Biology subjects

Opalko, H. E.

Publications and source records attributed to Opalko, H. E..

6 recordsLinked to original sources

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↗

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↗

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

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↗