bluuebunny/crossref_metadata_2025 huggingface dataset:
Sites in human nuclei where DNA damaged by ultraviolet light is repaired: visualization and localization relative to the nucleoskeleton
Authors: Jackson Dean A. , Balajee A. S. , Mullenders Leon , Cook Peter R.
Published: April 2021

The repair of damage induced in DNA by ultraviolet light involves excision of the damage and then repair synthesis to fill the gap. We investigated the sites of repair synthesis using MRC-5 fibroblasts and HeLa cells in G1 phase. Cells were encapsulated in agarose microbeads to protect them during manipulation, irradiated, incubated to allow repair to initiate, and permeabilized with streptolysin O to allow entry of labelled triphosphates; [32P]dTTP was incorporated into acid-insoluble material in a dose-dependent manner. Incubation with biotin-16-dUTP allowed sites of incorporation to be indirectly immunolabelled using a FITC-conjugated antibody; sites were not diffusely spread throughout nuclei but concentrated in discrete foci. This is similar to sites of S phase activity that are attached to an underlying nucleoskeleton. After treatment with an endonuclease, most repaired DNA electroeluted from beads with chromatin fragments; this was unlike nascent DNA made during S phase and suggests that repaired DNA is not as closely associated with the skeleton. However, the procedure destroyed repair activity, so repaired DNA might be attached in vivo through a polymerase that was removed electrophoretically. Therefore this approach cannot be used to determine decisively whether repair sites are associated with a skeleton in vivo.
Transfection of rat dermal papilla cells with a gene encoding a temperature-sensitive polyomavirus large T antigen generates cell lines retaining a differentiated phenotype
Authors: Filsell Wendy , Little Julie C. , Stones Amanda J. , Granger Stewart P. , Bayley Susan A.
Published: April 2021

The dermal papilla is a discrete group of cells at the base of the hair follicle and is implicated in controlling the hair growth cycle. Early passage dermal papilla cells can induce hair growth in vivo, but, upon further culturing, this property is lost. In order to study the events occurring in hair induction, a representative dermal papilla cell line was required. We have transfected passage 1 rat vibrissa dermal papilla cells with a polyomavirus large T gene encoding a temperature-sensitive T antigen, and generated permanent cell lines in which the immortalizing function can be switched off by temperature shift. The cells established without crisis, resembled cells in the starting population, and retained the aggregative properties of early passage dermal papilla cells. Growth studies were performed on the immortalized cell lines, which showed that transferring the cells to the restrictive temperature for the large T gene product resulted in cell senescence or quiescence, and changes in morphology. Implantation of cell pellets into the ears of immunologically compatible rats showed that the immortal cells retained hair-inductive ability.

Cytokines are believed to have an important role in the control of hair growth. The pattern of cytokine gene expression in the immortal cell lines was compared with early passage dermal papilla cells and a non-hair-inducing dermal papilla cell line, using reverse transcriptase-polymerase chain reaction. Epidermal growth factor, tumour necrosis factor, and interleukin-1a were detected in the immortalized and non-hair-inducing dermal papilla cell lines, but were absent in passage 2 dermal papilla cells. All other cytokines examined were detected in all the cell types under study.

These results demonstrate that the polyomavirus large Ttsa-immortalized dermal papilla cell lines are very similar to passage 2 dermal papilla cells and thus provide a good model for hair growth studies. Cytokine expression profiles indicate that the expression of several cytokines may be implicated in hair induction. Further studies are under way to investigate the relationship between cytokine expression and the hair growth cycle.
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

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

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

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

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

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

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

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

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

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

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

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.
Visualization and quantification of glycolipid polarity dynamics in the plasma membrane of the mammalian spermatozoon
Authors: Gadella B. M. , Gadella T. W. J. , Colenbrander B. , Golde L. M. G. van , Lopes-Cardozo M.
Published: April 2021

Seminolipid (sulphogalactosylalkylacylglycerol), the glycolipid that is specific for mammalian germ cells, is located exclusively in the outer leaflet of the sperm plasma membrane. In this study the lateral distribution of semi-nolipid on sperm heads has been investigated by indirect immunofluorescence labelling and detection with digital imaging fluorescence microscopy. In freshly ejaculated sperm cells this glycolipid was present primarily at the apical ridge subdomain of the plasma membrane of the sperm head. After binding the sperm cells to zona-coated coverslips seminolipid migrated, in 40 minutes, from the apical ridge to the equatorial subdomain of the plasma membrane. A similar redistribution of seminolipid was observed during capacitation of sperm cells in vitro induced by Ca2+ or bovine serum albumin. Comparable migration of seminolipid was also found after prolonged storage of ejaculated sperm cells, albeit at a much slower rate. Addition of arylsulphatase A, an enzyme present in seminal plasma that desulphates seminolipid, significantly enhanced the migration of seminolipid during storage of sperm cells. Its breakdown product desulphoseminolipid (galactosylalkylacylglycerol) appeared highly specifically at the equatorial segment. The measured fluorescence intensity over the sperm head surface correlated linearly with the spatial probe distribution as was checked by fluorescence lifetime imaging microscopy. This paper demonstrates and quantifies for the first time the polarity of semi-nolipid on the surface of the sperm cell and the dynamic alterations that occur in this polarity during post-ejaculatory events.
NSP-encoded reticulons, neuroendocrine proteins of a novel gene family associated with membranes of the endoplasmic reticulum
Authors: Velde Helgi J. K. van de , Roebroek Anton J. M. , Senden Nicole H. M. , Ramaekers Frans C. S. , Ven Wim J. M. Van de
Published: April 2021

The novel NSP gene was previously shown to encode, among a variety of neuroendocrine cell types, two 3′-over-lapping transcripts, a 3.4 kb one for NSP-A (776 amino acids) and a 1.8 kb one for NSP-C (208 amino acids). The deduced proteins, which were predicted to possess distinct amino-terminal regions, appeared to exhibit some archi-tectural resemblance to known neuroendocrine proteins. In this paper the biochemical characterization and subcellu-lar localization of the two proteins is addressed. In vitro translation of NSP-A and -C RNA produced proteins of about 135 and 23 kDa, respectively. Proteins of similar molecular mass were also detected in immunoprecipitation and western blot analyses of neural and endocrine cells using specific anti-NSP-A or -C antisera; some heterogen-eity of NSP-A was observed. NSP-A, but not NSP-C, appeared to be highly phosphorylated and preferentially on serine residues. In immunocytochemical studies, we demonstrated that NSP-A and -C are associated with the endoplasmic reticulum; NSP-A was found to co-localize with SERCA2b, a membrane-associated Ca2+-ATPase of the endoplasmic reticulum. In Purkinje cells, we found NSP-immunostaining in the perikaryon, the extensive dendritic tree and the axon, also suggesting association with the smooth endoplasmic reticulum. Biochemical studies of NSP-A provided evidence that NSP-A is strongly associated with microsomal membranes and analysis of deletion mutants of NSP-A revealed that the hydrophobic carboxy-terminal portion of the protein, which is also present in NSP-C, is critical for membrane binding. Through database searches, finally, we found two different NSP-related sequences, one in a sequenced region of human chromosome 19, and the second in a human, pan-creatic islet-derived partial cDNA, suggesting that the NSP gene is the prototype of a larger gene family. The results of our studies seem to indicate that the NSP-encoded proteins are novel, membrane-anchored components of the endo-plasmic reticulum for which we propose the name reticulons.
Association of kinesin with the Golgi apparatus in rat hepatocytes
Authors: Marks David L. , Larkin Janet M. , McNiven Mark A.
Published: April 2021

The Golgi apparatus is a dynamic membranous structure, which has been observed to alter its location and morphology during the cell cycle and after microtubule disruption. These dynamics are believed to be supported by a close structural interaction of the Golgi with the microtubule cytoskeleton and associated motor enzymes. One micro-tubule-dependent motor enzyme, kinesin, has been implicated in Golgi movement and function although direct evidence supporting this interaction is lacking. In this study, we utilized two well-characterized kinesin antibodies in conjunction with subcellular fractionation techniques, immunoblot analysis and immunofluorescence microscopy to conduct a detailed study on the association of kinesin with the Golgi and other membranous organelles in a polarized epithelial cell, the primary rat hepatocyte. We found that kinesin represents ∼0.3% of total protein in rat liver homogenates, with ∼30% membrane-associated and the remainder in the cytosol. Among membrane fractions, kinesin was concentrated markedly in Golgi-enriched fractions, which were prepared using two independent techniques. Kinesin was also abundant in fractions enriched in transcytotic carriers and secretory vesicles, with lower levels detected on fractions enriched in endosomes, endoplasmic reticulum, lysosomes and mito-chondria. Immunofluorescence microscopy showed that kinesin is concentrated on Golgi-like structures in both primary cultured hepatocytes and rat hepatocyte-derived clone 9 cells. Double-label immunofluorescence demonstrated that kinesin staining colocalizes with the Golgi marker, α-mannosidase II, in both cell types. These results provide compelling evidence showing that kinesin is associated with the Golgi complex in cells and implicate this motor enzyme in Golgi structure, function and dynamics.
Formation of high molecular mass DNA fragments is a marker of apoptosis in the human leukaemic cell line, U937
Authors: Bicknell Gareth R. , Snowden Roger T. , Cohen Gerald M.
Published: April 2021

Inhibitors of macromolecular synthesis and topoisomerases induce apoptosis in the human leukaemic cell line, U937. In this study, U937 cells were treated with the RNA synthesis inhibitor, actinomycin D (1 μM), the protein synthesis inhibitors, emetine (1 μM) and cycloheximide (100 μM), the topoisomerase II inhibitor, teniposide (5 μM), or the topoisomerase I inhibitor, camptothecin (1 μM). Apoptotic cell death was assessed both by flow cytometry and agarose gel electrophoresis, and was correlated to the appearance of large (20 to ≥580 kilobase pairs) DNA fragments, as assessed by field inversion gel electrophoresis. In all cases, the appearance of DNA fragments of 20-50 kilobase pairs accompanied the appearance of an apoptotic population and of internucleosomal cleavage. However, teniposide additionally induced a marked increase in fragmentation to ≥580 kilobase pairs. The co-treatment of cells with zinc (1 mM) inhibited the formation of all large DNA fragments, internucleosomal cleavage and the appearance of an apoptotic population.

We conclude that the generation of large DNA fragments is characteristic of apoptosis induced by various stimuli in U937, as has been found previously in rat thymocytes. However, unlike what occurs in rat thymocytes, zinc treatment does not dissociate the formation of large fragments from conventional markers of apoptosis.
Inhibition of apical but not basolateral endocytosis of ricin and folate in Caco-2 cells by cytochalasin D
Authors: Jackman Mark R. , Shurety Wenda , Ellis Juliet A. , Luzio J. Paul
Published: April 2021

Apical and basolateral endocytic pathways in polarised Caco-2 cells were investigated by following the uptake, recycling and transcytosis of the galactose-binding protein toxin ricin, as a membrane marker. Differences in the extent and kinetics of lectin uptake, recycling and transcy-tosis were observed at the apical and basolateral domains and altered with the age of the cell monolayer. Treatment of polarised Caco-2 cells with cytochalasin D showed a domain-specific, concentration-dependent inhibition of apical endocytosis of ricin. Inhibition of apical endocytosis by cytochalasin D was not due to a gross change in brush border morphology, although actin stress fibres within the cell body were disrupted. It is not clear whether inhibition of apical endocytosis in polarized epithelial cells by cytochalasin D is caused simply by disruption of a mechanochemical motor involving microvillar actin filaments. The cytochalasin D effect was also observed when measuring uptake of folate, suggesting apical domain-specific inhibition of caveolar, as well as clathrin-mediated, endocytic routes.
Utrophin actin binding domain: analysis of actin binding and cellular targeting
Authors: Winder S. J. , Hemmings L. , Maciver S. K. , Bolton S. J. , Tinsley J. M. , Davies K. E. , Critchley D. R. , Kendrick-Jones J.
Published: April 2021

Utrophin, or dystrophin-related protein, is an autosomal homologue of dystrophin. The protein is apparently ubiq-uitously expressed and in muscle tissues the expression is developmentally regulated. Since utrophin has a similar domain structure to dystrophin it has been suggested that it could substitute for dystrophin in dystrophic muscle. Like dystrophin, utrophin has been shown to be associated with a membrane-bound glycoprotein complex. Here we demonstrate that expressed regions of the predicted actin binding domain in the NH2 terminus of utrophin are able to bind to F-actin in vitro, but do not interact with G-actin. The utrophin actin binding domain was also able to associate with actin-containing structures, stress fibres and focal contacts, when microinjected into chick embryo fibroblasts. The expressed NH2-terminal 261 amino acid domain of utrophin has an affinity for skeletal F-actin (Kd 19±2.8 μM), midway between that of the corresponding domains of α-actinin (Kd 4 μM) and dystrophin (Kd 44 μM). Moreover, this utrophin domain binds to non-muscle actin with a ∼4-fold higher affinity than to skeletal muscle actin. These data (together with those of Matsumura et al. (1992)Nature, 360, 588-591) demonstrate for the first time that utrophin is capable of performing a functionally equivalent role to that of dystrophin. The NH2 terminus of utrophin binds to actin and the COOH terminus binds to the membrane associated glycoprotein complex, thus in non-muscle and developing muscle utrophin performs the same predicted ‘spacer’ or ‘shock absorber’ role as dystrophin in mature muscle tissues. These data suggest that utrophin could replace dystrophin functionally in dystrophic muscle.
In vitro differentiation of embryonic stem cells into glial cells and functional neurons
Authors: Fraichard A. , Chassande O. , Bilbaut G. , Dehay C. , Savatier P. , Samarut J.
Published: April 2021

Mouse embryonic stem cells were induced to differentiate in culture with retinoic acid. Putative precursors of neurons and glial cells (nestin-positive cells) were clearly identified as early as three days after the onset of differentiation. At day 6, neuron-like cells could be clearly identified, either as isolated cells or as cellular networks. Some of these cells were positive for astrocyteor oligodendrocyte-specific antigens (GFAP or O4 antigens, respectively). Other cells were positive for neuron-specific antigens (cytoskeleton proteins MAP2, MAP5 and NF200, as well as synaptophysin). Some neuronal-like cells were also positive for acetylcholinesterase activity or glutamic acid decarboxylase expression, indicating that ES cells could differentiate into GABAergic and possibly cholinergic neurons. Electrophysiological analyses performed in voltage clamp conditions showed that cell membranes contained voltagedependent channels. Overshooting action potentials could be triggered by current injection. Taken together, these data provide evidence that embryonic stem cells can differentiate first into neuron-glia progenitors, and later into glial cells and functional neurons, in vitro. This technique provides an unique system to study early steps of neuronal differentiation in vitro.
Does cytosolic free Ca2+ signal neutrophil chemotaxis in response to formylated chemotactic peptide?
Authors: Laffafian Iraj , Hallett M. B.
Published: April 2021

Cytosolic free Ca2+ concentration was measured and imaged in human neutrophils moving towards a source of formylated peptide in a micropipette held close to the cells. Under these conditions, neutrophils changed shape and displayed chemotaxis without significant or persistent global or localised elevations in cytosolic free Ca2+. A rearto-front persistent Ca2+ gradient of less than 0.5 nM/μm was present in the migrating neutrophils, until they reached the zone of higher peptide concentration, when an abrupt rise in cytosolic free Ca2+ concentration was triggered and chemotaxis stopped. Small localised rises in cytosolic free Ca2+, which were occasionally observed during neutrophil manoeuvring, were attributed to the effect of local deformation of the neutrophil membrane, since deformation of the membrane with a blunt micropipette caused similar Ca2+ changes. These data suggest that neutrophil chemotaxis towards a source of formylated peptide occurs without significant changes in Ca2+ signalling.
Targeted disruption of the Dictyostelium myosin essential light chain gene produces cells defective in cytokinesis and morphogenesis
Authors: Chen Tung-Ling L. , Kowalczyk Patricia A. , Ho Guyu , Chisholm Rex L.
Published: April 2021

We have previously demonstrated that the myosin essential light chain (ELC) is required for myosin function in a Dictyostelium cell line, 7-11, in which the expression of ELC was inhibited by antisense RNA overexpression. We have now disrupted the gene encoding the ELC (mlcE) in Dictyostelium by gene targeting. The mlcE−mutants provide a clean genetic background for phenotypic analysis and biochemical characterization by removing complications arising from the residual ELC present in 7-11 cells, as well as the possibility of mutations due to insertion of the antisense construct at multiple sites in the genome. The mlcE− mutants, when grown in suspension, exhibited the typical multinucleate phenotype observed in both myosin heavy chain mutants and 7-11 cells. This phenotype was rescued by introducing a construct that expressed the wildtype Dictyostelium ELC cDNA. Myosin purified from the mlcE− cells exhibited significant calcium ATPase activity, but the actin-activated ATPase activity was greatly reduced. The results obtained from the mlcE− mutants strengthen our previous conclusion based on the antisense cell line 7-11 that ELC is critical for myosin function. The proper localization of myosin in mlcE− cells suggests that its phenotypic defects primarily arise from defective contractile function of myosin rather than its mislocalization.

The enzymatic defect of myosin in mlcE− cells also suggests a possible mechanism for the observed chemotactic defect of mlcE− cells. We have shown that while mlcE− cells were able to respond to chemoattractant with proper directionality, their rate of movement was reduced. During chemotaxis, proper directionality toward chemoattractant may depend primarily on proper localization of myosin, while efficient motility requires contractile function. In addition, we have analyzed the morphogenetic events during the development of mlcE− cells using lacZ reporter constructs expressed from cell type specific promoters. By analyzing the morphogenetic patterns of the two major cell types arising during Dictyostelium development, prespore and prestalk cells, we have shown that the localization of prespore cells is more susceptible to the loss of ELC than prestalk cells, although localization of both cell types is abnormal when developed in chimeras formed by mixing equal numbers of wild-type and mutant cells. These results suggest that the morphogenetic events during Dictyostelium development have different requirements for myosin function.
Peripherin assembles into homopolymers in SW13 cells
Authors: Cui Cunqi , Stambrook Peter J. , Parysek Linda M.
Published: April 2021

The properties of full-length and mutant peripherins were studied in intermediate filament-less SW13 cells to define regions of peripherin that are essential for initiation of filament assembly. A full-length rat peripherin gene transfected into SW13 cells resulted in filament formation, consistent with the close structural relationship of peripherin to other type III intermediate filament proteins that readily form homopolymers. Translation of full-length rat peripherin is initiated predominantly at the second of two inframe AUGs. Deletions within the amino terminus of wildtype peripherin abolished its ability to form filaments in SW13 cells. In contrast, deletion of the entire carboxylterminal tail of peripherin did not affect its ability to form filamentous arrays in transfected SW13 cells. These results indicate that, of the intermediate filament proteins that are expressed in mature neurons, only peripherin and αinternexin are capable of making homopolymer intermediate filaments. In addition, mutations of the carboxyl tail of peripherin generally do not interfere with filament network formation.
Identification of an hsp70-related protein associated with the centrosome from dinoflagellates to human cells
Authors: Perret Eric , Moudjou Mohammed , Geraud Marie Line , Derancourt Jean , Soyer-Gobillard Marie-Odile , Bornens Michel
Published: April 2021

The monoclonal antibody CTR210 raised against isolated human centrosomes strongly decorates the centrosome and more weakly a domain congruent with the Golgi apparatus in several animal cells (HeLa, 3T3, CHO, PtK2). Both decorations resist Triton extraction in conditions which totally extract the Golgi apparatus, as judged by galactosyltransferase decoration. A 67 kDa centrosomal antigen can be demonstrated in human cells with this antibody. CTR210 also decorates the centrosome or associated structures in several systems, including unicellular eukaryotes such as dinoflagellates or ciliates. A 72 kDa antigen has been identified and purified from the dinoflagellate C. cohnii and its NH2-terminal sequence partially established. It shows a close homology with HSP70 proteins. The possibility that the 72 kDa antigen belongs to this chaperone family was further supported using a mAb reacting, in most species, with HSP70. A polyclonal antibody raised against the 72 kDa antigen from C. cohnii decorates the centrosome in human cells and reacts with the CTR210 centrosomal 67 kDa antigen. These results suggest that specific chaperone proteins are associated with the centrosome in eukaryotic cells. The centrosomal chaperones could participate in the microtubule nucleation reaction or in the process of centrosome assembly.
Tissue-specific and efficient expression of the human simple epithelial keratin 8 gene in transgenic mice
Authors: Casanova Llanos , Bravo Ana , Were Felipe , Ramírez Angel , Jorcano José J. , Vidal Miguel
Published: April 2021

Keratin 8 is a type II intermediate filament protein found in simple epithelia. We have introduced a 12 kb DNA fragment of the human K8 locus into the germ line of mice. The transgene, containing 1.1 kb of 5′ flanking sequences, 7.7 kb corresponding to the body of the gene and 3.2 kb of 3′ flanking sequences, was expressed in all six lines obtained. Immunolocalization and RNA analysis of adult tissues showed that the tissue-specific expression pattern of the transgene was almost indistinguishable from that of the endogenous gene. This pattern was found in organs containing single epithelial cell types, such as trachea, lung, stomach, intestine, liver, kidney, thymus and glands. The highest expressing line, however, also produced human K8 in tissues such as stratified epithelia, where it formed part of the pre-existing keratin cytoskeleton of basal cells. Steady state levels of human K8 RNA were proportional to the copy number of the transgene, but transgene expression was less efficient, per gene copy, than that of the endogenous gene. When in the 12 kb DNA fragment the exons and introns of the gene were replaced by the Escherichia coli lacZ gene, the resulting construct showed no expression in transgenic mice. This suggests that 5′ and 3′ flanking sequences, in the absence of intragenic sequences, are not sufficient for K8 expression and that important control elements are located in the body of the K8 gene.