UNC-98 links an integrin-associated complex to thick filaments in Caenorhabditis elegans muscle

Focal adhesions are multiprotein assemblages that link cells to the extracellular matrix. The transmembrane protein, integrin, is a key component of these structures. In vertebrate muscle, focal adhesion–like structures called costameres attach myofibrils at the periphery of muscle cells to the cell membrane. In Caenorhabditis elegans muscle, all the myofibrils are attached to the cell membrane at both dense bodies (Z-disks) and M-lines. Clustered at the base of dense bodies and M-lines, and associated with the cytoplasmic tail of β-integrin, is a complex of many proteins, including UNC-97 (vertebrate PINCH). Previously, we showed that UNC-97 interacts with UNC-98, a 37-kD protein, containing four C2H2 Zn fingers, that localizes to M-lines. We report that UNC-98 also interacts with the C-terminal portion of a myosin heavy chain. Multiple lines of evidence support a model in which UNC-98 links integrin-associated proteins to myosin in thick filaments at M-lines.

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Differential assembly of alpha- and gamma-filagenins into thick filaments in Caenorhabditis elegans

Journal of Cell Science ◽

10.1242/jcs.113.22.4001 ◽

2000 ◽

Vol 113 (22) ◽

pp. 4001-4012 ◽

Cited By ~ 1

Author(s):

F. Liu ◽

I. Ortiz ◽

A. Hutagalung ◽

C.C. Bauer ◽

R.G. Cook ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Immunoelectron Microscopy ◽

Body Wall ◽

Developmental Stages ◽

Body Wall Muscle ◽

Developmentally Regulated ◽

C Elegans ◽

Novel Proteins ◽

Thick Filaments ◽

Associated Proteins

Muscle thick filaments are highly organized supramolecular assemblies of myosin and associated proteins with lengths, diameters and flexural rigidities characteristic of their source. The cores of body wall muscle thick filaments of the nematode Caenorhabditis elegans are tubular structures of paramyosin sub-filaments coupled by filagenins and have been proposed to serve as templates for the assembly of native thick filaments. We have characterized alpha- and gamma-filagenins, two novel proteins of the cores with calculated molecular masses of 30,043 and 19,601 and isoelectric points of 10.52 and 11.49, respectively. Western blot and immunoelectron microscopy using affinity-purified antibodies confirmed that the two proteins are core components. Immunoelectron microscopy of the cores revealed that they assemble with different periodicities. Immunofluorescence microscopy showed that alpha-filagenin is localized in the medial regions of the A-bands of body wall muscle cells whereas gamma-filagenin is localized in the flanking regions, and that alpha-filagenin is expressed in 1.5-twofold embryos while gamma-filagenin becomes detectable only in late vermiform embryos. The expression of both proteins continues throughout later stages of development. C. elegans body wall muscle thick filaments of these developmental stages have distinct lengths. Our results suggest that the differential assembly of alpha- and gamma-filagenins into thick filaments of distinct lengths may be developmentally regulated.

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Purified thick filaments from the nematode Caenorhabditis elegans: evidence for multiple proteins associated with core structures.

The Journal of Cell Biology ◽

10.1083/jcb.106.6.1985 ◽

1988 ◽

Vol 106 (6) ◽

pp. 1985-1995 ◽

Cited By ~ 25

Author(s):

H F Epstein ◽

G C Berliner ◽

D L Casey ◽

I Ortiz

Keyword(s):

Caenorhabditis Elegans ◽

Body Wall ◽

Gradient Centrifugation ◽

Blue Staining ◽

Nematode Caenorhabditis Elegans ◽

C Elegans ◽

Thick Filaments ◽

Cell Biol ◽

Associated Proteins ◽

Body Wall Muscles

The thick filaments of the nematode, Caenorhabditis elegans, arising predominantly from the body-wall muscles, contain two myosin isoforms and paramyosin as their major proteins. The two myosins are located in distinct regions of the surfaces, while paramyosin is located within the backbones of the filaments. Tubular structures constitute the cores of the polar regions, and electron-dense material is present in the cores of the central regions (Epstein, H.F., D.M. Miller, I. Ortiz, and G.C. Berliner. 1985. J. Cell Biol. 100:904-915). Biochemical, genetic, and immunological experiments indicate that the two myosins and paramyosin are not necessary core components (Epstein, H.F., I. Ortiz, and L.A. Traeger Mackinnon. 1986. J. Cell Biol. 103:985-993). The existence of the core structures suggests, therefore, that additional proteins may be associated with thick filaments in C. elegans. To biochemically detect minor associated proteins, a new procedure for the isolation of thick filaments of high purity and structural preservation has been developed. The final step, glycerol gradient centrifugation, yielded fractions that are contaminated by, at most, 1-2% with actin, tropomyosin, or ribosome-associated proteins on the basis of Coomassie Blue staining and electron microscopy. Silver staining and radioautography of gel electrophoretograms of unlabeled and 35S-labeled proteins, respectively, revealed at least 10 additional bands that cosedimented with thick filaments in glycerol gradients. Core structures prepared from wild-type thick filaments contained at least six of these thick filament-associated protein bands. The six proteins also cosedimented with thick filaments purified by gradient centrifugation from CB190 mutants lacking myosin heavy chain B and from CB1214 mutants lacking paramyosin. For these reasons, we propose that the six associated proteins are potential candidates for putative components of core structures in the thick filaments of body-wall muscles of C. elegans.

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Myosin and paramyosin of Caenorhabditis elegans embryos assemble into nascent structures distinct from thick filaments and multi-filament assemblages

The Journal of Cell Biology ◽

10.1083/jcb.122.4.845 ◽

1993 ◽

Vol 122 (4) ◽

pp. 845-858 ◽

Cited By ~ 42

Author(s):

HF Epstein ◽

DL Casey ◽

I Ortiz

Keyword(s):

Caenorhabditis Elegans ◽

Functional Relationship ◽

Immunofluorescence Microscopy ◽

Myosin Heavy Chains ◽

Wild Type ◽

Linear Structures ◽

Heavy Chains ◽

Thick Filaments ◽

Vertebrate Muscle ◽

Charge Change

The organization of myosin heavy chains (mhc) A and B and paramyosin (pm) which are the major proteins of thick filaments in adult wild-type Caenorhabditis elegans were studied during embryonic development. As a probe of myosin-paramyosin interaction, the unc-15 mutation e73 which produces a glu342lys charge change in pm and leads to the formation of large paracrystalline multi-filament assemblages was compared to wild type. These three proteins colocalized in wild-type embryos from 300 to 550 min of development after first cleavage at 20 degrees C on the basis of immunofluorescence microscopy using specific monoclonal antibodies. Linear structures which were diversely oriented around the muscle cell peripheries appeared at 360 min and became progressively more aligned parallel to the embryonic long axis until distinct myofibrils were formed at 550 min. In the mutant, mhc A and pm were colocalized in the linear structures, but became progressively separated until they showed no spatial overlap at the myofibril stage. These results indicate that the linear structures represent nascent assemblies containing myosin and pm in which the proteins interact differently than in wild-type thick filaments of myofibrils. In e73, these nascent structures were distinct from the multi-filament assemblages. The overlapping of actin and mhc A in the nascent linear structures suggests their possible structural and functional relationship to the "stress fiber-like structures" of cultured vertebrate muscle cells.

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The Role of Cytoplasmic MEX-5/6 Polarity in Asymmetric Cell Division

Bulletin of Mathematical Biology ◽

10.1007/s11538-021-00860-0 ◽

2021 ◽

Vol 83 (4) ◽

Author(s):

Sungrim Seirin-Lee

Keyword(s):

Mathematical Model ◽

Cell Division ◽

Caenorhabditis Elegans ◽

Cell Membrane ◽

Mother Cell ◽

Asymmetric Cell Division ◽

Transmembrane Protein ◽

Cytoplasmic Protein ◽

Cell Geometry

AbstractIn the process of asymmetric cell division, the mother cell induces polarity in both the membrane and the cytosol by distributing substrates and components asymmetrically. Such polarity formation results from the harmonization of the upstream and downstream polarities between the cell membrane and the cytosol. MEX-5/6 is a well-investigated downstream cytoplasmic protein, which is deeply involved in the membrane polarity of the upstream transmembrane protein PAR in the Caenorhabditis elegans embryo. In contrast to the extensive exploration of membrane PAR polarity, cytoplasmic polarity is poorly understood, and the precise contribution of cytoplasmic polarity to the membrane PAR polarity remains largely unknown. In this study, we explored the interplay between the cytoplasmic MEX-5/6 polarity and the membrane PAR polarity by developing a mathematical model that integrates the dynamics of PAR and MEX-5/6 and reflects the cell geometry. Our investigations show that the downstream cytoplasmic protein MEX-5/6 plays an indispensable role in causing a robust upstream PAR polarity, and the integrated understanding of their interplay, including the effect of the cell geometry, is essential for the study of polarity formation in asymmetric cell division.

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Caenorhabditis elegans UNC-98, a C2H2 Zn Finger Protein, Is a Novel Partner of UNC-97/PINCH in Muscle Adhesion Complexes

Molecular Biology of the Cell ◽

10.1091/mbc.e02-10-0676 ◽

2003 ◽

Vol 14 (6) ◽

pp. 2492-2507 ◽

Cited By ~ 41

Author(s):

Kristina B. Mercer ◽

Denise B. Flaherty ◽

Rachel K. Miller ◽

Hiroshi Qadota ◽

Tina L. Tinley ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Nuclear Localization ◽

Nuclear Export ◽

Fluorescent Protein ◽

Nuclear Export Signal ◽

Focal Adhesions ◽

Cell Nuclei ◽

C Elegans ◽

Dense Bodies ◽

Green Fluorescent

To further understand the assembly and maintenance of the muscle contractile apparatus, we have identified a new protein, UNC-98, in the muscle of Caenorhabditis elegans. unc-98 mutants display reduced motility and a characteristic defect in muscle structure. We show that the major defect in the mutant muscle is in the M-lines and dense bodies (Z-line analogs). Both functionally and compositionally, nematode M-lines and dense bodies are analogous to focal adhesions of nonmuscle cells. UNC-98 is a novel 310-residue polypeptide consisting of four C2H2 Zn fingers and several possible nuclear localization signal and nuclear export signal sequences. By use of UNC-98 antibodies and green fluorescent protein fusions (to full-length UNC-98 and UNC-98 fragments), we have shown that UNC-98 resides at M-lines, muscle cell nuclei, and possibly at dense bodies. Furthermore, we demonstrated that 1) the N-terminal 106 amino acids are both necessary and sufficient for nuclear localization, and 2) the C-terminal (fourth) Zn finger is required for localization to M-lines and dense bodies. UNC-98 interacts with UNC-97, a C. elegans homolog of PINCH. We propose that UNC-98 is both a structural component of muscle focal adhesions and a nuclear protein that influences gene expression.

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Faculty Opinions recommendation of A conserved postsynaptic transmembrane protein affecting neuromuscular signaling in Caenorhabditis elegans.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1017785.203392 ◽

2004 ◽

Author(s):

Elior Peles

Keyword(s):

Caenorhabditis Elegans ◽

Transmembrane Protein

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Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle

Molecular Biology of the Cell ◽

10.1091/mbc.e07-03-0278 ◽

2007 ◽

Vol 18 (11) ◽

pp. 4317-4326 ◽

Cited By ~ 39

Author(s):

Hiroshi Qadota ◽

Kristina B. Mercer ◽

Rachel K. Miller ◽

Kozo Kaibuchi ◽

Guy M. Benian

Keyword(s):

Caenorhabditis Elegans ◽

Binding Assays ◽

Lim Domain ◽

Yeast Two Hybrid ◽

Lim Domains ◽

Thick Filaments ◽

Vitro Binding ◽

Two Hybrid ◽

Hybrid Screening

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

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Microtubules and microtubule-associated proteins from the nematode Caenorhabditis elegans: periodic cross-links connect microtubules in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.103.1.23 ◽

1986 ◽

Vol 103 (1) ◽

pp. 23-31 ◽

Cited By ~ 35

Author(s):

E J Aamodt ◽

J G Culotti

Keyword(s):

Caenorhabditis Elegans ◽

Apparent Molecular Weight ◽

Cross Link ◽

Nematode Caenorhabditis Elegans ◽

Microtubule Associated Proteins ◽

C Elegans ◽

Associated Proteins ◽

Cross Links ◽

The Cross

The nematode Caenorhabditis elegans should be an excellent model system in which to study the role of microtubules in mitosis, embryogenesis, morphogenesis, and nerve function. It may be studied by the use of biochemical, genetic, molecular biological, and cell biological approaches. We have purified microtubules and microtubule-associated proteins (MAPs) from C. elegans by the use of the anti-tumor drug taxol (Vallee, R. B., 1982, J. Cell Biol., 92:435-44). Approximately 0.2 mg of microtubules and 0.03 mg of MAPs were isolated from each gram of C. elegans. The C. elegans microtubules were smaller in diameter than bovine microtubules assembled in vitro in the same buffer. They contained primarily 9-11 protofilaments, while the bovine microtubules contained 13 protofilaments. The principal MAP had an apparent molecular weight of 32,000 and the minor MAPs were 30,000, 45,000, 47,000, 50,000, 57,000, and 100,000-110,000 mol wt as determined by SDS-gel electrophoresis. The microtubules were observed, by electron microscopy of negatively stained preparations, to be connected by stretches of highly periodic cross-links. The cross-links connected the adjacent protofilaments of aligned microtubules, and occurred at a frequency of one cross-link every 7.7 +/- 0.9 nm, or one cross-link per tubulin dimer along the protofilament. The cross-links were removed when the MAPs were extracted from the microtubules with 0.4 M NaCl. The cross-links then re-formed when the microtubules and the MAPs were recombined in a low salt buffer. These results strongly suggest that the cross-links are composed of MAPs.

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