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Feed-forward regulation ensures stability and rapid reversibility of a cellular state. A. Doncic1, J. Skotheim1; 1Biology, Stanford University, Stanford, CA
Cellular transitions are important for all life. Such transitions, including cell fate decisions, often employ positive feedback regulation to establish and stabilize the new cellular state. However, positive feedback is unlikely to underlie stable cell cycle arrest in yeast exposed to mating pheromone because the signaling pathway is linear, rather than bistable, over a broad range of extracellular pheromone concentration. We show that the stability of the pheromone arrested state results from coherent feed-forward regulation of the cell cycle inhibitor Far1, which is activated both by phosphorylation and transcription. Fast regulation by phosphorylation allows
rapid cell cycle arrest and reentry, whereas slow Far1 synthesis reinforces arrest. Importantly, feed-forward regulation achieves a stable arrest without altering signaling pathway output throughout the reversible transition. Since feed-forward regulation achieves the ostensibly conflicting aims of arrest stability and rapid reversibility without loss of signaling information, we expect its frequent implementation at reversible cellular transitions.
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Evolution and conservation of G1/S regulation in fungi and beyond. N. E. Buchler1; 1Biology and Physics, Duke University, Durham, NC
Cyclins are central regulators of cell cycle progression and form a large super-family, with multiple members in most eukaryotes. Recent work shows that the G1–S regulatory network (cyclins and transcription factors) and its associated dynamic properties in budding yeast and mammals are conserved, despite lack of sequence homology between many regulators. In particular, the origin of fungal G1 and B-type cyclins has remained enigmatic. To address this question, I have constructed a phylogeny of the cyclin superfamily in Fungi and beyond, anchored on Saccharomyces cerevisiae G1/S-type cyclins (CLN) and B-type cyclins (CLB). When compared to cyclins across the eukaryotic kingdom, the fungal CLB/CLN cyclins cluster closest to B-type cyclins found in plants and animals. In the fungal lineage leading to S. cerevisiae, the molecular phylogeny of G1/S-type cyclins (CLN) suggests they are derivatives of an ancient B-type cyclin (CLB) duplication that occurred in an early fungal ancestor. Evidence suggests that a fungal ancestor downsized and simplified its repertoire of cyclins. The zygomycetes contain only a single B-type cyclin (CLB) and no G1 cyclins (CLN), which may represent persistence of an ancestral state. The de novo evolution of fungal G1/S cyclins (CLN) from B-type cyclins (CLB) stands in stark contrast to the strong conservation of G1-S regulatory network between budding yeast and mammals. I also examine the phylogeny of fungal G1/S transcription factors (SWI6/SWI4/MBP1) and animal G1/S transcription factors (E2F/DP). Similar to cyclin phylogeny, early Fungi are characterized by the emergence of novel SWI6/SWI4/MBP1 transcription factors and the disappearance of the original E2F/DP transcription factors. These results suggest rapid evolution of the fungal cell cycle through which novel B-type cyclins and transcription factors are able to acquire and maintain the function and dynamics of G1-S regulatory network.
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Insertion of spindle pole bodies into the nuclear envelope.
J. Chen1, C. J. Smoyer1, J. R. Unruh1, B. D. Slaughter1, S. L. Jaspersen1,2; 1Stowers Institute for Medical Research, Kansas City, MO, 2Department of Molecular & Integrative Physiology,
University of Kansas Medical Center, Kansas City, KS
The defining feature of eukaryotic cells is the double lipid bilayer of the nuclear envelope (NE) that serves as a physical barrier separating the genome from the cytosol. Nuclear pore
complexes (NPCs) are embedded in the NE to facilitate transport of proteins and other
macromolecules into and out of the nucleus. In Saccharomyces cerevisiae where the NE does not completely breakdown during mitosis, the microtubule-organizing center, known as the spindle pole body (SPB), must also be inserted into the NE to facilitate organization of the mitotic spindle. Although most aspects of this process are unknown or unproven at a molecular level, it is thought that remodeling of NE lipds and stabilization of the resulting highly conserved pore membrane structure are involved in the formation of insertion sites for both the NPC and SPB.
Using a combination of genetics, live cell imaging and biochemistry we have analyzed the process of SPB insertion in order to better understand the functional role of membrane proteins in NE remodeling, such as the pore membrane proteins Pom34 and Pom152 that are required for NPC assembly, the shared NPC and SPB component Ndc1 and the yeast ortholog of the conserved SUN family of inner nuclear membrane proteins Mps3. Our results suggest that the NPC and SPB compete for a shared insertion factor that we propose to be Ndc1. Levels of Ndc1 at the NPC and SPB are regulated by its interactions with Pom152-Pom34 and with Mps3, respectively. A competition model between the NPC and SPB for a shared insertion factor could be a mechanism to restrict SPB duplication to G1 phase of the cell cycle and may explain why mutation of many SPB components results in diploidization.
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Molecular mechanism for proteasomal recognition of ubiquitinated substrates described by CryoEM.
G. C. Lander1, M. E. Matyskiela2, A. Martin2, E. Nogales1; 1Life Sciences Division, Lawrence Berkeley National Lab, Berkeley, CA, 2Molecular and Cellular Biology, UC Berkeley, Berkeley, CA
The proteasome is the major ATP-dependent protease in eukaryotic cells, degrading unwanted cellular proteins through recognition of covalently attached polyubiquitin chains. Highly discriminate recognition of ubiquitin chains by this multicatalytic complex is crucial to cellular function. Recent studies by cryo-electron microscopy (cryoEM) offer insight into the molecular organization of the regulatory particle, which is involved in substrate recognition, unfolding, and translocation, but the structural context by which the proteasome recognizes polyubiquitin chains remains poorly understood. Using cryoEM to visualize proteasome mutants lacking one or more of the ubiquitin receptors, we now have a detailed model for polyubiquitin reception by the proteasome. Upon receptor binding of a polyubiquitin chain, the proteasome regulatory particle undergoes a conformational reorganization in which portions of the lid serve as hinged attachment points to the core particle, while the remainder of the lid repositions the ATPases for substrate delivery to the proteolytic chamber. This offers an explanation for the proteasome’s dependence on the lid for efficient substrate degradation.
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Cell cycle coupled structural oscillation of centromeric nucleosomes and kinetochores in yeast.
M. Shivaraju1,2, J. R. Unruh1, B. Slaughter1, M. Mattingly1, J. Berman3, J. Gerton1,4; 1Stowers Institute for Medical Research, Kansas city, MO, 2The Open University, Milton Keynes, United Kingdom, 3Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 4Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas city, KS
The centromere is a specialized chromosomal structure that regulates chromosome segregation. Centromeres are marked by a histone H3 variant. In budding yeast, the histone H3 variant Cse4 is present in a single centromeric nucleosome. Experimental evidence supports several different models for the structure of centromeric nucleosomes. To investigate Cse4 copy number in live yeast we developed a new method coupling fluorescence correlation spectroscopy and calibrated imaging. We find that centromeric nucleosomes have one copy of Cse4 during most of the cell cycle, whereas two copies are detected at anaphase. The proposal of an anaphase coupled structural change is supported by Cse4-Cse4 interactions, incorporation of Cse4, and the absence of Scm3 in anaphase. Nucleosome reconstitution and
ChIP suggests both Cse4 structures contain H2A/H2B. The increase in Cse4 intensity and deposition at anaphase is also observed in Candida albicans. Using the same calibrated imaging approach for kinetochore proteins, we find that some components are dynamic in anaphase. Our experimental evidence supports a cell cycle coupled oscillation of centromeric nucleosome and kinetochore structure in yeast.
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Geometric control of cell division in fission yeast: one kinase – one substrate – two effects.
P. Bhatia1, O. Hachet1, S. A. Rincon2, M. Berthelot-Grosjean1, C. Bicho3, K. E. Sawin3, A. Paoletti2, S. G. Martin1; 1Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland, 2Institut Curie, Paris, France, 3Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
Spatial and temporal controls of cell division are essential to coordinate cell growth with division. In rod-shaped fission yeast cells, the DYRK-family Pom1 kinase couples cell size to cell cycle progression by forming plasma membrane-associated gradients emanating from the cell tips, which negatively regulate the medially localized SAD-family kinase Cdr2. Cdr2 promotes mitotic entry by inhibiting Wee1 and specifies the division plane at the cell middle by interaction with the anillin-like protein Mid1. While Cdr2 is a substrate for Pom1, the molecular mechanism underlying the negative regulation of Cdr2 by Pom1 remains elusive. In particular, pom1 deletion affects both the localization and the output of Cdr2, but the relationship between the two effects is unclear. In agreement with this, by quantitative analysis of Pom1 and Cdr2 cortical distributions, we observe that, as cells grow, Cdr2 levels increase at the cell middle, within a widening window of basal-level Pom1.
Using an analog-sensitive pom1-as allele, we find that Pom1 effects on Cdr2 activity and localization are separable. Similarly, we identified a separation-of-function pom1 allele, pom1- 305, which produces short cells but shows only minor effect on Cdr2 localization. Conversely, an active pom1 allele that lacks few autophosphorylation sites and spreads along cell sides, pom1-2A, produces long cells again with minor effects on Cdr2 localization. This suggests a differential threshold of Pom1-dependent phosphorylations on Cdr2 that separates the regulation of kinase activity from localization. We identified Pom1 phosphorylation sites on Cdr2 by SILAC and generated multiple phosphosite mutants spanning the entire Cdr2. Our experiments suggest that Pom1 regulates Cdr2 localization to the medial cortical region and its activity through phosphorylation of distinct domains. We show that Cdr2 membrane anchoring depends on a KA-1 domain, involved in phospholipid binding, and a polybasic motif, which is directly phosphorylated by Pom1. Ongoing work suggests other phosphosite mutants may specifically affect Cdr2 activity. These studies illustrate how one kinase may have two distinct effects on the same substrate to control timing and positioning of cell division.