bluuebunny/crossref_metadata_2025_split huggingface dataset:
Import of proteins into the trypanosome nucleus and their distribution at karyokinesis
Authors: Marchetti Maria A. , Tschudi Christian , Kwon Helen , Wolin Sandra L. , Ullu Elisabetta 
Published: April 2021

ABSTRACT
In all eukaryotic organisms proteins are targeted to the nucleus via a receptor-mediated mechanism that requires a specific nuclear localization sequence (NLS) in the protein. Little is known about this process in trypanosomatid protozoa that are considered amongst the earliest divergent eukaryotes. We have used the green fluorescent protein (gfp) and β-galactosidase reporters to identify the NLS of two trypanosomal proteins, namely the Trypanosoma brucei La protein homologue and histone H2B of T. cruzi. A monopartite NLS was demonstrated at the C terminus of the La protein, whereas a bipartite NLS was identified within the first 40 amino acids of histone H2B. Treatment of live trypanosomes with poisons of ATP synthesis resulted in exit of the La NLS-gfp fusion from the nucleus. Interestingly, this fusion protein accumulated at several discrete sites in the cytoplasm, rather than equilibrating between the nucleus and the cytoplasm. When ATP levels returned to normal, the protein reentered the nucleus, demonstrating that the process was energy dependent. Finally, using fusion proteins that localize to the nucleoplasm or the nucleolus, we identified a subpopulation of mitotic cells in which the chromosomes have segregated but the daughter nuclei remain connected by a thin thread-like structure. We propose that cells containing this structure represent a late stage in nuclear division that can be placed after chromosome segregation, but before completion of karyokinesis.
VEGF nuclear accumulation correlates with phenotypical changes in endothelial cells
Authors: Li Wenlu , Keller Gilbert-A. 
Published: April 2021

ABSTRACT
Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a prominent role in normal vascular biology and pathology. In an experimental wound model, the mechanical disruption of monolayers of cultured endothelial cells resulted in two phenotypically distinct cell subpopulations in which VEGF was internalized by alternative endocytotic pathways and delivered to different subcellular compartments. In the cells away from the wound, VEGF was internalized via the classical receptor-mediated endocytosis pathway and accumulated in the endosomal compartment, whereas in the cells situated at the edges of a wound, VEGF was rapidly taken up and translocated to the nucleus. VEGF internalization and subsequent nuclear accumulation only occurred for a short period of time after the wounding and was specifically abolished by antibodies that bind to the KDR binding site of VEGF.
In the cells with VEGF nuclear accumulation, the levels of wound healing related proteins, such as Factor VIII (FVIII), tissue factor (TF) and tissue plasminogen activator, rapidly and dramatically increased compared to the cells that internalized VEGF via the classical endocytotic pathway. The increase in FVIII and TF was abolished when the nuclear transport is blocked. These data suggest that nuclear VEGF accumulation may be involved in modulating the levels of the proteins of the coagulation and fibrinolysis pathways.
Cadherin repertoire determines partner-specific gap junctional communication during melanoma progression
Authors: Hsu Mei-Yu , Andl Thomas , Li Gang , Meinkoth Judith L. , Herlyn Meenhard 
Published: April 2021

ABSTRACT
Reduced gap junction activity has long been implicated in tumorigenesis. To elucidate the potential role of intercellular communication in melanoma development, we examined gap junctional capability of melanocytic cells from various stages of tumor progression in coculture models using dye transfer assays. Normal melanocytes coupled with keratinocytes by gap junctional formation, whereas melanoma cells did not. Instead, melanoma cells communicated among themselves and with fibroblasts. This switch in communication partners coincided with a shift from E-cadherin to N-cadherin expression during melanoma development. Forced expression of E-cadherin by adenoviral gene transfer in N-cadherin-expressing melanoma cells restored gap junctional compatibility with keratinocytes. Our data suggest that (1) melanocyte transformation is associated with loss of the pre-existing gap junctional activity with keratinocytes but a concomitant gain of communication with a newly juxtaposed cell type, the fibroblasts, (2) the specificity of gap junctional formation during melanoma development is determined by the cadherin profile on the melanocytic cells and (3) the overall gap junctional activity of melanocytic cells is not reduced with transformation.
A new specialized cell-matrix interaction in actively resorbing osteoclasts
Authors: Stenbeck Gudrun , Horton Michael A. 
Published: April 2021

ABSTRACT
We have identified a novel cell-matrix interaction in activated osteoclasts. Resorbing osteoclasts maintain a barrier adjacent to the bone surface that prevents the leakage of secreted protons and proteases from the resorption area. Using a series of fluorescent dyes of known molecular mass and different surface charge we established that negatively charged molecules with Mr up to 10,000 rapidly accumulate underneath actively resorbing osteoclasts. Live cell imaging shows that staining could be detected underneath the osteoclasts as early as 30 seconds after the addition of the low molecular mass markers. We provide evidence that the actin cytoskeleton and the adhesion substrate in contact with the cells are critically involved in the maintenance of the sealing barrier. These data taken together suggest that the accumulation under resorbing osteoclasts is by diffusion rather than transcytotic delivery. Our results indicate that the net concentration of secreted and resorbed components is a balance between generation rate and limited diffusion rather than the presence of an impermeable barrier as previously suggested. This dynamic osteoclast sealing zone may, thus, provide the mechanism by which osteoclast migration and resorption can occur simultaneously.
Senescence represses the nuclear localization of the serum response factor and differentiation regulates its nuclear localization with lineage specificity
Authors: Ding Wei , Gao Sizhi , Scott Robert E. 
Published: April 2021

ABSTRACT
The differentiation of cultured 3T3T mesenchymal stem cells into adipocytes represses growth factor responsiveness by limiting the nuclear localization of the serum response factor (SRF) that binds to and activates the promoters of growth control genes that contain the serum response elements (SRE), such as junB and c-fos. The regulation of SRF nuclear localization by adipocyte differentiation is specific, because we show that adipocyte differentiation does not repress the nuclear localization of six other transacting factors. To determine if repression of growth factor responsiveness that occurs during senescence also represses the nuclear localization of SRF, we studied normal human WI-38 fibroblasts at low versus high population doublings. The results show that SRF localizes to the nucleus of proliferative cells whereas in senescent cells SRF can not be detected in the nucleus. This result is apparent in both immunofluorescence assays and in western blot analysis. We next evaluated the cellular distribution of SRF in selected human tissues to determine whether the loss of proliferative potential in vivo could have a different effect on SRF nuclear localization. We found that in cells of the small bowel mucosa, differentiation modulates SRF nuclear localization in an opposite manner. Minimal SRF expression and nuclear localization is evident in undifferentiated cells at the base of crypts whereas increased SRF expression and nuclear localization is evident in differentiated cells at the surface tip of the villus. These results together establish that regulation of SRF expression and nuclear localization is important in senescence and differentiation in a lineage specific manner.
Novel roles for mammalian septins: from vesicle trafficking to oncogenesis
Authors: Kartmann Bettina , Roth Dagmar 
Published: April 2021

ABSTRACT
In recent years a convergence of various aspects of cell biology has become apparent, and yet investigators are only beginning to grasp the underlying unifying mechanisms. Among the proteins that participate in diverse aspects of cell biology are the septins. These are a group of novel GTPase proteins that are broadly distributed in many eukaryotes except plants. Although septins were originally identified as a protein family involved in cytokinesis in yeast, recent advances in the field have now ascribed additional functions to these proteins. In particular, the number of known mammalian septin family members has increased dramatically as more data has become available through genome analyses. We suggest a classification for the mammalian septins based on the sequence homologies in their highly divergent N- and C-termini. Recent work suggests novel functions for septins in vesicle trafficking, oncogenesis and compartmentalization of the plasma membrane. Given the ability of the septins to bind GTP and phosphatidylinositol 4,5-bisphosphate in a mutually exclusive manner, these proteins might be crucial elements for the spatial and/or temporal control of diverse cellular functions. As the functions of the septins become unraveled, our understanding of seemingly different cellular processes may move a step further.
The splice variants of vascular endothelial growth factor (VEGF) and their receptors
Authors: Robinson Christopher J. , Stringer Sally E. 
Published: April 2021

ABSTRACT
Vascular endothelial growth factor (VEGF) is a secreted mitogen highly specific for cultured endothelial cells. In vivo VEGF induces microvascular permeability and plays a central role in both angiogenesis and vasculogenesis. VEGF is a promising target for therapeutic intervention in certain pathological conditions that are angiogenesis dependent, most notably the neovascularisation of growing tumours. Through alternative mRNA splicing, a single gene gives rise to several distinct isoforms of VEGF, which differ in their expression patterns as well as their biochemical and biological properties. Two VEGF receptor tyrosine kinases (VEGFRs) have been identified, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). VEGFR-2 seems to mediate almost all observed endothelial cell responses to VEGF, whereas roles for VEGFR-1 are more elusive. VEGFR-1 might act predominantly as a ligand-binding molecule, sequestering VEGF from VEGFR-2 signalling. Several isoform-specific VEGF receptors exist that modulate VEGF activity. Neuropilin-1 acts as a co-receptor for VEGF165, enhancing its binding to VEGFR-2 and its bioactivity. Heparan sulphate proteoglycans (HSPGs), as well as binding certain VEGF isoforms, interact with both VEGFR-1 and VEGFR-2. HSPGs have a wide variety of functions, such as the ability to partially restore lost function to damaged VEGF165 and thereby prolonging its biological activity.
Growth and form of secretory granules involves stepwise assembly but not differential sorting of a family of secretory proteins in Paramecium
Authors: Vayssié Laurence , Loubresse Nicole Garreau de , Sperling Linda 
Published: April 2021

ABSTRACT
Paramecium trichocysts are voluminous secretory vesicles consisting of a spindle-shaped body surmounted by a tip that serves to anchor them at exocytotic sites in the plasma membrane. This constrained shape is conferred by the proteins stored in the vesicles, which form an insoluble three-dimensional crystalline array. The constituent polypeptides (Trichocyst Matrix Proteins, TMPs), which assemble during trichocyst biogenesis, are produced by proteolytic processing of soluble proproteins encoded by a large multigene family. In order to investigate the functional significance of the TMP multigene family, which assures the synthesis of a mixture of related polypeptides, we have designed synthetic genes for heterologous expression of three different mature polypeptides, which were used to obtain sequence-specific rabbit antisera. We used these antisera to carry out immunolocalization experiments with wild-type trichocysts at different stages of development and found that the trichocyst matrix consists of two concentric layers containing different TMPs, and that the assembly of each layer corresponds to a distinct phase of trichocyst growth. Examination of mutant trichocysts created by targeted gene silencing of different TMP genes showed that the layer containing the products of the silenced genes is specifically affected, as are all subsequently assembled parts of the structure, consistent with an ordered assembly pathway. This stepwise assembly is not controlled by differential sorting of the TMPs, as single and double label experiments provided evidence that the different TMPs are delivered together to post-Golgi vesicles and developing trichocysts. We present a model for trichocyst biogenesis in which TMP assembly is controlled by protein processing.
Dissection of receptor folding and ligand-binding property with functional minireceptors of LDL receptor-related protein
Authors: Obermoeller-McCormick Lynn M. , Li Yonghe , Osaka Hiroshi , FitzGerald David J. , Schwartz Alan L. , Bu Guojun 
Published: April 2021

ABSTRACT
The LDL receptor-related protein (LRP) is a large, multifunctional endocytic receptor that binds and endocytoses a variety of structurally and functionally distinct ligands. LRP contains four putative ligand-binding domains. However, only domains II, III and IV, but not domain I, bind the receptor-associated protein (RAP), a molecular chaperone and universal antagonist for LRP. In order to dissect the function of RAP in LRP folding and to examine the ligand-binding properties of LRP, we generated LRP minireceptors that represent each of the four putative ligand-binding domains (termed mLRP1, mLRP2, mLRP3 and mLRP4, respectively). We found that proper folding and trafficking of mLRP2, mLRP3, mLRP4, but not mLRP1, is facilitated by coexpression of RAP. When these mLRPs were stably expressed in Chinese
Hamster Ovary cells that lack the endogenous LRP, we found that each of these receptors was processed and traffics through the secretory pathway. Cell surface expression of these minireceptors was quantitatively examined by flow cytometric analyses. Using these minireceptor cell lines to map the ligand-binding domains, we found that although the majority of LRP ligands bind to both domain II and domain IV, Pseudomonas exotoxin A utilizes only domain IV for its binding to LRP. We conclude that while domains II and IV of LRP share many ligand-binding properties, each of the putative ligand-binding domains of LRP is unique in its contribution to ligand binding.
Mechanism for the transcriptional repression by c-Myc on PDGF β-receptor
Authors: Izumi Hiroto , Molander Catrin , Penn Linda Z. , Ishisaki Akira , Kohno Kimitoshi , Funa Keiko 
Published: April 2021

ABSTRACT
c-Myc plays a key role in the cell cycle dependent control of the PDGF β-receptor mRNA. The mouse platelet-derived growth factor (PDGF) β-receptor promoter contains a CCAAT motif, and NF-Y plays an essential role in its transcription. Coexpression of c-Myc represses PDGF β-receptor luciferase reporter activity, and the CCAAT motif in the promoter is indispensable for this repression. Here we show that c-Myc binds NF-Y subunits, YB and YC, by immunoprecipitation from cotransfected COS-1 cells. The in vitro-translated c-Myc also binds the glutathione S-transferase (GST)-NF-YB fusion protein and GST-NF-YC, but not GST-NF-YA. The most C-terminal region of HAP domains of NF-YB and NF-YC, and the Myc homology boxes, but not the C-terminal bHLHZip domain, are indispensable for the coimmunoprecipitation, and also for the repression of PDGF β-receptor. c-Myc binds NF-Y complex without affecting the efficiency of NF-Y binding to DNA. However, the expression of Myc represses the transcriptional activation of NF-YC when fused to the GAL4 DNA binding domain. Furthermore, this repression was seen only when Myc homology boxes are present, and NF-YC contains the c-Myc binding region.
Effects of mutations in potential phosphorylation sites on transcytosis of FcRn
Authors: McCarthy Kevin M. , Lam Michael , Subramanian Laura , Shakya Reena , Wu Zhen , Newton Estelle E. , Simister Neil E. 
Published: April 2021

ABSTRACT
The neonatal Fc receptor, FcRn, transports immunoglobulin G (IgG) across intestinal epithelial cells of suckling rats and mice from the lumenal surface to the serosal surface. In cell culture models FcRn transports IgG bidirectionally, but there are differences in the mechanisms of transport in the two directions. We investigated the effects of mutations in the cytoplasmic domain of FcRn on apical to basolateral and basolateral to apical transport of Fc across rat inner medullary collecting duct (IMCD) cells. Basolateral to apical transport did not depend upon determinants in the cytoplasmic domain. In contrast, an essentially tailless FcRn was markedly impaired in apical to basolateral transport. Using truncation and substitution mutants, we identified serine-313 and serine-319 as phosphorylation sites in the cytoplasmic domain of FcRn expressed in Rat1 fibroblasts. Mutations at Ser-319 did not affect transcytosis across IMCD cells. FcRn-S313A was impaired in apical to basolateral transcytosis to the same extent as tailless FcRn, whereas FcRn-S313D transported at wild-type levels. FcRn-S313A recycled more Fc to the apical medium than the wild-type receptor, suggesting that Ser-313 is required to allow FcRn to be diverted from an apical recycling pathway to a transcytotic pathway.
Identification and characterization of a novel human plant pathogenesis-related protein that localizes to lipid-enriched microdomains in the Golgi complex
Authors: Eberle Heike B. , Serrano Ramon L. , Füllekrug Joachim , Schlosser Andreas , Lehmann Wolf D. , Lottspeich Friedrich , Kaloyanova Dora , Wieland Felix T. , Helms J. Bernd 
Published: April 2021

Group 1 of plant pathogenesis-related proteins (PR-1) and a variety of related mammalian proteins constitute a superfamily of proteins that share structural similarities. Little is known about their function, but all the family members identified to date are co-translationally translocated to the lumen of the endoplasmic reticulum and are secreted as soluble proteins or are targeted to vacuoles. Here we report the identification of a novel family member that localizes to the cytosolic site of the endomembrane system in mammalian cells. After detergent solubilization of isolated Golgi membranes, a 17 kDa protein was found associated with a low-density detergent-insoluble fraction. The amino-acid sequence, determined by microsequencing and molecular cloning, revealed a significant homology with the superfamily of PR-1 proteins. Golgi-associated PR-1 protein (GAPR-1) showed a brefeldin-A-sensitive Golgi localization in immunofluorescence. Interestingly,the protein remained associated with the microdomain fraction in the presence of Brefeldin A.By mass spectrometry, GAPR-1 was shown to be myristoylated. Immunoprecipitation of GAPR- 1 from Golgi membranes resulted in the coimmunoprecipitation of caveolin-1, indicating a direct interaction between these two proteins. Myristoylation, together with protein-protein or electrostatic interactions at physiological pH owing to the highly basic pI of GAPR-1 (pI 9.4) could explain the strong membrane association of GAPR-1.Tissue screening revealed that GAPR-1 is not detectably expressed in liver,heart or adrenal glands. High expression was found in monocytes, leukocytes,lung, spleen and embryonic tissue. Consistent with the involvement of PR-1 proteins in the plant immune system, these data could indicate that GAPR-1 is involved in the immune system.
An ultrastructural and radioautographic investigation of the nucleolonemal component of plant interphase nucleoli
Authors: Lafontaine J.-G. , Lord A. 
Published: April 2021

ABSTRACTThe interphase nucleolus in Allhim porrum meristematic cells is characterized by the presence of 1–4 dense fibrillar zones of rather complex organization. Each such zone appears to consist essentially of a convoluted, evacuolated, filamentous structure approximately 1·5 μm in diameter. At the ultrastructural level, these structures exhibit an intricate array of lacunar spaces each of which is surrounded by a dense coating. These lacunae are filled with a loose fibrillar material and the largest ones sometimes also show a dense central core. In appropriate preparations, certain of the peripherally located lacunae are found to be continuous with segments of chromosomes. High-resolution radioautography reveals, moreover, that DNA is present within both the dense and lighter portions of the nucleolar loops. These observations add further support to the hypothesis that the convoluted filamentous structures in question correspond to loops of chromosomal origin and are thus related to the nucleolar organizer.
Ultrastructural aspects of the self-incompatibility mechanism in Lycopersicum Peruvianum mill
Authors: Nettancourt D. De , Devreux M. , Bozzini A. , Cresti M. , Pacini E. , Sarfatti G. 
Published: April 2021

ABSTRACT
The experimental results obtained show that the tip of the incompatible pollen tube bursts open after the outer-wall has considerably expanded in the intercellular spaces of the conducting tissue and the inner-wall has disappeared and numerous particles have accumulated in the tube cytoplasm. These particles, which measure approximately 0·2 μm in diameter and give a weak reaction to the test of Thiéry, differ in many respects from the vesicles normally present in compatible pollen tubes growing through the style; they appear to resemble, in some cases, the spheres which are discharged by the compatible pollen tubes after they have reached the embryo-sac.
It is considered that these observations support the current belief that the tube wall is the site of action for the incompatibility proteins and suggest that self-incompatibility is not a passive process resulting from lack of growth stimulation but an active event which leads to the destruction of the incompatible pollen tubes. The degradation mechanism involved appears similar to the one which enables the compatible pollen tube to release its contents in the degenerated synergid and presents some analogies with the lytic process taking place in virus-infected cells.
The general hypothesis is presented that the particles observed in the cytoplasm of self-incompatible pollen tubes consist of a mixture of incompatibility proteins and of basic constituents of the tube wall.
Pollen wall proteins: Pollen-Stigma interactions in ragweed and Cosmos (compositae)
Authors: Knox R. B. 
Published: April 2021

ABSTRACT
The release of wall-held materials from the pollen of ragweed (Ambrosia tenuifolia) and Cosmos bipimuxtus on to the stigma surface has been followed. When fresh stigmas were viewed by scanning electron microscopy, a fluid material was observed coating pollen grains, pollen tubes and adjacent stigmatic papillae. This fluid contained pollen-wall antigens, including the allergen Antigen E, detected by immunofluorescence, and proteins, lipids and carbohydrates detected by cytochemical methods.
In Cosmos, the fate of the antigens was much the same after both compatible and incompatible matings. In incompatible matings, pollen tubes were blocked with a polysaccharide considered to be callose. Callose particles appeared on neighbouring stigmatic papillae, and adjacent stigmatic hairs accumulated callose internally. This reaction may be an important one for the incompatibility response.