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Dynamic cytoplasmic fluidity at the mesoscale during morphogenesis in a human fungal pathogen

ABSTRACT: The molecular crowding of the cytoplasm impacts a range of cellular processes. Using a fluorescent microrheological probe (GEMs), we observed a striking increase in mesoscale diffusion during the yeast to filamentous growth transition in the human fungal pathogen Candida albicans. This increase in mesoscale diffusion is due to a decrease in ribosome concentration that results in part from an inhibition of ribosome biogenesis, combined with an increase in cytoplasmic volume, leading to a dilution of a major cytoplasmic crowder at the mesoscale. Moreover, our results suggest that inhibition of ribosome biogenesis is a trigger for C. albicans morphogenesis.

INSTRUMENT(S):

ORGANISM(S): Candida Albicans (yeast)

TISSUE(S): Fungal Cell

DISEASE(S): Phenotype

SUBMITTER: Ludwig Sinn

LAB HEAD: Markus Ralser

PROVIDER: PXD067792 | Pride | 2025-11-13

REPOSITORIES: Pride

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			Action	DRS
	20240327_Z2_LRS_001_30-0112_LudwigSinn-GEMS-Ribosomes_P00_QC-001.wiff	Wiff
	20240327_Z2_LRS_001_30-0112_LudwigSinn-GEMS-Ribosomes_P00_QC-001.wiff.scan	Wiff
	20240327_Z2_LRS_007_30-0112_LudwigSinn-GEMS-Ribosomes_P01_PY6414-90m-FCS-4.wiff	Wiff
	20240327_Z2_LRS_007_30-0112_LudwigSinn-GEMS-Ribosomes_P01_PY6414-90m-FCS-4.wiff.scan	Wiff
	20240327_Z2_LRS_013_30-0112_LudwigSinn-GEMS-Ribosomes_P00_QC-002.wiff	Wiff

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Project description:DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5’ end or subsequent scanning of the 5’UTR, but whether they perform distinct functions or act redundantly in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A mutant yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more pervasive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5’UTR length and propensity for secondary structure, implicating Ded1 in scanning though structured 5' UTRs. Reporter assays confirmed that cap- distal stem-loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs is strongly dependent on eIF4A, this dependence is significantly correlated with requirements for Ded1 and 5’UTR features characteristic of Ded1- dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5’UTRs; and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling.The study includes 32 samples, comprised of 16 mRNA-Seq samples and 16 ribosome footprint profiling samples, derived from biological replicates of 3 mutant strains, ded1-cs, ded1-ts and tif1-ts, and the corresponding wild-type strains. The tif1-ts mutant and its wild-type counterpart were analyzed at 30°C and 37°C.

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Dynamic cytoplasmic fluidity at the mesoscale during morphogenesis in a human fungal pathogen

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