bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of moesin in the sea urchin Lytechinus variegatus: redistribution to the plasma membrane following fertilization is inhibited by cytochalasin B
Authors: Bachman Eric Scott , McClay David R. 
Published: April 2021

ABSTRACT
We have investigated the distribution and function of an ezrin-radixin-moesin-like (ERM) molecule in the sea urchin. A sea urchin homologue of moesin was cloned that shares 75% amino acid similarity in the conserved N-terminal region to other moesin molecules. A 6.3 kb message is transcribed late in embryogenesis and is present in adult tissues. Polyclonal antibodies were generated to proteins expressed by a bacterial expression vector, and affinity purified. These antibodies recognize a single 75 kDa protein that is present throughout development in approximately equal abundance, and specifically they immuno-precipitate a single protein. We show by immunolocalization that SUmoesin has two predominant patterns during development. First, SUmoesin is rapidly redistributed after fertilization from a location throughout the egg cytoplasm to a location in the egg cortex. Later in embryogenesis, SUmoesin is localized to the apical ends of cells in the regions of cell-cell junctions. We show that SUmoesin is present in actin-rich regions of the embryo. Finally, we show that the location of SUmoesin requires an intact actin-based cytoskeleton. SUmoesin fails to localize to the plasma membrane after fertilization in the presence of cytocha-lasin B. Furthermore, SUmoesin loses its apical position in the region of cell-cell junctions in the presence of cytocha-lasin B in later stages of embryogenesis. This effect is reversible, and the microtubule inhibitor colchicine has no effect. These results show that SUmoesin becomes associated with apical plasma membrane structures early in development, and that SUmoesin is both coincident with actin and requires the assembly of actin filmaments to maintain its localization.
Molecular analysis of a cytoplasmic dynein light intermediate chain reveals homology to a family of ATPases
Authors: Hughes Sharon M. , Vaughan Kevin T. , Herskovits Jonathan S. , Vallee Richard B. 
Published: April 2021

ABSTRACT
Cytoplasmic dynein is a multi-subunit complex involved in retrograde organelle transport and some aspects of mitosis. In previous work we have cloned and sequenced cDNAs encoding the rat cytoplasmic dynein heavy and intermedi-ate chains. Here we report the cloning of the remaining class of cytoplasmic dynein subunits, which we refer to as the light intermediate chains (LICs: 53-59 kDa). Four LIC electrophoretic bands were resolved in purified bovine cytoplasmic dynein preparations by one-dimensional gel electrophoresis. These four bands were simplified to two bands (LIC53/55 and LIC57/59) by alkaline phosphatase treatment. N-terminal amino acid sequence was obtained from a total of 11 proteolytic peptides generated from both LIC53/55 and LIC57/59. Overlapping cDNA clones encoding LIC53/55 were isolated by oligonucleotide screening using probes based on the LIC53/55 peptide sequence. The cDNA sequence contained a 497 codon open reading frame encoding a polypeptide with a molecular mass of ∼55 kDa. Each of the LIC53/55 peptides was found within the deduced amino acid sequence, as well as four of the LIC57/59 peptides. Analysis of the LIC53/55 primary sequence revealed homology with the ABC transporter family of ATPases in the region surrounding the P-loop sequence element. Together these data identify the LICs as a novel family of dynein subunits with potential ATPase activity. They also reveal that the complexity of the LICs is due to both post-translational modification and the existence of at least two LIC polypeptides for which we propose the names LIC-1a and LIC-2.
Interaction of chromosome-6-encoded dystrophin related protein with the extracellular matrix
Authors: Khurana Tejvir S. , Kunkel Louis M. , Frederickson Alan D. , Carbonetto Salvatore , Watkins Simon C. 
Published: April 2021

ABSTRACT
Dystrophin-related protein/utrophin is a large, cytoskeletal protein that shares significant sequence similarity with dys-trophin. Dystrophin-related protein is known to be enriched where cell-extracellular matrix contacts are well defined; however, the mechanism of dystrophin-related protein enrichment and its functional role(s) at these sites are yet to be defined. Here, we demonstrate that dystrophin-related protein is concentrated in patches of astrocyte membrane in apposition with the extracellular matrix and that the distribution of dystrophin-related protein is temporally modulated by the extracellular matrix constituent laminin. Furthermore, we demonstrate the existence of a specific biochemical association between dystrophin-related protein and laminin in astrocytes. In these astrocytes, the depletion of dystrophin-related protein by the use of antisense dystrophin-related protein oligonucleotides causes marked reduction in the formation of functional substratum-membrane attachments. Taken together, these data suggest that dystrophin-related protein may function in the generation and maintenance of regional substratum-associated membrane specializations, such as those found at the blood-brain barrier.
rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis
Authors: Deretic Dusanka , Huber Lukas A. , Ransom Nancy , Mancini Michael , Simons Kai , Papermaster David S. 
Published: April 2021

ABSTRACT
Small GTP-binding protein rab8 regulates transport from the TGN to the basolateral plasma membrane in epithelial cells and to the dendritic plasma membrane in cultured hippocampal neurons. In our approach to identify proteins involved in rhodopsin transport and sorting in retinal pho-toreceptors, we have found, using [32P]GTP overlays of 2D gel blots, that six small GTP-binding proteins are tightly bound to the post-Golgi membranes immunoisolated with a mAb to the cytoplasmic domain of frog rhodopsin. We report here that one of these proteins is rab8. About 50% of photoreceptor rab8 is membrane associated and ∼13% is tightly bound to the post-Golgi vesicles. By confocal microscopy, antibody to rab8 specifically labels calycal processes and the actin bundles of the photoreceptor inner segment that extend inward to the junctional complexes that comprise the outer limiting membrane. Anti-rab8 shows a striking periodicity of high density labeling at 1±0.12 μm intervals along the actin bundles. Rhodopsin-bearing post-Golgi membranes cluster around the base of the cilium where rab8 and actin are also co-localized, as revealed by confocal microscopy of retinal sections double labeled with anti-rab8 and phalloidin. Microfilaments have been implicated in rod outer segment (ROS) disk morpho-genesis. Our data suggest that rab6, which we have previ-ously localized to the post-Golgi compartment, and rab8 associate with the post-Golgi membranes sequentially at different stages of transport. rab8 may mediate later steps that involve interaction of transport membranes with actin filaments and may participate in microfilament-dependent ROS disk morphogenesis.
Protein storage vacuoles form de novo during pea cotyledon development
Authors: Hoh Birgit , Hinz Giselbert , Jeong Byung-Kap , Robinson David G. 
Published: April 2021

ABSTRACT
We have investigated the formation of protein storage vacuoles in peas (Pisum sativum L.) in order to determine whether this organelle arises de novo during cotyledon development. A comparison of different stages in cotyledon development indicates that soluble protease activities decline and the amounts of storage proteins and the integral membrane protein of the protein body, α-TIP, increase during seed maturation. On linear sucrose density gradients we have been able to distinguish between two separate vesicle populations: one enriched in α-TIP, and one in TIP-Ma 27, a membrane protein characteristic of vegetative vacuoles. Both vesicle populations possess, however, PPase and V-ATPase activities. Conventionally fixed cotyledonary tissue at an intermediate stage in cotyledon development reveals the presence of a complex tubular-cisternal membrane system that seems to surround the pre-existing vacuoles. The latter gradually become compressed as a result of dilation of the former membrane system. This was confirmed immunocytochemically with the TIP-Ma 27 antiserum. Deposits of the storage proteins vicilin and legumin in the lumen, and the presence of α-TIP in the membranes of the expanding membrane system provide evidence of its identity as a precursor to the protein storage vacuole.
The isolation and long-term culture of normal human endometrial epithelium and stroma: Expression of mRNAs for angiogenic polypeptides basally and on oestrogen and progesterone challenges
Authors: Zhang Lyna , Rees Margaret C. P. , Bicknell Roy 
Published: April 2021

ABSTRACT
A highly reproducible and technically straightforward technique for the isolation and long-term culture of normal human endometrial epithelial cells is described. The essential conditions for long-term culture are that the cells be seeded onto a gelatin matrix and that ‘endothelial cell growth supplement’ be present in the culture medium. Normal endometrial epithelial cells express cytokeratins and oestrogen receptors. They may be passaged five to six times without change in properties. Growth of normal endometrial epithelial cells was stimulated by 17-β;-oestradiol and epidermal growth factor. Expression of the mRNA coding for seven polypeptide angiogenic factors, by normal endometrial epithelial, stromal and three endometrial carcinoma lines, was examined. The endometrial epithelial and stromal cells express mRNA for the polypeptide angio-genic factors, basic fibroblast growth factor, vascular endothelial cell growth factor, transforming growth factor-β;1 and pleiotrophin, as well as the cytokine midkine. Expression of the mRNA for both vascular endothelial growth factor and midkine by normal endometrial epithelial cells showed a 2-fold increase on treatment with a physiological dose of 17-β;-oestradiol (1010 M) while, in contrast, the mRNA of transforming growth factor-β;1 decreased 4-fold on treatment with 17-β;-oestradiol (1010 M) and was abolished by exposure to progesterone (5×109 M). Expression of the mRNAs for angiogenic polypeptides by the endometrial carcinoma lines was more restricted.
Association of vimentin intermediate filaments with the centrosome
Authors: Trevor Katrina T. , McGuire Jacqueline G. , Leonova Elena V. 
Published: April 2021

ABSTRACT
SW-13 cells that lack cytoplasmic intermediate filaments (IFs) were stably transfected with a human vimentin cDNA expression vector. Isolated subclones displayed two prevalent patterns of vimentin distribution as observed by indirect immuno-localization: (1) cytoplasmic filaments characteristic of a vimentin IF network; and (2) a distinct, juxtanuclear focus with limited filamentous extensions. Comparative analysis of two subclones that uniquely seg-regated these patterns of vimentin organization indicated that vimentin accumulated as a perinuclear focus in cells that expressed a 4-fold lower level of the protein. The observed variation in cellular organization was not due to detectable differences in vimentin protein modification, as determined by two-dimensional gel analysis. Increasing the amount of vimentin in a low expressing clone by a secondary transfection with human or mouse vimentin cDNA resulted in well-dispersed, cytoplasmic filaments, suggesting that the distinct juxtanuclear organization of vimentin arose due to lower cellular vimentin levels.
Employing anti-γ -tubulin and anti-vimentin antibodies, dual immunofluorescence together with confocal microscopy revealed that the juxtanuclear focus of vimentin was located in the centrosomal region. Electron microscopy showed a spheroidal, filamentous structure with at least some filaments closely associated with the peri-centriolar material (PCM). Because vimentin IF organiz-ation is at least partially dependent on microtubules, the effects of nocodazole and taxol on perinuclear vimentin foci were examined. Neither drug affected the juxtanuclear localization of foci, although taxol (10 μM, 5 hours) caused a release of pericentriolar γ -tubulin from the nuclear region in 50-60% of the cells. These studies indicate that lower, in vivo, levels of vimentin fail to form extended IFs but rather are organized as a perinuclear aggregate. Moreover, the PCM of the centrosome appears to possess attachment sites for vimentin IFs.
Three microtubule-organizing centres collaborate in a mouse cochlear epithelial cell during supracellularly coordinated control of microtubule positioning
Authors: Henderson Craig G. , Tucker John B. , Mogensen Mette M. , Mackie John B. , Chaplin Mark A. , Slepecky Norma B. , Leckie Lorna M. 
Published: April 2021

ABSTRACT
Large cell surface-associated microtubule bundles that include about 3,000 microtubules assemble in certain epithelial cells called inner pillar cells in the mouse organ of Corti. Microtubule-organizing centres (MTOCs) at both ends and near the middle of each cell act in concert during control of microtubule positioning. In addition, the three cell surface-associated microtubule-organizing centres are involved in coordinating the connection of bundle microtubules to cytoskeletal components in neighbouring cells and to a basement membrane.
The precisely defined locations of the three MTOCs specify the cell surface regions where microtubule ends will finally be anchored. The MTOCs are modified as anchorage proceeds. Substantial fibrous meshworks assemble at the surface sites occupied by the MTOCs and link microtubule ends to cell junctions. This procedure also connects the microtubule bundle to cytoskeletal arrays in neighbouring cells at two of the MTOC sites, and to the basilar membrane (a substantial basement membrane) in the case of the third site. A fourth meshwork that is not positioned at a major MTOC site is involved in connecting one side of the microtubule bundle to the cytoskeletons of two other cell neighbours. The term surfoskelosome is suggested for such concentrations of specialized cytoskeletal materials and junctions at cell surface anchorages for cytoskeletal arrays.
The large microtubule bundle in each cell is composed of two closely aligned microtubule arrays. Bundle assembly begins with nucleation of microtubules by a centrosomal MTOC that is attached to the apical cell surface. These microtubules elongate downwards and the plus ends of many of them are apparently captured by a basal MTOC that is attached to the plasma membrane at the bottom of the cell. In the lower portion of the cell, the microtubule bundle also includes a basal array of microtubules but these elongate in the opposite direction. This investigation provides evidence that they extend upwards from the basal MTOC to be captured by a medial MTOC which is attached to the plasma membrane and situated near the mid-level of the cell. However, there are sub-stantial indications that the basal array’s microtubules are also nucleated by the apically situated centrosomal MTOC, but escape from it, and are translocated downwards for capture of their plus ends by the basal MTOC. If this is the case, then these microtubules continue to elongate after translocation and extend back up to the medial MTOC, which captures their minus ends.
The gene b protein localises to the surface of Leishmania major parasites in the absence of metacyclic stage lipophosphoglycan
Authors: Rangarajan Desikan , Gokool Suzanne , McCrossan Maria V. , Smith Deborah F. 
Published: April 2021

ABSTRACT
The stage specific Gene B protein (GBP) of Leishmania major localises to the surface of infective metacyclic parasites, where it associates with the developmentally regulated surface glycoconjugate, lipophosphoglycan (LPG). This association has been proposed to aid maintenance of GBP on the parasite surface. In this paper, we show that the abundance of GBP on the extracellular meta-cyclic cell surface is in the order of 100,000 copies per cell. This level of expression is comparable to that seen in the intracellular amastigote stage, in which GBP is also localised to the surface, despite the lack of metacyclic stage specific LPG. Furthermore GBP expressed from an episome in avirulent parasites, which are unable to synthesise metacyclic LPG or endogenous GBP, also localises to the parasite surface. These data demonstrate that GBP can maintain a surface localisation in the absence of meta-cyclic LPG, suggesting that it is able to associate with other glycoconjugates on the surface of infective parasites.
Freeze-fracture replica electron microscopy combined with sds digestion for cytochemical labeling of integral membrane proteins: Application to the immunogold labeling of intercellular junctional complexes
Authors: Fujimoto Kazushi 
Published: April 2021

ABSTRACT
We propose a new electron microscopic method, the sodium dodecylsulphate (SDS)-digested freeze-fracture replica labeling technique, to study the two-dimensional distribution of integral membrane proteins in cellular membranes. Unfixed tissue slices were frozen with liquid helium, freeze-fractured, and replicated in a platinum/carbon evaporator. They were digested with 2.5% SDS to solubilize unfractured membranes and cytoplasm. While the detergent dissolved unfractured membranes and cytoplasm, it did not extract fractured membrane halves. After SDS-digestion, the platinum/carbon replicas, along with attached cytoplasmic and exoplasmic membrane halves, were processed for cyto-chemical labeling, followed by electron microscopic observation. As an initial screening, we applied this technique to the immunogold labeling of intercellular junction proteins: connexins (gap junction proteins), occludin (tight junction protein), desmoglein (desmosome protein), and E-cadherin (adherens junction protein). The immunogold labeling was seen superimposed on the image of a fracture face visualized by platinum/carbon shadowing. The immunoreaction was specific, and only the structures where the proteins were expected were labeled. For instance, anti-occludin immunogold complexes were observed immediately adjacent to the tight junction strands on the protoplasmic and exoplasmic fracture faces. No significant levels of gold label were associated with non-tight-junctional regions of plasma membranes. The procedures of the SDS-digested freeze-fracture replica labeling and its potential significance are discussed.
Xenopus lamin b3 has a direct role in the assembly of a replication competent nucleus: Evidence from cell-free egg extracts
Authors: Goldberg Martin , Jenkins Hazel , Allen Terry , Whitfield William G. F. , Hutchison Christopher J. 
Published: April 2021

ABSTRACT
Xenopus egg extracts which assemble replication competent nuclei in vitro were depleted of lamin B3 using monoclonal antibody L6 5D5 linked to paramagnetic beads. After depletion, the extracts were still capable of assembling nuclei around demembranated sperm heads. Using field emission in lens scanning electron microscopy (FEISEM) we show that most nuclei assembled in lamin B3-depleted extracts have continuous nuclear envelopes and well formed nuclear pores. However, several consistent differences were observed. Most nuclei were small and only attained diameters which were half the size of controls. In a small number of nuclei, nuclear pore baskets, normally present on the inner aspect of the nuclear envelope, appeared on its outer surface. Finally, the assembly of nuclear pores was slower in lamin B3-depleted extracts, indicating a slower overall rate of nuclear envelope assembly. The results of FEISEM were confirmed using conventional TEM thin sections, where again the majority of nuclei assembled in lamin B3-depleted extracts had well formed double unit membranes containing a high density of nuclear pores. Since nuclear envelope assembly was mostly normal but slow in these nuclei, the lamin content of ‘depleted’ extracts was investigated. While lamin B3 was recovered efficiently from cytosolic and membrane fractions by our procedure, a second minor lamin isoform, which has characteristics similar to those of the somatic lamin B2, remained in the extract. Thus it is likely that this lamin is necessary for nuclear envelope assembly. However, while lamin B2 did not co-precipitate with lamin B3 during immunodepletion experiments, several protein species did specifically associate with lamin B3 on para-magnetic immunobeads. The major protein species associated with lamin B3 migrated with molecular masses of 102 kDa and 57 kDa, respectively, on one-dimensional poly-acrylamide gels. On two-dimensional O’Farrell gels the mobility of the 102 kDa protein was identical to the mobility of a major nuclear matrix protein, indicating a specific association between lamin B3 and other nuclear matrix proteins. Nuclei assembled in lamin B3-depleted extracts did not assemble a lamina, judged by indirect immunofluorescence, and failed to initiate semi-conservative DNA replication. However, by reinoculating depleted extracts with purified lamin B3, nuclear lamina assembly and DNA replication could both be rescued. Thus it seems likely that the inability of lamin-depleted extracts to assemble a replication competent nucleus is a direct consequence of a failure to assemble a lamina.
The Aspergillus nidulans bime (Blocked-In-Mitosis) gene encodes multiple cell cycle functions involved in mitotic checkpoint control and mitosis
Authors: James Steven W. , Mirabito Peter M. , Scacheri Peter C. , Morris N. Ronald 
Published: April 2021

ABSTRACT
The bimE (blocked-in-mitosis) gene appears to function as a negative mitotic regulator because the recessive bimE7 mutation can override certain interphase-arresting treatments and mutations, causing abnormal induction of mitosis. We have further investigated the role of bimE in cell cycle checkpoint control by: (1) coordinately measuring mitotic induction and DNA content of bimE7 mutant cells; and (2) analyzing epistasis relationships between bimE7 and 16 different nim mutations. A combination of cytological and flow cytometric techniques was used to show that bimE7 cells at restrictive temperature (44°C) undergo a normal, although somewhat slower cell cycle prior to mitotic arrest. Most bimE7 cells were fully reversible from restrictive temperature arrest, indicating that they are able to enter mitosis normally, and therefore require bimE function in order to finish mitosis. Furthermore, epistasis studies between bimE7 and mutations in cdc2 pathway components revealed that the induction of mitosis caused by inactivation of bimE requires functional p34cdc2 kinase, and that mitotic induction by bimE7 depends upon several other nim genes whose functions are not yet known. The involvement of bimE in S phase function and mitotic checkpoint control was suggested by three lines of evidence. First, at restrictive temperature the bimE7 mutation slowed the cell cycle by delaying the onset or execution of S phase. Second, at permissive temperature (30°C) the bimE7 mutation conferred enhanced sensitivity to the DNA synthesis inhibitor hydroxyurea. Finally, the checkpoint linking M phase to the completion of S phase was abolished when bimE7 was combined with two nim mutations that cause arrest in G1 or S phase. A model for bimE function based on these findings is presented.