Nebulin and N-WASP cooperate to cause IGF-1-induced sarcomeric actin filament formation

Insulin-like growth factor 1 (IGF-1) induces skeletal muscle maturation and enlargement (hypertrophy). These responses require protein synthesis and myofibril formation (myofibrillogenesis). However, the signaling mechanisms of myofibrillogenesis remain obscure. We found that IGF-1-induced phosphatidylinositol 3-kinase-Akt signaling formed a complex of nebulin and N-WASP at the Z bands of myofibrils by interfering with glycogen synthase kinase-3β in mice. Although N-WASP is known to be an activator of the Arp2/3 complex to form branched actin filaments, the nebulin-N-WASP complex caused actin nucleation for unbranched actin filament formation from the Z bands without the Arp2/3 complex. Furthermore, N-WASP was required for IGF-1-induced muscle hypertrophy. These findings present the mechanisms of IGF-1-induced actin filament formation in myofibrillogenesis required for muscle maturation and hypertrophy and a mechanism of actin nucleation.     

Actin polymerization-driven molecular movement of mDia1 in living cells

mDia1, a Rho effector, belongs to the Formin family of proteins, which shares the conserved tandem FH1-FH2 unit structure. Formins including mDia1 accelerate actin nucleation while interacting with actin filament fast-growing ends. Here our single-molecule imaging revealed fast directional movement of mDia1 FH1-FH2 for tens of microns in living cells. The movement of mDia1 FH1-FH2 was blocked by actin-perturbing drugs, and the speed of mDia1 FH1-FH2 movement appeared to correlate with actin elongation rates. In vitro, mDia1 FH1-FH2 associated persistently with the growing actin barbed end. mDia1 probably moves processively along the growing end of actin filaments in cells, and Formins may be a molecular motility machinery that is independent from motor proteins.     

Structure of Arp2/3 complex in its activated state and in actin filament branch junctions

The seven-subunit Arp2/3 complex choreographs the formation of branched actin networks at the leading edge of migrating cells. When activated by Wiskott-Aldrich Syndrome protein (WASp), the Arp2/3 complex initiates actin filament branches from the sides of existing filaments. Electron cryomicroscopy and three-dimensional reconstruction of Acanthamoeba castellanii and Saccharomyces cerevisiae Arp2/3 complexes bound to the WASp carboxy-terminal domain reveal asymmetric, oblate ellipsoids. Image analysis of actin branches indicates that the complex binds the side of the mother filament, and Arp2 and Arp3 (for actin-related protein) are the first two subunits of the daughter filament. Comparison to the actin-free, WASp-activated complexes suggests that branch initiation involves large-scale structural rearrangements within Arp2/3.     

Requirement for coronin 1 in T lymphocyte trafficking and cellular homeostasis

The evolutionarily conserved actin-related protein (Arp2/3) complex is a key component of actin filament networks that is dynamically regulated by nucleation-promoting and inhibitory factors. Although much is known about actin assembly, the physiologic functions of inhibitory proteins are unclear. We generated coronin 1-/- mice and found that coronin 1 exerted an inhibitory effect on cellular steady-state F-actin formation via an Arp2/3-dependent mechanism. Whereas coronin 1 was required for chemokine-mediated migration, it was dispensable for T cell antigen receptor functions in T cells. Moreover, actin dynamics, through a mitochondrial pathway, was linked to lymphocyte homeostasis.     

Dynamic nuclear actin assembly by Arp2/3 complex and a baculovirus WASP-like protein

Diverse bacterial and viral pathogens induce actin polymerization in the cytoplasm of host cells to facilitate infection. Here, we describe a pathogenic mechanism for promoting dynamic actin assembly in the nucleus to enable viral replication. The baculovirus Autographa californica multiple nucleopolyhedrovirus induced nuclear actin polymerization by translocating the host actin-nucleating Arp2/3 complex into the nucleus, where it was activated by p78/83, a viral Wiskott-Aldrich syndrome protein (WASP)-like protein. Nuclear actin assembly by p78/83 and Arp2/3 complex was essential for viral progeny production. Recompartmentalizing dynamic host actin may represent a conserved mode of pathogenesis and reflect viral manipulation of normal functions of nuclear actin.     

Signal transduction. N-WASP regulation--the sting in the tail

Signaling proteins can be regulated by their interactions with other proteins and phospholipids. As Fawcett and Pawson discuss in their Perspective, activation of the N-WASP protein (which coordinates formation of actin filaments) is far more complex, depending on the interaction of N-WASP with both a protein and a phospholipid. The authors explain new results (Prehoda et al.) demonstrating that cooperative binding of the phospholipid PIP2 and the small GTPase Cdc42 to N-WASP results in its activation. The Arp2/3 complex is then able to bind to N-WASP and to proceed with its job of initiating the assembly of actin monomers into actin filaments.     

Rotational movement of the formin mDia1 along the double helical strand of an actin filament

Formin homology proteins (formins) elongate actin filaments (F-actin) by continuously associating with filament tips, potentially harnessing actin-generated pushing forces. During this processive elongation, formins are predicted to rotate along the axis of the double helical F-actin structure (referred to here as helical rotation), although this has not yet been definitively shown. We demonstrated helical rotation of the formin mDia1 by single-molecule fluorescence polarization (FL(P)). FL(P) of labeled F-actin, both elongating and depolymerizing from immobilized mDia1, oscillated with a periodicity corresponding to that of the F-actin long-pitch helix, and this was not altered by actin-bound nucleotides or the actin-binding protein profilin. Thus, helical rotation is an intrinsic property of formins. To harness pushing forces from growing F-actin, formins must be anchored flexibly to cell structures.     

Actin network architecture can determine myosin motor activity

The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture are maintained in a dynamic steady state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization, myosins selectively contracted and disassembled antiparallel actin structures, while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This "orientation selection" mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.     

Leiomodin is an actin filament nucleator in muscle cells

Initiation of actin polymerization in cells requires nucleation factors. Here we describe an actin-binding protein, leiomodin, that acted as a strong filament nucleator in muscle cells. Leiomodin shared two actin-binding sites with the filament pointed end-capping protein tropomodulin: a flexible N-terminal region and a leucine-rich repeat domain. Leiomodin also contained a C-terminal extension of 150 residues. The smallest fragment with strong nucleation activity included the leucine-rich repeat and C-terminal extension. The N-terminal region enhanced the nucleation activity threefold and recruited tropomyosin, which weakly stimulated nucleation and mediated localization of leiomodin to the middle of muscle sarcomeres. Knocking down leiomodin severely compromised sarcomere assembly in cultured muscle cells, which suggests a role for leiomodin in the nucleation of tropomyosin-decorated filaments in muscles.     

Rocket launcher mechanism of collaborative actin assembly defined by single-molecule imaging

Interacting sets of actin assembly factors work together in cells, but the underlying mechanisms have remained obscure. We used triple-color single-molecule fluorescence microscopy to image the tumor suppressor adenomatous polyposis coli (APC) and the formin mDia1 during filament assembly. Complexes consisting of APC, mDia1, and actin monomers initiated actin filament formation, overcoming inhibition by capping protein and profilin. Upon filament polymerization, the complexes separated, with mDia1 moving processively on growing barbed ends while APC remained at the site of nucleation. Thus, the two assembly factors directly interact to initiate filament assembly and then separate but retain independent associations with either end of the growing filament.     

小麦生理型雄性不育系微丝骨架和胼胝质的变化与其相关基因的表达分析

【目的】研究生理型雄性不育小麦花粉细胞内微丝和胼胝质的结构及其相关基因的表达,并揭示其与生理型雄性不育的关系,为进一步研究化学杂交剂SQ-1诱导小麦生理型雄性不育的机理提供一定的理论依据。【方法】以化学杂交剂SQ-1诱导的生理型雄性不育系ms(A)-西农1376及对应正常可育系(A)-西农1376为试材,用TRITC-phalloidin标记细胞内微丝,苯胺蓝标记胼胝质,qRT-PCR技术分别对肌动蛋白解聚因子TaADF（Actin depolymerizing factor）、类葡聚糖合成酶TaGSL（Glucan synthase-like）进行差异表达分析。【结果】(1)在减数分裂前期Ⅰ、中期Ⅰ、后期Ⅰ这三个时期,生理型雄性不育系花粉细胞的微丝结构与可育系没有显著差异：前期Ⅰ,微丝分布于整个细胞质中,细胞核区域也可见少量微丝环绕细胞核;中期Ⅰ,微丝分布在细胞质中,在形成纺锤体部位染色更深,形成纺锤体微丝,由细胞两极发出的纺锤体微丝伸向赤道板;后期Ⅰ,在向两极移动的染色体的中间部位染色较深,微丝分布较多。(2)在早末期Ⅰ,与可育系相比,不育系花粉细胞没有形成清晰且明显可见的中国灯笼状成膜体微丝结构,且在细胞中线部位亦没有清晰可见的微丝累积。(3)晚末期Ⅰ,可育系花粉细胞在形成细胞板的部位是线性的、平滑的,成膜体微丝消失,而不育系花粉细胞在形成细胞板的部位形成了很大的缝隙,同时,可育系胼胝质在细胞板处的沉积比较平滑,而不育系胼胝质在细胞板处的沉积较可育系相比缺乏,并且是褶皱的、有裂纹的。(4)四分体时期,可育系花粉可见围绕细胞核的辐射状微丝,不育系花粉细胞中微丝呈模糊状态,并且不育系中胼胝质染色的整体荧光强度较可育系减弱。利用实时荧光定量PCR技术分析肌动蛋白解聚因子TaADF和类葡聚糖合成酶TaGSL在减数分裂期的相对表达量,结果发现,不育系中TaADF的相对表达量是可育系的4.28倍,由于TaADF表达量上调,加剧了细胞内微丝解聚,微丝结构受到破坏,同时不育系中TaGSL表达量下降,只有可育系的0.83倍,胼胝质的沉积也受到影响。【结论】TaADF在不育系中上调表达,破坏了细胞内微丝的正常结构,使微丝不能正常行使其功能,进而可能导致花药发育中与育性相关的某些代谢通路等受到影响。与此同时,微丝结构的破坏导致细胞板形成出现异常也可能是引起胼胝质在细胞板处沉积受到影响的一个重要原因。因此,微丝和胼胝质的异常变化与化学杂交剂SQ-1诱导的生理型雄性不育密切相关。     

早春施肥对油松生长与针叶内色素含量、净光合速率的影响

为了研究施肥对油松幼苗生长、色素以及净光合速率的影响,笔者在田间条件下,设置4个施肥处理,即FH1~FH4,氮肥用量分别是15、30、45、60 kg/hm2,磷肥用量分别是30、30、50、50 kg/hm2,钾肥用量分别是40、60、60、40 kg/hm2,1个对照,3次重复。结果表明：油松幼苗高度随施氮量增加而增加,FH2~FH4之间无显著差异,显著高于对照;FH3和FH4的根茎叶、总干物质积累量之间无显著差异,显著高于对照,除根系外,FH1、FH2以及对照之间无显著差异;所有施肥处理叶绿素a含量无显著差异,显著高于对照;FH2~FH4的叶绿素b含量均无显著差异,显著高于对照;FH3和FH4的类胡萝卜素含量之间无显著差异,显著低于对照;总叶绿素含量所有施肥处理之间无显著差异;FH3与FH4的净光合速率之间无显著差异,显著高于其他处理和对照。综合分析认为,FH3处理为最佳的施肥配比,FH4与FH2次之,生产中应用FH3处理较好。     

早春施肥对油松生长与针叶色素含量、净光合速率的影响

为了研究施肥对油松幼苗生长、色素含量以及净光合速率的影响,在田间条件下,设置4个施肥处理,即FH1～FH4,氮肥用量分别是15、30、45、60kg/hm2,磷肥用量分别是30、30、50、50kg/hm2,钾肥用量分别是40、60、60、40kg/hm2,1个对照,3次重复。结果表明:油松幼苗高度随施氮量增加而增加,FH2～FH4之间无显著差异,显著高于对照;FH3和FH4的根茎叶、总干物质积累量之间无显著差异,显著高于对照,除根系外,FH1、FH2以及对照之间无显著差异;所有施肥处理叶绿素a含量无显著差异,显著高于对照;FH2～FH4的叶绿素b含量均无显著差异,显著高于对照;FH3和FH4的类胡萝卜素含量之间无显著差异,显著低于对照;总叶绿素含量所有施肥处理之间无显著差异;FH3与FH4的净光合速率之间无显著差异,显著高于其他处理和对照。综合分析认为,FH3处理为最佳的施肥配比,FH4与FH2次之,生产中应用FH3处理较好。     

Direct redox regulation of F-actin assembly and disassembly by Mical

Different types of cell behavior, including growth, motility, and navigation, require actin proteins to assemble into filaments. Here, we describe a biochemical process that was able to disassemble actin filaments and limit their reassembly. Actin was a specific substrate of the multidomain oxidation-reduction enzyme, Mical, a poorly understood actin disassembly factor that directly responds to Semaphorin/Plexin extracellular repulsive cues. Actin filament subunits were directly modified by Mical on their conserved pointed-end, which is critical for filament assembly. Mical posttranslationally oxidized the methionine 44 residue within the D-loop of actin, simultaneously severing filaments and decreasing polymerization. This mechanism underlying actin cytoskeletal collapse may have broad physiological and pathological ramifications.     

Integration of multiple signals through cooperative regulation of the N-WASP-Arp2/3 complex

The protein N-WASP [a homolog to the Wiskott-Aldrich syndrome protein (WASP)] regulates actin polymerization by stimulating the actin-nucleating activity of the actin-related protein 2/3 (Arp2/3) complex. N-WASP is tightly regulated by multiple signals: Only costimulation by Cdc42 and phosphatidylinositol (4,5)-bisphosphate (PIP2) yields potent polymerization. We found that regulation requires N-WASP's constitutively active output domain (VCA) and two regulatory domains: a Cdc42-binding domain and a previously undescribed PIP(2)-binding domain. In the absence of stimuli, the regulatory modules together hold the VCA-Arp2/3 complex in an inactive "closed" conformation. In this state, both the Cdc42- and PIP2-binding sites are masked. Binding of either input destabilizes the closed state and enhances binding of the other input. This cooperative activation mechanism shows how combinations of simple binding domains can be used to integrate and amplify coincident signals.     

Single-molecule speckle analysis of actin filament turnover in lamellipodia

Lamellipodia are thin, veil-like extensions at the edge of cells that contain a dynamic array of actin filaments. We describe an approach for analyzing spatial regulation of actin polymerization and depolymerization in vivo in which we tracked single molecules of actin fused to the green fluorescent protein. Polymerization and the lifetime of actin filaments in lamellipodia were measured with high spatial precision. Basal polymerization and depolymerization occurred throughout lamellipodia with largely constant kinetics, and polymerization was promoted within one micron of the lamellipodium tip. Most of the actin filaments in the lamellipodium were generated by polymerization away from the tip.     

Differential transmission of actin motion within focal adhesions

Cell migration requires the transmission of motion generated in the actin cytoskeleton to the extracellular environment through a complex assembly of proteins in focal adhesions. We developed correlational fluorescent speckle microscopy to measure the coupling of focal-adhesion proteins to actin filaments. Different classes of focal-adhesion structural and regulatory molecules exhibited varying degrees of correlated motions with actin filaments, indicating hierarchical transmission of actin motion through focal adhesions. Interactions between vinculin, talin, and actin filaments appear to constitute a slippage interface between the cytoskeleton and integrins, generating a molecular clutch that is regulated during the morphodynamic transitions of cell migration.     

gCap39, a calcium ion- and polyphosphoinositide-regulated actin capping protein

The polymerization of actin filaments is involved in growth, movement, and cell division. It has been shown that actin polymerization is controlled by gelsolin, whose interactions with actin are activated by calcium ion (Ca2+) and inhibited by membrane polyphosphoinositides (PPI). A smaller Ca2(+)- and PPI-regulated protein, gCap39, which has 49% sequence identity with gelsolin, has been identified by cDNA cloning and protein purification. Like gelsolin, gCap39 binds to the fast-growing (+) end of actin filaments. However, gCap39 does not sever actin filaments and can respond to Ca2+ and PPI transients independently, under conditions in which gelsolin is ineffective. The coexistence of gCap39 with gelsolin should allow precise regulation of actin assembly at the leading edge of the cell.     

Polarity and velocity of sliding filaments: control of direction by actin and of speed by myosin

Myosin filaments, which are responsible for a large repertoire of motile activities in muscle and nonmuscle cells, can translocate actin filaments both toward and away from their central bare zone. This bidirectional movement suggests that there is enough flexibility in the head portion of the tightly packed myosin molecules in the native myosin filaments to move actin filaments not only in the expected direction, but also in the direction opposite to that predicted by the regular structure of muscle--away from the center of the myosin filament.     

Assembly mechanism of the contractile ring for cytokinesis by fission yeast

Animals and fungi assemble a contractile ring of actin filaments and the motor protein myosin to separate into individual daughter cells during cytokinesis. We used fluorescence microscopy of live fission yeast cells to observe that membrane-bound nodes containing myosin were broadly distributed around the cell equator and assembled into a contractile ring through stochastic motions, after a meshwork of dynamic actin filaments appeared. Analysis of node motions and numerical simulations supported a mechanism whereby transient connections are established when myosins in one node capture and exert force on actin filaments growing from other nodes.