bluuebunny/crossref_metadata_2025_split huggingface dataset:
Depolarization of the actin cytoskeleton is a specific phenotype in Saccharomyces cerevisiae
Authors: Karpova Tatiana S. , Moltz Samuel L. , Riles Linda E. , Güldener Ulrich , Hegemann Johannes H. , Veronneau Steeve , Bussey Howard , Cooper John A. 
Published: April 2021

ABSTRACT
The yeast actin cytoskeleton is polarized during most of the cell cycle. Certain environmental factors and mutations are associated with depolarization of the actin cytoskeleton. Is depolarization of the actin cytoskeleton a specific response, or is it a nonspecific reaction to harsh conditions or poor metabolism? If depolarization is a nonspecific response, then any mutation that slows growth should induce depolarization. In addition, the number of genes with the depolarization phenotype should constitute a relatively large part of the genome. To address this question, we determined the effect of slow growth on the actin cytoskeleton, and we determined the frequency of mutations that affect the actin cytoskeleton. Eight mutants with slow growth showed no defect in actin polarization, indicating that slow growth alone is not sufficient to cause depolarization. Among 273 viable haploids disrupted for ORFs of chromosome I and VIII and 950 viable haploids with random genome disruptions, none had depolarization of the cytoskeleton. We conclude that depolarization of the actin cytoskeleton is a specific phenotype.
Flagellar mitochondrial association of the male-specific Don Juan protein in Drosophila spermatozoa
Authors: Santel Ansgar , Blümer Nicole , Kämpfer Matthias , Renkawitz-Pohl Renate 
Published: April 2021

ABSTRACT
The Drosophila don juan gene encodes a basic protein (Don Juan protein), which is solely expressed postmeiotically during spermiogenesis in elongated spermatids and in mature sperm. Transgenic expression of a GFP-tagged Don Juan protein (DJ-GFP) in the male germ line showed an association of the fusion protein with the sperm tail. Detailed examination of DJ-GFP localization revealed novel insights into its distinct temporal and spatial distribution along the sperm tail during the last phase of spermatid maturation. Co-localization of DJ-GFP with actin-labeled cysts demonstrated its emergence in elongated spermatids during individualization. Additionally, the endogenous Don Juan protein was detected with epitope-specific antibodies in finally elongated nuclei of spermatids. After completion of nuclear shaping Don Juan is no longer detectable in the sperm heads with the onset of individualization. Mislocalization of the DJ-GFP protein in flagella of a mutant with defective mitochondrial differentiation provides evidence of mitochondrial association of the fusion protein with flagellar mitochondrial arrays. Ectopically expressed DJ-GFP in premeiotic germ cells as well as salivary gland cells confirmed the capability of the fusion protein to associate with mitochondria. Therefore we suppose that Don Juan is a nuclear-encoded, germ-cell specifically expressed mitochondrial protein, which might be involved in the final steps of mitochondrial differentiation within the flagellum.
Actin ‘purse string’ filaments are anchored by E-cadherin-mediated adherens junctions at the leading edge of the epithelial wound, providing coordinated cell movement
Authors: Danjo Yukitaka , Gipson Ilene K. 
Published: April 2021

ABSTRACT
At the leading edge of healing embryonic epithelium, cables of actin filaments appear to extend from cell to cell, forming a ring around the wound circumference. It has been hypothesized that this actin filament cable functions as a contractile ‘purse string’ to facilitate wound closure. We have observed this cable in large, circular healing epithelial wounds in corneas of adult mice. To elucidate the role of the actin filament cable, we characterized the molecular components associated with the cell-cell junction where the actin filament cable inserts and with the actin filament cable itself, and we studied the effect of disruption of the cable using an E-cadherin function-blocking antibody, ECCD-1. Localization of E-cadherin and the direct association of catenins with actin filament cable at the cell-cell interface of the actin cable confirmed that the cell-cell junction associated with the actin filament cable is an adherens junction. The E-cadherin function-blocking antibody caused disruption of the actin filament cable and induction of prominent lamellipodial extensions on cells at the leading edge, leading to a ragged uneven epithelial wound margin. These data demonstrate that cell-to-cell associated E-cadherin molecules link the actin filament cable, forming a functional adherens junction, and that the actin filament cable plays a role in coordinating cell movement.
Conventional and novel PKC isoenzymes modify the heat-induced stress response but are not activated by heat shock
Authors: Holmberg Carina I. , Roos Pia M. K. , Lord Janet M. , Eriksson John E. , Sistonen Lea 
Published: April 2021

ABSTRACT
In mammalian cells, the heat-induced stress response is mediated by the constitutively expressed heat shock transcription factor 1 (HSF1). Upon exposure to elevated temperatures, HSF1 undergoes several post-translational modifications, including inducible phosphorylation or hyperphosphorylation. To date, neither the role of HSF1 hyperphosphorylation in regulation of the transcriptional activity of HSF1 nor the signaling pathways involved have been characterized. We have previously shown that the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), markedly enhances the heat-induced stress response, and in the present study we elucidate the mechanism by which PKC activation affects the heat shock response in human cells. Our results show that several conventional and novel PKC isoenzymes are activated during the TPA-mediated enhancement of the heat shock response and that the enhancement can be inhibited by the specific PKC inhibitor bisindolylmaleimide I. Furthermore, the potentiating effect of TPA on the heat-induced stress response requires an intact heat shock element in the hsp70 promoter, indicating that PKC-responsive pathways are able to modulate the activity of HSF1. We also demonstrate that PKC is not activated by heat stress per se. These results reveal that PKC exhibits a significant modulatory role of the heat-induced stress response, but is not directly involved in regulation of the heat shock response.
Entry into mitosis without Cdc2 kinase activation
Authors: Gowdy Patrick M. , Anderson Hilary J. , Roberge Michel 
Published: April 2021

ABSTRACT
Mouse FT210 cells at 39°C cannot enter mitosis but arrest in G2 phase, because they lack Cdc2 kinase activity as a result of a temperature-sensitive lesion in the cdc2 gene. Incubation of arrested cells with the protein phosphatase 1 and 2A inhibitor okadaic acid induces morphologically normal chromosome condensation. We now show that okadaic acid also induces two other landmark events of early mitosis, nuclear lamina depolymerization and centrosome separation, in the absence of Cdc2 kinase activity. Okadaic acid-induced entry into mitosis is accompanied by partial activation of Cdc25C and may be prevented by tyrosine phosphatase inhibitors and by the protein kinase inhibitor staurosporine, suggesting that Cdc25C and kinases distinct from Cdc2 are required for these mitotic events. Using in-gel assays, we show that a 45-kDa protein kinase normally activated at mitosis is also activated by okadaic acid independently of Cdc2 kinase. The 45-kDa kinase can utilize GTP, is stimulated by spermine and is inhibited by heparin. These properties are characteristic of the kinase CK2, but immunoprecipitation studies indicate that it is not CK2. The data underline the importance of a tyrosine phosphatase, possibly Cdc25C, and of kinases other than Cdc2 in the structural changes the cell undergoes at mitosis, and indicate that entry into mitosis involves the activation of multiple kinases working in concert with Cdc2 kinase.
α-COP can discriminate between distinct, functional di-lysine signals in vitro and regulates access into retrograde transport
Authors: Schröder-Köhne Stephan , Letourneur Francois , Riezman Howard 
Published: April 2021

ABSTRACT
Emp47p is a yeast Golgi transmembrane protein with a retrograde, Golgi to ER transport di-lysine signal in its cytoplasmic tail. Emp47p has previously been shown to recycle between the Golgi complex and the ER and to require its di-lysine signal for Golgi localization. In contrast to other proteins with di-lysine signals, the Golgi-localization of Emp47p has been shown to be preserved in ret1-1 cells expressing a mutant α-COP subunit of coatomer. Here we demonstrate by sucrose gradient fractionation and immunofluorescence analysis that recycling of Emp47p was unimpaired in ret1-1. Furthermore we have characterized three new alleles of ret1 and showed that Golgi localization of Emp47p was intact in cells with those mutant alleles. We could correlate the ongoing recycling of Emp47p in ret1-1 with preserved in vitro binding of coatomer from ret1-1 cells to immobilized GST-Emp47p-tail fusion protein. As previously reported, the di-lysine signal of Wbp1p was not recognized by ret1-1 mutant coatomer, suggesting a possible role for α-COP in the differential binding to distinct di-lysine signals. In contrast to results with α-COP mutants, we found that Emp47p was mislocalised to the vacuole in mutants affecting β’-, γ-, δ-, and ζ-COP subunits of coatomer and that the mutant coatomer bound neither to the Emp47p nor to the Wbp1p di-lysine signal in vitro. Therefore, the retrograde transport of Emp47p displayed a differential requirement for individual coatomer subunits and a special role of α-COP for a particular transport step in vivo.
Histone H3 phosphorylation is required for the initiation, but not maintenance, of mammalian chromosome condensation
Authors: Hooser Aaron Van , Goodrich David W. , Allis C. David , Brinkley B. R. , Mancini Michael A. 
Published: April 2021

ABSTRACT
The temporal and spatial patterns of histone H3 phosphorylation implicate a specific role for this modification in mammalian chromosome condensation. Cells arrest in late G2 when H3 phosphorylation is competitively inhibited by microinjecting excess substrate at mid-S-phase, suggesting a requirement for activity of the kinase that phosphorylates H3 during the initiation of chromosome condensation and entry into mitosis. Basal levels of phosphorylated H3 increase primarily in late-replicating/early-condensing heterochromatin both during G2 and when premature chromosome condensation is induced. The prematurely condensed state induced by okadaic acid treatment during S-phase culminates with H3 phosphorylation throughout the chromatin, but in an absence of mitotic chromosome morphology, indicating that the phosphorylation of H3 is not sufficient for complete condensation. Mild hypotonic treatment of cells arrested in mitosis results in the dephosphorylation of H3 without a cytological loss of chromosome compaction. Hypotonic-treated cells, however, complete mitosis only when H3 is phosphorylated. These observations suggest that H3 phosphorylation is required for cell cycle progression and specifically for the changes in chromatin structure incurred during chromosome condensation.
DNA-mediated transport of the intermediate filament protein vimentin into the nucleus of cultured cells
Authors: Hartig Roland , Shoeman Robert L. , Janetzko Alfred , Tolstonog Genrich , Traub Peter 
Published: April 2021

ABSTRACT
A number of characteristic properties of intermediate filament (IF) proteins, such as nucleic acid-binding activity, affinity for histones and structural relatedness to transcription factors and nuclear matrix proteins, in conjunction with the tight association of IFs with the nucleus, suggest that these proteins might also fulfill nuclear functions in addition to their structure-organizing and -stabilizing activities in the cytoplasm. Yet, cytoplasmic IF proteins do not possess nuclear localization signals. In a search for carriers capable of transporting the IF protein vimentin into the nucleus, complexes of FITC-vimentin with various DNAs were microinjected into the cytoplasm of cultured cells and the intracellular distribution of the protein was followed by confocal laser scanning microscopy. The single-stranded oligodeoxyribonucleotides oligo(dG)25, oligo[d(GT)12G] and oligo[d(G3T2A)4G] proved to be excellent nuclear carriers for vimentin. However, in fibroblasts, fluorescence-labeled vimentin taken up by the nuclei remained undetectable with affinity-purified, polyclonal anti-vimentin antibody, whereas it was readily identifiable in the nuclei of microinjected epithelial cells in this way. Moreover, when FITC-vimentin was preinjected into fibroblasts and allowed to assemble into the endogenous vimentin filament system, it was still transferred into the nucleus by post-injected oligo(dG)25, although to a lesser extent. Superhelical circular DNAs, like pBR322, SV40 and mitochondrial DNA, were also characterized by considerable capacities for nuclear vimentin transport; these transport potentials were totally destroyed by relaxation or linearization of the DNA molecules. Nevertheless, certain linear double-stranded DNA molecules with a high affinity for vimentin IFs, such as repetitive telomere and centromere or mobile long interspersed repeat (LINE) DNA, could carry FITC-vimentin into the nucleus. This was also true for a 375 bp extrachromosomal linear DNA fragment which occurs in the cytoplasm of mouse tumor cells and which is capable of immortalizing human lymphocytes. On the basis of these results, it appears very likely that cellular and viral products of reverse transcription as well as other extrachromosomal DNAs, which are circular, superhelical and apparently shuttling between the cytoplasm and the nucleus (eccDNA), are constantly loaded with vimentin in vimentin-positive cells. Since such DNAs are considered as markers of genomic instability, it is conceivable that vimentin directly participates as an architectural, chromatin-modifying protein in recombinatorial processes set off by these DNAs in the nucleus.
Myr 7 is a novel myosin IX-RhoGAP expressed in rat brain
Authors: Chieregatti Evelina , Gärtner Annette , Stöffler Hanns-Eugen , Bähler Martin 
Published: April 2021

ABSTRACT
Rho family GTPases are important regulators of neuronal morphology, but the proteins directly controlling their activity in neurons are still poorly defined. We report the identification of myr 7, a novel unconventional myosin IX-RhoGAP expressed in rat brain. Myr 7 is a multidomain protein related to myr 5, the first class IX myosin to be characterized. It exhibits a myosin head domain with an N-terminal extension and a large insertion at loop 2, an actin contact site and regulator of myosin ATPase rate. The myosin head domain is followed by a neck domain consisting of six unevenly spaced consecutive IQ motifs representing light chain binding sites. The tail domain contains a C6H2-zinc binding motif and a region that specifically stimulates the GTPase-activity of Rho followed by a short stretch predicted to adopt a coiled-coil structure. Five alternatively spliced regions, one in the 5′-noncoding region, two in the myosin head and two in the tail domain, were noted. Analysis of myr 7 and myr 5 expression in different tissues revealed that myr 7 is expressed at high levels in developing and adult brain tissue whereas myr 5 is expressed only at moderate levels in embryonic brain tissue and at even further reduced levels in adult brain tissue. Myr 5 is, however, highly expressed in lung, liver, spleen and testis. Myr 7 is expressed in all brain regions and is localized in the cytoplasm of cell bodies, dendrites and axons. Myr 5 exhibits an overlapping, but not identical cellular distribution. Finally, a myr 7 fusion protein encompassing the GAP domain specifically activates the GTPase-activity of Rho in vitro, and overexpression of myr 7 in HtTA1-HeLa cells leads to inactivation of Rho in vivo. These results are compatible with a role for myr 7 (and myr 5) in regulating Rho activity in neurons and hence in regulating neuronal morphology and function.
Key words: Unconventional myosin, RhoGAP, Neuron, Rat
The actin-binding proteins adseverin and gelsolin are both highly expressed but differentially localized in kidney and intestine
Authors: Lueck Andreas , Brown Dennis , Kwiatkowski David J. 
Published: April 2021

ABSTRACT
To understand the distinct functions of the closely related actin-severing proteins adseverin and gelsolin, we examined the expression of these proteins in detail during mouse and human development using a new highly sensitive and specific set of antibody reagents. Immunoblot analysis demonstrated that adseverin was highly expressed in mouse kidney and intestine at all stages of development and in human fetal and adult kidney. In contrast and as reported previously, gelsolin was expressed much more widely in both murine and human tissues.
Immunohistochemistry on murine kidney sections revealed a predominantly differential localization of adseverin and gelsolin. Adseverin was expressed in peripolar cells, thin limbs, thick ascending limbs, and principal cells of cortical and medullary collecting ducts where it was diffusely localized in the cytoplasm. Gelsolin was expressed in the distal convoluted tubule, intercalated cells and principal cells of cortical and medullary collecting ducts, and in ureter. In the distal convoluted tubule, gelsolin showed a diffuse distribution and in principal cells of collecting ducts a localization at the basolateral pole. In intercalated cells, gelsolin localization was heterogeneous, either at the apical pole or diffusely in the cytoplasm. In human fetal and adult kidney, adseverin was expressed only in collecting ducts whereas gelsolin was expressed in thick ascending limbs and collecting ducts.
In mouse and human intestine adseverin was expressed in enterocytes with a gradient of increasing expression from the duodenum to the colon, and from the crypt to the villus. The observations indicate high level expression of adseverin in specific cells of the kidney and colon, and suggest a previously unrecognized function of adseverin in epithelial cell function.
Coiled body numbers in the Arabidopsis root epidermis are regulated by cell type, developmental stage and cell cycle parameters
Authors: Boudonck Kurt , Dolan Liam , Shaw Peter J. 
Published: April 2021

ABSTRACT
We have used whole mount immunofluorescence labelling with the antibody 4G3, raised against the human snRNP-specific protein U2B″, and whole mount in situ hybridization with an anti-sense probe to a conserved region of U2 snRNA, in combination with confocal microscopy, to examine the organization of spliceosomal components throughout the development of the Arabidopsis thaliana root epidermis. We show that the number of coiled bodies, nuclear organelles in which splicing snRNPs and snRNAs concentrate, is developmentally regulated in the Arabidopsis root epidermis. Firstly, there is a progression from a small number of coiled bodies in the quiescent centre and initial cells, to a larger number in the cell division zone, returning to a lower number in the cell elongation and differentiation zone. Secondly, trichoblasts (root-hair forming epidermal cells) have on average 1.5 times more and often smaller coiled bodies than atrichoblasts (hairless epidermal cells). Moreover, we have shown that these differences in coiled body numbers are related to differences in cell cycle stage, cell type and developmental stage, but are not due to differences in nucleolar or general metabolic activity per se. We discuss possible explanations, including a model in which coiled bodies coalesce during interphase, for the developmental dynamics of coiled bodies.
A propagated wave of mpf activation accompanies surface contraction waves at first mitosis in xenopus
Authors: Pérez-Mongiovi Daniel , Chang Patrick , Houliston Evelyn 
Published: April 2021

ABSTRACT
During the period of mitosis, two surface contraction waves (SCWs) progress from the animal to vegetal poles of the Xenopus egg. It has been shown that these SCWs occur in parallel with the activation of MPF and with its subsequent inactivation in the animal and vegetal hemispheres, suggesting that they are responses to propagated waves of MPF activity across the egg. We have analysed the mechanism of MPF regulation in different regions of the egg in detail in relation to SCW progression. The distributions of histone HI kinase activity and of Cdc2 and cyclin B (the catalytic and regulatory subunits of MPF) were followed by dissection of intact eggs following freezing and in cultured fragments separated by ligation. Cdc2 was found to be distributed evenly throughout the egg cytoplasm. Loss of phosphorylated (inactive) forms of Cdc2 coincided spatially with the wave of MPF activation, while cyclin B2 accumulation occurred in parallel in animal and vegetal regions. In ligated vegetal pole fragments no MPF activation or Cdc2 dephosphorylation were detectable. A wave of cyclin B destruction that occurred in concert with the second SCW was also blocked. Taken together these results indicate that the triggering mechanism for MPF activation requires components specific to the animal cytoplasm, acting via Cdc2 dephosphorylation, and that MPF activation subsequently propagates autocatalytically across the egg.
SCW progression in the vegetal hemisphere was followed directly by time-lapse videomicroscopy of subcortical mitochondrial islands. The first SCW traversed the vegetal pole at the time of MPF activation in this region. Like MPF activation and inactivation, SCWs were blocked in the vegetal region by ligation. These observations reinforce the hypothesis that the first SCW is a direct consequence of the MPF activation wave. It may reflect depolymerisation of the subcortical microtubule network since it coincided exactly with the arrest of the microtubule-dependent movement of ‘cortical rotation’ and was related in direction in most eggs. The cyclin B destruction wave and associated cortical contraction of the second SCW may be localised downstream consequences of the MPF activation wave, or they may propagate independently from the animal cytoplasm.
The αvβ5 integrin functions as an endocytic receptor for vitronectin
Authors: Memmo Loretta M. , McKeown-Longo Paula 
Published: April 2021

ABSTRACT
Endocytosis and degradation of vitronectin by human skin fibroblasts are regulated by the β5 integrin. To determine whether the β5 integrin is directly mediating the internalization of vitronectin, both vitronectin and the β5 integrin were localized by indirect immunofluorescence during the endocytic process. This analysis showed that both vitronectin and β5 were found in intracellular vesicles within 5 minutes of the addition of exogenous vitronectin to fibroblast cell layers. By 15 minutes, approximately 20% of the vitronectin-containing vesicles stained positively for β5. In contrast, the β3 integrin was not found in any intracellular vesicles. Within 30 minutes, more than 50% of vitronectin-containing vesicles also stained for lamp-1, indicating that internalized vitronectin traveled to lysosomes. Inhibition of clathrin assembly by either potassium depletion or hypertonic buffer inhibited vitronectin internalization, suggesting that vitronectin internalization occurred through coated pits.
Confocal analysis confirmed the colocalization of vitronectin and αvβ5 in intracellular compartments and further demonstrated that the highest colocalization of the two proteins occurred within 1.8 μm from the ventral surface of the cell, suggesting endocytosis occurred at the substrate level. Pretreatment of cells with the PI-3 kinase inhibitor, wortmannin, resulted in a marked increase in the coincidence of vitronectin and β5 staining within vesicles and prevented the accumulation of vitronectin within lysosomes. This suggests that following internalization, vitronectin and the αvβ5 integrin are segregated to different cellular compartments. This study provides the first evidence that the αvβ5 vitronectin receptor directly mediates the internalization of vitronectin.