtubules confer mechanical support to cells and facilitate many cellular functions such as motility and division. Recent years have witnessed the development of a variety of molecular scaffolds that mimic such cytoskeletal filaments. Indeed, filaments that are programmable and compatible with biological systems may prove useful in studying or substituting such proteins. Here, we explore the use of ssRNA tiles to build and modify cytoskeletal-like filaments in vitro. We engineer a number of functionalities that are crucial to the function of natural cytoskeletal systems into the ssRNA tiles, including the abilities to assemble or disassemble filaments, to tune the filament stiffness, to induce membrane binding, and to attach binding proteins. The work paves the way for building dynamic cell-like cytoskeletal systems made out of rationally designed ssRNA tiles that can be transcribed in natural or synthetic cells.

synthetic biology↗

bioRxiv2022

Structural basis of CHMP2A-CHMP3 ESCRT-III polymer assembly and membrane cleavage

The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, the structural basis of ESCRT-III polymers stabilizing, constricting and cleaving negatively curved membranes is yet unknown. Here we present cryo electron microscopy structures of membrane-coated CHMP2A-CHMP3 filaments of two different diameters at 3.3 and 3.6 [A] resolution. The structures reveal helical filaments assembled by CHMP2A-CHMP3 heterodimers in the open ESCRT-III conformation, which generates a partially positive charged membrane interaction surface, positions short N-terminal motifs for membrane interaction and the C-terminal VPS4 target sequence towards the tube interior. Inter-filament interactions are electrostatic, which facilitate filament sliding upon VPS4-mediated polymer remodeling. Fluorescence microscopy as well as high speed atomic force microscopy imaging corroborate that CHMP2A-CHMP3 polymers and VPS4 can constrict and cleave narrow membrane tubes, thus acting as a minimal membrane fission machinery.

cell biology↗

bioRxiv2021

A synthetic membrane shaper for controlled liposome deformation

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 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.

synthetic biology↗

bioRxiv2021

The archaeal division protein CdvB1 assembles into polymers that are depolymerized by CdvC

The Cdv proteins constitute the cell-division system of the Crenarchaea, in a protein machinery that is closely related to the ESCRT system of eukaryotes. The CdvB paralog CdvB1 is believed to play a major role in the constricting ring that is the central actor in cell division in the crenarchaea. Here, we present an in vitro study of purified CdvB1 from the crenarchaeon M. sedula with a combination of TEM imaging and biochemical assays. We show that CdvB1 self-assembles into filamentous polymers that are depolymerized by the action of the Vps4-homolog ATPase CdvC. Using liposome flotation assays, we show that CdvB1 binds to negatively charged lipid membranes and can be detached from the membrane by the action of CdvC. Interestingly, we find that the polymerization and the membrane binding are mutually exclusive properties of the protein. Our findings provide novel insight into one of the main components of the archaeal cell division machinery.

biochemistry↗

bioRxiv2021

Optimized cDICE for efficient reconstitution of biological systems in giant unilamellar vesicles

Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of minimal systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (Protein synthesis Using Recombinant Elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.

synthetic biology↗

bioRxiv2017

ProLIF: a quantitative assay for investigating integrin cytoplasmic protein interactions and synergistic membrane effects on proteoliposomes

Integrin transmembrane heterodimeric receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. A diverse set of methods have been used to investigate cytoplasmic interactions between integrins and intracellular proteins. These predominantly consist of peptide-based pull-downs and biochemical immuno-isolations from detergent-solubilized cell lysates. However, quantitative methods to probe integrin-protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here we describe a technique called ProLIF (Protein-Liposome Iinenteractions by Flow cytometry) to reconstitute recombinant integrin transmembrane domain (TMD) and cytoplasmic tail (CT) fragments on liposomes as individual or {beta} subunits or as {beta} heterodimers and, using flow cytometry, to rapidly and quantitatively measure protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. By combining integrins with membrane lipids to generate proteoliposomes, the effects of membrane composition such as PI(4,5)P2 presence on protein recruitment to the integrin CTs can be analyzed. ProLIF requires no specific instrumentation, apart from a standard flow cytometer and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.

molecular biology↗