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1
Cano, M. L., D. A. Lauffenburger, and S. H. Zigmond. "Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution." Journal of Cell Biology 115, no. 3 (November 1, 1991): 677–87. http://dx.doi.org/10.1083/jcb.115.3.677.
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The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide-stimulated cells was examined. F-actin was quantified by the TRITC-labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar.
2
MORIYAMA, Kenji, and Ichiro YAHARA. "The actin-severing activity of cofilin is exerted by the interplay of three distinct sites on cofilin and essential for cell viability." Biochemical Journal 365, no. 1 (July 1, 2002): 147–55. http://dx.doi.org/10.1042/bj20020231.
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Cofilin/actin-depolymerizing factor is an essential and conserved modulator of actin dynamics. Cofilin binds to actin in either monomeric or filamentous form, severs and depolymerizes actin filaments, and speeds up their treadmilling. A high turnover rate of F-actin in actin-based motility seems driven largely by cofilin-mediated acceleration of directional subunit release, but little by fragmentation of the filaments. On the other hand, the filament-severing function of cofilin seems relevant for the healthy growth of cells. In this study, we have characterized three mutants of porcine cofilin to elucidate the molecular mechanism that underlies the filament-severing activity of cofilin. The first mutant could neither associate with actin filaments nor sever them, whereas it effectively accelerated their treadmilling and directional subunit release. The second mutant bound to actin filaments, but failed to sever them and to interfere with phalloidin binding to the filament. The third mutant could associate with actin filaments and sever them, although with a very reduced efficacy. Of these mutant proteins, only the last one was able to rescue Δcof1 yeast cells and to induce thick actin bundles in mammalian cells upon overexpression. Therefore, the actin-severing activity of cofilin is an essential element in its vital function and suggested to be exerted by co-operation of at least three distinct sites of cofilin.
3
Arikawa, K., J. L. Hicks, and D. S. Williams. "Identification of actin filaments in the rhabdomeral microvilli of Drosophila photoreceptors." Journal of Cell Biology 110, no. 6 (June 1, 1990): 1993–98. http://dx.doi.org/10.1083/jcb.110.6.1993.
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The phototransductive microvilli of arthropod photoreceptors each contain an axial cytoskeleton. The present study shows that actin filaments are a component of this cytoskeleton in Drosophila. Firstly, actin was detected in the rhabdomeral microvilli and in the subrhabdomeral cytoplasm by immunogold labeling with antiactin. Secondly, the rhabdomeres were labeled with phalloidin, indicating the presence of filamentous actin. Finally, the actin filaments were decorated with myosin subfragment-1. The characteristic arrowhead complex formed by subfragment-1 decoration points towards the base of the microvilli, so that the fast growing end of each filament is at the distal end of the microvillus, where it is embedded in a detergent-resistant cap. Each microvillus contains more than one actin filament. Decorated filaments extend the entire length of each microvillus and project into the subrhabdomeral cytoplasm. This organization is comparable to that of the actin filaments in intestinal brush border microvilli. Similar observations were made with the photoreceptor microvilli of the crayfish, Procambarus. Our results provide an indication as to how any myosin that is associated with the rhabdomeres might function.
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Braun, Tatjana, Albina Orlova, Karin Valegård, Ann-Christin Lindås, Gunnar F. Schröder, and Edward H. Egelman. "Archaeal actin from a hyperthermophile forms a single-stranded filament." Proceedings of the National Academy of Sciences 112, no. 30 (June 29, 2015): 9340–45. http://dx.doi.org/10.1073/pnas.1509069112.
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The prokaryotic origins of the actin cytoskeleton have been firmly established, but it has become clear that the bacterial actins form a wide variety of different filaments, different both from each other and from eukaryotic F-actin. We have used electron cryomicroscopy (cryo-EM) to examine the filaments formed by the protein crenactin (a crenarchaeal actin) from Pyrobaculum calidifontis, an organism that grows optimally at 90 °C. Although this protein only has ∼20% sequence identity with eukaryotic actin, phylogenetic analyses have placed it much closer to eukaryotic actin than any of the bacterial homologs. It has been assumed that the crenactin filament is double-stranded, like F-actin, in part because it would be hard to imagine how a single-stranded filament would be stable at such high temperatures. We show that not only is the crenactin filament single-stranded, but that it is remarkably similar to each of the two strands in F-actin. A large insertion in the crenactin sequence would prevent the formation of an F-actin-like double-stranded filament. Further, analysis of two existing crystal structures reveals six different subunit–subunit interfaces that are filament-like, but each is different from the others in terms of significant rotations. This variability in the subunit–subunit interface, seen at atomic resolution in crystals, can explain the large variability in the crenactin filaments observed by cryo-EM and helps to explain the variability in twist that has been observed for eukaryotic actin filaments.
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Stokes, DL, and DJ DeRosier. "The variable twist of actin and its modulation by actin-binding proteins." Journal of Cell Biology 104, no. 4 (April 1, 1987): 1005–17. http://dx.doi.org/10.1083/jcb.104.4.1005.
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Previous studies demonstrated that actin filaments have variable twist in which the intersubunit angles vary by approximately +/- 10 degrees within a filament. In this work we show that this variability was unchanged when different methods were used to prepare filaments for electron microscopy. We also show that actin-binding proteins can modulate the variability in twist. Three preparations of actin filaments were photographed in the electron microscope: negatively stained filaments, replicas of rapidly frozen, etched filaments, and frozen hydrated filaments. In addition, micrographs of actin + tropomyosin + troponin (thin filaments), of actin + myosin S1 (decorated filaments), and of filaments frayed from the acrosomal process of Limulus sperm (Limulus filaments) were obtained. We used two independent methods to measure variable twist based on Fourier transforms of single filaments. The first involved measuring layer line intensity versus filament length and the second involved measuring layer line position. We measured a variability in the intersubunit angle of actin filaments of approximately 12 degrees independent of the method of preparation or of measurement. Thin filaments have 15 degrees of variability, but the increase over pure actin is not statistically significant. Decorated filaments and Limulus filaments, however, have significantly less variability (approximately 2 and 1 degree, respectively), indicating a torsional stiffening relative to actin. The results from actin alone using different preparative methods are evidence that variable twist is a property of actin in solution. The results from actin filaments in the presence of actin-binding proteins suggest that the angular variability can be modulated, depending on the biological function.
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Bearer, E. L. "Direct observation of actin filament severing by gelsolin and binding by gCap39 and CapZ." Journal of Cell Biology 115, no. 6 (December 15, 1991): 1629–38. http://dx.doi.org/10.1083/jcb.115.6.1629.
Full textAbstract:
Dynamic behavior of actin filaments in cells is the basis of many different cellular activities. Remodeling of the actin filament network involves polymerization and depolymerization of the filaments. Proteins that regulate these behaviors include proteins that sever and/or cap actin filaments. This report presents direct observation of severing of fluorescently-labeled actin filaments. Coverslips coated with gelsolin, a multi-domain, calcium-dependent capping and severing protein, bound rhodamine-phalloidin-saturated filaments along their length in the presence of EGTA. Upon addition of calcium, attached filaments bent as they broke. Actophorin, a low molecular weight, monomer sequestering, calcium-independent severing protein did not sever phalloidin-saturated filaments. Both gCap 39, a gelsolin-like, calcium-dependent capping protein that does not sever filaments, and CapZ, a heterodimeric, non-calcium-dependent capping protein, bound the filaments by one end to the coverslip. Visualization of individual filaments also revealed severing activity present in mixtures of actin-binding proteins isolated by filamentous actin affinity chromatography from early Drosophila embryos. This activity was different from either gelsolin or actophorin because it was not inhibited by phalloidin, but was calcium independent. The results of these studies provide new information about the molecular mechanisms of severing and capping by well-characterized proteins as well as definition of a novel type of severing activity.
7
Hartwig, J. H., and P. Shevlin. "The architecture of actin filaments and the ultrastructural location of actin-binding protein in the periphery of lung macrophages." Journal of Cell Biology 103, no. 3 (September 1, 1986): 1007–20. http://dx.doi.org/10.1083/jcb.103.3.1007.
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A highly branched filament network is the principal structure in the periphery of detergent-extracted cytoskeletons of macrophages that have been spread on a surface and either freeze or critical point dried, and then rotary shadowed with platinum-carbon. This array of filaments completely fills lamellae extended from the cell and bifurcates to form 0.2-0.5 micron thick layers on the top and bottom of the cell body. Reaction of the macrophage cytoskeletons with anti-actin IgG and with anti-IgG bound to colloidal gold produces dense staining of these filaments, and incubation with myosin subfragment 1 uniformly decorates these filaments, identifying them as actin. 45% of the total cellular actin and approximately 70% of actin-binding protein remains in the detergent-insoluble cell residue. The soluble actin is not filamentous as determined by sedimentation analysis, the DNAase I inhibition assay, and electron microscopy, indicating that the cytoskeleton is not fragmented by detergent extraction. The spacing between the ramifications of the actin network is 94 +/- 47 nm and 118 +/- 72 nm in cytoskeletons prepared for electron microscopy by freeze drying and critical point drying, respectively. Free filament ends are rare, except for a few which project upward from the body of the network or which extend down to the substrate. Filaments of the network intersect predominantly at right angles to form either T-shaped and X-shaped overlaps having striking perpendicularity or else Y-shaped intersections composed of filaments intersecting at 120-130 degrees angles. The actin filament concentration in the lamellae is high, with an average value of 12.5 mg/ml. The concentration was much more uniform in freeze-dried preparations than in critical point-dried specimens, indicating that there is less collapse associated with the freezing technique. The orthogonal actin network of the macrophage cortical cytoplasm resembles actin gels made with actin-binding protein. Reaction of cell cytoskeletons and of an actin gel made with actin-binding protein with anti-actin-binding protein IgG and anti-IgG-coated gold beads resulted in the deposition of clusters of gold at points where filaments intersect and at the ends of filaments that may have been in contact with the membrane before its removal with detergent. In the actin gel made with actin-binding protein, 75% of actin-fiber intersections labeled, and the filament spacing between intersections is consistent with that predicted on theoretical grounds if each added actin-binding protein molecule cross-links two filaments to form an intersection in the gel.(ABSTRACT TRUNCATED AT 400 WORDS)
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Ijpma, Gijs, Ahmed M. Al-Jumaily, Simeon P. Cairns, and Gary C. Sieck. "Myosin filament polymerization and depolymerization in a model of partial length adaptation in airway smooth muscle." Journal of Applied Physiology 111, no. 3 (September 2011): 735–42. http://dx.doi.org/10.1152/japplphysiol.00114.2011.
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Length adaptation in airway smooth muscle (ASM) is attributed to reorganization of the cytoskeleton, and in particular the contractile elements. However, a constantly changing lung volume with tidal breathing (hence changing ASM length) is likely to restrict full adaptation of ASM for force generation. There is likely to be continuous length adaptation of ASM between states of incomplete or partial length adaption. We propose a new model that assimilates findings on myosin filament polymerization/depolymerization, partial length adaptation, isometric force, and shortening velocity to describe this continuous length adaptation process. In this model, the ASM adapts to an optimal force-generating capacity in a repeating cycle of events. Initially the myosin filament, shortened by prior length changes, associates with two longer actin filaments. The actin filaments are located adjacent to the myosin filaments, such that all myosin heads overlap with actin to permit maximal cross-bridge cycling. Since in this model the actin filaments are usually longer than myosin filaments, the excess length of the actin filament is located randomly with respect to the myosin filament. Once activated, the myosin filament elongates by polymerization along the actin filaments, with the growth limited by the overlap of the actin filaments. During relaxation, the myosin filaments dissociate from the actin filaments, and then the cycle repeats. This process causes a gradual adaptation of force and instantaneous adaptation of shortening velocity. Good agreement is found between model simulations and the experimental data depicting the relationship between force development, myosin filament density, or shortening velocity and length.
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Luo, Weibo, Benjamin Lin, Yingfei Wang, Jun Zhong, Robert O'Meally, Robert N. Cole, Akhilesh Pandey, Andre Levchenko, and Gregg L. Semenza. "PHD3-mediated prolyl hydroxylation of nonmuscle actin impairs polymerization and cell motility." Molecular Biology of the Cell 25, no. 18 (September 15, 2014): 2788–96. http://dx.doi.org/10.1091/mbc.e14-02-0775.
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Actin filaments play an essential role in cell movement, and many posttranslational modifications regulate actin filament assembly. Here we report that prolyl hydroxylase 3 (PHD3) interacts with nonmuscle actin in human cells and catalyzes hydroxylation of actin at proline residues 307 and 322. Blocking PHD3 expression or catalytic activity by short hairpin RNA knockdown or pharmacological inhibition, respectively, decreased actin prolyl hydroxylation. PHD3 knockdown increased filamentous F-actin assembly, which was reversed by PHD3 overexpression. PHD3 knockdown increased cell velocity and migration distance. Inhibition of PHD3 prolyl hydroxylase activity by dimethyloxalylglycine also increased actin polymerization and cell migration. These data reveal a novel role for PHD3 as a negative regulator of cell motility through posttranslational modification of nonmuscle actins.
10
Small, J. V., M. Herzog, and K. Anderson. "Actin filament organization in the fish keratocyte lamellipodium." Journal of Cell Biology 129, no. 5 (June 1, 1995): 1275–86. http://dx.doi.org/10.1083/jcb.129.5.1275.
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From recent studies of locomoting fish keratocytes it was proposed that the dynamic turnover of actin filaments takes place by a nucleation-release mechanism, which predicts the existence of short (less than 0.5 microns) filaments throughout the lamellipodium (Theriot, J. A., and T. J. Mitchison. 1991. Nature (Lond.). 352:126-131). We have tested this model by investigating the structure of whole mount keratocyte cytoskeletons in the electron microscope and phalloidin-labeled cells, after various fixations, in the light microscope. Micrographs of negatively stained keratocyte cytoskeletons produced by Triton extraction showed that the actin filaments of the lamellipodium are organized to a first approximation in a two-dimensional orthogonal network with the filaments subtending an angle of around 45 degrees to the cell front. Actin filament fringes grown onto the front edge of keratocyte cytoskeletons by the addition of exogenous actin showed a uniform polarity when decorated with myosin subfragment-1, consistent with the fast growing ends of the actin filaments abutting the anterior edge. A steady drop in filament density was observed from the mid-region of the lamellipodium to the perinuclear zone and in images of the more posterior regions of lower filament density many of the actin filaments could be seen to be at least several microns in length. Quantitative analysis of the intensity distribution of fluorescent phalloidin staining across the lamellipodium revealed that the gradient of filament density as well as the absolute content of F-actin was dependent on the fixation method. In cells first fixed and then extracted with Triton, a steep gradient of phalloidin staining was observed from the front to the rear of the lamellipodium. With the protocol required to obtain the electron microscope images, namely Triton extraction followed by fixation, phalloidin staining was, significantly and preferentially reduced in the anterior part of the lamellipodium. This resulted in a lower gradient of filament density, consistent with that seen in the electron microscope, and indicated a loss of around 45% of the filamentous actin during Triton extraction. We conclude, first that the filament organization and length distribution does not support a nucleation release model, but is more consistent with a treadmilling-type mechanism of locomotion featuring actin filaments of graded length. Second, we suggest that two layers of filaments make up the lamellipodium; a lower, stabilized layer associated with the ventral membrane and an upper layer associated with the dorsal membrane that is composed of filaments of a shorter range of lengths than the lower layer and which is mainly lost in Triton.
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JAMES, Marianne F., Nitasha MANCHANDA, Charo GONZALEZ-AGOSTI, John H. HARTWIG, and Vijaya RAMESH. "The neurofibromatosis 2 protein product merlin selectively binds F-actin but not G-actin, and stabilizes the filaments through a lateral association." Biochemical Journal 356, no. 2 (May 24, 2001): 377–86. http://dx.doi.org/10.1042/bj3560377.
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The neurofibromatosis 2 protein product merlin, named for its relatedness to the ezrin, radixin and moesin (ERM) family of proteins, is a tumour suppressor whose absence results in the occurrence of multiple tumours of the nervous system, particularly schwannomas and meningiomas. Merlin's similarity to ERMs suggests that it might share functions, acting as a link between cytoskeletal components and the cell membrane. The N-terminus of merlin has strong sequence identity to the N-terminal actin-binding region of ezrin; here we describe in detail the merlin–actin interaction. Employing standard actin co-sedimentation assays, we have determined that merlin isoform 2 binds F-actin with an apparent binding constant of 3.6μM and a stoichiometry of 1mol of merlin per 11.5mol of actin in filaments at saturation. Further, solid-phase binding assays reveal that merlin isoforms 1 and 2 bind actin filaments differentially, suggesting that the intramolecular interactions in isoform 1 might hinder its ability to bind actin. However, merlin does not bind G-actin. Studies of actin filament dynamics show that merlin slows filament disassembly with no influence on the assembly rate, indicating that merlin binds along actin filament lengths. This conclusion is supported by electron microscopy, which demonstrates that merlin binds periodically along cytoskeletal actin filaments. Comparison of these findings with those reported for ERM proteins reveal a distinct role for merlin in actin filament dynamics.
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Tilney, L. G., D. J. DeRosier, A. Weber, and M. S. Tilney. "How Listeria exploits host cell actin to form its own cytoskeleton. II. Nucleation, actin filament polarity, filament assembly, and evidence for a pointed end capper." Journal of Cell Biology 118, no. 1 (July 1, 1992): 83–93. http://dx.doi.org/10.1083/jcb.118.1.83.
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After Listeria, a bacterium, is phagocytosed by a macrophage, it dissolves the phagosomal membrane and enters the cytoplasm. The Listeria than nucleates actin filaments from its surface. These newly assembled actin filaments show unidirectional polarity with their barbed ends associated with the surface of the Listeria. Using actin concentrations below the pointed end critical concentration we find that filament elongation must be occurring by monomers adding to the barbed ends, the ends associated with the Listerial surface. If Listeria with tails are incubated in G actin under polymerizing conditions, the Listeria is translocated away from its preformed tail by the elongation of filaments attached to the Listeria. This experiment and others tell us that in vivo filament assembly must be tightly coupled to filament capping and cross-bridging so that if one process outstrips another, chaos ensues. We also show that the actin filaments in the tail are capped on their pointed ends which inhibits further elongation and/or disassembly in vitro. From these results we suggest a simple picture of how Listeria competes effectively for host cell actin. When Listeria secretes a nucleator, the host's actin subunits polymerize into a filament. Host cell machinery terminate the assembly leaving a short filament. Listeria overcomes the host control by nucleating new filaments and thus many short filaments assemble. The newest filaments push existing ones into a growing tail. Thus the competition is between nucleation of filaments caused by Listeria and the filament terminators produced by the host.
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Luther, P. K. "Three-dimensional reconstruction of a simple Z-band in fish muscle." Journal of Cell Biology 113, no. 5 (June 1, 1991): 1043–55. http://dx.doi.org/10.1083/jcb.113.5.1043.
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The three-dimensional structure of the Z-band in fish white muscle has been investigated by electron microscopy. This Z-band is described as simple, since in longitudinal sections it has the appearance of a single zigzag pattern connecting the ends of actin filaments of opposite polarity from adjacent sarcomeres. The reconstruction shows two pairs of links, the Z-links, between one actin filament and the facing four actin filaments in the adjacent sarcomere. The members of each pair have nearly diametrically opposed origins. In relation to one actin filament, one pair of links appears to bind along the final 10 nm of the actin filament (proximal site) and the other pair binds along a region extending from 5 to 20 nm from the filament end (distal site). Between one pair and the other, there is a rotation of approximately 80 degrees round the filament axis. A Z-link with a proximal site at the end of one actin filament attaches at a distal site on the oppositely oriented actin filaments of the facing sarcomere and vice versa. The length of each Z-link is consistent with the length of an alpha-actinin molecule. An additional set of links located 10-15 nm from the center of the Z-band occurs between actin filaments of the same polarity. These polar links connect the actin filaments along the same direction on each side of the Z-band. The three-dimensional structure appears to have twofold screw symmetry about the central plane of the Z-band. Only approximate twofold rotational symmetry is observed in directions parallel to the actin filaments. Previous models of the Z-band in which four identical and rotationally symmetrical links emanate from the end of one actin filament and span across to the ends of four actin filaments in the adjacent sarcomere are therefore incorrect.
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Russ, Misty, Daniel Croft, Omar Ali, Raquel Martinez, and Paul A. Steimle. "Myosin heavy-chain kinase A from Dictyostelium possesses a novel actin-binding domain that cross-links actin filaments." Biochemical Journal 395, no. 2 (March 28, 2006): 373–83. http://dx.doi.org/10.1042/bj20051376.
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Myosin heavy-chain kinase A (MHCK A) catalyses the disassembly of myosin II filaments in Dictyostelium cells via myosin II heavy-chain phosphorylation. MHCK A possesses a ‘coiled-coil’-enriched domain that mediates the oligomerization, cellular localization and actin-binding activities of the kinase. F-actin (filamentous actin) binding by the coiled-coil domain leads to a 40-fold increase in MHCK A activity. In the present study we examined the actin-binding characteristics of the coiled-coil domain as a means of identifying mechanisms by which MHCK A-mediated disassembly of myosin II filaments can be regulated in the cell. Co-sedimentation assays revealed that the coiled-coil domain of MHCK A binds co-operatively to F-actin with an apparent KD of approx. 0.5 μM and a stoichiometry of approx. 5:1 [actin/C(1–498)]. Further analyses indicate that the coiled-coil domain binds along the length of the actin filament and possesses at least two actin-binding regions. Quite surprisingly, we found that the coiled-coil domain cross-links actin filaments into bundles, indicating that MHCK A can affect the cytoskeleton in two important ways: (1) by driving myosin II-filament disassembly via myosin II heavy-chain phosphorylation, and (2) by cross-linking/bundling actin filaments. This discovery, along with other supporting data, suggests a model in which MHCK A-mediated bundling of actin filaments plays a central role in the recruitment and activation of the kinase at specific sites in the cell. Ultimately this provides a means for achieving the robust and highly localized disruption of myosin II filaments that facilitates polarized changes in cell shape during processes such as chemotaxis, cytokinesis and multicellular development.
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Yamagishi, Yuka, and Hiroshi Abe. "Reorganization of actin filaments by ADF/cofilin is involved in formation of microtubule structures during Xenopus oocyte maturation." Molecular Biology of the Cell 26, no. 24 (December 2015): 4387–400. http://dx.doi.org/10.1091/mbc.e15-01-0035.
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We examined the reorganization of actin filaments and microtubules during Xenopus oocyte maturation. Surrounding the germinal vesicle (GV) in immature oocytes, the cytoplasmic actin filaments reorganized to accumulate beneath the vegetal side of the GV, where the microtubule-organizing center and transient microtubule array (MTOC-TMA) assembled, just before GV breakdown (GVBD). Immediately after GVBD, both Xenopus ADF/cofilin (XAC) and its phosphatase Slingshot (XSSH) accumulated into the nuclei and intranuclear actin filaments disassembled from the vegetal side with the shrinkage of the GV. As the MTOC-TMA developed well, cytoplasmic actin filaments were retained at the MTOC-TMA base region. Suppression of XAC dephosphorylation by anti-XSSH antibody injection inhibited both actin filament reorganization and proper formation and localization of both the MTOC-TMA and meiotic spindles. Stabilization of actin filaments by phalloidin also inhibited formation of the MTOC-TMA and disassembly of intranuclear actin filaments without affecting nuclear shrinkage. Nocodazole also caused the MTOC-TMA and the cytoplasmic actin filaments at its base region to disappear, which further impeded disassembly of intranuclear actin filaments from the vegetal side. XAC appears to reorganize cytoplasmic actin filaments required for precise assembly of the MTOC and, together with the MTOC-TMA, regulate the intranuclear actin filament disassembly essential for meiotic spindle formation.
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Gokhin, David S., Roberta B. Nowak, Joseph A. Khoory, Alfonso de la Piedra, Ionita C. Ghiran, and Velia M. Fowler. "Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane." Molecular Biology of the Cell 26, no. 9 (May 2015): 1699–710. http://dx.doi.org/10.1091/mbc.e14-12-1583.
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Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here we show that a subpopulation of human RBC actin filaments is indeed dynamic, based on rhodamine-actin incorporation into filaments in resealed ghosts and fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (∼25–30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces an approximately twofold increase or ∼60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as by abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane.
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Drenckhahn, D., K. Engel, D. Höfer, C. Merte, L. Tilney, and M. Tilney. "Three different actin filament assemblies occur in every hair cell: each contains a specific actin crosslinking protein." Journal of Cell Biology 112, no. 4 (February 15, 1991): 641–51. http://dx.doi.org/10.1083/jcb.112.4.641.
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The apex of hair cells of the chicken auditory organ contains three different kinds of assemblies of actin filaments in close spatial proximity. These are (a) paracrystals of actin filaments with identical polarity in stereocilia, (b) a dense gellike meshwork of actin filaments forming the cuticular plate, and (c) a bundle of parallel actin filaments with mixed polarities that constitute the circumferential filament belt attached to the cytoplasmic aspect of the zonula adhaerens (ZA). Each different supramolecular assembly of actin filaments contains a specific actin filament cross-linking protein which is unique to that particular assembly. Thus fimbrin appears to be responsible for paracrystallin packing of actin filaments in stereocillia; an isoform of spectrin resides in the cuticular plate where it forms the whisker-like crossbridges, and alpha actinin is the actin crosslinking protein of the circumferential ZA bundle. Tropomyosin, which stabilizes actin filaments, is present in all the actin filament assemblies except for the stereocilia. Another striking finding was that myosin appears to be absent from the ZA ring and cuticular plate of hair cells although present in the ZA ring of supporting cells. The abundance of myosin in the ZA ring of the surrounding supporting cells means that it may be important in forming a supporting tensile cellular framework in which the hair cells are inserted.
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Qian, Yong, Joseph M. Baisden, Lidia Cherezova, Justin M. Summy, Anne Guappone-Koay, Xianglin Shi, Tom Mast, et al. "PKC Phosphorylation Increases the Ability of AFAP-110 to Cross-link Actin Filaments." Molecular Biology of the Cell 13, no. 7 (July 2002): 2311–22. http://dx.doi.org/10.1091/mbc.e01-12-0148.
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The actin filament-associated protein and Src-binding partner, AFAP-110, is an adaptor protein that links signaling molecules to actin filaments. AFAP-110 binds actin filaments directly and multimerizes through a leucine zipper motif. Cellular signals downstream of Src527F can regulate multimerization. Here, we determined recombinant AFAP-110 (rAFAP-110)-bound actin filaments cooperatively, through a lateral association. We demonstrate rAFAP-110 has the capability to cross-link actin filaments, and this ability is dependent on the integrity of the carboxy terminal actin binding domain. Deletion of the leucine zipper motif or PKC phosphorylation affected AFAP-110's conformation, which correlated with changes in multimerization and increased the capability of rAFAP-110 to cross-link actin filaments. AFAP-110 is both a substrate and binding partner of PKC. On PKC activation, stress filament organization is lost, motility structures form, and AFAP-110 colocalizes strongly with motility structures. Expression of a deletion mutant of AFAP-110 that is unable to bind PKC blocked the effect of PMA on actin filaments. We hypothesize that upon PKC activation, AFAP-110 can be cooperatively recruited to newly forming actin filaments, like those that exist in cell motility structures, and that PKC phosphorylation effects a conformational change that may enable AFAP-110 to promote actin filament cross-linking at the cell membrane.
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Suetsugu, Shiro, Hiroaki Miki, Hideki Yamaguchi, Takeshi Obinata, and Tadaomi Takenawa. "Enhancement of branching efficiency by the actin filament-binding activity of N-WASP/WAVE2." Journal of Cell Science 114, no. 24 (December 15, 2001): 4533–42. http://dx.doi.org/10.1242/jcs.114.24.4533.
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The actin-related protein (Arp) 2/3 complex is an essential regulator of de novo actin filament formation. Arp2/3 nucleates the polymerization of actin and creates branched actin filaments when activated by Arp2/3-complex activating domain (VCA) of Wiskott-Aldrich syndrome proteins (WASP family proteins). We found that the branching of actin filaments on pre-existing ADP filaments mediated by the Arp2/3 complex is twice as efficient when Arp2/3 was activated by wild-type neural WASP (N-WASP) or WASP-family verprolin-homologous protein (WAVE) 2 than when activated by the VCA domain alone. By contrast, there was no difference between wild-type N-WASP or WAVE2 and VCA in the branching efficiency on de novo filaments, which are thought to consist mainly of ADP-phosphate filaments. This increased branching efficiency on ADP filaments is due to the basic region located in the center of N-WASP and WAVE2, which was found to associate with ADP actin filaments. Actin filaments and phosphatidylinositol bisphosphate (PIP2) associate with N-WASP at different sites. This association of N-WASP and WAVE2 with actin filaments enhanced recruitment of Arp2/3 to the pre-existing filaments, presumably leading to efficient nucleation and branch formation on pre-existing filaments. These data together suggest that the actin filament binding activity of N-WASP and WAVE2 in the basic region increases the number of barbed ends created on pre-existing filaments. Efficient branching on ADP filaments may be important for initiation of actin-based motility.
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Tilney, Lewis G., Patricia S. Connelly, Linda Ruggiero, Kelly A. Vranich, and Gregory M. Guild. "Actin Filament Turnover Regulated by Cross-linking Accounts for the Size, Shape, Location, and Number of Actin Bundles in Drosophila Bristles." Molecular Biology of the Cell 14, no. 10 (October 2003): 3953–66. http://dx.doi.org/10.1091/mbc.e03-03-0158.
Full textAbstract:
Drosophila bristle cells are shaped during growth by longitudinal bundles of cross-linked actin filaments attached to the plasma membrane. We used confocal and electron microscopy to examine actin bundle structure and found that during bristle elongation, snarls of uncross-linked actin filaments and small internal bundles also form in the shaft cytoplasm only to disappear within 4 min. Thus, formation and later removal of actin filaments are prominent features of growing bristles. These transient snarls and internal bundles can be stabilized by culturing elongating bristles with jasplakinolide, a membrane-permeant inhibitor of actin filament depolymerization, resulting in enormous numbers of internal bundles and uncross-linked filaments. Examination of bundle disassembly in mutant bristles shows that plasma membrane association and cross-bridging adjacent actin filaments together inhibits depolymerization. Thus, highly cross-bridged and membrane-bound actin filaments turn over slowly and persist, whereas poorly cross-linked filaments turnover more rapidly. We argue that the selection of stable bundles relative to poorly cross-bridged filaments can account for the size, shape, number, and location of the longitudinal actin bundles in bristles. As a result, filament turnover plays an important role in regulating cytoskeleton assembly and consequently cell shape.
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McGough, Amy, Brian Pope, Wah Chiu, and Alan Weeds. "Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function." Journal of Cell Biology 138, no. 4 (August 25, 1997): 771–81. http://dx.doi.org/10.1083/jcb.138.4.771.
Full textAbstract:
Cofilin is an actin depolymerizing protein found widely distributed in animals and plants. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Cofilin binds filamentous (F)-actin cooperatively by bridging two longitudinally associated actin subunits. The binding site is centered axially at subdomain 2 of the lower actin subunit and radially at the cleft between subdomains 1 and 3 of the upper actin subunit. Our work has revealed a totally unexpected (and unique) property of cofilin, namely, its ability to change filament twist. As a consequence of this change in twist, filaments decorated with cofilin have much shorter ‘actin crossovers' (∼75% of those normally observed in F-actin structures). Although their binding sites are distinct, cofilin and phalloidin do not bind simultaneously to F-actin. This is the first demonstration of a protein that excludes another actin-binding molecule by changing filament twist. Alteration of F-actin structure by cofilin/ADF appears to be a novel mechanism through which the actin cytoskeleton may be regulated or remodeled.
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Sahoo, Nivedita, Wandy Beatty, John Heuser, David Sept, and L. David Sibley. "Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the Parasite Toxoplasma gondii." Molecular Biology of the Cell 17, no. 2 (February 2006): 895–906. http://dx.doi.org/10.1091/mbc.e05-06-0512.
Full textAbstract:
Toxoplasma is a protozoan parasite in the phylum Apicomplexa, which contains a number of medically important parasites that rely on a highly unusual form of motility termed gliding to actively penetrate their host cells. Parasite actin filaments regulate gliding motility, yet paradoxically filamentous actin is rarely detected in these parasites. To investigate the kinetics of this unusual parasite actin, we expressed TgACT1 in baculovirus and purified it to homogeneity. Biochemical analysis showed that Toxoplasma actin (TgACT1) rapidly polymerized into filaments at a critical concentration that was 3-4-fold lower than conventional actins, yet it failed to copolymerize with mammalian actin. Electron microscopic analysis revealed that TgACT1 filaments were 10 times shorter and less stable than rabbit actin. Phylogenetic comparison of actins revealed a limited number of apicomplexan-specific residues that likely govern the unusual behavior of parasite actin. Molecular modeling identified several key alterations that affect interactions between monomers and that are predicted to destabilize filaments. Our findings suggest that conserved molecular differences in parasite actin favor rapid cycles of assembly and disassembly that govern the unusual form of gliding motility utilized by apicomplexans.
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Zhang, Jun, Yuan-Liang Wang, Xin-Yong Chen, Chuang-Long He, Chao Cheng, and Yang Cao. "Preliminarily Investigating the Polymorphism of Self-organized Actin Filament in Vitro by Atomic Force Microscope." Acta Biochimica et Biophysica Sinica 36, no. 9 (September 1, 2004): 637–43. http://dx.doi.org/10.1093/abbs/36.9.637.
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Abstract With the atomic force microscope (AFM), we preliminarily investigated the large-scale structure of actin filaments formed in low concentration protein solution (5 μg/ml) via self-organization without the presence of any F-actin dynamic interfering factors (such as phalloidin) in vitro. It was found that the G-actin could be polymerized into ordered filamentous structures with different diameter from the slimmest filament of single F-actin to giant filament in tree-like branched aggregates. The observed polymerized actin filaments, to which our most intense attention was attracted, was discretely distributed and showed obvious polymorphism distinctly different from those in the presence of phalloidin or actin binding proteins (fimbrin, gelsolin, etc.) in previous experiments. Latter structures were mainly composed of single F-actin and/or multifilaments clearly consisting of several single F-actin. The experimental results clearly demonstrated that non-interference with the F-actin intrinsic dynamics in self-organizing could lead to the polymorphism of actin filamentous structures, and further analysis implied that the disturbance of normal F-actin dynamics by many factors could prevent the emergence of structural polymorphism, more often than not, give rise to formation of specific structures instead and different interference would bring about various particular structures under certain conditions.
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Belin, Brittany J., Beth A. Cimini, Elizabeth H. Blackburn, and R. Dyche Mullins. "Visualization of actin filaments and monomers in somatic cell nuclei." Molecular Biology of the Cell 24, no. 7 (April 2013): 982–94. http://dx.doi.org/10.1091/mbc.e12-09-0685.
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In addition to its long-studied presence in the cytoplasm, actin is also found in the nuclei of eukaryotic cells. The function and form (monomer, filament, or noncanonical oligomer) of nuclear actin are hotly debated, and its localization and dynamics are largely unknown. To determine the distribution of nuclear actin in live somatic cells and evaluate its potential functions, we constructed and validated fluorescent nuclear actin probes. Monomeric actin probes concentrate in nuclear speckles, suggesting an interaction of monomers with RNA-processing factors. Filamentous actin probes recognize discrete structures with submicron lengths that are excluded from chromatin-rich regions. In time-lapse movies, these actin filament structures exhibit one of two types of mobility: 1) diffusive, with an average diffusion coefficient of 0.06–0.08 μm2/s, or (2) subdiffusive, with a mobility coefficient of 0.015 μm2/s. Individual filament trajectories exhibit features of particles moving within a viscoelastic mesh. The small size of nuclear actin filaments is inconsistent with a role in micron-scale intranuclear transport, and their localization suggests that they do not participate directly in chromatin-based processes. Our results instead suggest that actin filaments form part of a large, viscoelastic structure in the nucleoplasm and may act as scaffolds that help organize nuclear contents.
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Jansen, Silvia, and Bruce L. Goode. "Tropomyosin isoforms differentially tune actin filament length and disassembly." Molecular Biology of the Cell 30, no. 5 (March 2019): 671–79. http://dx.doi.org/10.1091/mbc.e18-12-0815.
Full textAbstract:
Cellular actin networks exhibit diverse filamentous architectures and turnover dynamics, but how these differences are specified remains poorly understood. Here, we used multicolor total internal reflection fluorescence microscopy to ask how decoration of actin filaments by five biologically prominent Tropomyosin (TPM) isoforms influences disassembly induced by Cofilin alone, or by the collaborative effects of Cofilin, Coronin, and AIP1 (CCA). TPM decoration restricted Cofilin binding to pointed ends, while not interfering with Coronin binding to filament sides. Different isoforms of TPM provided variable levels of protection against disassembly, with the strongest protection by Tpm3.1 and the weakest by Tpm1.6. In biomimetic assays in which filaments were simultaneously assembled by formins and disassembled by CCA, these TPM isoform–specific effects persisted, giving rise to filaments with different lengths and treadmilling behavior. Together, our data reveal that TPM isoforms have quantitatively distinct abilities to tune actin filament length and turnover.
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Higashi-Fujime, S. "Unidirectional sliding of myosin filaments along the bundle of F-actin filaments spontaneously formed during superprecipitation." Journal of Cell Biology 101, no. 6 (December 1, 1985): 2335–44. http://dx.doi.org/10.1083/jcb.101.6.2335.
Full textAbstract:
I reported previously (Higashi-Fujime, S., 1982, Cold Spring Harbor Symp. Quant. Biol., 46:69-75) that active movements of fibrils composed of F-actin and myosin filaments occurred after superprecipitation in the presence of ATP at low ionic strengths. When the concentration of MgCl2 in the medium used in the above experiment was raised to 20-26 mM, bundles of F-actin filaments, in addition to large precipitates, were formed spontaneously both during and after superprecipitation. Along these bundles, many myosin filaments were observed to slide unidirectionally and successively through the bundle, from one end to the other. The sliding of myosin filaments continued for approximately 1 h at room temperature at a mean rate of 6.0 micron/s, as long as ATP remained in the medium. By electron microscopy, it was found that most F-actin filaments decorated with heavy meromyosin pointed to the same direction in the bundle. Myosin filaments moved actively not only along the F-actin bundle but also in the medium. Such movement probably occurred along F-actin filaments that did not form the bundle but were dispersed in the medium, although dispersed F-actin filaments were not visible under the microscope. In this case, myosin filament could have moved in a reverse direction, changing from one F-actin filament to the other. These results suggested that the direction of movement of myosin filament, which has a bipolar structure and the potentiality to move in both directions, was determined by the polarity of F-actin filament in action.
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Mabuchi, I. "Cleavage furrow: timing of emergence of contractile ring actin filaments and establishment of the contractile ring by filament bundling in sea urchin eggs." Journal of Cell Science 107, no. 7 (July 1, 1994): 1853–62. http://dx.doi.org/10.1242/jcs.107.7.1853.
Full textAbstract:
Cleavage furrow formation at the first cell division of sea urchin and sand dollar eggs was investigated in detail by fluorescence staining of actin filaments with rhodamine-phalloidin of either whole eggs or isolated egg cortices. Cortical actin filaments were clustered at anaphase and then the clusters became fibrillar at the end of anaphase. The timing when the contractile ring actin filaments appear was precisely determined in the course of mitosis: accumulation of the contractile ring actin filaments at the equatorial cell cortex is first noticed at the beginning of telophase (shortly before furrow formation), when the chromosomal vesicles are fusing with each other. The accumulated actin filaments were not well organized at the early stage but were organized into parallel bundles as the furrowing progressed. The bundles were finally fused into a tightly packed filament belt. Wheat germ agglutinin (WGA)-binding sites were distributed on the surface of the egg in a manner similar to the actin filaments after anaphase. The WGA-binding sites became accumulated in the contractile ring together with the contractile ring actin filaments, indicating an intimate relationship between these sites and actin filament-anchoring sites on the plasma membrane. Myosin also appeared in the contractile ring together with the actin filaments. The ‘cleavage stimulus’, a signal hypothesized by Rappaport (reviewed by R. Rappaport (1986) Int. Rev. Cytol. 105, 245–281) was suggested to induce aggregation or bundling of the actin filaments in the cortical layer.
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Pospich, Sabrina, Esa-Pekka Kumpula, Julian von der Ecken, Juha Vahokoski, Inari Kursula, and Stefan Raunser. "Near-atomic structure of jasplakinolide-stabilized malaria parasite F-actin reveals the structural basis of filament instability." Proceedings of the National Academy of Sciences 114, no. 40 (September 18, 2017): 10636–41. http://dx.doi.org/10.1073/pnas.1707506114.
Full textAbstract:
During their life cycle, apicomplexan parasites, such as the malaria parasite Plasmodium falciparum, use actomyosin-driven gliding motility to move and invade host cells. For this process, actin filament length and stability are temporally and spatially controlled. In contrast to canonical actin, P. falciparum actin 1 (PfAct1) does not readily polymerize into long, stable filaments. The structural basis of filament instability, which plays a pivotal role in host cell invasion, and thus infectivity, is poorly understood, largely because high-resolution structures of PfAct1 filaments were missing. Here, we report the near-atomic structure of jasplakinolide (JAS)-stabilized PfAct1 filaments determined by electron cryomicroscopy. The general filament architecture is similar to that of mammalian F-actin. The high resolution of the structure allowed us to identify small but important differences at inter- and intrastrand contact sites, explaining the inherent instability of apicomplexan actin filaments. JAS binds at regular intervals inside the filament to three adjacent actin subunits, reinforcing filament stability by hydrophobic interactions. Our study reveals the high-resolution structure of a small molecule bound to F-actin, highlighting the potential of electron cryomicroscopy for structure-based drug design. Furthermore, our work serves as a strong foundation for understanding the structural design and evolution of actin filaments and their function in motility and host cell invasion of apicomplexan parasites.
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Staiger, Christopher J., Michael B. Sheahan, Parul Khurana, Xia Wang, David W. McCurdy, and Laurent Blanchoin. "Actin filament dynamics are dominated by rapid growth and severing activity in the Arabidopsis cortical array." Journal of Cell Biology 184, no. 2 (January 26, 2009): 269–80. http://dx.doi.org/10.1083/jcb.200806185.
Full textAbstract:
Metazoan cells harness the power of actin dynamics to create cytoskeletal arrays that stimulate protrusions and drive intracellular organelle movements. In plant cells, the actin cytoskeleton is understood to participate in cell elongation; however, a detailed description and molecular mechanism(s) underpinning filament nucleation, growth, and turnover are lacking. Here, we use variable-angle epifluorescence microscopy (VAEM) to examine the organization and dynamics of the cortical cytoskeleton in growing and nongrowing epidermal cells. One population of filaments in the cortical array, which most likely represent single actin filaments, is randomly oriented and highly dynamic. These filaments grow at rates of 1.7 µm/s, but are generally short-lived. Instead of depolymerization at their ends, actin filaments are disassembled by severing activity. Remodeling of the cortical actin array also features filament buckling and straightening events. These observations indicate a mechanism inconsistent with treadmilling. Instead, cortical actin filament dynamics resemble the stochastic dynamics of an in vitro biomimetic system for actin assembly.
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Steinmetz, Michel O., Kenneth N. Goldie, and Ueli Aebi. "A Correlative Analysis of Actin Filament Assembly, Structure, and Dynamics." Journal of Cell Biology 138, no. 3 (August 11, 1997): 559–74. http://dx.doi.org/10.1083/jcb.138.3.559.
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The effect of the type of metal ion (i.e., Ca2+, Mg2+, or none) bound to the high-affinity divalent cation binding site (HAS) of actin on filament assembly, structure, and dynamics was investigated in the absence and presence of the mushroom toxin phalloidin. In agreement with earlier reports, we found the polymerization reaction of G-actin into F-actin filaments to be tightly controlled by the type of divalent cation residing in its HAS. Moreover, novel polymerization data are presented indicating that LD, a dimer unproductive by itself, does incorporate into growing F-actin filaments. This observation suggests that during actin filament formation, in addition to the obligatory nucleation– condensation pathway involving UD, a productive filament dimer, a facultative, LD-based pathway is implicated whose abundance strongly depends on the exact polymerization conditions chosen. The “ragged” and “branched” filaments observed during the early stages of assembly represent a hallmark of LD incorporation and might be key to producing an actin meshwork capable of rapidly assembling and disassembling in highly motile cells. Hence, LD incorporation into growing actin filaments might provide an additional level of regulation of actin cytoskeleton dynamics. Regarding the structure and mechanical properties of the F-actin filament at steady state, no significant correlation with the divalent cation residing in its HAS was found. However, compared to native filaments, phalloidin-stabilized filaments were stiffer and yielded subtle but significant structural changes. Together, our data indicate that whereas the G-actin conformation is tightly controlled by the divalent cation in its HAS, the F-actin conformation appears more robust than this variation. Hence, we conclude that the structure and dynamics of the Mg–F-actin moiety within the thin filament are not significantly modulated by the cyclic Ca2+ release as it occurs in muscle contraction to regulate the actomyosin interaction via troponin.
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Lu, Hailong, Patricia M. Fagnant, and Kathleen M. Trybus. "Unusual dynamics of the divergent malaria parasite PfAct1 actin filament." Proceedings of the National Academy of Sciences 116, no. 41 (September 23, 2019): 20418–27. http://dx.doi.org/10.1073/pnas.1906600116.
Full textAbstract:
Gliding motility and host cell invasion by the apicomplexan parasite Plasmodium falciparum (Pf), the causative agent of malaria, is powered by a macromolecular complex called the glideosome that lies between the parasite plasma membrane and the inner membrane complex. The glideosome core consists of a single-headed class XIV myosin PfMyoA and a divergent actin PfAct1. Here we use total internal reflection fluorescence microscopy to visualize growth of individual unstabilized PfAct1 filaments as a function of time, an approach not previously used with this actin isoform. Although PfAct1 was thought to be incapable of forming long filaments, filaments grew as long as 30 µm. Polymerization occurs via a nucleation–elongation mechanism, but with an ∼4 µM critical concentration, an order-of-magnitude higher than for skeletal actin. Protomers disassembled from both the barbed and pointed ends of the actin filament with similar fast kinetics of 10 to 15 subunits/s. Rapid treadmilling, where the barbed end of the filament grows and the pointed end shrinks while maintaining an approximately constant filament length, was visualized near the critical concentration. Once ATP has been hydrolyzed to ADP, the filament becomes very unstable, resulting in total dissolution in <40 min. Dynamics at the filament ends are suppressed in the presence of inorganic phosphate or more efficiently by BeFX. A chimeric PfAct1 with a mammalian actin D-loop forms a more stable filament. These unusual dynamic properties distinguish PfAct1 from more canonical actins, and likely contribute to the difficultly in visualizing PfAct1 filaments in the parasite.
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Uetake, Yukari, and R. Larry Peterson. "Changes in actin filament arrays in protocorm cells of the orchid species, Spiranthes sinensis, induced by the symbiotic fungus Ceratobasidium cornigerum." Canadian Journal of Botany 75, no. 10 (October 1, 1997): 1661–69. http://dx.doi.org/10.1139/b97-879.
Full textAbstract:
Seeds of the terrestrial orchid, Spiranthes sinensis, were germinated in vitro in association with the symbiotic fungus, Ceratobasidium cornigerum. Resulting colonized protocorms were prepared for light microscopy, transmission electron microscopy, and fluorescence labelling of actin filaments for examination with laser scanning confocal microscopy. Fungal hyphae invaded the suspensor end of embryos, formed typical hyphal coils (pelotons) within parenchyma cells, and then underwent lysis resulting in degraded hyphal masses. Hyphae and hyphal masses were enveloped by host-derived membrane. Changes in actin filament arrays accompanied fungal colonization. Uncolonized cells had a network of actin filaments and actin bundles (cables) located in the cortical region of the cell cytoplasm; some of these were associated with the nucleus and amyloplasts. Although actin filament arrays were still present in protocorm cell cytoplasm during fungal entry and peloton formation, most of the cortical network disappeared and instead actin filaments radiated from the periphery of developing pelotons towards the cell wall. Degraded hyphal masses also had actin filament arrays associated with them, again radiating toward the cell periphery; a network of cortical actin filaments reappeared in the protocorm cell cytoplasm at this stage. Actin filaments did not appear to have a close physical association with fungal hyphae except in the epidermal hairs that developed from protocorms; this differs from our previous observations on microtubules in this system. Key words: actin, actin filaments, orchids, mycorrhizas, laser scanning confocal microscopy.
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Lewis, A. K., and P. C. Bridgman. "Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity." Journal of Cell Biology 119, no. 5 (December 1, 1992): 1219–43. http://dx.doi.org/10.1083/jcb.119.5.1219.
Full textAbstract:
The organization and polarity of actin filaments in neuronal growth cones was studied with negative stain and freeze-etch EM using a permeabilization protocol that caused little detectable change in morphology when cultured nerve growth cones were observed by video-enhanced differential interference contrast microscopy. The lamellipodial actin cytoskeleton was composed of two distinct subpopulations: a population of 40-100-nm-wide filament bundles radiated from the leading edge, and a second population of branching short filaments filled the volume between the dorsal and ventral membrane surfaces. Together, the two populations formed the three-dimensional structural network seen within expanding lamellipodia. Interaction of the actin filaments with the ventral membrane surface occurred along the length of the filaments via membrane associated proteins. The long bundled filament population was primarily involved in these interactions. The filament tips of either population appeared to interact with the membrane only at the leading edge; this interaction was mediated by a globular Triton-insoluble material. Actin filament polarity was determined by decoration with myosin S1 or heavy meromyosin. Previous reports have suggested that the polarity of the actin filaments in motile cells is uniform, with the barbed ends toward the leading edge. We observed that the actin filament polarity within growth cone lamellipodia is not uniform; although the predominant orientation was with the barbed end toward the leading edge (47-56%), 22-25% of the filaments had the opposite orientation with their pointed ends toward the leading edge, and 19-31% ran parallel to the leading edge. The two actin filament populations display distinct polarity profiles: the longer filaments appear to be oriented predominantly with their barbed ends toward the leading edge, whereas the short filaments appear to be randomly oriented. The different length, organization and polarity of the two filament populations suggest that they differ in stability and function. The population of bundled long filaments, which appeared to be more ventrally located and in contact with membrane proteins, may be more stable than the population of short branched filaments. The location, organization, and polarity of the long bundled filaments suggest that they may be necessary for the expansion of lamellipodia and for the production of tension mediated by receptors to substrate adhesion molecules.
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Skau, Colleen T., Erin M. Neidt, and David R. Kovar. "Role of Tropomyosin in Formin-mediated Contractile Ring Assembly in Fission Yeast." Molecular Biology of the Cell 20, no. 8 (April 15, 2009): 2160–73. http://dx.doi.org/10.1091/mbc.e08-12-1201.
Full textAbstract:
Like animal cells, fission yeast divides by assembling actin filaments into a contractile ring. In addition to formin Cdc12p and profilin, the single tropomyosin isoform SpTm is required for contractile ring assembly. Cdc12p nucleates actin filaments and remains processively associated with the elongating barbed end while driving the addition of profilin-actin. SpTm is thought to stabilize mature filaments, but it is not known how SpTm localizes to the contractile ring and whether SpTm plays a direct role in Cdc12p-mediated actin polymerization. Using “bulk” and single actin filament assays, we discovered that Cdc12p can recruit SpTm to actin filaments and that SpTm has diverse effects on Cdc12p-mediated actin assembly. On its own, SpTm inhibits actin filament elongation and depolymerization. However, Cdc12p completely overcomes the combined inhibition of actin nucleation and barbed end elongation by profilin and SpTm. Furthermore, SpTm increases the length of Cdc12p-nucleated actin filaments by enhancing the elongation rate twofold and by allowing them to anneal end to end. In contrast, SpTm ultimately turns off Cdc12p-mediated elongation by “trapping” Cdc12p within annealed filaments or by dissociating Cdc12p from the barbed end. Therefore, SpTm makes multiple contributions to contractile ring assembly during and after actin polymerization.
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Marston, S. "Stoichiometry and stability of caldesmon in native thin filaments from sheep aorta smooth muscle." Biochemical Journal 272, no. 2 (December 1, 1990): 305–10. http://dx.doi.org/10.1042/bj2720305.
Full textAbstract:
Ca2(+)-regulated native thin filaments were extracted from sheep aorta smooth muscle. The caldesmon content determined by quantitative gel electrophoresis was 0.06 caldesmon molecule/actin monomer (1 caldesmon molecule per 16.3 actin monomers). Dissociation of caldesmon and tropomyosin from the thin filament and the depolymerization of actin was measured by sedimenting diluted thin filaments. Actin critical concentration was 0.05 microM at 10.1 and 0.13 at 10.05 compared with 0.5 microM for pure F-actin. Tropomyosin was tightly bound, with half-maximal dissociation at less than 0.3 microM thin filaments (actin monomer) under all conditions. Caldesmon dissociation was independent of tropomyosin and not co-operative. The concentration of thin filaments where 50% of the caldesmon was dissociated (CD50) ranged from 0.2 microM (actin monomer) at 10.03 to 8 microM at 10.16 in a 5 mM-MgCl2, pH 7.1, buffer. Mg2+, 25 mM at constant I, increased CD50 4-fold. CD50 was 4-fold greater at 10(-4) M-Ca2+ than at 10(-9) M-Ca2+. Aorta heavy meromyosin (HMM).ADP.Pi complex (2.5 microM excess over thin filaments) strongly antagonized caldesmon dissociation, but skeletal-muscle HMM.ADP.Pi did not. The behaviour of caldesmon in native thin filaments was indistinguishable from caldesmon in reconstituted synthetic thin filaments. The variability of Ca2(+)-sensitivity with conditions observed in thin filament preparations was shown to be related to dissociation of regulatory caldesmon from the thin filament.
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Taylor, Kenneth A., Dianne W. Taylor, and Fred Schachat. "Isoforms of α-Actinin from Cardiac, Smooth, and Skeletal Muscle Form Polar Arrays of Actin Filaments." Journal of Cell Biology 149, no. 3 (May 1, 2000): 635–46. http://dx.doi.org/10.1083/jcb.149.3.635.
Full textAbstract:
We have used a positively charged lipid monolayer to form two-dimensional bundles of F-actin cross-linked by α-actinin to investigate the relative orientation of the actin filaments within them. This method prevents growth of the bundles perpendicular to the monolayer plane, thereby facilitating interpretation of the electron micrographs. Using α-actinin isoforms isolated from the three types of vertebrate muscle, i.e., cardiac, skeletal, and smooth, we have observed almost exclusively cross-linking between polar arrays of filaments, i.e., actin filaments with their plus ends oriented in the same direction. One type of bundle can be classified as an Archimedian spiral consisting of a single actin filament that spirals inward as the filament grows and the bundle is formed. These spirals have a consistent hand and grow to a limiting internal diameter of 0.4–0.7 μm, where the filaments appear to break and spiral formation ceases. These results, using isoforms usually characterized as cross-linkers of bipolar actin filament bundles, suggest that α-actinin is capable of cross-linking actin filaments in any orientation. Formation of specifically bipolar or polar filament arrays cross-linked by α-actinin may require additional factors that either determine the filament orientation or restrict the cross-linking capabilities of α-actinin.
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Kołakowski, J., R. Makuch, D. Stepkowski, and R. Dabrowska. "Interaction of calponin with actin and its functional implications." Biochemical Journal 306, no. 1 (February 15, 1995): 199–204. http://dx.doi.org/10.1042/bj3060199.
Full textAbstract:
Titration of F-actin with calponin causes the formation of two types of complexes. One, at saturation, contains a lower ratio of calponin to actin (0.5:1) and is insoluble at physiological ionic strength. The another is soluble, with a higher ratio of calponin to actin (1:1). Electron microscopy revealed that the former complex consists of paracrystalline bundles of actin filaments, whereas the latter consists of separate filaments. Ca(2+)-calmodulin causes dissociation of bundles with simultaneous increase in the number of separate calponin-containing filaments. Further increase in the calmodulin concentration results in full release of calponin from actin filaments. In motility assays, calponin, when added together with ATP to actin filaments complexed with immobilized myosin, evoked a decrease in both the number and velocity of moving actin filaments. Addition of calponin to actin filaments before their binding to myosin resulted in a formation of actin filament bundles which were dissociated by ATP.
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Herrera, Ana M., Eliana C. Martinez, and Chun Y. Seow. "Electron microscopic study of actin polymerization in airway smooth muscle." American Journal of Physiology-Lung Cellular and Molecular Physiology 286, no. 6 (June 2004): L1161—L1168. http://dx.doi.org/10.1152/ajplung.00298.2003.
Full textAbstract:
Actin polymerization as part of the normal smooth muscle response to various stimuli has been reported. The actin dynamics are believed to be necessary for cytoskeletal remodeling in smooth muscle in its adaptation to external stress and strain and for maintenance of optimal contractility. We have shown in our previous studies in airway smooth muscle that myosins polymerized in response to contractile activation as well as to adaptation at longer cell lengths. We postulated that the same response could be elicited from actins under the same conditions. In the present study, actin filament formation was quantified electron microscopically in cell cross sections. Nanometer resolution allowed us to examine regional distribution of filaments in a cell cross section. Airway smooth muscle bundles were fixed in relaxed and activated states at two lengths; muscle preparations were also fixed after a period of oscillatory strain, a condition known to cause depolymerization of myosin filaments. The results indicate that contractile activation and increased cell length nonsynergistically enhanced actin polymerization; the extent of actin polymerization was substantially less than that of myosin polymerization. Oscillatory strain increased thin filament formation. Although thin filament density was found higher in cytoplasmic areas near dense bodies, contractile activation did not preferentially enhance actin polymerization in these areas. It is concluded that actin thin filaments are dynamic structures whose length and number are regulated by the cell in response to changes in extracellular environment and that polymerization and depolymerization of thin filaments occur uniformly across the whole cell cross section.
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WINDER, Steven J., Thomas JESS, and Kathryn R. AYSCOUGH. "SCP1 encodes an actin-bundling protein in yeast." Biochemical Journal 375, no. 2 (October 15, 2003): 287–95. http://dx.doi.org/10.1042/bj20030796.
Full textAbstract:
The association of F-actin (filamentous actin) with a large number of binding proteins is essential for cellular function. Actin-binding proteins control the dynamics of actin filaments, nucleate new filaments and facilitate formation of higher-order structures such as actin bundles. The yeast gene SCP1 encodes a small protein with significant homology to mammalian SM22/transgelin. We have investigated the role of Scp1p in budding yeast to probe the fundamental role of this family of proteins. Here, we demonstrate that Scp1p binds to F-actin and induces the formation of tight F-actin bundles in vitro. Deletion of SCP1 in yeast lacking the actin-bundling protein, fimbrin (Sac6p), exacerbates the disrupted actin phenotype and enhances latrunculin-A sensitivity. Furthermore, Scp1p co-localizes with actin in cortical patches and its localization is lost in the presence of latrunculin-A. Our data support a role for Scp1p in bundling actin filaments and, in concert with Sac6p, acting as a second actin-bundling activity crucial to the stability of the yeast actin cytoskeleton.
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Cramer, Louise P., Margaret Siebert, and Timothy J. Mitchison. "Identification of Novel Graded Polarity Actin Filament Bundles in Locomoting Heart Fibroblasts: Implications for the Generation of Motile Force." Journal of Cell Biology 136, no. 6 (March 24, 1997): 1287–305. http://dx.doi.org/10.1083/jcb.136.6.1287.
Full textAbstract:
We have determined the structural organization and dynamic behavior of actin filaments in entire primary locomoting heart fibroblasts by S1 decoration, serial section EM, and photoactivation of fluorescence. As expected, actin filaments in the lamellipodium of these cells have uniform polarity with barbed ends facing forward. In the lamella, cell body, and tail there are two observable types of actin filament organization. A less abundant type is located on the inner surface of the plasma membrane and is composed of short, overlapping actin bundles (0.25–2.5 μm) that repeatedly alternate in polarity from uniform barbed ends forward to uniform pointed ends forward. This type of organization is similar to the organization we show for actin filament bundles (stress fibers) in nonlocomoting cells (PtK2 cells) and to the known organization of muscle sarcomeres. The more abundant type of actin filament organization in locomoting heart fibroblasts is mostly ventrally located and is composed of long, overlapping bundles (average 13 μm, but can reach up to about 30 μm) which span the length of the cell. This more abundant type has a novel graded polarity organization. In each actin bundle, polarity gradually changes along the length of the bundle. Actual actin filament polarity at any given point in the bundle is determined by position in the cell; the closer to the front of the cell the more barbed ends of actin filaments face forward. By photoactivation marking in locomoting heart fibroblasts, as expected in the lamellipodium, actin filaments flow rearward with respect to substrate. In the lamella, all marked and observed actin filaments remain stationary with respect to substrate as the fibroblast locomotes. In the cell body of locomoting fibroblasts there are two dynamic populations of actin filaments: one remains stationary and the other moves forward with respect to substrate at the rate of the cell body. This is the first time that the structural organization and dynamics of actin filaments have been determined in an entire locomoting cell. The organization, dynamics, and relative abundance of graded polarity actin filament bundles have important implications for the generation of motile force during primary heart fibroblast locomotion.
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Dasbiswas, Kinjal, Shiqiong Hu, Frank Schnorrer, Samuel A. Safran, and Alexander D. Bershadsky. "Ordering of myosin II filaments driven by mechanical forces: experiments and theory." Philosophical Transactions of the Royal Society B: Biological Sciences 373, no. 1747 (April 9, 2018): 20170114. http://dx.doi.org/10.1098/rstb.2017.0114.
Full textAbstract:
Myosin II filaments form ordered superstructures in both cross-striated muscle and non-muscle cells. In cross-striated muscle, myosin II (thick) filaments, actin (thin) filaments and elastic titin filaments comprise the stereotypical contractile units of muscles called sarcomeres. Linear chains of sarcomeres, called myofibrils, are aligned laterally in registry to form cross-striated muscle cells. The experimentally observed dependence of the registered organization of myofibrils on extracellular matrix elasticity has been proposed to arise from the interactions of sarcomeric contractile elements (considered as force dipoles) through the matrix. Non-muscle cells form small bipolar filaments built of less than 30 myosin II molecules. These filaments are associated in registry forming superstructures (‘stacks’) orthogonal to actin filament bundles. Formation of myosin II filament stacks requires the myosin II ATPase activity and function of the actin filament crosslinking, polymerizing and depolymerizing proteins. We propose that the myosin II filaments embedded into elastic, intervening actin network (IVN) function as force dipoles that interact attractively through the IVN. This is in analogy with the theoretical picture developed for myofibrils where the elastic medium is now the actin cytoskeleton itself. Myosin stack formation in non-muscle cells provides a novel mechanism for the self-organization of the actin cytoskeleton at the level of the entire cell. This article is part of the theme issue ‘Self-organization in cell biology’.
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Verkhovsky, A. B., T. M. Svitkina, and G. G. Borisy. "Polarity sorting of actin filaments in cytochalasin-treated fibroblasts." Journal of Cell Science 110, no. 15 (August 1, 1997): 1693–704. http://dx.doi.org/10.1242/jcs.110.15.1693.
Full textAbstract:
The polarity of actin filaments is fundamental for the subcellular mechanics of actin-myosin interaction; however, little is known about how actin filaments are oriented with respect to myosin in non-muscle cells and how actin polarity organization is established and maintained. Here we approach these questions by investigating changes in the organization and polarity of actin relative to myosin II during actin filament translocation. Actin and myosin II reorganization was followed both kinetically, using microinjected fluorescent analogs of actin and myosin, and ultrastructurally, using myosin S1 decoration and immunogold labelling, in cultured fibroblasts that were induced to contract by treatment with cytochalasin D. We observed rapid (within 15 minutes) formation of ordered actin filament arrays: short tapered bundles and aster-like assemblies, in which filaments had uniform polarity with their barbed ends oriented toward the aggregate of myosin II at the base of a bundle or in the center of an aster. The resulting asters further interacted with each other and aggregated into bigger asters. The arrangement of actin in asters was in sharp contrast to the mixed polarity of actin filaments relative to myosin in non-treated cells. At the edge of the cell, actin filaments became oriented with their barbed ends toward the cell center; that is, the orientation was opposite to what was observed at the edge of nontreated cells. This rearrangement is indicative of relative translocation of actin and myosin II and of the ability of myosin II to sort actin filaments with respect to their polarity during translocation. The results suggest that the myosin II-actin system of non-muscle cells is organized as a dynamic network where actin filament arrangement is defined in the course of its interaction with myosin II.
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Lombardo, Andrew T., Shane R. Nelson, Guy G. Kennedy, Kathleen M. Trybus, Sam Walcott, and David M. Warshaw. "Myosin Va transport of liposomes in three-dimensional actin networks is modulated by actin filament density, position, and polarity." Proceedings of the National Academy of Sciences 116, no. 17 (April 9, 2019): 8326–35. http://dx.doi.org/10.1073/pnas.1901176116.
Full textAbstract:
The cell’s dense 3D actin filament network presents numerous challenges to vesicular transport by teams of myosin Va (MyoVa) molecular motors. These teams must navigate their cargo through diverse actin structures ranging from Arp2/3-branched lamellipodial networks to the dense, unbranched cortical networks. To define how actin filament network organization affects MyoVa cargo transport, we created two different 3D actin networks in vitro. One network was comprised of randomly oriented, unbranched actin filaments; the other was comprised of Arp2/3-branched actin filaments, which effectively polarized the network by aligning the actin filament plus-ends. Within both networks, we defined each actin filament’s 3D spatial position using superresolution stochastic optical reconstruction microscopy (STORM) and its polarity by observing the movement of single fluorescent reporter MyoVa. We then characterized the 3D trajectories of fluorescent, 350-nm fluid-like liposomes transported by MyoVa teams (∼10 motors) moving within each of the two networks. Compared with the unbranched network, we observed more liposomes with directed and fewer with stationary motion on the Arp2/3-branched network. This suggests that the modes of liposome transport by MyoVa motors are influenced by changes in the local actin filament polarity alignment within the network. This mechanism was supported by an in silico 3D model that provides a broader platform to understand how cellular regulation of the actin cytoskeletal architecture may fine tune MyoVa-based intracellular cargo transport.
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Sosnicki, A. A., K. E. Loesser, and L. C. Rome. "Myofilament overlap in swimming carp. I. Myofilament lengths of red and white muscle." American Journal of Physiology-Cell Physiology 260, no. 2 (February 1, 1991): C283—C288. http://dx.doi.org/10.1152/ajpcell.1991.260.2.c283.
Full textAbstract:
To assess myofilament overlap during locomotion, we estimated the length of myosin and actin filaments in axial red and white muscle of carp. Myosin filament lengths were 1.52 +/- 0.009 and 1.50 +/- 0.037 micron (means +/- SD) in the red and white muscle, respectively, as measured from thin sections. After correction for shrinkage (using the troponin-based 385-A axial periodicity), thin filaments were 0.96 +/- 0.009 and 0.97 +/- 0.023 micron in the red and white muscles, respectively. Filaments were also isolated from the white muscle and negatively stained. Myosin filaments were 1.56 +/- 0.025 microns, and actin filaments were 0.99 +/- 0.024 micron in length. The data from thin sections and isolated filaments agreed within 2% for actin and 4% for myosin filaments. The number of actin filament periods (24 for the red and white muscle) and the length of the filaments are the same as in frog. This suggests that the classic sarcomere length-tension curve of frog muscle may be used to estimate the functional properties of carp red and white muscle.
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Young, Michael E., John A. Cooper, and Paul C. Bridgman. "Yeast actin patches are networks of branched actin filaments." Journal of Cell Biology 166, no. 5 (August 30, 2004): 629–35. http://dx.doi.org/10.1083/jcb.200404159.
Full textAbstract:
Cortical actin patches are the most prominent actin structure in budding and fission yeast. Patches assemble, move, and disassemble rapidly. We investigated the mechanisms underlying patch actin assembly and motility by studying actin filament ultrastructure within a patch. Actin patches were partially purified from Saccharomyces cerevisiae and examined by negative-stain electron microscopy (EM). To identify patches in the EM, we correlated fluorescence and EM images of GFP-labeled patches. Patches contained a network of actin filaments with branches characteristic of Arp2/3 complex. An average patch contained 85 filaments. The average filament was only 50-nm (20 actin subunits) long, and the filament to branch ratio was 3:1. Patches lacking Sac6/fimbrin were unstable, and patches lacking capping protein were relatively normal. Our results are consistent with Arp2/3 complex-mediated actin polymerization driving yeast actin patch assembly and motility, as described by a variation of the dendritic nucleation model.
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Maul, Raymond S., Yuhong Song, Kurt J. Amann, Sachi C. Gerbin, Thomas D. Pollard, and David D. Chang. "EPLIN regulates actin dynamics by cross-linking and stabilizing filaments." Journal of Cell Biology 160, no. 3 (February 3, 2003): 399–407. http://dx.doi.org/10.1083/jcb.200212057.
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Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.
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Tsukada, N., and M. J. Phillips. "Bile canalicular contraction is coincident with reorganization of pericanalicular filaments and co-localization of actin and myosin-II." Journal of Histochemistry & Cytochemistry 41, no. 3 (March 1993): 353–63. http://dx.doi.org/10.1177/41.3.7679126.
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We examined the relationships between actin-myosin interaction and bile canalicular contraction using a new experimental model: cytoskeleton-enriched canalicular membranes (CCM). In CCM, the bile canaliculus compartment is isolated complete with membrane-attached pericanalicular actin filaments and the surrounding intermediate filament sheath. Immunofluorescence and immunoelectron microscopy showed that actin and myosin-II were distributed over pericanalicular microfilaments that insert into adherens (belt) junctions; intermediate filaments predominantly inserted into desmosomes. The addition of "contraction solution" (1 microM Ca2+, 1 mM ATP) resulted in closure of CCM lumens, which was interpreted as canalicular contraction. Contraction was also associated with shortening and/or twisting of canaliculi. Rearrangement of actin filaments and myosin-II with co-localization of actin and myosin was observed. Evidence is also provided for attachment of actin-myosin-II aggregates to intermediate filaments coincident with contraction, suggesting a key scaffold function for intermediate filaments of the canaliculus. Attention is drawn to the overall similarity of structure-function dynamics in hepatic apical membranes to those described in intestinal brush border membrane preparations. The results are consistent with dynamic actin-myosin interaction with co-localization of actin and myosin-II in filament clumps coincident with canalicular contraction.
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Schepers, Anna V., Charlotta Lorenz, Peter Nietmann, Andreas Janshoff, Stefan Klumpp, and Sarah Köster. "Multiscale mechanics and temporal evolution of vimentin intermediate filament networks." Proceedings of the National Academy of Sciences 118, no. 27 (June 29, 2021): e2102026118. http://dx.doi.org/10.1073/pnas.2102026118.
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The cytoskeleton, an intricate network of protein filaments, motor proteins, and cross-linkers, largely determines the mechanical properties of cells. Among the three filamentous components, F-actin, microtubules, and intermediate filaments (IFs), the IF network is by far the most extensible and resilient to stress. We present a multiscale approach to disentangle the three main contributions to vimentin IF network mechanics—single-filament mechanics, filament length, and interactions between filaments—including their temporal evolution. Combining particle tracking, quadruple optical trapping, and computational modeling, we derive quantitative information on the strength and kinetics of filament interactions. Specifically, we find that hydrophobic contributions to network mechanics enter mostly via filament-elongation kinetics, whereas electrostatics have a direct influence on filament–filament interactions.
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Bryce, Nicole S., Galina Schevzov, Vicki Ferguson, Justin M. Percival, Jim J. C. Lin, Fumio Matsumura, James R. Bamburg, et al. "Specification of Actin Filament Function and Molecular Composition by Tropomyosin Isoforms." Molecular Biology of the Cell 14, no. 3 (March 2003): 1002–16. http://dx.doi.org/10.1091/mbc.e02-04-0244.
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The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5NM1), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5NM1was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function.
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Moch, Marcin, and Rudolf E. Leube. "Hemidesmosome-Related Keratin Filament Bundling and Nucleation." International Journal of Molecular Sciences 22, no. 4 (February 21, 2021): 2130. http://dx.doi.org/10.3390/ijms22042130.
Full textAbstract:
The epithelial cytoskeleton encompasses actin filaments, microtubules, and keratin intermediate filaments. They are interconnected and attached to the extracellular matrix via focal adhesions and hemidesmosomes. To study their interplay, we inhibited actin and tubulin polymerization in the human keratinocyte cell line HaCaT by latrunculin B and nocodazole, respectively. Using immunocytochemistry and time-lapse imaging of living cells, we found that inhibition of actin and tubulin polymerization alone or in combination induced keratin network re-organization albeit differently in each situation. Keratin filament network retraction towards the nucleus and formation of bundled and radial keratin filaments was most pronounced in latrunculin-B treated cells but less in doubly-treated cells and not detectable in the presence of nocodazole alone. Hemidesmosomal keratin filament anchorage was maintained in each instance, whereas focal adhesions were disassembled in the absence of actin filaments. Simultaneous inhibition of actin and tubulin polymerization, therefore, allowed us to dissect hemidesmosome-specific functions for keratin network properties. These included not only anchorage of keratin filament bundles but also nucleation of keratin filaments, which was also observed in migrating cells. The findings highlight the fundamental role of hemidesmosomal adhesion for keratin network formation and organization independent of other cytoskeletal filaments pointing to a unique mechanobiological function.