bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nuclear transport of the U2 snRNP-specific U2B′′ protein is mediated by both direct and indirect signalling mechanisms
Authors: Kambach Christian , Mattaj Iain W. 
Published: April 2021

ABSTRACT
Experiments investigating the nuclear import of the U2 snRNP-specific B′′ protein (U2B′′) are presented. U2B′′ nuclear transport is shown to be able to occur independently of binding to U2 snRNA. The central segment of the protein (amino acids 90-146) encodes an unusual nuclear localization signal (NLS) that is related to that of the U1 snRNP-specific A protein. However, nuclear import of U2B′′ does not depend on this NLS. Sequences in the N-terminal RNP motif of the protein are sufficient to direct nuclear transport, and evidence is presented that the interaction of U2B′′ with the U2A′ protein mediates this effect. This suggests that U2B′′ can ‘piggy-back’ to the nucleus in association with U2A′, and thus be imported to the nucleus by two different mechanisms. U2A′ nuclear transport, on the other hand, can occur independently of both U2B′′ binding and of U2 snRNA.
Cell cycle-specific induction of an 89 kDa serine/threonine protein kinase activity in Trypanosoma brucei
Authors: Gale Michael , Carter Victoria , Parsons Marilyn 
Published: April 2021

ABSTRACT
The cell cycle compartmentalization of specific activities of the protozoan parasite Trypanosoma brucei has remained unexplored due to the lack of a cell synchronization protocol. We report here that stationary phase cells stimulated to enter the cell cycle showed significant synchrony through the first cycle. The pattern of tyrosine phosphorylated proteins, known to undergo alterations during try-panosome development, showed only moderate changes as quiescent cells entered the cycle, particularly an increase in a 77 kDa species. However, the activity of an 89 kDa protein kinase (SPK89), previously demonstrated to be restricted to the proliferative stages of the parasite’s life cycle, markedly increased as the population entered S phase. Cell sorting experiments demonstrated that SPK89 activity was highest in S phase cells and moderate in G2/M cells. The entry into S phase and increased SPK89 activity did not depend on serum factors but required protein synthesis for a discrete period after stimulation. Various modulators of protein phosphorylation were tested to determine their effects on progression to S and SPK89 activity. Only stau-rosporine and genistein were effective. However, both of these compounds inhibited virtually all protein phospho-rylation and protein synthesis in the parasites. Thus these drugs cannot be used as specific protein kinase inhibitors in trypanosomes.
Microtubule tracks can be detected in mouse oocytes with an antibody directed against a calcium transporter
Authors: Pennart Henri de , Cibert Christian , Petzelt Christian , Maro Bernard 
Published: April 2021

ABSTRACT
In metaphase II-arrested mouse oocytes, most micro-tubules are found in the meiotic spindle, a structure that remains stable for hours despite microtubule instability. Microtubule organizing centres (MTOCs) are present at the poles of the spindle and in the cytoplasm, but the latter nucleate very few microtubules. This particular organization of the microtubule network enabled us to observe the unexpected behaviour of a protein that can associate with microtubules. We compared the distribution of a mitosis-activated calcium transport system with that of the micro-tubule network, by immunofluorescence, using two mono-clonal antibodies, one directed against a component of the calcium transport system (7/13), and the other against the common tyrosinated form of α-tubulin (YL1/2). The 7/13 staining was associated with the spindle microtubules and with the kinetochore area. In addition, we observed many asters in the cytoplasm, around the cytoplasmic MTOCs. The majority of these asters were not stained with the anti-tubulin antibody. Moreover, these 7/13 asters either disappeared after nocodazole treatment or were enlarged after taxol treatment. Using a confocal microscope, we observed single fibres that were stained with both antibodies: the extremity furthest from the MTOC (corresponding to the + end of the microtubule) being detected by the 7/13 antibody only. All these observations suggest that the 7/13 antigen is associated with microtubule tracks that persist a few minutes after microtubule depolymerization. The possible role of these tracks in microtubule regrowth is discussed.
Structural diversity of band 4.1 superfamily members
Authors: Takeuchi Kosei , Kawashima Akiyoshi , Nagafuchi Akira , Tsukita Shoichiro 
Published: April 2021

ABSTRACT
Several proteins contain the domain homologous to the N-terminal half of band 4.1 protein, indicating the existence of a superfamily. The members of this ‘band 4.1’ super-family are thought to play crucial roles in the regulation of cytoskeleton-plasma membrane interaction just beneath plasma membranes. We examined the structural diversity of this superfamily by means of the polymerase chain reaction using synthesized mixed primers. We thus identified many members of the band 4.1 superfamily that were expressed in mouse teratocarcinoma F9 cells and mouse brain tissue. In total, 15 cDNA clones were obtained; 8 were identical to the corresponding parts of cDNAs for the known members, while 7 appeared to encode novel proteins (NBL1-7: novel band 4.1-like proteins). Sequence analyses of these clones revealed that the band 4.1 superfamily can be subdivided into 5 gene families; band 4.1 protein, ERM (ezrin/radixin/moesin/merlin/NBL6/NBL7), talin, PTPH1 (PTPH1/PTPMEG/NBL1-3), and NBL4 (NBL4/NBL5) families. The NBL4 family was first identified here, and the full-length cDNA encoding NBL4 was cloned. The deduced amino acid sequence revealed a myristoylation site, as well as phosphorylation sites for A-kinase and tyrosine kinases in its N-terminal half, suggesting its involvement in the phosphorylation-dependent regulation of cellular events just beneath the plasma membrane. In this study, we describe the initial characterization of these new members and discuss the evolution of the band 4.1 superfamily.
Identification of two N-terminal non-alpha-helical domain motifs important in the assembly of glial fibrillary acidic protein
Authors: Ralton Julie E. , Lu Xin , Hutcheson Aileen M. , Quinlan Roy A. 
Published: April 2021

ABSTRACT
The non-α-helical N-terminal domain of intermediate filament proteins plays a key role in filament assembly. Previous studies have identified a nonapeptide motif, SSYRRIFGG, in the non-α-helical N-terminal domain of vimentin that is required for assembly. This motif is also found in desmin, peripherin and the type IV intermediate filament proteins. GFAP is the only type III intermediate filament protein in which this motif is not readily identified. This study has identified two motifs in the non-α-helical N-terminal domain of mouse GFAP that play important roles in GFAP assembly. One motif is located at the very N terminus and has the consensus sequence, MERRRITS-ARRSY. It has some characteristics in common with the vimentin nonapeptide motif, SSYRRIFGG, including its location in the non-α-helical N-terminal domain and a concentration of arginine residues. Unlike the vimentin motif in which even conserved sequence changes affect filament assembly, the GFAP consensus sequence, MERRRITS-ARRSY, can be replaced by a completely unrelated sequence; namely, the heptapeptide, MVRANKR, derived from the λcII protein. When fused to GFAP sequences with sequential deletions of the N-terminal domain, the λcII heptapeptide was used to help identify a second motif, termed the RP-box, which is located just upstream of the GFAP α-helical rod domain. This RP-box affected the efficiency of filament assembly as well as protein-protein interactions in the filament, as shown by sedimentation assays and electron microscopy. These results are supported by previous data, which showed that the dramatic reorganization of GFAP within cells was due to phosphorylation-dephosphorylation of a site located in this RP-box. The results in this study suggest the RP-box motif to be a key modulator in the mechanism of GFAP assembly, and support a role for this motif in both the nucleation and elongation phases of filament assembly. The RP-box motif in GFAP has the consensus sequence, RLSL-RM-PP. Sequences similar to the GFAP RP-box motif are also to be found in vimentin, desmin and peripherin. Like GFAP, these include phosphorylation and proteolysis sites and are adjacent to the start of the central α-helical rod domain, suggesting that this motif is of general importance to type III intermediate filament protein assembly.
Ultrastructural visualization of cross-linked protein features in epidermal appendages
Authors: Rice Robert H. , Wong Viviana J. , Pinkerton Kent E. 
Published: April 2021

ABSTRACT
Upon vigorous extraction with ionic detergent under reducing conditions, the macroscopic structures of mammalian hair, bird feather and horny teeth of the hagfish became swollen and flexible but were substantially preserved. In each case, removal in this way of solubilizable constituents, such as disulfide-bonded keratins and associated matrix proteins, left a residue of ε-(γ-glutamyl)lysine cross-linked protein. Residual features in hair included cell envelopes in the cortex, cell envelopes and intracellular deposits in the medulla, and nearly the entire cuticle cells. In feather, extraction left largely intact the macroscopic barb structures but caused collapse of the rachis. In both rachis and barbs, considerable material resembling cell envelopes remained. In extracted hagfish teeth the cellular organization was clearly visible micro-scopically, including cell borders and remnant nuclei. Unlike the cornified envelopes of mature epidermal keratinocytes, which appear as doublets, reflecting their formation immediately beneath each plasma membrane of apposing cells, the borders of cells of hair cortex and hagfish teeth appeared single and continuous from one cell to the next. Thus the observed cross-linked features comprised four types: (i) condensation immediately beneath the plasma membrane (feather, hair medulla and cuticle) similar to cornified envelopes of epidermal keratinocytes; (ii) deposition between cells (hair cortex, horny teeth); (iii) cytoplasmic deposits (hair cuticle and medulla); and (iv) nuclear condensation (hair medulla, horny teeth). The results emphasize the importance of transglutaminases and their substrate proteins for the function of epidermal appendages and may provide a useful diagnostic test for perturbation of their normal structures.
Expression and localization of the phosphoglucomutase-related cytoskeletal protein, aciculin, in skeletal muscle
Authors: Belkin Alexey M. , Burridge Keith 
Published: April 2021

ABSTRACT
Recently, a 60/63 kDa cytoskeletal protein, highly homologous to the glycolytic enzyme phosphoglucomutase (PGM 1), was isolated from smooth muscle tissue and shown to localize in various adherens-type junctions of muscle and some nonmuscle cells. Since this protein, tentatively named ‘aciculin’, was enriched in muscle tissues and cells, we have attempted to study its expression and localization during myodifferentiation. C2C12 mouse myoblasts did not express any aciculin before cell fusion in culture. Immediately after cell fusion aciculin became detectable and its content continued to rise during myotube maturation. In early myotubes aciculin appeared first at cell tips and was predominantly localized to focal adhesions of immature myotubes. As myotubes matured in culture, aciculin became associated with growing myofibrils, and finally was found redistributed in striations, corresponding to sarcomere Z-discs. Immunoblotting showed that aciculin content in chicken breast skeletal muscle remained very low until day 11 of embryogenesis, but significantly increased in late prenatal and early postnatal development. By immunofluorescence, aciculin was not revealed in thigh skeletal muscle of day 11 chicken embryos, but was prominently localized at myotendinous junctions in thigh muscle of day 16 embryos. Myotendinous junctions appeared to be major sites of aciculin accumulation in developing and mature skeletal muscle fibers in vivo, suggesting some role for this protein in thin filament-membrane interactions and, potentially, in force transmission at these cell-matrix contacts. In adult skeletal muscle faint aciculin staining appeared at the sarcolemma and as striations in register with Z-discs. Since the protein was not identified in glycerinated myofibrils but was localized to striations in C2C12 myotubes and within the limited areas on skeletal muscle tissue sections, we conclude that aciculin is a component of skeletal muscle costameres. In cultured C2C12 myotubes we found some codistribution of aciculin with clusters of acetylcholine receptors, suggesting its presence at neuro-muscular junctions. However, we did not detect any significant concentration of aciculin at neuromuscular junctions in both embryonic and adult skeletal muscle. Taken together, our data show that aciculin expression in skeletal muscle is differentiation-dependent and upregulated during muscle development, and that this novel cytoskeletal protein is a component of various cell-matrix adherens junctions in muscle cells.
Salmonella typhimurium induces selective aggregation and internalization of host cell surface proteins during invasion of epithelial cells
Authors: Portillo Francisco Garcia-del , Pucciarelli M. Graciela , Jefferies Wilfred A. , Finlay B. Brett 
Published: April 2021

ABSTRACT
Salmonella interact with eucaryotic membranes to trigger internalization into non-phagocytic cells. In this study we examined the distribution of host plasma membrane proteins during S. typhimurium invasion of epithelial cells. Entry of S. typhimurium into HeLa epithelial cells produced extensive aggregation of cell surface class I MHC heavy chain, β2-microglobulin, fibronectin-receptor (α5β1 integrin), and hyaluronate receptor (CD-44). Other cell surface proteins such as transferrin-receptor or Thy-1 were aggregated by S. typhimurium to a much lesser extent. Capping of these plasma membrane proteins was observed in membrane ruffles localized to invading S. typhimurium and in the area surrounding these structures. In contrast, membrane ruffling induced by epidermal growth factor only produced minor aggregations of surface proteins, localized exclusively in the membrane ruffle. This result suggests that extensive redistribution of these proteins requires a signal related to bacterial invasion. This bacteria-induced process was associated with rearrangement of polymerized actin but not microtubules, since preincubation of epithelial cells with cytochalasin D blocked aggregation of these proteins while nocodazole treatment did not. Of the host surface proteins aggregated by S. typhimurium, only class I MHC heavy chain was predominantly present in the bacteria-containing vacuoles. No extensive aggregation of host plasma membrane proteins was detected when HeLa epithelial cells were infected with invasive bacteria that do not induce membrane ruffling, including Yersinia enterocolitica, a bacterium that triggers internalization via binding to β1 integrin, and a S. typhimurium invasion mutant that utilizes the Yersinia- internalization route. In contrast to the situation with S. typhimurium, class I MHC heavy chain was not selectively internalized into vacuoles containing these other bacteria. Extensive aggregation of host plasma membrane proteins was also not observed when other S. typhimurium mutants that are defective for invasion were used. The amount of internalized host plasma membrane proteins in the bacteria-containing vacuoles decreased over time with all invasive bacteria examined, indicating that modification of the composition of these vacuoles occurs. Therefore, our data show that S. typhimurium induces selective aggregation and internalization of host plasma membrane proteins, processes associated with the specific invasion strategy used by this bacterium to enter into epithelial cells.
An LI and ML motif in the cytoplasmic tail of the MHC-associated invariant chain mediate rapid internalization
Authors: Bremnes Bjørn , Madsen Toril , Gedde-Dahl Merete , Bakke Oddmund 
Published: April 2021

ABSTRACT
Invariant chain (Ii) is a transmembrane protein that associates with the MHC class II molecules in the endoplasmic reticulum. Two regions of the 30 residue cytoplasmic tail of Ii contain sorting information able to direct Ii to the endocytic pathway. The full-length cytoplasmic tail of Ii and the two tail regions were fused to neuraminidase (NA) forming chimeric proteins (INA). Ii is known to form trimers and when INA was transfected into COS cells it assembled as a tetramer like NA. The INA molecules were targeted to the endosomal pathway and cotransfection with Ii showed that both molecules appeared in the same vesicles. By labelling the INA fusion proteins with iodinated antibody it was found that molecules with either endocytosis signal were expressed at the plasma membrane and internalized rapidly. Point mutations revealed that an LI motif within the first region of the cytoplasmic tail and an ML motif in the second region were essential for efficient internalization. The region containing the LI motif is required for Ii to induce large endosomes but a functional LI internalization motif was not fundamental for this property. The cytoplasmic tail of Ii is essential for efficient targeting of the class II molecules to endosomes and the dual LI and ML motif may thus be responsible for directing these molecules to the endosomal pathway, possibly via the plasma membrane.
The RhoA-dependent assembly of focal adhesions in Swiss 3T3 cells is associated with increased tyrosine phosphorylation and the recruitment of both pp125FAK and protein kinase C-δ to focal adhesions
Authors: Barry Simon T. , Critchley David R. 
Published: April 2021

ABSTRACT
Mouse Swiss 3T3 fibroblasts cultured in serum-free medium lose their actin stress fibres and vinculin-containing focal adhesions, a process that can be reversed by the addition of serum, lysophosphatidic acid (LPA) or bombesin, and is mediated by rhoA (A. J. Ridley and A. Hall (1992) Cell 70, 389-399). We have shown that the addition of serum to these cells induces the recruitment of the cytoskeletal proteins talin, vinculin and paxillin, and the protein kinases pp125FAK and PKC-δ, to newly formed focal adhesions, and that α-actinin is distributed along the actin stress fibres associated with these structures. The newly formed focal adhesions stained heavily with an antibody to phosphotyrosine. A similar response was elicited by 100 ng/ml LPA. The effect of serum was rapid, with focal staining for paxillin largely restricted to cell margins seen within 2 minutes of serum addition, and preceding the assembly of actin filaments. Phosphotyrosine staining differed in that it was predominantly punctate and was widely distributed throughout the cell. By 5 minutes, the paxillin and phosphotyrosine staining was concentrated at the ends of actin filaments largely at the cell margins. The structures stained ranged from circular to oval, but by 10 minutes they more closely resembled the elongated focal adhesions found in cultured fibroblasts. Within 10 minutes, the addition of serum or LPA induced a marked increase in the levels of pp125FAK and paxillin immune-precipitated by an anti-phosphotyrosine antibody. The results suggest that both pp125FAK and paxillin undergo changes in tyrosine phosphorylation upon activation of rhoA, and that these changes are associated with the assembly of focal adhesions and actin stress fibres. The observation that formation of focal adhesions can be induced by the tyrosine phosphatase inhibitor vanadyl hydroperoxide is consistent with the direct involvement of tyrosine phosphorylation in the assembly process. The localisation of PKC-δ to newly formed focal adhesions suggests that serine/threonine phosphorylation may also be important in this regard.
Identification of coelomocyte unconventional myosin and its association with in vivo particle/vesicle motility
Authors: D’Andrea Lisanne , Danon Martha A. , Sgourdas George P. , Bonder Edward M. 
Published: April 2021

ABSTRACT
Sea urchin coelomocytes undergo an inducible structural transformation from petalloid to filopodial form during the ‘clotting’ response in sea urchins. Using a petalloid coelomocyte model, stimulated coelomocytes exhibited bidirectional particle/vesicle motility with a broad distribution of velocities, ranging from 0.02 to 0.12 μm s−1 in the outward bound direction. Coelomocytes treated with the microtubule-disrupting drug, nocodazole, continued to exhibit outward particle/vesicle movements along linear paths with an average velocity of 0.028±0.006 μm s−1. We partially purified a 110 kDa polypeptide possessing K+EDTA-, Ca2+-, Mg2+- and F-actin-activated Mg2+-ATPase activities characteristic of myosin-like motor proteins. The 110 kDa protein immuno-crossreacted with both affinity-purified, anti-brush border unconventional myosin-I polyclonal antibodies and anti-Acanthamoeba myosin head monoclonal antibodies. By indirect immunofluorescence, the 110 kDa unconventional myosin was localized to clusters of particles/vesicles within the perinuclear region of unstimulated coelomocytes, an area containing numerous mitochondria, acidic, lysosomal and Golgi organelles. Indirect immunofluorescence of partially transformed and filopodial coelomocytes detected a diminution of perinuclear staining with a concomitant appearance of stained linear arrays of particles/vesicles, enhanced staining of peripheral lamellae, and staining of the entire length of the filopodia. Subfractionation of unstimulated coelomocyte homogenates on linear sucrose gradients identified distinct peaks of ATPase activity associated with fractions containing conventional and 110 kDa unconventional myosin. Unconventional myosin-containing fractions were found to have numerous particles that stained with anti-brush border unconventional myosin-I antibodies and the lipophilic dye, DiOC6. Thus, coelomocytes demonstrate activatable movements of particles/vesicles in cells devoid of microtubules and possess an unconventional myosin, which may be the motor protein driving particle/vesicle translocation.