Multiscale modeling of cellular actin filaments: from atomistic molecular to coarse-grained dynamics

In this article, we present a computational multiscale model for the characterization of subcellular proteins. The model is encoded inside a simulation tool that builds coarse-grained (CG) force fields from atomistic simulations. Equilibrium molecular dynamics simulations on an all-atom model of the actin filament are performed. Then, using the statistical distribution of the distances between pairs of selected groups of atoms at the output of the MD simulations, the force field is parameterized using the Boltzmann inversion approach. This CG force field is further used to characterize the dynamics of the protein via Brownian dynamics simulations. This combination of methods into a single computational tool flow enables the simulation of actin filaments with length up to 400 nm, extending the time and length scales compared to state-of-the-art approaches. Moreover, the proposed multiscale modeling approach allows to investigate the relationship between atomistic structure and changes on the overall dynamics and mechanics of the filament and can be easily (i) extended to the characterization of other subcellular structures and (ii) used to investigate the cellular effects of molecular alterations due to pathological conditions.     

Regulation of gelsolin to plant actin filaments and its distribution in pollen

The effect of plasma gelsolin on plant microfilaments and its localization in plant cells were investigated. The results by using ultracentrifugation and electron microscopy showed that plant microfilaments could be severed into shorter fragments by gelsolin in a Ca2+-dependent manner. By measuring the binding ability of plasma gelsolin to pollen actin using the method of immunoprecipitation, it was shown that pollen actin could bind gelsolin at a ratio of 2.0±0.21 in the presence of Ca2+. Addition of EGTA could disassociate the actin-gelsolin complexes, reducing the ratio to 1.2±0.23, and the addition of PIP2 could further reduce the ratio to 0.8±0.1. The results indicate that plant actin has similar binding properties with plasma gelsolin as that of animal actin. By Western blotting we identified the existence of gelsolin in lily pollen. The results of immunolo-calization of gelsolin in pollen and pollen tube showed that gelsolin was mainly localized at the germinal furrow in pollen grains and at the cytoplasm in pollen tube, especially in the tip region.     

Regulation of gelsolin to plant actin filaments and its distribution in pollen

The dynamics of the actin cytoskeleton depends upon the unique constellation of ac- tin-binding proteins (ABPs), as well as their spatial distribution and local activation. However, the identification and characterization of actin-binding proteins in plant cells are still limited. At pre- sent, only a few plant ABPs have been identified in plant tissues, including profilin, ADF/cofilin, fimbrin, villin and several myosins. Compared with that in animals, there is still a long way for us …     

Subpopulations of tau interact with microtubules and actin filaments in various cell types

It has been demonstrated that microtubule-associated proteins (MAPs) interact with tubulin in vitro and in vivo. However, there is no clear evidence on the possible roles of the interactions of MAPs in vivo with other cytoskeletal components in maintaining the integrity of the cell architecture. To address this question we extracted the neuronal cytoskeleton from brain cells and studied the selective dissociation of specific molecular isospecies of tau protein under various experimental conditions. Tau, and in some cases MPA-2, were analysed by the use of anti-idiotypic antibodies that recognize epitopes on their tubulin binding sites. Fractions of microtubule-bound tau isoforms were extracted with 0.35 M NaCl or after the addition of nocodazole to allow microtubule depolymerization. Protein eluted with this inhibitor contained most of the assembled tubulin dimer pool and part of the remaining tau and MAP-2. When the remaining cytoskeletal pellet was treated with cytochalasin D to allow depolymerization of actin filaments, only tau isoforms were extracted. Immunoprecipitation studies along with immunolocalization experiments in cell lines containing tau-like components supported the findings on the roles of tau isospecies as linkers between tubulin in the microtubular structure with actin filaments. Interestingly, in certain types of cells, antibody-reactive tau isospecies were detected by immunofluorescence with a discrete distribution pattern along actin filaments, which was affected by cytochalasin disruption of the actin filament network. These results suggest the possible in vivo roles of subsets of tau protein in modulating the interactions between microtubules and actin filaments.     

RhoA/ROCK regulation of neuritogenesis via profilin IIa-mediated control of actin stability

Neuritogenesis, the first step of neuronal differentiation, takes place as nascent neurites bud from the immediate postmitotic neuronal soma. Little is known about the mechanisms underlying the dramatic morphological changes that characterize this event. Here, we show that RhoA activity plays a decisive role during neuritogenesis of cultured hippocampal neurons by recruiting and activating its specific kinase ROCK, which, in turn, complexes with profilin IIa. We establish that this previously uncharacterized brain-specific actin-binding protein controls neurite sprouting by modifying actin stability, a function regulated by ROCK-mediated phosphorylation. Furthermore, we determine that this novel cascade is switched on or off by physiological stimuli. We propose that RhoA/ROCK/PIIa-mediated regulation of actin stability, shown to be essential for neuritogenesis, may constitute a central mechanism throughout neuronal differentiation.     

Molecular dynamics simulations of wild type and mutant of Pin1 peptidyl-prolyl isomerase

Pin1 catalyses the intrinsically slow process of cis-trans isomerisation and has been identified as a possible drug target in many diseases. Recently, the wild type (WT) and the Cys113Asp mutant of the Pin1 peptidyl-prolyl isomerase (PPIase) domain were determined by nuclear magnetic resonance. In this article, the WT and Cys113Asp mutant of PPIase domain are studied by molecular dynamics simulations. The structural stability analysis shows that the Cys113Asp mutation leads to the higher fluctuation of hydrophobic core in PPIase domain. The intrinsic correlated motions are important for the catalytic function of Pin1, whereas the Cys113Asp mutant system loses pivotal dynamical properties and develops wider conformational states than those in WT system. The intramolecular hydrogen bonds play crucial roles in the structural stability of PPIase domain. The mutated residue Asp113 attracts the side chain of His59 in the Cys113Asp system, which unbalances the internal interactions inside the catalytic tetrad. Meanwhile, the conformational changes of PPIase domain affect the side chain orientations of Lys63 and Arg69, which limit their binding with substrates. The Cys113Asp mutation destabilises the whole binding region of Pin1 PPIase domain, so the catalysis activity is severely reduced. These results are consistent with experimental studies and may help to understand the isomerisation mechanisms of Pin1.     

Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement

Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.     

Roles of actin binding proteins in mammalian oocyte maturation and beyond

Actin nucleation factors, which promote the formation of new actin filaments, have emerged in the last decade as key regulatory factors controlling asymmetric division in mammalian oocytes. Actin nucleators such as formin-2, spire, and the ARP2/3 complex have been found to be important regulators of actin remodeling during oocyte maturation. Another class of actin-binding proteins including cofilin, tropomyosin, myosin motors, capping proteins, tropomodulin, and Ezrin-Radixin-Moesin proteins are thought to control actin cytoskeleton dynamics at various steps of oocyte maturation. In addition, actin dynamics controlling asymmetric-symmetric transitions after fertilization is a new area of investigation. Taken together, defining the mechanisms by which actin-binding proteins regulate actin cytoskeletons is crucial for understanding the basic biology of mammalian gamete formation and pre-implantation development.     

Molecular investigation of the mechanical properties of single actin filaments based on vibration analyses

The mechanical vibration properties of single actin filaments from 50 to 288 nm are investigated by the molecular dynamics simulation in this study. The natural frequencies obtained from the molecular simulations agree with those obtained from the analytical solution of the equivalent Euler–Bernoulli beam model. Through the convergence study of the mechanical properties with respect to the filament length, it was found that the Euler–Bernoulli beam model can only be reliably used when the single actin filament is of the order of hundreds of nanometre scale. This molecular investigation not only provides the evidence for the use of the continuum beam model in characterising the mechanical properties of single actin filaments, but also clarifies the criteria for the effective use of the Euler–Bernoulli beam model.     

Capping and dynamic relation between domains 1 and 2 of gelsolin

Gelsolin is a protein that severs and caps actin filaments. The two activities are located in the N-terminal half of the gelsolin molecules. Severing and subsequent capping requires the binding of domains 2 and 3 (S2–3) to the side of the filaments to position the N-terminal domain 1 (S1) at the barbed end of actin (actin subdomains 1 and 3). The results provide a structural basis for the gelsolin capping mechanism. The effects of a synthetic peptide derived from the sequence of a binding site located in gelsolin S2 on actin properties have been studied. CD and IR spectra indicate that this peptide presented a secondary structure in solution which would be similar to that expected for the native full length gelsolin molecule. The binding of the synthetic peptide induces conformational changes in actin subdomain 1 and actin oligomerization. An increase in the polymerization rate was observed, which could be attributed to a nucleation kinetics effect. The combined effects of two gelsolin fragments, the synthetic peptide derived from an S2 sequence and the purified segment 1 (S1), were also investigated as a molecule model. The two fragments induced nucleation enhancement and inhibited actin depolymerization, two characteristic properties of capping. In conclusion, for the first time it is reported that the binding of a small synthetic fragment is sufficient to promote efficient capping by S1 at the barbed end of actin filaments. ©1998 European Peptide Society and John Wiley & Sons, Ltd.     

Spatial associations between actin filaments, endoplasmic reticula, mitochondria and fungal hyphae in symbiotic cells of orchid protocorms

The relationships between endoplasmic reticula (ER), mitochondria, and actin filaments (Afs) were observed in uncolonized and colonized cells of symbiotic protocorms ofSpiranthes sinensis (Orchidaceae) germinated in the presence of the fungus,Ceratobasidium cornigerum. Mitochondria and ER were observed by transmission electron microscopy, and with the fluorescent probe DiOC₆ (3) (3,3′-dihexyloxacarbocyanine) combined with confocal laser scanning microscopy (CLSM). An indirect immunofluorescence method using CLSM and an indirect, pre-embedding immunogold method at the ultrastructural level were used for observation of Afs. In uncolonized cells, cortical ER showed a polygonal pattern and ER formed a network throughout the cytoplasm. In the cortex, a smooth face of ER contacted the plasma membrane. Mitochondria were associated with ER. Afs were in close proximity to ER, mitochondria and amyloplasts. Colonized cells retained cortical ER, and a smooth face of ER was also closely associated with the perifungal membrane. ER and mitochondria were present in the cytoplasmic channels bridging between the central peloton and the peripheral cytoplasm. This distribution of ER and mitochondria during fungal colonization and senescence coincided with that of Afs. The changes in the arrays of Afs accompanying symbiotic fungal colonization and senescence occurred concomitantly with the changes in ER.     

Molecular dynamics simulation of the nanofibrils formed by amyloid-based peptide amphiphiles

Abstract

Atomistic molecular dynamics simulations have been performed on the peptide amphiphiles (PAs) with four amyloid beta peptide fragments as head groups. The stable structures were monitored by the root mean square deviation with respect to the energy minimised initial structures. Random coil and β-sheet structures with hydrogen bonds along and perpendicular to the long axis of the nanofibre were obtained due to the different nature of the head groups. Influences of pH and capping ends on the nanofibre structures were investigated through variation of the protonation states of the ionic amino acids in the peptides. The peptides with opposite charges on both sides were found to have the fewest β-sheet structures, and the charges on the outer terminal tended to destruct the β-sheets while those at the inner side did not. The isolated charge in the centre of peptides was found to be able to promote the formation of regular β-sheets, while multiple charged residues could not support ordered β-sheet structures. When charge neutralisation occurred between adjacent residues, regular β-sheet laminates might also occur for systems with charges at the outer terminal. With the increase of β-sheet structures formed, the original twisted structures found for random coil structures of the PAs could be diminished by the hydrogen bonds.     

Structural modeling and molecular dynamics simulation of the actin filament

Actin is a major structural protein of the eukaryotic cytoskeleton and enables cell motility. Here, we present a model of the actin filament (F-actin) that not only incorporates the global structure of the recently published model by Oda et al. but also conserves internal stereochemistry. A comparison is made using molecular dynamics simulation of the model with other recent F-actin models. A number of structural determents such as the protomer propeller angle, the number of hydrogen bonds, and the structural variation among the protomers are analyzed. The MD comparison is found to reflect the evolution in quality of actin models over the last 6 years. In addition, simulations of the model are carried out in states with both ADP or ATP bound and local hydrogen-bonding differences characterized.     

Arabidopsis capping protein senses cellular phosphatidic acid levels and transduces these into changes in actin cytoskeleton dynamics

Plants respond rapidly and precisely to a broad spectrum of developmental, biotic and abiotic cues. In many instances, signaling cascades involved in transducing this information result in changes to the cellular architecture and cytoskeletal rearrangements. Based originally on paradigms for animal cell signaling, phospholipids have received increased scrutiny as key intermediates for transmitting information to the actin cytoskeleton. Significantly, a wealth of biochemical data for plant actin-binding proteins (ABPs) demonstrates that many of these interact with phosphoinositide lipids in vitro. Moreover, phosphatidic acid (PA) has been identified not only as an abundant structural lipid in plants, but also as an intermediary in developmental and stress signaling pathways that lead to altered actin organization. Several years ago, the heterodimeric capping protein (CP) from Arabidopsis was demonstrated to bind PA and is negatively regulated by this lipid in vitro. Whether this form of regulation occurs in cells, however, remained a mystery. A new study, that combines live-cell imaging of cytoskeletal dynamics with reverse-genetic analyses in Arabidopsis, provides compelling new evidence that CP is inhibited from binding filament ends in the presence of PA in vivo. This allows rapid actin polymerization and increases in filament abundance following stimulation and could be one key factor in the physiological responses of plant cells to environmental stimuli.     

Cofilin increases the torsional flexibility and dynamics of actin filaments

We have measured the effects of cofilin on the conformation and dynamics of actin filaments labeled at Cys374 with erythrosin-iodoacetemide (ErIA), using time-resolved phosphorescence anisotropy (TPA). Cofilin quenches the phosphorescence intensity of actin-bound ErIA, indicating that binding changes the local environment of the probe. The cofilin concentration-dependence of the phosphorescence intensity is sigmoidal, consistent with cooperative actin filament binding. Model-independent analysis of the anisotropies indicates that cofilin increases the rates of the microsecond rotational motions of actin. In contrast to the reduction in phosphorescence intensity, the changes in the rates of rotational motions display non-nearest-neighbor cooperative interactions and saturate at substoichiometric cofilin binding densities. Detailed analysis of the TPA decays indicates that cofilin decreases the torsional rigidity (C) of actin, increasing the thermally driven root-mean-square torsional angle between adjacent filament subunits from approximately 4 degrees (C = 2.30 x 10(-27) Nm2 radian(-1)) to approximately 17 degrees (C = 0.13 x 10(-27) Nm2 radian(-1)) at 25 degrees C. We favor a mechanism in which cofilin binding shifts the equilibrium between thermal ErIA-actin filament conformers, and facilitates two distinct structural changes in actin. One is local in nature, which affects the structure of actin's C terminus and is likely to mediate nearest-neighbor cooperative binding and filament severing. The second is a change in the internal dynamics of actin, which displays non-nearest-neighbor cooperativity and increases the torsional flexibility of filaments. The long-range effects of cofilin on the torsional dynamics of actin may accelerate P(i) release from filaments and modulate interactions with other regulatory actin filament binding proteins.     

Molecular dynamics simulation of content of bound water in ethylene glycol and glycerol solution

In this paper, the content of bound water was studied to evaluate the cryoprotective properties of ethylene glycol and glycerol solution. Molecular dynamic models for the solution were built, the classification principle and statistical methods of water molecules in solutions were presented, respectively. The content of bound water with various hydroxyl molarity at different temperatures was obtained through molecular dynamic simulation. The results reveal that the content of bound water increases with increasing hydroxyl molarity, but decreases with increasing temperature. It was found that, the content of bound water in ethylene glycol solution is always slightly more than that in glycerol solution, regardless of whether the temperature increases or not.     

Nuclear and cytoplasmic organization in Xenopus oocytes after disruption of actin filaments by latrunculin

Actin-containing filaments have been visualized inside Xenopus oocyte nuclei by a combination of fluorescence and transmission electron microscopy. It was shown that these filaments contact nucleoli, spherical bodies, and nuclear pore complexes. The incubation of oocytes with actin-depolymerizing agent, latrunculin, caused membrane vesiculation in cytoplasm and the disruption of nucleoplasm and the integrity of the nuclear envelope. We suggest that actin-containing filaments are important cell components involved in the regulation of nucleus-cytoplasm interactions, as well as of cellular transport of components during the growth of Xenopus oocytes.     

Structural insights into the tropomodulin assembly at the pointed ends of actin filaments

Tropomodulins are a family of important regulators of actin dynamics at the pointed ends of actin filaments. Four isoforms of tropomodulin, Tmod1‐Tmod4, are expressed in vertebrates. Binding of tropomodulin to the pointed end is dependent on tropomyosin, an actin binding protein that itself is represented in mammals by up to 40 isoforms. The understanding of the regulatory role of the tropomodulin/tropomyosin molecular diversity has been limited due to the lack of a three‐dimensional structure of the tropomodulin/tropomyosin complex. In this study, we mapped tropomyosin residues interacting with two tropomyosin‐binding sites of tropomodulin and generated a three‐dimensional model of the tropomodulin/tropomyosin complex for each of these sites. The models were refined by molecular dynamics simulations and validated via building a self‐consistent three‐dimensional model of tropomodulin assembly at the pointed end. The model of the pointed‐end Tmod assembly offers new insights in how Tmod binding ensures tight control over the pointed end dynamics.