Spectroscopic and Biophysical Methods to Determine Differential Salt-Uptake by Primitive Membraneless Polyester Microdroplets.

α-Hydroxy acids are prebiotic monomers that undergo dehydration synthesis to form polyester gels, which assemble into membraneless microdroplets upon aqueous rehydration. These microdroplets are proposed as protocells that can segregate and compartmentalize primitive molecules/reactions. Different primitive aqueous environments with a variety of salts could have hosted chemistries that formed polyester microdroplets. These salts could be essential cofactors of compartmentalized prebiotic reactions or even directly affect protocell structure. However, fully understanding polyester-salt interactions remains elusive, partially due to technical challenges of quantitative measurements in condensed phases. Here, spectroscopic and biophysical methods are applied to analyze salt uptake by polyester microdroplets. Inductively coupled plasma mass spectrometry is applied to measure the cation concentration within polyester microdroplets after addition of chloride salts. Combined with methods to determine the effects of salt uptake on droplet turbidity, size, surface potential and internal water distribution, it was observed that polyester microdroplets can selectively partition salt cations, leading to differential microdroplet coalescence due to ionic screening effects reducing electrostatic repulsion forces between microdroplets. Through applying existing techniques to novel analyses related to primitive compartment chemistry and biophysics, this study suggests that even minor differences in analyte uptake can lead to significant protocellular structural change.

Unbinding events of amino acids and peptides from water-pyrite interfaces: A case study of life's origin on mineral surfaces.

Selective binding of aqueous-phase amino acids to mineral surfaces is regarded as a plausible first step in oligopeptide formation on early Earth. To clarify the strength and underlying mechanism of amino acid binding to pyrite surfaces, we measured the unbinding (pull-off) force of ten amino acids and two oligo-peptides from water-pyrite interfaces using atomic force microscopy (AFM). The most probable unbinding force could be described by a linearly increasing function with the size of the amino acid and a characteristic offset. A good correlation was obtained between the most probable unbinding force and the residue volume, surface area and polarizability of samples suggesting at least a partial contribution of van der Waals (vdW) forces, especially the London dispersion force. These results are useful in analysis of adhesion phenomena of amino acids in the given environmental settings such as in this work.

Membraneless polyester microdroplets as primordial compartments at the origins of life.

Compartmentalization was likely essential for primitive chemical systems during the emergence of life, both for preventing leakage of important components, i.e., genetic materials, and for enhancing chemical reactions. Although life as we know it uses lipid bilayer-based compartments, the diversity of prebiotic chemistry may have enabled primitive living systems to start from other types of boundary systems. Here, we demonstrate membraneless compartmentalization based on prebiotically available organic compounds, α-hydroxy acids (αHAs), which are generally coproduced along with α-amino acids in prebiotic settings. Facile polymerization of αHAs provides a model pathway for the assembly of combinatorially diverse primitive compartments on early Earth. We characterized membraneless microdroplets generated from homo- and heteropolyesters synthesized from drying solutions of αHAs endowed with various side chains. These compartments can preferentially and differentially segregate and compartmentalize fluorescent dyes and fluorescently tagged RNA, providing readily available compartments that could have facilitated chemical evolution by protecting, exchanging, and encapsulating primitive components. Protein function within and RNA function in the presence of certain droplets is also preserved, suggesting the potential relevance of such droplets to various origins of life models. As a lipid amphiphile can also assemble around certain droplets, this further shows the droplets' potential compatibility with and scaffolding ability for nascent biomolecular systems that could have coexisted in complex chemical systems. These model compartments could have been more accessible in a "messy" prebiotic environment, enabling the localization of a variety of protometabolic and replication processes that could be subjected to further chemical evolution before the advent of the Last Universal Common Ancestor.

Size-Dependent Affinity of Glycine and Its Short Oligomers to Pyrite Surface: A Model for Prebiotic Accumulation of Amino Acid Oligomers on a Mineral Surface.

The interaction strength of progressively longer oligomers of glycine, (Gly), di-Gly, tri-Gly, and penta-Gly, with a natural pyrite surface was directly measured using the force mode of an atomic force microscope (AFM). In recent years, selective activation of abiotically formed amino acids on mineral surfaces, especially that of pyrite, has been proposed as an important step in many origins of life scenarios. To investigate such notions, we used AFM-based force measurements to probe possible non-covalent interactions between pyrite and amino acids, starting from the simplest amino acid, Gly. Although Gly itself interacted with the pyrite surface only weakly, progressively larger unbinding forces and binding frequencies were obtained using oligomers from di-Gly to penta-Gly. In addition to an expected increase of the configurational entropy and size-dependent van der Waals force, the increasing number of polar peptide bonds, among others, may be responsible for this observation. The effect of chain length was also investigated by performing similar experiments using l-lysine vs. poly-l-lysine (PLL), and l-glutamic acid vs. poly-l-glutamic acid. The results suggest that longer oligomers/polymers of amino acids can be preferentially adsorbed on pyrite surfaces.

Atomic force microscope as a nano- and micrometer scale biological manipulator: A short review.

The amazing capacity of atomic force microscope to let us touch the molecular and cellular level samples with a sharp probe stimulated its application to bio-medical field among others. In addition to topographical imaging of the sample surface, a direct mechanical manipulation has attracted innovative minds to develop new methodologies aiming at direct handling of proteins, DNA/RNA, and cells. Measurement of their mechanical properties brought about a vivid picture of their physical nature. Direct handling of individual molecules and cells prompted development of nano-medical applications. This short review summarized recent application of AFM for measurement of mechanical properties of biological samples and attempts to perform direct manipulations of nano-medicine.

Membrane wound healing at single cellular level.

We report a nano-technological method of creating a micrometer sized hole on the live cell membrane using atomic force microscope (AFM) and its resealing process at the single cellular level as a model of molecular level wound healing. First, the cell membrane was fluorescently labeled with Kusabira Orange (KO) which was tagged to a lipophilic membrane-sorting peptide. Then a glass bead glued on an AFM cantilever and modified with phospholipase A2 was made to contact the cell membrane. A small dark hole (4-14 µm2 in area) was created on the otherwise fluorescent cell surface often being accompanied by bleb formation. Refilling of holes with KO fluorescence proceeded at an average rate of ~0.014µm2s-1. The fluorescent lumps which initially surrounded the hole were gradually lost. We compared the present result with our previous ones on the repair processes of artificially damaged stress fibers (Graphical Abstract: Figure S2).

Spectrin-ankyrin interaction mechanics: A key force balance factor in the red blood cell membrane skeleton.

As major components of red blood cell (RBC) cytoskeleton, spectrin and F-actin form a network that covers the entire cytoplasmic surface of the plasma membrane. The cross-linked two layered structure, called the membrane skeleton, keeps the structural integrity of RBC under drastically changing mechanical environment during circulation. We performed force spectroscopy experiments on the atomic force microscope (AFM) as a means to clarify the mechanical characteristics of spectrin-ankyrin interaction, a key factor in the force balance of the RBC cytoskeletal structure. An AFM tip was functionalized with ANK1-62k and used to probe spectrin crosslinked to mica surface. A force spectroscopy study gave a mean unbinding force of ~30 pN under our experimental conditions. Two energy barriers were identified in the unbinding process. The result was related to the well-known flexibility of spectrin tetramer and participation of ankyrin 1-spectrin interaction in the overall balance of membrane skeleton dynamics.

Subunit unbinding mechanics of dimeric wheat germ agglutinin (WGA) studied by atomic force microscopy.

Wheat germ agglutinin (WGA) is an oligomeric lectin widely used as a model of sugar moieties in biochemistry. Subunit association is important for the crosslinking function of WGA, so we used atomic force microscopy to measure the subunit unbinding force of dimeric WGA. We found that the average unbinding force of dimeric WGA is ∼55 pN at ∼1 nN/s loading rate, whereas this unbinding force is increased at least up to 100 pN when WGA is bound to glycophorin A. Moreover, the dissociation rate constant of WGA was calculated to be 1­2 × 10(−2) s(−1), suggesting that dimer dissociation is relatively fast.

Requirement of LIM domains for the transient accumulation of paxillin at damaged stress fibres.

Cells recognize and respond to changes in intra- and extracellular mechanical conditions to maintain their mechanical homeostasis. Linear contractile bundles of actin filaments and myosin II known as stress fibres (SFs) mediate mechanical signals. Mechanical cues such as excessive stress driven by myosin II and/or external force may damage SFs and induce the local transient accumulation of SF-repair complexes (zyxin and VASP) at the damaged sites. Using an atomic force microscope mounted on a fluorescence microscope, we applied mechanical damage to cells expressing fluorescently tagged cytoskeletal proteins and recorded the subsequent mobilization of SF-repair complexes. We found that a LIM protein, paxillin, transiently accumulated at the damaged sites earlier than zyxin, while paxillin knockdown did not affect the kinetics of zyxin translocation. The C-terminal half of paxillin, comprising four-tandem LIM domains, can still translocate to damaged sites on SFs, suggesting that the LIM domain is essential for the mechanosensory function of paxillin. Our findings demonstrate a crucial role of the LIM domain in mechanosensing LIM proteins.

Fabricated cantilever for AFM measurements and manipulations: pre-stress analysis of stress fibers.

The atomic force microscope (AFM) is a highly successful instrument for imaging of nanometer-sized samples and measurement of pico- to nano-Newton forces acting between atoms and molecules, especially in liquid. Generally, commercial AFM cantilevers, which have a sharp tip, are used for AFM experiments. In this review, we introduce micro-fabricated AFM cantilevers and show several applications for cell biology. In manipulation of samples on a cellular scale with a force of tens to hundreds of nano-Newtons, attempts have been made to secure the formation of covalent/non-covalent linkages between the AFM probe and the sample surface. However, present chemistry-based modification protocols of cantilevers do not produce strong enough bonds. To measure the tensile strength and other mechanical properties of actin-based thin filaments in both living and semi-intact fibroblast cells, we fabricated a probe with a hooking function by focused ion beam technology and used it to capture, pull and eventually break a chosen thin filament, which was made visible through fusion with fluorescent proteins. Furthermore, we fabricated a microscoop cantilever specifically designed for pulling a microbead attached to a cell. The microscoop cantilevers can realize high-throughput measurements of cell stiffness.

Forced extension of delipidated red blood cell cytoskeleton with little indication of spectrin unfolding.

Force-extension curves obtained on intact human red blood cells (RBC) were compared with those of delipidated RBCs to assess the contribution of cytoskeletal flexibility to the extensibility of the intact membrane skeleton. The RBCs were first delipidated by treatment with phospholipase A2; tensile properties of the exposed cytoskeletal structures were measured using an atomic force microscope (AFM). The AFM probes were modified either with the Band 3 specific lectin, concanavalin A, (Con A) or anti-F-actin antibody, to localize the point of interaction between the probe and the cytoskeleton. Extension of the spectrin-based cytoskeleton reached up to 2-3 µm with a force less than 70 pN without showing any force peaks before the final rupture of the adhesive bonds. Our interpretation of the result is that the spectrin-based network was slack enough to allow the observed degree of extension without unfolding the tetrameric spectrin molecules. The force-extension curves obtained either on Band 3-ankyrin loci or on junction nodes of the cytoskeleton were not significantly different. Experimental results were verified by computer simulation of pulling mechanics of a network model of the RBC cytoskeleton. Our experimental results are also in agreement with the theoretical prediction of Mirijanian and Voth [2008; Proc Natl Acad Sci USA 105:1204-1208].

Nonlinear displacement of ventral stress fibers under externally applied lateral force by an atomic force microscope.

Actin-based stress fibers (SFs) have fundamental importance in the maintenance of mechanical stability of living cells. Several in vitro measurements of their elastic properties have therefore been made, but direct mechanical manipulation of individual SFs in vivo for the determination of their mechanical properties has not been attempted. No less important is a search for the possible formation of a global mechanical network involving SFs and other intracellular filamentous components. In this article, we present an application of atomic force microscopy to probe into a live cell and laterally push selected SFs in a fibroblast cells (VNOf 06 fibroblast-like cells derived from rat vomeronasal tissue) transfected with a green fluorescent protein-ß-actin gene. The transfected cells were transferred to a serum-depleted medium before the atomic force microscope manipulation. The lateral displacement of the SFs under a point loading condition recorded on a fluorescence microscope revealed both linear and nonlinear displacements against the contour distance from the point of force loading. The nonlinear displacements of the SFs were attributed to their association with a cortical actomyosin-cell membrane complex that effectively pulled them back as a 2D thin plate.

Direct detection of cellular adaptation to local cyclic stretching at the single cell level by atomic force microscopy.

The cellular response to external mechanical forces has important effects on numerous biological phenomena. The sequences of molecular events that underlie the observed changes in cellular properties have yet to be elucidated in detail. Here we have detected the responses of a cultured cell against locally applied cyclic stretching and compressive forces, after creating an artificial focal adhesion under a glass bead attached to the cantilever of an atomic force microscope. The cell tension initially increased in response to the tensile stress and then decreased within ∼1 min as a result of viscoelastic properties of the cell. This relaxation was followed by a gradual increase in tension extending over several minutes. The slow recovery of tension ceased after several cycles of force application. This tension-recovering activity was inhibited when cells were treated with cytochalasin D, an inhibitor of actin polymerization, or with (-)-blebbistatin, an inhibitor of myosin II ATPase activity, suggesting that the activity was driven by actin-myosin interaction. To our knowledge, this is the first quantitative analysis of cellular mechanical properties during the process of adaptation to locally applied cyclic external force.

Direct manipulation of intracellular stress fibres using a hook-shaped AFM probe.

Atomic force microscopy (AFM) is a highly successful technique for imaging nanometre-sized samples and measuring pico- to nano-newton forces acting between atoms and molecules. When it comes to the manipulation of larger samples with forces of tens and hundreds of nano-newtons, however, the present chemistry-based modification protocols for functionalizing AFM cantilevers to achieve the formation of covalent/non-covalent linkages between the AFM probe and the sample surface do not produce strong enough bonds. For the purpose of measuring the fracture strength and other mechanical properties of stress fibres (SFs) in living as well as semi-intact fibroblast cells, we fabricated an AFM probe with a hooking function by focused ion beam technology and used the AFM probe hook to capture, pull and eventually sever a chosen SF labelled with green or red fluorescent protein.

Molecular shape and binding force of Mycoplasma mobile's leg protein Gli349 revealed by an AFM study.

Recent studies of the gliding bacteria Mycoplasma mobile have identified a family of proteins called the Gli family which was considered to be involved in this novel and yet fairly unknown motility system. The 349kDa protein called Gli349 was successfully isolated and purified from the bacteria, and electron microscopy imaging and antibody experiments led to the hypothesis that it acts as the "leg" of M. mobile, responsible for attachment to the substrate as well as for gliding motility. However, more precise evidence of the molecular shape and function of this protein was required to asses this theory any further. In this study, an atomic force microscope (AFM) was used both as an imaging and a force measurement device to provide new information about Gli349 and its role in gliding motility. AFM images of the protein were obtained revealing a complex structure with both rigid and flexible parts, consistent with previous electron micrographs of the protein. Single-molecular force spectroscopy experiments were also performed, revealing that Gli349 is able to specifically bind to sialyllactose molecules and withstand unbinding forces around 70pN. These findings strongly support the idea that Gli349 is the "leg" protein of M. mobile, responsible for binding and also most probably force generation during gliding motility.

Atomic force microscopy for cellular level manipulation: imaging intracellular structures and DNA delivery through a membrane hole.

The atomic force microscope (AFM) is a versatile tool for imaging, force measurement and manipulation of proteins, DNA, and living cells basically at the single molecular level. In the cellular level manipulation, extraction, and identification of mRNA's from defined loci of a cell, insertion of plasmid DNA and pulling of membrane proteins, for example, have been reported. In this study, AFM was used to create holes at defined loci on the cell membrane for the investigation of viability of the cells after hole creation, visualization of intracellular structure through the hole and for targeted gene delivery into living cells. To create large holes with an approximate diameter of 5-10 microm, a phospholipase A(2) coated bead was added to the AFM cantilever and the bead was allowed to touch the cell surface for approximately 5-10 min. The evidence of hole creation was obtained mainly from fluorescent image of Vybrant DiO labeled cell before and after the contact with the bead and the AFM imaging of the contact area. In parallel, cells with a hole were imaged by AFM to reveal intracellular structures such as filamentous structures presumably actin fibers and mitochondria which were identified with fluorescent labeling with rhodamine 123. Targeted gene delivery was also attempted by inserting an AFM probe that was coated with the Monster Green Fluorescent Protein phMGFP Vector for transfection of the cell. Following targeted transfection, the gene expression of green fluorescent protein (GFP) was observed and confirmed by the fluorescence microscope.

Single molecular dynamic interactions between glycophorin A and lectin as probed by atomic force microscopy.

Glycophorin A (GpA) is one of the most abundant transmembrane proteins in human erythrocytes and its interaction with lectins has been studied as model systems for erythrocyte related biological processes. We performed a force measurement study using the force mode of atomic force microscopy (AFM) to investigate the single molecular level biophysical mechanisms involved in GpA-lectin interactions. GpA was mounted on a mica surface or natively presented on the erythrocyte membrane and probed with an AFM tip coated with the monomeric but multivalent Psathyrella velutina lectin (PVL) through covalent crosslinkers. A dynamic force spectroscopy study revealed similar interaction properties in both cases, with the unbinding force centering around 60 pN with a weak loading rate dependence. Hence we identified the presence of one energy barrier in the unbinding process. Force profile analysis showed that more than 70% of GpAs are free of cytoskeletal associations in agreement with previous reports.

Tensile mechanics of alanine-based helical polypeptide: force spectroscopy versus computer simulations.

In nature, an alpha-helix is commonly used to build thermodynamically stable and mechanically rigid protein conformations. In view of growing interest in the mechanical rigidity of proteins, we measured the tensile profile of an alanine-based alpha-helical polypeptide on an atomic-force microscope to investigate the basic mechanics of helix extension with minimal interference from side-chain interactions. The peptide was extended to its maximum contour length with much less force than in reported cases of poly-L-Glu or poly-L-Lys, indicating that chain stiffness strongly depended on the physicochemical properties of side chains, such as their bulkiness. The low tensile-force extension originated presumably in locally unfolded parts because of spontaneous structural fluctuations. In 50% trifluoroethanol, the well-known helix-promoting agent, the rigidity of the sample polypeptide was markedly increased. Computer simulations of the peptide-stretching process showed that a majority of constituent residues underwent a transition from an alpha-helical to an extended conformation by overcoming an energy barrier around psi approximately 0 degrees on the Ramachandran plot. The observed lability of an isolated helix signified the biological importance of the lateral bundling of helices to maintain a rigid protein structure.