Protein-mediated membrane remodeling

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Cell-cell fusion in animal development and in pathophysiology involves expansion of nascent fusion pores formed by protein fusogens to yield an open lumen of cell-size diameter. Earlier work on fusion mechanisms has been focused on the initial stages of the fusion pathways, which yield fusion pores of a few nanometers in diameter. In syncytium formation these pores expand to pores that are readily detectable by fluorescence microscopy (diameter >0.2 m) and finally yield an open lumen of cell-size diameter (10-15 m). Little is known about the properties of these larger pores and the mechanisms that underlie the enlargement of cytoplasmic bridges from early fusion pores to syncytia. For instance, we still do not know whether this enlargement is driven by the fusogens, proceeds spontaneously, or is driven by the cytoskeleton (Podbilewicz and White, 1994; Zheng and Chang, 1991), membrane tension (Knutton, 1980), or another, as-yet unidentified, cell machinery. [unreadable] [unreadable] In our most recent work we explored the enlargement of micron-scale pores in syncytium formation initiated by a well characterized fusogen baculovirus gp64. Low pH-triggered conformational change in gp64 results in a fast opening of fusion pores and inactivation of the fusogen (Markovic et al., 1998; Plonsky et al., 1999; Plonsky and Zimmerberg, 1996). Thus, this system has allowed effective uncoupling of fusogen-specific early fusion stages from the pore expansion stages. To visualize the three-dimensional morphological changes in the contact zone during the opening and expansion of the fusion pore(s), we labeled Sf9Op1D cells expressing gp64 with fluorescent lipids and imaged the cells with 3D time-lapse confocal microscopy as they underwent fusion. The development of fusion pores large enough to be detected by light microscopy was analyzed both in live-cell experiments and after fixing cells. [unreadable] [unreadable] The pores were roughly circular and expanded radially until they come close to one another, whence their shapes begin to distort. Fusion pore expansion within the zone of tight contact was accompanied by an increase in the cell contact area suggesting that fusion pores grow by displacement of membrane material towards the contact zone periphery. Pore growth is driven neither by membrane tension, nor by microtubule cytoskeleton but depends on cell metabolism and is accompanied by a local disassembly of the actin cortex under the pores. Polymerization and depolymerization of actin filaments respectively inhibited and promoted pore expansion and syncytium formation, indicating that the actin cytoskeleton restricts rather than drives the expansion of fusion pores. We propose that the growth of the strongly bent fusion pore rim is restricted by a dynamic resistance of the actin network and driven by membrane-bending proteins involved in the generation of highly curved intracellular membrane compartments. [unreadable] [unreadable] In another project, we have explored mechanisms by which arginine-rich cell-penetrating peptides (CPP) traverse cell membrane and reach cytosol and nucleus. These peptides have been shown to facilitate intracellular delivery of conjugated (or fused) macromolecules, while retaining their biological activity. In our earlier work we have established that cationic CPP enter cell by endocytosis. The mechanisms by which CPP cross endosomal membrane to be delivered into cytosol and nucleus remain unknown. Recent studies have indicated that cationic peptides such as oligoarginines and Tat 48-60 are rather inefficient in transporting uncharged oligonucleotide (ON) analogs such as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO) for a large part because CPP-conjugated material remained entrapped in endocytic vesicles. Since redirecting the splicing machinery through the hybridization of high affinity, RNase-incompetent PNA and PMO might lead to important clinical applications, our collaborators have recently explored efficiency of splicing correction by a number of PMO conjugates. Among these conjugates (R-Ahx-R)4 (Ahx standing for 6-aminohexanoic acid) was the most efficient in the tested group. Importantly, (R-Ahx-R)4-PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy.[unreadable] [unreadable] In our study we carried out structure-activity characterization of conjugated PMO aimed at identifying structural features facilitating their efficient nuclear delivery and uncovering limiting steps in the internalization pathway. A significant correlation between splicing correction efficiency, affinity for heparin and ability to destabilize model synthetic vesicles has been observed but no correlation with efficiency of cellular uptake has been found. To evaluate ability of CPP-PMO conjugates to escape from endosomes we employed liposome leakage assay. It is known that late endosomes are characterized by a rather unusual lipid composition enriched in bis(monooleoylglycero) phosphate and have acidified lumen with pH 5.5. We therefore prepared liposomes from the lipid mixture mimicking lipid composition of late endosomes and explored the effect of low pH on the CPP-PMO induced dye escape. In correlation with the data on splicing activity, (R-Ahx-R)4-PMO conjugate induced significantly more low pH-dependent leakage than other tested conjugates. Interestingly, more hydrophobic (R-AbuL-R)4-PMO (with Abu standing for 4-aminobutyric acid) induced considerably less leakage than (R-Ahx-R)4-PMO. Our findings indicate that efficiency of delivery the splice-redirecting ON analogs correlate with their ability to destabilize membranes with lipid composition mimicking that of the late endosomal membranes. [unreadable] [unreadable] Our work substantiates the hypothesis that entry of CPP proceeds through binding to cell surface heparin sulfates, endocytosis and escape for endosomal compartments with the latter stage to dramatically limit the efficiency of the entry. We expect future work on the mechanisms of low pH and lipid composition-dependent endosomal escape to bring about better cell-penetrating reagents required for drug delivery and provide new insights into intracellular trafficking of internalized material.