Parikh, A. N. | Search bioRxiv

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↗

bioRxiv2020

Recurrent Dynamics of Rupture Transitions of Single Giant Vesicles at Solid Surfaces

Single giant vesicles (GVs) rupture spontaneously from their salt-laden suspension onto solid surfaces. At hydrophilic surfaces, they rupture via a recurrent burst-heal dynamics: during burst, single pores nucleate at the contact boundary of the adhering vesicles facilitating asymmetric spreading and producing a "heart" shaped membrane patch. During the healing phase, the competing pore closure produces a daughter vesicle. At hydrophobic surfaces, by contrast, the GVs rupture via a distinctly different, yet recurrent, bouncing ball rhythm: Rendered tense by the substrate interactions, GVs porate and spread monomolecular layer on the hydrophobic surface in a symmetric manner. Here too, the competition from pore closure produces a daughter vesicle, which re-engages with the substrate. In both cases, the pattern of burst-reseal events repeats multiple times splashing and spreading the vesicular fragments as bilayer patches at the solid surface in a pulsatory manner. These remarkable recurrent dynamics arise not because of the elastic properties of the solid surface but because the competition between membrane spreading and pore healing, prompted by the surface-energy dependent adhesion, determine the course of the topological transition. STATEMENT OF SIGNIFICANCEGiant lipid vesicles adhering to a solid surface experience strong mechanical stresses. The contacting membrane segment loses thermal fluctuations and accumulates mechanical tension, the equilibration of which can give rise to global shape changes, lipid phase separation, and traction forces. Beyond a threshold tension, vesicles porate, unravel, and spread. Here, we find that a competition from pore-healing can make rupture iterative, rather than a single all-or-nothing event. During burst, single pores expand, spreading a lipid bilayer on the hydrophilic surface and a monolayer on the hydrophobic one. During heal, pore-healing can produce daughter vesicles. This burst-reseal event reiterates "splashing" portions of single vesicles at the solid surface and "bouncing" the remainder as a secondary vesicle in multiple steps.

biophysics↗