bluuebunny/crossref_metadata_2025_split huggingface dataset:
γ-Tubulin in mammalian cells: the centrosomal and the cytosolic forms
Authors: Moudjou Mohammed , Bordes Nicole , Paintrand Michel , Bornens Michel 
Published: April 2021

ABSTRACT
The centrosome is one of the cellular organelles for which the mechanism by which it operates still remains to be unravelled. The finding of the association with the centrosome of γ-tubulin, a protein which belongs to the tubulin superfamily, has provided a long sought after biochemical tool with which to address centrosome function. We have generated a specific anti-γ-tubulin polyclonal antibody to study the biochemical properties and the cellular distribution of the human lymphoblastic γ-tubulin. Using cell fractionation and mass isolation of centrosomes, we observed that in contrast to the figures suggested by immunofluorescence, a minimum figure of 80% of total γ-tubulin exists as a cytosolic form. The centrosomal form, for which at least half is not strongly associated with the centrosome, behaves in two-dimensional gel electrophoresis identically to the soluble form (as at least two spots of a pI of around 6). Post-embedding immunolocalization reveals that γ-tubulin is distributed in the pericentriolar matrix but is also closely associated with centrioles. Using a combination of gel filtration, ion exchange chromatography, equilibrium sucrose gradient centrifugation and immunoprecipitation, we show that the major part of cytosolic γ-tubulin might be involved in complexes heavier than the Tcp1 particle. We further demonstrate, by co-immunoprecipitation of γ-tubulin and Tcp1 with either anti-Tcp1 or anti-γ-tubulin antibodies, that a small part of γ-tubulin participates in Tcp1-γ-tubulin particles. Interestingly, the soluble form of γ-tubulin co-purifies with taxol-stabilized microtubules and its association with microtubules resisted salt, ATP and GTP treatments. The existence of a centrosomal form and a large pool of cytosolic γ-tubulin-containing complexes in somatic cells suggests that the overall γ-tubulin cellular distribution does not seem to be as straightforward as it was drawn earlier.
Transcriptional regulation of αB-crystallin in astrocytes: Analysis of HSF and AP1 activation by different types of physiological stress
Authors: Head Mark W. , Hurwitz Lina , Goldman James E. 
Published: April 2021

ABSTRACT
The coordinated cellular responses to physiological stress are known to be effected in part by the activation of heat shock factor 1, a transcriptional activator protein capable of binding to, and inducing transcription from genes containing heat shock elements. Other stress responsive signal transduction pathways also exist including the stress activated protein kinase cascade that regulates the activity of the transcription factor AP1. We have examined the expression of the low molecular stress proteins, heat shock protein 27 and αB-crystallin in astrocytes in response to physiological stress of different types and asked what component of this induction is effected at the transcriptional level and whether activation of heat shock factor 1 and AP1 might account for these events. We have found that stress regulated induction of αB-crystallin has a strong transcriptional component and that it may be effected by at least two different transcriptional mechanisms. In one set of phenomena, represented here by cadmium exposure, αB-crystallin and heat shock protein 27 are coordinately regulated and this occurs in the presence of activated heat shock factor 1. In the second series of phenomena, represented here by hypertonic stress, αB-crystallin is induced in the absence of heat shock factor activation and in the absence of any corresponding change in heat shock protein 27 expression. Although hypertonic stress does activate an AP1-like binding activity, the AP1 consensus binding site in the αB-crystallin promoter does not appear to be a target for this hypertonic stress inducible activity. These data suggest that the hypertonic stress response is effected through a heat shock factor independent mechanism and that hypertonic stress regulated induction of αB-crystallin does not directly depend on the SAPK pathway and AP1 activity.
Cytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule nucleation in HeLa cells
Authors: Gabrielli Brian G. , Souza Colin P. C. De , Tonks Ian D. , Clark Joanne M. , Hayward Nicholas K. , Ellem Kay A. O. 
Published: April 2021

ABSTRACT
The formation of the mitotic spindle is an essential prerequisite for successful mitosis. The dramatic changes in the level of microtubule (Mt) nucleation at the centrosomes and Mt dynamics that occur in prophase are presumed to be initiated through the activity of cdc2/cyclin B. Here we present data that the cdc25B isoform functions to activate the cytoplasmic pool of cdc2/cyclin B responsible for these events. In contrast to cdc25C, cdc25B is present at low levels in HeLa cells during interphase, but sharply increases in prophase, when cdc25B accumulation in the cytoplasm correlates with prophase spindle formation. Overexpression of wild type and dominant negative mutants of cdc25B and cdc25C shows that prophase Mt nucleation is a consequence of cytoplasmic cdc25B activity, and that cdc25C regulates nuclear G2/M events. Our data also suggest that the functional status of the centrosome can regulate nuclear mitotic events.
MAP2d promotes bundling and stabilization of both microtubules and microfilaments
Authors: Ferhat Lotfi , Represa Alfonso , Bernard Anne , Ben-Ari Yezekiel , Khrestchatisky Michel 
Published: April 2021

ABSTRACT
Two low molecular weight MAP2 variants have been described, MAP2c and MAP2d. These variants are produced from a single gene by alternative splicing, and in their C-terminal regions contain, respectively, 3 and 4 tandem repeats, some of which are known to be involved in binding to microtubules. Substantial differences in the developmental expression pattern of MAP2c and MAP2d suggest they have different functions in neural cells. In order to investigate the respective roles of these MAP2 variants, we have analyzed the effects of MAP2c and MAP2d expression on microtubule and microfilament organization in transiently transfected cells. Our results show that both variants stabilize microtubules, but only MAP2d stabilizes microfilaments.
Immunofluorescence studies of human fibroblasts demonstrate the presence of the complex of elongation factor-1βγd in the endoplasmic reticulum
Authors: Sanders Jo , Brandsma Margreet , Janssen George M. C. , Dijk Jan , Möller Wim 
Published: April 2021

ABSTRACT
The eukaryotic elongation factor-1 (EF-1) consists of four subunits, EF-1α, EF-1β, EF-1γ and EF-1d which induce efficient transfer of aminoacyltRNA to the ribosome. In this process EF-1α·GTP acts as the carrier of the aminoacyltRNA on its way to the ribosome. After release of aminoacyltRNA to the ribosome under concomitant hydrolysis of GTP, the inactive EF-1α·GDP form is recycled to EF-1α·GTP by EF-1βγd. In eukaryotic cells the concentration of EF-1α exceeds that of the complex βγd by a factor of 5-10. In order to delineate the intracellular localization of the different subunits of EF-1, antibodies against the EF-1 subunits have been elicited and indirect immunofluorescence microscopy experiments were performed.
In human fibroblasts, the guanine nucleotide exchange part of EF-1, EF-1βγd, was found to colocalize with the endoplasmic reticulum (ER), displaying a distinct finestructure in its staining pattern. The guanine nucleotidebinding subunit of EF-1, EF-1α, shows a more diffuse distribution throughout the cytoplasm and is, in addition, associated with the nucleus.
Trafficking of cell-surface amyloid β-protein precursor: I. Secretion, endocytosis and recycling as detected by labeled monoclonal antibody
Authors: Koo Edward H. , Squazzo Sharon L. , Selkoe Dennis J. , Koo Catherine H. 
Published: April 2021

ABSTRACT
Amyloid βprotein, the principal constituent of amyloid fibrils found in senile plaques and blood vessels in Alzheimer’s disease, is constitutively produced and released into medium of cultured cells. Amyloid βprotein is derived by proteolysis of the βamyloid precursor protein by unclear mechanisms. βamyloid precursor protein is a transmembrane protein which can be processed to release a large secretory product or processed in the endosomal/lysosomal pathway without secretion. Previous studies have shown that from the cell surface, βamyloid precursor protein may be released after cleavage or internalized without cleavage, the latter in a pathway that both produces amyloid βprotein and also targets some molecules to the lysosomal compartment. Analysis of βamyloid precursor protein trafficking is confounded by the concomitant secretion and internalization of molecules from the cell surface. To address this issue, we developed an assay, based on the binding of a radioiodinated monoclonal antibody, to measure the release and internalization of cell surface βamyloid precursor protein in transfected cells. With this approach, we showed that surface βamyloid precursor protein is either rapidly released or internalized, such that the duration at the cell surface is very short. Approximately 30% of cell surface βamyloid precursor protein molecules are released. Following internalization, a fraction of molecules are recycled while the majority of molecules are rapidly sorted to the lysosomal compartment for degradation. When the C terminus of βamyloid precursor protein is deleted, secretion is increased by approximately 2.5-fold as compared to wildtype molecules. There is a concomitant decrease in internalization in these mutant molecules as well as prolongation of the resident time on the cell surface. This observation is consistent with recent evidence that signals within the cytoplasmic domain mediate βamyloid precursor protein internalization.
Trafficking of cell-surface amyloid β-protein precursor: II. Endocytosis, recycling, and lysosomal targeting detected by immunolocalization
Authors: Yamazaki Tsuneo , Koo Edward H. , Selkoe Dennis J. 
Published: April 2021

ABSTRACT
Amyloid βprotein (Aβ) is a proteolytic fragment of the amyloid βprotein precursor (βPP). Progressive cerebral deposition of Aβ is an early and invariant feature of Alzheimer’s disease. The cellular trafficking of βPP is of particular interest because understanding the production of Aβ requires a comprehensive elucidation of the metabolic pathways of this protein. In addition, βPP is a type I integral membrane glycoprotein that belongs to a class of molecules with both full length and secreted products. Recent evidence suggests that βPP can be processed in an endosomal/lysosomal pathway. In the latter organelles, a number of βPP carboxyterminal derivatives are found, but the precise pathway and kinetics of βPP trafficking from the cell surface remain unclear. To address these questions, we visualized directly the βPP internalization pathway by following the localization and distribution of βPP monoclonal antibodies added to intact βPP-transfected Chinese hamster ovary cells. Using immunofluorescence and immunoelectron microscopy, βPP was shown to be rapidly internalized via coated pits and vesicles, after which the molecules were transported to endosomes, prelysosomes, and lysosomes. Using a modified immunodetection protocol, we demonstrated the rapid recycling of endocytosed βPP to the cell surface and its ultimate targeting to lysosomes. Because we recently found that endocytosis of cell surface βPP is one route for the constitutive production of Aβ, the recycling pathway for cell surface βPP demonstrated here is a probable route for production of the critical Aβ fragment.
A localisation signal in the 3′ untranslated region of c-myc mRNA targets c-myc mRNA and β-globin reporter sequences to the perinuclear cytoplasm and cytoskeletal-bound polysomes
Authors: Veyrune J.-L. , Campbell G. P. , Wiseman J. , Blanchard J.-M. , Hesketh J. E. 
Published: April 2021

ABSTRACT
There is increasing evidence that in mammalian cells some mRNAs are localised to specific parts of the cytoplasm and a proportion of mRNAs and polyribosomes are associated with the cytoskeleton. It has been shown previously that c-myc mRNA is present in the perinuclear cytoplasm and associated with the cytoskeleton, and that this localisation is dependent upon the 3′ untranslated region of the mRNA. The present studies show that in transfected fibroblasts the c-myc 3′ untranslated region is able to localise β-globin reporter sequences to the perinuclear cytoplasm. Studies with constructs containing deletions within the 3′ untranslated region identify the region between bases 194 and 280 as critical for localisation. Transfection of cells with constructs in which this region is linked to β-globin sequences showed that it was sufficient to localise the chimaeric tran-scripts to the perinuclear cytoplasm and to cytoskeletal-bound polyribosomes. Transfection with constructs containing a mutated AUUUA sequence within the 194-280 base region showed that this conserved AUUUA is required for targeting of both c-myc mRNA and a chimaeric tran-script of β-globin transcripts linked to the c-myc 3′ untranslated region. The region between bases 194 and 280 did not induce instability of β-globin transcripts and the AUUUA mutation had little effect upon mRNA stability. We propose that this 86 nt region of the 3′ untranslated region contains a localisation signal to target c-myc mRNA so that it is retained on cytoskeletal-bound polysomes in the perinuclear cytoplasm; a conserved AUUUA sequence appears to be a critical part of this signal.
Intracellular targeting of GLUT4 in transfected insulinoma cells: evidence for association with constitutively recycling vesicles distinct from synaptophysin and insulin vesicles
Authors: Thorens Bernard , Roth Jürgen 
Published: April 2021

ABSTRACT
In adipocytes and muscle cells, the GLUT4 glucose trans-porter isoform is present in intracellular vesicles which continuously recycle between an intracytoplasmic location and the plasma membrane. It is not clear whether the GLUT4-vesicles represent a specific kind of vesicle or resemble typical secretory granules or synaptic-like microvesicles. To approach this question, we expressed GLUT4 in the β cell line RINm5F and determined its intra-cellular localization by subcellular fractionation and by immunofluorescence and immunoelectron microscopy. GLUT4 was not found in insulin granules but was associated with a subpopulation of smooth-surface vesicles present in the trans-Golgi region and in vesicular structures adjacent to the plasma membrane. In the trans-Golgi region, GLUT4 did not colocalize with synaptophysin or TGN38. Incubation of the cells with horseradish peroxidase (HRP) led to colocalization of HRP and GLUT4 in some endosomal structures adjacent to the plasma membrane and in occasional trans-Golgi region vesicles. When cells were incubated in the presence of Bafilomycin A, analysis by confocal microscopy revealed GLUT4 in numerous large spots present throughout the cytoplasm, many of which costained for TGN38 and synaptophysin. By immunoelectron microscopy, numerous endosomes were observed which stained strongly for GLUT4. Together our data demonstrate that ectopic expression of GLUT4 in insulinoma cells reveals the presence of a subset of vesicular structures distinct from synaptic-like vesicles and insulin secretory granules. Furthermore, they indicate that GLUT4 constitutively recycles between the plasma membrane and its intracellular location by an endocytic route also taken by TGN38 and synaptophysin.
Syndecan-1 expression in mammary epithelial tumor cells is E-cadherin-dependent
Authors: Leppä Sirpa , Vleminckx Kris , Roy Frans Van , Jalkanen Markku 
Published: April 2021

ABSTRACT
E-cadherin is a Ca2+-dependent cell-cell adhesion molecule, which is mainly expressed in epithelial cells. Recent studies have shown that E-cadherin has an important role as an invasion suppressor molecule in epithelial tumor cells. Syndecan-1 is a cell surface proteoglycan that has been implicated in a number of cellular functions including cell-cell adhesion, cell-matrix anchorage and growth factor pre-sentation for signalling receptors. Its suppression has also been shown to be associated with malignant transforma-tion of epithelial cells. In order to better understand the coordinated regulation of cell-cell and cell-matrix interac-tions during malignant transformation, we have studied the expression of syndecan-1 in malignant mammary tumor cells genetically manipulated for E-cadherin expression. In invasive NM-e-ras-MAC1 cells, where E-cadherin was partially downregulated by specific antisense RNA, syndecan-1 expression was suppressed. Furthermore, transfection of E-cadherin cDNA into invasive NM-f-ras-TD cells resulted in the upregulation of syndecan-1 expression in association with decreased invasiveness. In both cases, regulation of syndecan-1 occurred post-tran-scriptionally, since syndecan-1 mRNA levels remained unchanged. Instead, a translational regulation is suggested, since syndecan-1 core protein synthesis was E-cadherin dependent. Another cell adhesion protein, β1-integrin was not affected by E-cadherin expression. The data provide an example of coordinated changes in the expression of two cell adhesion molecules, syndecan-1 and E-cadherin during epithelial cell transformation.
Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts
Authors: Strausfeld Ulrich P. , Howell Mike , Descombes Patrick , Chevalier Stephane , Rempel Rachel E. , Adamczewski Jörg , Maller James L. , Hunt Tim , Blow J. Julian 
Published: April 2021

ABSTRACT
Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define ‘S-phase promoting factor’ (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclu-sively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cdc2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.
The SAC3 gene encodes a nuclear protein required for normal progression of mitosis
Authors: Bauer Andreas , Kölling Ralf 
Published: April 2021

ABSTRACT
The SAC3 gene of Saccharomyces cerevisiae has been implicated in actin function by genetic experiments showing that a temperature sensitive mutation in the essential actin gene (act1-1) can be suppressed by mutations in SAC3. An involvement of SAC3 in actin function is further suggested by the observation that the actin cytoskeleton is altered in SAC3 mutants. Our fractionation experiments, however, point to a nuclear localization of Sac3p. On sucrose density gradients Sac3p co-fractionated with the nuclear proteins Ssb1p, Nop1p and Nop2p but not with the other organelle markers examined. Furthermore, Sac3p was enriched 10-fold in a nuclei preparation along with the nuclear protein Nop1p. In this report we further show that SAC3 function is required for normal progression of mitosis. SAC3 mutants showed a higher fraction of large-budded cells in culture, indicative of a cell cycle delay. The predominant population among the large-budded sac3 cells were cells with a single nucleus at the bud-neck and a short intranuclear spindle. This suggests that a cell cycle delay occurs in mitosis prior to anaphase. The observation that SAC3 mutants lose chromosomes with higher frequency than wild-type is another indication for a mitotic defect in SAC3 mutants. We further noticed that SAC3 mutants are more resistant against the microtubule destabilizing drug benomyl. This finding suggests that SAC3 is involved, directly or indirectly, in microtubule function. In summary, our data indicate that SAC3 is involved in a process which affects both the actin cytoskeleton and mitosis.
The kinetics of the B cyclin p56cdc13 and the phosphatase p80cdc25 during the cell cycle of the fission yeast Schizosaccharomyces pombe
Authors: Creanor J. , Mitchison J. M. 
Published: April 2021

ABSTRACT
The levels of the B cyclin p56cdc13 and the phosphatase p80cdc25 have been followed in selection-synchronised cultures of Schizosaccharomyces pombe wild-type and wee1 mutant cells. p56cdc13 has also been followed in induction-synchronised cells of the mutant cdc2-33. The main con-clusions are: (1) cdc13 levels in wild-type cells start to rise from base line at about mid-G2, reach a peak before mitosis and then fall slowly through G1. Cells exit mitosis with appreciable levels of cdc13. (2) cdc13 levels in wee1 cells fall to zero in interphase. They also start to rise at the beginning of G2, which may be related to the absence of a mitotic size control. (3) cdc25 starts to rise later and reaches a peak after mitosis. This is not what would be expected from a simple mitotic inducer and suggests that cdc25 has an important function at the end of mitosis. (4)
An upper (heavier) band of cdc25 peaks at the same time as the main band but rises and falls more rapidly. If this is a hyperphosphorylated form, its timing shows that it is most unlikely to function in the ways shown for such a form in eggs and mammalian cells. (5) Experiments with the mutant cdc10-129 and with hydroxyurea show that the initial signal to begin synthesis of cdc13 originates at Start. (6) In induction synchrony, where G2 spans across cell division, there is evidence that some events in one cycle cannot start in the previous one. (7) Revised timings are given for the times of mitosis in these cultures.