An actin-destabilizing factor is present in human plasma

Summary Plasma and serum of humans or experimental animals contain a factor which destabilizes F-actin. The factor has no DNAse or thrombin activity and after incubation with F-actin does not modify the position of the actin band on a SDS polyacrylamide gel. Hence it probably depolymerizes F-actin.Acknowledgment. This work was supported by the Swiss National Science Foundation, grant No.3.692-0.76. We thank Drs M. Crippa and C.A. Bouvier for measuring the DNAse and thrombin activities. The technical help of Mrs M. Redard and A. De Almeida and the photographic work of Mr J.-C. Rumbeli and Mr E. Denkinger are gratefully acknowledged.     

Swiprosin-1 modulates actin dynamics by regulating the F-actin accessibility to cofilin

Membrane protrusions, like lamellipodia, and cell movement are dependent on actin dynamics, which are regulated by a variety of actin-binding proteins acting cooperatively to reorganize actin filaments. Here, we provide evidence that Swiprosin-1, a newly identified actin-binding protein, modulates lamellipodial dynamics by regulating the accessibility of F-actin to cofilin. Overexpression of Swiprosin-1 increased lamellipodia formation in B16F10 melanoma cells, whereas knockdown of Swiprosin-1 inhibited EGF-induced lamellipodia formation, and led to a loss of actin stress fibers at the leading edges of cells but not in the cell cortex. Swiprosin-1 strongly facilitated the formation of entangled or clustered F-actin, which remodeled the structural organization of actin filaments making them inaccessible to cofilin. EGF-induced phosphorylation of Swiprosin-1 at Ser183, a phosphorylation site newly identified using mass spectrometry, effectively inhibited clustering of actin filaments and permitted cofilin access to F-actin, resulting in actin depolymerization. Cells overexpressing a Swiprosin-1 phosphorylation-mimicking mutant or a phosphorylation-deficient mutant exhibited irregular membrane dynamics during the protrusion and retraction cycles of lamellipodia. Taken together, these findings suggest that dynamic exchange of Swiprosin-1 phosphorylation and dephosphorylation is a novel mechanism that regulates actin dynamics by modulating the pattern of cofilin activity at the leading edges of cells.     

The neuronal p35 activator of Cdk5 is a novel F-actin binding and bundling protein

The neuronal Cdk5 activator p35 is involved in a multitude of neuronal activities, including cytoskeletal organization. We show here that p35 directly interacts with filamentous actin (F-actin) but not with monomeric actin (G-actin). Through binding, p35 induces the formation of actin bundles and stabilizes F-actin against dilution-induced depolymerization. p35 forms intermolecular self-associations, suggesting that p35 cross-links actin filaments into bundles via its intermolecular self-association. p35 dimerization and association with F-actin occur at the N-terminal region that is absent in the calpain-cleaved product p25, indicating that such p35 properties are lost by its truncation induced under neurotoxic conditions. Using p35 phosphorylated by Cdk5 and a mutational approach, we demonstrate that the phosphorylation of p35 promotes its homodimerization and p35-induced formation of F-actin bundles. In addition, the phosphorylation regulates p35 distribution to microtubule and actin cytoskeletons. Together, these observations define a novel function for p35 in cytoskeletal regulation.     

Calorimetric studies on monomeric and polymeric actin

Differential scanning calorimetry of polymeric F-actin at pH 8.0 showed that the polymer had a concentration-independent thermal profile with a single transition temperature of 81 degrees C. In contrast, the thermal profile of G-actin was concentration-dependent, and although it resembled the F-actin profile at lower concentrations, it was found to have a more complex profile at higher protein concentrations.     

Effect of phosphorylation of myosin light chains on interaction of heavy meromyosin with regulated F-actin in ghost fibers

The binding of phosphorylated heavy meromyosin to regulated F-actin in ghost fibers at high Ca2+ concentration increases, and at low Ca2+ concentration decreases, the anisotropy of intrinsic tryptophan fluorescence of F-actin. The effect is opposite to the effect of the binding of dephosphorylated heavy meromyosin.     

The turnover of F-actin-bound ADP in vivo.

A procedure for estimating the rate of turnover of F-actin-bound ADP in vivo is described. A turnover rate of 0.88 h-1 was determined for mouse muscle F-actin. The validity of the method when used to estimate the turnover rate of F-actin per se is discussed in relation to the possible exchange of F-actin-bound ADP.     

Effect of phosphorylation of myosin light chains on interaction of heavy meromyosin with regulated F-actin in ghost fibers

Summary The binding of phosphorylated heavy meromyosin to regulated F-actin in ghost fibers at high Ca²⁺ concentration increases, and at low Ca²⁺ concentration decreases, the anisotropy of intrinsic tryptophan fluorescence of F-actin. The effect is opposite to the effect of the binding of dephosphorylated heavy meromyosin.     

Calorimetric studies on monomeric and polymeric actin

Summary Differential scanning calorimetry of polymeric F-actin at pH 8.0 showed that the polymer had a concentration-independent thermal profile with a single transition temperature of 81 °C. In contrast, the thermal profile of G-actin was concentration-dependent, and although it resembled the F-actin profile at lower concentrations, it was found to have a more complex profile at higher protein concentrations.Acknowledgment. This research was carried out under grants to J.F.B. and P.J. from The Muscular Dystrophy Association of America.     

The turnover of F-actin-bound ADP in vivo

Summary A procedure for estimating the rate of turnover of F-actin-bound ADP in vivo is described. A turnover rate of 0.88 h^–1 was determined for mouse muscle F-actin. The validity of the method when used to estimate the turnover rate of F-actin per se is discussed in relation to the possible exchange of F-actin-bound ADP.Acknowledgments. The invaluable technical assistance of Mr L. Carrington is gratefully acknowledged.     

Effect in heavy meromyosin on conformation of F-actin

Summary Cooperative conformational changes of F-actin induced by heavy meromyosin (HMM) binding (in the absence of troponin and tropomyosin) were found by the method of polarized UV-fluorescence microscopy.     

连续性血液净化治疗对重症急性胰腺炎患者内皮细胞通透性的影响

目的探讨连续性血液净化治疗对重症急性胰腺炎(severeacutepancreatitis,SAP)患者内皮细胞的作用.方法人脐静脉内皮细胞(HumanUmbilicalVeinEndothelialCells,HUVEC)按实验分组分别用16例健康对照组血清,38例 SAP患者连续性血液净化(ContinuousBloodPurificantion,CBP)治疗前、治疗6h、治疗20h血清体外干预脐静脉内皮细胞5h.transwell小室观察内皮细胞通透性变化,激光共聚焦显微镜观察 F actin应力微丝的表达及分布.结果 SAP患者 CBP治疗前内皮细胞通透性较健康对照组明显增高(P<0.01),CBP治疗6h、治疗20h后通透性均降低(P<0.05),但治疗20h组与治疗6h组相比,差异无显著性(P>0.05).激光共聚焦结果显示,与健康对照组相比,CBP治疗前 SAP患者内皮细胞 F actin应力微丝的数量及密度均明显增加,CBP治疗6h后,F actin应力微丝形成减少,治疗20h后减少更加显著.结论重症急性胰腺炎患者内皮细胞通透性明显增加,CBP治疗可以显著降低 SAP患者内皮细胞通透性,其机制可能与调节 F actin应力微丝形成与分布有关     

The actin-binding domain of actin filament-associated protein (AFAP) is involved in the regulation of cytoskeletal structure

Actin filament-associated protein (AFAP) plays a critical role in the regulation of actin filament integrity, formation and maintenance of the actin network, function of focal contacts, and cell migration. Here, we show that endogenous AFAP was present not only in the cytoskeletal but also in the cytosolic fraction. Depolymerization of actin filaments with cytochalasin D or latrunculin A increased AFAP in the cytosolic fraction. AFAP harbors an actin-binding domain (ABD) in its C-terminus. AFAPΔABD, an AFAP mutant with selective ABD deletion, was mainly in the cytosolic fraction when overexpressed in the cells, which was associated with a disorganized cytoskeleton with reduced stress fibers, accumulation of F-actin on cellular membrane, and formation of actin-rich small dots. Cortactin, a well-known podosome marker, was colocalized with AFAPΔABD in these small dots at the ventral surface of the cell, indicating that these small dots fulfill certain criteria of podosomes. However, these podosome-like small dots did not digest gelatin matrix. This may be due to the reduced interaction between AFAPΔABD and c-Src. When AFAPΔABD-transfected cells were stimulated with phorbol ester, they formed podosome-like structures with larger sizes, less numerous and longer life span, in comparison with wild-type AFAP-transfected cells. These results indicate that the association of AFAP with F-actin through ABD is crucial for AFAP to regulate cytoskeletal structures. The AFAPΔABD, as cytosolic proteins, may be more accessible to the cellular membrane, podosome-like structures, and thus be more interactive for the regulation of cellular functions.     

Actin-based organelle movement

Evidence for actin-dependent organelle movement was first obtained from studies of cytoplasmic streaming in plants. These studies, together with cell-free organelle motility studies and biophysical analyses of muscle myosin, support a model whereby organelle-associated motor molecules utilize the energy of adenosine triphosphate binding and hydrolysis to drive movement along F-actin tracks Recent studies indicate that this mechanism for organelle movement may be responsible for organelle and vesicle movement during secretion, endocytosis and mitochondrial inheritance in a variety of eukaryotes.     

Effects of cytochalasin D on the actin cytoskeleton: association of neoformed actin aggregates with proteins involved in signaling and endocytosis

Cytochalasin D (CD) has been extensively used for assessing the role of the actin cytoskeleton in different biological processes. However, effects of CD have not always been consistent and CD-treated cells have been found to contain irregular spots of F-actin. By transfecting MCF-7 cells with an actin-enhanced yellow fluorescent protein fusion protein we show that, in vivo, CD induces actin aggregation de novo, while simultaneously depolymerizing preexisting actin cytoskeletal components. We also show that CD-induced actin aggregates bind the F-actin-selective drug phalloidin and associate with proteins involved in cell signaling as well as with receptors and endosomal markers (active MAP kinases, paxillin, erbB2, transferrin, Rab-5), but not with clathrin, protein kinase A, protein tyrosine phosphatase 1B, or tubulin. Thus, CD induces new sites of actin aggregation that selectively associate with several important regulatory proteins. Failure of CD to interupt a biological process may therefore not prove that the process is independent of actin aggregation.     

Nesprin interchain associations control nuclear size

Nesprins-1/-2/-3/-4 are nuclear envelope proteins, which connect nuclei to the cytoskeleton. The largest nesprin-1/-2 isoforms (termed giant) tether F-actin through their N-terminal actin binding domain (ABD). Nesprin-3, however, lacks an ABD and associates instead to plectin, which binds intermediate filaments. Nesprins are integrated into the outer nuclear membrane via their C-terminal KASH-domain. Here, we show that nesprin-1/-2 ABDs physically and functionally interact with nesprin-3. Thus, both ends of nesprin-1/-2 giant are integrated at the nuclear surface: via the C-terminal KASH-domain and the N-terminal ABD-nesprin-3 association. Interestingly, nesprin-2 ABD or KASH-domain overexpression leads to increased nuclear areas. Conversely, nesprin-2 mini (contains the ABD and KASH-domain but lacks the massive nesprin-2 giant rod segment) expression yields smaller nuclei. Nuclear shrinkage is further enhanced upon nesprin-3 co-expression or microfilament depolymerization. Our findings suggest that multivariate intermolecular nesprin interactions with the cytoskeleton form a lattice-like filamentous network covering the outer nuclear membrane, which determines nuclear size.     

A llama-derived gelsolin single-domain antibody blocks gelsolin–G-actin interaction

RNA interference has tremendously advanced our understanding of gene function but recent reports have exposed undesirable side-effects. Recombinant Camelid single-domain antibodies (VHHs) provide an attractive means for studying protein function without affecting gene expression. We raised VHHs against gelsolin (GsnVHHs), a multifunctional actin-binding protein that controls cellular actin organization and migration. GsnVHH-induced delocalization of gelsolin to mitochondria or the nucleus in mammalian cells reveals distinct subpopulations including free gelsolin and actin-bound gelsolin complexes. GsnVHH 13 specifically recognizes Ca²⁺-activated gelsolin (K _d ~10 nM) while GsnVHH 11 binds gelsolin irrespective of Ca²⁺ (K _d ~5 nM) but completely blocks its interaction with G-actin. Both GsnVHHs trace gelsolin in membrane ruffles of EGF-stimulated MCF-7 cells and delay cell migration without affecting F-actin severing/capping or actin nucleation activities by gelsolin. We conclude that VHHs represent a potent way of blocking structural proteins and that actin nucleation by gelsolin is more complex than previously anticipated.     

Myosin motor function: the ins and outs of actin-based membrane protrusions

Cells build plasma membrane protrusions supported by parallel bundles of F-actin to enable a wide variety of biological functions, ranging from motility to host defense. Filopodia, microvilli and stereocilia are three such protrusions that have been the focus of intense biological and biophysical investigation in recent years. While it is evident that actin dynamics play a significant role in the formation of these organelles, members of the myosin superfamily have also been implicated as key players in the maintenance of protrusion architecture and function. Based on a simple analysis of the physical forces that control protrusion formation and morphology, as well as our review of available data, we propose that myosins play two general roles within these structures: (1) as cargo transporters to move critical regulatory components toward distal tips and (2) as mediators of membrane-cytoskeleton adhesion.     

Cytoskeleton-mediated templating of complex cellulose-scaffolded extracellular structure and its association with oikosins in the urochordate <Emphasis Type="Italic">Oikopleura</Emphasis>

Oriented cellulose deposition is critical to plant patterning and models suggest microtubules constrain cellulose synthase movements through the plasma membrane. Though widespread in plants, urochordates are the only animals that synthesize cellulose. We characterized the distinctive cellulose microfibril scaffold of the larvacean house and its interaction with house structural proteins (oikosins). Targeted disruption of cytoskeletal elements, secretory pathways, and plasma membrane organization, suggested a working model for templating extracellular cellulose microfibrils from animal cells that shows both convergence and differences to plant models. Specialized cortical F-actin arrays template microfibril orientation and glycosylphosphatidylinositol-anchored proteins in lipid rafts may act as scaffolding proteins in microfibril elongation. Microtubules deliver and maintain cellulose synthase complexes to specific cell membrane sites rather than orienting their movement through the membrane. Oikosins are incorporated into house compartments directly above their corresponding cellular field of expression and interact with the cellulose scaffold to a variable extent.     

Reelin is a platelet protein and functions as a positive regulator of platelet spreading on fibrinogen

Abnormalities of platelet functions have been linked to reelin-impaired neuronal disorders. However, little attention has been given to understanding the interplay between reelin and platelet. In this study, reelin was found to present in the human platelets and megakaryocyte-like leukemic cells. Reelin-binding assays revealed that extracellular reelin can interact with platelets through the receptor belonging to the low density lipoprotein receptor gene family. The reelin-to-platelet interactions enhance platelet spreading on fibrinogen concomitant with the augmentation of lamellipodia formation and F-actin bundling. In contrast, reelin has no effect on integrin αIIbβ3 activation and agonist-induced platelet aggregation. Molecular analysis revealed that the up-regulation of Rac1 activity and the inhibition of protein kinase C δ-Thr505 phosphorylation are important for reelin-mediated enhancement of platelet spreading on fibrinogen. These findings demonstrate for the first time that reelin is present in platelets and the reelin-to-platelet interactions play a novel role in platelet signaling and functions.