Micron-scale, coexisting liquid-ordered (L) and liquid-disordered (L) phases are straightforward to observe in giant unilamellar vesicles (GUVs) composed of ternary lipid mixtures. Experimentally, uniform membranes undergo demixing when temperature is decreased: domains subsequently nucleate, diffuse, collide, and coalesce until only one domain of each phase remains. The sizes of these two domains are limited only by the size of the system. Under different conditions, vesicles exhibit smaller-scale domains of fixed sizes, leading to the question of what sets the length scale. In membranes with excess area, small domains are expected when coarsening is hindered or when a microemulsion or modulated phase arises. Here, we test predictions of how the size, morphology, and fluorescence levels of small domains vary with the membrane's temperature, tension, and composition. Using GUVs and cell-derived giant plasma membrane vesicles, we find that 1) the characteristic size of domains decreases when temperature is increased or membrane tension is decreased, 2) stripes are favored over circular domains for lipid compositions with low energy per unit interface, 3) fluorescence levels are consistent with domain registration across both monolayer leaflets of the bilayer, and 4) small domains form in GUVs composed of lipids both with and without ester-linked lipids. Our experimental results are consistent with several elements of current theories for microemulsions and modulated phases and inconsistent with others, suggesting a motivation to modify or enhance current theories.

Understanding the (de)mixing behavior of multicomponent lipid bilayers is an important step toward unraveling the nature of spatial composition heterogeneities in cellular membranes and their role in biological function. We use coarse-grained molecular dynamics simulations to study the composition phase diagram of a quaternary mixture of phospholipids and cholesterol. This mixture is known to exhibit both uniform and coexisting phases. We compare and combine different statistical measures of membrane structure to identify the onset of phase coexistence in composition space. An important element in our approach is the dependence of composition heterogeneities on the size of the system. While homogeneous phases can be structured and display long correlation lengths, the hallmark behavior of phase coexistence is the scaling of the apparent correlation length with system size. Because the latter cannot be easily varied in simulations, our method instead uses information obtained from observation windows of different sizes to accurately distinguish phase coexistence from structured homogeneous phases. This approach is built on very general physical principles, and will be beneficial to future studies of the phase behavior of multicomponent lipid bilayers.

Protein/lipid co-assembly is an understudied phenomenon that is important to the function of antimicrobial peptides as well as the pathological effects of amyloid. Here we study the co-assembly process of PAP 248-286 , a seminal peptide that displays both amyloid-forming and antimicrobial activity. PAP 248-286 is a fragment of prostatic acid phosphatase and has been reported to form amyloid fibrils, known as semen-derived enhancer of viral infection (SEVI), that enhance the viral infectivity of HIV. We find that in addition to forming amyloid, PAP 248-286 much more readily assembles with lipid vesicles into peptide/lipid co-aggregates that resemble amyloid fibrils in some important ways but are a distinct species.The formation of these co-aggregates, which we term "messicles", is controlled by the peptide:lipid (P:L) ratio and by the lipid composition. The optimal P:L ratio is around 1:10 and at least 70% anionic lipid is required for co-aggregate formation. Once formed, messicles are not disrupted by subsequent changes in P:L ratio. We propose that messicles form through a polyvalent assembly mechanism, where a critical surface density of PAP 248-286 on liposomes enables peptidemediated particle bridging into larger species. Even at ~100-fold lower PAP 248-286 concentrations, messicles form at least 10-fold faster than amyloid fibrils. It is therefore possible that, some or all of the biological activities assigned to SEVI, the amyloid form of PAP 248-286 , could instead be attributed to a PAP 248-286 /lipid co-aggregate. More broadly speaking, this work provides a potential framework for the discovery and characterization of peptide/lipid co-aggregates by other amyloid-forming proteins and antimicrobial peptides. Statement of SignificancePAP 248-286 , a fragment of prostatic acid phosphatase, forms amyloid thought to enhances the infectivity of many viruses, including HIV. This amyloid, termed semen-derived enhancer of viral infection (SEVI), has been assigned responsibility for all of PAP 248-286 's biological activities, while the monomer is thought to be inactive. However, SEVI formation is quite slow and requires very high concentrations of PAP 248-286 . Here, we show that PAP 248-286 can instead assemble much more rapidly with lipid membranes to form another species, mechanistically and morphologically distinct from both monomer and SEVI amyloid. We have characterized this new species, which could play a role in the biological activities currently ascribed to SEVI. Additionally, our proposed mechanism for peptide/lipid co-assembly could apply to other biologically important systems..

Cells migrate collectively to form tissues, to heal wounds, and, in cancer, to metastasize. During these biological processes, the collective migration exhibits a transition from a solid-like state, wherein cell positions remain fixed, to a fluid-like state, wherein cells flow freely and rearrange their positions with their neighbors. Recent theoretical models and experiments have demonstrated that this transition can be predicted by average cell shape, with cells having more elongated shapes and greater perimeters more easily sliding past their neighbors. At each cell-cell interface, it has been proposed that active actomyosin contraction generated within the cell cortex acts as an effective surface tension tending to reduce each cell's perimeter. Cell-cell adhesions have the opposite effect, tending to reduce the surface tension, thereby increasing the perimeter. It is unclear how cells regulate these competing factors in a confluent monolayer. Here, we investigate the factors affecting cell surface tension and cell perimeter, and we quantify the corresponding effects on collective migration. For this, we perturb actin and myosin-II using pharmacological treatments in a monolayer of Madin-Darby Canine Kidney cells and study the effects of the treatments on force, shape, and motion. To quantify the cells' active contractile forces, we employ traction force microscopy and monolayer stress microscopy; to measure cell perimeters and to quantify the relative amounts of cortical actin and adhesion molecules at cell-cell interfaces, we use fluorescent imaging; to characterize the cell migration, we analyze cell trajectories. By combining these experimental measurements, our study provides experimental testing of the theoretical models and establishes new principles relating cell force, shape, and motion.

5Protein/lipid co-assembly is an understudied phenomenon that is important to the function of antimicrobial peptides as 6 well as the pathological effects of amyloid. Here we study the co-assembly process of PAP 248-286 , a seminal peptide that 7 displays both amyloid-forming and antimicrobial activity. PAP 248-286 is a fragment of prostatic acid phosphatase and has 8 been reported to form amyloid fibrils, known as semen-derived enhancer of viral infection (SEVI), that enhance the viral 9 infectivity of HIV. We find that in addition to forming amyloid, PAP 248-286 much more readily assembles with lipid 10 vesicles into peptide/lipid co-aggregates that resemble amyloid fibrils in some important ways but are a distinct species. 11The formation of these co-aggregates, which we term "messicles", is controlled by the peptide:lipid (P:L) ratio and by the 12 lipid composition. The optimal P:L ratio is around 1:10 and at least 70% anionic lipid is required for co-aggregate 13 formation. Once formed, messicles are not disrupted by subsequent changes in P:L ratio. We propose that messicles form 14 through a polyvalent assembly mechanism, where a critical surface density of PAP 248-286 on liposomes enables peptide-15 mediated particle bridging into larger species. Even at ~100-fold lower PAP 248-286 concentrations, messicles form at least 16 10-fold faster than amyloid fibrils. It is therefore possible that, some or all of the biological activities assigned to SEVI, 17 the amyloid form of PAP 248-286 , could instead be attributed to a PAP 248-286 /lipid co-aggregate. More broadly speaking, this 18 work provides a potential framework for the discovery and characterization of peptide/lipid co-aggregates by other 19 amyloid-forming proteins and antimicrobial peptides. 20 Statement of Significance 21PAP 248-286 , a fragment of prostatic acid phosphatase, forms amyloid thought to enhances the infectivity of many 22 viruses, including HIV. This amyloid, termed semen-derived enhancer of viral infection (SEVI), has been assigned 23 responsibility for all of PAP 248-286 's biological activities, while the monomer is thought to be inactive. However, SEVI 24 formation is quite slow and requires very high concentrations of PAP . Here, we show that PAP 248-286 can instead 25 assemble much more rapidly with lipid membranes to form another species, mechanistically and morphologically distinct 26 from both monomer and SEVI amyloid. We have characterized this new species, which could play a role in the biological 27 activities currently ascribed to SEVI. Additionally, our proposed mechanism for peptide/lipid co-assembly could apply to 28 other biologically important systems. 29 30 Vane et al. PAP 248-286 /lipid co-aggregates 31Protein self-assembly has been important area of study for the past century. It is ubiquitous in biology -essential for 32 normal function and yet also underlying major diseases. Structural filaments like actin and tubulin microtubules, whose 33 assembly is critical for proper cellular motility, morphology, and trans...