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PubMed2006

Overlapping hand-over-hand mechanism of single molecular motility of cytoplasmic dynein.

Structural differences between dynein and kinesin suggest a unique molecular mechanism of dynein motility. Measuring the mechanical properties of a single molecule of dynein is crucial for revealing the mechanisms underlying its movement. We measured the step size and force produced by single molecules of active cytoplasmic dynein by using an optical trap and fluorescence imaging with a high temporal resolution. The velocity of dynein movement, 800 nm/s, is consistent with that reported in cells. The maximum force of 7-8 pN was independent of the ATP concentration and similar to that of kinesin. Dynein exhibited forward and occasional backwards steps of approximately 8 nm, independent of load. It is suggested that the large dynein heads take 16-nm steps by using an overlapping hand-over-hand mechanism.

Adenosine Triphosphate↗
PubMed1994

Cell localization and redistribution of the 67 kD laminin receptor and alpha 6 beta 1 integrin subunits in response to laminin stimulation: an immunogold electron microscopy study.

The 67 kDa laminin receptor (67LR), one of several cell surface laminin-binding proteins, is involved in the interactions between cancer cells and laminin during tumor invasion and metastasis. A 37 kDa polypeptide (37LRP), previously identified as the 67LR precursor, is abundantly present in the cytoplasm and has been implicated in polysome formation. To better understand the cellular localization of the 67LR and its precursor, transmission electron microscopic studies of human melanoma A2058 cells were carried out using immunogold labeling and a variety of antibodies: (a) affinity purified antibodies directed against 37LRP cDNA-derived synthetic peptides; (b) anti-67LR monoclonal antibodies raised against intact human small cell lung carcinoma cells; and (c) monoclonal antibodies against the subunits of the integrin laminin receptor, alpha 6 beta 1. Double-labeling immunocyto-chemistry revealed that anti-67LR monoclonal antibodies as well as anti-37 LRP antibodies recognized antigens that were localized in the cytoplasm in electron dense structures. As expected, cell membrane labeling was also observed. Surprisingly, alpha 6 and beta 1 integrin subunits were detected in the same cytoplasmic structures positive for the 67LR and the 37LRP. After addition of soluble laminin to A2058 cells in suspension, the number of labeled cytoplasmic structures increased especially in the vicinity of the plasma membrane, and were exported onto the cell surface. Neither fibronectin nor BSA induced such an effect. The data demonstrate that the 67 LR and the 37 LRP antibodies detect colocalized antigens that are in cytoplasmic structures with alpha 6 beta 1 integrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1975

Microfibrillar structures in the nucleus and cytoplasm of amoeba proteus.

The presence of microfibrillar structures in the nucleus and cytoplasm of Amoeba proteus has been described after glutaraldehyde and osmium fixation. The possible roles of cytoplasmic microfibrils in the contraction process of amoeba and nuclear microfibrils in the formation of the honeycomb nuclear lamina are discussed.

Amoeba↗
PubMed1975

Fine structure of granulocytes with cytoplasmic inclusions in pleural effusions from patients with rheumatoid pleuritis, tuberculous pleuritis and pleural carcinomatosis.

The fine structure of neutrophil granulocytes from pleural fluid obtained from patients with rheumatoid or tuberculous pleuritis or epidermoid carcinoma of the lung was identical irrespective of the primary disease of these patients. Homogenously stained lipid-like inclusions were present in the cytoplasm of the neutrophil granulocytes. They seemed to be formed de novo, as they lacked a distinct limiting unit membrane. The fine structure of the lipid-like inclusions gave no evidence to support the presence of immune complexes, these were probably located in the quantitative much fewer phagosomes of the granulocytes.

Adult↗
PubMed1995

Degradation of CD4 induced by human immunodeficiency virus type 1 Vpu protein: a predicted alpha-helix structure in the proximal cytoplasmic region of CD4 contributes to Vpu sensitivity.

The HIV-1-encoded Vpu protein induces a rapid and specific degradation of CD4 molecules in the endoplasmic reticulum (ER). In this study, Vpu-induced degradation of CD4 in the ER was investigated by quantitative immunoprecipitation of CD4 following cotransfection of COS-7 cells with CD4 and Vpu expressors in the presence of brefeldin A, a drug that blocks protein transport from the ER to the Golgi complex. In order to precisely define the sequence(s) or structural element(s) in the CD4 cytoplasmic domain necessary for Vpu-induced degradation, a panel of deletion and substitution mutants in the cytoplasmic domain of CD4 was generated and analyzed. In agreement with previous reports, our deletion analysis indicates that a region encompassing amino acids 411 to 419 (KRLLSEKKT) in the cytoplasmic domain of CD4 was required to confer Vpu sensitivity. However, six specific substitution mutations within this region did not confer CD4 resistance to Vpu, suggesting that neither the amino acid sequence nor the charge of the amino acids in this region was critical to Vpu-induced CD4 degradation. A dileucine motif that is important for internalization of CD4 and Nef-induced CD4 down-regulation was also not required for Vpu-induced CD4 degradation. Interestingly, two substitution mutants (CD4EMKL and CD4MK407,11PP) located in a more proximal cytoplasmic region of CD4 abolished Vpu-induced CD4 degradation. Computer-assisted analysis of the substitution and deletion mutants conferring CD4 resistance to Vpu-induced degradation indicated that these mutations disrupted a putative alpha-helix formed in the proximal cytoplasmic region of CD4. Taken together, these studies strongly suggest that a structural element in the proximal cytoplasmic region of CD4 contributes to Vpu sensitivity.

Amino Acid Sequence↗
PubMedDate not supplied

[Stress granules: RNP-containing cytoplasmic bodies springing up under stress. The structure and mechanism of organization].

In this review recent data describing stress granules are summarized. Stress granules are specific RNA-containing structures in the cytoplasm of living cells which arise under stress conditions (e. g. heat shock, UV irradiation, energy depletion and oxidative stress). It became evident that stress granules accumulate non-canonical 48S initiation complexes and contain mRNA with associated proteins, small ribosomal subunits and some initiation factors. Stress granules are depleted with ternary complex and large ribosomal subunit. It's proposed that eIF2alpha phosphorylation and ternary complex decrease can be a trigger for stress granule formation. Shuttling nuclear and cytoplasmic RNA-binding protein TIA-1 plays a crucial role in this process. It's proposed that TIA-1 forms prion-like aggregates, and these aggregates are scaffolds for other components of stress granules. Cytoskeletal structures facilitate the accumulation of stress granule components in local cytoplasmic sites. Investigation of process of stress granule formation is important for understanding of cell reaction to stress and translation regulation mechanisms.

Animals↗
PubMed2005

Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein.

The large majority of histidine kinases (HKs) are multifunctional enzymes having autokinase, phosphotransfer and phosphatase activities, and most of these are transmembrane sensor proteins. Sensor HKs possess conserved cytoplasmic phosphorylation and ATP-binding kinase domains. The different enzymatic activities require participation by one or both of these domains, implying the need for different conformational states. The catalytic domains are linked to the membrane through a coiled-coil segment that sometimes includes other domains. We describe here the first crystal structure of the complete cytoplasmic region of a sensor HK, one from the thermophile Thermotoga maritima in complex with ADPbetaN at 1.9 A resolution. The structure reveals previously unidentified functions for several conserved residues and reveals the relative disposition of domains in a state seemingly poised for phosphotransfer. The structure thereby inspires hypotheses for the mechanisms of autophosphorylation, phosphotransfer and response-regulator dephosphorylation, and for signal transduction through the coiled-coil segment. Mutational tests support the functional relevance of interdomain contacts.

Adenosine Triphosphate↗
PubMed1971

A Role for Zinc in the Structural Integrity of the Cytoplasmic Ribosomes of Euglena gacilis.

Zinc deficiency in dark-grown Euglena gracilis Klebs, Z strain Pringsheim, results in the disappearance of cytoplasmic ribosomes. In contrast, ribosomes in zinc-sufficient Euglena are conserved, do not undergo turnover, and can be demonstrated at any stage of growth. The zinc content of ribosomes from zinc-deficient Euglena just prior to ribosomal disappearance is 300 to 380 micrograms of zinc per gram rRNA as compared to 650 to 1280 micrograms of zinc per gram rRNA in ribosomes from zinc-sufficient cells. Ribosomal disappearance is believed to involve a generalized disintegration process related to the lower content of zinc in the ribosomes. Reappearance of ribosomes requires the addition of zinc. It is proposed that adequate zinc may be essential for normal tertiary and quaternary structure of the cytoplasmic ribosomes of Euglena.

Journal Article↗
PubMed1994

Structure, function and regulation of cytoplasmic dynein.

Molecular cloning studies have provided valuable structural information about the different subunits of cytoplasmic dynein and their relationships to their axonemal dynein counterparts. Recent unexpected findings regarding the role of this enzyme in mitosis have emerged from mutational analyses and microinjection experiments, while studies of organelle transport in vivo have revealed clues to mechanisms for physiological regulation of dynein activity.

Animals↗
PubMed1975

"Cytoplasmic" deoxyribonucleic acid polymerase. Structure and properties of the highly purified enzyme from human KB cells.

The freshly prepared crude cytoplasmic fraction of aqueously extracted KB cells contains a single major species of DNA polymerase activity (DNA polymerase C) that sediments homogeneously in low ionic strength sucrose gradients with a peak at 10.8 S. The enzyme activity from frozen crude extracts sediments heterogeneously under these conditions with peaks at 8.4 and 10 S. In 0.45 M salt-containing gradients all of the polymerase activity is recovered as a single 6.4 S species. When purified to a specific activity of 7,300, DNA polymerase C sediments in low ionic strength gradients as a single species of 6.5 S. From combined sedimentation and gel filtration analysis, we estimate the molecular weight of the active protomeric species of the polymerase to be about 170,000. Under no conditions of ionic strength does the enzyme disaggregate to active species smaller than 6.4 to 6.5 S. Sodium dodecyl sulfate-polyacrylamide gel analysis of the most highly purified enzyme fractions reveals two major protein bands of 87,000 and 175,000 daltons, respectively. These data suggest that DNA polymerase C contains an 87,000-dalton component and permit the interpretation that the active protomer of Mr equal 170,000 may be a dimer. The purified enzyme shows maximal activity with gapped duplex DNA and has an absolute requirement for 3'-hydroxyl termini. It utilizes initiated polydeoxynucleotide templates poorly and initiated polyribonucleotide templates not at all. Although the polymerase is inhibited by PPi it has only minimal ability to promote PPi exchange (0.8% of the polymerase activity). The purified enzyme is free of endonuclease and exonuclease activities (less than or equal to 0.003% of the polymerase activity) and demonstrates no primer-template-dependent conversion of substrate dNTP to free dNMP during the polymerization reaction. Finally, DNA polymerase C does not excise misparied primer termini from a synthetic homopolymer primer-template but can utilize such termini as initiation sites, although at a very slow rate.

Cell Line↗
PubMed1990

Role of intranuclear sacs, intrasaccate tubes, and cytoplasmic dense bodies in the structural completion of cytomegalovirus.

The addition of tegument or matrix proteins and the outermost membrane to human cytomegalovirus (strain AD 169) replicating in human cells appeared to occur differently in the nucleus than in the cytoplasm. In the nucleus cytomegalovirus was completed structurally within intranuclear sacs, as envelopment at the end of tubular structures, containing an electron-dense material, included tegument and outer membrane simultaneously. In the cytoplasm structural completion occurred in separate steps. Tegument could be added by association with dense bodies; then, interaction with the membranes of vacuoles or vesicles provided the outermost covering. Tubes originating in intranuclear sacs have been described previously only in guinea pig cells infected with guinea pig cytomegalovirus.

Antibodies, Monoclonal↗
PubMed2002

Cytoplasmic dynein-associated structures move bidirectionally in vivo.

Intracellular organelle transport is driven by motors that act upon microtubules or microfilaments. The microtubulebased motors, cytoplasmic dynein and kinesin, are believed to be responsible for retrograde and anterograde transport of intracellular cargo along microtubules. Many vesicles display bidirectional movement; however, the mechanism regulating directionality is unresolved. Directional movement might be accomplished by alternative binding of different motility factors to the cargo. Alternatively, different motors could associate with the same cargo and have their motor activity regulated. Although several studies have focused on the behavior of specific types of cargoes, little is known about the traffic of the motors themselves and how it correlates with cargo movement. To address this question, we studied cytoplasmic dynein dynamics in living Dictyostelium cells expressing dynein intermediate chain-green fluorescent protein (IC-GFP) fusion in an IC-null background. Dynein-associated structures display fast linear movement along microtubules in both minus-end and plus-end directions, with velocities similar to that of dynein and kinesin-like motors. In addition, dynein puncta often rapidly reverse their direction. Dynein stably associates with cargo moving in both directions as well as with those that rapidly reverse their direction of movement, suggesting that directional movement is not regulated by altering motor-cargo association but rather by switching activity of motors associated with the cargo. These observations suggest that both plus- and minus-end-directed motors associate with a given cargo and that coordinated regulation of motor activities controls vesicle directionality.

Animals↗
PubMed1986

A single gene from yeast for both nuclear and cytoplasmic polyadenylate-binding proteins: domain structure and expression.

Nuclear and cytoplasmic poly(A)-binding proteins have been purified from Saccharomyces cerevisiae, and antisera have been used to isolate a gene that encodes them. The gene occurs in a single copy on chromosome 5 and gives rise to a unique, unspliced 2.1 kb transcript. The nuclear protein appears to be derived from the cytoplasmic one by proteolytic cleavage into 53 and 17 kd polypeptides that remain associated during isolation. DNA sequence determination reveals four tandemly arrayed 90 amino acid regions of homology that probably represent poly(A)-binding domains. A 55 residue A-rich region upstream of the initiator methionine codon in the mRNA shows an affinity for poly(A)-binding protein comparable to that of poly(A)180-220, raising the possibility of feedback regulation of translation.

Amino Acid Sequence↗
PubMed1996

U6 snRNA maturation and stability.

The U6 snRNP is found as a monomer and as a heterodimer, complexed with the U4 snRNP (U4/U6). Northern blotting detects approximately equal amounts of U4/U6 heterodimer and U6 monomer in the nucleus but only U6 monomer in bona fide cytoplasm. In mammalian cells, newly synthesized U6 appears transiently in the cytoplasm before returning to the nucleus. Sedimentation analysis identifies cytoplasmic U6 in similarly sized structures as nuclear U4 and U6 and smaller structures than cytoplasmic U4. Inhibitor studies demonstrate that newly synthesized U6 can move from the cytoplasm into the nucleus in the absence of U4 synthesis. The nuclear half-life of U6 is significantly shorter than that of U4 and the other spliceosomal snRNAs. These data support a model in which U4 and U6 snRNAs undergo distinct cytoplasmic maturation pathways before returning to the nucleus, where the U4/U6 snRNP assembles.

Animals↗
PubMed1975

Comparison of canine distemper viral strains: an electron microscopic study.

A canine distemper (CD) viral strain, designated R252, originally obtained from a dog with demyelinating encephalomyelitis has been shown to reproduce this disease in gnotobiotic dogs in a high incidence in contradistinction to other CD viral strains which produce an acute fatal disease. Because comparision of R252 strain with the Snyder Hill (SH) and Onderstepoort (Ond) strains revealed differences in in vitro behavior, the 3 viruses were ultrastructurally investigated. The results revealed differences among the 3 viruses: Cytoplasmic nucleocapsid aggregates were characteristic of R252, diffuse aggregates of nucleocapsids were characteristic of SH, and although budding viral particles were recognized in Ond-infected cells, few nucleocapsids were seen in the cytoplasm. Cytoplasmic fibrillar structures were characteristic of R252- and Ond-infected cells. The budding viral particles observed with R252 and SH were similar, whereas Ond budding particles seemed to contain fewer nucleocapsids. Intranuclear inclusions recognized in R252- and SH-infected cultures appeared as areas of clearing of nucleoplasm along with disruption of the nucleolus. The presence of nucleocapsid-like structures was variable. Ultrastructurally, the cytoplasmic inclusions in cells infected with all 3 viruses progressed from focal aggregates of nucleocapsids to electron-dense bodies.

Animals↗
PubMed2004

NMR structural comparison of the cytoplasmic juxtamembrane domains of G-protein-coupled CB1 and CB2 receptors in membrane mimetic dodecylphosphocholine micelles.

The fourth cytoplasmic domain, the so-called C-terminal juxtamembrane segment or helix VIII, has been identified in numerous G-protein-coupled receptors and exhibits unique functional characteristics. Efforts have been devoted to studying the juxtamembrane segment in order to understand the biological importance of the segment in G-protein activation of the cannabinoid CB1 and CB2 receptors. Recent biochemical data revealed that the CB1 C-terminal juxtamembrane peptide fragment CB1-(401-417) can directly activate the G-protein and also showed that the specificity of the signal transduction activation by the C-terminal juxtamembrane region is unique to the CB1 receptor but not to the CB2 receptor (Mukhopadhyay, S., and Howlett, A. C. (2001) Eur. J. Biochem. 268, 499-505). However, there is experimental work, not yet reported, on the conformational analyses and structural comparison between the respective helix VIII segments of the two receptors. In the present study, we have examined the conformational specificities of the cytoplasmic helical domains for both cannabinoid receptors. Three-dimensional structural features of two synthetic CB1 and CB2 peptides, CB1I397-G418 and CB2I298-K319, respectively, in membrane mimetic DPC micelles were studied using a combined high resolution NMR and computer modeling approach. Comparisons of the NMR-determined structures of the two peptides as well as their correspondent mutant peptides revealed their conformational properties and salt bridge dissimilarity, which might help us to understand the different structural roles of the fourth cytoplasmic helices in the function and regulation of CB1 and CB2 receptors.

Cell Membrane↗
PubMed2001

A quantitative genetic analysis of nuclear-cytoplasmic male sterility in structured populations of Silene vulgaris.

Gynodioecy, the coexistence of functionally female and hermaphroditic morphs within plant populations, often has a complicated genetic basis involving several cytoplasmic male-sterility factors and nuclear restorers. This complexity has made it difficult to study the genetics and evolution of gynodioecy in natural populations. We use a quantitative genetic analysis of crosses within and among populations of Silene vulgaris to partition genetic variance for sex expression into nuclear and cytoplasmic components. We also use mitochondrial markers to determine whether cytoplasmic effects on sex expression can be traced to mitochondrial variance. Cytoplasmic variation and epistatic interactions between nuclear and cytoplasmic loci accounted for a significant portion of the variation in sex expression among the crosses. Source population also accounted for a significant portion of the sex ratio variation. Crosses among populations greatly enhanced the dam (cytoplasmic) effect, indicating that most among-population variance was at cytoplasmic loci. This is supported by the large among-population variance in the frequency of mitochondrial haplotypes, which also accounted for a significant portion of the sex ratio variance in our data. We discuss the similarities between the population structure we observed at loci that influence sex expression and previous work on putatively neutral loci, as well as the implications this has for what mechanisms may create and maintain population structure at loci that are influenced by natural selection.

Cell Nucleus↗
PubMed1985

Structural characterization of the cytoplasmic pole of band 3 from bovine erythrocyte membranes.

In an earlier study, we found that chymotryptic digestion of band 3 isolated from bovine erythrocyte membranes produces a 38,000-Da fragment in nonaethyleneglycol-n-dodecylether solution or a 50,000-Da fragment in deoxycholate solution as a primary fragment [Makino et al. (1984) J. Biochem. 95, 1019]. In the present study, these fragments were purified in an aqueous medium without detergent and their structural properties were examined. Several lines of evidence showed that the 50,000-Da fragment constitutes the entire cytoplasmic pole of bovine band 3 and that the 38,000-Da fragment is a subfragment of the 50,000-Da fragment. The large fragment was suggested to be divided into two distinct regions, the 12,000- and 38,000-Da portions, differing in their conformational thermal stability. However, attempts to identify the 12,000-Da portion as an isolable segment were without success. The cytoplasmic pole was characterized as a dimer which adopts an elongated gross conformation with helix of approximately 35%. Treatment of the fragments with dimethylmaleic anhydride dissociated the dimers into the monomers, accompanied by a significant conformational change of the 38,000-Da portion. Comparative studies suggested that the cytoplasmic domain of bovine band 3 has structurally different region(s) from that of human band 3, though their gross conformation shows extensive similarity.

Animals↗