Electrostimulated cell fusion in cell engineering

A survey of studies on reconstructions of animal and plant cells which apply a new physical method--electrostimulated fusion, is presented. Effects of different factors of the medium on the efficiency of electrofusion is discussed. A detailed account is given of the authors' studies on zygotes reconstruction by combined methods of microsurgery and electrostimulated cell fusion. Advantages of the latter as compared to the widely distributed methods of fusion by polyethylenglycol and Sendai virus are considered. This physical method can play an important role in the progress of cellular engineering.     

Physical characterization of ilv-lac fusions.

Electron microscopic heteroduplex analysis and comparative restriction digests have been used to characterize lambda p123(209), the complementary pair of phages used in the Casadaban technique of gene fusion. Derivatives of lambda 1(209) constructed to carry fusions of the lac genes to the control regions of the ilvC and ilvEDA operons were also analyzed. These physical maps have provided confirmation of the genetic models for these constructions and physical specifications important in interpreting the behavior of these ilv-lac fusions.     

Peptide models for the membrane destabilizing actions of viral fusion proteins.

The fusion of enveloped viruses to target membranes is promoted by certain viral fusion proteins. However, many other proteins and peptides stabilize bilayer membranes and inhibit membrane fusion. We have evaluated some characteristics of the interaction of peptides that are models of segments of measles and influenza fusion proteins with membranes. Our results indicate that these models of the fusogenic domains of viral fusion proteins promote conversion of model membrane bilayers to nonbilayer phases. This is opposite to the effects of peptides and proteins that inhibit viral fusion. A peptide model for the fusion segment of the HA protein of influenza increased membrane leakage as well as promoted the formation of nonbilayer phases upon acidification from pH 7-5. We analyze the gross conformational features of the peptides, and speculate on how these conformational features relate to the structures of the intact proteins and to their role in promoting membrane fusion.     

Multi-step formation of a hemifusion diaphragm for vesicle fusion revealed by all-atom molecular dynamics simulations

Membrane fusion is essential for intracellular trafficking and virus infection, but the molecular mechanisms underlying the fusion process remain poorly understood. In this study, we employed all-atom molecular dynamics simulations to investigate the membrane fusion mechanism using vesicle models which were pre-bound by inter-vesicle Ca^2 +-lipid clusters to approximate Ca^2 +-catalyzed fusion. Our results show that the formation of the hemifusion diaphragm for vesicle fusion is a multi-step event. This result contrasts with the assumptions made in most continuum models. The neighboring hemifused states are separated by an energy barrier on the energy landscape. The hemifusion diaphragm is much thinner than the planar lipid bilayers. The thinning of the hemifusion diaphragm during its formation results in the opening of a fusion pore for vesicle fusion. This work provides new insights into the formation of the hemifusion diaphragm and thus increases understanding of the molecular mechanism of membrane fusion. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Physical maturity in common bottlenose dolphins (Tursiops truncatus) from Sarasota Bay,Florida

Cetacean physical maturity is defined by growth cessation and complete fusion of epiphyses to vertebral bodies indicated by invisible sutures. Many studies have shown epiphyseal fusion is highly variable among individuals. In-depth examinations into fusion variability are lacking. We analyzed vertebrae of 37 (n = 21 female, n = 16 male) stranded common bottlenose dolphins (Tursiops truncatus) from the well-studied Gulf of Mexico, Sarasota Bay community. For each specimen, vertebrae were examined by vertebral region for degree of fusion anteriorly and posteriorly of each centrum and categorized from unfused to fused in five degrees. An ordinal logistic regression was used to estimate degree of fusion probability for each epiphysis. The model had fixed effects for age, number of offspring, sex, sexual maturity, and a random effect for epiphysis. Results show that age/reproductive status significantly explains an individual's degree of fusion. Adult females with fewer calves had more fusion than those with more reproductive experience across multiple ages. Access to long-term observational and sample data on the dolphins residing in the area served by Mote Marine Laboratory's Stranding Investigations Program offers a unique opportunity to examine the relationship between energetic demands of reproduction (calcium production/reproductive output) versus preconceived definitions of physical maturity (skeletal fusion) more closely.     

Structural insights into the molecular mechanism of Ca(2+)-dependent exocytosis

The fusion of vesicles with target membranes is controlled by a complex network of protein-protein and protein-lipid interactions. Recent structures of the SNARE complex, synaptotagmin III, nSec1, domains of NSF and its adaptor SNAP, along with Rab3 and some of its effectors, provide the framework for developing molecular models of vesicle fusion and for designing experiments to test these models. Ultimately, this knowledge of the structures of higher-order complexes and their dynamic behavior will allow us to obtain a full understanding of the vesicle fusion protein machinery.     

Nuclear fusion during yeast mating occurs by a three-step pathway

In Saccharomyces cerevisiae, mating culminates in nuclear fusion to produce a diploid zygote. Two models for nuclear fusion have been proposed: a one-step model in which the outer and inner nuclear membranes and the spindle pole bodies (SPBs) fuse simultaneously and a three-step model in which the three events occur separately. To differentiate between these models, we used electron tomography and time-lapse light microscopy of early stage wild-type zygotes. We observe two distinct SPBs in ~80% of zygotes that contain fused nuclei, whereas we only see fused or partially fused SPBs in zygotes in which the site of nuclear envelope (NE) fusion is already dilated. This demonstrates that SPB fusion occurs after NE fusion. Time-lapse microscopy of zygotes containing fluorescent protein tags that localize to either the NE lumen or the nucleoplasm demonstrates that outer membrane fusion precedes inner membrane fusion. We conclude that nuclear fusion occurs by a three-step pathway.     

Structural insights into the molecular mechanism of calcium-dependent vesicle-membrane fusion

The fusion of vesicles with target membranes is controlled by a complex network of protein-protein and protein-lipid interactions. Recently determined structures of the SNARE complex, synaptotagmin III, nSec1, domains of the NSF chaperone and its adaptor (SNAP), and Rab3 and some of its effectors provide the framework for developing molecular models of vesicle fusion and for designing experiments to test these models. Ultimately, knowledge of the structures of higher-order complexes and their dynamic behavior will be required to obtain a full understanding of the vesicle fusion protein machinery.     

Finite Element Analysis of Minimal Invasive Transforaminal Lumbar Interbody Fusion

The purpose of our study is to develop and validate three-dimensional finite element models of transforaminal lumbar interbody fusion, and explore the most appropriate method of fixation and fusion by comparing biomechanical characteristics of different fixation method. We developed four fusion models: bilateral pedicle screws fixation with a single cage insertion model (A), bilateral pedicle screws fixation with two cages insertion model (B), unilateral pedicle screws fixation with a single cage insertion model (C), and unilateral pedicle screws fixation with two cages insertion model (D); the models were subjected to different forces including anterior bending, posterior extension, left bending, right bending, rotation, and axial compressive. The von Mises stress of the fusion segments on the pedicle screw and cages was recorded. Angular variation and stress of pedicle screw and cage were compared. There were differences of Von Mises peak stress among four models, but were within the range of maximum force. The angular variation in A, B, C, and D decreased significantly compared with normal. There was no significant difference of angular variation between A and B, and C and D. Bilateral pedicle screws fixation had more superior biomechanics than unilateral pedicle screws fixation. In conclusion, the lumbar interbody fusion models were established using varying fixation methods, and the results verified that unilateral pedicle screws fixation with a single cage could meet the stability demand in minimal invasive transforaminal interbody fusion.     

Differences in 3D vs. 2D analysis in lumbar spinal fusion simulations

Lumbar interbody fusion is currently the gold standard in treating patients with disc degeneration or segmental instability. Despite it having been used for several decades, the non-union rate remains high. A failed fusion is frequently attributed to an inadequate mechanical environment after instrumentation. Finite element (FE) models can provide insights into the mechanics of the fusion process. Previous fusion simulations using FE models showed that the geometries and material of the cage can greatly influence the fusion outcome. However, these studies used axisymmetric models which lacked realistic spinal geometries. Therefore, different modeling approaches were evaluated to understand the bone-formation process.Three FE models of the lumbar motion segment (L4–L5) were developed: 2D, Sym-3D and Nonsym-3D. The fusion process based on existing mechano-regulation algorithms using the FE simulations to evaluate the mechanical environment was then integrated into these models. In addition, the influence of different lordotic angles (5, 10 and 15°) was investigated. The volume of newly formed bone, the axial stiffness of the whole segment and bone distribution inside and surrounding the cage were evaluated.In contrast to the Nonsym-3D, the 2D and Sym-3D models predicted excessive bone formation prior to bridging (peak values with 36 and 9% higher than in equilibrium, respectively). The 3D models predicted a more uniform bone distribution compared to the 2D model.The current results demonstrate the crucial role of the realistic 3D geometry of the lumbar motion segment in predicting bone formation after lumbar spinal fusion.     

Exocytotic fusion pore stability and topological defects in the membrane with orientational degree of ordering

Regulated exocytosis is a process that strongly depends on the formation and stability of the fusion pore. It was indicated experimentally and theoretically that narrow and highly curved fusion pore may be stabilized by accumulation of anisotropic membrane components possessing orientational ordering. On the other hand, narrow fusion pore may also undergo repetitive opening and closing, disruption in the so called kiss and run process or become completely opened in the process of full fusion of the vesicle with the membrane. In this paper we attempt to elucidate the subtle interplay between the stabilizing and destabilizing processes in the fusion neck. A possible physical mechanism which may lead to disruption of the stable fusion pore or complete fusion of the vesicle with the membrane is discussed. It is indicated that topologically driven defects of the in-plane orientational membrane ordering in the region of the fusion pore may disrupt the fusion. Alternatively, it may facilitate repetitive opening and closing of the fusion pore or induce full fusion of the vesicle with the target membrane.     

Liposome reconstitution of a minimal protein-mediated membrane fusion machine

Biological membrane fusion is dependent on protein catalysts to mediate localized restructuring of lipid bilayers. A central theme in current models of protein-mediated membrane fusion involves the sequential refolding of complex homomeric or heteromeric protein fusion machines. The structural features of a new family of fusion-associated small transmembrane (FAST) proteins appear incompatible with existing models of membrane fusion protein function. While the FAST proteins function to induce efficient cell-cell fusion when expressed in transfected cells, it was unclear whether they function on their own to mediate membrane fusion or are dependent on cellular protein cofactors. Using proteoliposomes containing the purified p14 FAST protein of reptilian reovirus, we now show via liposome-cell and liposome-liposome fusion assays that p14 is both necessary and sufficient for membrane fusion. Stoichiometric and kinetic analyses suggest that the relative efficiency of p14-mediated membrane fusion rivals that of the more complex cellular and viral fusion proteins, making the FAST proteins the simplest known membrane fusion machines.     

Single Vesicle Millisecond Fusion Kinetics Reveals Number of SNARE Complexes Optimal for Fast SNARE-mediated Membrane Fusion

SNAREs mediate membrane fusion in intracellular vesicle traffic and neuronal exocytosis. Reconstitution of membrane fusion in vitro proved that SNAREs constitute the minimal fusion machinery. However, the slow fusion rates observed in these systems are incompatible with those required in neurotransmission. Here we present a single vesicle fusion assay that records individual SNARE-mediated fusion events with millisecond time resolution. Docking and fusion of reconstituted synaptobrevin vesicles to target SNARE complex-containing planar membranes are distinguished by total internal reflection fluorescence microscopy as separate events. Docking and fusion are SNAP-25-dependent, require no Ca²⁺, and are efficient at room temperature. Analysis of the stochastic data with sequential and parallel multi-particle activation models reveals six to nine fast-activating steps. Of all the tested models, the kinetic model consisting of eight parallel reaction rates statistically fits the data best. This might be interpreted by fusion sites consisting of eight SNARE complexes that each activate in a single rate-limiting step in 8 ms.     

Molecular mechanism of mitochondrial membrane fusion

Mitochondrial fusion requires coordinated fusion of the outer and inner membranes. This process leads to exchange of contents, controls the shape of mitochondria, and is important for mitochondrial function. Two types of mitochondrial GTPases are essential for mitochondrial fusion. On the outer membrane, the fuzzy onions/mitofusin proteins form complexes in trans that mediate homotypic physical interactions between adjacent mitochondria and are likely directly involved in outer membrane fusion. Associated with the inner membrane, the OPA1 dynamin-family GTPase maintains membrane structure and is a good candidate for mediating inner membrane fusion. In yeast, Ugo1p binds to both of these GTPases to form a fusion complex, although a related protein has yet to be found in mammals. An understanding of the molecular mechanism of fusion may have implications for Charcot-Marie-Tooth subtype 2A and autosomal dominant optic atrophy, neurodegenerative diseases caused by mutations in Mfn2 and OPA1.     

V-ATPase, ScNhx1p and yeast vacuole fusion

Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos. It is a central cellular reaction that plays important roles in signal transduction, protein sorting and subcellular compartmentation. Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summarized in this article. It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhx1p are key components of the vacuole fusion machinery in yeast. Yeast ScNhx1p regulates vacuole fusion by controlling the luminal pH. V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast. Fission defects are epistatic to fusion defects. Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast, the fusion reaction does not need the transport activity but requires the physical presence of the proton pump. V0, the membrane-integral sector of the V-ATPase, forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the V0trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion.     

Replicating vesicles as models of primitive cell growth and division

Primitive cells, lacking the complex bio-machinery present in modern cells, would have had to rely on the self-organizing properties of their components and on interactions with their environment to achieve basic cellular functions such as growth and division. Many bilayer-membrane vesicles, depending on their composition and environment, can exhibit complex morphological changes such as growth, fusion, fission, budding, internal vesicle assembly and vesicle-surface interactions. The rich dynamic properties of these vesicles provide interesting models of how primitive cellular replication might have occurred in response to purely physical and chemical forces.     

What drives membrane fusion in eukaryotes?

The fusion of biological membranes is the terminal step of all vesicular trafficking reactions in eukaryotic cells. Therefore, this fusion is fundamental for the transfer of proteins and lipids between different compartments, for exocytosis and for the structural integrity of organelles. In the past decade, many parts of the molecular machinery involved in fusion have been uncovered. Although the mechanisms responsible for mutual recognition and binding of membranes inside eukaryotes are becoming reasonably well known, there is considerable uncertainty as to what causes the actual merging of the lipid bilayer. Two classes of mechanisms have been proposed. Proximity models postulate that very close apposition of membranes suffices to induce fusion. By contrast, pore models propose that continuous proteinaceous pores between apposed membranes could be the basis for fusion.     

Kiss-and-run, collapse and 'readily retrievable' vesicles

Two models of synaptic vesicle recycling have been intensely debated for decades: kiss‐and‐run, in which the vesicle opens and closes transiently, presumably through a small fusion pore, and full fusion, in which the vesicle collapses into the plasma membrane and is retrieved by clathrin‐coat‐dependent processes. Conceptually, it seems that kiss‐and‐run would be faster and would retrieve vesicles with greater fidelity. Is this the case? This review discusses recent evidence for both models. We conclude that both mechanisms allow for high fidelity of vesicle recycling. Also, the presence in the plasma membrane of a depot of previously fused vesicles that are already interacting with the endocytotic machinery (the ‘readily retrievable’ vesicles) allows full fusion to trigger quite fast endocytosis, further blurring the efficiency differences between the two models.