bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enhancement of the ncd
D microtubule motor mutant by mutants of αTub67C
Authors: Komma Donald J. , Endow Sharyn A. 
Published: April 2021

ABSTRACT
Ncd is a kinesin-related microtubule motor protein required for chromosome segregation in Drosophila oocytes and early embryos. In tests for interactions with other proteins, we find that mutants of αTub67C, which affect an oocyte- and early embryo-specific α-tubulin, enhance meiotic nondisjunction and zygotic loss of ncdD, a partial loss-of-function mutant of ncd. The enhancement is dominant and allele-specific with respect to αTub67C, and depends on the recessive effects of ncdD. Cytologically, embryos of αTub67C/+ show delayed meiotic divisions and defective female pronucleus formation, while meiotic spindle assembly is abnormal in embryos of ncdD/ncdD. Doubly mutant αTub67C ncdD/ncdD embryos are rescued for female pronucleus formation, but show delayed meiotic progression and defective pronuclear conjugation or fusion. Delayed completion of meiosis, together with failure of pronuclear fusion, prevents normal interactions of maternal with paternal chromosomes, enhancing the ncdD mutant phenotype. The genetics and cytology of doubly mutant embryos and the molecular defect of NcdD provide evidence for interaction of Ncd with αTub67C in vivo. These results imply that a specific α-tubulin isoform is required for normal cellular function of a kinesin motor protein.
Transcription-dependent redistribution of nuclear protein 4.1 to SC35-enriched nuclear domains
Authors: Lallena María-José , Correas Isabel 
Published: April 2021

ABSTRACT
Protein 4.1, originally identified as a component of the membrane-skeleton of the red blood cell, has also been localized in the nucleus of mammalian cells. To learn more about nuclear 4.1 protein, we have analyzed the nature of its association with the nuclear structure in comparison with SC35 and snRNP antigens, splicing proteins of the nuclear speckle domains. When MDCK or HeLa cells were digested with DNase I and washed in the presence of high salt (2 M NaCl), snRNP antigens were extracted whereas protein 4.1 and SC35 remained colocalizing in nuclear speckles. In cells treated with RNase A or heat shocked, nuclear 4.1 distribution also resembled that of SC35. Experiments carried out in transcriptionally active nuclei showed that protein 4.1 distributed in irregularly shaped speckles which appeared to be interconnected. During transcriptional inhibition, protein 4.1 accumulated in rounded speckles lacking interconnections. When cells were released from transcriptional inhibition, protein 4.1 redistributed back to the interconnected speckle pattern of transcriptionally active cells, as it was also observed for SC35. Finally, coprecipitation of 4.1 and SC35 proteins from RNase A digested HeLa nuclei further indicates that these two proteins are associated, forming part of the nuclear speckle domains to which they attach more tightly than snRNP antigens.
Localisation of the Schizosaccharomyces pombe rho1p GTPase and its involvement in the organisation of the actin cytoskeleton
Authors: Arellano Manuel , Duran Angel , Perez Pilar 
Published: April 2021

ABSTRACT
The Schizosaccharomyces pombe rholp GTPase directly activates the (1-3)β-D-glucan synthase and participates in the regulation of cell wall growth and morphogenesis in this fission yeast. Indirect immunofluorescence experiments using rho1p tagged with hemagglutinin have revealed that rho1p was located at the growing tips during interphase and at the septum prior to cytokinesis, localising to the same areas as actin patches. In S. pombe cdc10-129 mutant cells, arrested in G1, HA-rho1p accumulates at one tip whereas in cdc25-22 mutants, arrested in G2, HA-rho?p accumulates at both tips. In tea1-1 and tea2-1 cdc11-119 mutant cells, HA-rho?p is localised to the new growing tips. Overexpression of different rhol mutant alleles caused different effects on cortical actin patch distribution, (1-3)β-D-glucan synthase activation, and sensitivity to cell wall specific antifungal drugs. These results indicate that multiple cellular components are activated by rholp. Overexpression of the dominant negative rhol?20N allele was lethal as was the rho1+ deletion. Moreover, when rho1+ expression was repressed in actively growing S. pombe, cells died in about 10 to 12 hours. Under these conditions, normal cell morphology was maintained but the level of (1-3)β-D-glucan synthase activity decreased and the actin patches disappeared. Most cells lysed after cytokinesis during the process of separation, and lysis was not prevented by an osmotic stabiliser. We conclude that rho1p localisation is restricted to growth areas and regulated during the cell cycle and that rho1p is involved in cell wall growth and actin cytoskeleton organisation in S. pombe.
Extinction of immunoglobulin gene expression in B cells upon fusion with HeLa cells is preceded by rapid nuclear depletion of essential transcription factors and is accompanied by widespread inactivation of genes expressed in a B cell-specific manner
Authors: Junker Steffen , Lamm Martin , Nielsen Viggo , Matthias Patrick 
Published: April 2021

ABSTRACT
When immunoglobulin (Ig) expressing B cells are fused with non-B cells, Ig expression is rapidly suppressed at the level of transcription, a phenomenon termed extinction. Here we demonstrate that fusion of HeLa cells with either diploid or tetraploid B cells (Daudi) results in widespread extinction of several other B cell-encoded genes that are expressed in a B cell-specific manner. In contrast, expression of B cell-expressed genes that are not dependent on cell-specific controls is unaffected. We show that the molecular mechanism(s) underlying Ig gene extinction can be explained, at least in part, by a lack of transcription factors that are essential for Ig gene transcription. These transcription factors are either not produced due to block of transcription of their respective genes (Oct-2, OBF-1, PU.1), or are rendered inactive posttranslationally (NF-κB, E47). By isolating Daudi × HeLa heterokaryons a few hours after fusion, we have studied the initial fate of two B cellspecific transcription factors involved in Ig gene transcription, Oct-2 and NF-κB. This report provides the first demonstration that upon fusion with HeLa cells, the nuclear contents of B cell-expressed transcription factors are depleted within a few hours with kinetics that are as fast or faster than that of Ig gene extinction. Thus, the extinguishing mechanism is effective very early after fusion. We suggest that extinction of Ig genes is part of a global mechanism that suppresses the differentiation program foreign to the HeLa phenotype.
Commitment to differentiation and cell cycle re-entry are coincident but separable events in the transformation of African trypanosomes from their bloodstream to their insect form
Authors: Matthews Keith R. , Gull Keith 
Published: April 2021

ABSTRACT
African trypanosomes undergo extensive changes in cellular morphology, biochemistry and surface antigen expression as they differentiate from their bloodstream form to those forms that colonise the midgut of their tsetse fly vector. If initiated with stumpy-form cells, a nondividing sub-type of the bloodstream parasite, differentiation and cell cycle re-entry occur synchronously in the population and provide a means to dissect the respective controls of proliferation and transformation. We have exploited this synchrony to determine the respective importance and hierarchy of the known triggers for differen-tiation (cis aconitate, temperature drop) for individual components of both differentiation and the cell cycle. This has revealed the pre-eminence of cis aconitate as a primary trigger for parasite differentiation, and has allowed us to determine that the cellular commitment to both differentiation and cell-cycle re-entry are precisely co-incident processes
To bead or not to bead? Lens-specific intermediate filaments revisited
Authors: Georgatos Spyros D. , Gounari Fotini , Goulielmos George , Aebi Ueli 
Published: April 2021

ABSTRACT
For nearly three decades cytoplasmic intermediate filaments (IFs) have been described as 10 nm thick, unbranched ropes radiating from the cell nucleus and extending to the plasma membrane. This stereotype is now being challenged by the discovery and molecular characterization of the beaded filaments (BFs), a novel class of IFs composed of the lens-specific proteins filensin and phakinin. In contrast to ‘mainstream’ IFs, BFs have a distinctly nodular appearance and form a meshwork underneath the plasma membrane of the lens fiber cells. In vitro assembly studies, expression of filensin and phakinin in cultured cells, and analysis of the corresponding genes reveal that these proteins have evolved from two different subfamilies of IF proteins, thus yielding a unique structure. The new information provides a basis for understanding how the various forms of tissue-specific IF proteins might have developed adopting to the constraints of a specialized environment.
Distinct mechanisms of nuclear accumulation regulate the functional consequence of E2F transcription factors
Authors: Allen K. Elizabeth , Luna Susana de la , Kerkhoven Ron M. , Bernards René , Thangue Nicholas B. La 
Published: April 2021

ABSTRACT
Transcription factor E2F plays an important role in co-ordinating and integrating early cell cycle progression with the transcription apparatus. It is known that physiological E2F arises when a member of two families of proteins, E2F and DP, interact as E2F/DP heterodimers and that transcriptional activity is regulated through the physical association of pocket proteins such as pRb. However, little information is available regarding the mechanisms which control the levels of functional E2F. In this study, we have characterised one such mechanism which regulates the nuclear accumulation and activity of E2F. Specifically, we show that E2F proteins fall into two distinct categories according to their ability to accumulate in nuclei, one being exemplified by E2F-1 and the other by E2F-4 and -5. Thus, E2F-1 possesses an intrinsic nuclear localization signal whereas E2F-4 and -5 are devoid of such a signal. Furthermore, we find for E2F-4 and -5 that two distinct processes govern their nuclear accumulation whereby the nuclear localization signal is supplied in trans from either a DP heterodimer partner or a physically associated pocket protein. It is consistent with the role of pocket proteins in regulating nuclear accumulation that we find E2F-5 to be nuclear during early cell cycle progression with an increased cytoplasmic concentration in cycling cells. Our data show that the mechanism of nuclear accumulation determines the functional consequence of E2F on cell cycle progression: pocket protein-mediated accumulation impedes cell cycle progression, whereas DP-regulated nuclear accumulation promotes cell cycle progression. Moreover, the inactivation of pocket proteins by the adenovirus E1a protein, and subsequent release of E2F, failed to displace nuclear E2F. Our study identifies a new level of regulation in the control of E2F activity exerted at the level of nuclear accumulation where subunit composition and interaction with pocket proteins dictates the functional consequence on cell cycle progression.
Nuclear export signal of IκBα interferes with the Rev-dependent posttranscriptional regulation of human immunodeficiency virus type I
Authors: Bachelerie Françoise , Rodriguez Manuel S. , Dargemont Catherine , Rousset Dominique , Thomas Dominique , Virelizier Jean-Louis , Arenzana-Seisdedos Fernando 
Published: April 2021

ABSTRACT
De novo synthesized IκBα accumulates transiently in the nucleus where it inhibits NF-κB-dependent transcription and reduces nuclear NF-κB content. A sequence present in the C-terminal domain of IκBα and homologous to the HIV-1 Rev nuclear export signal (NES) has been recently defined as a functional NES conferring on IκBα the ability to export IκBα/NF-κB complexes. Rev utilises its RNA-binding activity and NES sequence to promote specifically the transport of unspliced and monospliced viral RNAs to the cytoplasm. The object of this work was to determine if nuclear IκBα could interfere with Revdependent transport of viral RNA from the nucleus to the cytoplasm. We report that accumulation of IκBα in the cell nucleus blocks viral replication. This effect could be dissociated from the capacity of IκBα to inhibit NF-κB-DNA-binding activity and required a functional IκBα NES motif. Indeed, mutation of the NES abrogated the capacity of IκBα to inhibit Rev-dependent mechanisms involved in the replication of either wild-type or NF-κB-mutated HIV-1 molecular clones. Nuclear accumulation of a reporter protein tagged with a nuclear localization signal (NLS) and fused to the IκBα NES motif (NLS-PK-NES) was sufficient to inhibit HIV-1 replication at a post-transcriptional level by specifically blocking the expression of a Rev-dependent gene. Furthermore, in cells pulsed with TNF, a treatment which favors nuclear accumulation of newly synthesized IκBα, NLS-PK-NES expression promoted sustained accumulation of nuclear NF-κB lacking DNA-binding activity. This NES-mediated accumulation of inactive nuclear NF-κB is likely the consequence of interference in the IκBα-mediated export of NF-κB. These findings indicate that IκBα and Rev compete for the same nuclear export pathway and suggest that nuclear accumulation of IκBα, which would occur during normal physiological cell activation process, may interfere with the Rev-NES-mediated export pathway of viral RNAs, thus inhibiting HIV-1 replication.
Contact guidance of CNS neurites on grooved quartz: influence of groove dimensions, neuronal age and cell type
Authors: Rajnicek Ann M. , Britland Stephen , McCaig Colin D. 
Published: April 2021

ABSTRACT
We used an in vitro system that eliminates competing guidance cues found in embryos to determine whether sub-stratum topography alone provides important neurite guidance information. Dissociated embryonic Xenopus spinal cord neurons and rat hippocampal neurons were grown on quartz etched with a series of parallel grooves. Xenopus neurites grew parallel to grooves as shallow as 14 nm and as narrow as 1 μm. Hippocampal neurites grew parallel to deep, wide grooves but perpendicular to shallow, narrow ones. Grooved substrata determined the sites at which neurites emerged from somas: Xenopus neurites sprouted from regions parallel to grooves but presumptive axons on rat hippocampal neurons emerged perpendicular to grooves and presumptive dendrites emerged parallel to them. Neurites grew faster in the favored direction of orientation and turned through large angles to align on grooves. The frequency of perpendicular alignment of hippocampal neurites depended on the age of the embryos from which neurons were isolated, suggesting that contact guidance is regulated in development. Collectively, the data indicate that substratum topography is a potent morphogenetic factor for developing CNS neurons and suggest that in addition to a role in pathfinding the geometry of the embryo assists in establishing neuronal polarity. In the companion paper (A. M. Rajnicek and C. D. McCaig (1997) J. Cell Sci. 110, 2915-2924) we explore the cellular mechanism for contact guidance of growth cones.
Guidance of CNS growth cones by substratum grooves and ridges: effects of inhibitors of the cytoskeleton, calcium channels and signal transduction pathways
Authors: Rajnicek Ann M. , McCaig Colin D. 
Published: April 2021

ABSTRACT
We exploited our observation that embryonic Xenopus spinal neurites align parallel to grooves in a quartz surface and that embryonic rat hippocampal neurites align perpendicular to shallow, narrow grooves (see companion paper: A. M. Rajnicek, S. Britland and C. D. McCaig, 1997) (J. Cell Sci. 110, 2905-2913) to investigate the mechanism of growth cone contact guidance. Substratum topography affected the pattern of growth cone filopodia and microtubules but parallel orientation of Xenopus neurites and perpendicular orientation of hippocampal neurites were unperturbed by cytochalasin B, which virtually eliminated filopodia. Hippocampal growth cone orientation and turning in response to grooves was unaffected by disruption of microtubules using taxol or nocodazole. Gross cytoskeletal reorganization on grooved substrata was therefore not required for growth cone steering.
Inhibitors were used to identify the signal transduction pathway for perpendicular alignment of hippocampal neurites. Alignment persisted in the presence of gadolinium chloride, a blocker of stretch-activated calcium channels, the G protein inhibitor pertussis toxin, the protein tyrosine kinase inhibitor genistein, the protein kinase A and G inhibitor HA1004, the protein kinase A inhibitor KT5720and the protein kinase G inhibitor KT5823. Low concentrations of the protein kinase C inhibitors staurosporine, bisindolylmaleimide or H-7 did not affect perpendicular orientation but higher concentrations inhibited it. The calcium channel blockers flunarizine, nifedipine and diltiazem also inhibited perpendicular orientation. Influx of calcium and protein kinase C activity therefore appear to be involved in perpendicular contact guidance.
A heterotrimeric G protein-phospholipase A2 signaling cascade is involved in the regulation of peroxisomal motility in CHO cells
Authors: Huber Christoph M. , Saffrich Rainer , Anton Markus , Paßreiter Michael , Ansorge Wilhelm , Gorgas Karin , Just Wilhelm W. 
Published: April 2021

ABSTRACT
Peroxisomal motility was studied in vivo in CHO cells following transfection with a green fluorescent protein construct containing the C-terminal peroxisomal targeting signal 1 (GFP-PTS1). Time-lapse imaging and evaluation of difference images revealed that peroxisomes attach to microtubules in a Ca2+ requiring step and are transported in an ATP-dependent manner.
Following microinjection of guanosine-5′-O-(3-thiotriphosphate) (GTPγS), peroxisomal movements were arrested, indicating regulation by GTP-binding proteins. The effect of GTPγS was mimicked by AlF4− and mastoparan, two drugs which are known to activate heterotrimeric G proteins. Pertussis toxin which prevents Gi/Go protein activation completely abolished the effect of GTPγS and mastoparan on peroxisomal motility suggesting that the G protein belongs to the Gi/Go class. At least one effector of the G protein is phospholipase A2 as demonstrated by the observation that the phospholipase A2 activating protein peptide efficiently blocks peroxisomal motility, and that the effect of mastoparan and AlF4− is largely abolished by various phospholipase A2 inhibitors.
In summary, these data provide evidence for a new type of regulation of organelle motility mediated by a Gi/Go- phospholipase A2 signaling pathway. This type of regulation has not been observed so far with other cell organelles such as mitochondria, the endoplasmic reticulum or axonal vesicles. Thus, motility is regulated individually for each cell organelle by distinct mechanisms enabling the cell to fulfill its vital functions.
2′,3′-Cyclic nucleotide 3′-phosphodiesterase is associated with mitochondria in diverse adrenal cell types
Authors: McFerran Brian W. , Burgoyne Robert D. 
Published: April 2021

ABSTRACT
In this study, we have examined the expression and intracellular localisation of the myelin protein 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) in bovine adrenal medullary chromaffin cell cultures. By immunoblotting, using two distinct anti-CNP monoclonal antibodies, CNP was detected in medullary cell cultures and expression of CNP was confirmed by reverse transcription and PCR amplification. CNP did not leak from digitonin-permeabilised chromaffin cells, suggesting that there is no cytosolic pool of this protein. Immunofluorescence studies with both antibodies showed that all cells in the medullary chromaffin cell culture were stained with a punctate appearance consistent with an intracellular localisation for CNP. More specifically it was demonstrated that CNP is colocalised with mitochondria. Various cell types in chromaffin cell cultures were stained with a mitochondrial pattern and CNP staining was co-localised with mitochondrial staining. These results show that CNP is a widely expressed protein that is associated with mitochondria and provides new clues as to its cellular function outside of myelin structures.
Ezrin: A protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures
Authors: Bretscher Anthony , Reczek David , Berryman Mark 
Published: April 2021

ABSTRACT
The cortical cytoskeleton of eucaryotic cells provides structural support to the plasma membrane and also contributes to dynamic processes such as endocytosis, exocytosis, and transmembrane signaling pathways. The ERM (ezrinradixin-moesin) family of proteins, of which ezrin is the best studied member, play structural and regulatory roles in the assembly and stabilization of specialized plasma membrane domains. Ezrin and related molecules are concentrated in surface projections such as microvilli and membrane ruffles where they link the microfilaments to the membrane.
The present knowledge about ezrin is discussed from an historical perspective. Both biochemical and cell biological studies have revealed that ezrin can exist in a dormant con-formation that requires activation to expose otherwise masked association sites. Current results indicate that activated ezrin monomers or head-to-tail oligomers associate directly with F-actin through a domain in its C terminus, and with the membrane through its N-terminal domain. The association of ezrin with transmembrane proteins can be direct, as in the case of CD44, or indirect through EBP50. Other binding partners, including the regulatory subunit of protein kinase A and rho-GDI, suggest that ezrin is an integral component of these signaling pathways. Although the membrane-cytoskeletal linking function is clear, further studies are necessary to reveal how the activation of ezrin and its association with different binding partners is regulated.
Dynamics of connexins, E-cadherin and α-catenin on cell membranes during gap junction formation
Authors: Fujimoto Kazushi , Nagafuchi Akira , Tsukita Shoichiro , Kuraoka Akio , Ohokuma Akiko , Shibata Yosaburo 
Published: April 2021

ABSTRACT
We examined the dynamics of connexins, E-cadherin and α-catenin during gap-junction disassembly and assembly in regenerating hepatocytes by immunofluorescence microscopy, and immunogold-electron microscopy using SDS-digested freeze-fracture replicas. The present findings suggest that during the disappearance of gap junctions most of the gap junction plaques are broken up into smaller aggregates, and then the gap junction proteins may be removed from the cell membrane, but some of the connexons or connexins remain dispersed in the plane of membrane as pure morphologically indistinguishable intramembrane proteins. Double-immunogold electron microscopy using a polyclonal antibody for connexins and a monoclonal antibody for E-cadherin or α-catenin revealed co-localization of these molecules at cell-to-cell contact sites during the reappearance of gap junction plaques. This implies that, at least in regenerating hepatocytes, the cadherin-catenin complex-mediated cell-to-cell contact sites act as foci for gap junction formation. In addition, connexin-immunoreactivity was also observed along tight junctional strands, suggesting that the gap junction may also form along the tight junctions.