bluuebunny/crossref_metadata_2025_split huggingface dataset:
High levels of actin tyrosine phosphorylation: Correlation with the dormant state of Dictyostelium spores
Authors: Kishi Yoshiro , Clements Chris , Mahadeo Dana C. , Cotter David A. , Sameshima Masazumi 
Published: April 2021

ABSTRACT
Upon removal of nutrients, the amoebae of the cellular slime mold Dictyostelium discoideum differentiate into dormant spores which survive starvation stress. In this study, we demonstrate that half of the actin molecules in the spores are tyrosine-phosphorylated. The phosphorylated actin is distributed around immobile crenate mitochondria and vesicles, as well as in the cytoplasm of the spores. The actin isolated from spore lysates contains phosphorylated and unphosphorylated forms at the same molar ratio as that of the original whole spore lysate. Under actin polymerizing conditions they form actin filaments and then they are completely depolymerized under actin depolymerizing conditions, indicating that tyrosine phosphorylation of actin may not prohibit actin polymerization nor stimulate depolymerization. The phosphorylation levels increase at the end of the culmination stage when spores have matured morphologically and physiologically, and reach maximum levels after an additional 12 hours of development. The levels are stable for 20 days following spore maturation, and decline to undetectable levels within the next 10 days.
Spores having high levels of phosphorylation show high viability, and vice versa. Following activation of spores with nutrient medium containing spore germination promoters, the phosphorylation levels quickly decrease with a half-life of about 5 minutes. After 20 minutes spores begin to swell. At this later time, most of the phosphorylated actin already has been dephosphorylated. Also, in heat-activated spores actin dephosphorylation occurs prior to spore swelling. However, addition of phosphatase inhibitors following heat-activation, prevented spore swelling and dephosphorylation of actin. Our data indicate that the high levels of actin tyrosine phosphorylation, specific to the spore stage, may be required for maintaining dormancy to withstand starvation stress. The rapid dephosphorylation of actin leads to a reactivated dynamic actin system which participates in spore swelling, vesicle movement, and mitochondrial shape changes during the spore germination process.
Anosmin-1 underlying the X chromosome-linked Kallmann syndrome is an adhesion molecule that can modulate neurite growth in a cell-type specific manner
Authors: Soussi-Yanicostas Nadia , Faivre-Sarrailh Catherine , Hardelin Jean-Pierre , Levilliers Jacqueline , Rougon Geneviève , Petit Christine 
Published: April 2021

ABSTRACT
Anosmin-1 is an extracellular matrix glycoprotein which underlies the X chromosome-linked form of Kallmann syndrome. This disease is characterized by hypogonadism due to GnRH deficiency, and a defective sense of smell related to the underdevelopment of the olfactory bulbs. This study reports that anosmin-1 is an adhesion molecule for a variety of neuronal and non-neuronal cell types in vitro. We show that cell adhesion to anosmin-1 is dependent on the presence of heparan sulfate and chondroitin sulfate glycosaminoglycans at the cell surface. A major cell adhesion site of anosmin-1 was identified in a 32 amino acid (32R1) sequence located within the first fibronectin-like type III repeat of the protein. The role of anosmin-1 as a substrate for neurite growth was tested on either coated culture dishes or monolayers of anosmin-1-producing CHO cells. In both experimental systems, anosmin-1 was shown to be a permissive substrate for the neurite growth of different types of neurons. Mouse P5 cerebellar neurons cultured on anosmin-1 coated wells developed long neurites; the 32R1 peptide was found to underly part of this neurite growth activity. When the cerebellar neurons were cultured on anosmin-1-producing CHO cells, neurite growth was reduced as compared to wild-type CHO cells; in contrast, no difference was observed for E18 hippocampal and P1 dorsal root ganglion neurons in the same experimental system. These results indicate that anosmin-1 can modulate neurite growth in a cell-type specific manner. Finally, anosmin-1 induced neurite fasciculation of P5 cerebellar neuron aggregates cultured on anosmin-1-producing CHO cells. The pathogenesis of the olfactory defect in the X-linked Kallmann syndrome is discussed in the light of the present results and the recent data reporting the immunohistochemical localisation of anosmin-1 during early embryonic development.
Messenger ribonucleoprotein complexes containing human ELAV proteins: interactions with cytoskeleton and translational apparatus
Authors: Antic Dragana , Keene Jack D. 
Published: April 2021

ABSTRACT
Mammalian ELAV proteins bind to polyadenylated messenger RNAs and have specificity for AU-rich sequences. Preferred binding sites in vitro include the AUUUA pentamer and related sequences present in the 3′ untranslated regions of many growth regulatory mRNAs. Human ELAV (hELAV) proteins have been implicated in post-transcriptional regulation of gene expression by their effects on the stability and translatability of growth regulatory mRNAs.
We have examined the intracellular localization of ELAV proteins in neurons and in tumor cells of neuronal origin using indirect immunofluorescence, confocal microscopy and biochemical separation. Mammalian neuronal ELAV proteins are found predominantly in the cytoplasm of cells in mRNP complexes termed α complexes which, when associated with polysomes, form large and high density β complexes, as assayed by glycerol and accudenz gradients, respectively. Puromycin, cytochalasin or EDTA treatments disrupt β complexes causing the release of α complexes, which then appear, by confocal microscopy, as large hELAV mRNP granules associated with microtubules. Association of partially purified hELAV mRNP α complexes with microtubules was confirmed by in vitro reconstitution assays. Furthermore, colchicine treatment of cells suggested that association of hELAV mRNP α complexes with microtubules is also necessary for the formation of β complexes.
Our data suggest a model in which a subset of mRNAs is associated with microtubules as ELAV mRNP particles (α complexes) which, in turn, associate with polysomes to form a translational apparatus (β complex) that is, through polysomes, associated with the microfilament cytoskeletal network. hELAV proteins in these mRNP granules may affect post-transcriptional regulation of gene expression via the intracellular transport, localization and/or translation of growth regulatory mRNAs.
Active nuclear pore complexes in Chironomus: visualization of transporter configurations related to mRNP export
Authors: Kiseleva Elena , Goldberg Martin W. , Allen Terence D. , Akey Christopher W. 
Published: April 2021

ABSTRACT
The Nuclear Pore Complex (NPC) regulates nucleocytoplasmic transport by providing small channels for passive diffusion and multiple docking surfaces that lead to a central translocation channel for active transport. In this study we have investigated by high resolution scanning and transmission electron microscopy the dynamics of NPC structure in salivary gland nuclei from Chironomus during Balbiani ring (BR) mRNP translocation, and present evidence of rearrangement of the transporter related to mRNP export. Analysis of the individual NPC components verified a strong evolutionary conservation of NPC structure between vertebrates and invertebrates. The transporter is an integral part of the NPC and is composed of a central short double cylinder that is retained within the inner spoke ring, and two peripheral globular assemblies which are tethered to the cytoplasmic and nucleoplasmic coaxial rings by eight conserved internal ring filaments.
Distinct stages of BR mRNP nuclear export through the individual NPC components were directly visualized and placed in a linear transport sequence. The BR mRNP first binds to the NPC basket, which forms an expanded distal basket ring. In this communication we present stages of BR mRNP transport through the nucleoplasmic, central and cytoplasmic transporter subunits, which change their conformation during mRNP translocation, and the emegence of mRNP into the cytoplasm. We propose that the reorganization of the basket may be driven, in part, by an active translocation process at the transporter. Furthermore, the images provide dramatic evidence that the transporter functions as a central translocation channel with transiently open discrete gates in its globular assemblies. A model of NPC transporter reorganization accompanied with mRNP translocation is discussed.
Truncations of the C-terminal cytoplasmic domain of MG160, a medial Golgi sialoglycoprotein, result in its partial transport to the plasma membrane and filopodia
Authors: Gonatas Jacqueline O. , Chen You-Jun , Stieber Anna , Mourelatos Zisimos , Gonatas Nicholas K. 
Published: April 2021

ABSTRACT
MG160, a type I cysteine-rich membrane sialoglycoprotein residing in the medial cisternae of the rat Golgi apparatus, is highly homologous to CFR, a fibroblast growth factor receptor, and ESL-1, an E-selectin ligand located at the cell surface of mouse myeloid cells and recently detected in the Golgi apparatus as well. The mechanism for the transport of MG160 from the Golgi apparatus to the cell surface is unknown.
In this study we found that differential processing of the carboxy-terminal cytoplasmic domain (CD), consisting of amino acids Arg1159 Ile Thr Lys Arg Val Thr Arg Glu Leu Lys Asp Arg1171, resulted in the partial transport of the protein to the plasma membrane and filopodia.
In Chinese hamster ovary cells (CHO), stably transfected with the entire cDNA encoding MG160, the protein was localized in the Golgi apparatus. However, when the terminal Arg1171 or up to nine distal amino acids were deleted, the protein was distributed to the plasma membrane and filopodia as well as the Golgi apparatus.
This report shows that the CD of an endogenous type I Golgi protein is important for its efficient retention and identifies a unique residue preference in this process. Cleavage within the CD of MG160 may constitute a regulatory mechanism for the partial export of the protein from the Golgi apparatus to the plasma membrane and filopodia.
Annexin VI defines an apical endocytic compartment in rat liver hepatocytes
Authors: Ortega David , Pol Albert , Biermer Michael , Jäckle Stefan , Enrich Carlos 
Published: April 2021

ABSTRACT
Annexin VI has been demonstrated previously to be a marker for hepatic endosomes. By western blotting with an affinity purified anti-annexin VI antibody it was shown that annexin VI was present in the three morphologically and functionally different endosomal fractions from rat liver. We have quantified the gold-labeled endosomes by immunoelectron microscopy in ultrathin Lowicryl sections of rat liver and now demonstrate that 80% of the total labeling with anti-annexin VI was associated with endocytic structures surrounding the bile canaliculus, the apical domain of hepatocytes, whereas only 20% was found in the subsinusoidal endosomes. In double immuno-gold labeling experiments 80% of the Rab5 positive apical endosomes were also labeled with anti-annexin VI antibodies. However, there was no significant colocalization with antibodies to the polymeric immunoglobulin receptor. Finally, we demonstrate that 50% of endosomes containing internalized gold-labeled transferrin were double labeled with anti-annexin VI antibodies. Thus, annexin VI becomes the first known structural protein at the apical ‘early’ endocytic compartment of the hepatocyte that may be involved in the receptor recycling and transport to late endocytic/lysosomal compartment pathways.
An image correlation analysis of the distribution of clathrin associated adaptor protein (AP-2) at the plasma membrane
Authors: Brown Claire M. , Petersen Nils O. 
Published: April 2021

ABSTRACT
Clathrin associated adaptor protein is involved in endocytosis at the plasma membrane (AP-2) and protein sorting at the Golgi membrane (AP-1). There is a great deal of information available on the structure, function and binding characteristics of AP-2, however, there is little quantitative data on the AP-2 distribution at the membrane. Image correlation spectroscopy is a technique which yields number counts from an autocorrelation analysis of intensity fluctuations within confocal microscopy images. Image correlation spectroscopy analysis of the indirect immunofluorescence from AP-2 at the plasma membrane of CV-1 cells shows that AP-2 is in a bimodal distribution consisting of large coated pit associated aggregates of ∼60 AP-2 molecules, and smaller aggregates containing ∼20 AP-2 molecules, which we propose are coated pit nucleation sites. Following hypertonic treatment 25% of the AP-2 molecules dissociate from the large AP-2 aggregates and form AP-2 dimers, leaving the remaining AP-2 as large aggregates with ∼45 molecules. The smaller AP-2 aggregates completely dissociate forming AP-2 dimers. Dispersion of AP-2 with hypertonic treatment is not seen qualitatively because the number of large AP-2 aggregates is unchanged, the aggregates are just 25% smaller. Change in temperature from 37°C to 4°C has no affect on the number of AP-2 aggregates or the AP-2 distribution between the two populations. These data and estimates of the coated pit size suggest that coated pits cover ∼0.9% of the cell membrane. Combination of image correlation spectroscopy analysis and measurements of the CV-1 cell surface area show that there are ∼6×105 AP-2 molecules per CV-1 cell with ∼2×105 AP-2 molecules within coated pit structures.
Polarized localizations of annexins I, II, VI and XIII in epithelial cells of intestinal, hepatic and pancreatic tissues
Authors: Massey-Harroche Dominique , Mayran Nathalie , Maroux Suzanne 
Published: April 2021

ABSTRACT
The cellular and subcellular localizations of annexins I, II, VI and XIII in the rabbit intestine, liver and pancreas were studied by performing immunofluorescence labeling on thin frozen tissue sections using specific monoclonal antibodies. The expression of annexins was found to be finely regulated. Annexins XIII and I were expressed exclusively in the small intestine and the colon, respectively, whereas annexin II was present in all the tissues tested and annexin VI specifically in the liver and pancreas. These different annexins were concentrated in the basolateral domain of polarized cells, and some of them had an extra-apical localization: annexin XIII was concentrated in the lower 3/4 of enterocyte brush border microvilli; annexin II was present in the upper part of the terminal web in intestinal absorbent cells as well as in the bile canalicular area in hepatocytes, whereas annexin VI was detected on some apical vesicles concentrated around the bile canaliculi. In pancreatic acinar cells, the presence of annexin II on some zymogen granules provides further evidence that annexin II may be involved in exocytic events. In conclusion, this study shows that the basolateral domain of polarized cells appears to be the main site where annexins are located, and they may therefore be involved in the important cellular events occurring at this level.
Centrosome dynamics in early embryos of Caenorhabditis elegans
Authors: Keating Heather H. , White John G. 
Published: April 2021

ABSTRACT
The early Caenorhabditis elegans embryo divides with a stereotyped pattern of cleavages to produce cells that vary in developmental potential. Differences in cleavage plane orientation arise between the anterior and posterior cells of the 2-cell embryo as a result of asymmetries in centrosome positioning. Mechanisms that position centrosomes are thought to involve interactions between microtubules and the cortex, however, these mechanisms remain poorly defined. Interestingly, in the early embryo the shape of the centrosome predicts its subsequent movement. We have used rhodamine-tubulin and live imaging techniques to study the development of asymmetries in centrosome morphology and positioning. In contrast to studies using fixed embryos, our images provide a detailed characterization of the dynamics of centrosome flattening. In addition, our observations of centrosome behavior in vivo challenge previous assumptions regarding centrosome separation by illustrating that centrosome flattening and daughter centrosome separation are distinct processes, and by revealing that nascent daughter centrosomes may become separated from the nucleus. Finally, we provide evidence that the midbody specifies a region of the cortex that directs rotational alignment of the centrosome-nucleus complex and that the process is likely to involve multiple interactions between microtubules and the cortex; the process of alignment involves oscillations and overshoots, suggesting a multiplicity of cortical sites that interact with microtubules.
VE-cadherin and desmoplakin are assembled into dermal microvascular endothelial intercellular junctions: A pivotal role for plakoglobin in the recruitment of desmoplakin to intercellular junctions
Authors: Kowalczyk Andrew P. , Navarro Pilar , Dejana Elisabetta , Bornslaeger Elayne A. , Green Kathleen J. , Kopp Daniel S. , Borgwardt Jeffrey E. 
Published: April 2021

ABSTRACT
Vascular endothelial cells assemble adhesive intercellular junctions comprising a unique cadherin, VE-cadherin, which is coupled to the actin cytoskeleton through cytoplasmic interactions with plakoglobin, β-catenin and α-catenin. However, the potential linkage between VE-cadherin and the vimentin intermediate filament cytoskeleton is not well characterized. Recent evidence indicates that lymphatic and vascular endothelial cells express desmoplakin, a cytoplasmic desmosomal protein that attaches intermediate filaments to the plasma membrane in epithelial cells. In the present study, desmoplakin was localized to intercellular junctions in human dermal microvascular endothelial cells. To determine if VE-cadherin could associate with desmoplakin, VE-cadherin, plakoglobin, and a desmoplakin amino-terminal polypeptide (DP-NTP) were co-expressed in L-cell fibroblasts. In the presence of VE-cadherin, both plakoglobin and DP-NTP were recruited to cell-cell borders. Interestingly, β-catenin could not substitute for plakoglobin in the recruitment of DP-NTP to cell borders, and DP-NTP bound to plakoglobin but not β-catenin in the yeast two-hybrid system. In addition, DP-NTP colocalized at cell-cell borders with α-catenin in the L-cell lines, and endogenous desmoplakin and α-catenin colocalized in cultured dermal microvascular endothelial cells. This is in striking contrast to epithelial cells, where desmoplakin and α-catenin are restricted to desmosomes and adherens junctions, respectively. These results suggest that endothelial cells assemble unique junctional complexes that couple VE-cadherin to both the actin and intermediate filament cytoskeleton.
Uptake by COPI-coated vesicles of both anterograde and retrograde cargo is inhibited by GTPγS in vitro
Authors: Nickel Walter , Malsam Jörg , Gorgas Karin , Ravazzola Mariella , Jenne Nicole , Helms J. Bernd , Wieland Felix T. 
Published: April 2021

ABSTRACT
On the basis of the cell surface protein CD8 we have constructed reporter molecules for both anterograde and retrograde transport from the Golgi complex. The cytoplasmic tail of CD8 was exchanged by a construct comprising a hemagglutinin (HA) epitope, the C-terminal sequence of the viral protein E19 (containing a KKXX retrieval signal) followed by a myc epitope (CD8-LT). Due to this masking of the KKXX retrieval signal CD8-LT is transported to the cell surface. Since the KKXX motif is joined to the myc epitope via a thrombin cleavage site, CD8-LT in isolated Golgi membranes can be proteolytically converted into an unmasked reporter molecule for retrograde transport (CD8-ST) in vitro. A CHO cell line stably expressing CD8-LT was generated and used for the isolation of Golgi membranes. These membranes were shown to contain CD8-LT en route to the cell surface. By addition of thrombin, CD8-LT could be efficiently converted into CD8-ST, and this allows us to study the sorting into coat protein COPI-coated vesicles of these different kinds of cargo on a comparative basis. COPI-coated vesicles were generated in vitro from Golgi membranes containing either CD8-LT or CD8-ST. When the incubation was performed in the presence of GTP, both CD8-LT and CD8-ST were packaged into COPI-coated vesicles. However, COPI-coated vesicles generated in the presence of the slowly hydrolyzable analogue of GTP, GTPγS contained strikingly lower amounts of CD8-LT and CD8-ST. While COPI-coated vesicles accumulated about 12-fold in the presence of GTPγS these vesicles together contained only one fifth of cargo compared to the few vesicles generated in the absence of GTPγS. These data indicate that cargo packaging into COPI-coated vesicles requires hydrolysis of GTP.
Cdc18p can block mitosis by two independent mechanisms
Authors: Greenwood Emma , Nishitani Hideo , Nurse Paul 
Published: April 2021

ABSTRACT
The DNA replication checkpoint is required to maintain the integrity of the genome, inhibiting mitosis until S phase has been successfully completed. The checkpoint preventing premature mitosis in Schizosaccharomyces pombe relies on phosphorylation of the tyrosine-15 residue on cdc2p to prevent its activation and hence mitosis. The cdc18 gene is essential for both generating the DNA replication checkpoint and the initiation of S phase, thus providing a key role for the overall control and coordination of the cell cycle. We show that the C terminus of the protein is capable of both initiating DNA replication and the checkpoint function of cdc18p. The C terminus of cdc18p acts upstream of the DNA replication checkpoint genes rad1, rad3, rad9, rad17, hus1 and cut5 and requires the wee1p/mik1p tyrosine kinases to block mitosis. The N terminus of cdc18p can also block mitosis but does so in the absence of the DNA replication checkpoint genes and the wee1p/mik1p kinases therefore acting downstream of these genes. Because the N terminus of cdc18p associates with cdc2p in vivo, we suggest that by binding the cdc2p/cdc13p mitotic kinase directly, it exerts an effect independently of the normal checkpoint control, probably in an unphysiological manner.
Protein synthesizing units in presynaptic and postsynaptic domains of squid neurons
Authors: Martin Rainer , Vaida Bianca , Bleher Reiner , Crispino Marianna , Giuditta Antonio 
Published: April 2021

ABSTRACT
Putative protein synthesizing domains, called plaques, are characterized in the squid giant synapse and axon and in terminals of squid photoreceptor neurons. Plaques are oval-shaped formations of about 1 μm in size, which (1) generate signals that have spectroscopic electron energy loss characteristics of ribosomes, (2) exhibit ribonuclease-sensitive binding of YOYO-1, a fluorescent RNA/DNA dye, and (3) in part hybridize with a poly(dT) oligonucleotide. In the giant synapse plaques are abundant in the postsynaptic area, but are absent in the presynaptic terminal. In the cortical layer of the optic lobes, plaques are localized in the large carrot-shaped presynaptic terminals of photoreceptor neurons, where they are surrounded by synaptic vesicles and mitochondria.
Biochemical and autoradiographic data have documented that the protein synthetic activity of squid optic lobe synaptosomes is largely due to the presynaptic terminals of the photoreceptor neurons. The identification of ribosomes and poly(A+)-mRNA in the plaques indicates that these structures are sites of local protein synthesis in synaptic domains.