Yifan Ge

Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures

There is growing recognition that lipid heterogeneities in cellular membranes play an important role in the distribution and functionality of membrane proteins. However, the detection and characterization of such heterogeneities at the cellular level remains challenging. Here we report on the poorly understood relationship between lipid bilayer asymmetry and membrane protein sequestering in raft-mimicking model membrane mixtures using a powerful experimental platform comprised of confocal spectroscopy XY-scan and photon-counting histogram analyses. This experimental approach is utilized to probe the domain-specific sequestering and oligomerization state of αvβ3 and α5β1 integrins in bilayers, which contain coexisting liquid-disordered/liquid-ordered (ld/lo) phase regions exclusively in the top leaflet of the bilayer (bottom leaflet contains ld phase). Comparison with previously reported integrin sequestering data in bilayer-spanning lo-ld phase separations demonstrates that bilayer asymmetry has a profound influence on αvβ3 and α5β1 sequestering behavior. For example, both integrins sequester preferentially to the lo phase in asymmetric bilayers, but to the ld phase in their symmetric counterparts. Furthermore, our data show that bilayer asymmetry significantly influences the role of native ligands in integrin sequestering.

N-cadherin-functionalized polymer-tethered multi-bilayer: a cell surface-mimicking substrate to probe cellular mechanosensitivity

Fate and function of anchorage-dependent cells depend on a variety of environmental cues, including those of mechanical nature. Previous progress in the understanding of cellular mechanosensitivity has been closely linked to the availability of artificial cell substrates of adjustable viscoelasticity, allowing for a direct correlation between substrate stiffness and cell response. Exemplary, polymeric gel substrates with polymer-conjugated cell-substrate linkers provided valuable insight into the role of mechanical signals during cell migration in an extracellular matrix environment. In contrast, less is known about the role of external mechanical signals across cell-cell interfaces, in part, due to the limitations of traditional polymeric substrates to mimic the remarkable dynamics of cell-cell linkages. To overcome this shortcoming, we introduce a cell surface-mimicking cell substrate of adjustable stiffness, which is comprised of a polymer-tethered lipid multi-bilayer stack with N-cadherin linkers. Unlike traditional polymeric cell substrates with polymer-conjugated linkers, this novel artificial cell substrate is able to replicate the dynamic assembly/disassembly of cadherin linkers into linker clusters and the long-range movements of cadherin-based cell-substrate linkages observed at cell-cell interfaces. Moreover, substrate stiffness can be changed by adjusting the number of bilayers in the multi-bilayer stack, thus enabling the analysis of cellular mechanosensitivity in the presence of artificial cell-cell linkages. The presented biomembrane-mimicking cell substrate provides a valuable tool to explore the functional role of mechanical cues from neighboring cells.

Changes in Cholesterol Level Alter Integrin Sequestration in Raft-Mimicking Lipid Mixtures

The influence of cholesterol (CHOL) level on integrin sequestration in raft-mimicking lipid mixtures forming coexisting liquid-ordered (lo) and liquid-disordered (ld) lipid domains is investigated using complementary, single-molecule-sensitive, confocal detection methods. Systematic analysis of membrane protein distribution in such a model membrane environment demonstrates that variation of CHOL level has a profound influence on lo-ld sequestration of integrins, thereby exhibiting overall ld preference in the absence of ligands and lo affinity upon vitronectin addition. Accompanying photon-counting histogram analysis of integrins in the different model membrane mixtures shows that the observed changes of integrin sequestration in response to variations of membrane CHOL level are not associated with altering integrin oligomerization states. Instead, our experiments suggest that the strong CHOL dependence of integrin sequestration can be attributed to CHOL-mediated changes of lipid packing and bilayer thickness in coexisting lo and ld domains, highlighting the significance of a biophysical mechanism of CHOL-mediated regulation of integrin sequestration. We envision that this model membrane study may help clarify the influence of CHOL in integrin functionality in plasma membranes, thus providing further insight into the role of lipid heterogeneities in membrane protein distribution and function in a cellular membrane environment.