bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microtubule-based peroxisome movement
Authors: Rapp Stephan , Saffrich Rainer , Anton Markus , Jäkle Ursula , Ansorge Wilhelm , Gorgas Karin , Just Wilhelm W. 
Published: April 2021

ABSTRACT
The association of peroxisomes with cytoskeletal structures was investigated both by electron microscopy and by kinetic analysis of peroxisome movement. The morphological studies indicated distinct interactions of peroxisomes with microtubules and frequently revealed multiple contact sites. The kinetic approach utilised microinjection and import of fluorescein-labeled luciferase in order to mark and track peroxisomes in vivo. Peroxisomal motility was analysed by time-lapse imaging and fluorescence microscopy. According to their movement peroxisomes were classified into two groups. Group 1 peroxisomes comprising the majority of organelles at 37°C moved slowly with an average velocity of 0.024±0.012 µm/second whereas the movement of group 2 peroxisomes, 10-15% of the total population, was saltatory exhibiting an average velocity of 0.26±0.17 µm/second with maximal values of more than 2 µm/second. Saltations were completely abolished by the microtubule-depolymerising drug nocodazole and were slightly reduced by about 25% by cytochalasin D which disrupts the actin microfilament system. Double fluorescence labeling of both peroxisomes and microtubules revealed peroxisome saltations linked to distinct microtubule tracks. Cellular depletion of endogenous levels of NTPs as well as the use of 5’-adenylylimidodiphosphate, a nonhydrolysable ATP analog, applied to a permeabilised cell preparation both completely blocked peroxisomal movement. These data suggest an ATPase dependent, microtubule-based mechanism of peroxisome movement. Both the intact and the permeabilised cell system presented in this paper for the first time allow kinetic measurements on peroxisomal motility and thus will be extremely helpful in the biochemical characterisation of the motor proteins involved.
rRNA distribution during microspore development in anthers of Beta vulgarisL.: quantitative in situ hybridization analysis
Authors: Majewska-Sawka Anna , Rodriguez-Garcia Maria Isabel 
Published: April 2021

ABSTRACT
We related changes in the ultrastructural organization of the nucleoli with the results of quantitative in situ hybridizations to characterize rRNA metabolism during the development of microspore mother cells in the sugar beet anther (Beta vulgaris L.). In the course of meiotic prophase and early postmeiotic interphase the morphological characteristics of the nucleoli are typical of low or no transcriptional activity and a low rate of rRNA processing.
However, we found evidence of an apparent increase in the relative numbers of 18 S rRNA transcripts in some stages of microsporogenesis. This was found in both the nucleoli and cytoplasm of pachytene meiocytes, and in later stages there was a spectacular accumulation of rRNA transcripts in nucleoli of the tetrad cells. Quantitative data are analyzed in the light of morphometric findings in the cells and their compartments to elucidate the degree to which changes in cell size are related to changes in labeling density and distribution. The results are discussed in terms of rRNA synthesis, transport and degradation as processes involved in the regulation of rRNA within microsporocytes and microspores.
Localization of kinesin superfamily proteins to the connecting cilium of fish photoreceptors
Authors: Beech Peter L. , Pagh-Roehl Kathryn , Noda Yasuko , Hirokawa Nobutaka , Burnside Beth , Rosenbaum Joel L. 
Published: April 2021

ABSTRACT
Kinesin superfamily proteins (KIFs) are probable motors in vesicular and non-vesicular transport along microtubular tracks. Since a variety of KIFs have been recently identified in the motile flagella of Chlamydomonas, we sought to ascertain whether KIFs are also associated with the connecting cilia of vertebrate rod photoreceptors. As the only structural link between the rod inner segment and the photosensitive rod outer segment, the connecting cilium is thought to be the channel through which all material passes into and out of the outer segment from the rod cell body. We have performed immunological tests on isolated sunfish rod inner-outer segments (RIS-ROS) using two antibodies that recognize the conserved motor domain of numerous KIFs (anti-LAGSE, a peptide antibody, and anti-Klp1 head, generated against the N terminus of Chlamydomonas Klp1) as well as an antibody specific to a neuronal KIF, KIF3A. On immunoblots of RIS-ROS, LAGSE antibody detected a prominent band at ∼117 kDa, which is likely to be kinesin heavy chain, and Klp1 head antibody detected a single band at ∼170 kDa; KIF3A antibody detected a polypeptide at ∼85 kDa which co-migrated with mammalian KIF3A and displayed ATP-dependent release from rod cytoskeletons. Immunofluorescence localizations with anti-LAGSE and anti-Klp1 head antibodies detected epitopes in the axoneme and ellipsoid, and immunoelectron microscopy with the LAGSE antibody showed that the connecting cilium region was particularly antigenic. Immunofluorescence with anti-KIF3A showed prominent labelling of the connecting cilium and the area surrounding its basal body; the outer segment axoneme and parts of the inner segment coincident with microtubules were also labelled. We propose that these putative kinesin superfamily proteins may be involved in the translocation of material between the rod inner and outer segments.
The vacuolar proton pump of Dictyostelium discoideum: Molecular cloning and analysis of the 100 kDa subunit
Authors: Liu Tongyao , Clarke Margaret 
Published: April 2021

ABSTRACT
The vacuolar proton pump is a highlyconserved multimeric enzyme that catalyzes the translocation of protons across the membranes of eukaryotic cells. Its largest subunit (95-116 kDa) occurs in tissue and organellespecific isoforms and thus may be involved in targeting the enyzme or modulating its function. In amoebae of Dictyostelium discoideum, proton pumps with a 100 kDa subunit are found in membranes of the contractile vacuole complex, an osmoregulatory organelle. We cloned the cDNA that encodes this 100 kDa protein and found that its sequence predicts a protein 45% identical (68% similar) to the corresponding mammalian proton pump subunit. Like the mammalian protein, the predicted Dictyostelium sequence contains six possible transmembrane domains and a single consensus sequence for N-linked glycosylation. Southern blot analysis detected only a single gene, which was designated vatM. Using genomic DNA and degenerate oligonucleotides based on conserved regions of the protein as primers, we generated products by polymerase chain reaction that included highly variable regions of this protein family. The cloned products were identical in nucleotide sequence to vatM, arguing that Dictyostelium cells contain only a single isoform of this proton pump subunit. Consistent with this interpretation, the amino acid sequences of peptides derived from a protein associated with endosomal membranes (Adessu et al. (1995) J. Cell Sci. 108, 3331-3337) match the predicted sequence of the protein encoded by vatM. Thus, a single isoform of the 100 kDa proton pump subunit appears to serve in both the contractile vacuole system and the endosomal/lysosomal system of Dictyostelium, arguing that this subunit is not responsible for regulating the differing abundance and function of proton pumps in these two compartments. Gene targeting experiments suggest that this subunit plays important (possibly essential) roles in Dictyostelium cells.
SCO-spondin: a new member of the thrombospondin family secreted by the subcommissural organ is a candidate in the modulation of neuronal aggregation
Authors: Gobron Stéphane , Monnerie Hubert , Meiniel Robert , Creveaux Isabelle , Lehmann Werner , Lamalle David , Dastugue Bernard , Meiniel Annie 
Published: April 2021

ABSTRACT
A number of cues are known to influence neuronal devel­opment including growth factors, cell-adhesion molecules, components of the extracellular matrix and guidance molecules. In this study, we present molecular and func­tional evidence that SCO-spondin, a novel relative of the thrombospondin family, could also be involved in neuronal development by modulating cell aggregative mechanisms. SCO-spondin corresponds to glycoproteins secreted by the subcommissural organ (SCO), an ependymal differen­tiation of the vertebrate brain located at the entrance to the Sylvian aqueduct. A cDNA clone of 2.6 kb, isolated from a bovine SCO cDNA library, was shown to be specifically and highly expressed in the bovine SCO by in situ hybridization and was subsequently sequenced. Analysis of the deduced amino acid sequence reveals the presence of four conserved domains known as thrombospondin (TSP) type I repeats. To account for the homology with thrombospondins and F- spondin, this secreted glycoprotein was called SCO- spondin. Two potent binding sites to glycosaminoglycan (BBXB) and to cytokine (TXWSXWS) are also found in the TSP type I repeats. The deduced amino acid sequence exhibits three other conserved domains called low density lipoprotein (LDL) receptor type A repeats. The possibility of SCO-spondin involvement in neuronal development as a component of the extracellular matrix is discussed regarding these molecular features. The idea of a modula­tion of cell-cell and/or cell-matrix interaction is further supported by the anti-aggregative effect observed on cultured neuronal cells of material solubilized from Reissner’s fiber. That Reissner’s fiber, the condensed secretory product of the SCO present along the whole spinal cord can be a potent morphogenetical structure is an important concept for the analysis of the molecular mech­anisms leading to spinal cord differentiation.
Activation of the Xenopuscyclin degradation machinery by full-length cyclin A
Authors: Jones Craig , Smythe Carl 
Published: April 2021

ABSTRACT
The entry into mitosis is dependent on the activation of mitotic forms of cdc2 kinase. In many cell types, cyclin A-associated kinase activity peaks just prior to that of cyclin B, although the precise role of cyclin A-associated kinase in the entry into mitosis is still unclear. Previous work has suggested that while cyclin B is capable of triggering cyclin destruction in Xenopus cellfree systems, cyclin A-associated kinase is not able to support this function. Here we have expressed a fulllength human cyclin A in Escherichia coli and purified the protein to homogeneity by virtue of an N-terminal histidine tag. We have found that when added to Xenopus cellfree extracts free of cyclin B and incapable of protein synthesis, the temporal pattern of cyclin A-associated cdc2 kinase activity showed distinct differences that were dependent on the concentration of cyclin A added. When cyclin A was added to a concentration that generated levels of cdc2 kinase activity capable of inducing nuclear envelope breakdown, the histone H1 kinase activity profile was biphasic, consisting of an activation phase followed by an inactivation phase. Inactivation was found to be due to cyclin destruction, which was prevented by mos protein.
Cyclin destruction was followed by nuclear reassembly and an additional round of DNA replication, indicating that there is no protein synthesis requirement for DNA replication in this embryonic system. It has been suggested that the evolutionary recruitment of cyclin A into an S phase function may have necessitated the loss of an original mitotic ability to activate the cyclin destruction pathway. The results presented here indicate that cyclin A has not lost the ability to activate its own destruction and that cyclin A-mediated activation of the cyclin destruction pathway permitted destruction of cyclin B1 as well as cyclin A, indicating that there are not distinct cyclin A and cyclin B destruction pathways. Thus the ordered progression of the cell cycle requires the careful titration of cyclin A concentration in order to avoid activation of the cyclin destruction pathway before sufficient active cyclin B/cdc2 kinase has accumulated.
Meiotic spindle organization in fertilized Drosophila oocyte: Presence of centrosomal components in the meiotic apparatus
Authors: Riparbelli Maria Giovanna , Callaini Giuliano 
Published: April 2021

ABSTRACT
We examined spindle reorganization during the completion of meiosis in fertilized and unfertilized oocytes of Drosophila using indirect immunofluorescence and laser scanning confocal microscopy. The results defined a complex pathway of spindle assembly during resumption of meiosis, and revealed a transient array of microtubules radiating from the equatorial region of the spindle towards discrete foci in the egg cortex. A monastral array of microtubules was observed between twin metaphase II spindles in fertilized and unfertilized eggs. These microtubules originated from diskshaped material stained with Rb188 antibody specific for an antigen associated with the centrosome of Drosophila embryos. The Drosophila egg, therefore, contains a maternal pool of centrosomal components undetectable in mature inactivated oocytes. These components nucleate microtubules in a monastral array after activation, but are unable to organize bipolar spindles.
Integrin dynamics on the tail region of migrating fibroblasts
Authors: Palecek Sean P. , Schmidt Christine E. , Lauffenburger Douglas A. , Horwitz Alan F. 
Published: April 2021

ABSTRACT
Cell migration is a complex process that can be considered as a repeated cycle of lamellipod extension and attachment, cytoskeletal contraction, and tail detachment. While lamellipodial and cytoskeletal phenomena are currently the focus of considerable research on cell migration, under many conditions locomotion appears to be ratelimited by events at the cell rear, especially release of cell/substratum adhesions. To study the mechanism of tail detachment, we have developed a novel experimental system that permits observation of integrin dynamics on the ventral surface of migrating fibroblasts. Photoactivatable caged fluorescein is coupled to a nonadhesionperturbing antiaviaN-β1 integrin subunit antibody, which labels integrins on chicken fibroblasts migrating on a laminincoated glass coverslip. Ultraviolet light is focused through a pinhole to photoactivate the caged fluorophore in a 10-µmdiameter spot at the rear of a polarized cell. The fate of integrins initially present in this spot is monitored using a cooled CCD camera to follow the movement of fluorescent intensity as a function of time over a 2 to 3 hour period.
We find that a substantial fraction of the integrins is left behind on the substratum as the cell detaches and locomotes, while another fraction collects into vesicles which are transported along the cell body as the cell migrates. As aggregates rip from the cell membrane, the integrincytoskeletal bonds are preferentially fractured resulting in 81±15% of the integrin remaining attached to the substratum. We additionally find that adhesions sometimes disperse into integrins which can form new adhesions at other locations in the cell. Adhesions along the cell edge can release from the substrate and translocate with the cell. They either disperse in the cell membrane, rip from the cell membrane and remain attached to the substratum, or form a new aggregate. These observations indicate that the behavior of integrins at the cell rear is much more dynamic than previously appreciated, suggesting that an important locus for regulation of motility may reside in this region.
A high molecular mass non-muscle tropomyosin isoform stimulates retrograde organelle transport
Authors: Pelham Robert J. , Lin Jim Jung-Ching , Wang Yuli 
Published: April 2021

ABSTRACT
Although nonmuscle tropomyosins (TM) have been implicated in various cellular functions, such as stabilization of actin filaments and possibly regulation of organelle transport, their physiological role is still poorly understood. We have probed the role of a high molecular mass isoform of human fibroblast TM, hTM3, in regulating organelle transport by microinjecting an excess amount of bacteriallyexpressed protein into normal rat kidney (NRK) epithelial cells. The microinjection induced the dramatic retrograde translocation of organelles into the perinuclear area. Microinjection of hTM5, a low molecular mass isoform, had no effect on organelle distribution. Fluorescent staining indicated that hTM3 injection stimulated the retrograde movement of both mitochondria and lysosomes. Moreover, both myosin I and cytoplasmic dynein were found to redistribute with the translocated organelles to the perinuclear area, indicating that these organelles were able to move along both microtubules and actin filaments. The involvement of microtubules was further suggested by the partial inhibiton of hTM3-induced organelle movement by the microtubuledepolymerizing drug nocodazole. Our results, along with previous genetic and antibody microinjection studies, suggest that hTM3 may be involved in the regulation of organelle transport.
Cell-free assembly of rough and smooth endoplasmic reticulum
Authors: Lavoie Christine , Lanoix Joël , Kan Frederick W. K. , Paiement Jacques 
Published: April 2021

ABSTRACT
Smooth endoplasmic reticulum assembly was studied in a cell-free system using thin-section and freeze-fracture electron microscopy. Incubation of rat hepatocyte rough and smooth microsomes in the presence of ATP, GTP, cytosol (Xenopus egg) and an ATP-regenerating system led to assembly of membrane networks comprising a central core of interconnecting smooth tubules continuous with peripherally located rough membrane cisternae. Glucose-6-phosphatase cytochemistry confirmed the endoplasmic reticulum origin of the reconstituted membranes. When both ATP and GTP were omitted from the incubation medium, or when GTP was replaced by a variety of nucleotide analogues, including GTPγS, membrane aggregates contained only unfused microsomes. The presence of GTP alone stimulated assembly of rough membrane cisternae but had no effect on smooth membranes. Smooth tubule formation occurred independent of cytosol and an ATP-regenerating system, but did require GTP and ATP.
Omission of ATP, or replacement of this nucleotide with a variety of analogues, including ATPγS, prevented tubule formation but did not affect the assembly of the rough membrane cisternae. Morphometric studies revealed sequential formation of rough membrane cisternae (0-60 minutes) followed by appearance of interconnecting smooth tubules (&gt;60 minutes). The amount of rough membrane cisternae per membrane network diminished with time after 60 minutes; that of smooth tubules increased. Thus GTP is required for reconstitution of rough membrane cisternae, both GTP and ATP are required for smooth tubule formation, and assembly of smooth tubules occurs as an outgrowth (i.e. via tubulation) from rough membranes.
The chicken lysozyme gene 5′ MAR and the Drosophila histone SAR are electroelutable from encapsulated and digested nuclei
Authors: Hempel Katrin , Strätling Wolf H. 
Published: April 2021

ABSTRACT
Cultured chicken cells were encapsulated in agarose microbeads, lysed in a near-physiological buffer and resulting encapsulated nuclei were digested with a restriction enzyme and electroeluted. After removal of ∼97% of the chromatin, the nuclear lamina, residual nucleoli and an internal nuclear network remained. The majority of nascent RNA was also recovered in digested and electroe-luted nuclei. Surprisingly, however, the chicken lysozyme gene 5′ MAR was quantitatively electroeluted from digested nuclei of expressing and non-expressing cells, as well as the promoter region and the coding sequence. When encapsulated nuclei were digested partially, the proportion of elutable 5′ MAR chromatin was comparable to that of elutable bulk chromatin. Furthermore, after digestion of encapsulated nuclei from Drosophila Kc cells, the histone SAR was electroeluted to the same extent as bulk chromatin. We conclude that the lysozyme gene 5′ MAR and the histone SAR are not permanently attached to a nuclear matrix or scaffold.
A cdc2-like kinase distinct from cdk5 is associated with neurofilaments
Authors: Starr Robyn , Hall Frederick L. , Monteiro Mervyn J. 
Published: April 2021

ABSTRACT
An immunoprecipitation assay was used to identify protein kinases which are physically associated with neurofilaments (NF) in mouse brain extracts. Using this approach, we show that a cdc2-related kinase is associated with NF. The cdc2-related kinase was found to be distinct from cdk5 and the authentic cdc2 by a number of criteria. Firstly, it has a molecular mass on SDS-PAGE gels of 34 kDa, similar to that of cdc2, but differing from cdk5 (31 kDa). Secondly, it is not recognized by an antibody specific for cdk5. Thirdly, it is recognized by an antibody raised against the C-terminal region of authentic cdc2, but not by an antibody specific for the PSTAIRE motif. Using immunoblotting, we further show that the cdc2-related kinase copurifies with NF isolated from rat tissues. In vitro kinase assays further demonstrated that immunoprecipitated cdc2-related kinase phosphorylates recombinant NF-H protein. Phos-phorylation of NF-H by the cdc2-like activity was not affected by 3 μM olomoucine but was inhibited by 10 μM of this kinase inhibitor. Phosphoamino acid analysis of in vitro phosphorylated NF-H indicates that the immunoprecipitated cdc2-related kinase phosphorylates serine residues.
Transport pathway, maturation, and targetting of the vesicular stomatitis virus glycoprotein in skeletal muscle fibers
Authors: Rahkila Paavo , Alakangas Anne , Väänänen Kalervo , Metsikkö Kalervo 
Published: April 2021

ABSTRACT
We have infected isolated skeletal muscle fibers with the vesicular stomatitis virus or the mutant tsO45, whose glycoprotein is blocked in the endoplasmic reticulum at 39°C. Immunofluorescence analysis for the viral glycoprotein indicated that the fibers were infected over their entire length at a virus dose of 109/ml. When we infected the myofibers with the tsO45 mutant at 39°C, the viral glyco-protein appeared to be localised to the terminal cisternae of the sarcoplasmic reticulum. Upon shifting the cultures to the permissive temperature, 32°C, in the presence of dinitrophenol, which blocks vesicular transport, the viral glycoprotein proceeded to completely fill the sarcoplasmic reticulum. Thus, both the endoplasmic reticulum located at the terminal cisternae of the sarcoplasmic reticulum, and the entire endoplasmic and sarcoplasmic reticulum appeared to be continuous. Shifting the culture temperature from 39°C to 20°C, resulted in prominent perinuclear staining throughout the fibers, accompanied by the appearance of distinct bright dots between the nuclei. Electron microscopic immunoperoxidase labeling indicated that these bright structures represented the Golgi apparatus. When either the tsO45-infected or wild-type virus-infected fibers were incubated at 32°C, the viral glycoprotein showed a staining pattern that consisted of double rows of punctate fluorescence. Immunogold labeling showed that the viral glycoprotein was present in both the transverse tubules as well as the endoplasmic/sarcoplasmic reticulum endomembranes. In addition, extensive viral budding was observed in the transverse tubules. Metabolic labeling experiments revealed that only half of the glycoprotein was processed in the Golgi, and this processed form had become incorporated into the budding viral particles. Thus, the processed viral glycoprotein was targeted to the transverse tubules. The other half of the glycoprotein remained endo-glycosidase H-sensitive, suggesting its retention in the endo-plasmic/sarcoplasmic reticulum endomembranes.