Mutagenic analysis of actin reveals the mechanism of His161 flipping that triggers ATP hydrolysis.

The dynamic assembly of actin is controlled by the hydrolysis of ATP, bound to the center of the molecule. Upon polymerization, actin undergoes a conformational change from the monomeric G-form to the fibrous F-form, which is associated with the flipping of the side chain of His161 toward ATP. His161 flipping from the gauche-minus to gauche-plus conformation leads to a rearrangement of the active site water molecules, including ATP attacking water (W1), into an orientation capable of hydrolysis. We previously showed that by using a human cardiac muscle α-actin expression system, mutations in the Pro-rich loop residues (A108G and P109A) and in a residue that was hydrogen-bonded to W1 (Q137A) affect the rate of polymerization and ATP hydrolysis. Here, we report the crystal structures of the three mutant actins bound to AMPPNP or ADP-Pi determined at a resolution of 1.35-1.55 Å, which are stabilized in the F-form conformation with the aid of the fragmin F1 domain. In A108G, His161 remained non-flipped despite the global actin conformation adopting the F-form, demonstrating that the side chain of His161 is flipped to avoid a steric clash with the methyl group of A108. Because of the non-flipped His161, W1 was located away from ATP, similar to G-actin, which was accompanied by incomplete hydrolysis. In P109A, the absence of the bulky proline ring allowed His161 to be positioned near the Pro-rich loop, with a minor influence on ATPase activity. In Q137A, two water molecules replaced the side-chain oxygen and nitrogen of Gln137 almost exactly at their positions; consequently, the active site structure, including the W1 position, is essentially conserved. This seemingly contradictory observation to the reported low ATPase activity of the Q137A filament could be attributed to a high fluctuation of the active site water. Together, our results suggest that the elaborate structural design of the active site residues ensures the precise control of the ATPase activity of actin.

Structures and mechanisms of actin ATP hydrolysis.

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) PG-O3B bond cleavage; 3) four concomitant events: W1-PO3- formation, OH- and proton cleavage, nucleophilic attack by the OH- against PG, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi-bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.

Keratinocyte-Like Cells Trans-Differentiated from Human Adipose-Derived Stem Cells, Facilitate Skin Wound Healing in Mice.

BACKGROUND: Mesenchymal stem cells (MSCs) have been reported to promote wound healing in both animal models and human studies. Among MSCs, adipose-derived stem cells (ADSCs) can be easily harvested in large quantities. OBJECTIVE: We investigated whether skin wound healing in mice can be facilitated by keratinocyte-like cells differentiated from ADSCs (KC-ADSCs). METHODS: For the wound contraction and epithelialization model, a 20 mm×20 mm fullthickness skin wound was made on the dorsum. For the wound epithelialization model, a 6 mm×6 mm full-thickness skin wound was made on the dorsum. A nitrile rubber stent with an inner diameter of 8 mm was sutured around the wounds to minimize wound contraction. Undifferentiated ADSCs (uADSCs) or KC-ADSCs was injected around the wound base in both models. To evaluate whether the injected ADSCs could enhance wound contraction in a skin wound, the contractile activity of ADSCs was assessed by an in vitro type I collagen gel contraction assay. Alpha-smooth muscle actin (αSMA) expressions in uADSCs and KC-ADSCs were also evaluated by flow cytometry and real-time polymerase chain reaction. RESULTS: In a wound contraction and epithelialization model, KC-ADSCs further facilitated wound healing compared with uADSCs. In a wound epithelialization model, KC-ADSCs also further facilitated wound epithelialization compared with uADSCs. The contractile activity of KC-ADSCs was lower than that of uADSCs. The uADSCs expressed high levels of αSMA, which decreased after the differentiation into keratinocyte-like cells. CONCLUSION: Our results suggest that the wound healing effect of KC-ADSCs depends primarily on re-epithelialization rather than wound contraction.

Structural Insights into the Regulation of Actin Capping Protein by Twinfilin C-terminal Tail.

Twinfilin is a conserved actin regulator that interacts with actin capping protein (CP) via C terminus residues (TWtail) that exhibits sequence similarity with the CP interaction (CPI) motif of CARMIL. Here we report the crystal structure of TWtail in complex with CP. Our structure showed that although TWtail and CARMIL CPI bind CP to an overlapping surface via their middle regions, they exhibit different CP-binding modes at both termini. Consequently, TWtail and CARMIL CPI restrict the CP in distinct conformations of open and closed forms, respectively. Interestingly, V-1, which targets CP away from the TWtail binding site, also favors the open-form CP. Consistently, TWtail forms a stable ternary complex with CP and V-1, a striking contrast to CARMIL CPI, which rapidly dissociates V-1 from CP. Our results demonstrate that TWtail is a unique CP-binding motif that regulates CP in a manner distinct from CARMIL CPI.

Crystal structure of human V-1 in the apo form.

V-1, also known as myotrophin, is a 13 kDa ankyrin-repeat protein that binds and inhibits the heterodimeric actin capping protein (CP), which is a key regulator of cytoskeletal actin dynamics. The crystal structure of V-1 in complex with CP revealed that V-1 recognizes CP via residues spanning several ankyrin repeats. Here, the crystal structure of human V-1 is reported in the absence of the specific ligand at 2.3 Šresolution. In the asymmetric unit, the crystal contains two V-1 monomers that exhibit nearly identical structures (Cα r.m.s.d. of 0.47 Å). The overall structures of the two apo V-1 chains are also highly similar to that of CP-bound V-1 (Cα r.m.s.d.s of <0.50 Å), indicating that CP does not induce a large conformational change in V-1. Detailed structural comparisons using the computational program All Atom Motion Tree revealed that CP binding can be accomplished by minor side-chain rearrangements of several residues. These findings are consistent with the known biological role of V-1, in which it globally inhibits CP in the cytoplasm.

D-Loop Mutation G42A/G46A Decreases Actin Dynamics.

Depolymerization and polymerization of the actin filament are indispensable in eukaryotes. The DNase I binding loop (D-loop), which forms part of the interface between the subunits in the actin filament, is an intrinsically disordered loop with a large degree of conformational freedom. Introduction of the double mutation G42A/G46A to the D-loop of the beta cytoskeletal mammalian actin restricted D-loop conformational freedom, whereas changes to the critical concentration were not large, and no major structural changes were observed. Polymerization and depolymerization rates at both ends of the filament were reduced, and cofilin binding was inhibited by the double mutation. These results indicate that the two glycines at the tip of the D-loop are important for actin dynamics, most likely by contributing to the large degree of conformational freedom.

Novel inter-domain Ca2+-binding site in the gelsolin superfamily protein fragmin.

Gelsolin superfamily proteins, consisting of multiple domains (usually six), sever actin filaments and cap the barbed ends in a Ca2+-dependent manner. Two types of evolutionally conserved Ca2+-binding sites have been identified in this family; type-1 (between gelsolin and actin) and type-2 (within the gelsolin domain). Fragmin, a member in the slime mold Physarum polycephalum, consists of three domains (F1-F3) that are highly similar to the N-terminal half of mammalian gelsolin (G1-G3). Despite their similarities, the two proteins exhibit a significant difference in the Ca2+ dependency; F1-F3 absolutely requires Ca2+ for the filament severing whereas G1-G3 does not. In this study, we examined the strong dependency of fragmin on Ca2+ using biochemical and structural approaches. Our co-sedimentation assay demonstrated that Ca2+ significantly enhanced the binding of F2-F3 to actin. We determined the crystal structure of F2-F3 in the presence of Ca2+. F2-F3 binds a total of three calcium ions; while two are located in type-2 sites within F2 or F3, the remaining one resides between the F2 long helix and the F3 short helix. The inter-domain Ca2+-coordination appears to stabilize F2-F3 in a closely packed configuration. Notably, the F3 long helix exhibits a bent conformation which is different from the straight G3 long helix in the presence of Ca2+. Our results provide the first structural evidence for the existence of an unconventional Ca2+-binding site in the gelsolin superfamily proteins.

Structural Polymorphism of Actin.

Information on the structural polymorphism of a protein is essential to understand the mechanisms of how it functions at an atomic level. Numerous studies on actin have accumulated substantial amounts of information about its polymorphism, and there are over 200 published atomic structures of different forms of actin using crystallography, fiber diffraction, and electron microscopy. To characterize all the reported structures, we proposed simple parameters based on the discrete rigid bodies within the actin molecule and identified four conformation groups by cluster analysis: the F-form in naked F-actin, the C-form in cofilactin, the O-form in profilin-actin, and the G-form in the majority of actin-containing crystal structures. The G-form group included the most variations, but each conformational variation was convertible via a thermal fluctuation, whereas the F- and C-forms were not accessible from the G-form. The convertibility and accessibility of the structures were evaluated using molecular dynamics simulations. Information about conformational conversion among each group is useful for understanding the mechanisms of actin function.

Analysis of finger vein variety in patients with various diseases using vein authentication technology.

In finger vein authentication technology, near-infrared rays penetrate the finger and are absorbed by the hemoglobin in blood. The veins appear as dark areas. The finger vein pattern images of patients with various diseases were acquired; a new evaluation method applying image processing technique ("E value") was developed, and it was examined whether the patterns have any characteristics differentiating them from those of healthy volunteers. As a result, low E values appeared in systemic sclerosis, mixed connective tissue disease, Sjögren's syndrome, and polymyositis/dermatomyositis. No statistical reduction in E value was shown in patients with rheumatoid arthritis, pernio (without rheumatic diseases), arteriosclerosis obliterans, diabetes, hypertension, hypothyroidism and alopecia areata. This technology could be used for screening and evaluation of some diseases and their conditions with impaired peripheral venous circulation. E value may be useful as an indicator of venous circulation.

Polymerization and depolymerization of actin with nucleotide states at filament ends.

Polymerization induces hydrolysis of ATP bound to actin, followed by γ-phosphate release, which helps advance the disassembly of actin filaments into ADP-G-actin. Mechanical understanding of this correlation between actin assembly and ATP hydrolysis has been an object of intensive studies in biochemistry and structural biology for many decades. Although actin polymerization and depolymerization occur only at either the barbed or pointed ends and the kinetic and equilibrium properties are substantially different from each other, characterizing their properties is difficult to do by bulk assays, as these assays report the average of all actin filaments in solution and are therefore not able to discern the properties of individual actin filaments. Biochemical studies of actin polymerization and hydrolysis were hampered by these inherent properties of actin filaments. Total internal reflection fluorescence (TIRF) microscopy overcame this problem by observing single actin filaments. With TIRF, we now know not only that each end has distinct properties, but also that the rate of γ-phosphate release is much faster from the terminals than from the interior of actin filaments. The rate of γ-phosphate release from actin filament ends is even more accelerated when latrunculin A is bound. These findings highlight the importance of resolving structural differences between actin molecules in the interior of the filament and those at either filament end. This review provides a history of observing actin filaments under light microscopy, an overview of dynamic properties of ATP hydrolysis at the end of actin filament, and structural views of γ-phosphate release.

Structural basis for cofilin binding and actin filament disassembly.

Actin depolymerizing factor (ADF) and cofilin accelerate actin dynamics by severing and disassembling actin filaments. Here, we present the 3.8 Å resolution cryo-EM structure of cofilactin (cofilin-decorated actin filament). The actin subunit structure of cofilactin (C-form) is distinct from those of F-actin (F-form) and monomeric actin (G-form). During the transition between these three conformations, the inner domain of actin (subdomains 3 and 4) and the majority of subdomain 1 move as two separate rigid bodies. The cofilin-actin interface consists of three distinct parts. Based on the rigid body movements of actin and the three cofilin-actin interfaces, we propose models for the cooperative binding of cofilin to actin, preferential binding of cofilin to ADP-bound actin filaments and cofilin-mediated severing of actin filaments.

Adipose-derived stem cells express higher levels of type VII collagen under specific culture conditions.

Type VII collagen (Col7) is a major component of the anchoring fibrils at the dermoepidermal junction. Adipose-derived stem cells (ADSCs) are a cell population highly useful in regenerative medicine because of their ease of isolation and their potential for multilineage differentiation. Based on the observations that K14 was expressed in undifferentiated ADSCs and the expression was downregulated after differentiation into adipocytes, we speculated that ADSCs are keratinocyte stem/progenitor cells. ADSCs were co-cultured with fibroblasts on type IV collagen in a medium containing all-trans retinoic acid and bone morphogenetic protein 4. At day 14 of culture in keratinocyte serum-free medium, the cells were harvested and subjected to immunofluorescence, flow cytometry, real-time PCR, and western blotting. Approximately, 45% of ADSCs were immunostained positively for anti-human cytokeratin 10, and approximately 80% were stained positively for Col7. Flow cytometry, real-time PCR, and western blotting also confirmed that differentiated ADSCs expressed higher levels of Col7. These findings support the therapeutic potential of ADSCs, not only for wound healing, but also for the correction of Col7 deficiencies.

Evidence for ESCRT- and clathrin-dependent microautophagy.

Microautophagy refers to a mode of autophagy in which the lysosomal or vacuolar membrane invaginates and directly engulfs target components. The molecular machinery of membrane dynamics driving microautophagy is still elusive. Using immunochemical monitoring of yeast vacuolar transmembrane proteins, Vph1 and Pho8, fused to fluorescent proteins, we obtained evidence showing an induction of microautophagy after a diauxic shift in the yeast Saccharomyces cerevisiae Components of the endosomal sorting complex required for transport machinery were found to be required for this process, and the gateway protein of the machinery, Vps27, was observed to change its localization onto the vacuolar membrane after a diauxic shift. We revealed the functional importance of Vps27's interaction with clathrin in this microautophagy that also contributed to uptake of lipid droplets into the vacuole. This study sheds light on the molecular mechanism of microautophagy, which does not require the core Atg proteins.

Autophagy-independent function of Atg8 in lipid droplet dynamics in yeast.

Dynamic features of lipid droplets include growth and degradation of the organelle. Autophagy, a system for the transport of cytoplasmic components to be degraded into the lysosome/vacuole, is regarded to be responsible for the degradation of lipid droplets. Atg8 protein in the yeast Saccharomyces cerevisiae is recruited to membrane structures synthesized during autophagy via a lipidation process. In this study, we report a novel function of Atg8 in lipid droplet dynamics. We found that loss of Atg8 specifically resulted in a decrease in the quantity of lipid droplets in cells at stationary phase. This protein was detected in a lipid droplet fraction independent of its lipidation. Loss of Atg8 hemifusion activity also caused a decrease in the quantity of lipid droplets. Consistent with these results, isolated lipid droplets underwent assembly into large clusters when incubated with Atg8 possessing hemifusion activity. The loss of Atg8 did not reduce the quantity of lipid droplets in a mutant defective in lipolysis, another system for lipid droplet degradation, which strongly suggests the function of Atg8 in antagonizing lipolysis. Together these results indicate a specific function of Atg8 in maintaining the quantity of lipid droplets that is independent of its autophagic function.

Direct observation of the actin filament by tip-scan atomic force microscopy.

Actin filaments, the actin-myosin complex and the actin-tropomyosin complex were observed by a tip-scan atomic force microscope (AFM), which was recently developed by Olympus as the AFM part of a correlative microscope. This newly developed AFM uses cantilevers of similar size as stage-scan AFMs to improve substantially the spatial and temporal resolution. Such an approach has previously never been possible by a tip-scan system, in which a cantilever moves in the x, y and z directions. We evaluated the performance of this developed tip-scan AFM by observing the molecular structure of actin filaments and the actin-tropomyosin complex. In the image of the actin filament, the molecular interval of the actin subunits (∼5.5 nm) was clearly observed as stripes. From the shape of the stripes, the polarity of the actin filament was directly determined and the results were consistent with the polarity determined by myosin binding. In the image of the actin-tropomyosin complex, each tropomyosin molecule (∼2 nm in diameter) on the actin filament was directly observed without averaging images of different molecules. Each tropomyosin molecule on the actin filament has never been directly observed by AFM or electron microscopy. Thus, our developed tip-scan AFM offers significant potential in observing purified proteins and cellular structures at nanometer resolution. Current results represent an important step in the development of a new correlative microscope to observe nm-order structures at an acceptable frame rate (∼10 s/frame) by AFM at the position indicated by the fluorescent dye observed under a light microscope.

Comprehensive analysis of motions in molecular dynamics trajectories of the actin capping protein and its inhibitor complexes.

The actin capping protein (CP) binds to actin filaments to block further elongation. The capping activity is inhibited by proteins V-1 and CARMIL interacting with CP via steric and allosteric mechanisms, respectively. The crystal structures of free CP, CP/V-1, and CP/CARMIL complexes suggest that the binding of CARMIL alters the flexibility of CP rather than the overall structure of CP, and this is an allosteric inhibition mechanism. Here, we performed molecular dynamics (MD) simulations of CP in the free form, and in complex with CARMIL or V-1. The resulting trajectories were analyzed exhaustively using Motion Tree, which identifies various rigid-body motions ranging from small local motions to large domain motions. After enumerating all the motions, CP flexibilities with different ligands were characterized by a list of frequencies for 20 dominant rigid-body motions, some of which were not identified in previous studies. The comparative analysis highlights the influence of the binding of the CARMIL peptide to CP flexibility. In free CP and the CP/V-1 complex, domain motions around a large crevice between the N-stalk and the CP-S domain occur frequently. The CARMIL peptide binds the crevice and suppresses the motions effectively. In addition, the binding of the CARMIL peptide enhances and alters local motions around the pocket that participates in V-1 binding. These newly identified motions are likely to suppress the binding of V-1 to CP. The observed changes in CP motion provide insights that describe the mechanism of allosteric regulation by CARMIL through modulating CP flexibility. Proteins 2016; 84:948-956. © 2016 Wiley Periodicals, Inc.

Enantioselective Total Synthesis of (+)-Neostenine.

A chirality transfer approach using acyclic polyol intermediates for the synthesis of (+)-neostenine (1) has been developed. The sequential Overman/Claisen rearrangement of an allylic 1,2-diol was especially useful, installing two contiguous stereocenters with complete diastereoselectivity in a one-pot sequence. The SmI2 -mediated cyclization and the subsequent chemoselective reduction of a lactam moiety accomplished the first enantioselective total synthesis of (+)-neostenine (1).

A defect of the vacuolar putative lipase Atg15 accelerates degradation of lipid droplets through lipolysis.

Lipid droplets (LDs) are the conserved organelles for the deposit of neutral lipids, and function as reservoirs of membrane and energy sources. To date, functional links between autophagy and LD dynamics have not been fully elucidated. Here, we report that a vacuolar putative lipase, Atg15, required for degradation of autophagic bodies, is crucial for the maintenance of LD amount in the yeast Saccharomyces cerevisiae in the stationary phase. Mutant analyses revealed that the putative lipase motif and vacuolar localization of Atg15 are important for the maintenance of LD amount. Loss of autophagosome formation by simultaneous deletion of core ATG genes cancelled the reduction in the LD amount in ATG15-deleted cells, indicating that degradation of autophagic bodies accounts for the functional involvement of Atg15 in LD dynamics. The reduced level of LDs in the mutant strain was dependent on Tgl3 and Tgl4, major lipases for lipolysis in S. cerevisiae. An altered phosphorylation status of Tgl3, higher accumulation of Tgl4, and closer associations of Tgl3 and Tgl4 with LDs were detected in the ATG15-deleted cells. Furthermore, increased levels of downstream metabolites of lipolysis in the mutant strain strongly suggested enhanced lipolytic activity caused by loss of ATG15. Our data provide evidence for a novel link between autophagic flux and LD dynamics integrated with Atg15 activity.

Dynactin 3D structure: implications for assembly and dynein binding.

The multisubunit protein complex, dynactin, is an essential component of the cytoplasmic dynein motor. High-resolution structural work on dynactin and the dynein/dynactin supercomplex has been limited to small subunits and recombinant fragments that do not report fully on either ≈1MDa assembly. In the present study, we used negative-stain electron microscopy and image analysis based on random conical tilt reconstruction to obtain a three-dimensional (3D) structure of native vertebrate dynactin. The 35-nm-long dynactin molecule has a V-shaped shoulder at one end and a flattened tip at the other end, both offset relative to the long axis of the actin-related protein (Arp) backbone. The shoulder projects dramatically away from the Arp filament core in a way that cannot be appreciated in two-dimensional images, which has implications for the mechanism of dynein binding. The 3D structure allows the helical parameters of the entire Arp filament core, which includes the actin capping protein, CP, to be determined for the first time. This structure exhibits near identity to F-actin and can be well fitted into the dynactin envelope. Molecular fitting of modeled CP-Arp polymers into the envelope shows that the filament contains between 7 and 9 Arp protomers and is capped at both ends. In the 7 Arp model, which agrees best with measured Arp stoichiometry and other structural information, actin capping protein (CP) is not present at the distal tip of the structure, unlike what is seen in the other models. The 3D structure suggests a mechanism for dynactin assembly and length specification.