Martin B. Forstner

Apparent Subdiffusion Inherent to Single Particle Tracking

Subdiffusion and its causes in both in vivo and in vitro lipid membranes have become the focus of recent research. We report apparent subdiffusion, observed via single particle tracking (SPT), in a homogeneous system that only allows normal diffusion (a DMPC monolayer in the fluid state). The apparent subdiffusion arises from slight errors in finding the actual particle position due to noise inherent in all experimental SPT systems. A model is presented that corrects this artifact, and predicts the time scales after which the effect becomes negligible. The techniques and results presented in this paper should be of use in all SPT experiments studying normal and anomalous diffusion.

Noncovalent Cell Surface Engineering: Incorporation of Bioactive Synthetic Glycopolymers into Cellular Membranes

The controlled addition of structurally defined components to live cell membranes can facilitate the molecular level analysis of cell surface phenomena. Here we demonstrate that cell surfaces can be engineered to display synthetic bioactive polymers at defined densities by exogenous membrane insertion. The polymers were designed to mimic native cell-surface mucin glycoproteins, which are defined by their dense glycosylation patterns and rod-like structures. End-functionalization with a hydrophobic anchor permitted incorporation into the membranes of live cultured cells. We probed the dynamic behavior of cell-bound glycopolymers bearing various hydrophobic anchors and glycan structures using fluorescence correlation spectroscopy (FCS). Their diffusion properties mirrored those of many natural membrane-associated biomolecules. Furthermore, the membrane-bound glycopolymers were internalized into early endosomes similarly to endogenous membrane components and were capable of specific interactions with protein receptors. This system provides a platform to study cell-surface phenomena with a degree of chemical control that cannot be achieved using conventional biological tools.

Fluorescence Imaging of Membrane Dynamics

Imaging membrane dynamics is an important goal, motivated by the abundance of biochemical and biophysical events that are orchestrated at, or by, cellular membranes. The short length scales, fast timescales, and environmental requirements of membrane phenomena present challenges to imaging experiments. Several technical advances offer means to overcome these challenges, and we describe here three powerful techniques applicable to membrane imaging: total internal reflection fluorescence (TIRF) microscopy, fluorescence interference contrast (FLIC) microscopy, and fluorescence correlation spectroscopy (FCS). For each, we discuss the physics underpinning the approach, its practical implementation, and recent examples highlighting its achievements in exploring the membrane environment.

A chemical approach to unraveling the biological function of the glycosylphosphatidylinositol anchor

The glycosylphosphatidylinositol (GPI) anchor is a C-terminal posttranslational modification found on many eukaryotic proteins that reside in the outer leaflet of the cell membrane. The complex and diverse structures of GPI anchors suggest a rich spectrum of biological functions, but few have been confirmed experimentally because of the lack of appropriate techniques that allow for structural perturbation in a cellular context. We previously synthesized a series of GPI anchor analogs with systematic deletions within the glycan core and coupled them to the GFP by a combination of expressed protein ligation and native chemical ligation [Paulick MG, Wise AR, Forstner MB, Groves JT, Bertozzi CR (2007) J Am Chem Soc 129:11543–11550]. Here we investigate the behavior of these GPI-protein analogs in living cells. These modified proteins integrated into the plasma membranes of a variety of mammalian cells and were internalized and directed to recycling endosomes similarly to GFP bearing a native GPI anchor. The GPI-protein analogs also diffused freely in cellular membranes. However, changes in the glycan structure significantly affected membrane mobility, with the loss of monosaccharide units correlating to decreased diffusion. Thus, this cellular system provides a platform for dissecting the contributions of various GPI anchor components to their biological function.

Measurement of diffusion in Langmuir monolayers by single-particle tracking

There is a great amount of literature available indicating that membranes are inhomogeneous, complex fluids. For instance, observation of diffusion in cell membranes demonstrated confined motion of membrane constituents and even subdiffusion. In order to circumvent the small dimensions of cells leading to weak statistics when investigating the diffusion properties of single membrane components, a technique based on optical microscopy employing Langmuir monolayers as membrane model systems has been developed in our lab. In earlier work, the motion of labeled single lipids was visualized. These measurements with long observation times, thus far only possible with this method, were combined with respective Monte-Carlo simulations. We could conclude that noise can lead in general to the assumption of subdiffusion while interpreting the results of single-particle-tracking (SPT) experiments within membranes in general. Since the packing density of lipids within monolayers at the air/water interface can be changed easily, inhomogeneity with regard to the phase state can be achieved by isothermal compression to coexistence regions. Surface charged polystyrene latexes were used as model proteins diffusing in inhomogeneous monolayers as biomembrane mimics. Epifluorescence microscopy coupled to SPT revealed that domain associated, dimensionally reduced diffusion can occur in these kinds of model systems. This was caused by an attractive potential generated by condensed domains within monolayers. Monte-Carlo simulations supported this view point. Moreover, long-time simulations show that diffusion coefficients of respective particles were dependent on the strength of the attractive potential present: a behavior reflecting altered dimensionality of diffusion. The widths of those potentials were also found to be affected by the domain size of the more ordered lipid phase. In biological membrane systems, cells could utilize these physical mechanisms to adjust diffusion properties of membrane components.

A Biosynthetic Membrane-Anchor/Protein System Based on a Genetically Encoded "Aldehyde Tag"

The covalent binding of an aldehyde side-chain containing protein to a lipid with an aminooxy -modified head group opens a versatile avenue to bio-functionalize lipid membranes without compromising function and dynamic properties of both the protein and the lipid membrane. It was recently found that the site-specific insertion of a 6 amino acid consensus sequence into a protein is sufficient to target it for post-translational modification by formylglycine-generating enzyme (FGE). FGE will enzymatically turn the cysteine in the consensus motif into a formylglycine, thus leading to the site-specific introduction of an aldehyde side chain for further chemical modification. We have engineered the consensus sequence to the C-terminus of an Enhanced Green Fluorescence Protein (EGFP) which was co-expressed with FGE in E. Coli. Lipids were chemically modified to bear a reactive aminooxy group and then conjugated with the aldehyde tagged EGFP. The resulting EGFP-lipid constructs were successfully incorporated into solid supported lipid bilayer as verified by fluorescence microscopy. Membrane integrity as well as protein and lipid motilities were investigated using both fluorescence recovery after photo-bleaching and fluorescence correlation spectroscopy. In order to determine integration efficiency, the surface concentration of EGFP-lipid constructs was monitored as a function of their solution concentration and incubation time. This site specific lipidation strategy promises to allow for the use of a variety of possible lipid anchors as well as to provide unprecedented freedom in the choice of the lipidation site on the protein.