The Contributions of the Amino and Carboxy Terminal Domains of Flightin to the Biomechanical Properties of Drosophila Flight Muscle Thick Filaments

Abstract The N-terminal domains (NTDs) of many members of the nuclear hormone receptor (NHR) family contain potent transcription-activating functions (AFs). Knowledge of the mechanisms of action of the NTD AFs has lagged, compared with that concerning other important domains of the NHRs. In part, this is because the NTD AFs appear to be unfolded when expressed as recombinant proteins. Recent studies have begun to shed light on the structure and function of the NTD AFs. Recombinant NTD AFs can be made to fold by application of certain osmolytes or when expressed in conjunction with a DNA-binding domain by binding that DNA-binding domain to a DNA response element. The sequence of the DNA binding site may affect the functional state of the AFs domain. If properly folded, NTD AFs can bind certain cofactors and primary transcription factors. Through these, and/or by direct interactions, the NTD AFs may interact with the AF2 domain in the ligand binding, carboxy-terminal portion of the NHRs. We propose models for the folding of the NTD AFs and their protein-protein interactions.

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Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster.

The Journal of Cell Biology ◽

10.1083/jcb.109.5.2157 ◽

1989 ◽

Vol 109 (5) ◽

pp. 2157-2167 ◽

Cited By ~ 78

Author(s):

J D Saide ◽

S Chin-Bow ◽

J Hogan-Sheldon ◽

L Busquets-Turner ◽

J O Vigoreaux ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Monoclonal Antibodies ◽

Flight Muscle ◽

Cross Reactivity ◽

Antigenic Determinants ◽

Immunogold Staining ◽

Thick Filaments ◽

The Matrix ◽

Flight Muscles

Twelve monoclonal antibodies have been raised against proteins in preparations of Z-disks isolated from Drosophila melanogaster flight muscle. The monoclonal antibodies that recognized Z-band components were identified by immunofluorescence microscopy of flight muscle myofibrils. These antibodies have identified three Z-disk antigens on immunoblots of myofibrillar proteins. Monoclonal antibodies alpha:1-4 recognize a 90-100-kD protein which we identify as alpha-actinin on the basis of cross-reactivity with antibodies raised against honeybee and vertebrate alpha-actinins. Monoclonal antibodies P:1-4 bind to the high molecular mass protein, projectin, a component of connecting filaments that link the ends of thick filaments to the Z-band in insect asynchronous flight muscles. The anti-projectin antibodies also stain synchronous muscle, but, surprisingly, the epitopes here are within the A-bands, not between the A- and Z-bands, as in flight muscle. Monoclonal antibodies Z(210):1-4 recognize a 210-kD protein that has not been previously shown to be a Z-band structural component. A fourth antigen, resolved as a doublet (approximately 400/600 kD) on immunoblots of Drosophila fibrillar proteins, is detected by a cross reacting antibody, Z(400):2, raised against a protein in isolated honeybee Z-disks. On Lowicryl sections of asynchronous flight muscle, indirect immunogold staining has localized alpha-actinin and the 210-kD protein throughout the matrix of the Z-band, projectin between the Z- and A-bands, and the 400/600-kD components at the I-band/Z-band junction. Drosophila alpha-actinin, projectin, and the 400/600-kD components share some antigenic determinants with corresponding honeybee proteins, but no honeybee protein interacts with any of the Z(210) antibodies.

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Molecular map of the desmosomal plaque

Journal of Cell Science ◽

10.1242/jcs.112.23.4325 ◽

1999 ◽

Vol 112 (23) ◽

pp. 4325-4336 ◽

Cited By ~ 13

Author(s):

A.J. North ◽

W.G. Bardsley ◽

J. Hyam ◽

E.A. Bornslaeger ◽

H.C. Cordingley ◽

...

Keyword(s):

Plasma Membrane ◽

Intermediate Filaments ◽

Protein Interactions ◽

Immunogold Labelling ◽

Molecular Approaches ◽

Molecular Map ◽

Carboxy Terminal ◽

Desmoglein 3 ◽

Terminal Domains

Recent biochemical and molecular approaches have begun to establish the protein interactions that lead to desmosome assembly. To determine whether these associations occur in native desmosomes we have performed ultrastructural localisation of specific domains of the major desmosomal components and have used the results to construct a molecular map of the desmosomal plaque. Antibodies directed against the amino- and carboxy-terminal domains of desmoplakin, plakoglobin and plakophilin 1, and against the carboxy-terminal domains of desmoglein 3, desmocollin 2a and desmocollin 2b, were used for immunogold labelling of ultrathin cryosections of bovine nasal epidermis. For each antibody, the mean distance of the gold particles, and thus the detected epitope, from the cytoplasmic surface of the plasma membrane was determined quantitatively. Results showed that: (i) plakophilin, although previously shown to bind intermediate filaments in vitro, is localised extremely close to the plasma membrane, rather than in the region where intermediate filaments are seen to insert into the desmosomal plaque; (ii) while the ‘a’ form of desmocollin overlaps with plakoglobin and desmoplakin, the shorter ‘b’ form may be spatially separated from them; (iii) desmoglein 3 extends across the entire outer plaque, beyond both desmocollins; (iv) the amino terminus of desmoplakin lies within the outer dense plaque and the carboxy terminus some 40 nm distant in the zone of intermediate filament attachment. This is consistent with a parallel arrangement of desmoplakin in dimers or higher order aggregates and with the predicted length of desmoplakin II, indicating that desmoplakin I may be folded or coiled. Thus several predictions from previous work were borne out by this study, but in other cases our observations yielded unexpected results. These results have significant implications relating to molecular interactions in desmosomes and emphasise the importance of applying multiple and complementary approaches to biological investigations.

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Alpha-helix in the carboxy-terminal domains of histones H1 and H5.

The EMBO Journal ◽

10.1002/j.1460-2075.1988.tb02784.x ◽

1988 ◽

Vol 7 (1) ◽

pp. 69-75 ◽

Cited By ~ 113

Author(s):

D. J. Clark ◽

C. S. Hill ◽

S. R. Martin ◽

J. O. Thomas

Keyword(s):

Alpha Helix ◽

Carboxy Terminal ◽

Terminal Domains

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Reconstitution Studies Using the Helical and Carboxy-Terminal Domains of Enzyme I of the Phosphoenolpyruvate:Sugar Phosphotransferase System

Biochemistry ◽

10.1021/bi991680p ◽

1999 ◽

Vol 38 (47) ◽

pp. 15470-15479 ◽

Cited By ~ 20

Author(s):

Peng-Peng Zhu ◽

Roman H. Szczepanowski ◽

Neil J. Nosworthy ◽

Ann Ginsburg ◽

Alan Peterkofsky

Keyword(s):

Phosphotransferase System ◽

Carboxy Terminal ◽

Enzyme I ◽

Terminal Domains

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Molecular and ultrastructural defects in a Drosophila myosin heavy chain mutant: differential effects on muscle function produced by similar thick filament abnormalities.

The Journal of Cell Biology ◽

10.1083/jcb.107.6.2601 ◽

1988 ◽

Vol 107 (6) ◽

pp. 2601-2612 ◽

Cited By ~ 65

Author(s):

P T O'Donnell ◽

S I Bernstein

Keyword(s):

Myosin Heavy Chain ◽

Heavy Chain ◽

Muscle Function ◽

Flight Muscle ◽

Thick Filament ◽

Differential Sensitivity ◽

Indirect Flight Muscle ◽

Molecular Defect ◽

Thick Filaments ◽

Nuclease Mapping

We have determined the molecular defect of the Drosophila melanogaster myosin heavy chain (MHC) mutation Mhc and the mutation's effect on indirect flight muscle, jump muscle, and larval intersegmental muscle. We show that the Mhc1 mutation is essentially a null allele which results in the dominant-flightless and recessive-lethal phenotypes associated with this mutant (Mogami, K., P. T. O'Donnell, S. I. Bernstein, T. R. F. Wright, C. P. Emerson, Jr. 1986. Proc. Natl. Acad. Sci. USA. 83:1393-1397). The mutation is a 101-bp deletion in the MHC gene which removes most of exon 5 and the intron that precedes it. S1 nuclease mapping indicates that mutant transcripts follow two alternative processing pathways. Both pathways result in the production of mature transcripts with altered reading frames, apparently yielding unstable, truncated MHC proteins. Interestingly, the preferred splicing pathway uses the more distal of two available splice donor sites. We present the first ultrastrutural characterization of a completely MHC-null muscle and show that it lacks any discernable thick filaments. Sarcomeres in these muscles are completely disorganized suggesting that thick filaments play a critical role in sarcomere assembly. To understand why the Mhc1 mutation severely disrupts indirect flight muscle and jump muscle function in heterozygotes, but does not seriously affect the function of other muscle types, we examined the muscle ultrastructure of Mhc1/+ heterozygotes. We find that these organisms have a nearly 50% reduction in the number of thick filaments in indirect flight muscle, jump muscle, and larval intersegmental muscle. In addition, aberrantly shaped thick filaments are common in the jump muscle and larval intersegmental muscle. We suggest that the differential sensitivity of muscle function to the Mhc1 mutation is a consequence of the unique myofilament arrays in each of these muscles. The highly variable myofilament array of larval intersegmental muscle makes its function relatively insensitive to changes in thick filament number and morphology. Conversely, the rigid double hexagonal lattice of the indirect flight muscle, and the organized lattice of the jump muscle cannot be perturbed without interfering with the specialized and evolutionarily more complex functions they perform.

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