Phalloidin Staining of Actin Filaments for Visualization of Muscle Fibers in Caenorhabditis elegans

During invasion, cells breach basement membrane (BM) barriers with actin-rich protrusions. It remains unclear, however, whether actin polymerization applies pushing forces to help break through BM, or whether actin filaments play a passive role as scaffolding for targeting invasive machinery. Here, using the developmental event of anchor cell (AC) invasion inCaenorhabditis elegans, we observe that the AC deforms the BM and underlying tissue just before invasion, exerting forces in the tens of nanonewtons range. Deformation is driven by actin polymerization nucleated by the Arp2/3 complex and its activators, whereas formins and cross-linkers are dispensable. Delays in invasion upon actin regulator loss are not caused by defects in AC polarity, trafficking, or secretion, as appropriate markers are correctly localized in the AC even when actin is reduced and invasion is disrupted. Overall force production emerges from this study as one of the main tools that invading cells use to promote BM disruption inC. elegans.

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Force-mediated cellular anisotropy and plasticity dictate the elongation dynamics of embryos

Science Advances ◽

10.1126/sciadv.abg3264 ◽

2021 ◽

Vol 7 (27) ◽

pp. eabg3264

Author(s):

Chao Fang ◽

Xi Wei ◽

Xueying Shao ◽

Yuan Lin

Keyword(s):

Plastic Deformation ◽

Caenorhabditis Elegans ◽

Muscle Contraction ◽

Dynamic Model ◽

Actin Filaments ◽

Actin Bundles ◽

Abnormal Embryo ◽

Intracellular Forces ◽

Kinetics Of ◽

Good Agreement

We developed a unified dynamic model to explain how cellular anisotropy and plasticity, induced by alignment and severing/rebundling of actin filaments, dictate the elongation dynamics of Caenorhabditis elegans embryos. It was found that the gradual alignment of F-actins must be synchronized with the development of intracellular forces for the embryo to elongate, which is then further sustained by muscle contraction–triggered plastic deformation of cells. In addition, we showed that preestablished anisotropy is essential for the proper onset of the process while defects in the integrity or bundling kinetics of actin bundles result in abnormal embryo elongation, all in good agreement with experimental observations.

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F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments.

The Journal of Cell Biology ◽

10.1083/jcb.135.5.1291 ◽

1996 ◽

Vol 135 (5) ◽

pp. 1291-1308 ◽

Cited By ~ 55

Author(s):

L G Tilney ◽

P Connelly ◽

S Smith ◽

G M Guild

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Modular Design ◽

Thin Sections ◽

Actin Bundle ◽

Actin Bundles ◽

Phalloidin Staining ◽

Filament Bundles ◽

End To End ◽

As If

The actin bundles in Drosophila bristles run the length of the bristle cell and are accordingly 65 microns (microchaetes) or 400 microns (macrochaetes) in length, depending on the bristle type. Shortly after completion of bristle elongation in pupae, the actin bundles break down as the bristle surface becomes chitinized. The bundles break down in a bizarre way; it is as if each bundle is sawed transversely into pieces that average 3 microns in length. Disassembly of the actin filaments proceeds at the "sawed" surfaces. In all cases, the cuts in adjacent bundles appear in transverse register. From these images, we suspected that each actin bundle is made up of a series of shorter bundles or modules that are attached end-to-end. With fluorescent phalloidin staining and serial thin sections, we show that the modular design is present in nondegenerating bundles. Decoration of the actin filaments in adjacent bundles in the same bristle with subfragment 1 of myosin reveals that the actin filaments in every module have the same polarity. To study how modules form developmentally, we sectioned newly formed and elongating bristles. At the bristle tip are numerous tiny clusters of 6-10 filaments. These clusters become connected together more basally to form filament bundles that are poorly organized, initially, but with time become maximally cross-linked. Additional filaments are then added to the periphery of these organized bundle modules. All these observations make us aware of a new mechanism for the formation and elongation of actin filament bundles, one in which short bundles are assembled and attached end-to-end to other short bundles, as are the vertical girders between the floors of a skyscraper.

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X-ray Diffraction Studies on the Structural Origin of Dynamic Tension Recovery Following Ramp-Shaped Releases in High-Ca Rigor Muscle Fibers

International Journal of Molecular Sciences ◽

10.3390/ijms21041244 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1244

Author(s):

Haruo Sugi ◽

Maki Yamaguchi ◽

Tetsuo Ohno ◽

Hiroshi Okuyama ◽

Naoto Yagi

Keyword(s):

Muscle Contraction ◽

Actin Filaments ◽

Myosin Head ◽

Muscle Fibers ◽

Skinned Fibers ◽

Myosin Filament ◽

X Ray Diffraction ◽

X Ray ◽

Tension Recovery ◽

Pass Through

It is generally believed that during muscle contraction, myosin heads (M) extending from myosin filament attaches to actin filaments (A) to perform power stroke, associated with the reaction, A-M-ADP-Pi → A-M + ADP + Pi, so that myosin heads pass through the state of A-M, i.e., rigor A-M complex. We have, however, recently found that: (1) an antibody to myosin head, completely covering actin-binding sites in myosin head, has no effect on Ca2+-activated tension in skinned muscle fibers; (2) skinned fibers exhibit distinct tension recovery following ramp-shaped releases (amplitude, 0.5% of Lo; complete in 5 ms); and (3) EDTA, chelating Mg ions, eliminate the tension recovery in low-Ca rigor fibers but not in high-Ca rigor fibers. These results suggest that A-M-ADP myosin heads in high-Ca rigor fibers have dynamic properties to produce the tension recovery following ramp-shaped releases, and that myosin heads do not pass through rigor A-M complex configuration during muscle contraction. To obtain information about the structural changes in A-M-ADP myosin heads during the tension recovery, we performed X-ray diffraction studies on high-Ca rigor skinned fibers subjected to ramp-shaped releases. X-ray diffraction patterns of the fibers were recorded before and after application of ramp-shaped releases. The results obtained indicate that during the initial drop in rigor tension coincident with the applied release, rigor myosin heads take up applied displacement by tilting from oblique to perpendicular configuration to myofilaments, and after the release myosin heads appear to rotate around the helical structure of actin filaments to produce the tension recovery.

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The two actin-interacting protein 1 genes have overlapping and essential function for embryonic development in Caenorhabditis elegans

Molecular Biology of the Cell ◽

10.1091/mbc.e10-12-0934 ◽

2011 ◽

Vol 22 (13) ◽

pp. 2258-2269 ◽

Cited By ~ 15

Author(s):

Shoichiro Ono ◽

Kazumi Nomura ◽

Sadae Hitosugi ◽

Domena K. Tu ◽

Jocelyn A. Lee ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Actin Filament ◽

Actin Filaments ◽

Body Wall ◽

The Body ◽

Body Wall Muscle ◽

Interacting Protein ◽

Adult Muscle ◽

Essential Function

Disassembly of actin filaments by actin-depolymerizing factor (ADF)/cofilin and actin-interacting protein 1 (AIP1) is a conserved mechanism to promote reorganization of the actin cytoskeleton. We previously reported that unc-78, an AIP1 gene in the nematode Caenorhabditis elegans, is required for organized assembly of sarcomeric actin filaments in the body wall muscle. unc-78 functions in larval and adult muscle, and an unc-78–null mutant is homozygous viable and shows only weak phenotypes in embryos. Here we report that a second AIP1 gene, aipl-1 (AIP1-like gene-1), has overlapping function with unc-78, and that depletion of the two AIP1 isoforms causes embryonic lethality. A single aipl-1–null mutation did not cause a detectable phenotype. However, depletion of both unc-78 and aipl-1 arrested development at late embryonic stages due to severe disorganization of sarcomeric actin filaments in body wall muscle. In vitro, both AIPL-1 and UNC-78 preferentially cooperated with UNC-60B, a muscle-specific ADF/cofilin isoform, in actin filament disassembly but not with UNC-60A, a nonmuscle ADF/cofilin. AIPL-1 is expressed in embryonic muscle, and forced expression of AIPL-1 in adult muscle compensated for the function of UNC-78. Thus our results suggest that enhancement of actin filament disassembly by ADF/cofilin and AIP1 proteins is critical for embryogenesis.

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The Caenorhabditis elegans unc-78 Gene Encodes a Homologue of Actin-Interacting Protein 1 Required for Organized Assembly of Muscle Actin Filaments

The Journal of Cell Biology ◽

10.1083/jcb.152.6.1313 ◽

2001 ◽

Vol 152 (6) ◽

pp. 1313-1320 ◽

Cited By ~ 69

Author(s):

Shoichiro Ono

Keyword(s):

Caenorhabditis Elegans ◽

Actin Filament ◽

Actin Filaments ◽

Body Wall ◽

Point Mutations ◽

The Body ◽

Body Wall Muscle ◽

Dynamic Regulation ◽

Interacting Protein ◽

Actin Organization

Assembly and maintenance of myofibrils require dynamic regulation of the actin cytoskeleton. In Caenorhabditis elegans, UNC-60B, a muscle-specific actin depolymerizing factor (ADF)/cofilin isoform, is required for proper actin filament assembly in body wall muscle (Ono, S., D.L. Baillie, and G.M. Benian. 1999. J. Cell Biol. 145:491–502). Here, I show that UNC-78 is a homologue of actin-interacting protein 1 (AIP1) and functions as a novel regulator of actin organization in myofibrils. In unc-78 mutants, the striated organization of actin filaments is disrupted, and large actin aggregates are formed in the body wall muscle cells, resulting in defects in their motility. Point mutations in unc-78 alleles change conserved residues within different WD repeats of the UNC-78 protein and cause less severe phenotypes than a deletion allele, suggesting that these mutations partially impair the function of UNC-78. UNC-60B is normally localized in the diffuse cytoplasm and to the myofibrils in wild type but mislocalized to the actin aggregates in unc-78 mutants. Similar Unc-78 phenotypes are observed in both embryonic and adult muscles. Thus, AIP1 is an important regulator of actin filament organization and localization of ADF/cofilin during development of myofibrils.

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Regeneration of Actin Filaments in Actin-Extracted Bumblebee Flight Muscle Fibers as Probed by X-ray Diffraction

Biophysical Journal ◽

10.1016/j.bpj.2015.11.1615 ◽

2016 ◽

Vol 110 (3) ◽

pp. 300a

Author(s):

Hiroyuki Iwamoto

Keyword(s):

Actin Filaments ◽

Flight Muscle ◽

Muscle Fibers ◽

X Ray Diffraction ◽

X Ray

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UNC-87 isoforms, Caenorhabditis elegans calponin-related proteins, interact with both actin and myosin and regulate actomyosin contractility

Molecular Biology of the Cell ◽

10.1091/mbc.e14-10-1483 ◽

2015 ◽

Vol 26 (9) ◽

pp. 1687-1698 ◽

Cited By ~ 5

Author(s):

Kanako Ono ◽

Takashi Obinata ◽

Sawako Yamashiro ◽

Zhongmei Liu ◽

Shoichiro Ono

Keyword(s):

Caenorhabditis Elegans ◽

Actin Filaments ◽

Striated Muscle ◽

Negative Regulator ◽

Actin Binding ◽

Binding Motif ◽

Actomyosin Contractility ◽

Somatic Gonad ◽

Nonmuscle Cells ◽

Related Proteins

Calponin-related proteins are widely distributed among eukaryotes and involved in signaling and cytoskeletal regulation. Calponin-like (CLIK) repeat is an actin-binding motif found in the C-termini of vertebrate calponins. Although CLIK repeats stabilize actin filaments, other functions of these actin-binding motifs are unknown. The Caenorhabditis elegans unc-87 gene encodes actin-binding proteins with seven CLIK repeats. UNC-87 stabilizes actin filaments and is essential for maintenance of sarcomeric actin filaments in striated muscle. Here we show that two UNC-87 isoforms, UNC-87A and UNC-87B, are expressed in muscle and nonmuscle cells in a tissue-specific manner by two independent promoters and exhibit quantitatively different effects on both actin and myosin. Both UNC-87A and UNC-87B have seven CLIK repeats, but UNC-87A has an extra N-terminal extension of ∼190 amino acids. Both UNC-87 isoforms bind to actin filaments and myosin to induce ATP-resistant actomyosin bundles and inhibit actomyosin motility. UNC-87A with an N-terminal extension binds to actin and myosin more strongly than UNC-87B. UNC-87B is associated with actin filaments in nonstriated muscle in the somatic gonad, and an unc-87 mutation causes its excessive contraction, which is dependent on myosin. These results strongly suggest that proteins with CLIK repeats function as a negative regulator of actomyosin contractility.

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Two Caenorhabditis elegans calponin-related proteins have overlapping functions to maintain cytoskeletal integrity and are essential for reproduction

10.1101/2020.04.29.069104 ◽

2020 ◽

Author(s):

Shoichiro Ono ◽

Kanako Ono

Keyword(s):

Caenorhabditis Elegans ◽

Actin Filaments ◽

Body Wall ◽

The Body ◽

Body Wall Muscle ◽

Related Protein ◽

Somatic Gonad ◽

Related Proteins

AbstractMulticellular organisms have multiple genes encoding calponins and calponin-related proteins, and some of these are known to regulate actin cytoskeletal dynamics and contractility. However, functional similarities and differences among these proteins are largely unknown. In the nematode Caenorhabditis elegans, UNC-87 is a calponin-related protein with seven calponin-like (CLIK) motifs and is required for maintenance of contractile apparatuses in muscle cells. Here, we report that CLIK-1, another calponin-related protein that also contains seven CLIK motifs, has an overlapping function with UNC-87 to maintain actin cytoskeletal integrity in vivo and has both common and different actin-regulatory activities in vitro. CLIK-1 is predominantly expressed in the body wall muscle and somatic gonad, where UNC-87 is also expressed. unc-87 mutation causes cytoskeletal defects in the body wall muscle and somatic gonad, whereas clik-1 depletion alone causes no detectable phenotypes. However, simultaneous depletion of clik-1 and unc-87 caused sterility due to ovulation failure by severely affecting the contractile actin networks in the myoepithelial sheath of the somatic gonad. In vitro, UNC-87 bundles actin filaments. However, CLIK-1 binds to actin filaments without bundling them and is antagonistic to UNC-87 in filament bundling. UNC-87 and CLIK-1 share common functions to inhibit cofilin binding and allow tropomyosin binding to actin filaments, suggesting that both proteins stabilize actin filaments. Thus, partially redundant functions of UNC-87 and CLIK-1 in ovulation is likely mediated by their common actin-regulatory activities, but their distinct activities in actin bundling suggest that they also have different biological functions.

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Muscle contraction as a Markov Process - II: x-ray interference data (M3 & M6 reflections) imply myosin cross-bridge motions are controlled by structural transitions along actin filaments

10.1101/2021.01.16.426939 ◽

2021 ◽

Author(s):

Clarence E Schutt ◽

Vladimir Gelfand ◽

Eli Paster

Keyword(s):

Muscle Contraction ◽

Actin Filaments ◽

Myosin Head ◽

Muscle Fibers ◽

Head Movements ◽

Published Data ◽

Structural Transitions ◽

X Ray ◽

Thick Filaments ◽

In Series

AbstractThe unit underlying the construction and functioning of muscle fibers is the sarcomere. Tension develops in fibers as thousands of sarcomeres arranged in series contract in unison. Shortening is due to the sliding of actin thin filaments along antiparallel arrays of myosin thick filaments. Remarkably, myosin catalytic heads situated across the center M-line of a sarcomere are separated by a distance that is a half integral of the 14.5 nm spacing between successive layers of myosin heads on the thick filaments. This results in the splitting of the 14.5 nm meridional reflection in X-ray diffraction patterns of muscle fibers. Following a quick drop in tension, changes in the relative intensities of the split meridional peaks provide a sensitive measure of myosin head movements. We use published data obtained with the x-ray interference method to validate a theory of muscle contraction in which cooperative structural transitions along force-generating actin filaments regulate the binding of myosin heads. The probability that an actin-bound myosin head will detach is represented here by a statistical function that yields a length-tension curve consistent with classical descriptions of the recovery of contracting muscle fibers subjected to millisecond drops in tension.

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