scholarly journals Alpha-Tubulin Acetylation in Trypanosoma cruzi: A Dynamic Instability of Microtubules Is Required for Replication and Cell Cycle Progression

2021 ◽  
Vol 11 ◽  
Author(s):  
Victoria Lucia Alonso ◽  
Mara Emilia Carloni ◽  
Camila Silva Gonçalves ◽  
Gonzalo Martinez Peralta ◽  
Maria Eugenia Chesta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Cruzi ◽  
Basal Body ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Dynamic Instability ◽  
Cycle Progression ◽  
Alpha Tubulin ◽  
Over Expression ◽  
Tubulin Acetylation

Trypanosomatids have a cytoskeleton arrangement that is simpler than what is found in most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body. Subpellicular, mitotic and axonemal microtubules are extensively acetylated inTrypanosoma cruzi. Acetylation on lysine (K) 40 of α-tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that K40 acetylation occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle in eukaryotes. Several tubulin posttranslational modifications, including acetylation of K40, have been cataloged in trypanosomatids, but the functional importance of these modifications for microtubule dynamics and parasite biology remains largely undefined. The primary tubulin acetyltransferase was recently identified in several eukaryotes as Mec-17/ATAT, a Gcn5-related N-acetyltransferase. Here, we report thatT. cruziATAT acetylates α-tubulinin vivoand is capable of auto-acetylation.TcATAT is located in the cytoskeleton and flagella of epimastigotes and colocalizes with acetylated α-tubulin in these structures. We have expressedTcATAT with an HA tag using the inducible vector pTcINDEX-GW inT. cruzi. Over-expression ofTcATAT causes increased levels of the alpha tubulin acetylated species, induces morphological and ultrastructural defects, especially in the mitochondrion, and causes a halt in the cell cycle progression of epimastigotes, which is related to an impairment of the kinetoplast division. Finally, as a result ofTcATAT over-expression we observed that parasites became more resistant to microtubule depolymerizing drugs. These results support the idea that α-tubulin acetylation levels are finely regulated for the normal progression ofT. cruzicell cycle.

scholarly journals Alpha-tubulin acetylation in Trypanosoma cruzi: a dynamic instability of microtubules is required for replication and cell cycle progression

2020 ◽  
Author(s):  
Victoria Lucia Alonso ◽  
Mara Emilia Carloni ◽  
Camila Silva Goncalves ◽  
Gonzalo Martinez Peralta ◽  
Maria Eugenia Chesta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Cruzi ◽  
Basal Body ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Dynamic Instability ◽  
Cycle Progression ◽  
Over Expression ◽  
Tubulin Acetylation

Trypanosomatids have a cytoskeleton arrangement that is simpler than what is found in most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body. Subpellicular, mitotic and axonemal microtubules are extensively acetylated in Trypanosoma cruzi. Acetylation on lysine (K) 40 of α-tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that K40 acetylation occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle in eukaryotes. Several tubulin posttranslational modifications, including acetylation of K40, have been catalogued in trypanosomatids, but the functional importance of these modifications for microtubule dynamics and parasite biology remains largely undefined. The primary tubulin acetyltransferase that delivers this modification was recently identified in several eukaryotes as Mec-17/ATAT, a Gcn5-related N-acetyltransferase. Here, we report that T. cruzi ATAT acetylates α-tubulin in vivo and is capable of auto-acetylation. TcATAT is located in the cytoskeleton and flagella of epimastigotes and colocalizes with acetylated α-tubulin in these structures. We have expressed TcATAT with an HA tag using the inducible vector pTcINDEX-GW in T. cruzi. Over-expression of TcATAT causes increased levels of the acetylated isoform, induces morphological and ultrastructural defects, especially in the mitochondrion, and causes a halt in the cell cycle progression of epimastigotes, which is related to an impairment of the kinetoplast division. Finally, as a result of TcATAT over-expression we observed that parasites became more resistant to microtubule depolymerizing drugs. These results support the idea that α-tubulin acetylation levels are finely regulated for the normal progression of T. cruzi cell cycle.

scholarly journals Centrin Gene Disruption Impairs Stage-specific Basal Body Duplication and Cell Cycle Progression inLeishmania

2004 ◽  
Vol 279 (24) ◽  
pp. 25703-25710 ◽  
Author(s):  
Angamuthu Selvapandiyan ◽  
Alain Debrabant ◽  
Robert Duncan ◽  
Jacqueline Muller ◽  
Poonam Salotra ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Basal Body ◽  
Gene Disruption ◽  
Cell Cycle Progression ◽  
Cycle Progression

The role of gamma-tubulin in mitotic spindle formation and cell cycle progression in Aspergillus nidulans

1997 ◽  
Vol 110 (5) ◽  
pp. 623-633 ◽  
Author(s):  
M.A. Martin ◽  
S.A. Osmani ◽  
B.R. Oakley
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Cytoplasmic Microtubule ◽  
Cycle Progression ◽  
Spindle Microtubules ◽  
Gamma Tubulin

gamma-Tubulin has been hypothesized to be essential for the nucleation of the assembly of mitotic spindle microtubules, but some recent results suggest that this may not be the case. To clarify the role of gamma-tubulin in microtubule assembly and cell-cycle progression, we have developed a novel variation of the gene disruption/heterokaryon rescue technique of Aspergillus nidulans. We have used temperature-sensitive cell-cycle mutations to synchronize germlings carrying a gamma-tubulin disruption and observe the phenotypes caused by the disruption in the first cell cycle after germination. Our results indicate that gamma-tubulin is absolutely required for the assembly of mitotic spindle microtubules, a finding that supports the hypothesis that gamma-tubulin is involved in spindle microtubule nucleation. In the absence of functional gamma-tubulin, nuclei are blocked with condensed chromosomes for about the length of one cell cycle before chromatin decondenses without nuclear division. Our results indicate that gamma-tubulin is not essential for progression from G1 to G2, for entry into mitosis nor for spindle pole body replication. It is also not required for reactivity of spindle pole bodies with the MPM-2 antibody which recognizes a phosphoepitope important to mitotic spindle formation. Finally, it does not appear to be absolutely required for cytoplasmic microtubule assembly but may play a role in the formation of normal cytoplasmic microtubule arrays.

scholarly journals Budding Yeast Bub2 Is Localized at Spindle Pole Bodies and Activates the Mitotic Checkpoint via a Different Pathway from Mad2

1999 ◽  
Vol 145 (5) ◽  
pp. 979-991 ◽  
Author(s):  
Roberta Fraschini ◽  
Elisa Formenti ◽  
Giovanna Lucchini ◽  
Simonetta Piatti
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Cycle Progression ◽  
Spindle Pole Bodies

The mitotic checkpoint blocks cell cycle progression before anaphase in case of mistakes in the alignment of chromosomes on the mitotic spindle. In budding yeast, the Mad1, 2, 3, and Bub1, 2, 3 proteins mediate this arrest. Vertebrate homologues of Mad1, 2, 3, and Bub1, 3 bind to unattached kinetochores and prevent progression through mitosis by inhibiting Cdc20/APC-mediated proteolysis of anaphase inhibitors, like Pds1 and B-type cyclins. We investigated the role of Bub2 in budding yeast mitotic checkpoint. The following observations indicate that Bub2 and Mad1, 2 probably activate the checkpoint via different pathways: (a) unlike the other Mad and Bub proteins, Bub2 localizes at the spindle pole body (SPB) throughout the cell cycle; (b) the effect of concomitant lack of Mad1 or Mad2 and Bub2 is additive, since nocodazole-treated mad1 bub2 and mad2 bub2 double mutants rereplicate DNA more rapidly and efficiently than either single mutant; (c) cell cycle progression of bub2 cells in the presence of nocodazole requires the Cdc26 APC subunit, which, conversely, is not required for mad2 cells in the same conditions. Altogether, our data suggest that activation of the mitotic checkpoint blocks progression through mitosis by independent and partially redundant mechanisms.

scholarly journals Association of human ubiquitin-conjugating enzyme CDC34 with the mitotic spindle in anaphase

2000 ◽  
Vol 113 (10) ◽  
pp. 1687-1694 ◽  
Author(s):  
F. Reymond ◽  
C. Wirbelauer ◽  
W. Krek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Intracellular Localization ◽  
Cycle Progression ◽  
Ubiquitin Conjugating Enzyme ◽  
Regulation Of Cell Cycle ◽  
Conjugating Enzyme

Present in organisms ranging from yeast to man, homologues of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme CDC34 have been shown to play important roles in the regulation of cell cycle progression and checkpoint function. Here we analyze the expression and intracellular localization of endogenous CDC34 during mammalian cell cycle progression. We find that CDC34 protein is constitutively expressed during all stages of the cell cycle. Immunofluorescence experiments reveal that during interphase, endogenous CDC34 is localized to distinct speckles in both the nucleus and the cytoplasm. The presence of CDC34 in these compartments has also been established by biochemical fractionation experiments. Interestingly, nuclear localization depends on the presence of specific carboxy-terminal CDC34 sequences that have previously been shown to be required for CDC34's cell cycle function in Saccharomyces cerevisiae. Finally, we find that in anaphase and not during early stages of mitosis, CDC34 colocalizes with (beta)-tubulin at the mitotic spindle, implying that it may contribute to spindle function at later stages of mitosis. Taken together, these results support a model in which CDC34 ubiquitin-conjugating enzyme functions in the regulation of nuclear and cytoplasmic activities as well as in the process of chromosome segregation at the onset of anaphase in mammalian cells.

TLE Corepressors as Novel Candidate AML Tumor Suppressor Genes That Regulate Wnt Signaling, Cell Cycle Progression, and Myeloid Differentiation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1601-1601
Author(s):  
Farshid Dayyani ◽  
Yuntian Zhang ◽  
Sayyed T. Zaidi ◽  
David A. Sweetser
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Wnt Signaling ◽  
Tumor Suppressor Genes ◽  
Cell Cycle Progression ◽  
Myeloid Cell ◽  
Myeloid Differentiation ◽  
Cd11b Expression ◽  
Cycle Progression ◽  
Over Expression

Abstract Deletion of the long arm of chromosome 9, del(9q), is one of the most common mutations associated with t(8;21) AML. Up to 50% of del(9q) AML is seen in association with t(8;21). The Runx1-ETO (AML1-ETO) fusion gene produced by t(8;21) is insufficient for leukemogenesis and loss of a critical gene(s) on chr9q apparently cooperates in leukemogenesis. We recently identified two members of the Groucho family of co-repressors, Transducin-like enhancer of split (TLE) 1 and TLE4, as candidate tumor suppressor genes, based on our mapping of the commonly deleted region in del(9q) AML. These proteins are known to inhibit Wnt signaling which has been implicated in hematopoietic stem cell renewal and they interact with hematopoietic transcription factors such as Runx1 and Pu.1. We have demonstrated that the expression of these two genes is specifically repressed in both del(9q) and t(8;21) AML samples. Knockdown of TLE1 or TLE4 mRNA using specific small interfering RNAs (siRNA) activates Wnt signaling in 293T cells as measured by TOPFLASH activity, while forced expression of TLE1 or 4 inhibits TOPFLASH. We show that siRNA against TLE4 using a specific siRNA increased cell cycle progression and cell division, while over-expression of TLE1 or TLE4 slowed cell cycle progression and lead to a pronounced growth disadvantage in THP-1 and HL60 myeloid cell lines. In addition to these effects on cell proliferation, these genes also affected myeloid cell differentiation. Over-expression of either TLE1 or TLE4 in monocytic THP-1 and promyelocytic HL-60 cell lines initiated myeloid differentiation as monitored by CD11b expression. These two genes have different effects on more terminal myeloid differentiation as TLE4, but not TLE1, was able to induce more terminal differentiation of THP-1 cells into monocytes as measured by CD14 expression, while TLE1 was able to induce the granulocytic marker CD15 in HL-60 cells. Furthermore, lentiviral delivery of siRNAs for either TLE1 or TLE4 to HL-60 cells inhibited induction of granulocytic and monocytic differentiation with all-trans retinoic acid (ATRA) or 1,25-dihydroxycholechalciferol (Vit.D3). TLE1 siRNA inhibited ATRA and Vit.D3 induced CD11b expression by more than 75%. While TLE4 siRNA decreased the induction of monocytic CD14 after Vit.D3 by 40%, TLE1 siRNA almost completely abrogated induction of CD14 with Vit.D3 or granulocytic CD15 with ATRA in HL-60 cells. Ex vivo culture of siRNA infected CD34 sorted human cord blood (CB) cells in semi-liquid media with differentiation inducing cytokines for seven days exhibited a decrease in the percentage of early CD33+ myeloid cells by 15% and 70% relative to controls in TLE1 and TLE4 siRNA infected CB cells, respectively. These results indicate that inhibition of TLE activity can promote cell cycle progression and inhibit myeloid differentiation. This study is the first to demonstrate a potential role for the TLEs in leukemogenesis and also indicates the TLEs may have non-redundant functions in myeloid differentiation. Understanding how various mutations work cooperatively to produce a malignant phenotype is one of the great challenges in oncology. The TLEs may represent an important cooperating mutation with Runx1-ETO in AML that links Wnt signaling and core binding protein transcription factors. Studies are currently underway to demonstrate this cooperativity.

scholarly journals Coordination of Cell Cycle Progression and Mitotic Spindle Assembly Involves Histone H3 Lysine 4 Methylation by Set1/COMPASS

Genetics ◽  
2016 ◽  
Vol 205 (1) ◽  
pp. 185-199 ◽  
Author(s):  
Traude H. Beilharz ◽  
Paul F. Harrison ◽  
Douglas Maya Miles ◽  
Michael Ming See ◽  
Uyen Minh Merry Le ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Histone H3 ◽  
Spindle Assembly ◽  
Cycle Progression ◽  
Mitotic Spindle Assembly

scholarly journals Low DLG2 gene expression, a link between 11q-deleted and MYCN-amplified neuroblastoma, causes forced cell cycle progression, and predicts poor patient survival.

2020 ◽  
Author(s):  
Simon Keane ◽  
Sophie Ameen ◽  
Angelica Lindlöf ◽  
Katarina Ejeskär
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Patient Survival ◽  
Fruit Fly ◽  
Tumor Stage ◽  
Expression Level ◽  
Mycn Amplification ◽  
Cycle Progression ◽  
Over Expression

Abstract Neuroblastoma (NB) is a childhood neural crest tumor. There are two groups of aggressive NBs, one with MYCN amplification, and another with 11q chromosomal deletion; these chromosomal aberrations are generally mutually exclusive. The DLG2 gene resides in the 11q-deleted region, thus makes it an interesting NB candidate tumor suppressor gene. Methods We evaluated the association of DLG2 gene expression in NB with patient outcomes, stage and MYCN status, using online microarray data combining independent NB patient data sets. Functional studies were also conducted using NB cell models and the fruit fly. Results Using the array data we concluded that higher DLG2 expression was positively correlated to patient survival. We could also see that expression of DLG2 was inversely correlated with MYCN status and tumor stage. Cell proliferation was lowered in both 11q-normal and 11q-deleted NB cells after DLG2 over expression, and increased in 11q-normal NB cells after DLG2 silencing. Higher level of DLG2 increased the percentage of cells in the G2/M phase and decreased the percentage of cells in the G1 phase. We detected increased protein levels of Cyclin A and Cyclin B in a fruit fly model over expressing dMyc or silencing dmDLG , indicating that both events results in enhanced cell cycling. Induced MYCN expression in NB cells, lowered DLG2 gene expression, this was confirmed in the fly, when dMyc was over expressed, the dmDLG protein level was lowered, indicating a link between Myc over expression and low dmDLG level. Conclusion We conclude that low DLG2 expression level forces cell cycle progression, and that it predicts poor NB patient survival. The low DLG2 expression level could be caused by either MYCN -amplification or 11q-deletion.

scholarly journals Subcellular localization and mitotic interactome analyses identify SIRT4 as a centrosomally localized and microtubule associated protein

2020 ◽  
Author(s):  
Laura Bergmann ◽  
Alexander Lang ◽  
Christoph Bross ◽  
Simone Altinoluk-Hambüchen ◽  
Iris Fey ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Suppressor ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Mitotic Progression ◽  
Cycle Progression ◽  
Functional Roles ◽  
The Family ◽  
Spindle Apparatus ◽  
Regulation Of Cell Cycle

AbstractThe stress-inducible and senescence-associated tumor suppressor SIRT4, a member of the family of mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), regulates bioenergetics and metabolism via NAD+-dependent enzymatic activities. Next to the known mitochondrial location, we found that a fraction of endogenous or ectopically expressed SIRT4, but not SIRT3, is located at the mitotic spindle apparatus in the cytosol. Confocal spinning disk microscopy revealed that SIRT4 localizes during the cell cycle dynamically at centrosomes with an intensity peak in G2 and early mitosis. Moreover, SIRT4 binds to microtubules and interacts with structural (α,β-tubulin, γ-tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by co-immunoprecipitation and mass spectrometric analyses of the mitotic SIRT4 interactome. Overexpression of SIRT4 resulted in a pronounced decrease of acetylated α-tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4(ΔN28), which is unable to translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. This study extends the functional roles of SIRT4 beyond mitochondrial metabolism, and suggests that SIRT4 acts as a novel centrosomal / microtubule-associated protein in the regulation of cell cycle progression. Thus, stress-induced SIRT4 may exert its role as tumor suppressor through mitochondrial as well as extramitochondrial functions, the latter associated with its localization at the mitotic spindle apparatus.

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