bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulation of intracellular transport of acidic fibroblast growth factor by mutations in the cytoplasmic receptor domain
Authors: Citores Lucía , Khnykin Denis , Sørensen Vigdis , Wesche Jørgen , Klingenberg Olav , Więdłocha Antoni , Olsnes Sjur 
Published: April 2021

ABSTRACT
Endocytic uptake and intracellular transport of acidic fibroblast growth factor (aFGF) was studied in cells transfected with FGF receptor 4 with mutations in the cytoplasmic part. Endocytic uptake in HeLa cells was reduced but not abolished when the tyrosine kinase of the receptor was inactivated by mutations or deletions. The tyrosine kinase-dependent endocytosis of aFGF was prevented by the expression of a dominant negative dynamin mutant that blocks endocytosis from coated pits and caveolae. However, more than half of the total endocytic uptake of aFGF was not affected under these conditions, indicating an endocytic uptake mechanism not involving coated pits or caveolae. Mutation or deletion of a putative caveolin-binding sequence did not prevent the localization of part of the receptors to a low density, caveolin-containing subcellular fraction. Whereas wild-type receptor transfers the growth factor from early endosomes to the recycling compartment, kinase negative, full length receptors were inefficient in this respect and the growth factor instead accumulated in lysosomes. By contrast, when most of the intracellular part of the receptor, including the kinase domain, was removed, aFGF was transported to the recycling compartment, as in cells that express wild-type receptors, suggesting the presence of a kinase-regulated targeting signal in the cytoplasmic tail.
Expression and localisation of synaptotagmin isoforms in endocrine β-cells: their function in insulin exocytosis
Authors: Gut Anne , Kiraly Catherine Eva , Fukuda Mitsunori , Mikoshiba Katsuhiko , Wollheim Claes B. , Lang Jochen 
Published: April 2021

ABSTRACT
Exocytosis of insulin containing Large Dense Core Vesicles (LDCVs) from pancreatic β-cells and derived cell lines is mainly controlled by Ca2+. Several lines of evidence have demonstrated a role of the Ca2+- and phospholipid-binding protein synaptotagmin (syt) in this event. Synaptotagmins form a large protein family with distinct affinities for Ca2+ determined by their two C2 domains (C2A/B). Except for the well-characterized isoforms I and II, their role is still unclear. We have used here insulin-secreting cells as a model system for LDCV exocytosis to gain insight into the function of synaptotagmins. Immunocytochemical analysis revealed that of the candidate Ca2+ sensors in LDCV exocytosis, syt III was not expressed in primary β-cells, whereas syt IV was only found adjacent to the TGN.
However, syt V-VIII isoforms were expressed at different levels in various insulin-secreting cells and in pancreatic islet preparations. In streptolysin-O permeabilized primary β-cells the introduction of recombinant peptides (100 nM) corresponding to the C2 domains of syt V, VII and VIII, but not of syt III, IV or VI, inhibited Ca2+-evoked insulin exocytosis by 30% without altering GTPγS-induced release. Our observations demonstrate that syt III and IV are not involved in the exocytosis of LDCVs from primary β-cells whereas V, VII and VIII may mediate Ca2+-regulation of exocytosis.
Nucleocytoplasmic transport in human astrocytes: decreased nuclear uptake of the HIV Rev shuttle protein
Authors: Neumann Markus , Afonina Elena , Ceccherini-Silberstein Francesca , Schlicht Stefan , Erfle Volker , Pavlakis George N. , Brack-Werner Ruth 
Published: April 2021

ABSTRACT
Astrocytes are cellular targets for the human immunodeficiency virus (HIV) that limit virus production, owing, at least in part, to the diminished functionality of the viral post-transcriptional stimulatory factor Rev. To understand the trafficking process in astrocytes, we compared nucleocytoplasmic transport of Rev and various proteins with well-characterized nucleocytoplasmic transport features in human astrocytes and control cells (HeLa). Localization and trafficking characteristics of several cellular and viral proteins, as well as nuclear trafficking of classical peptide signals upon microinjection were similar in both cell types, indicating maintenance of general features of nucleocytoplasmic transport in astrocytes. Quantification of fluorescence in living cells expressing Rev fused to green fluorescent protein (GFP) indicated a strong shift in intracellular distribution of Rev in astrocytes, with 50-70% of Rev in the cytoplasm, whereas the cytoplasmic proportion of Rev in HeLa cells is around 10%. The dynamics of nucleocytoplasmic trafficking of Rev were compared in astrocytes and Rev-permissive cells by monitoring migration of Rev-GFP in cell fusions using highly sensitive time-lapse imaging. Nuclear uptake of Rev was dramatically retarded in homo-polykaryons of astrocytes compared with control cells. Diminished nuclear uptake of Rev was also observed in hetero-polykaryons of Rev-permissive cells and astrocytes. These results indicate that astrocytes contain a cytoplasmic activity that interferes with nuclear uptake of Rev. Our studies suggest a model in which Rev is prevented from functioning efficiently in astrocytes by specific alterations of its nucleocytoplasmic trafficking properties.
Movies available on-line: http://www.biologists.com/JCS/movies/jcs1709.html
Rabphilin dissociated from Rab3 promotes endocytosis through interaction with Rabaptin-5
Authors: Coppola Thierry , Hirling Harald , Perret-Menoud Véronique , Gattesco Sonia , Catsicas Stefan , Joberty Gérard , Macara Ian G. , Regazzi Romano 
Published: April 2021

ABSTRACT
Rabphilin is a secretory vesicle protein that interacts with the GTP-bound form of the small GTPase Rab3. We investigated the involvement of Rabphilin in endocytosis using different point mutants of the protein. Overexpression of wild-type Rabphilin in the insulin-secreting cell line HIT-T15 did not affect receptor-mediated transferrin endocytosis. By contrast, Rabphilin V61A, a mutant that is unable to interact with Rab3, enhanced the rate of transferrin internalization. The effect of Rabphilin V61A was not mimicked by Rabphilin L83A, another mutant with impaired Rab3 binding. Careful analysis of the properties of the two mutants revealed that Rabphilin V61A and Rabphilin L83A are both targeted to secretory vesicles, have stimulatory activity on exocytosis, and bind equally well to α-actinin. However, Rabphilin L83A fails to interact with Rabaptin-5, an important component of the endocytotic machinery. These results indicate that Rabphilin promotes receptor-mediated endocytosis and that its action is negatively modulated by Rab3. We propose that the hydrolysis of GTP that is coupled to the exocytotic event disrupts the Rabphilin-Rab3 complex and permits the recruitment of Rabaptin-5 at the fusion site. Our data show that immediately after internalization the transferrin receptor and VAMP-2 colocalize on the same vesicular structures, suggesting that Rabphilin favors the rapid recycling of the components of the secretory vesicle.
Testase 1 (ADAM 24) a plasma membrane-anchored sperm protease implicated in sperm function during epididymal maturation or fertilization
Authors: Zhu Guo-Zhang , Myles Diana G. , Primakoff Paul 
Published: April 2021

ABSTRACT
Plasma membrane-anchored proteases have key roles in cell signaling, migration and refashioning the cell surface and its surroundings. We report the first example of a plasma membrane-anchored protease on mature sperm, testase 1 (ADAM 24). Unlike other studied sperm ADAMs (fertilin α and β, cyritestin) whose metalloprotease domains are removed during sperm development, we found testase 1 retains an active metalloprotease domain, suggesting it acts as a protease on mature sperm. Testase 1 is a glycoprotein (molecular mass 88 kDa), localized to the equatorial region of the plasma membrane of cauda epididymal sperm. Typically, proteolytic removal of the pro-domain is an initial activation step for ADAM proteases. The pro-domain of the testase 1 precursor (108 kDa) is proteolytically removed as sperm transit the caput epididymis to produce processed (mature) testase 1 (88 kDa). Testase 1 is unique among all studied ADAMs in that its proteolytic processing occurs on the sperm plasma membrane instead of at an intracellular site (the Golgi). Using GST-fusion proteins and a synthetic testase 1 C-terminal peptide, we found that the cytoplasmic tail of testase 1 could be phosphorylated in vitro by protein kinase C (PKC). Thus testase 1 apparently has a cytoplasmic PKC phosphorylation site(s). Protein kinase C is known to stimulate other ADAMs’ protease activity. Because events of the acrosome reaction include PKC activation, we speculate that testase 1 protease function could be important in sperm penetration of the zona pellucida after sperm PKC is activated during the acrosome reaction.
dally, aDrosophilamember of the glypican family of integral membrane proteoglycans, affects cell cycle progression and morphogenesis via a Cyclin A-mediated process
Authors: Nakato Hiroshi , Fox Bethany , Selleck Scott B. 
Published: April 2021

division abnormally delayed (dally) encodes an integral membrane proteoglycan of the glypican family that affects a number of patterning events during both embryonic and larval development. Earlier studies demonstrated that Dally regulates cellular responses to Wingless (Wg) and Decapentaplegic (Dpp) in a tissue-specific manner, consistent with its proposed role as a growth factor co-receptor. dally mutants also display cell cycle progression defects in specific sets of dividing cells in the developing optic lobe and retina. The affected cells in the retina and lamina show delays in completion of the G2-M segment of the cell cycle. We have investigated the molecular basis of dally-mediated cell division defects by examining the genetic interactions between dally and known cell cycle regulators.Reductions in cyclin A but not cyclin B or string expression, suppress dally cell division defects in the optic lobe. cycA mutations also dominantly rescue many dally adult morphological defects including lethality, phenotypes that are unaffected by reducing cycB function. dally mutants show abnormal Cyclin A expression in the dividing cells affected, with appreciable levels of Cyclin A remaining in late prophase and metaphase, stages where Cyclin A is normally absent. Given that Dally is known to regulate the activity of secreted growth factors our findings suggest that extracellular cues influence the degradation of Cyclin A in a manner that controls cell cycle progression and ultimately, cell division patterning.
GPI anchor transamidase ofTrypanosoma brucei: in vitro assay of the recombinant protein and VSG anchor exchange
Authors: Kang Xuedong , Szallies Alexander , Rawer Marc , Echner Hartmut , Duszenko Michael 
Published: April 2021

GPI8 from Trypanosoma brucei was cloned and expressed in Escherichia coli. TbGPI8 encodes a 37 kDa protein (35 kDa after removal of the putative signal sequence) with a pI of 5.5. It contains one potential N-glycosylation site near the N-terminus but no C-terminal hydrophobic region. Enzyme activity assays using trypanosomal lysates or recombinant TbGpi8 exhibited cleavage of the synthetic peptide acetyl-S-V-L-N-aminomethyl-coumarine, indicating that TbGpi8 is indeed directly involved in the proteolytic processing of the GPI anchoring signal. Intracellular localization of TbGpi8 within tubular structures, such as the endoplasmic reticulum, was observed by using specific anti-TbGpi8 antibodies.The transamidase mechanism of GPI anchoring was studied in bloodstream forms of Trypanosoma brucei using media containing hydrazine or biotinylated hydrazine. In the presence of the latter nucleophile, part of the newly formed VSG was linked to this instead of the GPI anchor and was not transferred to the cell surface. VSG-hydrazine-biotin was detected by streptavidin in western blots and intracellularly in Golgi-like compartments.
Life cycle of MTs: persistent growth in the cell interior, asymmetric transition frequencies and effects of the cell boundary
Authors: Komarova Yulia A. , Vorobjev Ivan A. , Borisy Gary G. 
Published: April 2021

Microtubule dynamics were investigated in CHO and NRK cells by novel experimental approaches designed to evaluate the microtubule behavior in the cell interior. These approaches were: (1) laser photobleaching of a path through the centrosome; (2) direct observation of microtubules in centrosome-containing cytoplasts; (3) GFP-CLIP-170 expression as a marker for microtubule plus end growth; and (iv) sequential subtraction analysis. The combination of these approaches allowed us to obtain data where the density of microtubules had previously prevented conventional methods to be applicable.In the steady state, nascent microtubules grew persistently from the centrosome towards the cell margin. Frequently, they arrived at the cell margin without undergoing any transition to the shortening phase. In contrast to the growth of microtubules, shortening of the plus ends from the periphery was non-persistent; that is, rescue was frequent and the extent of shortening showed a distribution of lengths reflecting a stochastic process. The combination of persistent growth and a cell boundary led to a difference in apparent microtubule behavior in the cell interior compared with that near the cell margin. Whereas microtubules in the cell interior showed asymmetric transition frequencies, their behavior near the cell margin showed frequent fluctuations between phases of shortening and growth. Complete microtubule turnover was accomplished by the relatively rare episodes of shortening back to the centrosome. Release from the centrosome with subsequent minus end shortening also occurred but was a minor mechanism for microtubule turnover compared with the plus end pathway.We propose a life cycle for a microtubule which consists of rapid growth from the centrosome to the cell margin followed by an indefinite period of fluctuations of phases of shortening and growth. We suggest that persistent growth and asymmetric transition frequencies serve the biological function of providing a mechanism by which microtubules may rapidly accommodate to the changing shape and advancing edge of motile cells.
Synthesis and secretion of growth hormone in the rat anterior pituitary: I. the intracellular pathway, its time course and energy requirements
Authors: Howell S. L. , Whitfield Margaret 
Published: April 2021

ABSTRACT
The intracellular processes involved in synthesis, transport and storage of newly synthesized proteins in the rat somatotroph, together with their time course and metabolic requirements, have been investigated in a quantitative electron-microscopic radioautography study of the tissue following pulse labelling with tritium-labelled amino acids and chase incubations in various conditions. Proteins are synthesized initially on the rough-surfaced elements of the endoplasmic reticulum and are transported within 10 min after their synthesis to transitional areas between the rough-surfaced endoplasmic reticulum and Golgi complex. Transfer to the Golgi lamellae is achieved, probably via transfer vesicles, within about 60 min after synthesis, while formation of mature storage granules occurs within 2 h following protein synthesis. Further experiments utilizing cycloheximide or ouabain during the chase incubations showed that the intracellular transport of newly synthesized protein and its time course are not significantly affected by inhibitors of protein synthesis, or by inhibition of sodium-potassium dependent ATPase by ouabain. Inhibitors of oxidative phosphorylation (250 μM 2,4-dinitrophenol) or of respiration (10 μM antimycin A) markedly reduced intracellular ATP levels and inhibited the intracellular transport processes. The requirement for ATP appeared to be operative at 2 stages: in the movement of transfer vesicles to the Golgi complex and in the formation of storage granules; possible roles of ATP in these processes are discussed.
The effect of spindle inhibitors applied before meiosis on meiotic chromosome pairing
Authors: Dover G. A. , Riley R. 
Published: April 2021

ABSTRACT
Injection of 0·5 % colchicine into immature tillers of genotypes of Triticum aestivum, T. aestivum × Aegilops mutica and T. aestivum x Secale cereale hybrids induces asynapsis at first meiotic metaphase irrespective of the homologous or homoeologous nature of the potential pairing chromosomes. The induction of asynapsis occurs at a time during and immediately following the last premeiotic mitosis of pollen mother cells. No disruption of synapsis and chiasma formation occurs in anthers having pollen mother cells originally at leptotene or immediately prior to leptotene when cultured in White’s medium plus colchicine. Tetraploid and octaploid pollen mother cells resulting from the disruption of premeiotic spindles by colchicine show pairing of chromosomes only in bivalents, in genotypes normally having a degree of multivalent pairing configurations. The induction of multipolar mitotic spindles with 0·01 % colchicine results in the development of pollen mother cell mosaics with different numbers of chromosomes. Such cells show high levels of chromosome pairing, including multivalents, in some genotypes that normally have very little chromosome pairing. The injection of 0·5 % chloral hydrate during the last premeiotic mitosis of the archesporium causes no disturbances of meiotic pairing. The results are discussed with reference to the hypothesis that the control mechanism of meiotic chromosome pairing involves centromeric microtubules of the spindle (not affected by chloral hydrate) that are responsible for the positional adjustment, during the last mitotic anaphase, of potential pairing partners.
The turnover of collagen in fibroblast cultures
Authors: Steinberg J. 
Published: April 2021

ABSTRACT
Collagen turnover was studied in mouse fibroblast cultures (3T6) by radioactive labelling and compartmental analyses. The incorporation of [14C]proline into protein during continuous labelling rapidly reached a maximum value which was directly proportional to the medium specific activity. Radioactivity appeared more slowly in hydroxyproline, and gradually accumulated as cultures became enriched in collagen and its breakdown products. In relation to total new protein synthesis, the proportional synthesis of collagen, as measured by the formation of [14C]hydroxyproline, was less in logarithmically growing than in stationary-phase cultures, and little was deposited in the cell layer.
Newly synthesized hydroxyproline was consistently present in all growth media. In stationary-phase cultures, media contained as much as 60 % of the total [14C]hydroxyproline in a form soluble in 0·5 M perchloric acid. Gel filtration chromatography confirmed that this was pre-dominantly free hydroxyproline, only 30 % appearing in small peptides whose degree of hydroxylation suggested their origin from larger collagen molecules. This acid-soluble compartment was taken as a convenient index of collagenolysis, which proved to be significant in both growth states, but was proportionately more important throughout logarithmic growth.
Reincubation of prelabelled cultures in fresh medium containing an excess of non-radioactive prohne (‘chase’ medium) was followed by the degradative loss of labelled cell layer protein. The released radioactivity could be quantitatively recovered in the growth medium for periods up to 6 days; the rate of its appearance was little influenced by the frequency of feeding. Despite extensive dilution of the proline precursor-pool specific activity, synthesis of [14C]-hydroxyproline continued in all chase cultures. The increment appeared largely as collagen breakdown products in the growth medium, and probably arose from 2 principal sources: (1) recently deposited collagen, and (2) the hydroxylation of peptidyl-[14C]proline residues in protocollagen. The balance between these contributions seemed to be dependent upon the extent to which ‘ageing’ of the cell layer collagen had occurred prior to initiating the chase. Radioactive hydroxyproline was rapidly lost from briefly prelabelled cell layers, but was well retained in a macromolecular form when the initial labelling period was sufficiently prolonged. It is proposed that the endogenous collagen-degradative apparatus attacks both young collagen and its polypeptide precursor, but that as the lability of the former substrate rapidly declines, enzyme activity continues to operate on protocollagen to yield [14C]hydroxyproline-containing breakdown products which gradually diminish as the latter substrate pool is exhausted.
The analysis of malignancy by cell fusion: IV. hybrids between tumour cells and a malignant l cell derivative
Authors: Wiener F. , Klein G. , Harris H. 
Published: April 2021

ABSTRACT
Previous experiments showed that when a range of different highly malignant mouse tumour cells were fused with L cell derivatives of low tumorigenicity, the resulting hybrid cells, so long as they retained something close to the complete chromosome sets of both parent cells, had little or no ability to grow progressively in vivo. Tumours arising from the injection of such hybrids were produced not by progressive growth of the cells injected, but by selective overgrowth of cells from which certain specific, but as yet unidentified, chromosomes had been eliminated. In the present experiments the same malignant mouse tumour cells were fused with a highly malignant L cell derivative selected from the wild type cell population by passage through the animal. In all cases the resulting hybrid cells were found to be highly malignant; and the chromosome constitutions of the tumours arising from the injection of these hybrids were not significantly different from those of the cells injected. These findings confirm the interpretation given to the previous work: the L cell derivatives of low tumorigenicity contribute to the hybrid cells some factor, linked to specific chromosomes, that suppresses the malignant character of the tumour cell, and this suppression is removed, with consequent reappearance of the malignant phenotype, when certain chromosomes are eliminated.
Cell multiplication in Tetrahymena cultures after addition of particulate material
Authors: Rasmussen L. , Modeweg-Hansen L. 
Published: April 2021

ABSTRACT
We have studied the effects of adding participate supplements to populations of Tetrahymena pyriformis in 2 % sterile-filtered proteose peptone broth which supports cell multiplication poorly (generation times in excess of 40 h). The tested compounds were: heat-sterilized suspensions of egg albumin, nutritionally inert particles of polystyrene, sulphopropyl and quarternary amino-ethyl substituted dextran (in concentrations of 4, 40 and 400 μg per ml). The particles had approximately the same size, but differed in their electric net charges. Particulate suspensions of 40 μg per ml or more greatly improved cell multiplication rates (generation times about 6 h). It is probable that the effect of the particles is to induce formation of food vacuoles without which cell multiplication and growth is very slow. The contribution of the food vacuole to nutrient uptake in Tetrahymena is discussed.