bluuebunny/crossref_metadata_2025_split huggingface dataset:
Involvement of the rfp tripartite motif in protein-protein interactions and subcellular distribution
Authors: Cao Tongyu , Borden Kathrine L. B. , Freemont Paul S. , Etkin Laurence D. 
Published: April 2021

ABSTRACT
The ret finger protein (rfp) is a member of the B box zinc finger gene family which possesses a tripartite motif consisting of a RING finger, B box finger, and a coiled-coil domain. Rfp is expressed at specific stages of spermatogenesis and in various adult mouse and human tissues. It becomes oncogenic when the tripartite domain is recombined with the tyrosine kinase domain of the ret protooncogene. Many of the B box family proteins function as homodimers, although the role of the individual components of the tripartite motif in this process remains unclear. We demonstrate that rfp homomultimerization occurs through the coiled-coil domains; however, while the B box is not an interacting interface itself, its structural integrity is necessary for this interaction to occur. This is the first evidence that the B box zinc finger domain is involved in regulating protein-protein interactions. Interestingly, we find that mutations of the RING finger and B box affect the subcellular compartmentalization of rfp in various cell lines. These results demonstrate that the interactions of rfp with itself and its association with specific subcellular compartments is dependent upon the function of all of the components of the tripartite motif. It is likely that these domains play a crucial role in the function of the rfp protein in normal cell differentiation and in its transformation potential in the recombined state.
Two proteins that cycle asynchronously between centrosomes and nuclear structures: Drosophila CP60 and CP190
Authors: Oegema Karen , Marshall Wallace F. , Sedat John W. , Alberts Bruce M. 
Published: April 2021

ABSTRACT
Both the nucleus and the centrosome are complex, dynamic structures whose architectures undergo cell cycle-specific rearrangements. CP190 and CP60 are two Drosophila proteins of unknown function that shuttle between centro-somes and nuclei in a cell cycle-dependent manner. These two proteins are associated in vitro, and localize to centrosomes in a microtubule independent manner. We injected fluorescently labeled, bacterially expressed CP190 and CP60 into living Drosophila embryos and followed their behavior during the rapid syncytial blastoderm divisions (nuclear cycles 10-13). Using quantitative 3-D wide-field fluorescence microscopy, we show that CP190 and CP60 cycle between nuclei and centrosomes asynchronously with the accumulation of CP190 leading that of CP60 both at centrosomes and in nuclei. During interphase, CP190 is found in nuclei. Immediately following nuclear envelope breakdown, CP190 localizes to centrosomes where it remains until telophase, thereafter accumulating in reforming nuclei. Unlike CP190, CP60 accumulates at centrosomes primarily during anaphase, where it remains into early interphase. During nuclear cycles 10 and 11, CP60 accumulates in nuclei simultaneous with nuclear envelope breakdown, suggesting that CP60 binds to an unknown nuclear structure that persists into mitosis. During nuclear cycles 12 and 13, CP60 accumulates gradually in nuclei during interphase, reaching peak levels just before nuclear envelope breakdown. Once in the nucleus, both CP190 and CP60 appear to form fibrous intranuclear networks that remain coherent even after nuclear envelope breakdown. The CP190 and CP60 networks do not co-localize extensively with each other or with DNA. This work provides direct evidence, in living cells, of a coherent protein network that may represent a nuclear skeleton.
Clusterin/ApoJ expression is associated with neuronal apoptosis in the olfactory mucosa of the adult mouse
Authors: Michel Denis , Moyse Emmanuel , Trembleau Alain , Jourdan François , Brun Gilbert 
Published: April 2021

ABSTRACT
The molecular events orchestrating neuronal degeneration and regeneration remain poorly understood. Attempts at identifying genes specifically expressed during these processes, have constantly led to the (re)isolation of the clusterin/ApoJ gene, whose expression is highly reactive to injury in a wide variety of tissues. To get insight into the function of clusterin in neuron loss, we have assessed in detail the clusterin gene expression in an experimental model of neurodegeneration, using the peripheral olfactory system of adult mouse. The sensory neurons of olfactory nasal mucosa can be massively induced to degenerate in vivo, by surgical removal of their only synaptic target: the olfactory bulb. We have previously shown that this neuron loss results from a near-synchronized induction of apoptosis genetic programs. We present here evidence that clusterin gene expression is tightly correlated to the onset of neuronal apoptoses in lesioned olfactory mucosae. The simultaneous preparation of DNA and RNA from the same tissue samples reveals that a strong clusterin mRNA accumulation coincides with the wave of nucleosome-sized DNA fragmentation. However, double detection of apoptotic nuclei by the TUNEL method and of clusterin messengers by in situ hybridization revealed that the clusterin gene expression is not induced in dying neurons, but in the glial sheath surrounding the axon bundles of degenerating olfactory neurons. Clusterin immunocytochemistry reveals that the clusterin protein accumulates not only in these producing cells, but also in the olfactory epithelium, suggesting the possibility of clusterin internalization by cells located at a distance from the synthesis loci. In view of this localization and of the activities of the clusterin protein reported so far, possible functions of clusterin in nervous plasticity are discussed.
A genetic screen reveals a role for the late G1-specific transcription factor Swi4p in diverse cellular functions including cytokinesis
Authors: Igual J. Carlos , Toone W. Mark , Johnston Leland H. 
Published: April 2021

ABSTRACT
The transcription factor Swi4p plays a crucial role in the control of the initiation of the cell cycle in budding yeast. To further understand Swi4p function, we set up a synthetic lethal screen for genes interacting with SWI4. Fourteen conditional mutations which resulted in lethality only in the absence of SWI4 have been isolated. Only two of them were suppressed by ectopic expression of CLN2, indicating that Swi4p is involved in diverse cellular processes in addition to its requirement for CLN1,2 regulation. In most of the mutants a cell cycle phenotype was observed, including defects in G1 progression, budding, the G2/M transition and cytokinesis. In addition, four of the mutations resulted in massive cell lysis at the restrictive temperature, indicating that Swi4p is involved in the maintenance of cell integrity.
One of the mutants, rsf1 swi4Δ, was characterized in detail and it is defective in cytokinesis at the restrictive temperature. Staining with Calcofluor revealed that the rsf1 swi4Δ mutant is impaired in chitin biosynthesis. rsf1 is allelic to the AGM1 gene, coding for N-acetylglucosaminephosphate mutase, an enzyme involved in the biosynthesis of chitin. A single copy of SWI4 suppressed the cytokinesis defect. The above data suggest that Swi4p has a role in cytokinesis and becomes essential in this process when chitin biosynthesis is compromised. As overexpression or ectopic expression of CLN did not suppress the rsf1 swi4Δ mutant phenotype, Swi4p must control some other gene(s) involved in cytokinesis.
HSP70 and HSP90 homologs are associated with tubulin in hetero-oligomeric complexes, cilia and the cortex of Tetrahymena
Authors: Williams Norman E. , Nelsen E. Marlo 
Published: April 2021

ABSTRACT
We show in the present study that homologs of hsp90 and hsp70 are induced by heat shocks in Tetrahymena and appear to form a high molecular mass complex (∼700 kDa) with tubulin. Three members of the hsp70 family (hsp72, 73, and 78) and one member of the hsp90 family (hsp82) have been identified by immunological or by a combination of immunological and sequencing methods. The known components of the 700 kDa complex and the conditions under which it can be recovered suggest that it may be an induced protective assemblage rather than a normal processing intermediate. Immunoblotting and immunofluorescence studies suggest further that large amounts of hsp73 and lesser amounts of hsp82 are associated with mature microtubules in both cilia and the cortex in this cell type. Some site-specific localizations of the identified heat shock proteins were also noted in non-microtubular components of the cell cortex.
Polarity sorting of actin filaments in cytochalasin-treated fibroblasts
Authors: Verkhovsky A. B. , Svitkina T. M. , Borisy G. G. 
Published: April 2021

ABSTRACT
The polarity of actin filaments is fundamental for the sub-cellular mechanics of actin-myosin interaction; however, little is known about how actin filaments are oriented with respect to myosin in non-muscle cells and how actin polarity organization is established and maintained. Here we approach these questions by investigating changes in the organization and polarity of actin relative to myosin II during actin filament translocation. Actin and myosin II reorganization was followed both kinetically, using microinjected fluorescent analogs of actin and myosin, and ultrastructurally, using myosin S1 decoration and immuno-gold labelling, in cultured fibroblasts that were induced to contract by treatment with cytochalasin D. We observed rapid (within 15 minutes) formation of ordered actin filament arrays: short tapered bundles and aster-like assemblies, in which filaments had uniform polarity with their barbed ends oriented toward the aggregate of myosin II at the base of a bundle or in the center of an aster. The resulting asters further interacted with each other and aggregated into bigger asters. The arrangement of actin in asters was in sharp contrast to the mixed polarity of actin filaments relative to myosin in non-treated cells. At the edge of the cell, actin filaments became oriented with their barbed ends toward the cell center; that is, the orientation was opposite to what was observed at the edge of non-treated cells. This rearrangement is indicative of relative translocation of actin and myosin II and of the ability of myosin II to sort actin filaments with respect to their polarity during translocation. The results suggest that the myosin II-actin system of non-muscle cells is organized as a dynamic network where actin filament arrangement is defined in the course of its interaction with myosin II.
A minimal region on the integrin β4 subunit that is critical to its localization in hemidesmosomes regulates the distribution of HD1/plectin in COS-7 cells
Authors: Niessen Carien M. , Hulsman Esther H. M. , Oomen Lauran C. J. M. , Kuikman I. , Sonnenberg Arnoud 
Published: April 2021

ABSTRACT
The integrin α6β4 is a major component of hemidesmosomes, in which it mediates firm adhesion to laminin 5. Previous studies have shown that the incorporation of α6β4 into hemidesmosomes requires a 303 amino acid stretch of the cytoplasmic domain of β4, comprising part of the first fibronectin type III (FNIII) repeat, the second FNIII repeat and the segment that connects the second to the third FNIII repeat (connecting segment). Now, we have further defined sequences within β4 that are critical for its localization in hemidesmosomes and we demonstrate that these sequences also induce the redistribution of HD1/plectin into junctional complexes containing the integrin α6β4 in COS-7 cells, transfected with cDNAs encoding α6A and β4. Truncation of the cytoplasmic domain of β4 after amino acids 1,382 or 1,355 in the connecting segment, by which a potential tyrosine activation motif (TAM) is removed, does not prevent the localization of α6β4 in hemidesmosomes in the rat bladder carcinoma cell line 804G and neither did it eliminate the ability of α 6β4 to change the subcellular dis-tribution of HD1/plectin in COS-7 cells. In contrast, β4 subunits in which the entire connecting segment had been deleted or which were truncated after amino acid 1,328, which removes almost the complete segment, had lost both of these functions. Furthermore, when β4 subunits with either a deletion of the second FNIII repeat or a small deletion in this repeat were co-expressed with α6, the integrins were not localized in hemidesmosomes and did not induce the redistribution of HD1/plectin in COS-7 cells. Finally, the fourth FNIII repeat of β4 could not replace the second in either of these activities. These findings establish that a region in β4, which encompasses the second FNIII repeat and a stretch of 27 amino acids (1,329-1,355) of the connecting segment, is critical for the localization of α6β4 in hemidesmosomes and that it regulates the distribution of HD1/plectin.
Apical sorting of influenza hemagglutinin by transcytosis in retinal pigment epithelium
Authors: Bonilha Vera L. , Marmorstein Alan D. , Cohen-Gould Leona , Rodriguez-Boulan Enrique 
Published: April 2021

ABSTRACT
The retinal pigment epithelium is endowed with a unique distribution of certain plasma membrane proteins. Na+,K+-ATPase, for instance, is polarized to the apical surface of RPE, rather than to the basolateral surface as in most other epithelia. To study the sorting pathways of RPE cells, we used temperature sensitive mutants of influenza and vesicular stomatitis virus (VSV) to synchronize the transport of hemagglutinin (HA) and VSV G protein (VSV G) along the biosynthetic pathway of the RPE cell line RPE-J. After HA and VSV G accumulated in the trans-Golgi network of RPE-J cells kept at 20°C, transfer to the permissive temperature (32°C) resulted in the transport of both HA and VSV G to the basolateral plasma membrane. Later, while VSV G remained basolateral, HA progressively reversed its polarity, eventually becoming apical. Further analysis demonstrated that the reversal of HA polarity was due to transcytosis of HA from the basolateral to the apical surface of RPE-J cells. To determine whether HA followed a transcytotic route in RPE in vivo, influenza and VSV were injected into the subretinal space of rat eyes. Again, both HA and VSV G were initially observed at the basolateral surface of RPE cells. However, whereas VSV G remained there, HA progressively redistributed to the apical surface. These findings demonstrated that RPE cells use a transcytotic pathway for the targeting of at least some apical proteins to their destination.
TLS (FUS) binds RNA in vivo and engages in nucleo-cytoplasmic shuttling
Authors: Zinszner Helene , Sok John , Immanuel David , Yin Yin , Ron David 
Published: April 2021

ABSTRACT
TLS, the product of a gene commonly translocated in liposarcomas (TLS), is prototypical of a newly identified class of nuclear proteins that contain a C-terminal domain with a distinct RNA recognition motif (RRM) surrounded by Arg-Gly-Gly (RGG) repeats. Its unique N terminus serves as an essential transforming domain for a number of fusion oncoproteins in human sarcomas and leukemias. In this study we use an in vivo UV crosslinking procedure to probe the interactions of TLS with RNA. TLS is found to bind RNA in vivo and the association of TLS with RNA is rapidly diminished by treating cells with transcriptional inhibitors. This suggests that the species bound by TLS turns over rapidly. Surprisingly, the RRM was found to be dispensable for RNA binding by TLS in vivo, suggesting that at any one time most of the interactions between TLS and RNA in the cell are not sequence specific. Analysis of inter specific heterokaryons formed between human and mouse or Xenopus cells revealed that TLS engages in rapid nucleocytoplasmic shuttling, a finding confirmed by the ability of anti-TLS antibodies to trap TLS when injected into the cytoplasm of HeLa cells. Cellular fractionation experiments suggest that TLS binds to RNA in both the nucleus and cytoplasm and support the hypothesis that TLS functions as a heterogeneous ribonuclear protein (hnRNP)-like chaperone of RNA. These findings are discussed in the context of the role altered forms of TLS play in cellular transformation.
A yeast heat shock transcription factor (Hsf1) mutant is defective in both Hsc82/Hsp82 synthesis and spindle pole body duplication
Authors: Zarzov Patrick , Boucherie Hélian , Mann Carl 
Published: April 2021

ABSTRACT
Cdc28 is a cyclin-dependent protein kinase of Saccharomyces cerevisiae that is required for the G1/S and G2/M transitions of the cell division cycle. All previously described cdc28 mutants aside from cdc28-1N arrest division specifically in the G1 phase. cdc28-1N arrests division in G2/mitosis. We show here that the cdc28-109 mutant exhibits a mixed cell division arrest at 37°C with cells in both the G1 and G2 phases. In order to identify proteins that functionally interact with Cdc28, we isolated mutants that are colethal with cdc28-109 at its permissive temperature. We describe here our phenotypic analysis of two such mutants, hsf1-82 and ydj1-10, that affect the heat shock transcription factor and a yeast dnaj-like protein chaperone, respectively. hsf1-82 and ydj1-10 temperaturesensitive mutants arrest the cell division cycle at several stages. However, one predominant class of cells in both mutants was arrested with a large bud and a single vertex of microtubules. Electron microscopic analysis of such hsf1-82 cells showed that they contained an unduplicated spindle pole body with an enlarged half-bridge. Twodimensional gel electrophoresis of total cell proteins revealed that the hsf1-82 cells were specifically defective in the expression of the Hsc82 and Hsp82 proteins. Furthermore, the hsf1-82 mutation was suppressed by the HSC82 gene on a multicopy plasmid that restored Hsc82 protein to high levels in these cells. These results show that Hsf1 is required for spindle pole body duplication at 37°C.
Structural and functional similarities between the human cytoskeletal protein zyxin and the ActA protein of Listeria monocytogenes
Authors: Golsteyn Roy M. , Beckerle Mary C. , Koay Tom , Friederich Evelyne 
Published: April 2021

ABSTRACT
The intracellular bacterial parasite Listeria monocytogenes produces ActA protein at its surface to facilitate the localized assembly of actin-filled comets that are required for movement. The organization of actin in Listeria comets shows striking similarity to the organization of actin at the plasma membrane of mammalian cells. Therefore we examined the possibility that an ActA-like protein is present in mammalian cells. By using antibodies directed against ActA, we identified zyxin as an ActA related protein in a number of cell types. We compared the functions of ActA and zyxin by transient expression of variants tagged with an inner plasma membrane localization sequence (a CAAX box). Targeting of the proline rich domain of zyxin to the plasma membrane disrupts the actin cytoskeleton and cell shape in a manner similar to that which occurs with membrane-targeted ActA sequences. A chimeric protein composed of the N-terminal domain of ActA fused to the N-terminal and central domains of zyxin induced a full ActA response in cells. Furthermore, zyxin and ActA exhibit common protein partners in vitro. On the basis of the shared properties of zyxin and ActA, we propose that zyxin enhances actin organizing activity in mammalian cells.
Glucose-induced microautophagy in Pichia pastoris requires the α-subunit of phosphofructokinase
Authors: Yuan Weiping , Tuttle Daniel L. , Shi Yu-Jiang , Ralph Gareth S. , Dunn William A. 
Published: April 2021

ABSTRACT
We have characterized biochemically, morphologically, and genetically two distinct pathways for the selective degradation of peroxisomes in Pichia pastoris. These pathways are independently regulated and analogous to microautophagy and macroautophagy that have been defined in mammalian cells. When P. pastoris is grown in methanol, cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized. During adaptation from methanol to glucose, these enzymes are rapidly and selectively degraded within the yeast vacuole by microautophagy. We have isolated gsa mutants that are defective in glucose-induced selective autophagy of peroxisomes. In this study, we have shown that gsa1 is unable to sequester peroxisomes into the yeast vacuole. In addition, we provide evidence that the glucose-induced selective autophagy 1 (GSA1) protein is the alpha subunit of the phosphofructokinase enzyme complex encoded by PFK1. First, we can rescue the gsa1 mutant by transformation with a vector containing PFK1. Second, cellular levels of both PFK1 mRNA and phosphofructokinase activity are dramatically reduced in gsa1 when compared to the parental GS115. Third, a PFK1 knockout (Δpfk1) is unable to degrade alcohol oxidase during glucose adaptation. As observed in gsa1, the peroxisomes in Δpfk1 remain outside the vacuole during adaptation. Our data are consistent with the concept that PFK1 protein is required for an event upstream of vacuole degradation (i.e. signaling, selection, or sequestration). However, the degradation of peroxisomes does not require a catalytically active phosphofructokinase. The inability of Δpfk1 cells to degrade alcohol oxidase can be rescued by transformation with either normal PFK1 or mutant pfk1 whose catalytic site had been inactivated by a single amino acid mutation. We propose that PFK1 protein directly modulates glucose-induced microautophagy independent of its ability to metabolize glucose intermediates.
Mitotic phosphoepitopes are expressed in Kc cells, neuroblasts and isolated chromosomes of Drosophila melanogaster
Authors: Bousbaa Hassan , Correia Luis , Gorbsky Gary J. , Sunkel Claudio E. 
Published: April 2021

ABSTRACT
The progression of cells from metaphase to anaphase is thought to be regulated by a checkpoint that delays entry into anaphase until all chromosomes reach a stable bi-polar attachment at the metaphase plate. Previous work has suggested that the 3F3/2 kinetochore phosphoepitopes are involved in this checkpoint system. We show that the 3F3/2 centromere phosphoepitopes are present in Kc cells, third instar larval neuroblasts and isolated chromosomes of Drosophila melanogaster. In tissue culture cells and neuroblasts isolated from third instar larvae, centromere labelling is detected from early prophase to the metaphaseanaphase transition but absent once cells enter anaphase. During anaphase, the antibody stains the spindle mid zone and during telophase the midbody is labelled until cells separate. In both cell types, the 3F3/2 antibody stains the centrosome from prophase to late telophase. The 3F3/2 staining is retained in Kc cells and third instar larval neuroblasts arrested at the prometaphase state with microtubule inhibitors. Also, two mitotic mutants that show abnormal spindle morphology retain the centromere labelling in a metaphase-like configuration, suggesting that they activate the metaphase-anaphase checkpoint. Finally, mitotic chromosomes isolated in the presence of a phosphatase inhibitor show phosphoepitopes at the primary constriction on the surface of each chromatid, however, chromosomes isolated in the absence of a phosphatase inhibitor do not. Incubation of these chromosomes with ATP causes the rephosphorylation of the phosphoepitopes at the centromere.