Biophysical Studies |
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Sarah Veatch Fluctuations and Liquid Immiscibility in Model Membranes and In Cells |
| In my previous work, I studied the phase behavior of multi-component lipid membranes containing cholesterol. Some researchers believe that phase separated liquid domains in model membranes resemble ‘raft’ domains in cell plasma membranes. Coexisting liquid phases are easily visualized in giant of unilamellar vesicles of synthetic lipid mixtures by fluorescence microscopy (see movie for transition with change of temperature). I have mapped phase diagrams of several different ternary lipid mixtures. One interesting feature of these diagrams is that they contain critical points, and critical fluctuations are observed in the vicinity of these points (Figure 1). |
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While phase separation in simple membranes is now well understood, it is not obvious how this knowledge can be applied to lipid organization in cells. In the Baird/Holowka laboratory, I am exploring phase separation in giant plasma membrane vesicles (GPMVs) blebbed directly from the plasma membrane of living cells. Previous members of the Baird/Holowka laboratory have demonstrated that coexisting phases are found in GPMVs at low temperature (Figure 2). I am interested in characterizing this miscibility transition, and exploring if critical fluctuations are present in GPMVs at higher temperatures. If present, critical fluctuations could provide mechanism for cells to control the size, composition, and lifetime of domains in their membranes |
Figure 1(above). Phase separation is easily observed in three component model membranes containing cholesterol by fluorescence microscopy. This phase behavior can be mapped into a phase diagram which contains a critical point, and micron-scale critical fluctuations are found in vesicles with near-critical lipid compositions. Figure 2 (right).Coexisting liquid phases can also be visualized in vesicles blebbed directly from cell plasma membranes at low temperatures. At higher temperatures, there is some evidence for fluctuations in domain boundaries consistent with being near a critical point. |
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Prabuddha Sengupta Investigation of the spatial organization of proteins and lipids in live cell membrane and plasma membrane vesicles with Adam Hammond and Marc Johnson |
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The functional organization of eukaryotic plasma membranes and the mechanisms of membrane remodeling during various physiological processes is incompletely understood, and it is a topic of much discussion and debate. Recent results indicate that the plasma membrane is compartmentalized on multiple spatial and temporal scales, and the current models of membrane architecture need to be revised into a more dynamic and complex representation. Using three independent approaches, we have been studying the steady state lateral distributions of its proteins and lipids in the plasma membrane. Our results provide new insights into the diverse mechanisms that drive functional plasma membrane compartmentalization. |
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Figure 1. Long chain, unsaturated acceptor DiI-C16 (black circles) show significantly higher FRET than equimolar amount of unsaturated acceptor DiI-C18:Δ9 (red triangles) with long chain, saturated donor DiO-C18, indicating nanoscopic heterogeneity in the lateral distribution of lipid probes in live cell membrane. |
Lateral organization of lipids in resting cell membrane Using a combination of homo and hetero FRET measurements, we find that carbocyanine lipid probes are laterally segregated in the plasma membrane of RBL mast cells based on their alkyl chain structure. The lateral heterogeneities detected by the carbocyanine probes are sensitive to Lo-perturbing agents, and the results point towards the presence of nanoscopic, Lo/Ld-related compositional fluctuations in the outer leaflet of the plasma membrane of live cells (Figure 1). |
Plasma membrane vesicles We recently observed that giant plasma membrane vesicles (GPMVs) spontaneously segregate into coexisting liquid order-like and liquid disorder-like phases (Figure 2). We have exploited these phase separated GPMVs to identify and characterize the structural features that determine the partitioning of proteins and lipids between coexisting fluid phases in compositionally complex biological membranes. Our results suggest that the partitioning of lipids is dictated by a complex balance between head-group and acyl chain mediated interactions with their membrane environment. The GPMVs provide a promising model system to characterize the association of proteins with distinct membrane environments, and they provide insights into the mechanisms of protein sorting and membrane compartmentalization. |
Figure 2. Giant plasma membrane vesicles segregate into coexisting fluid phases, with Napthopyrene (Lo-marker) and Rhodamine-DOPE (Ld-marker) showing complementary partitioning. |
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Figure 3. Backscatter scanning electron image of 5nm gold particles showing spatial distribution of GPI-anchored protein Thy1 on the surface of RBL-2H3 mast cells. Antimouse secondary antibody-5nm gold conjugate was used to label OX7-bound Thy1 on the surface of chemically fixed cells. |
Nanoscopic distribution of cell surface molecules and membrane reorganization during stimulation of cells and retro viral budding The size and exact composition of nanoscopic heterogeneities present on the cell membrane are still undefined and remain outstanding questions in the field. We are using a combination of secondary electron and backscatter scanning electron imaging to identify protein and lipid nano-clusters, and to distinguish between membrane microdomains of different dimensions and composition at the cell surface (Figure 3). The detailed characterization of the nanoscopic lateral distribution of cell surface molecules reveals a surprisingly complex picture of the plasma membrane organization and points towards an important role for protein-protein interactions in modulating the composition, size, and stability of membrane microdomains. |
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