Arf6 anchors Cdr2 nodes at the cell cortex to control cell size at division

Abstract

Fission yeast cells prevent mitotic entry until a threshold cell surface area is reached. The protein kinase Cdr2 contributes to this size control system by forming multiprotein nodes that inhibit Wee1 at the medial cell cortex. Cdr2 node anchoring at the cell cortex is not fully understood. Through a genomic screen, we identified the conserved GTPase Arf6 as a component of Cdr2 signaling. Cells lacking Arf6 failed to divide at a threshold surface area and instead shifted to volume-based divisions at increased overall size. Arf6 stably localized to Cdr2 nodes in its GTP-bound but not GDP-bound state, and its guanine nucleotide exchange factor (GEF), Syt22, was required for both Arf6 node localization and proper size at division. In arf6Δ mutants, Cdr2 nodes detached from the membrane and exhibited increased dynamics. These defects were enhanced when arf6Δ was combined with other node mutants. Our work identifies a regulated anchor for Cdr2 nodes that is required for cells to sense surface area.

The fission yeast cell size control system integrates pathways measuring cell surface area, volume, and time

“…We started our study by asking how changes to overall cell size impact surface area-based size control. We used wide ( rga4 Δ) and skinny ( rga2 Δ) mutants to uncouple the scaling of cell surface area, volume, and length, similar to previous work (Figure 1B) (Pan et al, 2014; Facchetti et al, 2019; Opalko et al, 2022). We designed a semi-automated image analysis pipeline to measure cell geometry (Figure S1A), which confirmed that wild type cells divide at a constant surface area while cdr2 Δ cells divide at a constant volume (Figures S1B-S1E).…”

“…Fission yeast cells were grown at 25°C in YE4S medium to logarithmic phase for imaging as previously described (Opalko et al, 2022). Cells were placed in a coverglass-bottom dish (P35G-1.5-20C; MatTek), and covered with a piece of YE4S agar prewarmed to 25°C.…”

“…The cortical density of nodes scales with cell surface area and could act as a surface area measurement system (Facchetti et al, 2019; Pan et al, 2014). Indeed, loss of Cdr2 causes cells to divide at an increased size and at a reproducible volume, as opposed to surface area (Facchetti et al, 2019; Opalko et al, 2022). However, it is not known if this shift from surface area-based to volume-based size control reflects changes in overall cell size or loss of Cdr2 signaling.…”

Regulation of cell size and Wee1 kinase by elevated levels of the cell cycle regulatory protein kinase Cdr2

“…Based on this working model, we sought additional information about these Cdr2 cytoplasmic clusters. Cdr2 recruits Cdr1 kinase, Arf6 GTPase, and anillin-like Mid1 to cortical nodes ( 17 , 18 , 29 , 30 ). We did not observe strong recruitment of either Cdr1 or Arf6 to cytoplasmic clusters in P Tet -cdr2 cells ( Fig.…”

Regulation of cell size and Wee1 by elevated levels of Cdr2

“…Based on this working model, we sought additional information about these Cdr2 cytoplasmic clusters. Cdr2 recruits Cdr1 kinase and Arf6 GTPase to cortical nodes (Martin and Berthelot-Grosjean, 2009; Moseley et al ., 2009; Opalko et al ., 2022), but we did not observe strong recruitment of either Cdr1 or Arf6 to cytoplasmic clusters in P Tet -cdr2 cells (Figure 4A-B). Thus, Cdr2 and Wee1 might be the primary components of these clusters.…”