Here we investigate the idea that there is a conserved mecha- nism by which Arp2/3complex activators additionally bind Ena/ VASP to maximize actin assembly. We show that this is true for WAVE and test the functional significance of the Ena/VASP-WAVE poly- peptide interaction. We further define what functional domains of Ena/VASP proteins are necessary for its effect on WAVE-based actin polymerization. For this study, we use a dual in vitro bead system/in vivo embryogenesis approach. In the in vitro system, cellular actin polymerization is reproduced on the surface of a bead in the form of an actin comet tail capable of propelling the bead forward, similar to the pushing out of the plasma membrane at the front of a moving cell (Wiesner et al., 2002; Plastino and Sykes, 2005). By changing what form of WAVE we absorb to the bead surface and what form of VASP we add to the motility mix, we address the functional conse- quences of the putative WAVE-VASP interaction and, in addition, which domains of VASP are required for its activity. In parallel, we ask the same questions in the ventral enclosure event of the devel- oping C. elegans embryo. Enclosure involves the formation of actin- filled protrusions by the ventral epidermal cells and their migration to the ventral midline of the embryo to seal the epithelial monolayer (Williams-Masson et al., 1997). As for lamellipodium formation in mammalian cells, WAVE and VASP (WVE-1 and UNC-34, respec- tively, in C. elegans vocabulary) are major players in ventral enclo- sure, with WAVE being the essential factor: when WAVE is removed, enclosure fails due to lack of migration of the epidermal cells (Patel
of VCA, at various VCA 䡠Arp2/3complex molar ratios in the load. A peak corresponding to free VCA appeared as soon as the VCA 䡠Arp2/3complex molar ratio in the load was higher than 1,
indicating that a single 1:1 VCA 䡠Arp2/3complex species was formed (Fig. 5A). Further addition of G-actin in increasing amounts in the load led to a single faster migrating species of FIGURE 5. Gel filtration analysis of the complexes of Arp2/3complex with VCA and actin. A, samples containing Arp2/3complex alone (3 M ) or supple- mented with VCA at indicated molar ratios were loaded and eluted on a Superdex 200 10 ⫻ 300 column at physiological ionic strength. B, elution patterns of samples containing Arp2/3complex alone or supplemented with VCA and actin at indicated ratios. Conditions as in A. C, SDS-PAGE analysis of the eluted VCA 䡠actin䡠Arp2/3 ternary complex and standards consisting of pre-mixed actin and Arp2/3 in a 1:1 molar ratio and at the indicated concentrations, from which the molar ratio of actin Arp2 and p34 in the eluted complex was derived. Standards and samples were electrophoresed on the same gel to establish the molar composition of VCA 䡠actin䡠Arp2/3complex rigorously. D, hydrodynamic radius and apparent molecular weight of the eluted complexes derived from the size-exclusion chromatography calibration with known protein standards. Stokes radius values are within 10% standard deviation.
Because assembly of actin filaments mediated by the Arp2/3complex has been shown to be an essential part of endocytosis in a wide range of organisms (for reviews see: Goley and Welch, 2006; Kaksonen et al., 2006; Gal- letta and Cooper, 2009), we stained C. albicans Arp2/3complex mutants with the lipophilic dye FM4-64, which is commonly used to visualize membrane internalization and endocytotic delivery to the vacuole (Vida and Emr, 1995). Both arp2D/D and arp2D/Darp3D/D mutants were clearly able to deliver FM4-64 to the vacuole as observed by the intracellular appearance of the dye after a 45 min chase period (Fig. 4A). In some Arp2/3 mutant cells, however, the vacuolar morphology appeared to be frag- mented, which sometimes resulted in staining throughout the vacuole. While in WT cells typically one to three vacu- oles were apparent, four or more smaller vacuoles could be observed in Arp2/3complex mutant cells. This frag- mented vacuolar morphology has also been described for
In addition to RhoGTPases, Nucleation Promoting Factors (NPFs) are involved in activating nucleators or maintaining their activity 19 . Arguably, the best studied NPF is the Wave regulatory complex (WRC), which consists of five subunits (SRA1, NAP1, Abi1, BRK1, WAVE2) and activates the Arp2/3complex to generate branched actin networks 19 . After the Wave complex detaches from Arp2/3, another NPF, cortactin, protects Arp2/3 against debranching 19 . Some NPFs can interact with multiple nucleators, making them prime candidates to mediate interplay. IQGAP1 can maintain the activity of mDia1 via its C-terminal DBR domain (Fig 1A) but also promote Arp2/3 activity by interacting with N-WASp and the Wave complex via its N-terminal Calponin Homology Domain (CHD) 20-22 . Another NPF, SPIN90/DIP/WISH/NCKIPSD, has been reported to interact with Arp2/3 in some studies and formins in others. SPIN90 forms a complex with Arp2/3 to stimulate formation of unbranched filaments 23 . In addition to providing the initial filament necessary for generation of dendritic networks by the WRC and Arp2/3 24 , very recent work proposes that SPIN90 competes with the WRC to modulate the degree of branching in networks 25 . Furthermore, SPIN90 can interact with the diaphanous related formins (DRF) mDia1 and mDia2 via its LRR and/or SH3 domains (Fig 1A) 26,27 and, surprisingly, it inhibits actin filament elongation by mDia2 but not mDia1 26 .
6 shell, dextran entry begins exactly at the site where the foci first appeared and spreads in the same pattern as the F-actin shell, continuing to lag the shell by ~30 s (Fig. 2A).
To test whether there is a causal relationship between these two processes, as suggested by their tight spatiotemporal correlation, we followed the kinetics of dextran entry in oocytes treated with CK-666 to block formation of the F-actin shell (Fig. 2B, C). Previous studies have shown that initial entry of small (25 kD) dextran occurs via NPC disassembly early in NEBD, when nuclear membranes are still intact, while entry of large (160 or 500 kD) dextran requires NE fragmentation . As expected, the initial entry kinetics of 25 kD dextran through NPCs prior to membrane fragmentation are not affected by CK-666 treatment (Fig. 2C, -5–0 minutes, gray lines), confirming that the F-actin shell and Arp2/3complex are not involved in NPC disassembly, which is driven by phosphorylation of nucleoporins . In contrast, entry of 160 kD dextran is considerably delayed in CK-666 treated oocytes (Fig. 2B, C, colored lines, Movie S2), implying a role for the F-actin shell in NE fragmentation. Interestingly, it is the rate of entry rather than the start time of entry that is affected, with a roughly 2-fold reduction in the peak entry rate (Fig. 2C, inset). To exclude any non-specific effects of CK-666, we confirmed this delay in dextran entry in oocytes treated with cytochalasin D or latrunculin B to prevent actin polymerization altogether (Fig. S2A). Finally, because those drugs may have side effects that damage the cell cortex, we also injected excess phalloidin into oocytes prior to NEBD to drive all cellular actin into stabilized cytoplasmic filament bundles and thereby prevent formation of any new F-actin structures without damaging the cell cortex. Phalloidin injection effectively blocks F-actin shell formation (data not shown) and delays entry of the 160 kD dextran in a manner indistinguishable from CK-666 treatment (Fig. 2B, C).
dishes fitted with German glass coverslips (“special dishes;” Goslin Amann, K.J., and Pollard, T.D. (2001). The Arp2/3complex nucleates and Banker, 1998) and coated with 1 mg/ml poly-D-lysine. Rat2 actin filament branches from the sides of pre-existing filaments. cells were grown in a low-bicarbonate medium as previously de- Nat. Cell Biol. 3, 306–310.
scribed (Bear et al., 2000). Neuronal cultures were plated as de- Arce, C.A., Hallak, M.E., Rodriguez, J.A., Barra, H.S., and Caputto, scribed above. For filopodia analysis, special dishes were trans- R. (1978). Capability of tubulin and microtubules to incorporate and ferred to a microscope humidified stage incubator that maintained to release tyrosine and phenylalanine and the effect of the incorpora- the cultures at 5% CO 2 . Time-lapse movies were made at 25 s tion of these amino acids on tubulin assembly. J. Neurochem. 31,
Currently, the available high-resolution structures of mammalian Arp2/3 cannot address the role of isoform-specified diversity: using natively purified proteins, only a single isoform combination - ARPC1B-ARPC5 - of mammalian Arp2/3 is visualised in these structures (for example, the first structure described by (Robinson et al., 2001); this combination was shown to have intermediate nucleation activity (Abella et al., 2016). To begin to understand how subunit composition affects the properties of human Arp2/3 complexes, we used recombinant protein expression and cryo-electron microscopy (cryo-EM) to determine the structure of the most active human Arp2/3complex, containing ARPC1B and ARPC5L subunits, referred to here as Arp2/3-C1B-C5L. We compared it with the structure of a complex containing ARPC1A and ARPC5 (Arp2/3-C1A-C5), which has the lowest activity (Abella et al., 2016). Our structures – the first sub-nanometre resolution reconstructions of any Arp2/3complex determined by cryo-EM – show isoform-specific differences in the N- terminus of ARPC5/5L and suggest that these structural variations mediate different
In this paper, we investigate the case of the expressions 3𝑥/3 and 3(𝑥/3) for 𝑥 a binary floating-point num- ber. Using a mixture of classical Rounding Analysis and systematic study of the elementary operations “3𝑥” and “𝑥/3”, we determinate for each possible value of 𝑥 the sign and the magnitude of the error in computing 3𝑥/3 and 3(𝑥/3). This study is used in a forthcoming article [ 5 ] to prove inclusion properties of some inter- val trisection operators. We expect it to be useful in the study of the many other algorithms that rely on a multiplication or a division by 3 (see, e.g., de Dinechin et al.’s work [ 1 ]).
We can look at a subring of the ring of Cheeger-Simons characters on X which are the “holo- morphic” characters (that is the G-cohomology defined in [Es, § 4], or equivalently the restricted differential characters defined in [Br, § 2]). This subring can be mapped onto the Deligne co- homology ring, but not in an injective way in general. When E is a holomorphic vector bundle with a compatible connection, the Cheeger-Simons theory (see [Ch-Si]) produces Chern classes with values in this subring. It is known that these classes are the same as the Deligne classes (see [Br] in the algebraic case and [Zu, § 5] for the general case). When E is topologically trivial, this construction gives the so-called secondary classes with values in the intermediate jacobians of X (see [Na] for a different construction, and [Ber] who proves the link with the generalized Abel-Jacobi map). The intermediate jacobians of X have been constructed in the K¨ahler case by Griffiths. They are complex tori (see [Vo 1, Ch. 12 § 1], and [Es-Vi, § 7 and 8]). If X is not K¨ahler, intermediate jacobians can still be defined but they are no longer complex tori.
Table 2 Some Early Classification of Systems
In each of these lists, each successive item increases in complexity, and to some degree incorporates the preceding entries. In addition, Bertalanffy suggests the “theories and models” useful in each level of his hierarchy. Although this is the kind of utility desired, both of these frameworks fail the first criterion as they do not differentiate among the systems of interest. All of the “test bed systems” are similar combinations of the last three levels in both hierarchies and then only if we assume complex human-designed systems are included in these categories.
But although their results are visually similar to ours, they are usually not based on a theory of Discrete Complex Analysis as solid and thorough as the present one, with the notable exception of , where the link to our theory is still not com- pletely clear. While most geometry processing papers are concerned with efficient algorithms producing beautiful pictures such as  and not primarily on the the- oretical side of the question, it is quite the contrary in this document, and the following numerics are therefore more a proof of concept and not optimized, the linear algebra library (JTEM) we used is very basic and we sticked to rough double precision.
arXiv:1608.05541v1 [math.DG] 19 Aug 2016
BO BERNDTSSON, DARIO CORDERO-ERAUSQUIN, BO’AZ KLARTAG, YANIR A. RUBINSTEIN
A BSTRACT . We introduce complex generalizations of the classical Legendre transform, operating on Kähler metrics on a compact complex manifold. These Legendre transforms give explicit local isometric symmetries for the Mabuchi metric on the space of Kähler metrics around any real analytic Kähler metric, answering a question originating in Semmes’ work.
1 (S = 1) ? 1 A 1 (S = 0)). Therefore, for isolated mole cules in solution, one can question if the lack of molecular interac tions does not forbid reverse LIESST photo switching.
In this Letter, we study the relaxation of the photo excited [Fe(2 CH 3 phen) 3 ] (BF 4 ) 2 (phen = 1,10 phenanthroline) complex dis solved in acetonitrile (CH 3 CN) by means of ultrafast pump probe time resolved optical absorption spectroscopy. This complex is of particular interest since, at room temperature, both HS (80%) and LS (20%) states are populated [23 26] . We ﬁrst excited the solution with picosecond pulses centered at k p = 500 nm. In good agreement with our previous study of [Fe(phen) 3 ] (BF 4 ) 2 complex
The Koszul duality theory of associative algebras is based on a chain complex, called the Koszul
complex, built on the tensor product of chain complexes. The notions of algebras, operads and
properads are “associative” notions in the sense that they are all monoids in a monoidal category. This makes the generalization of the Koszul complex to operads and properads possible. To define a Koszul duality theory for algebras over an operad P, we have to find a good generalization for this Koszul complex in a non-associative setting.
structure: SHELXL97 (Sheldrick, 1997); molecular graphics: ORTEP-3 ( Farrugia, 1997) and Mercury (CCDC); software used to prepare material for publication: SHELXL97 (Sheldrick, 1997).
Figure 1. The structure of complex (II), showing the atom-numbering scheme and displacement ellipsoids at the 30% probability level. For clarity, only the manganese atom and the atoms belonging to the silole ligand have been labelled. (nans1)
Our aim in this paper is to extend the structure of a g-complex on C • (R, M )
to the much richer structure of a calculus structure. The notion of a calculus structure originated in Hochschild cohomology theory [DTT] (in fact, the defini- tion in [DTT] differs from ours by some signs). It is defined as a representation (ι · , L · ) of a Gerstenhaber (=odd Poisson) algebra G on a complex (Ω, d), such
4 Our Remote Visualization System
Our visualization system is based on a client/server architecture. The server precomputes and stores the lighting structures and the level of detail (LOD) of each complex geometry represented as a progressive mesh. The server sends either new geometric (cf. Sec- tion 4.2) or lighting (cf. Section 4.1) level of detail depending on the client requests. After each data reception, the client performs some processes on the illumination structure and on the progressive mesh before uploading them on the GPU. Moreover, our approach allows to interleave geometric and lighting data when transmitting the scene from the server to the client. This offers a very smooth progressive visualization until the desired quality is reached. 4.1 Irradiance Vector Grid Streaming