Analysis of the H-Ras mobility pattern in vivo shows cellular heterogeneity inside epidermal tissue

Developments in single-molecule microscopy (SMM) have enabled imaging individual proteins in biological systems, focusing on the analysis of protein mobility patterns inside cultured cells. In the present study, SMM was applied in vivo, using the zebrafish embryo model. We studied dynamics of the membrane protein H-Ras, its membrane-anchoring domain, C10H-Ras, and mutants, using total internal reflection fluorescence microscopy (TIRFM). Our results consistently confirm the presence of fast- and slow-diffusing subpopulations of molecules, which confine to microdomains within the plasma membrane. The active mutant H-RasV12 exhibits higher diffusion rates and is confined to larger domains than the wild-type H-Ras and its inactive mutant H-RasN17. Subsequently, we demonstrate that the structure and composition of the plasma membrane have an imperative role in modulating H-Ras mobility patterns. Ultimately, we establish that differences between cells within the same embryo largely contribute to the overall data variability. Our findings agree with a model where the cell architecture and the protein activation state determine protein mobility, underlining the importance of SMM imaging to study factors influencing protein dynamics in an intact living organism.

Dual RNA modulation of protein mobility and stability within phase-separated condensates

One of the key mechanisms employed by cells to control their spatiotemporal organization is the formation and dissolution of phase-separated condensates. Such balance between condensate assembly and disassembly can be critically regulated by the presence of RNA. In this work, we use a novel chemically accurate coarse-grained model for proteins and RNA to unravel the impact of poly-uridine RNA in modulating the protein mobility and stability within different biomolecular condensates. We explore the behavior of FUS, hnRNPA1 and TDP-43 proteins along with that of their corresponding prion-like domains and RNA-recognition motifs, from absence to moderately high RNA concentration. By characterising the phase diagram, key molecular interactions, surface tension and viscoelastic properties, we report a dual RNA-induced behavior: On the one hand, poly-uridine enhances phase separation at low concentration, whilst at high concentration, it inhibits the ability of proteins to self-assemble. On the other hand, as a consequence of such stability modulation, the viscoelastic liquid properties of the condensates are significantly enhanced at moderately high RNA concentration, as long as the length of poly-uridine strands is comparable or moderately shorter than those of the proteins, whereas protein self-diffusion barely depends on poly-uridine length. On the whole, our work elucidates the different routes by which RNA can regulate phase separation and condensate dynamics, as well as the subsequent aberrant rigidification implicated in the emergence of various neuropathologies and age-related diseases.

Measurement of Protein Mobility in Listeria monocytogenes Reveals a Unique Tolerance to Osmotic Stress and Temperature Dependence of Diffusion

Protein mobility in the cytoplasm is essential for cellular functions, and slow diffusion may limit the rates of biochemical reactions in the living cell. Here, we determined the apparent lateral diffusion coefficient (DL) of GFP in Listeria monocytogenes as a function of osmotic stress, temperature, and media composition. We find that DL is much less affected by hyperosmotic stress in L. monocytogenes than under similar conditions in Lactococcus lactis and Escherichia coli. We find a temperature optimum for protein diffusion in L. monocytogenes at 30°C, which deviates from predicted trends from the generalized Stokes-Einstein equation under dilute conditions and suggests that the structure of the cytoplasm and macromolecular crowding vary as a function of temperature. The turgor pressure of L. monocytogenes is comparable to other Gram-positive bacteria like Bacillus subtilis and L. lactis but higher in a knockout strain lacking the stress-inducible sigma factor SigB. We discuss these findings in the context of how L. monocytogenes survives during environmental transmission and interaction with the human host.

Influence of Hydrogen Ion Concentrations on the Protein Mobility in Native-PAGE Buffers

The study demonstrates the effect of hydrogen ion concentrations (pH) of Tank Buffer (TB) and resolving gel buffer (RGB) in a native-PAGE system on protein samples. A pH range of 7.8 to 9.3 for RGB and 8.3 to 9.3 for TB were used. Proteins in the samples under native-PAGE at varying pH of RGB such as 7.8, 8.3 and 8.8 with pH of TB, 8.3 and 8.8 were resolved well. The total running time for the samples to reach the end of the dye front ranged between 2.30 h to 4.30 h at the above pH combinations. It was observed as 3.30 h as total running time under normal pH conditions (RGB, pH 8.8; TB, pH 8.3). At the same time, buffers at higher pH or the combination of extreme pH (7.8 vs. 9.3) in the buffers were not favored good protein mobility/resolution, and bands were diffused. Longer running time was observed in various combinations of pH of RGB and pH of TB with 18.00 h being a longest with a pH of 8.3 and 9.3 for RGB and TB, respectively. This indicated the importance of pH of electrophoresis buffers for ionization of various proteins for better separation.

A stochastic hybrid model for DNA replication incorporating protein mobility dynamics

SummaryDNA replication, the basis of genetic information maintenance, is a remarkably robust yet highly stochastic process. We present a computational model that incorporates experimental genome structures and protein mobility dynamics to mechanistically describe the stochastic foundations of DNA replication. Analysis of about 300,000 in silico profiles for fission yeast indicates that the number of firing factors is rate-limiting and dominates completion time. Incorporating probabilistic activation and binding, a full-genome duplication was achieved with at least 300 firing factors, with the only assumption that factors get recycled upon replication fork collision. Spatial patterns of replication timing were reproduced only when firing factors were explicitly activated proximally to the spindle pole body. Independent in vivo experiments validate that the spindle pole body acts as a replication activator, driving origin firing. Our model provides a framework to realistically simulate full-genome DNA replication and investigate the effects of nuclear architecture.