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"Some cycle others do not" : new views on cyclin function in the multinucleate hyphae of "A. gossypii"

Hungerbühler, Anne Katrin. "Some cycle others do not" : new views on cyclin function in the multinucleate hyphae of "A. gossypii". 2007, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_7903

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Abstract

Abstract Chapter I: Cyclin characterization in A. gossypii:
The nuclear division cycle in multinucleated cells generally occurs synchronously, due to the free exchange of cell cycle regulators between nuclei sharing the same cytoplasm. The filamentous fungus, Ashbya gossypii however, undergoes asynchronous nuclear division in that neighboring nuclei are in different cell cycle stages despite close physical proximity. The behaviour of typically oscillating proteins such as cyclins was investigated in this model organism, where each nucleus has its own rhythm. As a first step towards a better understanding how such proteins function in a multinucleated cell, the deletion of the five cyclins was characterized in A. gossypii. Three of the five cyclins were essential and most interestingly the deletion of the S‑phase cyclin AgCLB5/6 revealed a lethal phenotype. This is in contrast to yeast, where the double deletion of Scclb5Δclb6Δ was viable with a delay in the timely initiation of DNA replication. Based on the lethality of the deletion of the S-phase cyclin, its key involvement in cell cycle regulation of A. gossypii is hypothesized.
A reduced amount of redundancy between the five cyclins of A. gossypii may explain the severe deletion phenotypes observed. This lack of redundancy may be the result of differences in substrate specificity, timing, levels of protein expression or localization between the different cyclins in A. gossypii and will be investigated in more detail in Chapter II and III.
Abstract Chapter II: Regulation of cell cycle progression by noncycling cyclins in the multinucleated, filamentous fungus A. gossypii:
The cell cycle process has been conserved throughout eukaryotes and requires temporally regulated expression, localization and degradation of cyclins and other regulatory proteins. Cyclin localization and oscillation has been investigated in great detail in many uninucleated cells, however nearly no data existed on cyclin behaviour in multinucleated organisms. Characterization of cyclins in A. gossypii revealed that neighboring nuclei did not differ significantly in their patterns of cyclin protein localization such that both G1 and the mitotic cyclin AgClb1/2p were present regardless of cell cycle stage, suggesting that complete destruction of cyclins is not occurring in this system. Indeed, the expression of mitotic cyclin lacking N-terminal destruction box sequences did not block cell cycle progression. Nuclei remain independent in this system even though mitotic cyclin protein transcribed from one nucleus can diffuse to and enter neighboring nuclei. Furthermore, displacing a fraction of mitotic cyclin AgClb1/2p into the cytoplasm with two exogenous nuclear export signals (NESs) led to no change in asynchrony. Cells lacking AgSic1p, a predicted cyclin-dependent kinase (CDK) inhibitor, however, showed aberrant multipolar spindles and fragmented nuclei that are indicative of flawed mitoses. We hypothesize that the continuous cytoplasm in these cells promoted the evolution of nuclear division in which CDK inhibitors primarily control CDK activity rather than oscillating mitotic cyclin proteins.
Abstract Chapter III: The S‑phase cyclin as a major cell cycle regulator in A. gossypii:
Progression through the eukaryotic cell cycle is mediated by the action of different cyclins which bind and activate a cyclin-dependent kinase to control distinct events in each stage of the cell cycle. We wanted to understand how asynchronous mitosis is controlled in the multinucleate fungus A. gossypii. Despite sharing a common cytoplasm, each nucleus seems to be independent of its neighbors and has its own cell cycle rhythm. Therefore we wanted to know, whether each nucleus contains different proteins according to its nuclear stage. Both G1 -and the mitotic cyclin AgClb1/2p had been shown to be present and nuclear during the entire division cycle. The essential S-phase cyclin AgClb5/6p was an alternative candidate for nuclear cycle dependent protein oscillation and was therefore chosen to investigate.
AgClb5/6p was localized in the nucleus of G1, S, G2 and metaphase nuclei, however during anaphase, no AgClb5/6p could be detected. Changes in protein abundance due to coordinated nuclear export could have been an explanation for its absence during anaphase. The addition of two exogenous NLSs did not reveal any effect on cell cycle progression and therefore regulation through changes in localization of the S-phase cyclin seems unlikely. However, the addition of two exogenous NESs raised the levels of synchrony in these nuclei, suggesting more diffusion and thereby interaction between nuclei.
The absence of nuclear AgClb5/6p in anaphase is likely based on cyclin degradation. Two potential degradation motifs have been identified in the N-terminus of AgClb5/6p, and one of them has been shown to cause S‑phase cyclin degradation during metaphase. Therefore we hypothesize that A. gossypii has evolved a mechanism to ensure proper cell cycle progression where only few key cell cycle proteins are oscillating and others are rather regulated by their activity through Cdk‑inhibitors such as AgSic1p.
Abstract Chapter IV: Characterization of Karyopherins and Nucleoporins in A. gossypii:
Nuclear division in the multinucleate ascomycete A. gossypii is asynchronous meaning that nuclei of different spindle stages co-exist and proceed independently of each other through the nuclear cycle. Although proteins from every nuclear stage are translated in the common cytoplasm, they do not seem to interfere with nuclear progression of nuclei in a different stage. Therefore we speculate that nuclear autonomy may be based on cyclic alterations in the uptake of proteins from this common cytoplasm.
Nuclear proteins translated in the common cytoplasm need to be guided by transport factors called karyopherins into the nucleus. Bidirectional exchange of macromolecules between the nucleus and the cytoplasm takes place through the nuclear pore, which is built by nucleoporins. As a first step to find out whether asynchrony is maintained by controlling the uptake of protein, various karyopherins and nucleoporins have been investigated. A. gossypii and S. cerevisiae sequences were compared by applying bioinformatics, gene deletions and GFP fusions of several candidate karyopherins and nucleoporins were constructed in A. gossypii and a complementation assay between A. gossypii and S. cerevisiae was made to get fundamental knowledge on the control of cell cycle progression in the multinucleate fungus A. gossypii.
Advisors:Philippsen, Peter
Committee Members:Barral, Yves and Pieters, Jean
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Applied Microbiology (Philippsen)
UniBasel Contributors:Philippsen, Peter and Pieters, Jean
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:7903
Thesis status:Complete
Number of Pages:167
Language:English
Identification Number:
edoc DOI:
Last Modified:02 Aug 2021 15:05
Deposited On:13 Feb 2009 16:03

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