bluuebunny/crossref_metadata_2025_split huggingface dataset:
The mammalian ARF-like protein 1 (Arl1) is associated with the Golgi complex
Authors: Lowe Stephen Loucian , Wong Siew Heng , Hong Wanjin 
Published: April 2021

ABSTRACT
A rat cDNA clone was isolated which encodes a protein displaying characteristics of a ras-like small GTPase. The deduced amino acid sequence shows the highest amino acid identity (79%) with the Drosophila ARF-like protein 1 (dArl1) among all the known members of the ras-like small GTPase superfamily. The encoded protein was tentatively named rat Arl1 (rArl1). Northern blotting analysis revealed that the rArl1 gene is ubiquitously expressed in rat tissues. Recombinant rArl1 fused to glutathione-S-transferase (GST) to create GST-rArl1 binds GTP-γ-S in a dose-dependent manner. Polyclonal antibodies raised against two unique rArl1 peptides recognized a 22 kDa protein in total NRK cell lysate. Immunofluorescence microscopy of NRK cells revealed discrete perinuclear labelling that could be competed out by GST-rArl1 but not GST. Examination of 8 additional cell lines revealed a similar labelling, suggesting that the antigen recognised by the antibodies is conserved and widely-expressed. Co-localization experiments in NRK cells with antibodies to mannosidase II and a newly identified cis-Golgi protein, p28, showed that rArl1 is localized to the Golgi complex. When cells were treated with nocodazole, the Golgi complex marked by mannosidase II and p28 was fragmented into punctate structures scattered throughout the cell, in which rArl1 was colocalized. Treatment with brefeldin A (BFA) resulted in the redistribution of rArl1 and mannosidase II into the cytoplasm and endoplasmic reticulum, respectively. The kinetics of the redistribution of rArl1 in response to BFA differ from those of ARF and β-COP, two components of non-clathrin coated vesicles.
Annexins II, IV, V and VI relocate in response to rises in intracellular calcium
Authors: Barwise Jennifer L. , Walker John H. 
Published: April 2021

ABSTRACT
Annexins are a family of proteins implicated in a number of cellular processes involving calcium. We studied annexins I, II, IV, V and VI and found that they are all present in human foreskin fibroblasts and, from immunocytochemical studies, have distinct locations in the cell. Only annexin IV and annexin V have unstructured cytoplasmic staining patterns consistent with predominantly cytosolic locations. Annexin VI partially colocalizes with the endoplasmic reticulum. In contrast, annexins I and II are both associated with the plasma membrane with annexin II having a very homogeneous staining compared with the punctate pattern observed for annexin I. Annexins I, IV and V are all present in the nucleus at higher concentrations than in the cytoplasm. Treatment of cells with the calcium ionophore A23187 to raise intracellular calcium, results in relocations of annexin II, IV, V and VI. Intranuclear annexins IV and V relocate to the nuclear membrane whereas the cytosolic pools of these annexins relocate to the plasma membrane. Annexin II relocates to granular structures at the plasma membrane whereas annexin VI relocates to a more homogeneous distribution on the plasma membrane. These results are consistent with an important role for annexins in mediating the calcium signal at the plasma membrane and within the nuclei of fibroblasts.
Calcium-independent regulation of pigment granule aggregation and dispersion in teleost retinal pigment epithelial cells
Authors: King-Smith Christina , Chen Paul , Garcia Dana , Rey Homero , Burnside Beth 
Published: April 2021

ABSTRACT
In the eyes of teleosts and amphibians, melanin pigment granules of the retinal pigment epithelium (RPE) migrate in response to changes in light conditions. In the light, pigment granules disperse into the cells’ long apical projections, thereby shielding the rod photoreceptor outer segments and reducing their extent of bleach. In darkness, pigment granules aggregate towards the base of the RPE cells. In vitro, RPE pigment granule aggregation can be induced by application of nonderivatized cAMP, and pigment granule dispersion can be induced by cAMP washout. In previous studies based on RPE-retina cocultures, extracellular calcium was found to influence pigment granule migration. To examine the role of calcium in regulation of RPE pigment granule migration in the absence of retinal influences, we have used isolated RPE sheets and dissociated, cultured RPE cells. Under these conditions depletion of extracellular or intracellular calcium ([Ca2+]o, [Ca2+]i) had no effect on RPE pigment granule aggregation or dispersion. Using the intracellular calcium dye fura-2 and a new dye, fura-pe3, to monitor calcium dynamics in isolated RPE cells, we found that [Ca2+]i did not change from basal levels when pigment granule aggregation was triggered by cAMP, or dispersion was triggered by cAMP washout. Also, no change in [Ca2+]i was detected when dispersion was triggered by cAMP washout in the presence of 10 µM dopamine, a treatment previously shown to enhance dispersion. In addition, elevation of [Ca2+]i by addition of ionomycin neither triggered pigment movements, nor interfered with pigment granule motility elicited by cAMP addition or washout. Since other studies have indicated that actin plays a role in both pigment granule dispersion and aggregation in RPE, our findings suggest that RPE pigment granule migration depends on an actin-based motility system that is not directly regulated by calcium.
Assembly and function of Chlamydomonas flagellar mastigonemes as probed with a monoclonal antibody
Authors: Nakamura Shogo , Tanaka Genta , Maeda Tsukasa , Kamiya Ritsu , Matsunaga Tsukasa , Nikaido Osamu 
Published: April 2021

ABSTRACT
Mastigonemes are hair-like projections on the flagella of various kinds of lower eukaryotes. We obtained a monoclonal antibody (mAb-MAST1) to mastigonemes of Chlamydomonas reinhardtii, and found that it reacts with a single flagellar glycoprotein of about 230 kDa. Interestingly, immunofluorescence microscopy demonstrated that mAb-MAST1 recognizes not only the flagellar mastigonemes but also a ring composed of 10 or more particles located in the anterior end of the cell body close to the flagellar bases. The ring structure may be the pool of the mastigoneme protein. When the flagella are amputated, they regenerate to their original length in 90-120 minutes. We found that mastigonemes appear on the new flagellar surface as early as 15 minutes after deflagellation, and that new mastigonemes are mostly assembled onto the distal region of the flagellar surface. Mastigonemes thus appear to be inserted into the membrane only in the distal region of the flagellum. Alternatively, mastigonemes may be inserted at the base and transported very rapidly to the distal portion where they are trapped. When live cells are treated with mAb-MAST1, mastigonemes disappear from the flagellar surface. In these mAb-MAST1 treated cells, the swimming velocity decreases to 70-80% of the normal value, although the flagellar beat frequency increases to ∼110% of the control. These findings demonstrate vectorial transport of mastigonemes to their assembly sites, and show that mastigonemes function to increase flagellar propulsive force by increasing the effective surface of the flagellum.
Functional analysis of the MAP2 repeat domain
Authors: Ludin Beat , Ashbridge Kevin , Fünfschilling Ursula , Matus Andrew 
Published: April 2021

ABSTRACT
The neuronal microtubule-associated protein MAP2 binds to microtubules via a domain near its C terminus containing a set of 3 or 4 imperfect repeats of a 31 amino acid motif. Using naturally occurring and mutated forms of the molecule containing between 1 and 4 repeats we have examined the contribution that these repeats make to MAP2 function and explored the significance of their repetition. The experiments utilised the short 3- and 4-repeat splice variants MAP2c and MAP2d that are expressed in developing neurons and in glia respectively, and mutant 1- and 2-repeat versions that were produced by using in vitro mutagenesis to remove further 31 amino acid units while leaving the rest of the molecule unaltered. The properties of these MAP2 variants were compared both with respect to their influence on microtubules in transfected nonneuronal cells and their ability to promote microtubule assembly in vitro. We found that each of the known effects of MAP2, including the bundling of microtubules and induction of process formation in living cells, are expressed by the 1-repeat form MAP2c3, which contains only the third repeat (R3). A second 1-repeat form, MAP2c4, which contains only R4, interacts more weakly with tubulin in vitro and does not bind to microtubules in transfected cells. The microtubule-related properties of MAP2 thus arise mainly from a single predominant repeat unit, R3. In vitro assembly experiments showed that the primary effect of all the repeats is to lower the critical concentration of tubulin required for microtubule assembly but that they differ greatly in potency. The results did not reveal a separate function related to the repetition of the repeat motifs, but instead suggest that its purpose is to tailor the efficiency of MAP2 to the cellular environment in which it has to function.
Nuclear accumulation of the E2F heterodimer regulated by subunit composition and alternative splicing of a nuclear localization signal
Authors: Luna Susana de la , Burden Morwenna J. , Lee Chang-Woo , Thangue Nicholas B. La 
Published: April 2021

ABSTRACT
The cellular transcription factor E2F plays a critical role in integrating cell cycle progression with the transcription apparatus by virtue of a physical interaction and control by key regulators of the cell cycle, such as pRb, cyclins and cyclin-dependent kinases. Generic E2F DNA binding activity arises when a member of two families of proteins, E2F and DP, form heterodimeric complexes, an interaction which results in co-operative transcriptional and DNA binding activity. Here, we characterise a new and hitherto unexpected mechanism of control influencing the activity of E2F which is mediated at the level of intracellular location through a dependence on heterodimer formation for nuclear translocation. Nuclear accumulation is dramatically influenced by two distinct processes: alternative splicing of a nuclear localization signal and subunit composition of the E2F heterodimer. These data define a new level of control in the E2F transcription factor whereby interplay between subunits dictates the levels of nuclear DNA binding activity.
Neurofilament triplet protein interactions: evidence for the preferred formation of NF-L-containing dimers and a putative function for the end domains
Authors: Carpenter David A. , Ip Wallace 
Published: April 2021

ABSTRACT
In this report we examine the molecular interactions that lead to formation of neurofilaments, the intermediate filaments in neurons. Using the yeast two-hybrid system, we found that the rod domains of all three NF triplet proteins interacted strongly with one another and with rod domains of the Type III IF proteins, vimentin and desmin. A slight preference toward NF-L-containing dimers was observed over ones not containing NF-L. Interactions among the full length NF triplet proteins exhibited more specificity. Full length NF-L had only a relatively weak interaction with another full length NF-L molecule, but reacted more robustly with full length NF-M or NF-H lacking only part of the head domain. No homologous or heterologous dimerization of NF-M and NF-H was detectable. These results support the hypothesis that neurofilaments are obligate heteropolymers and that heterodimeric subunits are the preferred building blocks. They further suggest that the mechanism that specifies heterodimeric interaction among the NF triplet proteins resides in the end domains.
Cyclic GMP-dependent protein kinase is required for thrombospondin and tenascin mediated focal adhesion disassembly
Authors: Murphy-Ullrich Joanne E. , Pallero Manuel A. , Boerth Nancy , Greenwood Jeffrey A. , Lincoln Thomas M. , Cornwell Trudy L. 
Published: April 2021

ABSTRACT
Focal adhesions are specialized regions of cell membranes that are foci for the transmission of signals between the outside and the inside of the cell. Intracellular signaling events are important in the organization and stability of these structures. In previous work, we showed that the counter-adhesive extracellular matrix proteins, thrombospondin, tenascin, and SPARC, induce the disassembly of focal adhesion plaques and we identified the active regions of these proteins. In order to determine the mechanisms whereby the anti-adhesive matrix proteins modulate cytoskeletal organization and focal adhesion integrity, we examined the role of protein kinases in mediating the loss of focal adhesions by these proteins. Data from these studies show that cGMP-dependent protein kinase is necessary to mediate focal adhesion disassembly triggered by either thrombospondin or tenascin, but not by SPARC. In experiments using various protein kinase inhibitors, we observed that selective inhibitors of cyclic GMP-dependent protein kinase, KT5823 and Rp-8-Br-cGMPS, blocked the effects of both the active sequence of thrombospondin 1 (hep I) and the alternatively-spliced segment (TNfnA-D) of tenascin-C on focal adhesion disassembly. Moreover, early passage rat aortic smooth muscle cells which have high levels of cGMP-dependent protein kinase were sensitive to hep I treatment, in contrast to passaged cGMP-dependent protein kinase deficient cells which were refractory to hep I or TNfnA-D treatment, but were sensitive to SPARC. Transfection of passaged smooth muscle cells with the catalytic domain of PKG Iα restored responsiveness to hep I and TNfnA-D. While these studies show that cGMP-dependent protein kinase activity is necessary for thrombospondin and tenascin-mediated focal adhesion disassembly, kinase activity alone is not sufficient to induce disassembly as transfection of the catalytic domain of the kinase in the absence of additional stimuli does not result in loss of focal adhesions.
Laminin-5 and hemidesmosomes: role of the α3 chain subunit in hemidesmosome stability and assembly
Authors: Baker Scott E. , Hopkinson Susan B. , Fitchmun Mark , Andreason Grai L. , Frasier Francine , Plopper George , Quaranta Vito , Jones Jonathan C. R. 
Published: April 2021

ABSTRACT
Hemidesmosomes are complex macromolecular structures which integrate elements of the extracellular matrix and the cytoskeleton of epithelial cells. To characterize cell-matrix interactions in the hemidesmosome, we have made use of 804G cells which possess the unusual ability to assemble hemidesmosomes in vitro. During the course of our studies, we have raised a set of monoclonal antibodies against rat laminin-5, the major structural element comprising 804G matrix. One of these, termed CM6, recognizes the 150 kDa α chain of rat laminin-5 and binds the globular (G) domain of intact laminin-5 molecules as determined by rotary shadowing. CM6 antibodies perturb formed hemidesmosomes in 804G cells. In particular, within 1 hour of incubation of 804G cells with CM6 antibodies, colocalization of laminin-5 and α6β4 integrin is lost and by 2 hours, staining generated by hemidesmosomal antibodies appears primarily cytoplasmic in the perinuclear zone. Ultrastructurally, CM6 antibodies first appear to induce detachment of hemidesmosomes from the underlying matrix. Next, portions of the basal cell surface invaginate to form vesicles whose cytoplasmic-facing surface is coated with hemidesmosomes still associated with keratin intermediate filaments. Anchoring filaments extend into the inside compartment of the vesicles. We have also studied the impact of CM6 antibodies on a model system in which the matrix of 804G cells induces de novo assembly of hemidesmosomes in human keratinocytes. This process involves the plasma membrane reorganization of the hemidesmosome associated integrin α6β4 as well as a redistribution of other hemidesmosome components such as the 230 kDa bullous pemphigoid antigen. Pretreatment of 804G matrix with CM6 antibodies blocks such plasma membrane reorganization of hemidesmosome components and inhibits hemidesmosome formation. Our studies indicate a crucial role for the G domain of the α chain of laminin-5 in both nucleation of hemidesmosome assembly as well as maintenance of hemidesmosome structural integrity.
Increased expression of extracellular matrix proteins and decreased expression of matrix proteases after serial passage of glomerular mesangial cells
Authors: Schnaper H. William , Kopp Jeffrey B. , Poncelet Anne Christine , Hubchak Susan C. , Stetler-Stevenson William G. , Klotman Paul E. , Kleinman Hynda K. 
Published: April 2021

ABSTRACT
The cellular events causing pathological extracellular matrix (ECM) accumulation in vivo are not well understood. Prolonged serial passage of several cell types in culture leads to increased production of extracellular matrix (ECM) proteins, but the mechanism for these putative fibrotic changes is not known. Here, human fetal glomerular mesangial cells were subjected to serial passage (P) in culture and the expression of ECM proteins, proteases and protease inhibitors was comprehensively evaluated. From P11 through P14, a series of phenotypic changes occurred. Steady-state expression of mRNA for α1 chains of type III and type IV (but not type I) collagen, and for laminin β1 and γ1, increased 2-to 8-fold, while expression of mRNA for interstitial collagenase (MMP-1) and gelatinase A (MMP-2) virtually ceased. Expression of tissue-type plasminogen activator (tPA) mRNA also decreased markedly. Expression of mRNA for the tissue inhibitor of metalloproteinases (TIMP)-1, and of the smaller of two mRNA species for the PA inhibitor PAI-1, ceased by P14. There was a switch in expression of the two species of TIMP-2 mRNA: whereas the ratio of signal intensity comparing the 3.5 kb mRNA species to the 1.0 kb species was 5:1 up to P11, it was reversed (1:5) at P14 and later. Serial passage also led to changes in protein expression, with increased type IV collagen and laminin, but decreased interstitial collagenase and gelatinase A. The cells showed a progressive increase in staining for type IV collagen. These findings define the appearance of a matrixaccumulating phenotype in later-passage mesangial cells.
Matrix expansion in vivo has been associated with increased transforming growth factor (TGF)-β synthesis; the cells were found to show at least 5-fold increased expression of TGF-β1 mRNA from P8 to P16. However, treatment of P9 or P10 cells with graded doses of TGF-β1 increased expression of both collagen IV and gelatinase A mRNA and did not alter the ratio of signal intensity for TIMP-2 mRNA species. Thus, assumption of a matrixaccumulating phenotype by these cultured fetal glomerular mesangial cells is not accelerated by exogenous TGF-β. These data describe an in vitro model of mesangial cell matrix turnover in which matrix accumulation could result from a concerted increase in ECM synthesis and decrease in ECM degradation.
Testicular biosynthesis and epididymal endoproteolytic processing of rat sperm surface antigen 2B1
Authors: Jones Roy , Ma Alicia , Hou Sheng-Tao , Shalgi Ruth , Hall Len 
Published: April 2021

ABSTRACT
Binding of mammalian spermatozoa to the zona pellucida of homologous eggs is mediated by specific molecules on their surface membranes. In the present investigation we describe the biogenesis, epididymal processing and cellular distribution of a plasma membrane antigen (2B1) on rat spermatozoa that has a potential role in mediating zona binding. 2B1 is expressed postmeiotically in the testis as a precursor glycoprotein (∼60 kDa) that first appears on the plasma membrane of stage 6 to 8 round spermatids. Northern and western blot analyses show that there is a close correlation between the timing of transcription and expression of the glycoprotein on the cell surface. During spermatid elongation 2B1 is excluded from the head domain and is sequestered onto the sperm tail. As spermatozoa pass through the caput epididymidis 2B1 is endoproteolytically cleaved at a specific arginine residue (Arg 312) to produce a heterodimeric glycoprotein (∼40 kDa and ∼19 kDa) containing intramolecular disulphide bridges. Endoproteolysis at Arg 312 also takes place during culture of washed testicular or caput spermatozoa in vitro and can be prevented by serine proteinase inhibitors or enhanced by trypsinisation. However, neither processing in vivo or in vitro has any effect on the domain organisation of 2B1 antigen i.e. it remains localised to the tail. These results support the hypothesis that sperm antigens that are important for fertilization are synthesized as precursor molecules in the testis and are then ‘activated’ during epididymal maturation and capacitation, thereby ensuring that they only become fully functional at the site of fertilization.
Serum albumin is a specific inhibitor of apoptosis in human endothelial cells
Authors: Zoellner Hans , Höfler Manfred , Beckmann Renate , Hufnagl Peter , Vanyek Erika , Bielek Edith , Wojta Johannes , Fabry Astrid , Lockie Samantha , Binder Bernd R. 
Published: April 2021

ABSTRACT
Excess blood vessels are removed by apoptosis of endothelial cells, however, the signals responsible for this have not been defined. Apoptosis of cultured human umbilical vein endothelial cells is induced by deprivation of serum or adhesion. In this paper, apoptosis in human umbilical vein and microvascular endothelium was induced by deprivation of serum and or adhesion. Apoptosis was confirmed on the basis of morphology, ultrastructure and internucleo-somal cleavage of DNA. Loss of endothelial adhesion was found to be an early event in cultured endothelial cell apoptosis and was exploited to quantitate apoptosis. The effect of: bovine serum albumin; human serum albumin; recombinant human albumin; dithiothreitol reduced human and bovine albumin; CNBr treated human and bovine albumin as well as ovalbumin upon endothelial apoptosis was determined. Native bovine and human albumin as well as recombinant human material inhibited apoptosis at physiological concentrations with identical dose response curves in both umbilical vein and micro-vascular cells. Dithiothreitol treatment destroyed all protective activity while bovine but not human albumin was partially inactivated by CNBr treatment. The unrelated protein ovalbumin was not protective. Albumin did not inhibit apoptosis if cells were also deprived of adhesion. The data suggest that albumin is a specific inhibitor of human endothelial apoptosis but does not protect cells also deprived of adhesion. Reduced supply of albumin to endothelium in poorly perfused blood vessels may provide a mechanism for the removal of excess blood vessels in remodelling tissues. Also, the failure of albumin to protect endothelial cells deprived of adhesion from apoptosis may reflect the need to remove potentially micro-embolic cells detached due to trauma.