Final Report Summary - MEMBRANE NANOTUBES (Determining the molecular basis for the formation of membrane nanotubes between immune cells)
The project set out to study the molecular mechanisms of membrane nanotube formation through comparison to filopodia formation. Membrane nanotubes are recently described thin, actin-containing membrane tubes that connect cells. Our data demonstrates that a filopodial tip marker myosin-10 is enriched also in the tips of the membrane nanotubes forming between Jurkat cells. Since myosin-10 preferentially associates to bundled actin on the basis of repeat distance of actin in bundles and accumulates at the ends of structures containing bundled actin (such as filopodia) [Brawley and Rock, 2009, Proc. Natl. Acad. Sci. USA 106: 9685], the result strongly suggests that Jurkat cell nanotubes contain well-organised, bundled actin. More quantitative analysis demonstrated that GFP-tagged myosin-10 heavy meromyosin construct (GFP-MYO10-HMM) that lacks the tail domains [Berg and Cheney, 2002, Nat. Cell Biol. 4: 246] is enriched in the tips of nanotubes and filopodia to nearly similar extent. GFP-MYO10-HMM overexpression increased the frequency of filopodia formation approx. 5. 3-fold compared to Jurkat transfected with only GFP. This is consistent with an earlier report of approx. 4. 9-fold increase in filopodia numbers in COS7 cells [Tokuo et al., 2007, J. Cell Biol. 179: 229]. Importantly however, the frequency of nanotube formation was unaffected by the overexpression of GFP-MYO10-HMM. We have also overexpressed in Jurkat cells other proteins (IRSp53-I-BAR, IRSp53-full, MIM) that are known to induce filopodia, but found no effect on the numbers of nanotubes. This demonstrates for the first time that there must be differences in the fundamental mechanism of nanotube and filopodia formation, though both appear to contain a backbone of bundled actin filaments. A methodological paper on nanotube work has been published [Sowinski, Alakoskela,, Jolly, and Davis, 2010, Methods in press], and the work on nanotubes in the host laboratory continues.
Perhaps the most successful part of the project has been the development and exploitation of new tools for quantitative assessment of size-based segregation in live cell conjugate immunological synapses. We tested the exclusion of fluorescent nanoparticles from synaptic clefts of immunological synapses, and our results show that nanoparticles larger than the intermembrane gap of central synaptic cleft become excluded from the central zone of the synapse. The width of the gap is defined by the lengths of the extracellular domains of immune cell receptors ligated to their ligands on target cells. We assessed the exclusion of membrane-attached fluorescent nanoparticles (diameters 6, 10, 15, 22, and 28 nm) from the central region of immunological synapse formed between an NK cell-like YTS cells transfected to express inhibitory NK cell receptor KIR2DL1 and 721. 221 B cell lymphoma cell line transfected to express HLA-Cw6-GFP. The intermembrane distance is expected to be approx. 15 nm, and, accordingly, membrane bending elasticity-originated forces are expected to drive the exclusion of particles larger than 15 nm from the central zone of the synapse, with the level of exclusion increasing with the size of the particle, in agreement with our results. We estimated the effect of lateral crowding from scaled particle theory [Chatelier and Minton, 1996, Biophys. J. 71: 2367] and found that it cannot explain the exclusion of large particles. Our results demonstrate that size-based exclusion in immune synapses is not dependent on the interactions of protein cytoplasmic tails with actin cytoskeleton, but that the size of extracellular domain is sufficient to drive segregation, as predicted by membrane bending elasticity considerations playing important role in kinetic-segregation model of immune cell activation (Davis and van der Merwe, 2006, Nat. Immunol. 7: 803). A manuscript of the results is currently in preparation for a top specialist journal, and is expected to be submitted during November 2010.
Perhaps the most successful part of the project has been the development and exploitation of new tools for quantitative assessment of size-based segregation in live cell conjugate immunological synapses. We tested the exclusion of fluorescent nanoparticles from synaptic clefts of immunological synapses, and our results show that nanoparticles larger than the intermembrane gap of central synaptic cleft become excluded from the central zone of the synapse. The width of the gap is defined by the lengths of the extracellular domains of immune cell receptors ligated to their ligands on target cells. We assessed the exclusion of membrane-attached fluorescent nanoparticles (diameters 6, 10, 15, 22, and 28 nm) from the central region of immunological synapse formed between an NK cell-like YTS cells transfected to express inhibitory NK cell receptor KIR2DL1 and 721. 221 B cell lymphoma cell line transfected to express HLA-Cw6-GFP. The intermembrane distance is expected to be approx. 15 nm, and, accordingly, membrane bending elasticity-originated forces are expected to drive the exclusion of particles larger than 15 nm from the central zone of the synapse, with the level of exclusion increasing with the size of the particle, in agreement with our results. We estimated the effect of lateral crowding from scaled particle theory [Chatelier and Minton, 1996, Biophys. J. 71: 2367] and found that it cannot explain the exclusion of large particles. Our results demonstrate that size-based exclusion in immune synapses is not dependent on the interactions of protein cytoplasmic tails with actin cytoskeleton, but that the size of extracellular domain is sufficient to drive segregation, as predicted by membrane bending elasticity considerations playing important role in kinetic-segregation model of immune cell activation (Davis and van der Merwe, 2006, Nat. Immunol. 7: 803). A manuscript of the results is currently in preparation for a top specialist journal, and is expected to be submitted during November 2010.