Structural effects of cofilin on longitudinal contacts in F-actin

Structural effects of yeast cofilin on skeletal muscle and yeast actin were examined in solution. Cofilin binding to native actin was non-cooperative and saturated at a 1:1 molar ratio, with K(d)相似文献   

Cofilin induced conformational changes in F-actin expose subdomain 2 to proteolysis

Cofilin/ADF affects strongly the structure of actin filaments and especially the intermolecular contacts of the DNase I binding loop (D-loop) in subdomain 2. In G-actin, the D-loop is cleaved by subtilisin between Met47 and Gly48, while in F-actin this cleavage is inhibited. Here, we report that yeast cofilin, which is resistant to both subtilisin and trypsin, accelerates greatly the rate of subtilisin cleavage of this loop in F-actin at pH 6.8 and at pH 8.0. Similarly, cofilin accelerates strongly the tryptic cleavage in F-actin of loop 60-69 in subdomain 2, at Arg62 and Lys68. The acceleration of the loops' proteolysis cannot be attributed to an increased treadmilling of F-actin for the following reasons: (i) the rate of subtilisin cleavage is independent of pH between pH 6.8 and 8.0, unlike F-actin depolymerization, which is pH-dependent; (ii) at high concentrations of protease the cleavage rate of F-actin in the presence of cofilin is faster than the rate of monomer dissociation from the pointed end of TRC-labeled F-actin, which limits the rate of treadmilling; and (iii) cofilin also accelerates the rate of subtilisin cleavage of F-actin in which the treadmilling is blocked by interprotomer cross-linking of the D-loop to the C terminus on an adjacent protomer. This suggests a substantial flexibility of the D-loop in the cross-linked F-actin. The increased cleavage rates of the D-loop and loop 60-69 reveal extensive exposure of subdomain 2 in F-actin to proteolytic enzymes by cofilin.     

Cofilin cross-bridges adjacent actin protomers and replaces part of the longitudinal F-actin interface

ADF/cofilins are abundant actin binding proteins critical to the survival of eukaryotic cells. Most ADF/cofilins bind both G and F-actin, sever the filaments and accelerate their treadmilling. These effects are linked to rearrangements of interprotomer contacts, changes in the mean twist, and filament destabilization by ADF/cofilin. Paradoxically, it was reported that under certain in vitro and in vivo conditions cofilin may stabilize actin filaments and nucleate their formation. Here, we show that yeast cofilin and human muscle cofilin (cofilin-2) accelerate the nucleation and elongation of ADP-F-actin and stabilize such filaments. Moreover, cofilin rescues the polymerization of the assembly incompetent tethramethyl rhodamine (TMR)-actin and T203C/C374S yeast mutant actin. Filaments of cofilin-decorated TMR-actin and unlabeled actin are indistinguishable, as revealed by electron microscopy and three-dimensional reconstruction. Our data suggest that ADF/cofilins play an active role in establishing new interprotomer interfaces in F-actin that substitute for disrupted (as in TMR-actin and mutant actin) or weakened (as in ADP-actin) longitudinal contacts in filaments.     

Cooperative effects of cofilin (ADF) on actin structure suggest allosteric mechanism of cofilin function

Using site-specific fluorescence probes and cross-linking we demonstrated that cofilin (ADF), a key regulator of actin cellular dynamics, weakens longitudinal contacts in F-actin in a cooperative manner. Differential scanning calorimetry detected a dual nature of cofilin effects on F-actin conformation. At sub-stoichiometric cofilin to actin ratios, cofilin stabilized sterically and non-cooperatively protomers at the points of attachment, and destabilized allosterically and cooperatively protomers in the cofilin-free parts of F-actin. This destabilizing effect had a long range, with one cofilin molecule affecting more than 100 protomers, and concentration-dependent amplitude that reached maximum at about 1:2 molar ratio of cofilin to actin. In contrast to existing models, our results suggest an allosteric mechanism of actin depolymerization by cofilin. We propose that cofilin is less likely to sever actin filaments at the points of attachment as thought previously. Instead, due to its dual structural effect, spontaneous fragmentation occurs most likely in cofilin-free segments of filaments weakened allosterically by nearby cofilin molecules.     

Determining the differences in actin binding by human ADF and cofilin.

The actin-depolymerizing factor (ADF)/cofilin family of proteins play an essential role in actin dynamics and cytoskeletal re-organization. Human tissues express two isoforms in the same cells, ADF and cofilin, and these two proteins are more than 70% identical in amino acid sequence. We show that ADF is a much more potent actin-depolymerizing agent than cofilin: the maximum level of depolymerization at pH 8 by ADF is about 20 microM compared to 5 microM for cofilin, but little depolymerization occurs at pH 6.5 with either protein. However, we find little difference between the two proteins in their binding to filaments, their severing activities or their activation of subunit release from the pointed ends of filaments. Likewise, they show no significant differences in their affinities for monomeric actin: both bind 15-fold more tightly to actin.ADP than to actin.ATP. Complexes between actin.ADP and ADF or cofilin associate with both barbed and pointed ends of filaments at similar rates (close to those of actin.ATP and much higher than those of actin.ADP). This explains why high concentrations of both proteins reverse the activation of subunit release at pointed ends. The major difference between the two proteins is that the nucleating activity of cofilin-actin.ADP complexes is twice that of ADF-actin.ADP complexes and this, in turn, is twice that of actin.ATP alone. It is this weaker nucleating potential of ADF-actin.ADP that accounts for the much higher steady-state depolymerizing activity. The pH-sensitivity is due to the nucleating activity of complexes being greater at pH 6.5 than at pH 8. Sequence analysis of mammalian and avian isoforms shows a consistent pattern of charge differences in regions of the protein associated with F-actin-binding that may account for the differences in activity between ADF and cofilin.     

Polymerisation of chemically cross-linked actin:thymosin beta(4) complex to filamentous actin: alteration in helical parameters and visualisation of thymosin beta(4) binding on F-actin.

The beta-thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin beta(4) with F-actin. Thymosin beta(4) at 200 microM was chemically cross-linked to F-actin. In the presence of phalloidin, the chemically cross-linked actin:thymosin beta(4) complex was incorporated into F-actin. These mixed filaments were of normal appearance when inspected by conventional transmission electron microscopy after negative staining. We purified the chemically cross-linked actin:thymosin beta(4) complex, which polymerised only when phalloidin and the gelsolin:2-actin complex were present simultaneously. Using scanning transmission electron microscopy, the mass-per-length of control and actin:thymosin beta(4) filaments was found to be 16.0(+/-0.8) kDa/nm and 18.0(+/-0.9) kDa/nm, respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis of the helical parameters revealed an increase of the crossover spacing of the two right-handed long-pitch helical strands from 36.0 to 40.5 nm. Difference map analysis of 3-D helical reconstruction of control and actin:thymosin beta(4) filaments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible with published data of the atomic structure of thymosin beta(4). The deduced binding sites of thymosin beta(4) to actin were in agreement with those identified previously. However, parts of the difference map might represent subtle conformational changes of both proteins occurring upon complex formation.     

Dolastatin 11 connects two long-pitch strands in F-actin to stabilize microfilaments

Dolastatin 11, a drug isolated from the Indian Ocean sea hare Dolabella auricularia, arrests cytokinesis in vivo and increases the amount of F-actin to stabilize F-actin in vitro, like phalloidin and jasplakinolide. However, according to the previous biochemical study, the binding of dolastatin 11 to F-actin does not compete with that of phalloidin, suggesting that the binding sites are different. To understand the mechanism of F-actin stabilization by dolastatin 11, we determined the position of bound dolastatin 11 in F-actin using the X-ray fiber diffraction from oriented filament sols. Our analysis shows that the position of dolastatin 11 is clearly different from that of phalloidin. However, these bound drugs are present in the gap between the two long-pitch F-actin strands in a similar way. The result suggests that the connection between the two long-pitch F-actin strands might be a key for the control of F-actin stabilization.     

The F-actin distribution during microsporogenesis in Magnolia soulangeana Soul. (Magnoliaceae)

Summary F-actin distribution during male meiosis in Magnolia soulangeana was studied by means of fluorescence microscopy following staining with rhodaminephalloidin. Actin filaments were observed to persist during all of the developmental stages of meiosis. Four main types of configurations were recognized: (1) peripheral filaments underlying the plasma membrane (cortical network); (2) filaments dispersed throughout the inner cytoplasm (central cytoplasmic network); (3) filaments associated with the meiotic spindles; (4) filaments associated with the phragmoplasts. The cortical and central cytoplasmic filaments exhibited different behaviours. Whereas the cortical network remained present in an apparently unchanged form during all of the meiotic stages, the central cytoplasmic filaments, although they never completely disappeared, were reduced and concentrated around the nucleus at the end of prophase. At metaphase, fluorescent spindles consisting of filament bundles running from pole to pole or being interrupted at the equatorial zone could be seen. At the end of both the first and second division of meiosis, fluorescent bands of filaments (disks) appeared at the level of the cell division planes (equatorial regions) where cleavage furrows were constituted. These cleavage furrows did not form when floral buds were cultivated in a cytochalasin-containing medium. Our results show that during microsporogenesis in M. soulangeana the actin filaments constitute a highly complex and dynamic system that is involved in particular in cytoplasm cleavage of the meiocytes.     

Non-muscle tropomyosin (Tpm3) is crucial for asymmetric cell division and maintenance of cortical integrity in mouse oocytes

Tropomyosins are actin-binding cytoskeletal proteins that play a pivotal role in regulating the function of actin filaments in muscle and non-muscle cells; however, the roles of non-muscle tropomyosins in mouse oocytes are unknown. This study investigated the expression and functions of non-muscle tropomyosin (Tpm3) during meiotic maturation of mouse oocytes. Tpm3 mRNA was detected at all developmental stages in mouse oocytes. Tpm3 protein was localized at the cortex during the germinal vesicle and germinal vesicle breakdown stages. However, the overall fluorescence intensity of Tpm3 immunostaining was markedly decreased in metaphase II oocytes. Knockdown of Tpm3 impaired asymmetric division of oocytes and spindle migration, considerably reduced the amount of cortical actin, and caused membrane blebbing during cytokinesis. Expression of a constitutively active cofilin mutant and Tpm3 overexpression confirmed that Tpm3 protects cortical actin from depolymerization by cofilin. The data indicate that Tpm3 plays crucial roles in maintaining cortical actin integrity and asymmetric cell division during oocyte maturation, and that dynamic regulation of cortical actin by Tpm3 is critical to ensure proper polar body protrusion.     

Microfilaments (F-actin) in generative cells and sperm: an evaluation

Summary Disagreement has arisen over the presence of actin-containing microfilaments (Mfs) in angiosperm generative cells and sperm (GSP). In order to address this issue, we subjected GSP of Tradescantia virginiana, Nicotiana tabacum and Rhododendron laetum to a series of localizations using different antiactins, rhodamine phalloidin and antimyosin. Coordinate staining with antitubulin and Hoechst 33258 defined the status of the microtubule (Mt) cytoskeleton and stages of generative cell division. Additional experiments utilized cytochalasin D (CD). In no instance could Mfs be detected in GSP of the three species. Instead, Mfs seen at the periphery of GSP appear to be continuous with vegetative Mfs and thus are in the vegetative cytoplasm. Mfs are not seen in the constriction zone of dividing T. virginiana generative cells, nor are they indicated in the phragmoplast of N. tabacum and R. laetum. Myosin localizations reveal punctate staining in the vegetative cytoplasm and a thin line of fluorescence around the the outside of the generative cell. While CD seems to delay generative cell division, cytokinesis still takes place. CD-induced Mf fragments are evident in the vegetative cytoplasm but not in GSP. The weight of evidence therefore indicates that GSP do not contain Mfs. The implications of this conclusion for the behavior of GSP and the mechanism of cytokinesis in dividing generative cells are considerable.     

Mammalian CAP (Cyclase-associated protein) in the world of cell migration

Cell migration is essential for a variety of fundamental biological processes such as embryonic development, wound healing, and immune response. Aberrant cell migration also underlies pathological conditions such as cancer metastasis, in which morphological transformation promotes spreading of cancer to new sites. Cell migration is driven by actin dynamics, which is the repeated cycling of monomeric actin (G-actin) into and out of filamentous actin (F-actin). CAP (Cyclase-associated protein, also called Srv2) is a conserved actin-regulatory protein, which is implicated in cell motility and the invasiveness of human cancers. It cooperates with another actin regulatory protein, cofilin, to accelerate actin dynamics. Hence, knockdown of CAP1 slows down actin filament turnover, which in most cells leads to reduced cell motility. However, depletion of CAP1 in HeLa cells, while causing reduction in dynamics, actually led to increased cell motility. The increases in motility are likely through activation of cell adhesion signals through an inside-out signaling. The potential to activate adhesion signaling competes with the negative effect of CAP1 depletion on actin dynamics, which would reduce cell migration. In this commentary, we provide a brief overview of the roles of mammalian CAP1 in cell migration, and highlight a likely mechanism underlying the activation of cell adhesion signaling and elevated motility caused by depletion of CAP1.     

External Morphology and Muscle Arrangement of Brachionus urceolaris, Floscularia ringens, Hexarthra mira and Notommata glyphura (Rotifera, Monogononta)

We studied four monogonont rotifers (Brachionus urceolaris, Floscularia ringens, Hexarthra mira, Notommata glyphura) using two different techniques of microscopy: (1) the presence of filamentous actin was examined using phalloidin-fluorescent labelled specimens and a confocal laser scanning microscope (CLSM); (2) external morphology was investigated using a scanning electron microscope (SEM). B. urceolaris, F. ringens, and N. glyphura showed similar patterns of muscle distribution: a set of longitudinal muscles acting as head and foot retractors, and a set of circular muscles. However, the size and distribution of circular muscles differed among these species. H. mira differed from the other species in that it lacked circular muscles but possessed strong muscles that extended into each arm. The study showed that using both CLSM and SEM provides better resolution of the anatomy and external morphology of rotifers than using one of these techniques alone. This can facilitate better understanding of the complicated anatomy of these animals.     

Actin Depolymerizing Factor (ADF/Cofilin) Enhances the Rate of Filament Turnover: Implication in Actin-based Motility

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.     

UNC-60B, an ADF/cofilin family protein, is required for proper assembly of actin into myofibrils in Caenorhabditis elegans body wall muscle

The Caenorhabditis elegans unc-60 gene encodes two functionally distinct isoforms of ADF/cofilin that are implicated in myofibril assembly. Here, we show that one of the gene products, UNC-60B, is specifically required for proper assembly of actin into myofibrils. We found that all homozygous viable unc-60 mutations resided in the unc-60B coding region, indicating that UNC-60B is responsible for the Unc-60 phenotype. Wild-type UNC-60B had F-actin binding, partial actin depolymerizing, and weak F-actin severing activities in vitro. However, mutations in UNC-60B caused various alterations in these activities. Three missense mutations resulted in weaker F-actin binding and actin depolymerizing activities and complete loss of severing activity. The r398 mutation truncated three residues from the COOH terminus and resulted in the loss of severing activity and greater actin depolymerizing activity. The s1307 mutation in a putative actin-binding helix caused greater activity in actin-depolymerizing and severing. Using a specific antibody for UNC-60B, we found varying protein levels of UNC-60B in mutant animals, and that UNC-60B was expressed in embryonic muscles. Regardless of these various molecular phenotypes, actin was not properly assembled into embryonic myofibrils in all unc-60 mutants to similar extents. We conclude that precise control of actin filament dynamics by UNC-60B is required for proper integration of actin into myofibrils.     

Mammalian CAP (Cyclase-associated protein) in the world of cell migration: Roles in actin filament dynamics and beyond

Cell migration is essential for a variety of fundamental biological processes such as embryonic development, wound healing, and immune response. Aberrant cell migration also underlies pathological conditions such as cancer metastasis, in which morphological transformation promotes spreading of cancer to new sites. Cell migration is driven by actin dynamics, which is the repeated cycling of monomeric actin (G-actin) into and out of filamentous actin (F-actin). CAP (Cyclase-associated protein, also called Srv2) is a conserved actin-regulatory protein, which is implicated in cell motility and the invasiveness of human cancers. It cooperates with another actin regulatory protein, cofilin, to accelerate actin dynamics. Hence, knockdown of CAP1 slows down actin filament turnover, which in most cells leads to reduced cell motility. However, depletion of CAP1 in HeLa cells, while causing reduction in dynamics, actually led to increased cell motility. The increases in motility are likely through activation of cell adhesion signals through an inside-out signaling. The potential to activate adhesion signaling competes with the negative effect of CAP1 depletion on actin dynamics, which would reduce cell migration. In this commentary, we provide a brief overview of the roles of mammalian CAP1 in cell migration, and highlight a likely mechanism underlying the activation of cell adhesion signaling and elevated motility caused by depletion of CAP1.     

Immunocytochemistry of the neuromuscular systems of Loxosomella vivipara and L. parguerensis (Entoprocta: Loxosomatidae)

Little detailed information exists on the anatomy of the nervous system and the musculature of Entoprocta. Herein we describe the distribution of the neurotransmitters RFamide and serotonin as well as the myo-anatomy of adults and asexually produced budding stages of the solitary entoproct species Loxosomella vivipara and L. parguerensis using immunocytochemistry and epifluorescence as well as confocal microscopy. The development of the RFamidergic and serotonergic nervous system starts in early budding stages. In the adults, RFamide is present in the bilateral symmetric cerebral ganglion, a pair of oral nerves that innervate two pairs of nerve cell clusters in the heel of the foot, a pair of aboral nerves, the paired lateral nerves, the calyx nerves, the atrial ring nerve, the tentacle nerves, the stomach nerves, and the rectal nerves. Serotonin is only found in the cerebral ganglion, the oral nerves, and in the tentacle nerves. Some differences in the distribution of both neurotransmitters were found between L. vivipara and L. parguerensis and are most obvious in the differing number of large serotonergic perikarya associated with the oral nerves. Nerves arising from the cerebral ganglion and running in a ventral direction have not been described for Entoprocta before, and the homology of these to the ventral nerve cords of other Spiralia is considered possible. The body musculature of both Loxosomella species comprises longitudinal and diagonal muscles in the foot, the stalk, and the calyx. We found several circular muscles in the calyx. The stalk and parts of the foot and the calyx are surrounded by a fine outer layer of ring muscles. In addition to the congruent details regarding the myo-anatomy of both species, species-specific muscle structures could be revealed. The comparison of our data with recent findings of the myo-anatomy of two Loxosoma species indicates that longitudinal and diagonal body muscles, atrial ring muscles, tentacle muscles, esophageal and rectal ring muscles, as well as intestinal and anal sphincters are probably part of the ancestral entoproct muscle bauplan.     

Prolyl Hydroxylase Domain (PHD) 2 Affects Cell Migration and F-actin Formation via RhoA/Rho-associated Kinase-dependent Cofilin Phosphorylation

Cells are responding to hypoxia via prolyl-4-hydroxylase domain (PHD) enzymes, which are responsible for oxygen-dependent hydroxylation of the hypoxia-inducible factor (HIF)-1α subunit. To gain further insight into PHD function, we generated knockdown cell models for the PHD2 isoform, which is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. Induction of a PHD2 knockdown in tetracycline-inducible HeLa PHD2 knockdown cells resulted in increased F-actin formation as detected by phalloidin staining. A similar effect could be observed in the stably transfected PHD2 knockdown cell clones 1B6 and 3B7. F-actin is at least in part responsible for shaping cell morphology as well as regulating cell migration. Cell migration was impaired significantly as a consequence of PHD2 knockdown in a scratch assay. Mechanistically, PHD2 knockdown resulted in activation of the RhoA (Ras homolog gene family member A)/Rho-associated kinase pathway with subsequent phosphorylation of cofilin. Because cofilin phosphorylation impairs its actin-severing function, this may explain the F-actin phenotype, thereby providing a functional link between PHD2-dependent signaling and cell motility.     

Interaction of pollen F-actin with rabbit muscle myosin and its subfragments (HMM,S1)

Summary Actin purified from maize pollen grains can be polymerized into F-actin which increased the ATPase activities of proteolytic fragments (HMM, S₁) of rabbit muscle myosin. The values of K_app is 232 M for HMM and 290 M for S₁, which are six- and seven-fold higher than those of rabbit muscle F-actin under the same conditions. Pollen actin and rabbit muscle myosin form hybrid actomyosin showing increase in viscosity and turbidity of solution. Viscosity and turbidity of the actomyosin dropped and then increased again with addition of ATP. Polymerized pollen actin can be decorated in vitro with both rabbit muscle HMM and S₁ to form an arrowhead-shaped structure like that observed in living plant cells. The results show that pollen actin is similar to muscle actin at a qualitative level. But there are differences between them at a quantitative level.Abbreviations HMM heavy meromyosin - S₁ myosin subfragment 1 - ATP adenosine-5-triphosphate     

Functional morphology of the muscles in Philodina sp. (Rotifera: Bdelloidea)

Whole-mounts of Philodina sp., a bdelloid rotifer, were stained with fluorescent-labeled phalloidin to visualize the musculature. Several different muscle types were identified including incomplete circular bands, coronal retractors and foot retractors. Based on the position of the larger muscle bands in the body wall, their function during creeping locomotion and tun formation was inferred. Bdelloid creeping begins with the contraction of incomplete circular muscle bands against the hydrostatic pseudocoel, resulting in an anterior elongation of the body. One or more sets of ventral longitudinal muscles then contract bringing the rostrum into contact with the substrate, where it presumably attaches via adhesive glands. Different sets of ventral longitudinal muscles, foot and trunk retractors, function to pull the body forward. These same longitudinal muscle sets are also used in `tun' formation, in which the head and foot are withdrawn into the body. Three sets of longitudinal muscles supply the head region (anterior head segments) and function in withdrawal of the corona and rostrum. Two additional pairs of longitudinal muscles function to retract the anterior trunk segments immediately behind the head, and approximately five sets of longitudinal retractors are involved in the withdrawal of the foot and posterior toes. To achieve a greater understanding of rotifer behavior, it is important to elucidate the structural complexity of body wall muscles in rotifers. The utility of fluorescently-labeled phalloidin for the visualization of these muscles is discussed and placed in the context of rotifer functional morphology.     

Patterns of arrangement of lateral appendages on axes of Stigmaria ficoides (Sternberg) Brongniart

The arrangement of lateral insertions has been examined on axes of Stigmaria ficoides . The arrangement can be considered as a phyllotactic pattern made up of parastichies and orthostichies. Orthodox phyllotactic patterns, based on parastichy numbers from the Fibonacci or common accessory series, do not occur. Instead spiral (or multijugate) arrangements occur based on pairs of parastichy numbers such as x and x + 1, x and x + 2, x and x + 3, etc. and x ranges from 12 to 19. Whorled arrangements occur relatively infrequently. Individual axes commonly show frequent changes in pattern. The observations are used to make deductions about the growth and homology of stigmarian axes.