scholarly journals NIMA-related kinase 1 (NEK1) regulates the localization and phosphorylation of α-Adducin (ADD1) and Myosin X (MYO10) during meiosis

2017 ◽  
Author(s):  
Miguel A. Brieño-Enríquez ◽  
Stefannie L. Moak ◽  
J. Kim Holloway ◽  
Paula E. Cohen
Keyword(s):  
Meiotic Spindle ◽  
Mitotic Spindles ◽  
Spindle Formation ◽  
Protein Levels ◽  
Chromosome Congression ◽  
Multipolar Spindles ◽  
Summary Statement ◽  
Myosin X ◽  
And Function ◽  
Meiosis I

Summary statement:NEK1 kinase regulates the assembly and function of the meiosis I spindle by phosphorylating α-adducin (ADD1) and thereby facilitating its interaction with Myosin X (MYO10)Abstract:NIMA-related kinase 1 (NEK1) is a serine/threonine and tyrosine kinase that is highly expressed in mammalian germ cells. Mutations in Nek1 induce anemia, polycystic kidney and infertility. In this study we evaluated the role of NEK1 in meiotic spindle formation in both male and female gametes. Our results show that the lack of NEK1 provokes an abnormal organization of the meiosis I spindle characterized by elongated and/or multipolar spindles, and abnormal chromosome congression. The aberrant spindle structure is concomitant with the disruption in localization and protein levels of myosin X (MYO10) and α-adducin (ADD1), both of which are implicated in the regulation of spindle formation during mitosis. Interaction of ADD1 with MYO10 is dependent on phosphorylation, whereby phosphorylation of ADD1 enables its binding to MYO10 on mitotic spindles. Reduction in ADD1 protein in NEK1 mutant mice is associated with hyperphosphorylation of ADD1, thereby preventing the interaction with MYO10 during meiotic spindle formation. Our results reveal a novel regulatory role for NEK1 in the regulation of spindle architecture and function during meiosis.

scholarly journals BUB-1 targets PP2A:B56 to regulate chromosome congression during meiosis I in C. elegans oocytes

2020 ◽  
Author(s):  
Laura Bel Borja ◽  
Flavie Soubigou ◽  
Samuel J.P. Taylor ◽  
Conchita Fraguas Bringas ◽  
Jacqueline Budrewicz ◽  
...  
Keyword(s):  
Kinase Domain ◽  
Meiotic Spindle ◽  
Dependent Manner ◽  
Female Meiosis ◽  
Linear Motif ◽  
Interaction Domain ◽  
C Elegans ◽  
Chromosome Congression ◽  
B Subunits ◽  
Meiosis I

ABSTRACTProtein Phosphatase 2A (PP2A) is an heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits with various key roles during cell division. While A and C subunits form the core enzyme, the diversity generated by interchangeable B subunits dictates substrate specificity. Within the B subunits, B56-type subunits play important roles during meiosis in yeast and mice by protecting centromeric cohesion and stabilising the kinetochore-microtubule attachments. These functions are achieved through targeting of B56 subunits to centromere and kinetochore by Shugoshin and BUBR1. In the nematode Caenorhabditis elegans (C. elegans) the closest BUBR1 ortholog lacks the B56 interaction domain and the Shugoshin orthologue is not required for normal segregation during oocyte meiosis. Therefore, the role of PP2A in C. elegans female meiosis is not known. Here, we report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. Specifically, B56 subunits PPTR-1 and PPTR-2 associate with chromosomes during prometaphase I and regulate chromosome congression. The chromosome localization of B56 subunits does not require shugoshin orthologue SGO-1. Instead we have identified the kinase BUB-1 as the key B56 targeting factor to the chromosomes during meiosis. PP2A BUB-1 recruits PP2A:B56 to the chromosomes via dual mechanism: 1) PPTR-1/2 interacts with the newly identified LxxIxE short linear motif (SLiM) within a disordered region in BUB-1 in a phosphorylation-dependent manner; and 2) PPTR-2 can also be recruited to chromosomes in a BUB-1 kinase domain-dependent manner. Our results highlight a novel, BUB-1-dependent mechanism for B56 recruitment, essential for recruiting a pool of PP2A required for proper chromosome congression during meiosis I.

scholarly journals The Nek6 and Nek7 Protein Kinases Are Required for Robust Mitotic Spindle Formation and Cytokinesis

2009 ◽  
Vol 29 (14) ◽  
pp. 3975-3990 ◽  
Author(s):  
Laura O'Regan ◽  
Andrew M. Fry
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Spindles ◽  
Mitotic Progression ◽  
Serine Threonine Kinase ◽  
Spindle Formation ◽  
Spindle Poles ◽  
And Function ◽  
Interfering Rna ◽  
Reduced Activity

ABSTRACT Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.

scholarly journals Adducin-1 is essential for mitotic spindle assembly through its interaction with myosin-X

2013 ◽  
Vol 204 (1) ◽  
pp. 19-28 ◽  
Author(s):  
Po-Chao Chan ◽  
Rosaline Y.C. Hsu ◽  
Chih-Wei Liu ◽  
Chien-Chen Lai ◽  
Hong-Chen Chen
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Actin Binding ◽  
Cyclin Dependent Kinase ◽  
Mitotic Spindles ◽  
Mitotic Progression ◽  
Filamentous Actin ◽  
Multipolar Spindles ◽  
Mitotic Spindle Assembly ◽  
Myosin X

Mitotic spindles are microtubule-based structures, but increasing evidence indicates that filamentous actin (F-actin) and F-actin–based motors are components of these structures. ADD1 (adducin-1) is an actin-binding protein that has been shown to play important roles in the stabilization of the membrane cortical cytoskeleton and cell–cell adhesions. In this study, we show that ADD1 associates with mitotic spindles and is crucial for proper spindle assembly and mitotic progression. Phosphorylation of ADD1 at Ser12 and Ser355 by cyclin-dependent kinase 1 enables ADD1 to bind to myosin-X (Myo10) and therefore to associate with mitotic spindles. ADD1 depletion resulted in distorted, elongated, and multipolar spindles, accompanied by aberrant chromosomal alignment. Remarkably, the mitotic defects caused by ADD1 depletion were rescued by reexpression of ADD1 but not of an ADD1 mutant defective in Myo10 binding. Together, our findings unveil a novel function for ADD1 in mitotic spindle assembly through its interaction with Myo10.

scholarly journals Xorbit/CLASP links dynamic microtubules to chromosomes in the Xenopus meiotic spindle

2006 ◽  
Vol 172 (1) ◽  
pp. 19-25 ◽  
Author(s):  
Eva Hannak ◽  
Rebecca Heald
Keyword(s):  
Spindle Assembly ◽  
Dominant Negative ◽  
Meiotic Spindle ◽  
Chromosome Congression ◽  
Biochemical Interaction ◽  
Egg Extracts ◽  
Important Player ◽  
Centromeric Protein ◽  
And Function ◽  
Functional Mechanisms

A family of microtubule (MT)-binding proteins, Orbit/multiple asters/cytoplasmic linker protein–associated protein, has emerged as an important player during mitosis, but their functional mechanisms are poorly understood. In this study, we used meiotic egg extracts to gain insight into the role of the Xenopus laevis homologue Xorbit in spindle assembly and function. Xorbit immunodepletion or its inhibition by a dominant-negative fragment resulted in chromosome alignment defects and aberrant MT structures, including monopolar and small spindles. Xorbit-depleted extracts failed to nucleate MTs around chromatin-coated beads, indicating its essential requirement for spindle assembly in the absence of centrosomes and kinetochores. Xorbit's MT stabilizing effect was most apparent during anaphase, when spindle MTs depolymerized rapidly upon Xorbit inhibition. Biochemical interaction between a COOH-terminal Xorbit fragment and the kinetochore-associated kinesin centromeric protein E may contribute to Xorbit's role in chromosome congression. We propose that Xorbit tethers dynamic MT plus ends to kinetochores and chromatin, providing a stabilizing activity that is crucial for spindle assembly and chromosome segregation.

scholarly journals Telomeres and centromeres have interchangeable roles in promoting meiotic spindle formation

2015 ◽  
Vol 208 (4) ◽  
pp. 415-428 ◽  
Author(s):  
Alex Fennell ◽  
Alfonso Fernández-Álvarez ◽  
Kazunori Tomita ◽  
Julia Promisel Cooper
Keyword(s):  
Fission Yeast ◽  
Nuclear Membrane ◽  
Meiotic Prophase ◽  
Meiotic Spindle ◽  
The Sun ◽  
Spindle Formation ◽  
Domain Protein ◽  
Bipolar Spindles ◽  
Sun Domain ◽  
Meiosis I

Telomeres and centromeres have traditionally been considered to perform distinct roles. During meiotic prophase, in a conserved chromosomal configuration called the bouquet, telomeres gather to the nuclear membrane (NM), often near centrosomes. We found previously that upon disruption of the fission yeast bouquet, centrosomes failed to insert into the NM at meiosis I and nucleate bipolar spindles. Hence, the trans-NM association of telomeres with centrosomes during prophase is crucial for efficient spindle formation. Nonetheless, in approximately half of bouquet-deficient meiocytes, spindles form properly. Here, we show that bouquet-deficient cells can successfully undergo meiosis using centromere–centrosome contact instead of telomere–centrosome contact to generate spindle formation. Accordingly, forced association between centromeres and centrosomes fully rescued the spindle defects incurred by bouquet disruption. Telomeres and centromeres both stimulate focal accumulation of the SUN domain protein Sad1 beneath the centrosome, suggesting a molecular underpinning for their shared spindle-generating ability. Our observations demonstrate an unanticipated level of interchangeability between the two most prominent chromosomal landmarks.

scholarly journals The Kinesinlike Protein Subito Contributes to Central Spindle Assembly and Organization of the Meiotic Spindle in Drosophila Oocytes

2005 ◽  
Vol 16 (10) ◽  
pp. 4684-4694 ◽  
Author(s):  
J. K. Jang ◽  
T. Rahman ◽  
K. S. McKim
Keyword(s):  
Meiotic Spindle ◽  
Spindle Formation ◽  
Present Evidence ◽  
Homologous Chromosomes ◽  
Central Spindle ◽  
Bipolar Spindle ◽  
Passenger Proteins ◽  
Bipolar Spindles ◽  
Meiosis I

In the oocytes of many species, bipolar spindles form in the absence of centrosomes. Drosophila melanogaster oocyte chromosomes have a major role in nucleating microtubules, which precedes the bundling and assembly of these microtubules into a bipolar spindle. Here we present evidence that a region similar to the anaphase central spindle functions to organize acentrosomal spindles. Subito mutants are characterized by the formation of tripolar or monopolar spindles and nondisjunction of homologous chromosomes at meiosis I. Subito encodes a kinesinlike protein and associates with the meiotic central spindle, consistent with its classification in the Kinesin 6/MKLP1 family. This class of proteins is known to be required for cytokinesis, but our results suggest a new function in spindle formation. The meiotic central spindle appears during prometaphase and includes passenger complex proteins such as AurB and Incenp. Unlike mitotic cells, the passenger proteins do not associate with centromeres before anaphase. In the absence of Subito, central spindle formation is defective and AurB and Incenp fail to properly localize. We propose that Subito is required for establishing and/or maintaining the central spindle in Drosophila oocytes, and this substitutes for the role of centrosomes in organizing the bipolar spindle.

scholarly journals BUB-1 targets PP2A:B56 to regulate chromosome congression during meiosis I in C. elegans oocytes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Laura Bel Borja ◽  
Flavie Soubigou ◽  
Samuel J P Taylor ◽  
Conchita Fraguas Bringas ◽  
Jacqueline Budrewicz ◽  
...  
Keyword(s):  
Meiotic Spindle ◽  
Dependent Manner ◽  
Female Meiosis ◽  
Interaction Domain ◽  
C Elegans ◽  
Chromosome Congression ◽  
Phosphatase 2A ◽  
Main Chromosome ◽  
Meiosis I

Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by Shugoshin and BUBR1 to protect centromeric cohesion and stabilise kinetochore–microtubule attachments in yeast and mouse meiosis. In Caenorhabditis elegans, the closest BUBR1 orthologue lacks the B56-interaction domain and Shugoshin is not required for meiotic segregation. Therefore, the role of PP2A in C. elegans female meiosis is unknown. We report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. BUB-1 is the main chromosome-targeting factor for B56 subunits during prometaphase I. BUB-1 recruits PP2A:B56 to the chromosomes via a newly identified LxxIxE motif in a phosphorylation-dependent manner, and this recruitment is important for proper chromosome congression. Our results highlight a novel mechanism for B56 recruitment, essential for recruiting a pool of PP2A involved in chromosome congression during meiosis I.

scholarly journals Restoration of Noradrenergic Function in Parkinson’s Disease Model Mice

ASN NEURO ◽  
2021 ◽  
Vol 13 ◽  
pp. 175909142110097
Author(s):  
Kui Cui ◽  
Fan Yang ◽  
Turan Tufan ◽  
Muhammad U. Raza ◽  
Yanqiang Zhan ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Transcription Factors ◽  
Disease Model ◽  
Neuronal Loss ◽  
Mrna Levels ◽  
Gene Therapies ◽  
Protein Levels ◽  
Dopaminergic Systems ◽  
And Function

Dysfunction of the central noradrenergic and dopaminergic systems is the primary neurobiological characteristic of Parkinson’s disease (PD). Importantly, neuronal loss in the locus coeruleus (LC) that occurs in early stages of PD may accelerate progressive loss of dopaminergic neurons. Therefore, restoring the activity and function of the deficient noradrenergic system may be an important therapeutic strategy for early PD. In the present study, the lentiviral constructions of transcription factors Phox2a/2b, Hand2 and Gata3, either alone or in combination, were microinjected into the LC region of the PD model VMAT2 Lo mice at 12 and 18 month age. Biochemical analysis showed that microinjection of lentiviral expression cassettes into the LC significantly increased mRNA levels of Phox2a, and Phox2b, which were accompanied by parallel increases of mRNA and proteins of dopamine β-hydroxylase (DBH) and tyrosine hydroxylase (TH) in the LC. Furthermore, there was considerable enhancement of DBH protein levels in the frontal cortex and hippocampus, as well as enhanced TH protein levels in the striatum and substantia nigra. Moreover, these manipulations profoundly increased norepinephrine and dopamine concentrations in the striatum, which was followed by a remarkable improvement of the spatial memory and locomotor behavior. These results reveal that over-expression of these transcription factors in the LC improves noradrenergic and dopaminergic activities and functions in this rodent model of PD. It provides the necessary groundwork for the development of gene therapies of PD, and expands our understanding of the link between the LC-norepinephrine and dopamine systems during the progression of PD.

scholarly journals Genetic Relationships in the Toxin-Producing Fungal Endophyte, Alternaria oxytropis Using Polyketide Synthase and Non-Ribosomal Peptide Synthase Genes

2021 ◽  
Vol 7 (7) ◽  
pp. 538
Author(s):  
Rebecca Creamer ◽  
Deana Baucom Hille ◽  
Marwa Neyaz ◽  
Tesneem Nusayr ◽  
Christopher L. Schardl ◽  
...  
Keyword(s):  
Amino Acid ◽  
Polyketide Synthase ◽  
Genetic Relationships ◽  
Protein Sequences ◽  
Fungal Endophyte ◽  
Melanin Synthesis ◽  
Melanin Biosynthesis ◽  
Protein Levels ◽  
Oxytropis Sericea ◽  
And Function

The legume Oxytropis sericea hosts a fungal endophyte, Alternaria oxytropis, which produces secondary metabolites (SM), including the toxin swainsonine. Polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) enzymes are associated with biosynthesis of fungal SM. To better understand the origins of the SM, an unannotated genome of A. oxytropis was assessed for protein sequences similar to known PKS and NRPS enzymes of fungi. Contigs exhibiting identity with known genes were analyzed at nucleotide and protein levels using available databases. Software were used to identify PKS and NRPS domains and predict identity and function. Confirmation of sequence for selected gene sequences was accomplished using PCR. Thirteen PKS, 5 NRPS, and 4 PKS-NRPS hybrids were identified and characterized with functions including swainsonine and melanin biosynthesis. Phylogenetic relationships among closest amino acid matches with Alternaria spp. were identified for seven highly conserved PKS and NRPS, including melanin synthesis. Three PKS and NRPS were most closely related to other fungi within the Pleosporaceae family, while five PKS and PKS-NRPS were closely related to fungi in the Pleosporales order. However, seven PKS and PKS-NRPS showed no identity with fungi in the Pleosporales or the class Dothideomycetes, suggesting a different evolutionary origin for those genes.

scholarly journals DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα

2013 ◽  
Vol 451 (3) ◽  
pp. 453-461 ◽  
Author(s):  
Claudia C. S. Chini ◽  
Carlos Escande ◽  
Veronica Nin ◽  
Eduardo N. Chini
Keyword(s):  
Breast Cancer ◽  
Nuclear Receptor ◽  
Clock Genes ◽  
Mrna Levels ◽  
Protein Levels ◽  
The Stability ◽  
And Function ◽  
Interfering Rna ◽  
In Cells

The nuclear receptor Rev-erbα has been implicated as a major regulator of the circadian clock and integrates circadian rhythm and metabolism. Rev-erbα controls circadian oscillations of several clock genes and Rev-erbα protein degradation is important for maintenance of the circadian oscillations and also for adipocyte differentiation. Elucidating the mechanisms that regulate Rev-erbα stability is essential for our understanding of these processes. In the present paper, we report that the protein DBC1 (Deleted in Breast Cancer 1) is a novel regulator of Rev-erbα. Rev-erbα and DBC1 interact in cells and in vivo, and DBC1 modulates the Rev-erbα repressor function. Depletion of DBC1 by siRNA (small interfering RNA) in cells or in DBC1-KO (knockout) mice produced a marked decrease in Rev-erbα protein levels, but not in mRNA levels. In contrast, DBC1 overexpression significantly enhanced Rev-erbα protein stability by preventing its ubiquitination and degradation. The regulation of Rev-erbα protein levels and function by DBC1 depends on both the N-terminal and C-terminal domains of DBC1. More importantly, in cells depleted of DBC1, there was a dramatic decrease in circadian oscillations of both Rev-erbα and BMAL1. In summary, our data identify DBC1 as an important regulator of the circadian receptor Rev-erbα and proposes that Rev-erbα could be involved in mediating some of the physiological effects of DBC1.

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