The cyclin Cln1 controls polyploid titan cell formation following a stress-induced G2 arrest in Cryptococcus

Abstract

The pathogenic yeast Cryptococcus neoformans produces polyploid titan cells in response to the host lung environment that are critical for host adaptation and subsequent disease. We analyzed the in vivo and in vitro cell cycles to identify key aspects of the C. neoformans cell cycle that are important for the formation of titan cells. We identified unbudded 2C cells, referred to as a G2 arrest, produced both in vivo and in vitro in response to various stresses. Deletion of the non-essential cyclin Cln1 resulted in over-production of titan cells in vivo, and transient morphology defects upon release from stationary phase in vivo. Using a copper-repressible promoter PCTR4-CLN1 strain and a two-step in vitro titan cell formation assay, our in vitro studies revealed Cln1 functions after the G2 arrest. These studies highlight unique cell cycle alterations in C. neoformans that ultimately promote genomic diversity and virulence in this important fungal pathogen.

An in vitro method for inducing titan cells reveals novel features of yeast-to-titan switching in the human fungal pathogen Cryptococcus gattii

“…However, it is intriguing that CLN1 is partially upregulated at day 5 and 7. In C. neoformans, CLN1 forms a critical balance between DNA replication and cell division [25]. Our data suggest that CLN1 is involved in the regulation of cell division during the first 24 hr of induction and in DNA replication during the unbudded phases at 5 and 7 days.…”

“…Expression is shown relative to the housekeeping gene GAPDH. Genes quantified were A) CDC11 ( CNBG_5339) , involved in bud formation; B) CLN1 (CNBG_4803) , associated with balancing cell division and DNA replication [25]; C) MCM6 (CNBG_5506 ), involved in DNA replication; and D) BUB2 (CNBG_4446) , involved in G2 arrest. The graphs represent 3 biological repeats (3 technical replicates each), with error bars depicting the standard deviaion of delta - delta CT values.…”

“…CLN1 is required for releasing C. neoformans titan cells from G2 arrest (during cell cycle progression) [25] and therefore it is not surprising for that CLN1 is downregulated at day 3 (Fig. 5B) when the budding index drops significantly.…”

“…In C. neoformans , large unbudded G2 cells have been shown to emerge during a stationary growth phase [44]. Recently, while scrutinizing the cell cycle regulation of titan cells in C. neoformans , Altamirano et al [25] described a two-step process of titanisation: a) typically-sized cells duplicate DNA to 2C and arrest in G2 as unbudded cells; and b) then the cells are released (by the combined influence of the cell cycle gene Cyclin Cln1 and “stress signals”) to form polyploid titan cells. Contrary to this phenomenon, R265 cells exhibit an actively budding phase in the early period of induction where the majority of the cells display 1C DNA content consistent with G1 and then undergo DNA replication to form G2 arrested unbudded polyploid titan cells at the later time point (day 5 onwards) where the 1C DNA content is totally lost (Fig.…”

“…Consequently, the budding and mitosis genes, CDC11 and CLN1, were expressed early and peaked at 24hrs (a point at which budding is prevalent and most cells have a 1C DNA content), suggesting that the cells were predominantly in either G1 or M phase. CLN1 is required for releasing C. neoformans titan cells from G2 arrest (during cell cycle progression) [25] and therefore it is not surprising for that CLN1 is downregulated at day 3 (Fig. 5B) when the budding index drops significantly.…”