AAA domains and organization of the dynein motor unit

2000 ◽  
Vol 113 (14) ◽  
pp. 2521-2526 ◽  
Author(s):  
S.M. King
Keyword(s):  
Heavy Chain ◽  
Structural Information ◽  
Microscopic Analysis ◽  
Motor Units ◽  
Holliday Junctions ◽  
Electron Microscopic ◽  
Heavy Chains ◽  
Dynein Motor ◽  
Microtubule Motor ◽  
Globular Head

Dyneins contain one-three microtubule motor units that are each derived from the C-terminal globular head of a heavy chain. The N-terminal regions of the heavy chains form stems that are required for intra-dynein associations. The microtubule-binding sites are located at the terminus of a short stalk that emanates from each globular head. Recent electron microscopic analysis indicates that the dynein head has a heptameric toroidal organization. This finding is echoed by the identification of six AAA (ATPases associated with cellular activities) domains and a seventh unrelated unit within this heavy chain region. At least two of these AAA domains can bind nucleotide, although only one appears able to hydrolyze ATP. Several other AAA domain proteins exhibit a similar annular organization of six AAA units. Detailed structural information is available for several AAA proteins, including N-ethylmaleimide-sensitive vesicle-fusion protein and the RuvB motor involved in DNA migration and resolution of Holliday junctions. The resulting structural parallels allow intriguing predictions to be made concerning dynein organization and motor function.

scholarly journals Ifm(2)2 is a myosin heavy chain allele that disrupts myofibrillar assembly only in the indirect flight muscle of Drosophila melanogaster.

1988 ◽  
Vol 107 (6) ◽  
pp. 2613-2621 ◽  
Author(s):  
M Chun ◽  
S Falkenthal
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Flight Muscle ◽  
Microscopic Analysis ◽  
Thick Filament ◽  
Indirect Flight Muscle ◽  
Chain Gene ◽  
Wild Type ◽  
Electron Microscopic ◽  
Sarcomere Assembly

Using a combination of molecular and genetic techniques we demonstrate that Ifm(2)2 is an allele of the single-copy sarcomeric myosin heavy chain gene. Flies homozygous for this allele accumulate wild-type levels of mRNA and protein in tubular muscle of adults, but fail to accumulate detectable amounts of myosin heavy chain mRNA or protein in the indirect flight muscle. We propose that the mutation interferes with either transcription of the gene or splicing of the primary transcript in the indirect flight muscle and not in other muscle tissues. Biochemical and electron microscopic analysis of flies homozygous for this mutation has revealed that thick filament assembly is abolished in the indirect flight muscle resulting in the instability of wild-type thick filament proteins. In contrast, thin filament and Z disc assembly are marginally affected. We discuss a working hypothesis for sarcomere assembly and define and experimental approach to test the predictions of this proposed pathway for sarcomere assembly.

scholarly journals The substructure of isolated and in situ outer dynein arms of sea urchin sperm flagella.

1985 ◽  
Vol 101 (4) ◽  
pp. 1400-1412 ◽  
Author(s):  
W S Sale ◽  
U W Goodenough ◽  
J E Heuser
Keyword(s):  
Sea Urchin ◽  
Heavy Chain ◽  
Heavy Chains ◽  
Low Salt ◽  
Spherical Head ◽  
Dynein Arms ◽  
Sea Urchin Sperm ◽  
Globular Head

Outer-arm dynein from the sperm of the sea urchin S. purpuratus was adsorbed to mica flakes and visualized by the quick-freeze, deep-etch technique. Replicas reveal particles comprised of two globular heads joined by two irregularly shaped stems which make contact along their length. One head is pear-shaped (18.5 X 12.5 nm) and the other is spherical (14.5-nm diam). The stems are decorated by a complex of bead-like subunits. The same two-headed protein is found in the 21S dynein-1 fraction of sucrose gradients. The beta-heavy chain/intermediate chain 1 (beta/IC-1) dynein subfraction, produced by low-salt dialysis and zonal centrifugation of the high-salt-extracted dynein-1, contains only single-headed molecules with single stems. These heads are predominantly pear-shaped (18.5 X 12.5 nm). Since 21S dynein-1 contains two heavy chains (alpha and beta), and the beta/IC-1 subfraction is comprised of only the beta-heavy chain (Tang et al., 1982, J. Biol. Chem. 257: 508-515), we conclude that each head is formed by a heavy chain, that the pear-shaped head contains the beta-heavy chain, and that the spherical head contains the alpha-heavy chain. The in situ outer dynein arms of demembranated sperm were also studied by the quick-freeze, deep-etch method. When frozen in reactivation buffer devoid of ATP, each arm consists of a large globular head that attaches to the A-microtubule by distally skewed subunits and attaches to the B-microtubule by a slender stalk. In ATP, this head shifts its orientation such that it can be seen to be constructed from two globular domains. We offer possible correlates between the in situ and the in vitro images, and we compare the structure of sea-urchin dynein with dynein previously described from Chlamydomonas and Tetrahymena.

scholarly journals Insights into the Structural Organization of the I1 Inner Arm Dynein from a Domain Analysis of the 1β Dynein Heavy Chain

2000 ◽  
Vol 11 (7) ◽  
pp. 2297-2313 ◽  
Author(s):  
Catherine A. Perrone ◽  
Steven H. Myster ◽  
Raqual Bower ◽  
Eileen T. O'Toole ◽  
Mary E. Porter
Keyword(s):  
Heavy Chain ◽  
Structural Organization ◽  
Microscopic Analysis ◽  
Transcription Unit ◽  
Light Chains ◽  
Flagellar Motility ◽  
Electron Microscopic ◽  
Dynein Heavy Chain ◽  
Complex Transformation ◽  
Motility Mutant

To identify domains in the dynein heavy chain (Dhc) required for the assembly of an inner arm dynein, we characterized a new motility mutant (ida2-6) obtained by insertional mutagenesis.ida2-6 axonemes lack the polypeptides associated with the I1 inner arm complex. Recovery of genomic DNA flanking the mutation indicates that the defects are caused by plasmid insertion into theDhc10 transcription unit, which encodes the 1β Dhc of the I1 complex. Transformation with Dhc10 constructs encoding <20% of the Dhc can partially rescue the motility defects by reassembly of an I1 complex containing an N-terminal 1β Dhc fragment and a full-length 1α Dhc. Electron microscopic analysis reveals the location of the missing 1β Dhc motor domain within the axoneme structure. These observations, together with recent studies on the 1α Dhc, identify a Dhc domain required for complex assembly and further demonstrate that the intermediate and light chains are associated with the stem regions of the Dhcs in a distinct structural location. The positioning of these subunits within the I1 structure has significant implications for the pathways that target the assembly of the I1 complex into the axoneme and modify the activity of the I1 dynein during flagellar motility.

scholarly journals Drosophila cytoplasmic dynein, a microtubule motor that is asymmetrically localized in the oocyte.

1994 ◽  
Vol 126 (6) ◽  
pp. 1475-1494 ◽  
Author(s):  
M Li ◽  
M McGrail ◽  
M Serr ◽  
T S Hays
Keyword(s):  
Heavy Chain ◽  
Maternal Effect ◽  
Nurse Cell ◽  
Cytoplasmic Dynein ◽  
Cdna Clones ◽  
Dynein Heavy Chain ◽  
Dynein Motor ◽  
Maternal Effect Gene ◽  
Microtubule Motor ◽  
Egg Chambers

The unidirectional movements of the microtubule-associated motors, dyneins, and kinesins, provide an important mechanism for the positioning of cellular organelles and molecules. An intriguing possibility is that this mechanism may underlie the directed transport and asymmetric positioning of morphogens that influence the development of multicellular embryos. In this report, we characterize the Drosophila gene, Dhc64C, that encodes a cytoplasmic dynein heavy chain polypeptide. The primary structure of the Drosophila cytoplasmic dynein heavy chain polypeptide has been determined by the isolation and sequence analysis of overlapping cDNA clones. Drosophila cytoplasmic dynein is highly similar in sequence and structure to cytoplasmic dynein isoforms reported for other organisms. The Dhc64C dynein transcript is differentially expressed during development with the highest levels being detected in the ovaries of adult females. Within the developing egg chambers of the ovary, the dynein gene is predominantly transcribed in the nurse cell complex. In contrast, the encoded dynein motor protein displays a striking accumulation in the single cell that will develop as the oocyte. The temporal and spatial pattern of dynein accumulation in the oocyte is remarkably similar to that of several maternal effect gene products that are essential for oocyte differentiation and axis specification. This distribution and its disruption by specific maternal effect mutations lends support to recent models suggesting that microtubule motors participate in the transport of these morphogens from the nurse cell cytoplasm to the oocyte.

scholarly journals A Ras-like domain in the light intermediate chain bridges the dynein motor to a cargo-binding region

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Courtney M Schroeder ◽  
Jonathan ML Ostrem ◽  
Nicholas T Hertz ◽  
Ronald D Vale
Keyword(s):  
G Proteins ◽  
Heavy Chain ◽  
Structural Information ◽  
Cytoplasmic Dynein ◽  
Protein Fold ◽  
Binding Region ◽  
Aromatic Residues ◽  
Dynein Motor ◽  
Insight Into ◽  
Intermediate Chain

Cytoplasmic dynein, a microtubule-based motor protein, transports many intracellular cargos by means of its light intermediate chain (LIC). In this study, we have determined the crystal structure of the conserved LIC domain, which binds the motor heavy chain, from a thermophilic fungus. We show that the LIC has a Ras-like fold with insertions that distinguish it from Ras and other previously described G proteins. Despite having a G protein fold, the fungal LIC has lost its ability to bind nucleotide, while the human LIC1 binds GDP preferentially over GTP. We show that the LIC G domain binds the dynein heavy chain using a conserved patch of aromatic residues, whereas the less conserved C-terminal domain binds several Rab effectors involved in membrane transport. These studies provide the first structural information and insight into the evolutionary origin of the LIC as well as revealing how this critical subunit connects the dynein motor to cargo.

Proximal Tubulopathies Associated With Monoclonal Light Chains: The Spectrum of Clinicopathologic Manifestations and Molecular Pathogenesis

2014 ◽  
Vol 138 (10) ◽  
pp. 1365-1380 ◽  
Author(s):  
Guillermo A. Herrera
Keyword(s):  
Proximal Tubule ◽  
Heavy Chain ◽  
Light Chain ◽  
Molecular Mechanisms ◽  
Light Chains ◽  
Clinical Settings ◽  
Electron Microscopic ◽  
Heavy Chains ◽  
Cast Nephropathy ◽  
Diagnostic Difficulties

Context.—Lesions associated with monoclonal light and heavy chains display a variety of glomerular, tubular interstitial, and vascular manifestations. While some of the entities are well recognized, including light and heavy chain deposition diseases, AL (light chain) and AH (heavy chain) amyloidosis, and light chain (“myeloma”) cast nephropathy, other lesions centered on proximal tubules are much less accurately identified, properly diagnosed, and adequately understood in terms of pathogenesis and molecular mechanisms involved. These proximal tubule–centered lesions are typically associated with monoclonal light chains and have not been reported in patients with circulating monoclonal heavy chains. Objective.—To determine the incidence of proximal tubulopathies in a series of patients with monoclonal light chain–related renal lesions and characterize them with an emphasis on clinical correlations and elucidation of molecular mechanisms involved in their pathogenesis. Design.—A study of 5410 renal biopsies with careful evaluation of light microscopic, immunofluorescence, and electron microscopic findings was conducted to identify these monoclonal light/heavy chain–related lesions. In selected cases, ultrastructural immunolabeling was performed to better illustrate and understand molecular mechanisms involved or to resolve specific diagnostic difficulties. Results.—In all, 2.5% of the biopsies were diagnosed as demonstrating renal pathology associated with monoclonal light or heavy chains. Of these, approximately 46% were classified as proximal tubule–centered lesions, also referred to as monoclonal light chain–associated proximal tubulopathies. These proximal tubulopathies were divided into 4 groups defined by characteristic immunomorphologic manifestations associated with specific clinical settings. Conclusions.—These are important lesions whose recognition in the different clinical settings is extremely important for patients' clinical management, therapeutic purposes, and prognosis. These entities have been segregated into 4 distinct variants, conceptualized morphologically and clinically. Specific mechanisms involved in their pathogenesis are proposed.

scholarly journals A Chlamydomonas outer arm dynein mutant with a truncated beta heavy chain

1993 ◽  
Vol 122 (3) ◽  
pp. 653-661 ◽  
Author(s):  
H Sakakibara ◽  
S Takada ◽  
SM King ◽  
GB Witman ◽  
R Kamiya
Keyword(s):  
Cross Sections ◽  
Heavy Chain ◽  
Swimming Velocity ◽  
Wild Type ◽  
Terminal Part ◽  
Electron Microscopic ◽  
Heavy Chains ◽  
Dynein Arms ◽  
Alpha Beta ◽  
Arm Function

A new allele of the Chlamydomonas oda4 flagellar mutant (oda4-s7) possessing abnormal outer dynein arms was isolated. Unlike the previously described oda4 axoneme lacking all three (alpha, beta, and gamma) outer-arm dynein heavy chains, the oda4-s7 axoneme contains the alpha and gamma heavy chains and a novel peptide with a molecular mass of approximately 160 kD. The peptide reacts with a mAb (18 beta B) that recognizes an epitope on the NH2-terminal part of the beta heavy chain. These observations indicate that this mutant has a truncated beta heavy chain, and that the NH2-terminal part of the beta heavy chain is important for the stable assembly of the outer arms. In averaged electron microscopic images of outer arms from cross sections of axonemes, the mutant outer arm lacks its mid-portion, producing a forked appearance. Together with our previous finding that the mutant oda11 lacks the alpha heavy chain and the outermost portion of the arm (Sakakibara, H., D. R. Mitchell, and R. Kamiya. 1991. J. Cell Biol. 113:615-622), this result defines the approximate locations of the three outer arm heavy chains in the axonemal cross section. The swimming velocity of oda4-s7 is 65 +/- 8 microns/s, close to that of oda4 which lacks the entire outer arm (62 +/- 8 microns/s) but significantly lower than the velocities of wild type (194 +/- 23 microns/s) and oda11 (119 +/- 17 microns/s). Thus, the lack of the beta heavy chain impairs outer-arm function more seriously than does the lack of the alpha heavy chain, suggesting that the alpha and beta chains play different roles in outer arm function.

Preparatory Procedures for Electron Microscopic Analysis at the Molecular Level of Resolution

1978 ◽  
Vol 36 (3) ◽  
pp. 516-520
Author(s):  
F.J. Sjostrand
Keyword(s):  
Electron Microscope ◽  
Molecular Level ◽  
Microscopic Analysis ◽  
Resolving Power ◽  
Cellular Structures ◽  
Electron Microscopic ◽  
Systematic Analysis ◽  
Technical Developments ◽  
Thin Sectioning ◽  
Obvious Reason

In the 1940's and 1950's electron microscopy conferences were attended with everybody interested in learning about the latest technical developments for one very obvious reason. There was the electron microscope with its outstanding performance but nobody could make very much use of it because we were lacking proper techniques to prepare biological specimens. The development of the thin sectioning technique with its perfectioning in 1952 changed the situation and systematic analysis of the structure of cells could now be pursued. Since then electron microscopists have in general become satisfied with the level of resolution at which cellular structures can be analyzed when applying this technique. There has been little interest in trying to push the limit of resolution closer to that determined by the resolving power of the electron microscope.

Direct visualization of phase-separated domains in lipid membranes

1978 ◽  
Vol 36 (2) ◽  
pp. 290-291
Author(s):  
S. W. Hui ◽  
T. P. Stewart
Keyword(s):  
Lipid Membranes ◽  
Structural Information ◽  
Freeze Fracture ◽  
Biological Molecules ◽  
Vacuum Drying ◽  
Direct Visualization ◽  
X Ray Diffraction ◽  
Electron Microscopic ◽  
X Ray ◽  
Hydrocarbon Chains

Direct electron microscopic study of biological molecules has been hampered by such factors as radiation damage, lack of contrast and vacuum drying. In certain cases, however, the difficulties may be overcome by using redundent structural information from repeating units and by various specimen preservation methods. With bilayers of phospholipids in which both the solid and fluid phases co-exist, the ordering of the hydrocarbon chains may be utilized to form diffraction contrast images. Domains of different molecular packings may be recgnizable by placing properly chosen filters in the diffraction plane. These domains would correspond to those observed by freeze fracture, if certain distinctive undulating patterns are associated with certain molecular packing, as suggested by X-ray diffraction studies. By using an environmental stage, we were able to directly observe these domains in bilayers of mixed phospholipids at various temperatures at which their phases change from misible to inmissible states.

Structure of Myosin Subfragment-1 from Electron Microscopy and Crystallography

1988 ◽  
Vol 46 ◽  
pp. 156-157
Author(s):  
Donald A. Winkelmann
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Actin Filaments ◽  
Light Chains ◽  
Myosin Filament ◽  
Primary Role ◽  
Heavy Chains ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Globular Head

The primary role of the interaction of actin and myosin is the generation of force and motion as a direct consequence of the cyclic interaction of myosin crossbridges with actin filaments. Myosin is composed of six polypeptides: two heavy chains of molecular weight 220,000 daltons and two pairs of light chains of molecular weight 17,000-23,000. The C-terminal portions of the myosin heavy chains associate to form an α-helical coiled-coil rod which is responsible for myosin filament formation. The N-terminal portion of each heavy chain associates with two different light chains to form a globular head that binds actin and hydrolyses ATP. Myosin can be fragmented by limited proteolysis into several structural and functional domains. It has recently been demonstrated using an in vitro movement assay that the globular head domain, subfragment-1, is sufficient to cause sliding movement of actin filaments.The discovery of conditions for crystallization of the myosin subfragment-1 (S1) has led to a systematic analysis of S1 structure by x-ray crystallography and electron microscopy. Image analysis of electron micrographs of thin sections of small S1 crystals has been used to determine the structure of S1 in the crystal lattice.

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