bluuebunny/crossref_metadata_2025_split huggingface dataset:
The intracellular mobility of a viral membrane glycoprotein measured by confocal microscope fluorescence recovery after photobleaching
Authors: Storrie Brian , Pepperkok Rainer , Stelzer Ernst H. K. , Kreis Thomas E. 
Published: April 2021

ABSTRACT
Fluorescence recovery after photobleaching (FRAP) has been a powerful tool for characterizing the mobility of cell surface membrane proteins. However, the application of FRAP to the study of intracellular membrane proteins has been hampered by the lack of specific probes and their physical inaccessibility in the cytoplasm. We have measured the mobility of a model transmembrane protein, the temperature-sensitive vesicular stomatitis viral membrane glycoprotein (ts-O45-G), in transit from the endoplasmic reticulum (ER) to the Golgi complex. ts-O45-G accumulates in the ER at nonpermissive temperature (39.5°C) and is transported via the Golgi complex to the surface upon shifting cells to the permissive temperature (31°C). Rhodamine-labeled Fab fragments against a cyto-plasmic epitope of ts-O45-G (rh-P5D4-Fabs) were microin-jected into cells to visualize the intracellular viral membrane protein and to determine its mobility by FRAP with a confocal microscope. Moreover, we have measured the effects of microinjected antibodies against β-COP on the mobility of ts-O45-G following release of the temperature block. FRAP was essentially complete when rh-P5D4-Fab-injected cells were bleached either following release of labeled ts-O45-G from the ER or upon its accumulation at 20°C in the trans-Golgi network (TGN). In contrast, recovery was reduced by about one third when infected cells had been injected with antibodies that bind to β-COP in vivo. The diffusion constant of mobile ts-O45-G under all conditions was ∼10×10−10cm2/s. These results validate the feasibility of FRAP for the study of an intracellular transmembrane protein and provide the first evidence that such a protein is highly mobile.
Modulation of tight junction structure in blood-brain barrier endothelial cells Effects of tissue culture, second messengers and cocultured astrocytes
Authors: Wolburg Hartwig , Neuhaus Jochen , Kniesel Uwe , Krauß Bodo , Schmid Eva-Maria , Öcalan Mücella , Farrell Catherine , Risau Werner 
Published: April 2021

ABSTRACT
Tight junctions between endothelial cells of brain capillar- ies are the most important structural elements of the blood- brain barrier. Cultured brain endothelial cells are known to loose tight junction-dependent blood-brain barrier char- acteristics such as macromolecular impermeability and high electrical resistance. We have directly analyzed the structure and function of tight junctions in primary cultures of bovine brain endothelial cells using quantitative freeze-fracture electron microscopy, and ion and inulin permeability. The complexity of tight junctions, defined as the number of branch points per unit length of tight junc- tional strands, decreased 5 hours after culture but there- after remained almost constant. In contrast, the association of tight junction particles with the cytoplasmic leaflet of the endothelial membrane bilayer (P-face) decreased continu- ously with a major drop between 16 hours and 24 hours. The complexity of tight junctions could be increased by elevation of intracellular cAMP levels while phorbol esters had the opposite effect. On the other hand, the P-face asso- ciation of tight junction particles was enhanced by elevation of cAMP levels and by coculture of endothelial cells with astrocytes or exposure to astrocyte-conditioned medium. The latter effect on P-face association was induced by astrocytes but not fibroblasts. Elevation of cAMP levels together with astrocyte-conditioned medium synergistically increased transendothelial electrical resis- tance and decreased inulin permeability of primary cultures, thus confirming the effects on tight junction structure and barrier function. P-face association of tight junction particles in brain endothelial cells may therefore be a critical feature of blood-brain barrier function that can be specifically modulated by astrocytes and cAMP levels. Our results suggest an important functional role for the cytoplasmic anchorage of tight junction particles for brain endothelial barrier function in particular and probably paracellular permeability in general.
Identification and characterization of tropomodulin and tropomyosin in the adult rat lens
Authors: Woo Mary K. , Fowler Velia M. 
Published: April 2021

ABSTRACT
The lens fiber cells express all the major components of the erythrocyte membrane skeleton including spectrin, protein 4.1 and ankyrin. We have used immunoblot and immuno-precipitation analyses, as well as immunofluorescence localization to identify and characterize two additional components of the membrane skeleton in the rat lens: tropomyosin and the tropomyosin-binding protein tropomodulin. In the erythrocyte, tropomyosin and tropomodulin are proposed to stabilize and limit the lengths of the short actin filaments of the spectrin-actin network, thus influencing the organization and mechanical properties of the erythrocyte membrane skeleton.
Antibodies directed against erythrocyte tropomodulin specifically recognize a Mr43,000 polypeptide from rat lens that comigrates with erythrocyte tropomodulin on SDS-gels. A non-muscle isoform of tropomyosin is also present in the lens. This tropomyosin isoform migrates on SDS-gels with a Mrof ∼28,000 and is distinct from the two erythrocyte isoforms of tropomyosin (Mr27,000 and 29,000). Indirect immunofluorescence staining of 5 μm cryosections of adult rat lens reveals that both tropomodulin and tropomyosin colocalize with rhodamine phalloidin staining for actin filaments on fiber cell plasma membranes. Lens tropomodulin exhibits many characteristics that are similar to its erythrocyte counterpart. For example, lens tropomodulin binds tropomyosin in a solid-phase blot binding assay, and extraction experiments with Triton X-100, urea and NaOH show that the membrane-bound tropomodulin in the lens is a tightly associated peripheral membrane protein that is a component of the Triton-insoluble cytoskeleton. However, unlike the erythrocyte, there are approximately 2000 actin monomers per tropo-modulin in the lens. This differs greatly from the 16/1 ratio seen in the erythrocyte, where actin, tropomodulin and tropomyosin associate to form the short actin filaments in the membrane skeleton. In addition, we have identified a large soluble pool of actin and tropomodulin in the lens, which does not exist in erythrocytes. These differences imply the presence of distinct pools of cytoskeletal components in the lens. The membrane-bound portion of tropomodulin and tropomyosin may associate with that portion of lens actin that is associated with spectrin and protein 4.1 to form a membrane skeleton in the lens fiber cell, whereas the soluble pools of tropomodulin and actin may serve as precursors necessary for rapid assembly of the membrane skeleton during fiber cell elongation. The membrane skeleton may play a role in the maintenance of cell shape and deformability during visual accommodation as well as in the generation of plasma membrane domains during fiber cell differentiation.
Cell adhesion and gap junction formation in the early mouse embryo are induced prematurely by 6-DMAP in the absence of E-cadherin phosphorylation
Authors: Aghion Joël , Gueth-Hallonet Catherine , Antony Claude , Gros Daniel , Maro Bernard 
Published: April 2021

ABSTRACT
Compaction of the mouse embryo, which takes place at the 8-cell stage, is dependent upon the adhesion molecule E-cadherin (uvomurulin), but does not require protein synthesis, suggesting that post-translational modification(s) is (are) implicated in the setting up of this phenomenon. The demonstration recently that E-cadherin is phosphorylated at the 8-cell stage just before compaction supports this theory. In this work we used 6-dimethylaminopurine, a serine-threonine kinase inhibitor, to investigate the role of protein phosphorylation in compaction of mouse embryos. 6-dimethylaminopurine is able to induce cell flattening and gap junction formation prematurely at the 4-cell stage; however, it does not induce cell surface polarization, as occurs during normal compaction. 6-dimethylaminopurine-induced premature flattening is inhibited when the embryos are cultured in the presence of an anti-E-cadherin antibody or without extra-cellular Ca2+, demonstrating that this process requires functional E-cadherin; whereas cell flattening and gap junction formation take place in the absence of E-cadherin phosphorylation, suggesting that its phosphorylation is not required normally for these events. The relationship between E-cadherin-mediated cell flattening and gap junction formation during compaction is discussed.
Expression of neural cell adhesion molecule (N-CAM) in rat islets and its role in islet cell type segregation
Authors: Cirulli V. , Baetens D. , Rutishauser U. , Halban P. A. , Orci L. , Rouiller D. G. 
Published: April 2021

ABSTRACT
Endocrine cell types are non-randomly distributed within pancreatic islets of Langerhans. In the rat, insulin- secreting B-cells occupy the core of the islets and are sur- rounded by A-, D- and PP-cells, secreting glucagon, somatostatin and pancreatic polypeptide, respectively. Furthermore, dissociated islet cells have the ability in vitro to form aggregates with the same cell-type organization as native islets (pseudoislets). These observations suggest that a differential expression of cell adhesion molecules (CAMs) might characterize B- and non-B-cells (A-, D- and PP-cells), and be in part responsible for the establishment and main- tenance of islet architecture. Indirect immunofluorescence using antibodies against CAMs and islet hormones was performed on serial sections of the splenic and duodenal parts of the rat pancreas. Staining for the Ca2+-dependent CAM E-cadherin was detected on both exocrine and endocrine tissue and was uniform over the entire islet section, in both pancreatic regions. By contrast, staining for the Ca2+-independent neural CAM (N-CAM) was restricted to endocrine tissue and nerve endings. Further- more, N-CAM staining of endocrine cells was stronger in the islet periphery, a region composed mostly of non-B-cells. Serial sections demonstrate that cells staining strongly for N-CAM in the splenic part correspond to glucagon cells and in the duodenal part to pancreatic polypeptide cells. Within pseudoislets in vitro a stronger staining for N-CAM was also observed on peripheral cells, corresponding to non-B-cells.These data suggest that a dif- ference in the expression of N-CAM might play a role in the segregation between islet cells. To verify this hypothe- sis, isolated islet cells were allowed to form aggregates in culture, in the presence of non-immune or anti-N-CAM Fab fragments. Inhibition of N-CAM function by specific Fab fragments prevented the segregation between cell types that normally occurs within 5 days. In conclusion, higher levels of N-CAM characterize peripheral islets cells, irrespective of their secretory type, and anti-N-CAM Fab fragments prevent islet cell type segregation within pseudoislets in vitro. These results suggest that differences in N-CAM expression between cell types contribute to the characteristic distribution of cells within islets of Langer- hans.
Differential effects of glycosaminoglycans on neurite outgrowth from hippocampal and thalamic neurones
Authors: Fernaud-Espinosa I. , Nieto-Sampedro M. , Bovolenta P. 
Published: April 2021

ABSTRACT
Chondroitin sulphate proteoglycans are expressed in a temporally restricted pattern from embryonic day 17 to postnatal day 0 in both the thalamus and the cortical subplate, to which thalamic neurones transiently project. To study whether chondroitin sulphate proteoglycans could be specifically involved in the modulation of thalamic axon outgrowth, we compared neurite outgrowth from cultured rat embryonic hippocampal and thalamic neurones, in the presence of chondroitin sulphate type C (isolated from shark cartilage) and chondroitin sulphate type B (dermatan sulphate; isolated from bovine mucosa). When added to the culture medium, both types of gly- cosaminoglycan lowered the adhesion to laminin and polylysine of both hippocampal and thalamic neurones. However, only chondroitin sulphate specifically modified the pattern of thalamic but not hippocampal neurone outgrowth, promoting axon growth. The morphological changes induced by chondroitin sulphate were concentra- tion dependent and correlated with the selective binding of chondroitin sulphate to the neuronal plasma membrane and its subsequent internalisation. Chondroitin sulphate loosely bound to the surface of hippocampal neurones, but was not internalised. These results indicate that proteogly- cans, and in particular the glycosaminoglycan component of these molecules, can differentially modulate neurite outgrowth, depending on their biochemical composition and on the type of neurones they bind to; this would be a possible mechanism of controlling axon guidance in vivo.
Apoptosis is induced in post-mitotic rat sympathetic neurons by arabinosides and topoisomerase II inhibitors in the presence of NGF
Authors: Tomkins Chris E. , Edwards Susan N. , Tolkovsky Aviva M. 
Published: April 2021

ABSTRACT
Sympathetic neurons depend on nerve growth factor (NGF) for their survival and die by apoptosis when NGF is withdrawn, despite their post-mitotic state. Martin et al. (1990, J. Neurosci. 10, 184-193) showed that cytosine ara- binoside, but no other arabinofuranosyl nucleoside, could induce cell death in the presence of NGF and they suggested that it may block a critical step in the NGF-sig- nalling pathway. We show that cytosine arabinoside is not the only nucleoside capable of inducing apoptosis in sym- pathetic neurons in the presence of NGF. In newly isolated neurons from P0 rat pups cultured in the presence of NGF, all the arabinose nucleosides (adenine, cytosine, guanine and thymine) induce apoptosis at 10 μM when combined with 5-fluorodeoxyuridine treatment. Because 1-β-arabi- nofuranosylcytosine is associated with double-strand breaks and chromosomal abberrations, we examined whether topoisomerase II inhibitors, which also cause double-strand breaks by stabilising the enzyme-DNA ‘cleavable complex’, were capable of promoting apoptosis in these neurons. Although P0 rat neurons are strictly post- mitotic, topoisomerase II inhibitors teniposide and mitox- antrone induced them to die by apoptosis in the presence of NGF with the same apparent time-course as arabinose treatment or NGF withdrawal. By contrast, ICRF 193, a catalytic inhibitor of topoisomerase II, reduced the extent of apoptosis induced by mitoxantrone or teniposide by 80% if added simultaneously with the latter but by 2 hours it had no rescue effect, suggesting that topoisomerase II is highly active in these neurons. ICRF 193 also partially reduced the induction of fluorodeoxyuridine-dependent apoptosis by the arabinose nucleosides. These data suggest that induction of double-strand breaks in the DNA or NGF withdrawal activate similar apoptotic programmes in sym- pathetic neurons. We propose that treatment with FdUr results in dUTP incorporation in the neurons, that the presence of arabinose nucleosides causes disruption of the repair system that involves topoisomerase II and this causes double-strand breaks and thus apoptosis.
Kinesin-related proteins in eukaryotic flagella
Authors: Fox Laura A. , Sawin Kenneth E. , Sale Winfield S. 
Published: April 2021

ABSTRACT
To identify kinesin-related proteins that are important for ciliary and eukaryotic flagellar functions, we used affinity-purified, polyclonal antibodies to synthetic peptides corresponding to conserved sequences in the motor domain of kinesin (Sawin et al. (1992)J. Cell Sci. 101, 303-313). Using immunoblot analysis, two antibodies to distinct sequences (LNLVDLAGSE, ‘LAGSE’ and, HIPYRESKLT, ‘HIPYR’) reveal a family of proteins in flagella and axonemes isolated from Chlamydomonas. Similar analysis of axonemes from mutant Chlamydomonas strains or fractionated axonemes indicates that none of the immunoreactive proteins are associated with dynein arm or spoke structures. In contrast, one protein, ∼110 kDa, is reduced in axonemes from mutant strains defective in the central pair apparatus. Immunoreactive proteins with masses of 96 and 97 kDa (the ‘97 kDa’ proteins) are selectively solubilized from isolated axonemes in 10 mM ATP. The 97 kDa proteins co-sediment in sucrose gradients at about 9 S and bind to axonemes or purified microtubules in a nucleotidedependent fashion characteristic of kinesin. These results reveal that flagella contain kinesin-related proteins, which may be involved in axonemal central pair function and flagellar motility, or directed transport involved in morphogenesis or mating responses in Chlamydomonas.
Brush border myosin-I microinjected into cultured cells is targeted to actin- containing surface structures
Authors: Footer Matthew , Bretscher Anthony 
Published: April 2021

ABSTRACT
The isolated intestinal microvillus cytoskeleton (core) consists of four major proteins: actin, villin, fimbrin and brush border myosin-I. These proteins can assemble in vitro into structures resembling native microvillus cores. Of these components, villin and brush border myosin-I show tissue-specific expression, so they may be involved in the morphogenesis of intestinal microvilli. When intro- duced into cultured cells that normally lack the protein, villin induces a reorganization of the actin filaments to generate large surface microvilli. Here we examine the con- sequences of microinjecting brush border myosin-I either alone or together with villin into cultured fibroblasts. Injection of brush border myosin-I has no discernible effect on the overall morphology of the cells, but does become localized to either normal or villin-induced microvilli and other surface structures containing an actin cytoskeleton. Since some endogenous myosin-Is have been found associ- ated with cytoplasmic vesicles, these results show that brush border myosin-I has a domain that specifically targets it to the plasma membrane in both intestinal and cultured cell systems. Ultrastructural examination of microvilli on control cultured cells revealed that they contain a far more highly ordered bundle of microfilaments than had been previously appreciated. The actin filaments in microvilli of villin-injected cells appeared to be more tightly cross-linked when examined by thin-section electron microscopy. In intestinal microvilli, the core bundle is separated from the plasma membrane by about 30 nm due to the presence of brush border myosin-I. As this same 30 nm separation is seen in microvilli of control cells and we see more prominent links in the myosin-I-injected cells, it seems likely that cultured cells contain an immunologically distinct, but functionally similar, myosin-I that links the microfilament core bundle to the plasma membrane.
Cyclic AMP-regulated AChR assembly is independent of AChR subunit phosphorylation by PKA
Authors: Jayawickreme Shiranthi P. , Green William N. , Claudio Toni 
Published: April 2021

ABSTRACT
Forskolin treatment of cells expressing Torpedo acetyl- choline receptors leads to enhanced assembly efficiency of subunits, which correlates with increased phosphorylation of the γ subunit. To determine the role of the two potential protein kinase A sites of the γ subunit in receptor assembly, cell lines expressing different mutant receptors were estab- lished. Mouse fibroblast cell lines stably expressing wild- type Torpedo acetylcholine receptor α, β, δ subunits plus one of three γ subunit mutations (S353A, S354A, or S353,354A) were established to identify the protein kinase A phosphorylation sites of γ in vivo, and to determine if increased phosphorylation of the γ subunit leads to enhanced expression of receptors. We found that both serines (353, 354) in γ are phosphorylated in vivo by protein kinase A, however, phosphorylation of either or both of these sites does not lead to increased assembly efficiency. We established a cell line expressing α, β, and γ(S353,354A) subunits only (no δ), and found that the presence of δ (or its phosphorylation) is also not necessary for the observed stimulation by forskolin. αβγ, αγ, and βγ associations were stimulated by forskolin but αβ and αδ interactions were not. These data imply that the presence of γ is necessary for forskolin action. We postulate that forskolin may stimulate acetylcholine receptor expression through a cellular protein that is involved in the folding and/or assembly of protein complexes, and that forskolin may regulate the action of such a protein through phosphorylation.
Association of Rap1a and Rap1b proteins with late endocytic/phagocytic compartments and Rap2a with the Golgi complex
Authors: Pizon Véronique , Desjardins Michel , Bucci Cecilia , Parton Robert G. , Zerial Marino 
Published: April 2021

ABSTRACT
Among the small GTPases of the Ras family, Rap proteins exhibit the highest homology with p21Ras. The four Rap proteins so far identified constitute two subgroups, comprising the Rap1(A,B) and the Rap2(A,B) proteins. The intracellular location of Rap1A, Rap1B and Rap2A proteins was investigated in mammalian cells by confocal immunofluorescence microscopy. Using a specific anti-Rap1 affinity-purified antibody, both Rap1A and Rap1B proteins were localized to late endocytic compartments (late endosomes/lysosomes) in fibroblasts. The localization of the Rap1A and B proteins transiently overexpressed with the vaccinia T7 system was identical to that observed for endogenous Rap1 proteins. In contrast, epitope-tagged Rap2A protein colocalized with several markers of the Golgi complex, thus indicating that its site of function was distinct from that of Rap1A. In addition, morphological and subcellular fractionation studies provided evidence for the association of Rap1 proteins with phagosomes displaying biochemical features of late endocytic structures in J774 macrophages. Thus, the localization of Rap1A and Rap1B implicates their involvement in late endocytic/phagocytic processes.
Comparison of human CAP and CAP2, homologs of the yeast adenylyl cyclase-associated proteins
Authors: Yu Gang , Swiston John , Young Dallan 
Published: April 2021

ABSTRACT
We previously reported the identification of human CAP, a protein that is related to the Saccharomyces cerevisiae and Schizosaccharomyces pombe adenylyl cyclase-associated CAP proteins. The two yeast CAP proteins have similar functions: the N-terminal domains are required for the normal function of adenylyl cyclase, while loss of the C-terminal domains result in morphological and nutritional defects that are unrelated to the cAMP pathways. We have amplified and cloned cDNAs from a human glioblastoma library that encode a second CAP-related protein, CAP2. The human CAP and CAP2 proteins are 64% identical. Expression of either human CAP or CAP2 in S. cerevisiae cap− strains suppresses phenotypes associated with deletion of the C-terminal domain of CAP, but does not restore hyper-activation of adenylyl cyclase by RAS2val19. Similarly, expression of either human CAP or CAP2 in S. pombe cap− strains suppresses the morphological and temperature-sensitive phenotypes associated with deletion of the C-terminal domain of CAP in this yeast. In addition, expression of human CAP, but not CAP2, suppresses the propensity to sporulate due to deletion of the N-terminal domain of CAP in S. pombe. This latter observation suggests that human CAP restores normal adenylyl cyclase activity in S. pombe cap− cells. Thus, functional properties of both N-terminal and C-terminal domains are conserved between the human and S. pombe CAP proteins.