Alcaide Gavilan, M. | Search bioRxiv

bioRxiv2025

Uncoupling of nutrient sensing and cell size control by specific defects in ceramide structure

Ceramides are essential structural lipids whose chemical diversity arises from variations in acyl-chain length and sphingoid base modifications, yet how these structural features couple metabolic state to growth regulation remains unclear. In Saccharomyces cerevisiae, the TORC2-Ypk1/2 signaling axis coordinates plasma membrane homeostasis with cellular growth; however, the precise lipid-derived signals that modulate this pathway remain incompletely characterized. Here, we establish that the structural integrity of very-long-chain fatty acid (VLCFA)-containing ceramides is a critical determinant of nutrient-dependent cell size modulation and TORC2 activity. Disruption of VLCFA elongation elevates Ypk1/2 pT662, consistent with increased TORC2 output, in both nutrient conditions. We further discovered that yeast mutants in VLCFA elongation (elo3{Delta}), fail to modulate their cell size in response to nutrient deprivation. To separate VLCFA elongation from ceramide production, we expressed mammalian ceramide synthases with defined acyl-chain preferences. Ceramides with C24-C26 chains (CerS3) supported near-normal nutrient-dependent size modulation, whereas production of C22-C24 ceramides (CerS2) phenocopied elongation mutants, leading to hyperactive TORC2 signaling and defective size regulation. Remarkably, cells producing C18 ceramides retained size control despite elevated TORC2 activity, revealing that basal signaling and size regulation can be uncoupled. Furthermore, we identify a distinct role for hydroxylation. While sur2{Delta} mutants exhibited size regulation defects resembling elongation mutants, they retained normal nutrient-responsive TORC2 signaling. Conversely, scs7{Delta} mutants maintained normal size regulation but displayed reduced basal TORC2 activity. This striking uncoupling suggests that while the TORC2 pathway senses acyl chain length, sphingoid base hydroxylation is biophysically required downstream for the execution of cell size remodeling. Our findings demonstrate that distinct structural features of ceramides differentially regulate nutrient sensing, signaling intensity, and the mechanical execution of cell size control.

cell biology↗

bioRxiv2024

Mechanisms of growth-dependent regulation of the Gin4 kinase

AbstractCell cycle progression is dependent upon cell growth. Cells must therefore translate growth into a proportional signal that can be used to determine when there has been sufficient growth for cell cycle progression. In budding yeast, the protein kinase Gin4 is required for normal control of cell growth and undergoes gradual hyperphosphorylation and activation that are dependent upon growth and proportional to the extent of growth, which suggests that Gin4 could function in mechanisms that measure cell growth. However, the molecular mechanisms that drive hyperphosphorylation of Gin4 are poorly understood. Here, we used biochemical reconstitution and genetic analysis to test hypotheses for the mechanisms that drive phosphorylation of Gin4. We ruled out a previous model in which phosphatidylserine delivered to sites of plasma membrane growth binds Gin4 to initiate autophosphorylation. Instead, we show that Elm1, a homolog of the mammalian Lkb1 tumor suppressor kinase, is sufficient to promote hyperphosphorylation of Gin4 in vitro, likely via initiation of Gin4 autophosphorylation. Furthermore, we show that casein kinase I is required for growth-dependent hyperphosphorylation of Gin4 and also for normal regulation of Elm1. Together, these discoveries lead to new insight into mechanisms that link cell cycle progression to cell growth.

cell biology↗