Myomerger Induces Membrane Hemifusion and Regulates Fusion Pore Expansion.

Membrane fusion is a crucial mechanism in a wide variety of important events in cell biology from viral infection to exocytosis. However, despite many efforts and much progress, cell-cell fusion has remained elusive to our understanding. Along the life of the fusion pore, large conformational changes take place from the initial lipid bilayer bending, passing through the hemifusion intermediates, and ending with the formation of the first nascent fusion pore. In this sense, computer simulations are an ideal technique for describing such complex lipid remodeling at the molecular level. In this work, we studied the role played by the muscle-specific membrane protein Myomerger during the formation of the fusion pore. We have conducted µs length atomistic and coarse-grained molecular dynamics, together with free-energy calculations using ad hoc collective variables. Our results show that Myomerger favors the hemifusion diaphragm-stalk transition, reduces the nucleation-expansion energy difference, and promotes the formation of nonenlarging fusion pores.

Selection of a novel cell-internalizing RNA aptamer specific for CD22 antigen in B cell acute lymphoblastic leukemia.

Despite improvements in B cell acute lymphoblastic leukemia (B-ALL) treatment, a significant number of patients experience relapse of the disease, resulting in poor prognosis and high mortality. One of the drawbacks of current B-ALL treatments is the high toxicity associated with the non-specificity of chemotherapeutic drugs. Targeted therapy is an appealing strategy to treat B-ALL to mitigate these toxic off-target effects. One such target is the B cell surface protein CD22. The restricted expression of CD22 on the B-cell lineage and its ligand-induced internalizing properties make it an attractive target in cases of B cell malignancies. To target B-ALL and the CD22 protein, we performed cell internalization SELEX (Systematic Evolution of Ligands by EXponential enrichment) followed by molecular docking to identify internalizing aptamers specific for B-ALL cells that bind the CD22 cell-surface receptor. We identified two RNA aptamers, B-ALL1 and B-ALL2, that target human malignant B cells, with B-ALL1 the first documented RNA aptamer interacting with the CD22 antigen. These B-ALL-specific aptamers represent an important first step toward developing novel targeted therapies for B cell malignancy treatments.

α-Synuclein is required for sperm exocytosis at a post-fusion stage.

The sperm acrosome is a large dense-core granule whose contents are secreted by regulated exocytosis at fertilization through the opening of numerous fusion pores between the acrosomal and plasma membranes. In other cells, the nascent pore generated when the membrane surrounding a secretory vesicle fuses with the plasma membrane may have different fates. In sperm, pore dilation leads to the vesiculation and release of these membranes, together with the granule contents. α-Synuclein is a small cytosolic protein claimed to exhibit different roles in exocytic pathways in neurons and neuroendocrine cells. Here, we scrutinized its function in human sperm. Western blot revealed the presence of α-synuclein and indirect immunofluorescence its localization to the acrosomal domain of human sperm. Despite its small size, the protein was retained following permeabilization of the plasma membrane with streptolysin O. α-Synuclein was required for acrosomal release, as demonstrated by the inability of an inducer to elicit exocytosis when permeabilized human sperm were loaded with inhibitory antibodies to human α-synuclein. The antibodies halted calcium-induced secretion when introduced after the acrosome docked to the cell membrane. Two functional assays, fluorescence and transmission electron microscopies revealed that the stabilization of open fusion pores was responsible for the secretion blockage. Interestingly, synaptobrevin was insensitive to neurotoxin cleavage at this point, an indication of its engagement in cis SNARE complexes. The very existence of such complexes during AE reflects a new paradigm. Recombinant α-synuclein rescued the inhibitory effects of the anti-α-synuclein antibodies and of a chimeric Rab3A-22A protein that also inhibits AE after fusion pore opening. We applied restrained molecular dynamics simulations to compare the energy cost of expanding a nascent fusion pore between two model membranes and found it higher in the absence than in the presence of α-synuclein. Hence, our results suggest that α-synuclein is essential for expanding fusion pores.

Intrinsic Disorder in α-Synuclein Regulates the Exocytotic Fusion Pore Transition.

Today, it is widely accepted that intrinsic disorder is strongly related to the cell cycle, during mitosis, differentiation, and apoptosis. Of particular interest are hybrid proteins possessing both structured and unstructured domains that are critical in human health and disease, such as α-synuclein. In this work, we describe how α-synuclein interacts with the nascent fusion pore as it evolves toward expansion. We unveil the key role played by its intrinsically disordered region as a thermodynamic regulator of the nucleation-expansion energy barrier. By analyzing a truncated variant of α-synuclein that lacks the disordered region, we find that the landscape of protein interactions with PIP2 and POPS lipids is highly altered, ultimately increasing the energy cost for the fusion pore to transit from nucleation to expansion. We conclude that the intrinsically disordered region in full-length α-synuclein recognizes and allocates pivotal protein:lipid interactions during membrane remodeling in the first stages of the fusion pore.

Enhanced Expansion and Reduced Kiss-and-Run Events in Fusion Pores Steered by Synaptotagmin-1 C2B Domains.

The fusion pore controls the release of exocytotic vesicle contents through a precise orchestration of lipids from the fusing membranes and proteins. There is a major lipid reorganization during the different stages in life of the fusion pore (membrane fusion, nucleation, and expansion) that can be scrutinized thermodynamically. In this work, using umbrella sampling simulations we describe the expansion of the fusion pore. We have calculated free energy profiles to drive a nascent, just nucleated, fusion pore to its expanded configuration. We have quantified the effects on the free energy of one and two Synaptotagmin-1 C2B domains in the cytosolic space. We show that C2B domains cumulatively reduce the cost for expansion, favoring the system to evolve toward full fusion. Finally, by conducting thousands of unbiased molecular dynamics simulations, we show that C2B domains significantly decrease the probability of kiss-and-run events.

Synaptotagmin-1 C2B domains cooperatively stabilize the fusion stalk via a master-servant mechanism.

Synaptotagmin-1 is a low-affinity Ca2+ sensor that triggers synchronous vesicle fusion. It contains two similar C2 domains (C2A and C2B) that cooperate in membrane binding, being the C2B domain mainly responsible for the membrane fusion process due to its polybasic patch KRLKKKKTTIKK (321-332). In this work, a master-servant mechanism between two identical C2B domains is shown to control the formation of the fusion stalk in a calcium-independent manner. Two regions in C2B are essential for the process, the well-known polybasic patch and a recently described pair of arginines (398 399). The master domain shows strong PIP2 interactions with its polybasic patch and its pair of arginines. At the same time, the servant analogously cooperates with the master to reduce the total work to form the fusion stalk. The strategic mutation (T328E, T329E) in both master and servant domains disrupts the cooperative mechanism, drastically increasing the free energy needed to induce the fusion stalk, however, with negligible effects on the master domain interactions with PIP2. These data point to a difference in the behavior of the servant domain, which is unable to sustain its PIP2 interactions neither through its polybasic patch nor through its pair of arginines, and in the end, losing its ability to assist the master in the formation of the fusion stalk.