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.     

Role of formins in actin assembly: nucleation and barbed-end association

Nucleation of branched actin filaments by the Arp2/3 complex is a conserved process in eukaryotic cells, yet the source of unbranched actin filaments has remained obscure. In yeast, formins stimulate assembly of actin cables independently of Arp2/3. Here, the conserved core of formin homology domains 1 and 2 of Bni1p (Bni1pFH1FH2) was found to nucleate unbranched actin filaments in vitro. Bni1pFH2 provided the minimal region sufficient for nucleation. Unique among actin nucleators, Bni1pFH1FH2 remained associated with the growing barbed ends of filaments. This combination of properties suggests a direct role for formins in regulating nucleation and polarization of unbranched filamentous actin structures.     

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.     

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.     

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.     

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.     

Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lamina

The passage of large-sized herpesviral capsids through the nuclear lamina and the inner nuclear membrane to leave the nucleus requires a dissolution of the nuclear lamina. Here, we report on the functions of M50/p35, a beta-herpesviral protein of murine cytomegalovirus. M50/p35 inserts into the inner nuclear membrane and is aggregated by a second viral protein, M53/p38, to form the capsid docking site. M50/p35 recruits the cellular protein kinase C for phosphorylation and dissolution of the nuclear lamina, suggesting that herpesviruses target a critical element of nuclear architecture.     

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.     

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.     

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.     

Virus-specific splicing inhibitor in extracts from cells infected with HIV-1

Human immunodeficiency virus type 1 (HIV-1), in contrast with most other retroviruses, encodes trans-regulatory proteins for virus gene expression. It is shown in this study, by means of an in vitro splicing system, that nuclear extracts obtained from cells infected with HIV-1 contain a factor (or factors) that specifically inhibits splicing of a synthetic SP6/HIV pre-messenger RNA (pre-mRNA)-containing donor and acceptor splice sites in the coding region for the envelope protein. It is also shown that the SP6/HIV pre-mRNA is not capable of assembly in a ribonucleoprotein complex, spliceosome, in extracts from infected cells. These findings raise the possibility that specific inhibition of pre-mRNA splicing in the envelope protein coding region by HIV-1 trans-regulatory factors might be one control mechanism for efficient production of structural viral proteins and virion assembly.     

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.     

SRC mediates a switch from microtubule- to actin-based motility of vaccinia virus

The cascade of events that leads to vaccinia-induced actin polymerization requires Src-dependent tyrosine phosphorylation of the viral membrane protein A36R. We found that a localized outside-in signaling cascade induced by the viral membrane protein B5R is required to potently activate Src and induce A36R phosphorylation at the plasma membrane. In addition, Src-mediated phosphorylation of A36R regulated the ability of virus particles to recruit and release conventional kinesin. Thus, Src activity regulates the transition between cytoplasmic microtubule transport and actin-based motility at the plasma membrane.     

Salmonella SipA polymerizes actin by stapling filaments with nonglobular protein arms

Like many bacterial pathogens, Salmonella spp. use a type III secretion system to inject virulence proteins into host cells. The Salmonella invasion protein A (SipA) binds host actin, enhances its polymerization near adherent extracellular bacteria, and contributes to cytoskeletal rearrangements that internalize the pathogen. By combining x-ray crystallography of SipA with electron microscopy and image analysis of SipA-actin filaments, we show that SipA functions as a "molecular staple," in which a globular domain and two nonglobular "arms" mechanically stabilize the filament by tethering actin subunits in opposing strands. Deletion analysis of the tethering arms provides strong support for this model.     

拟南芥HCF243蛋白参与叶绿体PSⅡ稳定调控机制的研究

核基因编码的调控因子在光系统Ⅱ（PSⅡ）的组装过程中起着关键的调控或辅助作用。已有研究证明,HCF243蛋白是一个重要的核基因编码的调控因子,推测它可能作为一个辅因子通过与D1蛋白的直接相互作用来维持后者在PSⅡ蛋白复合物中的稳定。为了探究HCF243蛋白在光抑制条件下在PSⅡ核心亚基尤其D1蛋白周转过程中的作用机制,首先利用TAIL-PCR的方法鉴定了hcf243突变体T-DNA的插入位点,并通过半定量RT-PCR对该基因（At3g15095）进行了表达模式分析,发现其表达量在叶中最高并随发育阶段逐渐上调,不同胁迫条件下的表达量也有变化。利用体内蛋白质同位素标记实验,发现hcf243突变体中D1蛋白组装到PSⅡ复合体中的速度明显低于野生型;进一步通过蛋白免疫印记分析证实,在光抑制条件下,相比野生型,hcf243突变体中PSⅡ复合物核心亚基D1降解速度显著加快,可能是造成PSⅡ复合体组装速率降低的原因。因此,通过上述实验推测HCF243蛋白作为一个辅因子,在PSⅡ反应中心D1蛋白的周转尤其降解过程中起着关键的调控作用。     

细胞松弛素B对牛卵母细胞体外成熟过程中微管、微丝及染色体的影响

细胞松弛素B(Cytochalasin B,CB)是一种抑制微丝聚合的药物,能抑制微丝的组装从而阻止胞质分裂和极体排放,是研究细胞分裂器形成与变化的重要药物。在牛卵母细胞体外成熟培养过程中加入7.5 μg/mL的CB进行处理,分析CB对减数分裂过程中细胞骨架形态、染色体的排列与分离等方面的影响。结果显示,CB处理后卵母细胞第一极体的排放受到了抑制,染色体的排列和分离受到了影响,出现了同源染色体分离不完全或分离不均匀及分离后又聚在一起等异常情况,形成许多二倍体卵母细胞;纺锤体微管的形态发生了变化,出现了两个纺锤体、巨大纺锤体和多极纺锤体等异常结构;微丝的正常分布受到了影响,染色体周围没有或少有微丝分布,皮质下的微丝分布也变得少而不均匀;这说明微丝与微管在减数分裂过程中是协同作用的,CB通过影响微丝的动态变化,改变了纺锤体微管的形态结构,最终抑制了极体的排放。     

A p53 amino-terminal nuclear export signal inhibited by DNA damage-induced phosphorylation

The p53 protein is present in low amounts in normally growing cells and is activated in response to physiological insults. MDM2 regulates p53 either through inhibiting p53's transactivating function in the nucleus or by targeting p53 degradation in the cytoplasm. We identified a previously unknown nuclear export signal (NES) in the amino terminus of p53, spanning residues 11 to 27 and containing two serine residues phosphorylated after DNA damage, which was required for p53 nuclear export in colloboration with the carboxyl-terminal NES. Serine-15-phosphorylated p53 induced by ultraviolet irradiation was not exported. Thus, DNA damage-induced phosphorylation may achieve optimal p53 activation by inhibiting both MDM2 binding to, and the nuclear export of, p53.