bluuebunny/crossref_metadata_2025_split huggingface dataset:
Timing of calcium and protein synthesis requirements for the first mitotic cell cycle in fertilised Xenopus eggs
Authors: Beckhelling C. , Penny C. , Clyde S. , Ford C. 
Published: April 2021

ABSTRACT
Mitosis is governed by the activity of the M-phase promoting factor (MPF). In some systems, particularly early embryos, transient increases in calcium concentration have been shown to be necessary for mitosis and regulate its timing. By microinjection of the calcium buffer, dibromoBAPTA, into fertilised Xenopus eggs, we have assessed whether calcium events are required to initiate MPF activation and inactivation. Since initial experiments showed that this buffer inhibited protein synthesis, we measured when mitosis and cleavage became independent of translation. We found that, after a period of protein synthesis essential for cleavage, there was a phase during which continued translation affected the timing of cleavage, but was not essential for its occurrence. Measurement of MPF activity in single embryos injected with calcium buffer at different times in the first cell cycle, showed that there were two sensitive periods. The first period of sensitivity blocked MPF activation and coincided with the time at which cleavage became completely independent of protein synthesis. The second sensitive period occurred just before histone kinase activity peaked, and was necessary for kinase inactivation. Preventing inactivation in this way arrested egg extracts in mitosis. These results support the view that transient increases in free calcium concentration contribute to mitotic progression by first triggering MPF activation and subsequently, with elevated MPF activity, inducing its inactivation.
Membrane flow through the Golgi apparatus: specific disassembly of the cis-Golgi network by ATP depletion
Authors: Valle Mercedes del , Robledo Yolanda , Sandoval Ignacio V. 
Published: April 2021

ABSTRACT
Incubation of NRK cells for 30 to 45 minutes with 50 mM 2-deoxy-D-glucose (DOG) in glucose and pyruvate-free medium results in depletion of the cellular ATP pool and in specific disassembly of the cis-Golgi network (CGN), with the stack of Golgi cisternae (SGC) and the trans-Golgi network (TGN) remaining intact and sensitive to BFA. The disassembly of the CGN is mediated by long tubular structures extending outwards from the Golgi complex and involves microtubules. Upon removal of DOG and addition of glucose and pyruvate to the culture medium, the morphology of the CGN is slowly reestablished. Reconstruction of the CGN involves COPI/COPII-positive vesicles that resume the transport of proteins and in particular of CGN membrane proteins out of the ER. Exit of CGN membrane proteins from the ER is insensitive to BFA. In cells pretreated with nocodazole, the CGN membrane proteins are transported to the vicinity of the SGC fragments dispersed throughout the cytoplasm. Ultrastructural studies of cells engaged in the reconstruction of the CGN revealed that the CGN cisterna emerge as tubular structures extending from 0.2-0.3 µm uncoated vesicles prior to their organization on the cis-side of the SGC.
Function of a minus-end-directed kinesin-like motor protein in mammalian cells
Authors: Matuliene Jurgita , Essner Russell , Ryu Jung-Hwa , Hamaguchi Yukihisa , Baas Peter W. , Haraguchi Tokuko , Hiraoka Yasushi , Kuriyama Ryoko 
Published: April 2021

ABSTRACT
CHO2 is a mammalian minus-end-directed kinesin-like motor protein present in interphase centrosomes/nuclei and mitotic spindle fibers/poles. Expression of HA- or GFP-tagged subfragments in transfected CHO cells revealed the presence of the nuclear localization site at the N-terminal tail. This domain becomes associated with spindle fibers during mitosis, indicating that the tail is capable of interaction with microtubules in vivo. While the central stalk diffusely distributes in the entire cytoplasm of cells, the motor domain co-localizes with microtubules throughout the cell cycle, which is eliminated by mutation of the ATP-binding consensus motif from GKT to AAA. Overexpression of the full-length CHO2 causes mitotic arrest and spindle abnormality. The effect of protein expression was first seen around the polar region where microtubule tended to be bundled together. A higher level of protein expression induces more elongated spindles which eventually become disorganized by loosing the structural integrity between microtubule bundles. Live cell observation demonstrated that GFP-labeled microtubule bundles underwent continuous changes in their relative position to one another through repeated attachment and detachment at one end; this results in the formation of irregular number of microtubule focal points in mitotic arrested cells. Thus the primary action of CHO2 appears to cross-link microtubules and move toward the minus-end direction to maintain association of the microtubule end at the pole. In contrast to the full-length of CHO2, overexpression of neither truncated nor mutant polypeptides resulted in significant effects on mitosis and mitotic spindles, suggesting that the function of CHO2 in mammalian cells may be redundant with other motor molecules during cell division.
Epidermal tendon cells require Broad Complex function for correct attachment of the indirect flight muscles in Drosophila melanogaster
Authors: Sandstrom David J. , Restifo Linda L. 
Published: April 2021

ABSTRACT
Drosophila Broad Complex, a primary response gene in the ecdysone cascade, encodes a family of zinc-finger transcription factors essential for metamorphosis. Broad Complex mutations of the rbp complementation group disrupt attachment of the dorsoventral indirect flight muscles during pupal development. We previously demonstrated that isoform BRC-Z1 mediates the muscle attachment function of rbp+ and is expressed in both developing muscle fibers and their epidermal attachment sites. We now report two complementary studies to determine the cellular site and mode of action of rbp+ during maturation of the myotendinous junctions of dorsoventral indirect flight muscles. First, genetic mosaics, produced using the paternal loss method, revealed that the muscle attachment phenotype is determined primarily by the genotype of the dorsal epidermis, with the muscle fiber and the ventral epidermis exerting little or no influence. When the dorsal epidermis was mutant, the vast majority of muscles detached or chose ectopic attachment sites, regardless of the muscle genotype. Conversely, wild-type dorsal epidermis could support attachment of mutant muscles. Second, ultrastructural analysis corroborated and extended these results, revealing defective and delayed differentiation of rbp mutant epidermal tendon cells in the dorsal attachment sites. Tendon cell processes, the stress-bearing links between the epidermis and muscle, were reduced in number and showed delayed appearance of microtubule bundles. In contrast, mutant muscle and ventral epidermis resembled the wild type. In conclusion, BRC-Z1 acts in the dorsal epidermis to ensure differentiation of the myotendinous junction. By analogy with the cell-cell interaction essential for embryonic muscle attachment, we propose that BRC-Z1 regulates one or more components of the epidermal response to a signal from the developing muscle.
Peroxisome degradation in Saccharomyces cerevisiae is dependent on machinery of macroautophagy and the Cvt pathway
Authors: Hutchins Maria U. , Veenhuis Marten , Klionsky Daniel J. 
Published: April 2021

ABSTRACT
Organelle biogenesis and turnover are necessary to maintain biochemical processes that are appropriate to the needs of the eukaryotic cell. Specific degradation of organelles in response to changing environmental cues is one aspect of achieving proper metabolic function. For example, the yeast Saccharomyces cerevisiae adjusts the level of peroxisomes in response to differing nutritional sources. When cells are grown on oleic acid as the sole carbon source, peroxisome biogenesis is induced. Conversely, a subsequent shift to glucose-rich or nitrogen-limiting conditions results in peroxisome degradation. The degradation process, pexophagy, requires the activity of vacuolar hydrolases. In addition, peroxisome degradation is specific. Analyses of cellular marker proteins indicate that peroxisome degradation under these conditions occurs more rapidly and to a greater extent than mitochondrial, Golgi, or cytosolic protein delivery to the vacuole by the non-selective autophagy pathway. To elucidate the molecular mechanism of selective peroxisome degradation, we examined pexophagy in mutants that are defective in autophagy (apg) and the selective targeting of aminopeptidase I to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway. Inhibition of peroxisome degradation in cvt and apg mutants indicates that these pathways overlap and that peroxisomes are delivered to the vacuole by a mechanism that utilizes protein components of the Cvt/autophagy pathways.
Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a retro-translocation complex mediating protein transport for ER degradation
Authors: Plemper Richard K. , Bordallo Javier , Deak Peter M. , Taxis Christof , Hitt Reiner , Wolf Dieter H. 
Published: April 2021

ABSTRACT
The endoplasmic reticulum contains a quality control system that subjects misfolded or unassembled secretory proteins to rapid degradation via the cytosolic ubiquitin proteasome system. This requires retrograde protein transport from the endoplasmic reticulum back to the cytosol. The Sec61 pore, the central component of the protein import channel into the endoplasmic reticulum, was identified as the core subunit of the retro-translocon as well. As import of mutated proteins into the endoplasmic reticulum lumen is successfully terminated, a new targeting mechanism must exist that mediates re-entering of misfolded proteins into the Sec61 pore from the lumenal side de novo. The previously identified proteins Der3p/Hrd1p and, as we show here, Hrd3p of the yeast Saccharomyces cerevisiae, are localised in the endoplasmic reticulum membrane and are essential for the degradation of several substrates of the endoplasmic reticulum degradation machinery. Based on genetic studies we demonstrate that they functionally interact with each other and with Sec61p, probably establishing the central part of the retro-translocon. In the absence of Hrd3p, the otherwise stable protein Der3p/Hrd1p becomes rapidly degraded. This depends on a functional ubiquitin proteasome system and the presence of substrate molecules of the endoplasmic reticulum degradation system. When overexpressed, Der3p/Hrd1p accelerates CPY* degradation in hrd3 cells. Our data suggest a recycling process of Der3p/Hrd1p through Hrd3p. The retro-translocon seems to be build up at least by the Sec61 pore, Der3p/Hrd1p and Hrd3p and mediates both retrograde transport and ubiquitination of substrate molecules.
The heterotrimeric Gi3 protein acts in slow but not in fast exocytosis of rat melanotrophs
Authors: Kreft Marko , Gasman Stéphane , Chasserot-Golaz Sylvette , Kuster Vlasta , Rupnik Marjan , Sikdar Sujit K. , Bader Marie-France , Zorec Robert 
Published: April 2021

ABSTRACT
Besides having a role in signal transduction some trimeric G-proteins may be involved in a late stage of exocytosis. Using immunocytochemistry and confocal microscopy we found that Gi3-protein resides mainly in the plasma membrane, whereas Gi1/2-protein is preferentially associated with secretory granules. To study the function of trimeric Gi3- and Gi1/2-proteins, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. We report here that mastoparan, an activator of trimeric G-proteins, enhances calcium-induced secretory activity in rat melanotrophs. The introduction of synthetic peptides corresponding to the C-terminal domain of the α-subunit of Gi3- and Gi1/2-proteins indicated that Gi3 peptide specifically blocked the mastoparan-stimulated secretory activity, which indicates an involvement of a trimeric Gi3-protein in mastoparan-stimulated secretory activity. Flash photolysis of caged Ca2+-elicited biphasic capacitance increases consisting of a fast and a slower component. Injection of anti-Gi3 antibodies selectively inhibited the slow but not the fast component of secretory activity in rat melanotrophs. We propose that the plasma membrane-bound Gi3-protein may be involved in regulated secretion by specifically controlling the slower kinetic component of exocytosis.
Fission yeast msp1 is a mitochondrial dynamin-related protein
Authors: Pelloquin Laetitia , Belenguer Pascale , Menon Yoann , Gas Nicole , Ducommun Bernard 
Published: April 2021

ABSTRACT
We recently identified Msp1p, a fission yeast Schizosaccharomyces pombe dynamin-related protein, which is essential for the maintenance of mitochondrial DNA. The Msp1p sequence displays typical features of a mitochondrial protein. Here we report in vitro and in vivo data that validate that prediction. We demonstrate that the targeting sequence of Msp1p is processed by recombinant mitochondrial processing peptidase and that Msp1p is imported into S. pombe mitochondria in vitro in the presence of cellular extracts. We show that the first 109 residues of Msp1p encompass a functional peptide signal that is sufficient to direct chimera to mitochondria. Immunofluorescence studies indicate that Msp1p staining colocalises with a mitochondrial marker and electron microscopy shows that the protein is located inside the mitochondria. Mitochondrial enrichment and fractionation further confirm that localisation and show that Msp1p is anchored to the matrix side of the mitochondrial inner membrane. Finally, we report that overexpression of the Msp1 protein results in gross alteration of the mitochondrial structure and function. All together our results suggest that Msp1p is an essential component for mitochondrial maintenance.
Regulation of fibrillin carboxy-terminal furin processing by N-glycosylation, and association of amino- and carboxy-terminal sequences
Authors: Ashworth Jane L. , Kelly Vicky , Rock Matthew J. , Shuttleworth C. Adrian , Kielty Cay M. 
Published: April 2021

ABSTRACT
The molecular mechanisms of fibrillin assembly into microfibrils are poorly understood. In this study, we investigated human fibrillin-1 carboxy-terminal processing and assembly using a recombinant approach. Processing of carboxy-terminal fibrillin-1 was strongly influenced by N-glycosylation at the site immediately downstream of the furin site, and by association with calreticulin. The carboxy terminus of fibrillin-2 underwent less efficient processing than carboxy-terminal fibrillin-1 under identical conditions. Size fractionation of the amino-terminal region of fibrillin-1, and of unprocessed and furin-processed carboxy-terminal region of fibrillin-1, revealed that the amino terminus formed abundant disulphide-bonded aggregates. Some association of unprocessed carboxy-terminal fibrillin-1 was also apparent, but processed carboxy-terminal sequences remained monomeric unless amino-terminal sequences encoded by exons 12-15 were present. These data indicate the presence of fibrillin-1 molecular recognition sequences within the amino terminus and the extreme carboxy-terminal sequence downstream of the furin site, and a specific amino- and carboxy-terminal association which could drive overlapping linear accretion of furin-processed fibrillin molecules in the extracellular space. Differences in processing of the two fibrillin isoforms may reflect differential abilities to assemble in the extracellular space.
FYVE-finger proteins – effectors of an inositol lipid
Authors: Stenmark Harald , Aasland Rein 
Published: April 2021

ABSTRACT
The binding of cytosolic proteins to specific intracellular membranes containing phosphorylated derivatives of phosphatidylinositol (PtdIns) is a common theme in vital cellular processes, such as cytoskeletal function, receptor signalling and membrane trafficking. Recently, several potential effectors of the phosphoinositide 3-kinase product PtdIns 3-phosphate (PtdIns(3)P) have emerged through the observation that a conserved zinc-finger-like domain, the FYVE-finger, binds specifically to this lipid. Here we review current knowledge about the structural basis for the FYVE-PtdIns(3)P interaction, its role in membrane recruitment of proteins and the functions of FYVE-finger proteins in membrane trafficking and other cellular processes.
Relationship between Na+,K+-ATPase and cell attachment
Authors: Contreras R. G. , Shoshani L. , Flores-Maldonado C. , Lázaro A. , Cereijido M. 
Published: April 2021

ABSTRACT
A prolonged ouabain blockade of the Na+,K+-ATPase detaches cells from each other and from the substrate. This suggests the existence of a link between pump (P) and attachment (A). In the present work, we report that MDCK-W cells treated with ouabain increase tyrosine phosphorylation and content of active MAP kinase, redistribute molecules involved in cell attachment (occludin, ZO-1, desmoplakin, cytokeratin, α;-actinin, vinculin and actin), and detach. Genistein and UO126, inhibitors of protein tyrosine kinase and of MAP kinase kinase, respectively, block this detachment. The content of P190Rho-GAP, a GTPase activating protein of the Rho small G-protein subfamily, is increased by ouabain, suggesting that both the Rho/Rac and MAPK pathways are involved.
Another clone of MDCK cells whose Na+,K+-ATPase has a negligible affinity for the drug, show none of the effects described for MDCK-W and remain attached. Ma104 cells, a line that has a high affinity for ouabain and stops pumping, fail to modify phosphorylation, as well as the pattern of distribution of attaching molecules, and remain in the monolayer. Taken together, these results suggest that there is a mechanism (P⟶A) that transduces a blockade of the pump in a detachment of the cell from neighbors and substrate, in which Ma104 cells are faulty.
Occludin proteolysis and increased permeability in endothelial cells through tyrosine phosphatase inhibition
Authors: Wachtel Marco , Frei Karl , Ehler Elisabeth , Fontana Adriano , Winterhalter Kaspar , Gloor Sergio M. 
Published: April 2021

ABSTRACT
Regulation of epithelial and endothelial permeability is essential for proper function of compartmentalized organisms, and tyrosine phosphorylation plays an important role in this process. We analyzed the impact of protein tyrosine phosphatase (PTP) inhibition on the structure of endothelial junctional proteins. In human umbilical vein endothelial cells (HUVECs) the PTP inhibitors phenylarsine oxide (PAO) and pervanadate induced proteolysis of the tight junction protein occludin. Occludin proteolysis was inhibited by the metalloproteinase inhibitor 1,10-phenanthroline (PHEN), but not by inhibitors against other types of proteases. The junctional proteins ZO-1, cadherin and β-catenin were not cleaved. Under conditions of occludin proteolysis, PAO treatment elevated permeability for FITC-dextran. Simultaneous incubation of HUVECs with PAO and PHEN inhibited the rise in permeability by more than 60%. PAO treatment lead to progressive disappearance of occludin from the cell periphery. In contrast, ZO-1, cadherin and β-catenin retained their positions at the sites of intercellular contact. Simultaneous administration of PAO and PHEN greatly prevented the redistribution of occludin. These results demonstrate a selective cleavage of occludin by a metalloproteinase and suggest that this process can contribute to the control of paracellular permeability in endothelial cells.
Polyglutamylation and polyglycylation of α- and β-tubulins during in vitro ciliated cell differentiation of human respiratory epithelial cells
Authors: Million Karine , Larcher Jean-Christophe , Laoukili Jamila , Bourguignon David , Marano Francelyne , Tournier Frédéric 
Published: April 2021

ABSTRACT
Tubulins are the major proteins within centriolar and axonemal structures. In all cell types studied so far, numerous α- and β-tubulin isoforms are generated both by expression of a multigenic family and various post-translational modifications. We have developed a primary culture of human nasal epithelial cells where the ciliated cell differentiation process has been observed and quantified. We have used this system to study several properties concerning polyglutamylation and polyglycylation of tubulin. GT335, a monoclonal antibody directed against glutamylated tubulins, stained the centriole/basal bodies and the axonemes of ciliated cells, and the centrioles of non-ciliated cells. By contrast, axonemal but not centriolar tubulins were polyglycylated. Several polyglutamylated and polyglycylated tubulin isotypes were detected by two-dimensional electrophoresis, using GT335 and a specific monoclonal antibody (TAP952) directed against short polyglycyl chains. Immunoelectron microscopy experiments revealed that polyglycylation only affected axonemal tubulin. Using the same technical approach, polyglutamylation was shown to be an early event in the centriole assembly process, as gold particles were detected in fibrogranular material corresponding to the first cytoplasmic structures involved in centriologenesis. In a functional assay, GT335 and TAP952 had a dose-dependent inhibitory effect on ciliary beat frequency. TAP952 had only a weak effect while GT335 treatment led to a total arrest of beating. These results strongly suggest that in human ciliated epithelial cells, tubulin polyglycylation has only a structural role in cilia axonemes, while polyglutamylation may have a function both in centriole assembly and in cilia activity.
Cell lysis induces redistribution of the GPI-anchored variant surface glycoprotein on both faces of the plasma membrane of Trypanosoma brucei
Authors: Almeida M. Lucia Cardoso de , Geuskens Maurice , Pays Etienne 
Published: April 2021

ABSTRACT
African trypanosomes are coated by 10 million copies of a single variant specific glycoprotein (VSG) which are anchored in the plasma membrane by glycosylphosphatidylinositol (GPI). A GPI-specific phospholipase C (GPI-PLC) triggers fast VSG release upon cell lysis but in vivo it is safely controlled and topologically concealed from its substrate by being intracellular. One enigmatic aspect of GPI-PLC action therefore consists of how it could gain access to the VSG in the exoplasmic leaflet of the membrane. The data presented herewith disclose an unexpected possible solution for this puzzle: upon cell rupture the VSG invades the cytoplasmic face of the plasma membrane which thus becomes double coated. This unusual VSG rearrangement was stable in ruptured plasma membrane from GPI-PLC null mutant trypanosomes but transiently preceded VSG release in wild-type parasites. The formation of double coat membrane (DCM) was independent of the presence or activation of GPI-PLC, occurred both at 4°C and 30°C and was unaffected by the classical inhibitor of VSG release, p-choromercuryphenylsulfonic acid (PCM). DCMs conserved the same coat thickness and association with subpellicular microtubules as in intact cells and were prone to form vesicles following gradual detachment of the latter. Our data also demonstrate that: (i) GPI-PLC expressed by one trypanosome only targets its own plasma membrane, being unable to release VSG of another parasite; (ii) DCMs concomitantly formed from trypanosomes expressing different VSGs do not intermix, an indication that DCM might be refractory to membrane fusion.