The single Cdk1-G1 cyclin of Cryptococcus neoformans is not essential for cell cycle progression, but plays important roles in the proper commitment to DNA synthesis and bud emergence in this yeast

Cryptococcus neoformans is the major cause of fungal meningitis in HIV-infected patients. Several studies have highlighted the important contributions of Ca2+ signaling and homeostasis to the virulence of C. neoformans. Here, we identify the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) and demonstrate its role in Ca2+ homeostasis, bud emergence, cell cycle progression, and virulence. We also show that Ncs1 function is regulated by the calcineurin/Crz1 signaling cascade. Our work provides evidence of a link between Ca2+ homeostasis and cell cycle progression in C. neoformans.

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Calcium binding protein Ncs1 is calcineurin-regulated in Cryptococcus neoformans and essential for cell division and virulence

10.1101/2020.07.23.218974 ◽

2020 ◽

Author(s):

Eamim Daidrê Squizani ◽

Júlia Catarina Vieira Reuwsaat ◽

Sophie Lev ◽

Heryk Motta ◽

Julia Sperotto ◽

...

Keyword(s):

Cell Cycle ◽

Cryptococcus Neoformans ◽

Cell Cycle Progression ◽

Calcium Sensor ◽

Cycle Progression ◽

Bud Emergence ◽

Fungal Meningitis ◽

Ef Hand ◽

Signalling Cascade ◽

Calcineurin Pathway

AbstractIntracellular calcium (Ca2+) is crucial for signal transduction in Cryptococcus neoformans, the major cause of fatal fungal meningitis. The calcineurin pathway is the only Ca2+-requiring signalling cascade implicated in cryptococcal stress adaptation and virulence, with Ca2+-binding mediated by the EF-hand domains of the Ca2+ sensor protein calmodulin. In this study, we identified the cryptococcal ortholog of neuronal calcium sensor-1 (Ncs1) as a member of the EF-hand superfamily. We demonstrated that Ncs1 has a role in Ca2+ homeostasis under stress and non-stress conditions, as the ncs1Δ mutant is sensitive to a high Ca2+ concentration and has an elevated basal Ca2+ level that correlates with increased expression of the Ca2+ transporter genes, CCH1 and MID1. Furthermore, NCS1 expression is induced by Ca2+, with the Ncs1 protein adopting a punctate subcellular distribution. We also demonstrate that, in contrast to Saccharomyces cerevisiae, NCS1 expression in C. neoformans is regulated by the calcineurin pathway via the transcription factor Crz1, as NCS1 expression is reduced by FK506 treatment and CRZ1 deletion. Moreover, the ncs1Δ mutant shares a high temperature and high Ca2+ sensitivity phenotype with the calcineurin and calmodulin mutants (cna1Δ and cam1Δ) and the NCS1 promoter contains two calcineurin/Crz1-dependent response elements (CDRE1). Ncs1-deficency coincided with reduced growth, characterized by delayed bud emergence and aberrant cell division, and hypovirulence in a mouse infection model. In summary, our data shows that Ncs1 plays distinct roles in Ca2+ sensing in C. neoformans despite widespread functional conservation of Ncs1 and other regulators of Ca2+ homeostasis.ImportanceCryptococcus neoformans is the major cause of fungal meningitis in HIV infected patients. Several studies have highlighted the important contribution of Ca2+ signalling and homeostasis to the virulence of C. neoformans. Here, we identify the cryptococcal ortholog of neuronal calcium sensor-1 (Ncs1) and demonstrate its role in Ca2+ homeostasis, bud emergence, cell cycle progression and virulence. We also show that Ncs1 function is regulated by the calcineurin/Crz1 signalling cascade. Our work provides evidence of a link between Ca2+ homeostasis and cell cycle progression in C. neoformans.

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Landomycin a inhibits DNA synthesis and cell cycle progression

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/s0960-894x(99)00261-9 ◽

1999 ◽

Vol 9 (12) ◽

pp. 1663-1666 ◽

Cited By ~ 40

Author(s):

Robert T Crow ◽

Betty Rosenbaum ◽

Roger Smith ◽

Yu Guo ◽

Kenneth S Ramos ◽

...

Keyword(s):

Cell Cycle ◽

Dna Synthesis ◽

Cell Cycle Progression ◽

Cycle Progression ◽

Landomycin A

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The human histone gene expression regulator HBP/SLBP is required for histone and DNA synthesis, cell cycle progression and cell proliferation in mitotic cells

Journal of Cell Science ◽

10.1242/jcs.01523 ◽

2004 ◽

Vol 117 (25) ◽

pp. 6043-6051 ◽

Cited By ~ 43

Author(s):

X. Zhao

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cell Proliferation ◽

Dna Synthesis ◽

Cell Cycle Progression ◽

Histone Gene ◽

Cycle Progression ◽

Histone Gene Expression ◽

Mitotic Cells

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Cellular Ras and Cyclin D1 Are Required during Different Cell Cycle Periods in Cycling NIH 3T3 Cells

Molecular and Cellular Biology ◽

10.1128/mcb.19.7.4623 ◽

1999 ◽

Vol 19 (7) ◽

pp. 4623-4632 ◽

Cited By ~ 50

Author(s):

Masahiro Hitomi ◽

Dennis W. Stacey

Keyword(s):

Cell Cycle ◽

Dna Synthesis ◽

Cyclin D1 ◽

Cell Cycle Progression ◽

Time Lapse ◽

S Phase ◽

3T3 Cells ◽

Cycle Progression ◽

Nih 3T3 ◽

Nih 3T3 Cells

ABSTRACT Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cycle position of individual cells in an asynchronous culture at the time of inhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibited entry into S phase only when introduced into cells prior to the preceding mitosis, several hours before the beginning of S phase. Anti-cyclin D1, on the other hand, was an efficient inhibitor when introduced up until just before the initiation of DNA synthesis. Cycloheximide treatment, like anti-cyclin D1 microinjection, was inhibitory throughout G1 phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry into S phase only during the first hour following mitosis. Kinetic analysis and a novel dual-labeling technique were used to confirm the differences in cell cycle requirements for Ras, cyclin D1, and cycloheximide. These studies demonstrate a fundamental difference in mitogenic signal transduction between quiescent and cycling NIH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.

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The second phase activation of protein kinase C δ at late G1 is required for DNA synthesis in serum-induced cell cycle progression

Genes to Cells ◽

10.1046/j.1365-2443.2003.00635.x ◽

2003 ◽

Vol 8 (4) ◽

pp. 311-324 ◽

Cited By ~ 10

Author(s):

Koichi Kitamura ◽

Keiko Mizuno ◽

Akiko Etoh ◽

Yoshiko Akita ◽

Akitomo Miyamoto ◽

...

Keyword(s):

Cell Cycle ◽

Protein Kinase C ◽

Protein Kinase ◽

Dna Synthesis ◽

Cell Cycle Progression ◽

Second Phase ◽

Cycle Progression ◽

Kinase C

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Capsule Growth in Cryptococcus neoformans Is Coordinated with Cell Cycle Progression

mBio ◽

10.1128/mbio.00945-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 42

Author(s):

Rocío García-Rodas ◽

Radames J. B. Cordero ◽

Nuria Trevijano-Contador ◽

Guilhem Janbon ◽

Frédérique Moyrand ◽

...

Keyword(s):

Cell Cycle ◽

Cryptococcus Neoformans ◽

Mutant Strain ◽

Virulence Factor ◽

Cell Cycle Progression ◽

Regulatory Elements ◽

Pathogenic Yeast ◽

Cycle Progression ◽

Content Type ◽

Polysaccharide Capsule

ABSTRACT The fungal pathogen Cryptococcus neoformans has several virulence factors, among which the most important is a polysaccharide capsule. The size of the capsule is variable and can increase significantly during infection. In this work, we investigated the relationship between capsular enlargement and the cell cycle. Capsule growth occurred primarily during the G1 phase. Real-time visualization of capsule growth demonstrated that this process occurred before the appearance of the bud and that capsule growth arrested during budding. Benomyl, which arrests the cells in G2/M, inhibited capsule growth, while sirolimus (rapamycin) addition, which induces G1 arrest, resulted in cells with larger capsule. Furthermore, we have characterized a mutant strain that lacks a putative G1/S cyclin. This mutant showed an increased capacity to enlarge the capsule, both in vivo (using Galleria mellonella as the host model) and in vitro. In the absence of Cln1, there was a significant increase in the production of extracellular vesicles. Proteomic assays suggest that in the cln1 mutant strain, there is an upregulation of the glyoxylate acid cycle. Besides, this cyclin mutant is avirulent at 37°C, which correlates with growth defects at this temperature in rich medium. In addition, the cln1 mutant showed lower intracellular replication rates in murine macrophages. We conclude that cell cycle regulatory elements are involved in the modulation of the expression of the main virulence factor in C. neoformans. IMPORTANCE Cryptococcus neoformans is a pathogenic fungus that has significant incidence worldwide. Its main virulence factor is a polysaccharide capsule that can increase in size during infection. In this work, we demonstrate that this process occurs in a specific phase of the cell cycle, in particular, in G1. In agreement, mutants that have an abnormal longer G1 phase show larger capsule sizes. We believe that our findings are relevant because they provide a link between capsule growth, cell cycle progression, and virulence in C. neoformans that reveals new aspects about the pathogenicity of this fungus. Moreover, our findings indicate that cell cycle elements could be used as antifungal targets in C. neoformans by affecting both the growth of the cells and the expression of the main virulence factor of this pathogenic yeast.

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Regulation of cell cycle and cyclins by 16alpha-hydroxyestrone in MCF-7 breast cancer cells

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0270293 ◽

2001 ◽

Vol 27 (3) ◽

pp. 293-307 ◽

Cited By ~ 13

Author(s):

JS Lewis ◽

TJ Thomas ◽

CM Klinge ◽

MA Gallo ◽

T Thomas

Keyword(s):

Breast Cancer ◽

Cell Cycle ◽

Dna Synthesis ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Cell Cycle Progression ◽

Fold Increase ◽

Cycle Progression ◽

Synchronized Cells ◽

Mcf 7

It has been suggested that alterations in estradiol (E(2)) metabolism, resulting in increased production of 16alpha-hydroxyestrone (16alpha-OHE(1)), is associated with an increased risk of breast cancer. In the present study, we examined the effects of 16alpha-OHE(1)on DNA synthesis, cell cycle progression, and the expression of cell cycle regulatory genes in MCF-7 breast cancer cells. G(1) synchronized cells were treated with 1 to 25 nM 16alpha-OHE(1) for 24 and 48 h. [(3)H]Thymidine incorporation assay showed that 16alpha-OHE(1) caused an 8-fold increase in DNA synthesis compared with that of control cells, whereas E(2) caused a 4-fold increase. Flow cytometric analysis of cell cycle progression also demonstrated the potency of 16alpha-OHE(1) in stimulating cell growth. When G(1) synchronized cells were treated with 10 nM 16alpha-OHE(1) for 24 h, 62+/-3% of cells were in S phase compared with 14+/-3% and 52+/-2% of cells in the control and E(2)-treated groups respectively. In order to explore the role of 16alpha-OHE(1) in cell cycle regulation, we examined its effects on cyclins (D1, E, A, B1), cyclin dependent kinases (Cdk4, Cdk2), and retinoblastoma protein (pRB) using Western and Northern blot analysis. Treatment of cells with 10 nM 16alpha-OHE(1) resulted in 4- and 3-fold increases in cyclin D1 and cyclin A, respectively, at the protein level. There was also a significant increase in pRB phosphorylation and Cdk2 activation. In addition, transient transfection assay using an estrogen response element-driven luciferase reporter vector showed a 15-fold increase in estrogen receptor-mediated transactivation compared with control. These results show that 16alpha-OHE(1) is a potent estrogen capable of accelerating cell cycle kinetics and stimulating the expression of cell cycle regulatory proteins.

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