bluuebunny/crossref_metadata_2025_split huggingface dataset:
Roles of plakoglobin end domains in desmosome assembly
Authors: Palka Helena L. , Green Kathleen J. 
Published: April 2021

ABSTRACT
Plakoglobin, a member of the armadillo family of proteins, is a component of intercellular adhesive junctions. The central domain of plakoglobin comprises a highly conserved series of armadillo repeats that facilitate its association with either desmosomal or classic cadherins, or with cytosolic proteins such as the tumor suppressor gene product adenomatous polyposis coli. Sequences in the N- and C-terminal domains of plakoglobin are less highly conserved, and their possible roles in regulating plakoglo-bin’s subcellular distribution and junction assembly are still unclear. Here we have examined the role of plakoglo-bin end domains by stably expressing constructs lacking the N and/or C terminus of plakoglobin in A-431 cells. Our results demonstrate that myc-tagged plakoglobin lacking either end domain is still able to associate with the desmo-somal cadherin desmoglein and incorporate into desmo-somes. In cell lines that express an N-terminal truncation of plakoglobin, an increase in the cytosolic pool of en-dogenous and ectopic plakoglobin was observed that may reflect an increase in the stability of the protein. Deletion of the N terminus did not have a dramatic effect on the structure of desmosomes in these cells. On the other hand, striking alterations in desmosome morphology were observed in cells expressing C-terminal truncations of plakoglobin. In these cell lines, ectopic plakoglobin incorporated into desmosomes, and extremely long junctions or groups of tandemly linked desmosomes which remained well attached to keratin intermediate filaments, were observed. Together, these results suggest that plakoglobin end domains play a role in regulating its subcellular distribution, and that the presence of the C terminus limits the size of desmosomes, perhaps through regulating proteinprotein interactions required for assembly of the desmosomal plaque.
Expression of a minus-end-directed motor protein induces Sf9 cells to form axon-like processes with uniform microtubule polarity orientation
Authors: Sharp David J. , Kuriyama Ryoko , Essner Russell , Baas Peter W. 
Published: April 2021

ABSTRACT
Neurons extend two types of processes with distinct morphologies and patterns of microtubule polarity orientation. Axons are thin cylindrical processes containing microtubules that are uniformly oriented with their plus-ends-distal to the cell body while dendrites are stout tapering processes that contain nonuniformly oriented microtubules. We have proposed that these distinct microtubule patterns are established by molecular motors that transport microtubules into each type of process with the appropriate orientation. To test the feasibility of this proposal, we have embarked on a series of studies involving the expression of vertebrate motors in insect Sf9 cells. We previously focused on a kinesin-related protein termed CHO1/MKLP1, which localizes to the midzone of the mitotic spindle, and which has been shown to have the appropriate properties to transport microtubules of opposite orientation relative to one another. Expression of a fragment of CHO1/MKLP1 containing its motor domain induces Sf9 cells to extend processes with a stout tapering morphology and a nonuniform microtubule polarity pattern similar to dendrites. Here we focus on a minus-end-directed kinesin-related motor protein termed CHO2, which localizes to the nonoverlapping regions of the mitotic spindle, and which has been shown to have the appropriate properties to transport microtubules with plus-ends-leading. Sf9 cells induced to express a fragment of CHO2 containing its motor domain extend processes with a long cylindrical morphology and a uniformly plus-end-distal microtubule polarity pattern similar to axons. These results show that motor proteins have the capacity to organize distinct patterns of microtubule polarity orientation during process outgrowth, and that these patterns are intimately related to the unique morphological characteristics of the processes. Moreover, mutation of three amino acids corresponding to the ATP binding site necessary for motor function suppresses the capacity of the CHO2 fragment to induce process formation and microtubule reorganization, indicating that at least in the case of CHO2, the transport properties of the motor are essential for it to elicit these effects.
When rDNA transcription is arrested during mitosis, UBF is still associated with non-condensed rDNA
Authors: Gébrane-Younès Jeannine , Fomproix Nathalie , Hernandez-Verdun Danièle 
Published: April 2021

ABSTRACT
The mechanisms that control inactivation of ribosomal gene (rDNA) transcription during mitosis is still an open question. To investigate this fundamental question, the precise timing of mitotic arrest was established. In PtK1 cells, rDNA transcription was still active in prophase, stopped in prometaphase until early anaphase, and activated in late anaphase. Because rDNA transcription can still occur in prophase and late anaphase chromosomes, the kinetics of rDNA condensation during mitosis was questioned. The conformation of the rDNA was analyzed by electron microscopy from the G2/M transition to late anaphase in the secondary constriction, the chromosome regions where the rDNAs are clustered. Whether at transcribing or non-transcribing stages, non-condensed rDNA was observed in addition to axial condensed rDNA. Thus, the persistence of this non-condensed rDNA during inactive transcription argues in favor of the fact that mitotic inactivation is not the consequence of rDNA condensation. Analysis of the three-dimensional distribution of the rDNA transcription factor, UBF, revealed that it was similar at each stage of mitosis in the secondary constriction. In addition, the colocalization of UBF with non-condensed rDNA was demonstrated. This is the first visual evidence of the association of UBF with non-condensed rDNA. As we previously reported that the rDNA transcription machinery remained assembled during mitosis, the colocalization of rDNA fibers with UBF argues in favor of the association of the transcription machinery with certain rDNA copies even in the absence of transcription. If this hypothesis is correct, it can be assumed that condensation of rDNA as well as dissociation of the transcription machinery from rDNA cannot explain the arrest of rDNA transcription during mitosis. It is proposed that modifications of the transcription machinery occurring in prometaphase could explain the arrest of transcription, while reverse modifications in late anaphase could explain activation.
Microtubule depolymerization inhibits clathrin coated-pit internalization in non-adherent cell lines while interleukin 2 endocytosis is not affected
Authors: Subtil Agathe , Dautry-Varsat Alice 
Published: April 2021

ABSTRACT
The microtubule cytoskeleton is generally not considered to be essential for the first steps of clathrin-mediated endocytosis of membrane receptors. Its role in clathrin-inde-pendent endocytosis has not been investigated. We have previously shown that the cytokine interleukin 2 (IL2) is internalized in lymphoid cells expressing its receptors when clathrin-dependent endocytosis is inhibited. Here we compare the internalization of IL2 and of transferrin, a marker of clathrin-dependent endocytosis, after microtubule disruption. In hemopoietic cell lines, which express IL2 receptors, transferrin receptor entry was inhibited by about 40%. However, in adherent cell lines, transferrin entry was unaffected by microtubule disruption, as previously reported. Unlike the case for transferrin, internalization of IL2 receptors was not affected by depolymerization of the microtubule cytoskeleton in hemopoietic cell lines. These results show that IL2 and transferrin receptors do not have the same endocytic properties and support our previous conclusion that these receptors follow different pathways of endocytosis.
Reduced expression of tissue transglutaminase in a human endothelial cell line leads to changes in cell spreading, cell adhesion and reduced polymerisation of fibronectin
Authors: Jones R. A. , Nicholas B. , Mian S. , Davies P. J. A. , Griffin M. 
Published: April 2021

ABSTRACT
Tissue transglutaminase (tTgase, type II) is a Ca2+-dependent GTP binding protein which crosslinks proteins via ε(γ-glutamyl)lysine bridges. Although essentially a cytosolic enzyme there is increasing evidence to suggest the enzyme is externalised where it may play a role in extracellular matrix organisation. To investigate the function of this enzyme in a human umbilical endothelial cell line ECV304 tTgase expression was reduced in these cells by up to 90% by stable transfection with a 1.1. kb antisense construct in the plasmid vector pSG5. Two clones showing a reduction in expression of tTgase activity of 70 and 90% have been isolated and characterised. These clones show a number of phenotypic differences when compared to the parent cell line and the transfected controls which include reduced cell spreading and a decreased adhesion of cells on different substrata as measured by their susceptibility to removal by trypsin. Reduced cell spreading in the antisense transfected clones was accompanied by a decrease in the crosslinking of fibronectin into polymeric multimers which could be correlated to the amount of tTgase externalised by cells. A novel assay was developed to measure externalised tTgase activity which is cell mediated, inhibited by preincubation of cells with anti-tTgase antibody and relies on the incorporation of biotinylated cadaverine into fibronectin. The results of these experiments suggest that externalised tTgase may play a key role in a number of cell behavioural patterns which might be related to the enzymes ability to bind and crosslink fibronectin.
Fluid-phase uptake by macropinocytosis in Dictyostelium
Authors: Hacker Ulrike , Albrecht Richard , Maniak Markus 
Published: April 2021

ABSTRACT
To study fluid-phase endocytosis in living cells and its relationship to changes in the cell cortex, we have used a green fluorescent protein (GFP)-tagged version of coronin, an actin-associated protein that localises to dynamic regions of the Dictyostelium cell cortex. In the confocal microscope, internalisation of fluorescently labelled dextran as a fluid-phase marker can be recorded simultaneously with the recruitment of the coronin-GFP fusion-protein from the cytoplasm of the phagocyte. At crown-shaped surface protrusions, extracellular medium is taken up into vesicles with an average diameter of 1.6 µm, which is significantly larger than the 0.1 µm diameter of clathrin-coated pinosomes. The observed frequency of macropinosome formation can account for a large portion, if not all, of the fluid-phase uptake. The redistribution of coronin-GFP strongly resembles cytoskeletal rearrangements during phagocytosis. Scanning-electron micrographs indicate that crown-shaped cell-surface extensions can undergo shape changes, without a particle bound, that are similar to shape changes that occur during phagocytosis. In quantitative assays, the uptake of particles and fluid are about equally dependent on F-actin and coronin.
The expression of endothelial tissue plasminogen activator in vivo: a function defined by vessel size and anatomic location
Authors: Levin Eugene G. , Santell Lydia , Osborn Kent G. 
Published: April 2021

ABSTRACT
Plasma tissue plasminogen activator (tPA) has long been considered to be the product of the endothelial cells that line the various parts of the vascular system regardless of vessel size or location. To determine whether this was truly the case in vivo, the distribution of tPA in the endothelium of the mouse lung and other tissues was evaluated. Immunohistochemical analysis of normal lung tissue showed positive staining limited to the endothelial cells of the bronchial arteries regardless of size with few cells of the pulmonary circulation associated with tPA. The pulmonary vessels that did contain endothelial cell-derived tPA were consistently between 7 and 30 µm in diameter. No capillary or large vessel pulmonary endothelium ever stained positive. These results were also observed in primate lung tissue where the bronchial endothelium of all vessels, even down to capillary size, contained tPA while none of the pulmonary endothelium did. Prolonged exposure of mice to hyperoxic conditions promotes acute lung injury and associated inflammation. Using this model, the effect of inflammation on endothelial cell tPA expression was evaluated. A 4.5-fold increase in the number of pulmonary vessels staining positive for tPA was observed after 66 hours with all of these vessels having a diameter between 7 and 30 µm. Again, none of the endothelium of large arteries or veins nor the capillaries had tPA. Whole tissue tPA mRNA increased dramatically with hyperoxia and in situ hybridization analysis showed tPA mRNA in the endothelium of the same types of vessels as antigen. The tPA localized to both the bronchial and pulmonary endothelium was active with neither tPA-PAI-1 complexes nor urokinase found in perfused lung tissue. These results indicate that endothelial cell tPA expression, either constitutive or induced by a pathologic event, is a function of a highly select group of endothelial cells which are defined by their association with vessels of discrete size and/or anatomic location. Thus, the widely held concept that the steady state level of plasma tPA is maintained through its constitutive production by all endothelial cells of the vascular system is invalid. Also suggested is the possibility that endothelial cell tPA might play a broader role than simply maintaining vessel patency as a component of the fibrinolytic pathway and contribute to complex dynamic processes such as inflammation.
The subcellular distribution of the high molecular mass protein, HD1, is determined by the cytoplasmic domain of the integrin β4 subunit
Authors: Sánchez-Aparicio Paloma , Martínez de Velasco Ana M. , Niessen Carien M. , Borradori Luca , Kuikman Ingrid , Hulsman Esther H. M. , Fässler Reinhard , Owaribe Katsushi , Sonnenberg Arnoud 
Published: April 2021

ABSTRACT
The high molecular mass protein, HD1, is a structural protein present in hemidesmosomes as well as in distinct adhesion structures termed type II hemidesmosomes. We have studied the distribution and expression of HD1 in the GD25 cells, derived from murine embryonal stem cells deficient for the β1 integrin subunit. We report here that these cells possess HD1 but not BP230 or BP180, two other hemidesmosomal constituents, and express only traces of the α6β4 integrin. By immunofluorescence and interference reflection microscopy HD1 was found together with vinculin at the end of actin filaments in focal contacts. In OVCAR-4 cells, derived from a human ovarian carcinoma which, like GD25 cells, only weakly express α6β4, HD1 was also localized in focal contacts. Upon transfection of both GD25 and OVCAR-4 cells with cDNA for the human β4 subunit the subcellular distribution of HD1 changed significantly. HD1 is then no longer present in focal contacts but in other structures at cell-substrate contacts, colocalized with α6β4. These junctional complexes are probably the equivalent of the type II hemidesmosomes. Transfection of GD25 cells with β1 cDNA did not affect the distribution of HD1, which indicates that the localization of HD1 in focal contacts was not due to the absence of β1. Moreover, in GD25 cells transfected with cDNA encoding a β4/β1 chimera, in which the cytoplasmic domain of β4 was replaced by that of β1, the distribution of HD1 was unaffected. Our findings indicate that the cytoplasmic domain of β4 determines the subcellular distribution of HD1 and emphasize the important role of α6β4 in the assembly of hemidesmosomes and other junctional adhesive complexes containing HD1.
LK6, a short lived protein kinase in Drosophila that can associate with microtubules and centrosomes
Authors: Kidd Deborah , Raff Jordan W. 
Published: April 2021

ABSTRACT
A number of polyclonal mouse sera were raised against Drosophila proteins that bound to microtubules in vitro (Kellogg et al. (1989)J. Cell Biol. 109, 2977-2991). Some of these sera recognised centrosomes in vivo, and we have been using these to screen expression libraries to isolate cDNAs encoding these putative centrosomal microtubule-associated proteins. Here we report the cloning of one such cDNA that encodes a novel serine/threonine protein kinase called LK6. The protein appears to exist in two forms: an abundant 185 kDa form and a rarer ∼220 kDa form that interacts with microtubules. At least some of the LK6 protein is located in centrosomes at all stages of the cell cycle in fly embryos. Interestingly, the protein contains a PEST-like sequence and is rapidly turned over in vivo. Constitutive overexpression of LK6 is deleterious to flies and causes defects in microtubule organisation in both eggs and early embryos, whereas constitutive overexpression of a mutant form containing a point mutation that severely impairs the kinase activity is without effect. These findings suggest that LK6 may play a role in regulating microtubule function.
A simple method for measurement of cell-substrate attachment forces: application to HIV-1 Tat
Authors: Channavajjala Lakshmi S. , Eidsath Alec , Saxinger W. Carl 
Published: April 2021

ABSTRACT
In order to understand the importance of cell attachment to HIV-1 Tat, we quantified the strength of cell attachment to immobilized Tat in microtiter plate wells by the application of buoyant force. By replacing the attachment medium with dense medium, and subjecting the attached cells in the microtiter plates to centrifugal force in the conventional upright position, weakly binding and strongly binding cells could be discriminated (and separated) by varying the centrifugal speed. The strength of attachment of HT1080 cells to Tat was compared with that of the well-known extracellular matrix (ECM) proteins fibronectin and vitronectin. We observed that all three proteins mediated significant attachment of HT1080 cells both at 4°C and 37°C. However, unlike the ECM proteins, Tat was unable to engage in higher strength binding when the temperature was raised to 37°C. The relatively weak binding of HT1080 cells to Tat (in the order of 3.0 µdynes/picomole of coated Tat) and lack of strengthening of binding to Tat at physiologic temperature suggests that this protein does not mimic adhesion molecule function. We anticipate that the methodology developed and described here will be useful in a wide variety of cell-matrix and cell-cell interaction studies.
Analysis of the expression, distribution and function of cyclin dependent kinase 5 (cdk5) in developing cerebellar macroneurons
Authors: Pigino Gustavo , Paglini Gabriela , Ulloa Luis , Avila Jesús , Cáceres Alfredo 
Published: April 2021

ABSTRACT
Cultures of cerebellar macroneurons were used to study the expression, activity, subcellular localization, and function of cdk5 during neuronal morphogenesis. The results obtained indicate that in non-polarized neurons cdk5 is restricted to the cell body but as soon as polarity is established it becomes highly concentrated at the distal tip of growing axons where it associates with microtubules and the subcortical cytoskeleton. In addtion, we show that laminin, an extracellular matrix molecule capable of stimulating axonal extension and promoting MAP1b phosphorylation (DiTella et al., 1996), accelerates the redistribution of cdk5 to the axonal tip and dramatically increases its activity. Finally, our results indicate that cdk5 suppression by antisense oligonucleotide treatment selectively reduces axonal elongation and decreases the phosphorylation status of MAP1b, as well as its binding to microtubules. Taken collectively, our observations suggest that cdk5 may serve as an important regulatory linker between enviromental signals (e.g. laminin) and constituents of the intracellular machinery (e.g. MAP1b) involved in axonal formation.
GST-lamin fusion proteins act as dominant negative mutants in Xenopusegg extract and reveal the function of the lamina in DNA replication
Authors: Ellis Darren J. , Jenkins Hazel , Whitfield William G. F. , Hutchison Christopher J. 
Published: April 2021

ABSTRACT
A cDNA encoding Xlamin B1 was cloned from a whole ovary mRNA by RT-PCR. GST-lamin fusion constructs were generated from this cDNA by first creating convenient restriction sites within the Xlamin B1 coding sequence, using PCR directed mutagenesis, and then sub-cloning relevant sequences into pGEX-4T-3. Two expression constructs were made, the first, termed Δ2+ lacked sequences encoding the amino-terminal ‘head domain’ of lamin B1 but included sequences encoding the nuclear localization signal sequence (NLS). The second expression construct, termed Δ2-, lacked sequences encoding the amino-terminal ‘head domain’ as well as sequences encoding the NLS. Purified fusion proteins expressed from these constructs, when added to egg extracts prior to sperm pronuclear assembly, formed hetero-oligomers with the endogenous lamin B3. The Δ2+ fusion protein prevented nuclear lamina assembly but not nuclear membrane assembly. The resulting nuclei were small (∼10 µm in diameter), did not assemble replication centers and failed to initiate DNA replication. When the Δ2-fusion protein was added to egg extracts prior to sperm pronuclear assembly, lamina assembly was delayed but not prevented. The resulting nuclei although small (∼12 µm), did form replication centers and initiated DNA replication. When added to egg extracts after sperm pronuclear assembly was completed Δ2+, but not Δ2-, entered the pre-formed nuclei causing lamina disassembly. However, the disassembly of the lamina by Δ2+ did not result in the disruption of replication centers and indeed these centres remained functional. These results are consistent with the hypothesis that lamina assembly precedes and is required for the formation of replication centers but does not support those centers directly.
Assembly of A- and B-type lamins studied in vivo with the baculovirus system
Authors: Klapper Markus , Exner Kay , Kempf Anja , Gehrig Claudia , Stuurman Nico , Fisher Paul A. , Krohne Georg 
Published: April 2021

ABSTRACT
We have expressed an A-type lamin (Xenopus lamin A), a probable A-type lamin (Drosophila lamin C), two B-type lamins (Xenopus lamin LI, Drosophila lamin Dmo), and two mutants of Xenopus lamin A in Sf9 cells. All proteins were synthesized at high levels resulting in formation of paracrystals with an axial repeat of 18.5-20.0 nm by A-type lamins; in contrast B-type lamins assembled into aggregates with a fibrillar ultrastucture. Of the four wild-type proteins analyzed only lamin Dmo was found in the nuclear compartment of Sf9 cells in association with the lamina whereas the three other lamins assembled into polymers localized in the cytoplasm as well as the nucleoplasm.
The Xenopus lamin A mutant lacking the complete carboxy-terminal tail assembled in the cytoplasm into long filament bundles consisting of fibrils of less than 6 nm diameter. In vitro the non-helical amino-terminal head domain of lamins is required for the formation of ‘head-to-tail’ polymers. A lamin A mutant lacking this domain could be efficiently extracted from Sf9 cells with physiological buffers containing Triton X-100, demonstrating the importance of this domain for lamin assembly in vivo.