Spherical Buds Research Articles

Study of impacts of two types of cellular aging on the yeast bud morphogenesis.

Understanding the mechanisms of the cellular aging processes is crucial for attempting to extend organismal lifespan and for studying age-related degenerative diseases. Yeast cells divide through budding, providing a classical biological model for studying cellular aging. With their powerful genetics, relatively short cell cycle, and well-established signaling pathways also found in animals, yeast cells offer valuable insights into the aging process. Recent experiments suggested the existence of two aging modes in yeast characterized by nucleolar and mitochondrial declines, respectively. By analyzing experimental data, this study shows that cells evolving into those two aging modes behave differently when they are young. While buds grow linearly in both modes, cells that consistently generate spherical buds throughout their lifespan demonstrate greater efficacy in controlling bud size and growth rate at young ages. A three-dimensional multiscale chemical-mechanical model was developed and used to suggest and test hypothesized impacts of aging on bud morphogenesis. Experimentally calibrated model simulations showed that during the early stage of budding, tubular bud shape in one aging mode could be generated by locally inserting new materials at the bud tip, a process guided by the polarized Cdc42 signal. Furthermore, the aspect ratio of the tubular bud could be stabilized during the late stage as observed in experiments in this work. The model simulation results suggest that the localization of new cell surface material insertion, regulated by chemical signal polarization, could be weakened due to cellular aging in yeast and other cell types, leading to the change and stabilization of the bud aspect ratio.

Delineating the shape of COat Protein complex-II coated membrane bud.

Curvature-generating proteins that direct membrane trafficking assemble on the surface of lipid bilayers to bud transport intermediates, which move protein and lipid cargoes from one cellular compartment to another. However, it remains unclear what controls the overall shape of the membrane bud once curvature induction has begun. In vitro experiments showed that excessive concentrations of the COPII protein Sar1 promoted the formation of membrane tubules from synthetic vesicles, while COPII-coated transport intermediates in cells are generally more spherical or lobed in shape. To understand the origin of these morphological differences, we employ atomistic, coarse-grained (CG), and continuum mesoscopic simulations of membranes in the presence of multiple curvature-generating proteins. We first characterize the membrane-bending ability of amphipathic peptides derived from the amino terminus of Sar1, as a function of interpeptide angle and concentration using an atomistic bicelle simulation protocol. Then, we employ CG simulations to reveal that Sec23 and Sec24 control the relative spacing between Sar1 protomers and form the inner-coat unit through an attachment with Sar1. Finally, using dynamical triangulated surface simulations based on the Helfrich Hamiltonian, we demonstrate that the uniform distribution of spacer molecules among curvature-generating proteins is crucial to the spherical budding of the membrane. Overall, our analyses suggest a new role for Sec23, Sec24, and cargo proteins in COPII-mediated membrane budding process in which they act as spacers to preserve a dispersed arrangement of Sar1 protomers and help determine the overall shape of the membrane bud.

Isolation of Rhizobial Extracellular Membrane Vesicles from Bacteroids.

Extracellular-membrane vesicles (EMVs) are spherical buds of the extracellular membrane, commonly produced by Gram-negative bacteria, known to mediate intricate inter-kingdom communication. In this context, comprehensive research dissecting the role of EMVs in one of the most complex nature-occurring molecular dialogues, rhizobium-legume symbiosis, has been so far neglected. During the different stages of the symbiotic process, rhizobia and their host plants establish a very specific and controlled intercellular trafficking of signal molecules. Thus, as conveyors of a broad range of molecules into the target cell, EMVs are gaining weight in the field. Here, we describe a detailed protocol to isolate EMVs from bacteroids of legume nodules, opening a new door for discovering new authors of the symbiotic process.

Influence of the Ethanol Content of Adduct on the Comonomer Incorporation of Related Ziegler-Natta Catalysts in Propylene (Co)polymerizations.

The aim of this work is to investigate the influence of the ethanol content of adducts on the catalytic behavior of related Ziegler-Natta (ZN) catalysts in propylene homo- and copolymerizations (with 1-hexene comonomer) in terms of activity, isotacticity, H2 response, and comonomer incorporation. For this purpose, three MgCl2.nEtOH adducts with n values of 0.7, 1.2, and 2.8 were synthesized and used in the synthesis of related ZN catalysts. The catalysts were thoroughly characterized using XRD, BET, SEM, EDX, N2 adsorption-desorption, and DFT techniques. Additionally, the microstructure of the synthesized (co)polymers was distinguished via DSC, SSA, and TREF techniques. Their activity was found to enhance with the adduct's ethanol content in both homo- and copolymerization experiments, and the increase was more pronounced in homopolymerization reactions in the absence of H2. Furthermore, the catalyst with the highest ethanol content provided a copolymer with a lower isotacticity index, a shorter meso sequence length, and a more uniform distribution of comonomer within the chains. These results were attributed to the higher total surface area and Ti content of the corresponding catalyst, as well as its lower average pore diameter, a larger proportion of large pores compared to the other two catalysts, and its spherical open bud morphology. It affirms the importance of catalyst/support ethanol-content control during the preparation process. Then, molecular simulation was employed to shed light on the iso-specificity of the polypropylene produced via synthesized catalysts.

Bio-Inspired Drug Delivery Systems: From Synthetic Polypeptide Vesicles to Outer Membrane Vesicles

Nanomedicine is a broad field that focuses on the development of nanocarriers to deliver specific drugs to targeted sites. A synthetic polypeptide is a kind of biomaterial composed of repeating amino acid units that are linked by peptide bonds. The multiplied amphiphilicity segment of the polypeptide could assemble to form polypeptide vesicles (PVs) under suitable conditions. Different from polypeptide vesicles, outer membrane vesicles (OMVs) are spherical buds of the outer membrane filled with periplasmic content, which commonly originate from Gram-negative bacteria. Owing to their biodegradability and excellent biocompatibility, both PVs and OMVs have been utilized as carriers in delivering drugs. In this review, we discuss the recent drug delivery research based on PVs and OMVs. These related topics are presented: (1) a brief introduction to the production methods for PVs and OMVs; (2) a thorough explanation of PV- and OMV-related applications in drug delivery including the vesicle design and biological assessment; (3) finally, we conclude with a discussion on perspectives and future challenges related to the drug delivery systems of PVs and OMVs.

Outer membrane vesicles as molecular biomarkers for Gram-negative sepsis: Taking advantage of nature’s perfect packages

Sepsis is an often life-threatening response to infection, occurring when host proinflammatory immune responses become abnormally elevated and dysregulated. To diagnose sepsis, the patient must have a confirmed or predicted infection, as well as other symptoms associated with the pathophysiology of sepsis. However, a recent study found that a specific causal organism could not be determined in the majority (70.1%) of sepsis cases, likely due to aggressive antibiotics or localized infections. The timing of a patient’s sepsis diagnosis is often predictive of their clinical outcome, underlining the need for a more definitive molecular diagnostic test. Here, we outline the advantages and challenges to using bacterial outer membrane vesicles (OMVs), nanoscale spherical buds derived from the outer membrane of Gram-negative bacteria, as a diagnostic biomarker for Gram-negative sepsis. Advantages include OMV abundance, their robustness in the presence of antibiotics, and their unique features derived from their parent cell that could allow for differentiation between bacterial species. Challenges include the rigorous purification methods required to isolate OMVs from complex biofluids and the additional need to separate OMVs from similarly sized extracellular vesicles, which can share physical properties with OMVs.

Membrane shape deformation induced by curvature-inducing proteins consisting of chiral crescent binding and intrinsically disordered domains.

Curvature-inducing proteins containing a bin/amphiphysin/Rvs domain often have intrinsically disordered domains. Recent experiments have shown that these disordered chains enhance curvature sensing and generation. Here, we report on the modification of protein-membrane interactions by disordered chains using meshless membrane simulations. The protein and bound membrane are modeled together as a chiral crescent protein rod with two excluded-volume chains. As the chain length increases, the repulsion between them reduces the cluster size of the proteins. It induces spindle-shaped vesicles and a transition between arc-shaped and circular protein assemblies in a disk-shaped vesicle. For flat membranes, an intermediate chain length induces many tubules owing to the repulsion between the protein assemblies, whereas longer chains promote perpendicular elongation of tubules. Moreover, protein rods with zero rod curvature and sufficiently long chains stabilize the spherical buds. For proteins with a negative rod curvature, an intermediate chain length induces a rugged membrane with branched protein assemblies, whereas longer chains induce the formation of tubules with periodic concave-ring structures.

Binding of curvature-inducing proteins onto biomembranes

In this paper, we review the theoretical analyses and simulations of the interactions between curvature-inducing proteins and biomembranes. Laterally isotropic proteins induce spherical budding, whereas anisotropic proteins such as Bin/Amphiphysin/Rvs (BAR) superfamily proteins, induce tubulation. Both types of proteins can sense the membrane curvature. We describe the theoretical analyzes of various transitions of protein binding accompanied by a change in various properties, such as the number of buds, the radius of membrane tubes, and the nematic order of anisotropic proteins. Moreover, we explain the membrane-mediated interactions and protein assembly. Many types of membrane shape transformations (spontaneous tubulation, formation of polyhedral vesicles, polygonal tubes, periodic bumps, network structures, etc.) have been demonstrated by coarse-grained simulations. Furthermore, traveling waves and turing patterns under the coupling of reaction-diffusion dynamics and membrane deformation are described.

Mem3DG: Modeling membrane mechanochemical dynamics in 3D using discrete differential geometry

Biomembranes adopt varying morphologies that are vital to cellular functions. Many studies use computational modeling to understand how various mechanochemical factors contribute to membrane shape transformations. Compared with approximation-based methods (e.g., finite element method [FEM]), the class of discrete mesh models offers greater flexibility to simulate complex physics and shapes in three dimensions; its formulation produces an efficient algorithm while maintaining coordinate-free geometric descriptions. However, ambiguities in geometric definitions in the discrete context have led to a lack of consensus on which discrete mesh model is theoretically and numerically optimal; a bijective relationship between the terms contributing to both the energy and forces from the discrete and smooth geometric theories remains to be established. We address this and present an extensible framework, Mem3DG, for modeling 3D mechanochemical dynamics of membranes based on discrete differential geometry (DDG) on triangulated meshes. The formalism of DDG resolves the inconsistency and provides a unifying perspective on how to relate the smooth and discrete energy and forces. To demonstrate, Mem3DG is used to model a sequence of examples with increasing mechanochemical complexity: recovering classical shape transformations such as 1) biconcave disk, dumbbell, and unduloid; and 2) spherical bud on spherical, flat-patch membrane; investigating how the coupling of membrane mechanics with protein mobility jointly affects phase and shape transformation. As high-resolution 3D imaging of membrane ultrastructure becomes more readily available, we envision Mem3DG to be applied as an end-to-end tool to simulate realistic cell geometry under user-specified mechanochemical conditions.

Triggering toxicity: How antibiotics can enhance the release of outer membrane vesicles from Escherichia coli during sepsis

OMVs are spherical buds derived from the outer membrane of Gram‐negative bacteria, containing lipopolysaccharide (LPS) and phospholipids, proteins, nucleic acids, peptidoglycan, and other cellular molecules. Some of those molecules, such as LPS and peptidoglycan associated lipoprotein (Pal), have been shown to be toxic and to contribute to sepsis‐related inflammation. While OMVs are released constitutively from Gram‐negative bacteria, environmental factors like temperature changes, stress, and antibiotics can enhance OMV release. We proposed that some antibiotics, such as beta lactams that target the peptidoglycan layer of the cell, enhance OMV release more than other types of antibiotics. We used ultracentrifugation, immunoblotting, and nanoparticle tracking analysis to purify, detect, and roughly quantify OMV release from Escherichia coli in the presence of different antibiotics. Preliminary results support our hypothesis, although data collection is ongoing. Finally, we propose a novel use of released OMVs as molecular biomarkers for bacterial sepsis.

Mem3DG: modeling membrane mechanochemical dynamics in 3D using discrete differential geometry

Biomembranes adopt varying morphologies that are vital to cellular functions. Many studies use computational modeling to understand how various mechanochemical factors contribute to membrane shape transformations. Compared to approximation-based methods (e.g., finite element method), the class of discrete mesh models offers greater flexibility to simulate complex physics and shapes in three dimensions; its formulation produces an efficient algorithm while maintaining coordinate-free geometric descriptions. However, ambiguities in geometric definitions in the discrete context have led to a lack of consensus on which discrete mesh model is theoretically and numerically optimal; a bijective relationship between the terms contributing to both the energy and forces from the discrete and smooth geometric theories remains to be established. We address this and present an extensible framework, $\texttt{Mem3DG}$, for modeling 3D mechanochemical dynamics of membranes based on Discrete Differential Geometry (DDG) on triangulated meshes. The formalism of DDG resolves the inconsistency and provides a unifying perspective on how to relate the smooth and discrete energy and forces. To demonstrate, $\texttt{Mem3DG}$ is used to model a sequence of examples with increasing mechanochemical complexity: recovering classical shape transformations such as 1) biconcave disk, dumbbell, and unduloid and 2) spherical bud on spherical, flat-patch membrane; investigating how the coupling of membrane mechanics with protein mobility jointly affects phase and shape transformation. As high-resolution 3D imaging of membrane ultrastructure becomes more readily available, we envision Mem3DG to be applied as an end-to-end tool to simulate realistic cell geometry under user-specified mechanochemical conditions.

Vesicle budding induced by binding of curvature-inducing proteins.

Vesicle budding induced by protein binding that generates an isotropic spontaneous curvature is studied using a mean-field theory. Many spherical buds are formed via protein binding. As the binding chemical potential increases, the proteins first bind to the buds and then to the remainder of the vesicle. For a high spontaneous curvature and/or high bending rigidity of the bound membrane, it is found that a first-order transition occurs between a small number of large buds and a large number of small buds. These two states coexist around the transition point. The proposed scheme is simple and easily applicable to many interaction types, so we investigate the effects of interprotein interactions, the protein-insertion-induced changes in area, the variation of the saddle-splay modulus, and the area-difference-elasticity energy. The differences in the preferred curvatures for curvature sensing and generation are also clarified.

Nanoscale Curvature Promotes High Yield Spontaneous Formation of Cell-Mimetic Giant Vesicles on Nanocellulose Paper.

To date, techniques for the assembly of phospholipid films into cell-like giant unilamellar vesicles (GUVs) use planar surfaces and require the application of electric fields or dissolved molecules to obtain adequate yields. Here, we present the use of nanocellulose paper, which are surfaces composed of entangled cylindrical nanofibers, to promote the facile and high yield assembly of GUVs. Use of nanocellulose paper results in up to a 100 000-fold reduction in costs while increasing yields compared to extant surface-assisted assembly techniques. Quantitative measurements of yields and the distributions of sizes using large data set confocal microscopy illuminates the mechanism of assembly. We present a thermodynamic "budding and merging", BNM, model that offers a unified explanation for the differences in the yields and sizes of GUVs obtained from surfaces of varying geometry and chemistry. The BNM model considers the change in free energy due to budding by balancing the elastic, adhesion, and edge energies of a section of a surface-attached membrane that transitions into a surface-attached spherical bud. The model reveals that the formation of GUVs is spontaneous on hydrophilic surfaces consisting of entangled cylindrical nanofibers with dimensions similar to nanocellulose fibers. This work advances understanding of the effects of surface properties on the assembly of GUVs. It also addresses practical barriers that currently impede the promising use of GUVs as vehicles for the delivery of drugs, for the manufacturing of synthetic cells, and for the assembly of artificial tissues at scale.

Local sensitivity analysis of the “membrane shape equation” derived from the Helfrich energy

The Helfrich energy is commonly used to model the elastic bending energy of lipid bilayers in membrane mechanics. The governing differential equations for certain geometric characteristics of the shape of the membrane can be obtained by applying variational methods (minimization principles) to the Helfrich energy functional and are well studied in the axisymmetric framework. However, the Helfrich energy functional and the resulting differential equations involve a number of parameters, and there is little explanation of the choice of parameters in the literature, particularly with respect to the choice of the “spontaneous curvature” term that appears in the functional. In this paper, we present a careful analytical and numerical study of certain aspects of parametric sensitivity of Helfrich’s model. Using simulations of specific model systems, we demonstrate the application of our scheme to the formation of spherical buds and pearled shapes in membrane vesicles.

Cryptococcus laurentii: An unusual cause for atypical pneumonia in hematological malignancy

Cryptococcus laurentii, one of the nonneoformans Cryptococcus is a known biopesticide to control fruit rot, previously considered to be nonpathogenic to humans, is now have been reported as a cause of opportunistic infections. Here, we present a case of pulmonary cryptococcosis due to C. laurentii in a patient with chronic lymphocytic leukemia and prolymphocytic leukemia. Sputum microscopy showed Gram-positive spherical capsulated budding yeast cells, culture of which revealed it as C. laurentii. The patient was promptly put on fluconazole, but he died on the subsequent day after initiation of therapy. A high index of suspicion and improvement of techniques of culture and identification is essential for early diagnosis and treatment of unusual fungal infections.

Silver Nanoflower Decorated Graphene Oxide Sponges for Highly Sensitive Variable Stiffness Stress Sensors.

Soft conductive materials should enable large deformation while keeping high electrical conductivity and elasticity. The graphene oxide (GO)-based sponge is a potential candidate to endow large deformation. However, it typically exhibits low conductivity and elasticity. Here, the highly conductive and elastic sponge composed of GO, flower-shaped silver nanoparticles (AgNFs), and polyimide (GO-AgNF-PI sponge) are demonstrated. The average pore size and porosity are 114 µm and 94.7%, respectively. Ag NFs have thin petals (8-20 nm) protruding out of the surface of a spherical bud (300-350 nm) significantly enhancing the specific surface area (2.83 m2 g-1 ). The electrical conductivity (0.306 S m-1 at 0% strain) of the GO-AgNF-PI sponge is increased by more than an order of magnitude with the addition of Ag NFs. A nearly perfect elasticity is obtained over a wide compressive strain range (0-90%). The strain-dependent, nonlinear variation of Young's modulus of the sponge provides a unique opportunity as a variable stiffness stress sensor that operates over a wide stress range (0-10 kPa) with a high maximum sensitivity (0.572 kPa-1 ). It allows grasping of a soft rose and a hard bottle, with the minimal object deformation, when attached on the finger of a robot gripper.

Formation and Stability of Membrane Necks from Molecular Simulation

In their fluid state, lipid membranes are able to execute a variety of shape transformations. One intriguing example is provided by vesicle budding, i.e., by the expulsion of a small, spherical bud connected to the mother vesicle by a narrow membrane neck. The formation of such necks provides an essential step of many biological processes (cellular uptake, secretion as well as cell division or cytokinesis). In the framework of curvature elasticity, formation and stability of such a narrow neck are determined by local stability conditions, which depend both on the local mean curvature and on the spontaneous curvature of the membrane. However, these conditions ignore the molecular structure of the membrane, which may affect the shape of the neck. Furthermore, because of their locality, the stability conditions cannot describe the overall response of a neck to mechanical perturbations. In order to address these questions, we use molecular simulations to study the neck formation. Budding and neck formation are induced by small solutes such as monosaccharides that adsorb onto the bilayer membranes. If the two leaflets of a membrane are exposed to different solute concentrations, the membrane becomes asymmetric and acquires a certain spontaneous curvature. We start from a spherical vesicle that is exposed to a certain solute concentration in the exterior solution. When we reduce the volume of this vesicle, we observe formation of narrow membrane necks provided the concentration exceeds a certain threshold value. We induce mechanical perturbation using a geometry that resembles micropipette aspiration on the micrometer scale to observe neck opening for which we calculate the associated free energy landscape.

Development of the Rabbit (Oryctolagus cuniculus) Metanephros

Thirty-seven rabbit embryos at em- bryonic days (E) 10-30 were used to study the morphogenesis of the metanephros by light microscope. The primordium of the metanephros appears at E 12 within the pelvic cavity as a spherical ureteric bud capped by metanephrogenic tissue. The nephrogenic spherules and the renal vesicles develop at E 15 while formation of the renal tubules and differentiation of the glomerular capsule take place at E 16. At E 18, differentiation of the glomerular ca- pillaries occurs and the proximal and distal convoluted tubules are clearly first time demarcated. Positive alkaline phosphatase activity and PAS-positive material are confined to the metanephric corpuscles and the luminal border of the proxi- mal convoluted tubules. In addition, the collecting tubules show large masses of intracytoplasmic PAS- positive granules. The rabbit metanephros is smooth unipapillary with indistinct renal columns and pyramids. It undergoes migration until reaches its permanent position at E 30 where the right metanephros is located opposite to the level of the 2nd – 4th lumbar vertebrae and the left one is situated opposite to the level of the 3rd – 5th lumbar verte- brae. This migration is accompanied by rotation for about 135 degrees resulting in a dorsomedially located hilus. In conclusion, the stages of development of the rabbit metanephros are similar to those in other mammals but they maintain a de- layed sequence.

Self-Assembly of Mesoscale Artificial Clathrin Mimics.

Fluidic control and sampling in complex environments is an important process in biotechnology, materials synthesis, and microfluidics. An elegant solution to this problem has evolved in nature through cellular endocytosis, where the dynamic recruitment, self-assembly, and spherical budding of clathrin proteins allows cells to sample their external environment. Yet despite the importance and utility of endocytosis, artificial systems which can replicate this dynamic behavior have not been developed. Guided by clathrin's unusual structure, we created simplified metallic microparticles that capture the three-legged shape, particle curvature, and interfacial attachment characteristics of clathrin. These artificial clathrin mimics successfully recreate biomimetic analogues of clathrin's recruitment, assembly, and budding, ultimately forming extended networks at fluid interfaces and invaginating immiscible phases into spheres under external fields. Particle curvature was discovered to be a critical structural motif, greatly limiting irreversible aggregation and inducing the legs' selective tip-to-tip attraction. This architecture provides a template for a class of active self-assembly units to drive structural and dimensional transformations of liquid-liquid interfaces and microscale fluidic sampling.

Construction of Nuclear Envelope Shape by a High-Genus Vesicle with Pore-Size Constraint

Nuclear pores have an approximately uniform distribution in the nuclear envelope of most living cells. Hence, the morphology of the nuclear envelope is a spherical stomatocyte with a high genus. We have investigated the morphology of high-genus vesicles under pore-size constraint using dynamically triangulated membrane simulations. Bending-energy minimization without volume or other constraints produces a circular-cage stomatocyte, where the pores are aligned in a circular line on an oblate bud. As the pore radius is reduced, the circular-pore alignment is more stabilized than a random pore distribution on a spherical bud. However, we have clarified the conditions for the formation of a spherical stomatocyte: a small perinuclear volume, osmotic pressure within nucleoplasm, and repulsion between the pores. When area-difference elasticity is taken into account, the formation of cylindrical or budded tubules from the stomatocyte and discoidal stomatocyte is found.