Division Of Vesicles Research Articles

Stabilization of Prebiotic Vesicles by Peptides Depends on Sequence and Chirality: A Mechanism for Selection of Protocell-Associated Peptides.

Cells require oligonucleotides and polypeptides with specific, homochiral sequences to perform essential functions, but it is unclear how such oligomers were selected from random sequences at the origin of life. Cells were probably preceded by simple compartments such as fatty acid vesicles, and oligomers that increased the stability, growth, or division of vesicles could have thereby increased in frequency. We therefore tested whether prebiotic peptides alter the stability or growth of vesicles composed of a prebiotic fatty acid. We find that three of 15 dipeptides tested reduce salt-induced flocculation of vesicles. All three contain leucine, and increasing their length increases the efficacy. Also, leucine-leucine but not alanine-alanine increases the size of vesicles grown by multiple additions of micelles. In a molecular simulation, leucine-leucine docks to the membrane, with the side chains inserted into the hydrophobic core of the bilayer, while alanine-alanine fails to dock. Finally, the heterochiral forms of leucine-leucine, at a high concentration, rapidly shrink the vesicles and make them leakier and less stable to high pH than the homochiral forms do. Thus, prebiotic peptide-membrane interactions influence the flocculation, growth, size, leakiness, and pH stability of prebiotic vesicles, with differential effects due to sequence, length, and chirality. These differences could lead to a population of vesicles enriched for peptides with beneficial sequence and chirality, beginning selection for the functional oligomers that underpin life.

Abstract 5723: Selectivity profiling of small molecule kinesin inhibitors using microplate-based ATPase activity assay

Abstract Kinesin superfamily proteins (KIFs) are a large family of molecular motor proteins, that share a highly conserved motor domain. KIFs play an important role in cell division and transport of vesicles and organelles within cells. Mitotic kinesins hydrolyze ATP through its ATPase activity to move along the spindle microtubules and carry out several functions during mitosis to facilitate precise chromosome segregation. Altered expressions of several kinesins are shown in various cancers, fueling the aggressive nature of cancers leading to genomic instability. KIF11/Eg5 overexpression is shown in various cancers including hepatic carcinoma, lung, prostate, colorectal, gastric, and pancreatic cancers. Small molecule inhibitors of KIF11/Eg5 and CENP-E are shown to result in mitotic arrest leading to apoptosis in tumor cell lines. Despite the poor clinical outcome of several Eg5 and CENP-E inhibitors entered in clinical trials, targeting kinesins remains an attractive anti-cancer therapeutic approach. Recently, an inhibitor of KIF18A has entered clinical trial for treatment of solid tumors with high chromosomal instability. Here we show development of a microplate based biochemical screening assays for a panel of kinesin motor domain proteins. We summarize kinetic characterization of kinesin proteins and study the selectivity of various previously reported KIF18A, Eg5, CENP-E inhibitors across a kinesin selectivity panel. Citation Format: Brenna P. Lee, Sung Won Oh, Kurumi Y. Horiuchi, Safnas F. AbdulSalam. Selectivity profiling of small molecule kinesin inhibitors using microplate-based ATPase activity assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5723.

Concepts of a synthetic minimal cell: Information molecules, metabolic pathways, and vesicle reproduction.

How do the living systems emerge from non-living molecular assemblies? What physical and chemical principles supported the process? To address these questions, a promising strategy is to artificially reconstruct living cells in a bottom-up way. Recently, the authors developed the "synthetic minimal cell" system showing recursive growth and division cycles, where the concepts of information molecules, metabolic pathways, and cell reproduction were artificially and concisely redesigned with the vesicle-based system. We intentionally avoided using the sophisticated molecular machinery of the biological cells and tried to redesign the cells in the simplest forms. This review focuses on the similarities and differences between the biological cells and our synthetic minimal cell concerning each concept of cells. Such comparisons between natural and artificial cells will provide insights on how the molecules should be assembled to create living systems to the wide readers in the field of synthetic biology, artificial cells, and protocells research. This review article is an extended version of the Japanese article "Growth and division of vesicles coupled with information molecules," published in SEIBUTSU-BUTSURI vol. 61, p. 378-381 (2021).

Mitochondrial dynamics caused by QoIs and SDHIs fungicides depended on FgDnm1 in Fusarium graminearum

Fusarium head blight (FHB) caused by Fusarium graminearum is a devastating fungal disease on small grain cereal crops, because it reduces yield and quality and causes the mycotoxin contamination to the grain. Dynamins and dynamin-related proteins (DRPs) are large GTPase superfamily members, which are typically involved in the budding and division of vesicles in eukaryotic cells, but their roles in Fusarium spp. remain unexplored. Here, we found that FgDnm1, a DRP and homolog to Dnm1 in Saccharomyces cerevisiae, contributes to the normal fungal growth, sexual reproduction and sensitivity to fungicides. In addition, we found FgDnm1 co-localizes with mitochondria and is involved in toxisome formation and deoxynivalenol (DON) production. Several quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) cause fragmentated morphology of mitochondria. Importantly, the deletion of FgDnm1 displays filamentous mitochondria and blocks the mitochondrial fragmentation induced by QoIs and SDHIs. Taken together, our studies uncover the effect of mitochondrial dynamics in fungal normal growth and how such events link to fungicides sensitivity and toxisome formation. Thus, we concluded that altered mitochondrial morphology induced by QoIs and SDHIs depends on FgDnm1.

Shape Deformation, Budding and Division of Giant Vesicles and Artificial Cells: A Review.

The understanding of the shape-change dynamics leading to the budding and division of artificial cells has gained much attention in the past few decades due to an increased interest in designing stimuli-responsive synthetic systems and minimal models of biological self-reproduction. In this respect, membranes and their composition play a fundamental role in many aspects related to the stability of the vesicles: permeability, elasticity, rigidity, tunability and response to external changes. In this review, we summarise recent experimental and theoretical work dealing with shape deformation and division of (giant) vesicles made of phospholipids and/or fatty acids membranes. Following a classic approach, we divide the strategies used to destabilise the membranes into two different types, physical (osmotic stress, temperature and light) and chemical (addition of amphiphiles, the addition of reactive molecules and pH changes) even though they often act in synergy when leading to a complete division process. Finally, we review the most important theoretical methods employed to describe the equilibrium shapes of giant vesicles and how they provide ways to explain and control the morphological changes leading from one equilibrium structure to another.

Optical Membrane Control with Red Light Enabled by Red-Shifted Photolipids.

Photoswitchable phospholipids, or "photolipids", that harbor an azobenzene group in their lipid tails are versatile tools to manipulate and control lipid bilayer properties with light. So far, the limited ultraviolet-A/blue spectral range in which the photoisomerization of regular azobenzene operates has been a major obstacle for biophysical or photopharmaceutical applications. Here, we report on the synthesis of nano- and micrometer-sized liposomes from tetra-ortho-chloro azobenzene-substituted phosphatidylcholine (termed red-azo-PC) that undergoes photoisomerization on irradiation with tissue-penetrating red light (≥630 nm). Photoswitching strongly affects the fluidity and mechanical properties of lipid membranes, although small-angle X-ray scattering and dynamic light scattering measurements reveal only a minor influence on the overall bilayer thickness and area expansion. By controlling the photostationary state and the photoswitching efficiency of red-azo-PC for specific wavelengths, we demonstrate that shape transitions such as budding or pearling and the division of cell-sized vesicles can be achieved. These results emphasize the applicability of red-azo-PC as a nanophotonic tool in synthetic biology and for biomedical applications.

Remodeling of Membrane Shape and Topology by Curvature Elasticity and Membrane Tension.

Cellular membranes exhibit a fascinating variety of different morphologies, which are continuously remodeled by transformations of membrane shape and topology. This remodeling is essential for important biological processes (cell division, intracellular vesicle trafficking, endocytosis) and can be elucidated in a systematic and quantitative manner using synthetic membrane systems. Here, recent insights obtained from such synthetic systems are reviewed, integrating experimental observations and molecular dynamics simulations with the theory of membrane elasticity. The study starts from the polymorphism of biomembranes as observed for giant vesicles by optical microscopy and small nanovesicles in simulations. This polymorphism reflects the unusual elasticity of fluid membranes and includes the formation of membrane necks or fluid 'worm holes'. The proliferation of membrane necks generates stable multi-spherical shapes, which can form tubules and tubular junctions. Membrane necks are also essential for the remodeling of membrane topology via membrane fission and fusion. Neck fission can be induced by fine-tuning of membrane curvature, which leads to the controlled division of giant vesicles, and by adhesion-induced membrane tension as observed for small nanovesicles. Challenges for future research include the interplay of curvature elasticity and membrane tension during membrane fusion and the localization of fission and fusion processes within intramembrane domains.

Growth and Division of Vesicles Coupled with Information Molecules

Growth and Division of Vesicles Coupled with Information Molecules

Controlled division of cell-sized vesicles by low densities of membrane-bound proteins

The proliferation of life on earth is based on the ability of single cells to divide into two daughter cells. During cell division, the plasma membrane undergoes a series of morphological transformations which ultimately lead to membrane fission. Here, we show that analogous remodeling processes can be induced by low densities of proteins bound to the membranes of cell-sized lipid vesicles. Using His-tagged fluorescent proteins, we are able to precisely control the spontaneous curvature of the vesicle membranes. By fine-tuning this curvature, we obtain dumbbell-shaped vesicles with closed membrane necks as well as neck fission and complete vesicle division. Our results demonstrate that the spontaneous curvature generates constriction forces around the membrane necks and that these forces can easily cover the force range found in vivo. Our approach involves only one species of membrane-bound proteins at low densities, thereby providing a simple and extendible module for bottom-up synthetic biology.

Dynamics of Growth and Form in Prebiotic Vesicles.

The growth, form, and division of prebiotic vesicles, membraneous bags of fluid of varying components and shapes is hypothesized to have served as the substrate for the origin of life. The dynamics of these out-of-equilibrium structures is controlled by physicochemical processes that include the intercalation of amphiphiles into the membrane, fluid flow across the membrane, and elastic deformations of the membrane. To understand prebiotic vesicular forms and their dynamics, we construct a minimal model that couples membrane growth, deformation, and fluid permeation, ultimately couched in terms of two dimensionless parameters that characterize the relative rate of membrane growth and the membrane permeability. Numerical simulations show that our model captures the morphological diversity seen in extant precursor mimics of cellular life, and might provide simple guidelines for the synthesis of these complex shapes from simple ingredients.

Budding and Division of Giant Vesicles Linked to Phospholipid Production

The self-reproduction of supramolecular assemblies based on the synthesis and self-assembly of building blocks is a critical step towards the construction of chemical systems with autonomous, adaptive, and propagation properties. In this report, we demonstrate that giant vesicles can grow and produce daughter vesicles by synthesizing and incorporating phospholipids in situ from ad-hoc precursors. Our model involves acyl chain elongation via copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition reaction and the ensuing production of synthetic phospholipids to induce budding and division. In addition, the growth and budding of giant vesicles were compatible with the encapsulation and transfer of macromolecules as large as lambda phage DNA to the buds. This chemical system provides a useful model towards the implementation of cell-like compartments capable of propagation and transport of biological materials.

Role of Inverse-Cone-Shape Lipids in Temperature-Controlled Self-Reproduction of Binary Vesicles

We investigate a temperature-driven recursive division of binary giant unilamellar vesicles (GUVs). During the heating step of the heating-cooling cycle, the spherical mother vesicle deforms to a budded limiting shape using up the excess area produced by the chain melting of the lipids and then splits off into two daughter vesicles. Upon cooling, the daughter vesicle opens a pore and recovers the spherical shape of the mother vesicle. Our GUVs are composed of DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine) and DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine). During each cycle, vesicle deformation is monitored by a fast confocal microscope and the images are analyzed to obtain the time evolution of reduced volume and reduced monolayer area difference as the key geometric parameters that quantify vesicle shape. By interpreting the deformation pathway using the area-difference elasticity theory, we conclude that vesicle division relies on (1) a tiny asymmetric distribution of DLPE within the bilayer, which controls the observed deformation from the sphere to the budded shape; and (2) redistribution of DLPE during the deformation-division stage, which ensures that the process is recursive. The spontaneous coupling between membrane curvature and PE lipid distribution is responsible for the observed recursive division of GUVs. These results shed light on the mechanisms of vesicle self-reproduction.

Synapsin Selectively Controls the Mobility of Resting Pool Vesicles at Hippocampal Terminals

Presynaptic terminals are specialized sites for information transmission where vesicles fuse with the plasma membrane and are locally recycled. Recent work has extended this classical view, with the observation that a subset of functional vesicles is dynamically shared between adjacent terminals by lateral axonal transport. Conceptually, such transport would be expected to disrupt vesicle retention around the active zone, yet terminals are characterized by a high-density vesicle cluster, suggesting that counteracting stabilizing mechanisms must operate against this tendency. The synapsins are a family of proteins that associate with synaptic vesicles and determine vesicle numbers at the terminal, but their specific function remains controversial. Here, using multiple quantitative fluorescence-based approaches and electron microscopy, we show that synapsin is instrumental for resisting vesicle dispersion and serves as a regulatory element for controlling lateral vesicle sharing between synapses. Deleting synapsin disrupts the organization of presynaptic vesicle clusters, making their boundaries hard to define. Concurrently, the fraction of vesicles amenable to transport is increased, and more vesicles are translocated to the axon. Importantly, in neurons from synapsin knock-out mice the resting and recycling pools are equally mobile. Synapsin, when present, specifically restricts the mobility of resting pool vesicles without affecting the division of vesicles between these pools. Specific expression of synapsin IIa, the sole isoform affecting synaptic depression, rescues the knock-out phenotype. Together, our results show that synapsin is pivotal for maintaining synaptic vesicle cluster integrity and that it contributes to the regulated sharing of vesicles between terminals.

Tubulin acetyltransferase discovered: Ciliary role in the ancestral eukaryote expanded to neurons in metazoans

The emergence of α- and β-tubulins from an FtsZ-like prokaryotic homolog represented a defining event in eukaryogenesis. It permitted, together with actin and molecular motors, the establishment of a cytoskeleton able to support dynamic events such as cell division and trafficking of vesicles and proteins. It also served as the foundation for the microtubule-based cilium, an organelle with motility and sensory functions that replaced the less versatile prokaryotic flagellum (1). As it turns out, a mechanism to stabilize the ciliary axoneme, involving acetylation of α-tubulin, evolved in the ancestral eukaryote (2–4). This reversible modification situated inside the microtubule lumen still operates in extant ciliated protists and is exploited by metazoans to support ancillary microtubule functions, particularly in neurons (5). The concurrent discovery of an α-tubulin acetyltransferase by two separate groups, published in PNAS (3) and in Nature (4), now paves the way for a detailed understanding of the cellular and physiological roles conferred by tubulin acetylation.

Inhibition of Kinesin Motor Proteins by Adociasulfate-2

Kinesin motor proteins are involved in cell division and intracellular transport of vesicles and organelles, and as such, they play a role in neurological disease, cancer, and developmental disorders. Inhibitors of kinesin would be valuable as probes of cell physiology and as potential therapeutics. Adociasulfate-2 (AS-2) is the only known natural product inhibitor of kinesins, but its mechanism of action is unknown. We utilized kinetic studies, dynamic light scattering, and transmission electron microscopy to investigate the inhibitory action of AS-2. Our data suggest that AS-2 is not a classical 1:1 inhibitor. Instead, a rodlike aggregate that mimics microtubules is complexed with kinesin and inhibits its ATPase activity. An intriguing implication of this hypothesis is that aggregates of a chiral natural product can have interesting and biologically relevant properties. This mode of action might represent one way in which a small molecule can disrupt a protein-protein interaction.

Mechanical analyses of morphological and topological transformation of liposomes

Liposomes are micro-compartments made of lipid bilayer membranes possessing the characteristics quite similar to those of biological membranes. To form artificial cell-like structures, we made liposomes that contained subunit proteins of cytoskeletons: tubulin or actin. Spherical liposomes were transformed into bipolar or cell-like shapes by mechanical forces generated by the polymerization of encapsulated subunits of microtubules. On the other hand, disk- or dumbbell-shaped liposomes were developed by the polymerization of encapsulated actin. Dynamic processes of morphological transformations of liposomes were visualized by high intensity dark-field light microscopy. Topological changes, such as fusion and division of membrane vesicles, play an essential role in cellular activities. To investigate the mechanism of these processes, we visualized the liposomes undergoing topological transformation in real time. A variety of novel topological transformations were found, including the opening-up of liposomes and the direct expulsion of inner vesicles.

Morphological and topological transformation of membrane vesicles.

Liposomes are micro-compartments made of lipid bilayer membranes withcharacteristics quite similar to those of biological membranes. To formartificial cell-like structures, we generated liposomes that containedsubunit proteins of cytoskeletons: tubulin or actin. Spherical liposomeswere transformed into bipolar or cell-like shapes by mechanical forcesgenerated by the polymerization of encapsulated subunits of microtubules.Disk- or dumbbell-shaped liposomes were developed by the polymerizationof encapsulated actin. Dynamic processes of morphological transformationsof liposomes were visualized by high intensity dark-field lightmicroscopy.Topological changes, such as fusion and division of membrane vesicles,play an essential role in cellular activities. To investigate themechanism of these processes, we visualized in real time the liposomesundergoing topological transformation. A variety of novel topologicaltransformations were found, including the opening-up of liposomes and thedirect expulsion of inner vesicles.

The synaptology of the granule cells of the olfactory bulb.

ABSTRACT The synapses related to the granule cells of the olfactory bulb of rat brain have been studied in aldehyde-fixed material. The synapses can be divided into three classes: (1) those which have asymmetrical synaptic membrane thickenings and spheroidal synaptic vesicles; (2) those with symmetrical synaptic thickenings and flattened vesicles; and (3) the reciprocal synapses, one half of which (from mitral to granule cell) has an asymmetrical synaptic thickening associated with spheroidal vesicles, while the other half (from granule to mitral cell) has a symmetrical synaptic thickening and flattened vesicles. Qualitative observations, supported by preliminary quantitative measurements, suggest that it may be possible to divide both the spheroidal and flattened-vesicle types into two further varieties, on the basis of size, The smaller variety of spheroidal vesicles is found in most axon terminals, while the larger spheroidal vesicles are present in mitral cell dendrites and in some of the axon terminals. The flattened vesicles associated with symmetrical synapses which are oriented on to the granule cells are smaller than the spheroidal vesicles, but the flattened vesicles in the spines and gemmules of the granule cells are the same size or larger than the spheroidal vesicles. The division of flattened vesicles into two sizes is supported by statistical analysis of measurements of these vesicles, but because of difficulty in identifying the axon terminals with asymmetrical synapses there is no quantitative evidence for such a division of spheroidal vesicles. The asymmetrical synapses are found predominantly on spines, gemmules, and dendritic varicosities, although they are occasionally present on shafts of dendrites and on the cell somata. The symmetrical synapses are almost completely restricted to the shafts of the peripheral processes and the deep dendrites, and to the cell somata; only very rarely are synapses of this type found on spines, and then always in conjunction with an asymmetrical synapse.