scholarly journals Actin crosslinker competition and sorting drive emergent GUV size-dependent actin network architecture

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yashar Bashirzadeh ◽  
Steven A. Redford ◽  
Chatipat Lorpaiboon ◽  
Alessandro Groaz ◽  
Hossein Moghimianavval ◽  
...  
Keyword(s):  
Network Architecture ◽  
Molecular Size ◽  
Coarse Grained ◽  
General Mechanism ◽  
Complex Structures ◽  
Actin Bundles ◽  
Size Dependent ◽  
Apparent Stiffness ◽  
Coarse Grained Simulations

AbstractThe proteins that make up the actin cytoskeleton can self-assemble into a variety of structures. In vitro experiments and coarse-grained simulations have shown that the actin crosslinking proteins α-actinin and fascin segregate into distinct domains in single actin bundles with a molecular size-dependent competition-based mechanism. Here, by encapsulating actin, α-actinin, and fascin in giant unilamellar vesicles (GUVs), we show that physical confinement can cause these proteins to form much more complex structures, including rings and asters at GUV peripheries and centers; the prevalence of different structures depends on GUV size. Strikingly, we found that α-actinin and fascin self-sort into separate domains in the aster structures with actin bundles whose apparent stiffness depends on the ratio of the relative concentrations of α-actinin and fascin. The observed boundary-imposed effect on protein sorting may be a general mechanism for creating emergent structures in biopolymer networks with multiple crosslinkers.

scholarly journals Actin crosslinker competition and sorting drive emergent GUV size-dependent actin network architecture

2020 ◽  
Author(s):  
Yashar Bashirzadeh ◽  
Steven A. Redford ◽  
Chatipat Lorpaiboon ◽  
Alessandro Groaz ◽  
Thomas Litschel ◽  
...  
Keyword(s):  
Network Architecture ◽  
Coarse Grained ◽  
General Mechanism ◽  
Actin Networks ◽  
Cellular Processes ◽  
Size Dependent ◽  
Sensory Projections ◽  
Coarse Grained Simulations ◽  
Cytoskeletal Networks

AbstractRobust spatiotemporal organization of cytoskeletal networks is crucial, enabling cellular processes such as cell migration and division. α-Actinin and fascin are two actin crosslinking proteins localized to distinct regions of eukaryotes to form actin bundles with optimized spacing for cell contractile machinery and sensory projections, respectively. In vitro reconstitution assays and coarse-grained simulations have shown that these actin bundling proteins segregate into distinct domains with a bundler size-dependent competition-based mechanism, driven by the minimization of F-actin bending energy. However, it is not known how physical confinement imposed by the cell membrane contributes to sorting of actin bundling proteins and the concomitant reorganization of actin networks in intracellular environment. Here, by encapsulating actin, α-actinin, and fascin in giant unilamellar vesicles (GUVs), we show that the size of such a spherical boundary determines equilibrated structure of actin networks among three typical structures: single rings, astral structures, and star-like structures. We show that α-actinin bundling activity and its tendency for clustering actin is central to the formation of these structures. By analyzing physical features of crosslinked actin networks, we show that spontaneous sorting and domain formation of α-actinin and fascin are intimately linked to the resulting structures. We propose that the observed boundary-imposed effect on sorting and structure formation is a general mechanism by which cells can select between different structural dynamical steady states.

scholarly journals Permeability properties of monolayers of the human trophoblast cell line BeWo

1997 ◽  
Vol 273 (5) ◽  
pp. C1596-C1604 ◽  
Author(s):  
Fei Liu ◽  
Michael J. Soares ◽  
Kenneth L. Audus
Keyword(s):  
Linoleic Acid ◽  
Cell Line ◽  
Morphological Changes ◽  
Molecular Size ◽  
Bewo Cell ◽  
Human Trophoblast ◽  
In Vitro System ◽  
Size Dependent ◽  
Bewo Cell Line

The BeWo cell line (b30 clone) has been examined as a potential in vitro system to study transplacental transport. At the light and electron microscope level, the cells were observed to form confluent monolayers on polycarbonate filters in ∼5 days and morphologically resembled the typical human trophoblast. BeWo monolayers developed a modest transepithelial electrical resistance and a molecular size-dependent permeability to hydrophilic passive diffusion markers, fluorescein, and selected fluorescein-labeled dextrans. Linoleic acid permeation across BeWo monolayers was asymmetric, saturable, and inhibited by low temperature and excess competing fatty acid. Forskolin and 8-bromoadenosine 3′,5′-cyclic monophosphate treatments stimulated morphological changes in BeWo cultures and enhanced the asymmetric passage of linoleic acid across the BeWo monolayers while having minimal effects on passive permeability, affirming that the differentiation state of the cells can influence membrane transporters and transmonolayer permeability. The basic permeability properties of the BeWo monolayers suggest that the cells grown on permeable supports may be examined as a convenient in vitro system to evaluate some transplacental transport mechanisms.

scholarly journals Molecular details of protein condensates probed by microsecond-long atomistic simulations

2020 ◽  
Author(s):  
Wenwei Zheng ◽  
Gregory L. Dignon ◽  
Xichen Xu ◽  
Roshan M. Regy ◽  
Nicolas L. Fawzi ◽  
...  
Keyword(s):  
Phase Separation ◽  
Intrinsically Disordered Proteins ◽  
Low Complexity ◽  
Coarse Grained ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Residue Interaction ◽  
Coarse Grained Simulations ◽  
Protein Density

AbstractThe formation of membraneless organelles in cells commonly occurs via liquid-liquid phase separation (LLPS), and is in many cases driven by multivalent interactions between intrinsically disordered proteins (IDPs). Molecular simulations can reveal the specific amino acid interactions driving LLPS, which is hard to obtain from experiment. Coarse-grained simulations have been used to directly observe the sequence determinants of phase separation but have limited spatial resolution, while all-atom simulations have yet to be applied to LLPS due to the challenges of large system sizes and long time scales relevant to phase separation. We present a novel multiscale computational framework by obtaining initial molecular configurations of a condensed protein-rich phase from equilibrium coarse-grained simulations, and back mapping to an all-atom representation. Using the specialized Anton 2 supercomputer, we resolve microscopic structural and dynamical details of protein condensates through microsecond-scale all-atom explicit-solvent simulations. We have studied two IDPs which phase separate in vitro: the low complexity domain of FUS and the N-terminal disordered domain of LAF-1. Using this approach, we explain the partitioning of ions between phases with low and high protein density, demonstrate that the proteins are remarkably dynamic within the condensed phase, identify the key residue-residue interaction modes stabilizing the dense phase, all while showing good agreement with experimental observations. Our approach is generally applicable to all-atom studies of other single and multi-component systems of proteins and nucleic acids involved in the formation of membraneless organelles.

scholarly journals A helical assembly of human ESCRT-I scaffolds reverse-topology membrane scission

2020 ◽  
Author(s):  
Thomas G. Flower ◽  
Yoshinori Takahashi ◽  
Arpa Hudait ◽  
Kevin Rose ◽  
Nicholas Tjahjono ◽  
...  
Keyword(s):  
Membered Ring ◽  
Coarse Grained ◽  
Virus Release ◽  
Cell Processes ◽  
Membrane Scission ◽  
Coarse Grained Simulations ◽  
Gag Assembly ◽  
Late Stages ◽  
Hiv 1

AbstractThe ESCRT complexes drive membrane scission in HIV-1 release, autophagosome closure, MVB biogenesis, cytokinesis, and other cell processes. ESCRT-I is the most upstream complex and bridges the system to HIV-1 Gag in virus release. The crystal structure of the headpiece of human ESCRT-I comprising TSG101:VPS28:VPS37B:MVB12A was determined, revealing an ESCRT-I helical assembly with a 12 molecule repeat. Electron microscopy confirmed that ESCRT-I subcomplexes form helical filaments in solution. Mutation of VPS28 helical interface residues blocks filament formation in vitro and autophagosome closure and HIV-1 release in human cells. Coarse grained simulations of ESCRT assembly at HIV-1 budding sites suggest that formation of a 12-membered ring of ESCRT-I molecules is a geometry-dependent checkpoint during late stages of Gag assembly and HIV-1 budding, and templates ESCRT-III assembly for membrane scission. These data show that ESCRT-I is not merely a bridging adaptor, but has an essential scaffolding and mechanical role in its own right.

Grand-Reaction Method for Simulations of Ionization Equilibria and Ion Partitioning in a Broad Range of pH and Ionic Strength

2019 ◽  
Author(s):  
Jonas Landsgesell ◽  
Oleg Rud ◽  
Pascal Hebbeker ◽  
Raju Lunkad ◽  
Peter Košovan ◽  
...  
Keyword(s):  
Mean Field ◽  
Coarse Grained ◽  
Reactive System ◽  
Acid Base ◽  
Buffer Solution ◽  
Reaction Method ◽  
Ionization Equilibria ◽  
Coarse Grained Simulations ◽  
Ph Range ◽  
Weak Polyelectrolytes

We introduce the grand-reaction method for coarse-grained simulations of acid-base equilibria in a system coupled to a reservoir at a given pH and concentration of added salt. It can be viewed as an extension of the constant-pH method and the reaction ensemble, combining explicit simulations of reactions within the system, and grand-canonical exchange of particles with the reservoir. Unlike the previously introduced methods, the grand-reaction method is applicable to acid-base equilibria in the whole pH range because it avoids known artifacts. However, the method is more general, and can be used for simulations of any reactive system coupled to a reservoir of a known composition. To demonstrate the advantages of the grand-reaction method, we simulated a model system: A solution of weak polyelectrolytes in equilibrium with a buffer solution. By carefully accounting for the exchange of all constituents, the method ensures that all chemical potentials are equal in the system and in the multi-component reservoir. Thus, the grand-reaction method is able to predict non-monotonic swelling of weak polyelectrolytes as a function of pH, that has been known from mean-field predictions and from experiments but has never been observed in coarse-grained simulations. Finally, we outline possible extensions and further generalizations of the method, and provide a set of guidelines to enable safe usage of the method by a broad community of users.<br><br>

scholarly journals Native-like membrane models of E. coli polar lipid extract shed light on the importance of lipid composition complexity

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Kristyna Pluhackova ◽  
Andreas Horner
Keyword(s):  
Polar Lipid ◽  
Lipid Extract ◽  
Coarse Grained ◽  
In Vitro Experiments ◽  
E Coli ◽  
Water Permeation ◽  
Aquaporin 1 ◽  
Lipid Component ◽  
Membrane Models

Abstract Background Lipid-protein interactions stabilize protein oligomers, shape their structure, and modulate their function. Whereas in vitro experiments already account for the functional importance of lipids by using natural lipid extracts, in silico methods lack behind by embedding proteins in single component lipid bilayers. However, to accurately complement in vitro experiments with molecular details at very high spatio-temporal resolution, molecular dynamics simulations have to be performed in natural(-like) lipid environments. Results To enable more accurate MD simulations, we have prepared four membrane models of E. coli polar lipid extract, a typical model organism, each at all-atom (CHARMM36) and coarse-grained (Martini3) representations. These models contain all main lipid headgroup types of the E. coli inner membrane, i.e., phosphatidylethanolamines, phosphatidylglycerols, and cardiolipins, symmetrically distributed between the membrane leaflets. The lipid tail (un)saturation and propanylation stereochemistry represent the bacterial lipid tail composition of E. coli grown at 37∘C until 3/4 of the log growth phase. The comparison of the Simple three lipid component models to the complex 14-lipid component model Avanti over a broad range of physiologically relevant temperatures revealed that the balance of lipid tail unsaturation and propanylation in different positions and inclusion of lipid tails of various length maintain realistic values for lipid mobility, membrane area compressibility, lipid ordering, lipid volume and area, and the bilayer thickness. The only Simple model that was able to satisfactory reproduce most of the structural properties of the complex Avanti model showed worse agreement of the activation energy of basal water permeation with the here performed measurements. The Martini3 models reflect extremely well both experimental and atomistic behavior of the E. coli polar lipid extract membranes. Aquaporin-1 embedded in our native(-like) membranes causes partial lipid ordering and membrane thinning in its vicinity. Moreover, aquaporin-1 attracts and temporarily binds negatively charged lipids, mainly cardiolipins, with a distinct cardiolipin binding site in the crevice at the contact site between two monomers, most probably stabilizing the tetrameric protein assembly. Conclusions The here prepared and validated membrane models of E. coli polar lipids extract revealed that lipid tail complexity, in terms of double bond and cyclopropane location and varying lipid tail length, is key to stabilize membrane properties over a broad temperature range. In addition, they build a solid basis for manifold future simulation studies on more realistic lipid membranes bridging the gap between simulations and experiments.

scholarly journals Anti-TNF Alpha Antibody Humira with pH-dependent Binding Characteristics: A constant-pH Molecular Dynamics, Gaussian Accelerated Molecular Dynamics, and In Vitro Study

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 334
Author(s):  
Shih-Ting Hong ◽  
Yu-Cheng Su ◽  
Yu-Jen Wang ◽  
Tian-Lu Cheng ◽  
Yeng-Tseng Wang
Keyword(s):  
Molecular Dynamics ◽  
Tnf Alpha ◽  
Complex Structures ◽  
Binding Characteristics ◽  
Accelerated Molecular Dynamics ◽  
Constant Ph ◽  
Ph Dependent ◽  
Constant Ph Molecular Dynamics ◽  
Antibody Drug

Humira is a monoclonal antibody that binds to TNF alpha, inactivates TNF alpha receptors, and inhibits inflammation. Neonatal Fc receptors can mediate the transcytosis of Humira–TNF alpha complex structures and process them toward degradation pathways, which reduces the therapeutic effect of Humira. Allowing the Humira–TNF alpha complex structures to dissociate to Humira and soluble TNF alpha in the early endosome to enable Humira recycling is crucial. We used the cytoplasmic pH (7.4), the early endosomal pH (6.0), and pKa of histidine side chains (6.0–6.4) to mutate the residues of complementarity-determining regions with histidine. Our engineered Humira (W1-Humira) can bind to TNF alpha in plasma at neutral pH and dissociate from the TNF alpha in the endosome at acidic pH. We used the constant-pH molecular dynamics, Gaussian accelerated molecular dynamics, two-dimensional potential mean force profiles, and in vitro methods to investigate the characteristics of W1-Humira. Our results revealed that the proposed Humira can bind TNF alpha with pH-dependent affinity in vitro. The W1-Humira was weaker than wild-type Humira at neutral pH in vitro, and our prediction results were close to the in vitro results. Furthermore, our approach displayed a high accuracy in antibody pH-dependent binding characteristics prediction, which may facilitate antibody drug design. Advancements in computational methods and computing power may further aid in addressing the challenges in antibody drug design.

scholarly journals Enzymic characterization in vitro of recombinant proprotein convertase PC4

1999 ◽  
Vol 343 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Ajoy BASAK ◽  
Bakary B. TOURÉ ◽  
Claude LAZURE ◽  
Majambu MBIKAY ◽  
Michel CHRÉTIEN ◽  
...  
Keyword(s):  
Synthetic Peptides ◽  
Serine Proteases ◽  
Active Form ◽  
Molecular Size ◽  
Maximal Activity ◽  
Transition Metal Ions ◽  
Proprotein Convertase ◽  
Major Band ◽  
Insulin Growth Factor

Proprotein convertase PC4A, a member of the subtilisin/kexin family of serine proteases, was obtained in enzymically active form following expression of vaccinia virus recombinant rat (r)PC4A in GH4C1 cells. It displayed maximal activity at pH 7.0 and a Ca2+ concentration of 2.0 mM. Using PC4-specific antibodies, Western blot analysis of the medium revealed a major band at ≈ 54 kDa, corresponding to the molecular size of mature rPC4A. Among the various peptidyl-[4-methylcoumarin 7-amide (MCA)] substrates tested, the one that was preferred the most by rPC4A was acetyl (Ac)-Arg-Lys-Lys-Arg-MCA, which is cleaved 9 times faster (as judged from Vmax/Km measurements) than the best furin and PC1 substrate, pGlu-Arg-Thr-Lys-Arg-MCA. Recombinant rPC4A, along with human (h)furin and hPC1, cleaved a 17-amino-acid synthetic peptide, YQTLRRRVKR↓ SLVVPTD (where ↓ denotes site of cleavage, and the important basic residues are shown in bold), encompassing the junction between the putative pro-segment of rPC4A and the active enzyme, suggesting a possible auto-activation of the enzyme. In an effort to identify potential physiological substrates for PC4, studies were performed with pro-[insulin-growth-factor (IGF)]-derived synthetic peptides, namely Ac-PAKSAR↓ SVRA (IGF-I66-75) and Ac-PAKSER↓ DVST (IGF-II63-72), as well as two lysine mutants [(IGF-I66-75Lys70) and (IGF-II63-72Lys67)]. Unlike PC1 and furin, rPC4A cleaved efficiently both IGF-I66-75 and IGF-II63-72, suggesting a possible role of PC4 in the maturation of IGF-I and -II. In contrast, the peptides with a position 2 (P2) lysine mutation, IGF-I66-75Lys70 and IGF-II63-72Lys67, were cleaved more efficiently by PC1 and furin compared with rPC4A. Furthermore, using synthetic peptides containing the processing sites of pituitary adenylate-cyclase-activating polypeptide (PACAP)-38, we were able to confirm that, of the two testicular enzymes PC4 and PC7, PC4 is the best candidate enzyme for maturation of PACAP. Our data suggest that rPC4A is a functionally active convertase, with a substrate specificity somewhat different from that of other convertases, namely KXXR↓ (where X denotes any other residue). As expected, p-chloromercuribenzoic acid and metal chelators such as EDTA, EGTA and trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid inhibit the proteolytic activity of rPC4A, whereas it is activated by dithiothreitol. PC4A was also inhibited by transition-metal ions (Cu2+>Hg2+>Zn2+ Ni2+>Co2+), as well as by small peptide semicarbazones (SCs), such as Arg-Lys-Lys-Arg-SC (Ki 0.75 μM) and Arg-Ser-Lys-Arg-SC (Ki 11.4 μM).

Sign in / Sign up

Export Citation Format

Share Document