scholarly journals Auxin/AID versus conventional knockouts: distinguishing the roles of CENP-T/W in mitotic kinetochore assembly and stability

Open Biology ◽  
2016 ◽  
Vol 6 (1) ◽  
pp. 150230 ◽  
Author(s):  
Laura Wood ◽  
Daniel G. Booth ◽  
Giulia Vargiu ◽  
Shinya Ohta ◽  
Flavia deLima Alves ◽  
...  
Keyword(s):  
Protein Function ◽  
Mitotic Chromosomes ◽  
Protein Levels ◽  
Kinetochore Assembly ◽  
Rnai Technology ◽  
Gradual Loss ◽  
A Cell ◽  
Stable Association ◽  
Or Gene ◽  
Regulated Gene Expression

Most studies using knockout technologies to examine protein function have relied either on shutting off transcription (conventional conditional knockouts with tetracycline-regulated gene expression or gene disruption) or destroying the mature mRNA (RNAi technology). In both cases, the target protein is lost at a rate determined by its intrinsic half-life. Thus, protein levels typically fall over at least 1–3 days, and cells continue to cycle while exposed to a decreasing concentration of the protein. Here we characterise the kinetochore proteome of mitotic chromosomes isolated from a cell line in which the essential kinetochore protein CENP-T is present as an auxin-inducible degron (AID) fusion protein that is fully functional and able to support the viability of the cells. Stripping of the protein from chromosomes in early mitosis via targeted proteasomal degradation reveals the dependency of other proteins on CENP-T for their maintenance in kinetochores. We compare these results with the kinetochore proteome of conventional CENP-T/W knockouts. As the cell cycle is mostly formed from G1, S and G2 phases a gradual loss of CENP-T/W levels is more likely to reflect dependencies associated with kinetochore assembly pre-mitosis and upon entry into mitosis. Interestingly, a putative super-complex involving Rod-Zw10-zwilch (RZZ complex), Spindly, Mad1/Mad2 and CENP-E requires the function of CENP-T/W during kinetochore assembly for its stable association with the outer kinetochore, but once assembled remains associated with chromosomes after stripping of CENP-T during mitosis. This study highlights the different roles core kinetochore components may play in the assembly of kinetochores (upon entry into mitosis) versus the maintenance of specific components (during mitosis).

scholarly journals CENP-C Is Involved in Chromosome Segregation, Mitotic Checkpoint Function, and Kinetochore Assembly

2007 ◽  
Vol 18 (6) ◽  
pp. 2155-2168 ◽  
Author(s):  
Mi-Sun Kwon ◽  
Tetsuya Hori ◽  
Masahiro Okada ◽  
Tatsuo Fukagawa
Keyword(s):  
Mitotic Checkpoint ◽  
Conditional Knockout ◽  
Mitotic Chromosomes ◽  
Interphase Nuclei ◽  
Kinetochore Assembly ◽  
Checkpoint Pathway ◽  
Interphase Cells ◽  
Mitotic Delay ◽  
A Cell ◽  
Tetracycline Inducible System

CENP-C is a conserved inner kinetochore component. To understand the precise roles of CENP-C in the kinetochore, we created a cell line with a conditional knockout of CENP-C with the tetracycline-inducible system in which the target protein is inactivated at the level of transcription. We found that CENP-C inactivation causes mitotic delay. However, observations of living cells showed that CENP-C-knockout cells progressed to the next cell cycle without normal cell division after mitotic delay. Interphase cells with two nuclei before subsequent cell death were sometimes observed. We also found that ∼60% of CENP-C–deficient cells had no Mad2 signals even after treatment with nocodazole, suggesting that lack of CENP-C impairs the Mad2 spindle checkpoint pathway. We also observed significant reductions in the signal intensities of Mis12 complex proteins at centromeres in CENP-C–deficient cells. CENP-C signals were also weak in interphase nuclei but not in mitotic chromosomes of cells with a knockout of CENP-K, a member of CENP-H complex proteins. These results suggest that centromere localization of CENP-C in interphase nuclei occurs upstream of localization of the Mis12 complex and downstream of localization of the CENP-H complex.

Nutrient-regulated gene expression in eukaryotes

2006 ◽  
Vol 73 ◽  
pp. 85-96 ◽  
Author(s):  
Richard J. Reece ◽  
Laila Beynon ◽  
Stacey Holden ◽  
Amanda D. Hughes ◽  
Karine Rébora ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Control ◽  
Direct Interaction ◽  
Signalling Pathways ◽  
A Cell ◽  
One Step ◽  
Higher Eukaryotes ◽  
Regulated Gene Expression

The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.

Mechanism of estrogen receptor-dependent transcription in a cell-free system

1990 ◽  
Vol 10 (12) ◽  
pp. 6607-6612
Author(s):  
J F Elliston ◽  
S E Fawell ◽  
L Klein-Hitpass ◽  
S Y Tsai ◽  
M J Tsai ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Rna Polymerase Ii ◽  
Expression System ◽  
Steroid Receptor ◽  
Free Expression ◽  
Independent Manner ◽  
Free System ◽  
A Cell ◽  
Regulated Gene Expression ◽  
Cell Free Expression System

RNA synthesis was stimulated directly in a cell-free expression system by crude preparations of recombinant mouse estrogen receptor (ER). Receptor-stimulated transcription required the presence of estrogen response elements (EREs) in the test template and could be specifically inhibited by addition of competitor oligonucleotides containing EREs. Moreover, polyclonal antibodies directed against the DNA-binding region of ER inhibited ER-dependent transcription. In our cell-free expression system, hormone-free ER induced transcription in a hormone-independent manner. Evidence is presented suggesting that ER acts by facilitating the formation of a stable preinitiation complex at the target gene promoter and thus augments the initiation of transcription by RNA polymerase II. These observations lend support to our current understanding of the mechanism of steroid receptor-regulated gene expression and suggest strong conservation of function among members of the steroid receptor superfamily.

scholarly journals Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Encarnación Medina-Carmona ◽  
Rogelio J. Palomino-Morales ◽  
Julian E. Fuchs ◽  
Esperanza Padín-Gonzalez ◽  
Noel Mesa-Torres ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Protein Function ◽  
Conformational Dynamics ◽  
Loss Of Function ◽  
Quinone Oxidoreductase ◽  
Protein Levels ◽  
Terminal Domain ◽  
Directional Preference

Abstract Protein dynamics is essential to understand protein function and stability, even though is rarely investigated as the origin of loss-of-function due to genetic variations. Here, we use biochemical, biophysical, cell and computational biology tools to study two loss-of-function and cancer-associated polymorphisms (p.R139W and p.P187S) in human NAD(P)H quinone oxidoreductase 1 (NQO1), a FAD-dependent enzyme which activates cancer pro-drugs and stabilizes several oncosuppressors. We show that p.P187S strongly destabilizes the NQO1 dimer in vitro and increases the flexibility of the C-terminal domain, while a combination of FAD and the inhibitor dicoumarol overcome these alterations. Additionally, changes in global stability due to polymorphisms and ligand binding are linked to the dynamics of the dimer interface, whereas the low activity and affinity for FAD in p.P187S is caused by increased fluctuations at the FAD binding site. Importantly, NQO1 steady-state protein levels in cell cultures correlate primarily with the dynamics of the C-terminal domain, supporting a directional preference in NQO1 proteasomal degradation and the use of ligands binding to this domain to stabilize p.P187S in vivo. In conclusion, protein dynamics are fundamental to understanding loss-of-function in p.P187S and to develop new pharmacological therapies to rescue this function.

scholarly journals Effects of cell-cycle-dependent expression on random fluctuations in protein levels

2016 ◽  
Vol 3 (12) ◽  
pp. 160578 ◽  
Author(s):  
Mohammad Soltani ◽  
Abhyudai Singh
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Protein Levels ◽  
Stochastic Component ◽  
Stochastic Expression ◽  
A Cell ◽  
Intrinsic Stochasticity ◽  
Cycle Dependent ◽  
Random Fluctuations ◽  
Cell To Cell Variability

Expression of many genes varies as a cell transitions through different cell-cycle stages. How coupling between stochastic expression and cell cycle impacts cell-to-cell variability (noise) in the level of protein is not well understood. We analyse a model where a stable protein is synthesized in random bursts, and the frequency with which bursts occur varies within the cell cycle. Formulae quantifying the extent of fluctuations in the protein copy number are derived and decomposed into components arising from the cell cycle and stochastic processes. The latter stochastic component represents contributions from bursty expression and errors incurred during partitioning of molecules between daughter cells. These formulae reveal an interesting trade-off: cell-cycle dependencies that amplify the noise contribution from bursty expression also attenuate the contribution from partitioning errors. We investigate the existence of optimum strategies for coupling expression to the cell cycle that minimize the stochastic component. Intriguingly, results show that a zero production rate throughout the cell cycle, with expression only occurring just before cell division, minimizes noise from bursty expression for a fixed mean protein level. By contrast, the optimal strategy in the case of partitioning errors is to make the protein just after cell division. We provide examples of regulatory proteins that are expressed only towards the end of the cell cycle, and argue that such strategies enhance robustness of cell-cycle decisions to the intrinsic stochasticity of gene expression.

Gli proteins encode context-dependent positive and negative functions: implications for development and disease

Development ◽  
1999 ◽  
Vol 126 (14) ◽  
pp. 3205-3216 ◽  
Author(s):  
A. Ruiz i Altaba
Keyword(s):  
Nuclear Localization ◽  
Protein Function ◽  
Hedgehog Signaling ◽  
Functional Divergence ◽  
Dominant Negative ◽  
Full Length ◽  
Gli Proteins ◽  
A Cell ◽  
Context Dependent ◽  
Modified Forms

Several lines of evidence implicate zinc finger proteins of the Gli family in the final steps of Hedgehog signaling in normal development and disease. C-terminally truncated mutant GLI3 proteins are also associated with human syndromes, but it is not clear whether these C-terminally truncated Gli proteins fulfil the same function as full-length ones. Here, structure-function analyses of Gli proteins have been performed using floor plate and neuronal induction assays in frog embryos, as well as induction of alkaline phosphatase (AP) in SHH-responsive mouse C3H10T1/2 (10T1/2) cells. These assays show that C-terminal sequences are required for positive inducing activity and cytoplasmic localization, whereas N-terminal sequences determine dominant negative function and nuclear localization. Analyses of nuclear targeted Gli1 and Gli2 proteins suggest that both activator and dominant negative proteins are modified forms. In embryos and COS cells, tagged Gli cDNAs yield C-terminally deleted forms similar to that of Ci. These results thus provide a molecular basis for the human Polydactyly type A and Pallister-Hall Syndrome phenotypes, derived from the deregulated production of C-terminally truncated GLI3 proteins. Analyses of full-length Gli function in 10T1/2 cells suggest that nuclear localization of activating forms is a regulated event and show that only Gli1 mimics SHH in inducing AP activity. Moreover, full-length Gli3 and all C-terminally truncated forms act antagonistically whereas Gli2 is inactive in this assay. In 10T1/2 cells, protein kinase A (PKA), a known inhibitor of Hh signaling, promotes Gli3 repressor formation and inhibits Gli1 function. Together, these findings suggest a context-dependent functional divergence of Gli protein function, in which a cell represses Gli3 and activates Gli1/2 prevents the formation of repressor Gli forms to respond to Shh. Interpretation of Hh signals by Gli proteins therefore appears to involve a fine balance of divergent functions within each and among different Gli proteins, the misregulation of which has profound biological consequences.

scholarly journals Functional Analysis of Kinetochore Assembly in Caenorhabditis elegans

2001 ◽  
Vol 153 (6) ◽  
pp. 1209-1226 ◽  
Author(s):  
Karen Oegema ◽  
Arshad Desai ◽  
Sonja Rybina ◽  
Matthew Kirkham ◽  
Anthony A. Hyman
Keyword(s):  
Caenorhabditis Elegans ◽  
Chromosome Segregation ◽  
Mitotic Chromosome ◽  
Mitotic Chromosomes ◽  
Cell Stage ◽  
Kinetochore Assembly ◽  
Chromosomal Passenger ◽  
Complete Failure ◽  
Spindle Microtubules ◽  
The One

In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, we use live and fixed assays in the one-cell stage Caenorhabditis elegans embryo. We compare the consequences of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical “kinetochore null” phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A–containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.

scholarly journals Cardiac fibroblasts require focal adhesion kinase for normal proliferation and migration

2009 ◽  
Vol 296 (3) ◽  
pp. H627-H638 ◽  
Author(s):  
Ana Maria Manso ◽  
Seok-Min Kang ◽  
Sergey V. Plotnikov ◽  
Ingo Thievessen ◽  
Jaewon Oh ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Adult Mouse ◽  
Cardiac Fibroblasts ◽  
Focal Adhesions ◽  
Protein Levels ◽  
Tyrosine Kinase 2 ◽  
A Cell ◽  
And Migration ◽  
Proliferation And Migration

Migration and proliferation of cardiac fibroblasts (CFs) play an important role in the myocardial remodeling process. While many factors have been identified that regulate CF growth and migration, less is known about the signaling mechanisms involved in these processes. Here, we utilized Cre-LoxP technology to obtain focal adhesion kinase (FAK)-deficient adult mouse CFs and studied how FAK functioned in modulating cell adhesion, proliferation, and migration of these cells. Treatment of FAKflox/flox CFs with Ad/Cre virus caused over 70% reduction of FAK protein levels within a cell population. FAK-deficient CFs showed no changes in focal adhesions, cell morphology, or protein expression levels of vinculin, talin, or paxillin; proline-rich tyrosine kinase 2 (Pyk2) expression and activity were increased. Knockdown of FAK protein in CFs increased PDGF-BB-induced proliferation, while it reduced PDGF-BB-induced migration. Adhesion to fibronectin was not altered. To distinguish between the function of FAK and Pyk2, FAK function was inhibited via adenoviral-mediated overexpression of the natural FAK inhibitor FAK-related nonkinase (FRNK). Ad/FRNK had no effect on Pyk2 expression, inhibited the PDGF-BB-induced migration, but did not change the PDGF-BB-induced proliferation. FAK deficiency had only modest effects on increasing PDGF-BB activation of p38 and JNK MAPKs, with no alteration in the ERK response vs. control cells. These results demonstrate that FAK is required for the PDGF-BB-induced migratory response of adult mouse CFs and suggest that FAK could play an essential role in the wound-healing response that occurs in numerous cardiac pathologies.

scholarly journals Immunolocalization of the Bcl-2 protein within hematopoietic neoplasms

Blood ◽  
1991 ◽  
Vol 78 (4) ◽  
pp. 1062-1068 ◽  
Author(s):  
M Zutter ◽  
D Hockenbery ◽  
GA Silverman ◽  
SJ Korsmeyer
Keyword(s):  
Cell Survival ◽  
Large Cell ◽  
Hematologic Malignancies ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Protein Levels ◽  
Stage Of Development ◽  
T Cell Lymphomas ◽  
A Cell ◽  
Reactive Hyperplasia

Abstract The Bcl-2 proto-oncogene was discovered at the t(14;18) breakpoint found in most follicular B-cell lymphomas and some diffuse large-cell lymphomas. Bcl-2 is unique among proto-oncogenes, being localized to mitochondria and extending cell survival by blocking programmed cell death. We examined Bcl-2 protein expression in 82 hematologic malignancies and reactive lymphoid processes. All lymphomas with Bcl-2 rearrangement demonstrated high levels of Bcl-2 protein. However, most follicular and diffuse lymphomas without Bcl-2 rearrangement also displayed intense Bcl-2 staining. In these cases, mechanisms other than classic translocation may be deregulation Bcl-2. The pattern of Bcl-2 staining in follicular lymphoma is the inverse of the pattern in reactive hyperplasia, confirming a role for Bcl-2 immunolocalization in routine diagnosis. Small lymphocytic malignancies, including small lymphocytic lymphoma, mantle zone lymphoma, and chronic lymphocytic leukemia, expressed intermediate levels of Bcl-2. Bcl-2 protein varied in plasma cell dyscrasias. Bcl-2 protein levels in T-cell lymphomas reflected their corresponding stage of development. No substantial Bcl- 2 was present in the Reed-Sternberg cells of nodular sclerosing Hodgkin's disease. Chronic myelogenous leukemia was strongly positive for Bcl-2, consistent with the presence of Bcl-2 in normal myeloid progenitors. Immunohistochemistry identified an expanded spectrum of hematopoietic neoplasms in which Bcl-2 may provide a cell survival advantage.

scholarly journals Cell Cycle-dependent Expression and Nucleolar Localization of hCAP-H

2001 ◽  
Vol 12 (11) ◽  
pp. 3527-3537 ◽  
Author(s):  
Olga A. Cabello ◽  
Elena Eliseeva ◽  
WeiGong He ◽  
Hagop Youssoufian ◽  
Sharon E. Plon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Histone H3 ◽  
Chromosome Condensation ◽  
Nucleolar Localization ◽  
Mitotic Chromosomes ◽  
Proliferating Cells ◽  
Protein Levels ◽  
Sister Chromatids ◽  
H3 Phosphorylation ◽  
Histone H3 Phosphorylation

Condensin is a conserved 13S heteropentamer composed of two nonidentical structural maintenance of chromosome (SMC) family proteins, in Xenopus XCAP-C and XCAP-E, and three regulatory subunits, XCAP-D2, XCAP-G, and XCAP-H. Both biochemical and genetic analyses have demonstrated an essential role for the 13S condensin complex in mitotic chromosome condensation. Further, a potential requirement for condensin in completion of chromatid arm separation in early anaphase is demonstrated by the mutational phenotypes of the Drosophila homologues ofXCAP-H, barren and XCAP-C,DmSMC4. In this study we have investigated the expression and subcellular distribution of hCAP-H, the human homolog of XCAP-H, in order to better understand its cellular functions. Transcription of hCAP-H was restricted to proliferating cells with highest expression during the G2 phase of the cell cycle. In contrast, cellular hCAP-H protein levels were constant throughout the cell cycle. hCAP-H was found to be associated with mitotic chromosomes exhibiting a nonuniform but symmetric distribution along sister chromatids. The symmetry of hCAP-H association with sister chromatids suggests that there are sequence-dependent domains of condensin aggregation. During interphase hCAP-H, -C, and -E, have distinct punctate nucleolar localization, suggesting that condensin may associate with and modulate the conformation and function of rDNA. hCAP-H association with condensed chromatin was not observed in the early phase of chromosome condensation when histone H3 phosphorylation has already taken place. This finding is consistent with the hypothesis that histone H3 phosphorylation precedes condensin-mediated condensation.

Sign in / Sign up

Export Citation Format

Share Document