Direct observation of oriented behavior of actin filaments interacting with desmin intermediate filaments.

BACKGROUND: Associations between actin filaments (AFs) and intermediate filaments (IFs) are frequently observed in living cells. The crosstalk between these cytoskeletal components underpins cellular organization and dynamics; however, the molecular basis of filamentous interactions is not fully understood. Here, we describe the mode of interaction between AFs and desmin IFs (DIFs) in a reconstituted in vitro system. METHODS: AFs (rabbit skeletal muscle) and DIFs (chicken gizzard) were labeled with fluorescent dyes. DIFs were immobilized on a heavy meromyosin (HMM)-coated collodion surface. HMM-driven AFs with ATP hydrolysis was assessed in the presence of DIFs. Images of single filaments were obtained using fluorescence microscopy. Vector changes in the trajectories of single AFs were calculated from microscopy images. RESULTS: AF speed transiently decreased upon contact with DIF. The difference between the incoming and outgoing angles of a moving AF broadened upon contact with a DIF. A smaller incoming angle tended to result in a smaller outgoing angle in a nematic manner. The percentage of moving AFs decreased with an increasing DIF density, but the speed of the moving AFs was similar to that in the no-desmin control. An abundance of DIFs tended to exclude AFs from the HMM-coated surfaces. CONCLUSIONS: DIFs agitate the movement of AFs with the orientation. DIFs can bind to HMMs and weaken actin-myosin interactions. GENERAL SIGNIFICANCE: The study indicates that apart from the binding strength, the accumulation of weak interactions characteristic of filamentous structures may affect the dynamic organization of cell architecture.

Cloning and structural basis of fluorescent protein color variants from identical species of sea anemone, Diadumene lineata.

Diadumene lineata is a colorful sea anemone with orange stripe tissue of the body column and plain tentacles with red lines. We subjected Diadumene lineata to expression cloning and obtained genes encoding orange (OFP: DiLiFP561) and red fluorescent proteins (RFPs: DiLiFP570 and DiLiFP571). These proteins formed obligatory tetramers. All three proteins showed bright fluorescence with the brightness of 58.3 mM-1·cm-1 (DiLiFP561), 43.9 mM-1·cm-1 (DiLiFP570), and 31.2 mM-1·cm-1 (DiLiFP571), which were equivalent to that of commonly used red fluorescent proteins. Amplitude-weighted average fluorescence lifetimes of DiLiFP561, DiLiFP570 and DiLiFP571 were determined as 3.7, 3.6 and 3.0 ns. We determined a crystal structure of DiLiFP570 at 1.63 Å resolution. The crystal structure of DiLiFP570 revealed that the chromophore has an extended π-conjugated structure similar to that of DsRed. Most of the amino acid residues surrounding the chromophore were common between DiLiFP570 and DiLiFP561, except M159 of DiLiFP570 (Lysine in DiLiFP561), which is located close to the chromophore hydroxyl group. Interestingly, a similar K-to-M substitution has been reported in a red-shifted variant of DsRed (mRFP1). It is a striking observation that the naturally evolved color-change variants are consistent with the mutation induced via protein engineering processes. The newly cloned proteins are promising as orange and red fluorescent markers for imaging with long fluorescence lifetime.

Fluorescence microscopic imaging of single desmin intermediate filaments elongated by the presence of divalent cations in vitro.

The formation of intermediate filaments (IFs), a paradigmatic assembly system in biological macromolecules, depends on cations. Herein, to explore the combined effect of ionic strength and divalent cations, we used fluorescence microscopy and examined the in vitro effects of MgCl2, CaCl2, and SrCl2 on the KCl concentration-dependent growth of desmin IFs. Fluorescently-labeled desmin IF assembly initiated by KCl and 5 mM divalent cations led to the formation of single desmin IFs in the KCl concentration range of 25-50 mM. Addition of divalent cations resulted in increased fluorescence intensity in the filament images. KCl concentrations lower or higher than the aforementioned range resulted in the induction of networks of entangled IFs, which were visualized at high resolution via direct stochastic optical reconstruction microscopy. These findings provide insights into the versatility of the IF assembly mechanism and the optimization of fluorescence microscopy of single desmin IFs.

Polymer-Carrying Ability of Actin Filaments Interacting with Myosin Motors in a Biological Motility System In Vitro.

The reconstituted motility system of actin-myosin is expected to be used in bioinspired transport devices, in which carried materials are attached to either moving actin filaments or walking myosin molecules. However, the dependence of the ability to transport on the size of the attached materials is still inadequately understood. Here, as carried materials, polyethylene glycols (PEGs) of various sizes are covalently bound to actin filaments, and the motility of PEG-attached filaments on a heavy meromyosin (HMM) immobilized on a glass surface is observed via fluorescence microscopy. Full attachment of 2 kDa PEG, with an approximately 2 nm gyration radius, decreases the velocity and fraction of moving actin filaments by approximately 10% relative to unattached filaments. For the 5 kDa PEG, the fraction of moving filaments is decreased by approximately 70% even when the filaments contain only 20% PEG-attached actin. The attachment of both sizes of PEGs suppresses the actin-activated ATPase activity at the same level. These results suggest that actin filaments can carry PEGs up to 2 kDa having the same size as actin monomers, while the rate of ATP hydrolysis is limited. The size dependence may provide a criterion for material delivery via actin filaments in nanotransport applications.

Selective association of desmin intermediate filaments with a phospholipid layer in droplets.

Desmin, an intermediate filament protein expressed in muscle cells, plays a key role in the integrity and regulation of the contractile system. Furthermore, the distribution of desmin in cells and its interplay with plasma and organelle membranes are crucial for cell functions; however, the fundamental properties of lipid-desmin interactions remain unknown. Using a water-in-oil method for a limited space system in vitro, we examined the distribution of desmin in three types of phospholipid droplets: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS). When fluorescent-labeled desmin was observed for 60 min after desmin assembly was initiated by adding 25 mM KCl, desmin accumulated on both the DOPE and DOPS layers; however, it did not accumulate on the DOPC layer of droplets. An increase in salt concentration did not moderate the accumulation. The initial form of either oligomer or mature filament affected the accumulation on each lipid layer. When liposomes were included in the droplets, desmin was associated with DOPE but not on DOPC liposomes. These results suggest that desmin has the potential for association with phospholipids concerning desmin form and lipid shape. The behavior and composition of living membranes may affect the distribution of desmin networks.

Lysozyme-induced suppression of enzymatic and motile activities of actin-myosin: Impact of basic proteins.

Electrostatic interactions between actin filaments and myosin molecules, which are ubiquitous proteins in eukaryotes, are crucial for their enzymatic activity and motility. Nonspecific electrostatic interactions between proteins are unavoidable in cells; therefore, it is worth exploring how ambient proteins, such as polyelectrolytes, affect actin-myosin functions. To understand the effect of counterionic proteins on actin-myosin, we examined ATPase activity and sliding velocity via actin-myosin interactions in the presence of the basic model protein hen egg lysozyme. In an in vitro motility assay with ATP, the sliding velocity of actin filaments on heavy meromyosin (HMM) decreased with increasing lysozyme concentrations. Actin filaments were completely stalled at a lysozyme concentration above 0.08 mg/mL. Lysozyme decreased the ATP hydrolysis rate of the actin-HMM complex but not that HMM alone. Co-sedimentation assays revealed that lysozyme enhanced the binding of HMM to actin filaments in the presence of ATP. Additionally, lysozyme could bind to actin and myosin filaments. The inhibitory effect of poly-l-lysine, histone mixture, and lactoferrin on the motility of actin-myosin was higher than that of lysozyme. Thus, nonspecific electrostatic interactions of basic proteins are involved in the bundling of actin filaments and modulation of essential functions of the actomyosin complex.

Condensed desmin and actin cytoskeletal communication in lipid droplets.

The interplay between intermediate filaments (IFs) and other cytoskeletal components is important for the integrity and motility of cells. The impact of IF assembly on other components and cell morphology is not yet fully understood. Therefore, we examined the effects of combined desmin and actin assembly on cytoskeletal network arrangement in artificial cell-sized droplets. Fluorescently labeled desmin, with or without actin, was enclosed in droplets prepared with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) using the water-in-oil method. Protein networks were observed using fluorescence microscopy in the presence of 150 mM KCl, 20 mM imidazole-HCl (pH 7.4), 2 mM MgCl2 , and 1 mM adenosine 5'-triphosphate for both desmin and actin assembly. As desmin alone can assemble into filaments within seconds, desmin networks mainly localizing at the inner margins of the droplets were observed within 10 min after assembly initiation. Subsequently, deformations of droplets appeared. Furthermore, a portion of droplets formed desmin-rich protrusions of several micrometers. Notably, actin alone rarely formed protrusions under the same conditions. When 1,2-dioleoyl-sn-glycero-3-phosphocholine was used instead of DOPE, protrusions became less frequent. The combination of desmin and actin increased the number of deformed droplets in which the proteins were considerably colocalized. The assembly process of desmin facilitated colocalization. Atomic force microscopy failed to reveal interactions between the two filament types. These results suggest that the mechanical properties of desmin networks may influence the behavior of actin networks, as well as membrane morphology, possibly reflecting the mechanical function of desmin filaments in muscle cells.

Inorganic polyphosphate hydrolysis catalyzed by skeletal muscular actomyosin complexes is uncoupled with motility.

Hydrolysis of the triphosphate moiety of ATP, catalyzed by myosin, induces alterations in the affinity of the myosin heads for actin filaments via conformational changes, thereby causing motility of the actomyosin complexes. To elucidate the contribution of the triphosphate group attached to adenosine, we examined the enzymatic activity of heavy meromyosin (HMM) with actin filaments for inorganic tripolyphosphate (3PP) using a Malachite green method and evaluated using fluorescence microscopy the effects of 3PP on actin filament motility on HMM-coated glass slides. In the presence of MgCl2, HMM hydrolyzed 3PP at a maximum rate of 0.016 s-1 HMM-1, which was four times lower than the hydrolysis rate of ATP. Tetrapolyphosphate (4PP) was hydrolyzed at a rate similar to that of 3PP hydrolysis. The hydrolysis rates of 3PP and 4PP were enhanced by roughly 10-fold in the presence of actin filaments. In motility assays, the presence of polyphosphates did not lead to the sliding movement of actin filaments. Moreover, in the presence of ATP at low concentrations, the sliding velocity of actin filaments decreased as the concentration of added polyphosphate increased, indicating a competitive binding of polyphosphate to myosin heads with ATP. These results suggested that the energy produced by standalone triphosphate hydrolysis did not induce the unidirectional motion of actomyosin and that the link between triphosphate and adenosine was crucial for motility.

Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense.

Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT)14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense. We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein "ember" from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 105 M-1·cm-1. The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.

Alignment of actin filament streams driven by myosin motors in crowded environments.

BACKGROUND: Cellular dynamics depend on cytoskeletal filaments and motor proteins. Collective movements of filaments driven by motor proteins are observed in the presence of dense filaments in in vitro systems. As multiple macromolecules exist within cells and the physiological ionic conditions affect their interactions, crowding might contribute to ordered cytoskeletal architecture because of collective behavior. METHODS: Using an in vitro reconstituted system, we observed the emergence of stripe patterns resulting from collective actin filament streaming driven by myosin motors in the presence of the crowding agent, methylcellulose (MC). RESULTS: Although at high KCl concentrations (150mM), actin filaments tended to dissociate from a myosin-coated surface, 1% MC prevented this dissociation and enabled filament movement on myosin molecules. At concentrations of actin filaments above 0.2mg/mL, the moving filaments accumulated and progressively formed long, dense bands. The bands were spaced at about 10-µm intervals. Increasing the KCl concentration up to 300mM resulted in narrowing of the spacing between the aligned bands. On the other hand, low KCl concentrations (≤25mM) induced broad streams, where actin filaments exhibited bidirectional movement. CONCLUSIONS: These results suggest that crowded environments can promote spatial patterning of the actin cytoskeleton, depending on the intensity of the myosin driving force and filament velocity, both modulated by the ionic strength. GENERAL SIGNIFICANCE: The mutual contribution of packing and driving forces provides insight into cytoskeleton organization in living cells, in which various macromolecules mingle.

Switching of actin-myosin motors by voltage-induced pH bias in vitro.

ATP-driven motor proteins, which function in cell motility and organelle transport, have potential applications as bio-inspired micro-devices; however, their control remains unsatisfactory. Here, we show rapid-velocity control of actin filaments interacting with myosin motors using voltage applied to Pt electrodes in an in vitro motility system, by which immediate increases and decreases in velocity were induced beside the cathode and anode, respectively. Indicator dye revealed pH changes after voltage application, and alternate voltage switching allowed actin filaments to cyclically alter their velocity in response to these changes. This principle provides a basis for on-demand control of not only motor proteins but also pH-sensitive events at a microscopic level.

Temperature control of the motility of actin filaments interacting with myosin molecules using an electrically conductive glass in the presence of direct current.

The motility of actin filaments interacting with heavy meromyosin molecules was directly observed on indium tin oxide-coated glass (ITO-glass), over which a surface current flowed. Because the increase in surface current applied to ITO-glass increases the temperature, we focused on the temperature-dependence of the sliding velocity and the effect of the current flow on the orientation of filament motion. Using high precision fluorescence measurements, the displacement vectors of filaments were collected at intervals of 1/30 s. The direction of filament motion was independent to that of current flow up to 0.17 A (7.7 A/m of surface current density); however, the velocity increased by approximately 2-fold when the surface temperature increased from 25 °C to 37 °C. The moving actin filaments exhibited a broader velocity distribution at high temperature than at low temperature. Collectively, these data suggest that using ITO-glass with a surface current to generate a well-controlled temperature change may serve to evaluate temperature-dependent transient responses in protein activity under a microscope, without interference from electrical effects.

Effect of urea and trimethylamine N-oxide on the binding between actin molecules.

Urea and trimethylamine N-oxide (TMAO) are known to denature and stabilize proteins, respectively. We examined two actin-binding processes, namely, end-to-end annealing of actin filaments (F-form) and the polymerization of actin monomers (G-form) into filaments, in the presence of urea, TMAO, and both solutes. Fluorescence microscopy for direct observation of actin filaments bound by fluorescent phalloidin revealed that the annealing rate constant decreased as the concentrations of urea or TMAO increased. Fluorescence spectroscopy with pyrene-labeled actin monomers showed that urea decreased the polymerization rate, whereas TMAO enhanced the rate. The decrease in the polymerization rate constant and thermal stability induced by 0.6M urea was almost completely ameliorated by the addition of 0.3M TMAO. These results suggest that TMAO-dependent stabilization of actin structure facilitates the binding of G-form actin to the ends of F-form actin filaments. Conversely, the binding between ends of mature filaments was impaired by TMAO.

The excluded volume effect induced by poly(ethylene glycol) modulates the motility of actin filaments interacting with myosin.

To examine the motility of actomyosin complexes in the presence of high concentrations of polymers, we investigated the effect of poly(ethylene glycol) on the sliding velocities of actin filaments and regulated thin filaments on myosin molecules in the presence of ATP. Increased concentrations and relative molecular masses of poly(ethylene glycol) decreased the sliding velocities of actin and regulated thin filaments. The decreased ratio of velocity in regulated thin filaments at - log[Ca(2+) ] of 4 was higher than that of actin filaments. Furthermore, in the absence of Ca(2+) , regulated thin filaments were moderately motile in the presence of poly(ethylene glycol). The excluded volume change (∆V), defined as the change in water volume surrounding actomyosin during the interactions, was estimated by determining the relationship between osmotic pressure exerted by poly(ethylene glycol) and the decreased ratio of the velocities in the presence and absence of poly(ethylene glycol). The ∆V increased up to 3.7 × 10(5) Å(3) as the Mr range of poly(ethylene glycol) was increased up to 20,000. Moreover, the ∆V for regulated thin filaments was approximately two-fold higher than that of actin filaments. This finding suggests that differences in the conformation of filaments according to whether troponin-tropomyosin complexes lie on actin filaments alter the ∆V during interactions of actomyosin complexes and influence motility.

Trimethylamine N-oxide suppresses the activity of the actomyosin motor.

BACKGROUND: During actomyosin interactions, the transduction of energy from ATP hydrolysis to motility seems to occur with the modulation of hydration. Trimethylamine N-oxide (TMAO) perturbs the surface of proteins by altering hydrogen bonding in a manner opposite to that of urea. Hence, we focus on the effects of TMAO on the motility and ATPase activation of actomyosin complexes. METHODS: Actin and heavy meromyosin (HMM) were prepared from rabbit skeletal muscle. Structural changes in HMM were detected using fluorescence and circular dichroism spectroscopy. The sliding velocity of rhodamine-phalloidin-bound actin filaments on HMM was measured using an in vitro motility assay. ATPase activity was measured using a malachite green method. RESULTS: Although TMAO, unlike urea, stabilized the HMM structure, both the sliding velocity and ATPase activity of acto-HMM were considerably decreased with increasing TMAO concentrations from 0-1.0M. Whereas urea-induced decreases in the structural stability of HMM were recovered by TMAO, TMAO further decreased the urea-induced decrease in ATPase activation. Urea and TMAO were found to have counteractive effects on motility at concentrations of 0.6M and 0.2M, respectively. CONCLUSIONS: The excessive stabilization of the HMM structure by TMAO may suppress its activities; however, the counteractive effects of urea and TMAO on actomyosin motor activity is distinct from their effects on HMM stability. GENERAL SIGNIFICANCE: The present results provide insight into not only the water-related properties of proteins, but also the physiological significance of TMAO and urea osmolytes in the muscular proteins of water-stressed animals.

Transport of actin-decorated liposomes along myosin molecules in vitro.

We examined whether actin filaments bound to positively charged liposomes could interact with myosin molecules and induce liposome motility. When liposomes were constructed from the mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cationic N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), actin filaments bound to the liposomes. The actin-bound liposomes exhibited movement on myosin molecules in the presence of adenosine-5'-triphosphate (ATP). The displacement was almost linearly increased with time and the behavior differed from that of Brownian motion. Furthermore, the presence of 30% DOTAP in liposomes was most effective for transport. These data show that the actomyosin system was successfully integrated into the liposomes and possesses the ability to actively transport useful agents enclosed within the liposomes.

Effects of urea and guanidine hydrochloride on the sliding movement of actin filaments with ATP hydrolysis by myosin molecules.

To evaluate the role of the hydration layer on the protein surface of actomyosin, we compared the effects of urea and guanidine-HCl on the sliding velocities and ATPase activities of the actin-heavy meromyosin (HMM) system. Both chemicals denature proteins, but only urea perturbs the hydration layer. Both the sliding velocity of actin filaments and actin-activated ATPase activity decreased with increasing urea concentrations. The sliding movement was completely inhibited at 1.0 M urea, while actin filaments were bound to HMM molecules fixed on the glass surface. Guanidine-HCl (0-0.05 M) drastically decreased both the sliding velocity and ATPase activation of acto-HMM complexes. Under this condition, actin filaments almost detached from HMM molecules. In contrast, the ATPase activity of HMM without actin filaments was almost independent of urea concentrations <1.0 M and guanidine-HCl concentrations <0.05 M. An increase in urea concentrations up to 2.0 M partly induced changes in the ternary structure of HMM molecules, while the actin filaments were stable in this concentration range. Hydration changes around such actomyosin complexes may alter both the stability of part of the myosin molecules, and the affinity for force transmission between actin filaments and myosin heads.

Slowly modulating fluctuations as mesoscopic distortions occurring on an actin filament.

An actin filament sliding on myosin molecules exhibits fluctuating or staggered movements as responding to changes in the ATP concentration. We previously observed that fluctuations in the sliding velocity enhanced in a manner being independent of the magnitude of the velocity. The present study focused upon a single actin filament bound to a glass surface through avidin-biotin bonding to examine those fluctuations inherent to the filament in the presence of heavy meromyosin. The auto-correlation analysis revealed that the relaxation time of fluctuations in the filamental displacement obtains its maximum value at about 100microM of the ATP concentration in the ambient, while the magnitude of the fluctuations gradually increased with an increase of the concentration. Furthermore, the measurement of the fluorescence intensity from the markers fixed on the filament demonstrated an enhancement of the negative correlation between the measured peak intensity and the spatial spreading of its intensity over the range of 0-200microM of the ATP concentration, as indicating both development and mitigation of local distortions occurring within the filament.

Relationship between the flexibility and the motility of actin filaments: effects of pH.

Both the sliding velocity of fluorescently labeled actin filament and its persistence length as an index of the bending flexibility of the filament were examined in the motility assay as varying the pH values of the solution for preparing actin filaments. When the pH value was varied from 5.0 to 9.0 in the solution in which actin filaments were formed from the constituent monomers, the motile performance of Mg(2+) bound actin filaments (Mg-F-actin) was apparently suppressed compared to the case of Ca(2+) bound ones (Ca-F-actin). The persistence length for Ca-F-actin gradually increased with the increase of the pH value while the similar length for Mg-F-actin remained rather independent of the value. The largest sliding velocity of the filament, on the other hand, obtained at the persistence length of roughly 6 microm for both cases of Mg-F-actin and Ca-F-actin.

Evolving lipid vesicles in prebiotic hydrothermal environments.

We compared three different kinds of lipid vesicles made of saturated fatty acids, unsaturated fatty acids, and phospholipids for their evolutionary capabilities in a simulated hydrothermal environment. Encapsulation of the glycine monomers enhanced the oligomerization of peptides in all cases. Fatty acid vesicles remained stable at higher temperatures and efficiently utilized heat energy for this synthetic reaction. Phospholipid vesicles were destabilized by higher temperatures, and thus were found to be better suited to enhance synthetic reactions at lower temperatures.