Phase Separation of Shell Protein and Enzyme: An Insight into the Biogenesis of a Prokaryotic Metabolosome

Bacterial microcompartments are substrate specific metabolic modules that are conditionally expressed in certain bacterial species. These all protein structures have size in the range of 100-150 nm and are formed by the self-assembly of thousands of protein subunits, all encoded by genes belonging to a single operon. The operon contains genes that encode for both enzymes and shell proteins. The shell proteins self-assemble to form the outer coat of the compartment and enzymes are encapsulated within. A perplexing question in MCP biology is to understand the mechanism which governs the formation of these small yet complex assemblages of proteins. In this work we use 1,2-propanediol utilization microcompartments (PduMCP) as a paradigm to identify the factors that drive the self-assembly of MCP proteins. We find that a major shell protein PduBB tend to self-assemble under macromolecular crowded environment and suitable ionic strength. Microscopic visualization and biophysical studies reveal phase separation to be the principle mechanism behind the self-association of shell protein in the presence of salts and macromolecular crowding. The shell protein PduBB interacts with the enzyme diol-dehydratase PduCDE and co-assemble into phase separated liquid droplets. The co-assembly of PduCDE and PduBB results in the enhancement of catalytic activity of the enzyme. A combination of spectroscopic and biochemical techniques shows the relevance of divalent cation Mg2+ in providing stability to intact PduMCP in vivo. Together our results suggest a combination of protein-protein interactions and phase separation guiding the self-assembly of Pdu shell protein and enzyme in solution phase.

Live 3D imaging and mapping of shear stresses within tissues using incompressible elastic beads

To investigate the role of mechanical constraints in morphogenesis and development, we develop a pipeline of techniques based on incompressible elastic sensors. These techniques combine the advantages of incompressible liquid droplets, which have been used as precise in situ shear stress sensors, and of elastic compressible beads, which are easier to tune and to use. Droplets of a polydimethylsiloxane (PDMS) mix, made fluorescent through specific covalent binding to a rhodamin dye, are produced by a microfluidics device. The elastomer rigidity after polymerization is adjusted to the tissue rigidity. Its mechanical properties are carefully calibrated in situ, for a sensor embedded in a cell aggregate submitted to uniaxial compression. Thelocal shear stress tensor is retrieved from the sensor shape, accurately reconstructed through an active contour method. In vitro, within cell aggregates, and in vivo, in the prechordal plate of the Zebrafish embryo during gastrulation,our pipeline of techniques demonstrates its efficiency to directly measure the three dimensional shear stress repartition within a tissue.

Cis-clustering of cadherin-23 controls the kinetics of cell-cell adhesion

Cis and trans-interactions in cadherins are the foundations of multicellularity. While the trans-interaction mediate cell-cell adhesion, the cis-interaction is postulated as strengthening to trans by clustering. The well-accepted model in cadherin-adhesion is that the trans precedes cis via a diffusion-trap kinetic model. Here we report that cadherin-23, a non-classical cadherin with an extended extracellular region, undergoes clustering in solution via lateral interactions independent of trans and phase separate as liquid droplets. In cellulo using fluorescence-recovery after the photobleaching, we noticed a significantly slow-diffusion of cadherin-23 at the intercellular junctions, indicating the diffusion of a cluster. The cis-clustering accelerates the cell-cell adhesion and, thus, kinetically controls cell-adhesion via cis precedes trans model. Though the connection of cis-clustering with the rapid adhesion is yet to explore, M2-macrophages that predominantly express cadherin-23 undergo fast attachments to circulatory tumor cells during metastasis.

Laser Fabrication of Titanium Alloy-Based Photothermal Responsive Slippery Surface

In recent years, biomimetic materials inspired from natural organisms have attracted great attention due to their promising functionalities and cutting-edge applications, emerging as an important research topic. For example, how to reduce the reflectivity of the solid surface and increase the absorption of the substrate surface is essential for developing light response smart surface. Suitable solutions to this issue can be found in natural creatures; however, it is technologically challenging. In this work, inspired from butterfly wings, we proposed a laser processing technology to prepare micro nanostructured titanium alloy surfaces with anti-reflection properties. The reflectivity is significantly suppressed, and thus, the light absorption is improved. Consequently, the anti-reflection titanium alloy surface can be further employed as a photothermal substrate for developing light-responsive slippery surface. The sliding behavior of liquid droplets on the smart slippery surface can be well controlled via light irradiation. This method facilitates the preparation of low-reflection and high-absorption metallic surfaces towards bionic applications.

Electrostatic tweezer for droplet manipulation

Various physical tweezers for manipulating liquid droplets based on optical, electrical, magnetic, acoustic, or other external fields have emerged and revolutionized research and application in medical, biological, and environmental fields. Despite notable progress, the existing modalities for droplet control and manipulation are still limited by the extra responsive additives and relatively poor controllability in terms of droplet motion behaviors, such as distance, velocity, and direction. Herein, we report a versatile droplet electrostatic tweezer (DEST) for remotely and programmatically trapping or guiding the liquid droplets under diverse conditions, such as in open and closed spaces and on flat and tilted surfaces as well as in oil medium. DEST, leveraging on the coulomb attraction force resulting from its electrostatic induction to a droplet, could manipulate droplets of various compositions, volumes, and arrays on various substrates, offering a potential platform for a series of applications, such as high-throughput surface-enhanced Raman spectroscopy detection with single measuring time less than 20 s.

Features of the Flow of a Model Liquid Into a Medium with a Variable Degree of Rarefaction

Experimental results of observing ethanol micro-jets expiring into a highly rarefied medium (vacuum) through a nozzle are presented. The study of the process was carried out both at the horizontal and vertical liquid stream from the source compared to the direction of gravity The residual background gas pressure in the vacuum chamber was maintained at a level much lower than the saturated vapor pressure of the working fluid at a given outlet temperature. The possibility of modeling complex processes of micro-fluids expiring into a medium with a given rarefied atmosphere on a compact vacuum gas-dynamic stand is shown. It is established that the long-term flow from a thin capillary or a small-diameter hole into a vacuum or a highly rarefied gas medium differs significantly from the well-studied flow modes into a dense gas medium, as well as from the pulsed flow modes into a vacuum. The paper describes the main features of the flow and the conditions for the occurrence of instability. It is shown that the long-term flow of a liquid micro-jet in a vacuum has a high degree of surface instability, with a large number of sudden changes in the direction, structure, and observed density. An explanation of the reasons for the destruction of the micro-jet is proposed. The formation of surface gas caverns causing explosive destruction of the micro-jet with the release of vapor-liquid droplets is established.

Implementing Complementary Approaches to Shape the Mechanism of α-Synuclein Oligomerization as a Model of Amyloid Aggregation

The aggregation of proteins into amyloid fibers is linked to more than forty still incurable cellular and neurodegenerative diseases such as Parkinson’s disease (PD), multiple system atrophy, Alzheimer’s disease and type 2 diabetes, among others. The process of amyloid formation is a main feature of cell degeneration and disease pathogenesis. Despite being methodologically challenging, a complete understanding of the molecular mechanism of aggregation, especially in the early stages, is essential to find new biological targets for innovative therapies. Here, we reviewed selected examples on α-syn showing how complementary approaches, which employ different biophysical techniques and models, can better deal with a comprehensive study of amyloid aggregation. In addition to the monomer aggregation and conformational transition hypothesis, we reported new emerging theories regarding the self-aggregation of α-syn, such as the alpha-helix rich tetramer hypothesis, whose destabilization induce monomer aggregation; and the liquid-liquid phase separation hypothesis, which considers a phase separation of α-syn into liquid droplets as a primary event towards the evolution to aggregates. The final aim of this review is to show how multimodal methodologies provide a complete portrait of α-syn oligomerization and can be successfully extended to other protein aggregation diseases.