bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cholesterol requirement for cation-independent mannose 6-phosphate receptor exit from multivesicular late endosomes to the Golgi
Authors: Miwako Ishido , Yamamoto Akitsugu , Kitamura Toshio , Nagayama Kuniaki , Ohashi Masato 
Published: April 2021

ABSTRACT
The regulation of endocytic traffic of receptors has central importance in the fine tuning of cell activities. Here, we provide evidence that cholesterol is required for the exit of cation-independent mannose 6-phosphate receptor (CI-MPR) from the endosomal carrier vesicle/multivesicular bodies (ECV/MVBs) to the Golgi. A previously established Chinese hamster ovary cell mutant, LEX2, exhibits arrested ECV/MVBs in which CI-MPR and lysosomal glycoprotein-B (lgp-B) are accumulated. The abnormal accumulation of CI-MPR within the ECV/MVBs in LEX2 cells was corrected in a post-translational manner by the supplementation of medium with cholesterol. Furthermore, it was shown that, by expression cloning using LEX2 mutant, the introduction of the NAD(P)H steroid dehydrogenase-like protein, an enzyme involved in the later stage of cholesterol biosynthesis, allows the exit of CI-MPR from the MVBs to the Golgi and reduces the number of arrested ECV/MVBs in LEX2 cells. The recovery of the exit transport of CI-MPR from the ECV/MVBs was associated with the restoration of the normal cellular free cholesterol level and segregation between CI-MPR and lgp-B, both of which had been localized at the internal small vesicles of the arrested ECV/MVBs. By contrast, the restoration of cholesterol failed to correct the defective processing of endocytosed LDL to a degradative compartment in LEX2 cells. These results suggest that cholesterol is required for ECV/MVB reorganization that drives the sorting/transport of materials destined for the Golgi out of the pathways towards lysosomes.
The tyrosine motifs of Lamp 1 and LAP determine their direct and indirect targetting to lysosomes
Authors: Obermüller Stefanie , Kiecke Christina , von Figura Kurt , Höning Stefan 
Published: April 2021

Lamp 1 and lysosomal acid phosphatase (LAP) are lysosomal membrane proteins that harbour a tyrosine-based sorting motif within their short cytoplasmic tails. Lamp 1 is delivered from the trans-Golgi network (TGN) via endosomes directly to lysosomes bypassing the plasma membrane, whereas LAP is indirectly transported to lysosomes and recycles between endosomes and the plasma membrane before being delivered to lysosomes.By analysing truncated forms of LAP and chimeras in which the cytoplasmic tail or part of the cytoplasmic tails of LAP and Lamp 1 were exchanged, we were able to show that the YRHV tyrosine motif of LAP is necessary and sufficient to mediate recycling between endosomes and the plasma membrane. When peptides corresponding to the cytoplasmic tails of LAP and Lamp 1 and chimeric or mutant forms of these tails were assayed for in vitro binding of AP1 and AP2, we found that AP2 bound to LAP- and Lamp-1-derived peptides, whereas AP1 bound only to peptides containing the YQTI tyrosine motif of Lamp 1. Residues +2 and +3 of the tyrosine motif were critical for the differential binding of adaptors. LAP in which these residues (–HV) were substituted for those of Lamp 1 (–TI) was transported directly to lysosomes, whereas a chimera carrying the Lamp 1 tail in which residues +2 and +3 were substituted for those of LAP (–HV) gained the ability to recycle. In conclusion, the residues +2 and +3 of the tyrosine motifs determine the sorting of Lamp 1 and LAP in endosomes, mediating either the direct or the indirect pathway to lysosomes.
Multi-parameter analysis of the kinetics of NF-κB signalling and transcription in single living cells
Authors: Nelson Glyn , Paraoan Luminita , Spiller David G. , Wilde Geraint J. C. , Browne Mark A. , Djali Peter K. , Unitt John F. , Sullivan Elaine , Floettmann Eike , White Michael R. H. 
Published: April 2021

Proteins of the NF-κB transcription factor family normally reside in the cytoplasm of cells in a complex with IκB inhibitor proteins. Stimulation with TNFα leads to proteosomal degradation of the IκB proteins and nuclear translocation of the NF-κB proteins. Expression of p65 and IκBα fused to fluorescent proteins was used to measure the dynamics of these processes in transfected HeLa cells. Simultaneous visualisation of p65-dsRed translocation and IκBα-EGFP degradation indicated that in the presence of dual fluorescent fusion protein expression,the half-time of IκBα-EGFP degradation was reduced and that of p65 translocation was significantly increased when compared with cells expressing the single fluorescent fusion proteins. These results suggest that the ratio of IκBα and p65 determine the kinetics of transcription factor translocation into the nucleus and indicate that the complex of p65 and IκBα is the true substrate for TNFα stimulation in mammalian cells.
When cells were treated with the CRM-1-dependent nuclear export inhibitor,leptomycin B (LMB), there was nuclear accumulation of IκBα-EGFP and p65-dsRed, with IκBα-EGFP accumulating more rapidly. No NF-κB-dependent transcriptional activation was seen in response to LMB treatment. Following 1 hour treatment with LMB, significant IκBα-EGFP nuclear accumulation, but low levels of p65-dsRed nuclear accumulation, was observed. When these cells were stimulated with TNFα, degradation of IκBα-EGFP was observed in both the cytoplasm and nucleus. A normal transient transcription response was observed in the same cells using luminescence imaging of NF-κB-dependent transcription. These observations suggest that both normal activation and post-induction repression of NF-κB-dependent transcription occur even when nuclear export of NF-κB is inhibited. The results provide functional evidence that other factors, such as modification of p65 by phosphorylation, or interaction with other proteins such as transcriptional co-activators/co-repressors, may critically modulate the kinetics of transcription through this signalling pathway.
Association of the tetraspanin CD151 with the laminin-binding integrinsα3β1, α6β1, α6β4 and α7β1 in cells in culture and in vivo
Authors: Sterk Lotus M. T. , Geuijen Cecile A. W. , van den Berg José G. , Claessen Nike , Weening Jan J. , Sonnenberg Arnoud 
Published: April 2021

CD151 is a cell surface protein that belongs to the tetraspanin superfamily. It forms complexes with the laminin-binding integrinsα3β1, α6β1 and α6β4 and is codistributed with these integrins in many tissues at sites of cell-matrix interactions. In this study we show that CD151 can also form stable complexes with the laminin-binding integrin α7β1. The strength of this interaction is comparable to that between CD151 and α3β1. Complexes ofα3β1, α6β1 and α7β1 with CD151 are equally well formed with all splice variants of the α3, α6 and α7 subunits, and complex formation is not affected by mutations that prevent the cleavage of the integrin α6 subunit. Like the expression ofα3β1 and α6β1, expression of α7β1 in K562 cells results in increased levels of CD151 at its surface. Two non-integrin laminin receptors, dystroglycan and the polypeptide on which the Lutheran blood group antigens are expressed, are also often colocalized with CD151, but no association with CD151-α3β1 complexes was found with biochemical analysis.The anti-CD151 antibody TS151R detects an epitope at a site at which CD151 interacts with integrins, and therefore it cannot react with CD151 when it is bound to an integrin. Comparison of the straining patterns produced by TS151R with that by of an anti-CD151 antibody recognizing an epitope outside the binding site (P48) revealed that most tissues expressing one or more laminin-binding integrins reacted with P48 but not with TS151R. However,smooth muscle cells that express α7β1 and renal tubular epithelial cells that express α6β1 were stained equally well by TS151R and P48. These results suggest that the interactions between CD151 and laminin-binding integrins are subject to cell-type-specific regulation.
Increased responsiveness of hypoxic endothelial cells to FGF2 is mediated by HIF-1α-dependent regulation of enzymes involved in synthesis of heparan sulfate FGF2-binding sites
Authors: Li Jian , Shworak Nicholas W. , Simons Michael 
Published: April 2021

Binding of basic fibroblast growth factor (FGF2) to its high affinity receptors requires the presence of specific heparan sulfate (HS) moieties on the cell surface that act as coreceptors. To determine the contribution of cell-surface HS to modulation of FGF2-dependent cell growth, we studied the changes in the cell mass and FGF2 binding of endothelial cell HS under normoxic and hypoxic conditions in vitro. Both large vein and cardiac microvascular endothelial cells cultured under hypoxic conditions demonstrated an increase in the ratio of cell-surface HS to chondroitin sulfate (CS), as well as an increase in the number of low affinity (HS-associated) binding sites for FGF2 with no change in the apparent Kd. This increase in the number of HS-FGF2 binding sites, in the absence of a significant change in FGF receptor expression, resulted in enhanced responsiveness of hypoxic,compared with normoxic, endothelial cells to FGF2 stimulation.Gene expression studies demonstrated increased expression of the key regulatory enzyme responsible for HS chain synthesis, 1,4 GlcNAc transferase(GlcNAcT-I), as well as increased expression of 2-O sulfotransferase (HS2ST),the enzyme responsible for sulfation of IdoA, a crucial part of the HS-FGF2 binding site. Transduction of cells with an adenovirus encoding a HIF-1αexpression construct resulted in a similar increase in GlcNAcT-I and HS2ST expression. We conclude that hypoxia increases endothelial cell responsiveness to FGF2 by promoting preferential synthesis of HS rather than CS chains and increasing the number of FGF2-binding sites on HS chains. Both of these events are mediated by a HIF-1α-dependent increase in expression of the enzymes GlnNAcT-I and HS2ST. This shift in cell-surface HS composition results in enhanced cell sensitivity to FGF2-induced growth stimulation.
Ca2+-binding proteins of cilia and infraciliary lattice ofParamecium tetraurelia: their phosphorylation by purified endogenous Ca2+-dependent protein kinases
Authors: Kim Kwanghee , Son Min , Peterson Joan B. , Nelson David L. 
Published: April 2021

We purified two small, acidic calcium-binding proteins(ParameciumCa2+-binding proteins, PCBP-25α and PCBP-25β) from Paramecium tetraurelia by Ca2+-dependent chromatography on phenyl-Sepharose and by anion-exchange chromatography. The proteins were immunologically distinct. Monoclonal antibodies against PCBP-25β did not react with PCBP-25α, and antibodies against centrin from Chlamydomonas reacted with PCBP-25α but not with PCBP-25β. Like the centrins described previously, both PCBPs were associated with the infraciliary lattice (ICL), a fibrillar cytoskeletal element in Paramecium. Both were also present in isolated cilia, from which they could be released (with dynein) by a high-salt wash, and both PCBPs cosedimented with dynein in a sucrose gradient. PCBP-25β was especially prominent in cilia and in the deciliation supernatant, a soluble fraction released during the process of deciliation. The results of immunoreactivity and localization experiments suggest that PCBP-25α is a Paramecium centrin and that PCBP-25β is a distinct Ca2+-binding protein that confers Ca2+ sensitivity on some component of the cilium, ciliary basal body or ICL.We characterized these proteins and Paramecium calmodulin as substrates for two Ca2+-dependent protein kinases purified from Paramecium. PCBP-25α and calmodulin were in vitro substrates for one of the two Ca2+-dependent protein kinases (CaPK-2), but only PCBP-25α was phosphorylated by CaPK-1. These results raise the possibility that the biological activities of PCBP-25α and calmodulin are regulated by phosphorylation.
Meiosis in Coprinus: IV. morphology and behaviour of spindle pole bodies
Authors: Raju N. B. , Lu B. C. 
Published: April 2021

ABSTRACT
Meiotic synchrony in the genus Coprinus has permitted the sequential study of spindle pole body (SPB) behaviour through the meiotic process. The SPBs are monoglobular in the young basidia immediately after the last premeiotic mitosis. From 10 to 15 h before karyogamy until pachytene, spindle pole bodies are not found. They become conspicuous in diplotene and persist until the completion of meiosis. During diplotene, metaphase, anaphase and telophase stages the spindle pole bodies are monoglobular but at late diplotene they duplicate and become diglobular with an isthmus connecting the 2 globular elements. The spindle pole bodies remain in a diglobular state until diakinesis when the isthmus breaks separating the 2 daughter spindle pole bodies. The diglobular SPBs in late diplotene and prophase II are believed to represent the duplicated form of the monoglobular state. The spindle pole bodies in Coprinus contain no centrioles. In thin sections the SPBs appear to be fibrillar amorphous structures with a dense inner core surrounded by a less-dense outer zone.
The early development of muscle spindles in the rat
Authors: Milburn Alice 
Published: April 2021

ABSTRACT
The morphogenesis of muscle spindles in rat lower hind-limb muscles has been investigated using the electron microscope. The earliest detectable spindles are seen in the ig-5-day foetus and consist of a single myotube bearing simple nerve terminals of the large primary afferent axon from nearby unmyelinated intramuscular nerve trunks. The capsule forms by an extension of the perineural epithelium of the supplying nerve fasciculus, and is confined initially to the innervated zone. Myonuclei accumulate in this region, so that the first intrafusal muscle fibre to develop is a nuclear-bag fibre. Myoblasts, present within the capsule of the spindle throughout its development, fuse to form a smaller less-differentiated myotube by the 20-day foetal stage. This new myotube matures by close association with the initial fibre, and by birth (21–22 days gestation) has formed the smaller, intermediate bag fibre, that has been identified histochemically and ultra-structurally in the adult. The nuclear-chain fibres develop in the same way; myoblasts fuse to form satellite myotubes that mature in pseudopodial apposition to one of the other fibres within its basement membrane. This apposition consists of extensions of sarcoplasm from the developing myotube into the supporting fibre. By the 4-day postnatal stage the full adult complement of 4 intrafusal muscle fibres is present, although ultra-structural variations, seen in the adult, are not differentiated. The fusimotor innervation begins to arrive at birth, but is not mature until the 12th postnatal day, when the myofibrillar ultra-structural differentiation, including the loss of the M-line in the large-diameter bag fibre, is complete. The periaxial space appears at the same time. It is suggested that the sequential development of the intrafusal fibres is a reflexion of the decreasing morphogenetic effect of the afferent innervation, whereas the role of the fusimotor innervation is in ultra-structural, myofibrillar differentiation.
Functional development of the reticular system in an insect muscle with synchronously differentiating cells
Authors: Tyrer N. M. 
Published: April 2021

ABSTRACT
In contrast to many developing muscles which contain a spectrum of cells at different stages of differentiation, cell development in the locust abdominal intersegmental muscle tends to be synchronous, so that at any stage of development the cells are at the same stage of differentiation. This makes it feasible to relate the properties of the developing tissue as a whole to changes occurring within the cells.
Muscle contraction in the abdominal dorsal muscles of locust embryos and young hoppers has been related to ultra-structural changes within the muscle. Early in development both contraction and relaxation rates are very slow and the muscle shortens very little. The maximum registered tension is achieved relatively early while the rates of contraction and relaxation remain slow. Contraction and relaxation become more rapid as the embryo develops.
These changes can be related to the development of the reticular system, various components of which mature at different rates. When the muscle is first able to develop tension the myofibres contain scattered clusters of myofilaments while the transverse tubule system (T-system), the cisternae and the sarcoplasmic reticulum (SR) are not well developed. The myofibres reach their final size and the filaments are fully formed while the T-system is still irregular and the SR is sparse. The T-system and the cisternae become well developed before the SR. During the time that the relaxation time decreases the SR becomes increasingly prominent.
The substructure of ciliary microtubules
Authors: Warner F. D. , Satir P. 
Published: April 2021

ABSTRACT
Gill cilia from the freshwater mussel Elliptio complanatus were examined after negative staining with phosphotungstic acid. The doublet and central pair microtubules typically collapse on the grid supporting film so that the protofilaments making up the wall of each tubule spread into a visible array. Thirteen protofilaments can be seen to form both the central pair microtubules and subfibre A of each doublet. Sub fibre B of the doublet consists of 10 proto-filaments.
As viewed in both normal and thiourea-treated cross-sectioned cilia, subfibre A appears as a complete microtubule with a circular profile and 26 nm diameter. Subfibre B is an incomplete, elliptically profiled tubule that shares, as a common wall or partition, 4 or 5 protofilaments of the A subfibre. The organization of the 3·5-nm thick protofilaments within the microtubules and their relationship to the various doublet appendages is discussed.
Microfilaments and cytoplasmic streaming: Inhibition of streaming with cytochalasin
Authors: Bradley M. O. 
Published: April 2021

ABSTRACT
Cytochalasin B reversibly inhibits cytoplasmic streaming in both Nitella and Avena cells. Colchicine, on the other hand, has no effect on streaming in either plant; nor does colchicine prevent the recovery of streaming after cytochalasin is withdrawn. The inhibition of protein synthesis by cycloheximide has no effect on either streaming itself or on the recovery of streaming after cytochalasin withdrawal. All this suggests that microfilaments may provide one component of the structure that generates the streaming force and that microtubules play little, if any, role in the process.
Ultrastructural studies of Nitella demonstrate that microfilaments are localized at the boundary of the streaming endoplasm and the stationary ectoplasm. Microfilaments are organized in discrete bundles, with possible cross-bridges between individual filaments in each bundle. These bundles are closely associated with the extensive endoplasmic reticulum.
Cytochalasin B does not cause ultrastructural changes in Nitella microfilaments as it does in some animal-cell filaments. Since the molecular mechanism of cytochalasin’s action is unknown, there may be no necessary correlation between functional inhibition by the drug and altered microfilament morphology.
A model is advanced which proposes that streaming is generated by an interaction between microfilaments and the endoplasmic reticulum.
An ultrastructural and radioautographic study of early oogenesis in the toad Xenopus Laevis
Authors: Coggins Lesley Watson 
Published: April 2021

ABSTRACT
Early oogenesis in the toad Xenopus laevis has been investigated at the ultrastructural level, with particular reference to the formation of extrachromosomal DNA. Thymidine incorporation was localized by electron microscope radioautography. In oogonia, the nucleus is irregular in outline and may contain several nucleoli. Oocytes, from premeiotic interphase to late pachytene, are found in cell nests which are estimated to consist of about 16 cells each. Adjacent oocytes within a nest are connected by intercellular bridges and develop synchronously. Each premeiotic interphase-leptotene oocyte has a round nucleus which contains one or two centrally located, spherical nucleoli. Electron-microscope radioautography showed that all nuclei in a cell nest incorporate thymidine synchronously during premeiotic S-phase. In zygotene oocytes, axial cores and synaptonemal complexes are observed in the nucleus and abut against the inner nuclear membrane in the region nearest the centre of the cell nest. The nucleolus is still more- or-less round in outline, but is asymmetrically positioned in the nucleus. It lies near the nuclear envelope on the side of the nucleus furthest away from the attachment of the chromosome ends, that is, nearest the outside of the cell nest. Each nucleolus is surrounded by a fibrillar ‘halo’ of nucleolus-associated chromatin into which a low level of thymidine incorporation occurs during zygotene. This is thought to represent the start of the major period of amplification of the ribosomal DNA. Pachytene is characterized by the presence of synaptonemal complexes in the nucleus. The nucleolus becomes very irregular in outline. The fibrillar area around it, which represents the extrachromosomal DNA, increases in size and thymidine is incorporated over the whole of this region. In late pachytene, many small fibrogranular bodies, the multiple nucleoli, are formed in it. The members of a cell nest become separated from one another at this time and begin to develop asynchronously. In diplotene, synaptonemal complexes are no longer observed in the nucleus. The most prominent structures in the nucleus are now the multiple nucleoli, which increase greatly in number in early diplotene. A large increase in cytoplasmic volume occurs and the oocyte grows in size.