Biology subjects

Moseley, J. B.

Publications and source records attributed to Moseley, J. B..

14 recordsLinked to original sources

bioRxiv2026

The role of cell growth rate on accumulation of the mitotic cyclin Cdc13 in fission yeast

Eukaryotic cells control their size by coordinating growth and division. Fission yeast divide at a reproducible cell size due to regulated activation of the cyclin-dependent kinase Cdk1. The nuclear concentration of mitotic cyclin Cdc13 increases in a time-dependent manner to promote Cdk1 activation as cells grow. Here, we show that interphase Cdc13 is stable against degradation and nuclear export, but is diluted by cell growth. Low glucose reduced cell growth rate but not time-dependent accumulation of Cdc13. Uncoupling the rates of cell growth and Cdc13 accumulation resulted in higher concentrations of nuclear Cdc13 despite reduced cell size. This change coincided with reduced activating phosphorylation of Cdk1-T167. Mathematical modeling and experiments showed that cells maintain size homeostasis under these conditions. In contrast to low glucose, poor nitrogen reduced both cell growth rate and Cdc13 accumulation rate. Therefore, Cdc13 accumulation is independent of cell growth rate but can be altered by nutrient-specific mechanisms.

cell biology↗

bioRxiv2025

Fission yeast Pdk1 kinase regulates cytokinesis and eisosomes

The conserved phosphoinositide-dependent protein kinase PDK1 regulates cell growth and stress signaling in eukaryotes. In the fission yeast S. pombe, Pdk1 has been linked to cytokinesis, which could point to new functions for this kinase family. Here, we discovered that Pdk1 localizes to eisosomes, which create invaginations in the plasma membrane, in addition to the spindle pole body (SPB). Pdk1 promotes phosphorylation of the core eisosome protein Pil1 and regulates eisosome length. Dysregulated eisosomes are not responsible for cytokinesis defects previously observed in pdk1{Delta} cells. Instead, we found that Pdk1 regulates localization of the anillin-like protein Mid1 and the protein kinase Sid2, which promotes cytokinesis as part of the septation initiation network (SIN). Our combined results provide insights into the role of Pdk1 in eisosomes and cytokinesis, which extend the functions of this conserved protein kinase family beyond canonical growth control pathways.

cell biology↗

bioRxiv2024

PP2A-B56 regulates Mid1 protein levels for proper cytokinesis in fission yeast

Protein phosphorylation regulates many steps in the cell division process including cytokinesis. In fission yeast cells, the anillin-like protein Mid1 sets the cell division plane and is regulated by phosphorylation. Multiple protein kinases act on Mid1, but no protein phosphatases have been shown to regulate Mid1. Here, we discovered that the conserved protein phosphatase PP2A-B56 is required for proper cytokinesis by promoting Mid1 protein levels. We find that par1{Delta} cells lacking the primary B56 subunit divide asymmetrically due to the assembly of misplaced cytokinetic rings that slide towards cell tips. These par1{Delta} mutants have reduced whole-cell levels of Mid1 protein, leading to reduced Mid1 at the cytokinetic ring. Restoring proper Mid1 expression suppresses par1{Delta} cytokinesis defects. This work identifies a new PP2A-B56 pathway regulating cytokinesis through Mid1, with implications for control of cytokinesis in other organisms.

cell biology↗

bioRxiv2023

Design principles of Cdr2 node patterns in fission yeast cells

Pattern forming networks have diverse roles in cell biology. Rod-shaped fission yeast cells use pattern formation to control the localization of mitotic signaling proteins and the cytokinetic ring. During interphase, the kinase Cdr2 forms membrane-bound multiprotein complexes termed nodes, which are positioned in the cell middle due in part to the node inhibitor Pom1 enriched at cell tips. Node positioning is important for timely cell cycle progression and positioning of the cytokinetic ring. Here, we combined experimental and modeling approaches to investigate pattern formation by the Pom1-Cdr2 system. We found that Cdr2 nodes accumulate near the nucleus, and Cdr2 undergoes nucleocytoplasmic shuttling when cortical anchoring is reduced. We generated particle-based simulations based on tip inhibition, nuclear positioning, and cortical anchoring. We tested model predictions by investigating Pom1-Cdr2 localization patterns after perturbing each positioning mechanism, including in both anucleate and multinucleated cells. Experiments show that tip inhibition and cortical anchoring alone are sufficient for the assembly and positioning of nodes in the absence of the nucleus, but that the nucleus and Pom1 facilitate the formation of unexpected node patterns in multinucleated cells. These findings have implications for spatial control of cytokinesis by nodes and for spatial patterning in other biological systems.

cell biology↗

bioRxiv2022

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

Eukaryotic cells tightly control their size, but the relevant aspect of size is unknown in most cases. Fission yeast divide at a threshold cell surface area due in part to the protein kinase Cdr2. We find that fission yeast cells only divide by surface area under a size threshold but shift to volume-based divisions when they reach a larger size. The size threshold for changing from surface area to volume-based control is set by ploidy. Within this size control system, we identified the mitotic activator Cdc25 as a volume-based sizer molecule, while the mitotic cyclin Cdc13 accumulates as a timer. We propose an integrated model for cell size control based on multiple signaling pathways that report on distinct aspects of cell size and growth, including cell surface area (Cdr2), cell volume (Cdc25), and time (Cdc13). Combined modeling and experiments show how this system can generate both sizer and adder-like properties. HIGHLIGHTSO_LIFission yeast use surface area or volume-based cell size control depending on overall size and ploidy C_LIO_LIMitotic activator Cdc25 exhibits properties of a volume-based sizer molecule C_LIO_LIMitotic cyclin Cdc13 accumulates in the nucleus dependent on time, not size C_LIO_LICombined modeling and experiments identify conditions for sizer versus adder behavior C_LI

cell biology↗

bioRxiv2022

Regulation of cell size and Wee1 by elevated levels of Cdr2

Many cell cycle regulatory proteins catalyze cell cycle progression in a concentration-dependent manner. In fission yeast S. pombe, the protein kinase Cdr2 promotes mitotic entry by organizing cortical oligomeric nodes that lead to inhibition of Wee1, which itself inhibits Cdk1. cdr2{Delta} cells lack nodes and divide at increased size due to overactive Wee1, but it has not been known how increased Cdr2 levels might impact Wee1 and cell size. Using a Tetracycline-inducible expression system, we found that a 6X increase in Cdr2 expression caused hyperphosphorylation of Wee1 and reduction in cell size. This overexpressed Cdr2 formed clusters that sequestered Wee1 adjacent to the nuclear envelope. Cdr2 mutants that disrupt either kinase activity or clustering ability failed to sequester Wee1 and to reduce cell size. We propose that Cdr2 acts as a dosage-dependent regulator of cell size by sequestering its substrate Wee1 away from Cdk1 in the nucleus. This mechanism has implications for other clustered kinases, which may act similarly by sequestering substrates.

cell biology↗

bioRxiv2021

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

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 GEF (guanine nucleotide exchange factor) Syt22 was required for both Arf6 node localization and proper size at division. In arf6{Delta} mutants, Cdr2 nodes detached from the membrane and exhibited increased dynamics. These defects were enhanced when arf6{Delta} was combined with other node mutants. Our work identifies a regulated anchor for Cdr2 nodes that is required for cells to sense surface area.

cell biology↗

bioRxiv2021

Pak1 kinase regulates ribonucleoprotein granules through Sts5 for polarized growth and the glucose starvation response

Fission yeast cells maintain a rod shape due to conserved signaling pathways that organize the cytoskeleton for polarized growth. We discovered a mechanism linking the conserved protein kinase Pak1 with cell shape through the RNA-binding protein Sts5. Pak1 (also called Shk1 and Orb2) prevents Sts5 association with P bodies by directly phosphorylating its intrinsically disordered region (IDR). Pak1 and the cell polarity kinase Orb6 both phosphorylate the Sts5 IDR but at distinct residues. Mutations preventing phosphorylation in the Sts5 IDR cause increased P body formation and defects in cell shape and polarity. Unexpectedly, when cells encounter glucose starvation, PKA signaling triggers Pak1 recruitment to stress granules with Sts5. Through retargeting experiments, we reveal that Pak1 localizes to stress granules to promote rapid dissolution of Sts5 upon glucose addition. Our work reveals a new role for Pak1 in regulating cell shape through ribonucleoprotein granules during normal and stressed growth conditions.

cell biology↗

bioRxiv2020

Sequestration of the exocytic SNARE Psy1 into multiprotein nodes reinforces polarized morphogenesis in fission yeast

Polarized morphogenesis is achieved by targeting or inhibiting growth at distinct regions. Rod-shaped fission yeast cells grow exclusively at their ends by restricting exocytosis and secretion to these sites. This growth pattern implies the existence of mechanisms that prevent exocytosis and growth along non-growing cell sides. We previously identified a set of 50-100 megadalton-sized node structures along the sides of fission yeast cells that contain the interacting proteins Skb1 and Slf1. Here, we show that Skb1-Slf1 nodes contain the syntaxin-like SNARE Psy1, which mediates exocytosis in fission yeast. Psy1 localizes in a diffuse pattern at cell tips where it likely promotes exocytosis and growth, but Psy1 is sequestered in Skb1-Slf1 nodes at cell sides where growth does not occur. Mutations that prevent node assembly or inhibit Psy1 localization to nodes lead to aberrant exocytosis at cell sides and increased cell width. Genetic results indicate that this Psy1 node mechanism acts in parallel to actin cables and Cdc42 regulation. Our work suggests that sequestration of syntaxin-like Psy1 at non-growing regions of the cell cortex reinforces cell morphology by restricting exocytosis to proper sites of polarized growth.

cell biology↗

bioRxiv2019

A mechanism for how Cdr1/Nim1 kinase promotes mitotic entry by inhibiting Wee1

To enter into mitosis, cells must shut off the cell cycle inhibitor Wee1. SAD family protein kinases regulate Wee1 signaling in yeast and humans. In S. pombe, two SAD kinases (Cdr1/Nim1 and Cdr2) act as upstream inhibitors of Wee1. Previous studies found that S. pombe Cdr1/Nim1 directly phosphorylates and inhibits Wee1 in vitro, but different results were obtained for budding yeast and human SAD kinases. Without a full understanding of Cdr1 action on Wee1, it has been difficult to assess the in vivo relevance and conservation of this mechanism. Here, we show that both Cdr1 and Cdr2 promote Wee1 phosphorylation in cells, but only Cdr1 inhibits Wee1 kinase activity. Inhibition occurs when Cdr1 phosphorylates a cluster of serine residues linking -helices G and H of the Wee1 kinase domain. This region is highly divergent among different Wee1 proteins, consistent with distinct regulatory mechanisms. A wee(4A) mutant that impairs phosphorylation by Cdr1 delays mitotic entry and causes elongated cells. By disrupting and re-targeting Cdr1 localization, we show that Cdr1 inhibition of Wee1 occurs in cells at cortical nodes formed by Cdr2. Based on our results, we propose a two-step model for inhibition of Wee1 by Cdr1 and Cdr2 at nodes.

cell biology↗

bioRxiv2019

Defining how Pak1 regulates cell polarity and cell division in fission yeast

Protein kinases direct polarized growth by regulating the cytoskeleton in time and space, and could play similar roles in cell division. We found that the Cdc42-activated polarity kinase Pak1 colocalizes with the assembling contractile actomyosin ring (CAR) and remains at the division site during septation. Mutations in pak1 led to defects in CAR assembly and genetic interactions with cytokinesis mutants. Through a phosphoproteomic screen, we identified novel Pak1 substrates that function in polarized growth and cytokinesis. For cytokinesis, we found that Pak1 regulates the localization of its substrates Mid1 and Cdc15 to the CAR. Mechanistically, Pak1 phosphorylates the Mid1 N-terminus to promote its association with cortical nodes that act as CAR precursors. Defects in Pak1-Mid1 signaling lead to misplaced and defective division planes, but these phenotypes can be rescued by synthetic tethering of Mid1 to cortical nodes. Our work defines a new signaling mechanism driven by a cell polarity kinase that promotes CAR assembly in the correct time and place. SummaryMagliozzi et al. show that fission yeast cell polarity kinase Pak1 regulates cytokinesis. Through a phosphoproteomic screen and subsequent mutant analysis, their work uncovers direct targets and mechanisms for Pak1 activity during cell division.

cell biology↗

bioRxiv2019

The phosphatase inhibitor Sds23 regulates cell division symmetry in fission yeast

Animal and fungal cells divide through the assembly, anchoring, and constriction of a contractile actomyosin ring (CAR) during cytokinesis. The timing and position of the CAR must be tightly controlled to prevent defects in cell division, but many of the underlying signaling events remain unknown. The conserved heterotrimeric protein phosphatase PP2A controls the timing of events in mitosis, and upstream pathways including Greatwall-Ensa regulate PP2A activity. A role for PP2A in CAR regulation has been less clear, although loss of PP2A in yeast causes defects in cytokinesis. Here, we report that Sds23, an inhibitor of PP2A family protein phosphatases, promotes the symmetric division of fission yeast cells through spatial control of cytokinesis. We found that sds23{Delta} cells divide asymmetrically due to misplaced CAR assembly, followed by sliding of the CAR away from its assembly site. These mutant cells exhibit delayed recruitment of putative CAR anchoring proteins including the glucan synthase Bgs1. Our observations likely reflect a broader role for regulation of PP2A in cell polarity and cytokinesis because sds23{Delta} phenotypes were exacerbated when combined with mutations in the fission yeast Ensa homolog, Igo1. These results identify the PP2A regulatory network as a critical component in the signaling pathways coordinating cytokinesis.

cell biology↗

bioRxiv2019

Stable Pom1 clusters form a glucose-modulated concentration gradient that regulates mitotic entry

Control of cell size requires molecular size sensors that are coupled to the cell cycle. Rod-shaped fission yeast cells divide at a threshold size partly due to Cdr2 kinase, which forms nodes at the medial cell cortex where it inhibits the Cdk1-inhibitor Wee1. Pom1 kinase phosphorylates and inhibits Cdr2, and forms cortical concentration gradients from cell poles. Pom1 inhibits Cdr2 signaling to Wee1 specifically in small cells, but the time and place of their regulatory interactions were unclear. We show that Pom1 forms stable oligomeric puncta that dynamically sample the cell cortex. Binding frequency is patterned into a concentration gradient by the polarity landmarks Tea1 and Tea4. Pom1 puncta colocalize with Cdr2 nodes, forming a glucose-modulated inhibitory threshold against node activation. Our work reveals how Pom1-Cdr2-Wee1 operates in multiprotein clusters at the cell cortex to promote mitotic entry at a specific size that can be modified by nutrient availability.

cell biology↗

bioRxiv2017

Cell size-dependent regulation of Wee1 localization by Cdr2 cortical nodes

Cell size control requires mechanisms that link cell growth with Cdk1 activity. In fission yeast, the protein kinase Cdr2 forms cortical nodes that include the Cdk1 inhibitor Wee1, along with the Wee1-inhibitory kinase Cdr1. We investigated how nodes inhibit Wee1 during cell growth. Biochemical fractionation revealed that Cdr2 nodes were megadalton structures enriched for activated Cdr2, which increases in level during interphase growth. In live-cell TIRF movies, Cdr2 and Cdr1 remained constant at nodes over time, but Wee1 localized to nodes in short bursts. Recruitment of Wee1 to nodes required Cdr2 kinase activity and the noncatalytic N-terminus of Wee1. Bursts of Wee1 localization to nodes increased 20-fold as cells doubled in size throughout G2. Size-dependent signaling was due in part to the Cdr2 inhibitor Pom1, which suppressed Wee1 node bursts in small cells. Thus, increasing Cdr2 activity during cell growth promotes Wee1 localization to nodes, where inhibitory phosphorylation of Wee1 by Cdr1 and Cdr2 kinases promotes mitotic entry.\n\nSummaryCells turn off the mitotic inhibitor Wee1 to enter into mitosis. This study shows how cell growth progressively inhibits fission yeast Wee1 through dynamic bursts of localization to cortical node structures that contain Wee1 inhibitory kinases.

cell biology↗