Huang, W. Y. C. | bioRxiv Science

bioRxiv2021

Cytoplasmic organization promotes protein diffusion

The cytoplasm is highly organized. However, the extent to which this organization influences the dynamics of cytoplasmic proteins is not well understood. Here, we used Xenopus laevis egg extracts as a model system to study diffusion dynamics in organized versus disorganized cytoplasm. Such extracts are initially homogenized and disorganized, and will self-organize into cell-like units over the course of 20-60 min. Using fluorescence correlation spectroscopy, we observed that self-organization is accompanied by changes in protein diffusivity; as the extract organizes, proteins diffuse about twice as quickly over a length scale of a few hundred nanometers. Even though the ordered cytoplasm contained organelles and cytoskeletal elements that might be expected to interfere with diffusion, after self-organization took place, the speed of protein diffusion approached that of organelle-depleted cytosolic extracts. This finding suggests that subcellular organization optimizes protein diffusivity. The effect of organization on diffusion varies with molecular size, with the effects being largest for protein-sized molecules. These results show that cytoplasmic organization promotes the efficient diffusion of protein molecules in a densely packed environment.

biophysics↗

bioRxiv2020

Coupling between lipid miscibility and phosphotyrosine driven protein condensation at the membrane

Lipid miscibility phase separation has long been considered to be a central element of cell membrane organization. More recently, protein condensation phase transitions, into three-dimensional droplets or in two-dimensional lattices on membrane surfaces, have emerged as another important organizational principle within cells. Here, we reconstitute the LAT:Grb2:SOS protein condensation on the surface of giant unilamellar vesicles capable of undergoing lipid phase separations. Our results indicate that assembly of the protein condensate on the membrane surface can drive lipid phase separation. This phase transition occurs isothermally and is governed by tyrosine phosphorylation on LAT. Furthermore, we observe that the induced lipid phase separation drives localization of the SOS substrate, K-Ras, into the LAT:Grb2:SOS protein condensate. Statement of SignificanceProtein condensation phase transitions are emerging as an important organizing principles in cells. One such condensate plays a key role in T cell receptor signaling. Immediately after receptor activation, multivalent phosphorylation of the adaptor protein LAT at the plasma membrane leads to networked assembly of a number of signaling proteins into a two-dimensional condensate on the membrane surface. In this study, we demonstrate that LAT condensates in reconstituted vesicles are sufficient to drive lipid phase separation. This lipid reorganization drives another key downstream signaling molecule, Ras, into the LAT condensates. These results show that the LAT condensation phase transition, which is actively controlled by phosphorylation reactions, extends its influence to control lipid phase separation in the underlying membrane.

biophysics↗