Polarity is a fundamental feature of almost all cells. It generally
refers to the asymmetric organization of several cellular
components. The plasma membrane, for example, exhibits both a
transbilayer and a lateral asymmetry in most eukaryotic cells.
Lipids are asymmetrically distributed between the cytoplasmic and
the extracellular leaflet of the membrane and segregate laterally
together with specific proteins to form dynamic nanoscale
assemblies, known as rafts. Polarity can also specifically describe
the asymmetric distribution of key molecules within a cell. These
molecules, known as polarity determinants, can orient a multitude
of specialized cellular functions, such as cell shape, cell
division and fate determination. In the framework of this thesis,
we aimed to reconstitute essential features of membrane unmixing
and cell polarity with a "bottom-up" synthetic biology approach. We
worked with both: pure lipid systems, whose unmixing is driven by
the asymmetric distribution of lipids in the two leaflets, and a
lipid-protein system, whose polarization is instead due to
reaction-diffusion mechanisms. In both cases, we used Giant
Unilamellar Vesicles (GUVs) and Sup- ported Lipid Bilayers (SLBs)
to model biological membranes and employed modern biophys- ical
techniques, such as fluorescence correlation spectroscopy, to
quantitatively characterize lipid bilayers and protein-lipid
interactions. In the pure lipid systems, we first reconstituted
membrane transbilayer asymmetry, applying a cyclodextrin-mediated
lipid exchange method, which enables us to enrich membranes with
lipids of choice. The enrichment of the membrane with sphingomyelin
and/or cholesterol triggers the segregation of lipids into two
coexisting asymmetric phases both in SLBs and GUVs, whereas
exchanging different amounts of phosphatidylglycerol with the outer
leaflet of the GUV membranes controls vesicle shape. Tuning the
lipid content of model membranes revealed that small changes in the
composition of one leaflet affect the overall lipid miscibility of
the bilayer and that membrane shape transformations are possible
also in absence of a protein machinery and as a consequence of the
lipid redistribution in the membrane. In the protein-lipid system,
we aimed to reconstitute a minimal polarization system inspired by
the C. elegans embryo at one-cell stage, which polarize along the
anterior-posterior axis by sorting the PARtitioning defective (PAR)
proteins into two distinct cortical domains. In this system
polarity is maintained by the mutual inhibition between anterior
(aPARs: PAR-3, PAR-6 and PKC-3) and posterior (pPARs: PAR-1, PAR-2
and LGL-1) PARs, which reciprocally antagonize their binding to the
cortex, mutually excluding each other. We focused on LGL-1, which
acts directly on PAR-6. Submitting LGL-1 to model membranes allowed
us to identify a conserved region of the protein that binds
negatively-charged membranes and to determine its lipid binding
affinity and specificity. Selected LGL-1 mutants were then gen-
erated to better understand the electrostatic mechanism involved in
the membrane binding. LGL-1 was finally combined with PKC-3 to
generate a functional membrane binding switch.
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