bluuebunny/crossref_metadata_2025_split huggingface dataset:
The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner
Authors: Zhao Jian , Tenev Tencho , Martins Luis M. , Downward Julian , Lemoine Nicholas R. 
Published: April 2021

ABSTRACT
Survivin, a human inhibitor of apoptosis protein (IAP), plays an important role in both cell cycle regulation and inhibition of apoptosis. Survivin is expressed in cells during the G2/M phase of the cell cycle, followed by rapid decline of both mRNA and protein levels at the G1 phase. It has been suggested that cell cycle-dependent expression of survivin is regulated at the transcriptional level.
In this study we demonstrate involvement of the ubiquitin-proteasome pathway in post-translational regulation of survivin. Survivin is a short-lived protein with a half-life of about 30 minutes and proteasome inhibitors greatly stabilise survivin in vivo. Expression of the survivin gene under the control of the CMV promoter cannot block cell cycle-dependent degradation of the protein. Proteasome inhibitors can block survivin degradation during the G1 phase and polyubiquitinated derivatives can be detected in vivo. Mutation of critical amino acid residues within the baculovirus IAP repeat (BIR) domain or truncation of the N terminus or the C terminus sensitises survivin to proteasome degradation. Together, these results indicate that the ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner and structural changes greatly destabilise the survivin protein.
How motor proteins influence microtubule polymerization dynamics
Authors: Hunter Andrew W. , Wordeman Linda 
Published: April 2021

ABSTRACT
The interplay between microtubules and microtubule-based motors is fundamental to basic aspects of cellular function, such as the intracellular transport of organelles and alterations in cellular morphology during cell locomotion and division. Motor proteins are unique in that they couple nucleotide hydrolysis to force production that can do work. The force transduction by proteins belonging to the kinesin and dynein superfamilies has been thought only to power movement of these motors along the surface of microtubules; however, a growing body of evidence, both genetic and biochemical, suggests that motors can also directly influence the polymerization dynamics of microtubules. For example, at the vertebrate kinetochore, motors interact directly with microtubule ends and modulate polymerization dynamics to orchestrate chromosome movements during mitosis. Although a role for motors in regulating microtubule length has been established, the mechanisms used by motors to promote microtubule growth or shrinkage are unclear, as is an understanding of why cells might choose motors to control dynamics rather than a variety of non-motor proteins known to affect microtubule stability. Elucidation of the exact mechanisms by which motors alter the exchange of tubulin subunits at microtubule ends in vitro may shed light on how microtubule stability is regulated to produce the array of dynamic behavior seen in cells.
Functional visualization of the separate but interacting calcium stores sensitive to NAADP and cyclic ADP-ribose
Authors: Lee Hon Cheung , Aarhus Robert 
Published: April 2021

ABSTRACT
Cells possess multiple Ca2+ stores and their selective mobilization provides the spatial-temporal Ca2+ signals crucial in regulating diverse cellular functions. Except for the inositol trisphosphate (IP3)-sensitive Ca2+ stores, the identities and the mechanisms of how these internal stores are mobilized are largely unknown. In this study, we describe two Ca2+ stores, one of which is regulated by cyclic ADP-ribose (cADPR) and the other by nicotinic acid adenine dinucleotide phosphate (NAADP). We took advantage of the large size of the sea urchin egg and stratified its organelles by centrifugation. Using photolysis to produce either uniform or localized increases of cADPR and NAADP from their respective caged analogs, the two separate stores could be visually identified by Ca2+ imaging and shown to be segregated to the opposite poles of the eggs. The cADPR-pole also contained the IP3-sensitive Ca2+ stores, the egg nucleus and the endoplasmic reticulum (ER); the latter was visualized using Bodipy-thapsigargin. On the other hand, the mitochondria, as visualized by rhodamine 123, were segregated to the opposite pole together with the NAADP-sensitive calcium stores. Fertilization of the stratified eggs elicited a Ca2+ wave starting at the cADPR-pole and propagating toward the NAADP-pole. These results provide the first direct and visual evidence that the NAADP-sensitive Ca2+ stores are novel and distinct from the ER. During fertilization, communicating signals appear to be transmitted from the ER to NAADP-sensitive Ca2+ stores, leading to their activation.
Human Cdc5, a regulator of mitotic entry, can act as a site-specific DNA binding protein
Authors: Lei Xiang-He , Shen Xun , Xu Xiao-Qin , Bernstein Harold S. 
Published: April 2021

ABSTRACT
G2/M progression requires coordinated expression of many gene products, but little is known about the transcriptional regulators involved. We recently identified human Cdc5, a positive regulator of G2/M in mammalian cells. We also demonstrated the presence of a latent activation domain in its carboxyl terminus, suggesting that human Cdc5 regulates G2/M through transcriptional activation. Despite the presence of a DNA binding domain, studies by others have failed to identify a preferential binding site for Cdc5 family members. In addition, Cdc5 recently has been associated with the splicesome in several organisms, suggesting that it may not act through DNA binding. We now report the identification of a 12 bp sequence to which human Cdc5 binds specifically and with high affinity through its amino terminus. We show that this DNA-protein interaction is capable of activating transcription. We also used a selection system in yeast to identify human genomic fragments that interact with human Cdc5. Several of these contained sequences similar to the binding site. We demonstrate that these bind human Cdc5 with similar specificity and affinity. These experiments provide the first evidence that Cdc5 family members can act as site-specific DNA binding proteins, and that human Cdc5 may interact with specific, low abundance sequences in the human genome. This raises the possibility that Cdc5 proteins may participate in more than one process necessary for regulated cell division.
Yeast myosin light chain, Mlc1p, interacts with both IQGAP and Class II myosin to effect cytokinesis
Authors: Boyne James R. , Yosuf Hirzun Mohd , Bieganowski Pawel , Brenner Charles , Price Clive 
Published: April 2021

ABSTRACT
MLC1 (myosin light chain) acts as a dosage suppressor of a temperature sensitive mutation in the gene encoding the S. cerevisiae IQGAP protein. Both proteins localize to the bud neck in mitosis although Mlc1p localisation precedes Iqg1p. Mlc1p is also found at the incipient bud site in G1 and the growing bud tip during S and G2 phases of the cell cycle. A dominant negative GST-Mlc1p fusion protein specifically blocks cytokinesis and prevents Iqg1p localisation to the bud neck, as does depletion of Mlc1p. These data support a direct interaction between the two proteins and immunoprecipitation experiments confirm this prediction. Mlc1p is also shown to interact with the class II conventional myosin (Myo1p). All three proteins form a complex, however, the interaction between Mlc1p and Iqg1p can be separated from the Mlc1p/Myo1p interaction. Mlc1p localisation and maintenance at the bud neck is independent of actin, Myo1p and Iqg1p. It is proposed that Mlc1p therefore functions to recruit Iqg1p and in turn actin to the actomyosin ring and that it is also required for Myo1p function during ring contraction.
Identification of an α-tubulin mutant of fission yeast from γ-tubulin-interacting protein screening: genetic evidence for α-/γ-tubulin interaction
Authors: Takeoka Aya , Shimizu Miyuki , Horio Tetsuya 
Published: April 2021

ABSTRACT
γ-Tubulin has been determined to be a central element of microtubule nucleation and, thus, indispensable for cellular organization of the microtubule. Utilizing the fact that human γ-tubulin can function in the fission yeast Schizosaccharomyces pombe, we have generated a unique mutant screening procedure which can specifically select mutants of genes encoding γ-tubulin-interacting proteins. One of the isolated mutants, cs76, turned out to carry a mutation in the α1-tubulin gene (nda2+). This result suggests a direct interaction between the α- and γ-tubulins. We located the mutation site in the nda2 gene and characterized the mutant phenotype. Our results demonstrate the importance of the α-/γ-tubulin interaction in microtubule nucleation and should complement previous knowledge.
A PAK-like protein kinase is required for maturation of young hyphae and septation in the filamentous ascomycete Ashbya gossypii
Authors: Ayad-Durieux Yasmina , Knechtle Philipp , Goff Stephen , Dietrich Fred , Philippsen Peter 
Published: April 2021

ABSTRACT
Filamentous fungi grow by hyphal extension, which is an extreme example of polarized growth. In contrast to yeast species, where polarized growth of the tip of an emerging bud is temporally limited, filamentous fungi exhibit constitutive polarized growth of the hyphal tip. In many fungi, including Ashbya gossypii, polarized growth is reinforced by a process called hyphal maturation. Hyphal maturation refers to the developmental switch from slow-growing hyphae of young mycelium to fast-growing hyphae of mature mycelium. This process is essential for efficient expansion of mycelium. We report for the first time on the identification and characterization of a fungal gene important for hyphal maturation. This novel A. gossypii gene encodes a presumptive PAK (p21-activated kinase)-like kinase. Its closest homolog is the S. cerevisiae Cla4 protein kinase; the A. gossypii protein is therefore called AgCla4p. Agcla4 deletion strains are no longer able to perform the developmental switch from young to mature hyphae, and GFP (green fluorescent protein)-tagged AgCla4p localizes with much higher frequency in mature hyphal tips than in young hyphal tips. Both results support the importance of AgCla4p in hyphal maturation. AgCla4p is also required for septation, indicated by the inability of Agcla4 deletion strains to properly form actin rings and chitin rings. Despite the requirement of AgCla4p for the development of fast-growing hyphae, AgCla4p is not necessary for actin polarization per se, because tips enriched in cortical patches and hyphae with a fully developed network of actin cables can be seen in Agcla4 deletion strains. The possibility that AgCla4p may be involved in regulatory mechanisms that control the dynamics of the actin patches and/or actin cables is discussed.
Distribution of GPI-anchored proteins in the protozoan parasite Leishmania, based on an improved ultrastructural description using high-pressure frozen cells
Authors: Weise Frank , Stierhof York-Dieter , Kühn Christina , Wiese Martin , Overath Peter 
Published: April 2021

ABSTRACT
The cellular distribution of two glycosyl-phosphatidylinositol (GPI)-anchored proteins and a trans-membrane protein and the compartments involved in their trafficking were investigated in the insect stage of Leishmania mexicana, which belongs to the phylogenetically old protozoan family Trypanosomatidae. Electron microscopy of sections from high-pressure frozen and freeze-substituted cells allowed a detailed description of exo- and endocytic structures located in the vesicle-rich, densly packed anterior part of the spindle-shaped cell. A complex of tubular clusters/translucent vesicles is the prominent structure between the trans-side of the single Golgi apparatus and the flagellar pocket, the only site of endo- and exocytosis. A tubulovesicular compartment lined by one or two distinct microtubules and extending along the length of the cell is proposed to be a post-Golgi and probably late endosomal/lysosomal compartment. Using biotinylation experiments, FACS analysis and quantitative immunoelectron microscopy it was found that, at comparable expression levels, 73-75% of the two GPI-anchored proteins but only 13% of the trans-membrane protein are located on the cell surface. The tubulovesicular compartment contains 46%, the ER 5%, the Golgi complex 1.9% and the tubular cluster/translucent vesicle complex 3.6% of the intracellular fraction of the GPI-anchored protease, GP63. The density of GP63 was found to be 23-fold higher on the plasma/flagellar pocket membrane than on the ER and about tenfold higher than on membranes of the Golgi complex or of endo- or exocytic vesicles. These results indicate that there is a considerable concentration gradient of GPI-anchored proteins between the plasma/flagellar pocket membrane and the ER as well as structures involved in exo- or endocytosis. Possible mechanisms how this concentration gradient is established are discussed.
Interactions between FcεRI and lipid raft components are regulated by the actin cytoskeleton
Authors: Holowka David , Sheets Erin D. , Baird Barbara 
Published: April 2021

ABSTRACT
Previous studies showed that crosslinking of IgE-FcεRI complexes on RBL-2H3 mast cells causes their association with isolated detergent-resistant membranes, also known as lipid rafts, in a cholesterol-dependent process that precedes initiation of signaling by these receptors. To investigate these interactions on intact cells, we examined the co-redistribution of raft components with crosslinked IgE-FcεRI using confocal microscopy. After several hours of crosslinking at 4°C, the glycosylphosphatidylinositol-linked protein Thy-1 and the Src-family tyrosine kinase Lyn co-redistribute with IgE-FcεRI in large patches at the plasma membrane. Under these conditions, F-actin also undergoes dramatic co-segregation with FcεRI and raft components but is dispersed following a brief warm-up to 37°C. When crosslinking of IgE-FcεRI is initiated at higher temperatures, co-redistribution of raft components with patched FcεRI is not readily detected unless stimulated F-actin polymerization is inhibited by cytochalasin D. In parallel, cytochalasin D converts transient antigen-stimulated tyrosine phosphorylation to a more sustained response. Sucrose gradient analysis of lysed cells reveals that crosslinked IgE-FcεRI remains associated with lipid rafts throughout the time course of the transient phosphorylation response but undergoes a time-dependent shift to higher density that is prevented by cytochalasin D. Our results indicate that interactions between Lyn and crosslinked IgE-FcεRI are regulated by stimulated F-actin polymerization, and this is best explained by a segregation of anchored raft components from more mobile ones.
Evidence for the coincident initiation of homolog pairing and synapsis during the telomere-clustering (bouquet) stage of meiotic prophase
Authors: Bass Hank W. , Riera-Lizarazu Oscar , Ananiev Evgueni V. , Bordoli Stefano J. , Rines Howard W. , Phillips Ronald L. , Sedat John W. , Agard David A. , Cande W. Zacheus 
Published: April 2021

ABSTRACT
To improve knowledge of the prerequisites for meiotic chromosome segregation in higher eukaryotes, we analyzed the spatial distribution of a pair of homologs before and during early meiotic prophase. Three-dimensional images of fluorescence in situ hybridization (FISH) were used to localize a single pair of homologs in diploid nuclei of a chromosome-addition line of oat, oat-maize9b. The system provided a robust assay for pairing based on cytological colocalization of FISH signals. Using a triple labeling scheme for simultaneous imaging of chromatin, telomeres and the homolog pair, we determined the timing of pairing in relation to the onset of three sequential hallmarks of early meiotic prophase: chromatin condensation (the leptotene stage), meiotic telomere clustering (the bouquet stage) and the initiation of synapsis (the zygotene stage). We found that the two homologs were mostly unpaired up through middle leptotene, at which point their spherical cloud-like domains began to transform into elongated and stretched-out domains. At late leptotene, the homologs had completely reorganized into long extended fibers, and the beginning of the bouquet stage was conspicuously marked by the de novo clustering of telomeres at the nuclear periphery. The homologs paired and synapsed during the bouquet stage, consistent with the timing of pairing observed for several oat 5S rDNA loci. In summary, results from analysis of more than 100 intact nuclei lead us to conclude that pairing and synapsis of homologous chromosomes are largely coincident processes, ruling out a role for premeiotic pairing in this system. These findings suggest that the genome-wide remodeling of chromatin and telomere-mediated nuclear reorganization are prerequisite steps to the DNA sequence-based homology-search process in higher eukaryotes.
A novel mutant allele of the chromatin-bound fission yeast checkpoint protein Rad17 separates the DNA structure checkpoints
Authors: Griffiths Dominic , Uchiyama Masashi , Nurse Paul , Wang Teresa S.-F. 
Published: April 2021

ABSTRACT
To further dissect the genetic differences between the checkpoint pathway following S-phase cdcarrest versus DNA damage, a genetic screen was performed for checkpoint mutants that were unable to arrest mitosis following cell-cycle arrest with a temperature-sensitive DNA polymerase ε mutant, cdc20-M10. One such checkpoint mutant, rad17-d14, was found to display the cutphenotype following S-phase arrest by cdc20-M10, but not by the DNA synthesis inhibitor hydroxyurea, reminiscent of the chk1Δ mutant. Unlike chk1Δ, rad17-d14was not sensitive to UV irradiation. Interestingly, the ionising radiation sensitivity of rad17-d14was only at higher doses, and cells were found to be defective in properly arresting cell division following irradiation in S phase, but not G2phase. Biochemical analysis attributes the checkpoint defects of rad17-d14to the failure to phosphorylate the checkpoint effector Chk1p. To investigate if Rad17p monitors the genome for abnormal DNA structures specifically during DNA synthesis, chromatin association of Rad17p was analysed. Rad17p was found to be chromatin associated throughout the cell cycle, not just during S phase. This interaction occurred irrespective of the arrest with cdc20-M10and, surprisingly, was also independent of the other checkpoint Rad proteins, and the cell-cycle effectors Chk1p and Cds1p.
Formation of hemidesmosome-like structures in the absence of ligand binding by the α6β4 integrin requires binding of HD1/plectin to the cytoplasmic domain of the β4 integrin subunit
Authors: Nievers Mirjam G. , Kuikman Ingrid , Geerts Dirk , Leigh Irene M. , Sonnenberg Arnoud 
Published: April 2021

ABSTRACT
Hemidesmosomes are adhesion structures that mediate anchorage of epithelial cells to the underlying basement membrane. We have previously shown that the α6β4 integrin can induce the assembly of these multi-protein structures independent of binding to its ligand laminin-5 (ligand-independent formation of hemidesmosomes). Our results suggested a role for HD1/plectin, which binds to the cytoplasmic domain of the β4 integrin subunit, in controlling the clustering of hemidesmosomal components at the basal side of the cell. Using keratinocytes derived from patients lacking HD1/plectin, we now show that ligand-independent formation of hemidesmosomal clusters indeed requires HD1/plectin, in contrast to the ligand-dependent assembly of hemidesmosomes. No clustering of the α6β4 integrin, or of the bullous pemphigoid antigens BP180 and BP230, was seen when HD1/plectin-deficient keratinocytes were plated on fibronectin or type IV collagen. In β4-deficient keratinocytes, expression of an interleukin 2 receptor (IL2R) transmembrane chimera containing the β4 cytoplasmic tail with the mutation R1281W, which abrogates HD1/plectin binding, resulted in a diffuse distribution of the chimeric receptor. In contrast, a β4R1281Wmutant that can associate with α6 and bind ligand, was found to be directed to the basal surface of the cells, at sites where laminin-5 was deposited. In addition, this mutant induced clustering of BP180 and BP230 at these sites. Together, these results show that the formation of hemidesmosomes requires binding of either ligand or HD1/plectin to the β4 integrin subunit. Intriguingly, we found that IL2R/β4 chimeras become localized in pre-existing hemidesmosomes of HD1/plectin-deficient keratinocytes, and that this localization requires a domain in the β4 cytoplasmic tail that is also required for HD1/plectin binding (residues 1115-1356). Because this part of β4 lacks the BP180 binding site, and since we show in this study that it is unable to interact with the same part on another β4 molecule, we suggest that the chimera becomes incorporated into hemidesmosomes of HD1/ plectin-deficient keratinocytes by interacting with an as yet unidentified hemidesmosomal component.
p39 activates cdk5 in neurons, and is associated with the actin cytoskeleton
Authors: Humbert Sandrine , Dhavan Rani , Tsai Li-Huei 
Published: April 2021

ABSTRACT
Cyclin-dependent kinase 5 (cdk5) is a small serine/ threonine kinase that displays close sequence homology to the mitotically active cyclin-dependent kinases. Cdk5 has been shown to play an essential role in the development of the nervous system, including neuronal migration and neurite outgrowth. Cdk5 activation requires the presence of a regulatory activator such as p35. cdk5 −/− mice have much more extensive defects in the development of the nervous system than p35 −/− mice, leading to the speculation that other regulatory activators of cdk5 exist. Indeed, p39 is a p35 related protein isolated by sequence homology to p35. We show here that p39 associates with cdk5 in brain lysates, and that this complex is active in phosphorylation of histone H1. By extensive characterization of p39 subcellular localization in different cell types, we demonstrate the presence of p39 in lamellipodial and fillopodial structures of cells and in growth cones of neurons. We show that p39 colocalizes with actin, and cofractionates with the detergent insoluble cytoskeleton from brain. Further, p39 coimmunoprecipitates with actin in brain lysates. Finally, disruption of the actin cytoskeleton alters p39 subcellular localization as well as kinase activity of the p39/cdk5 complex. Therefore, our results reveal the existence of the p39/cdk5 complex in vivo and suggest that it might play a role in regulating actin cytoskeletal dynamics in cells.
Inhibiting cadherin function by dominant mutant E-cadherin expression increases the extent of tight junction assembly
Authors: Troxell Megan L. , Gopalakrishnan Shobha , McCormack Joanne , Poteat Bradley A. , Pennington Janice , Garringer Sean M. , Schneeberger Eveline E. , Nelson W. James , Marrs James A. 
Published: April 2021

ABSTRACT
Previous studies have shown that induction of cadherin-mediated cell-cell adhesion leads to tight junction formation, and that blocking cadherin-mediated cell-cell adhesion inhibits tight junction assembly. Here we report analysis of tight junction assembly in MDCK cells overexpressing a mutant E-cadherin protein that lacks an adhesive extracellular domain (T151 cells). Mutant E-cadherin overexpression caused a dramatic reduction in endogenous cadherin levels. Despite this, tight junction assembly was extensive. The number of tight junction strands observed by freeze-fracture electron microscopy significantly increased in T151 cells compared to that in control cells. Our data indicate that the hierarchical regulation of junctional complex assembly is not absolute, and that inhibition of cadherin function has both positive and negative effects on tight junction assembly.