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1.  GDI-Mediated Cell Polarization in Yeast Provides Precise Spatial and Temporal Control of Cdc42 Signaling 
PLoS Computational Biology  2013;9(12):e1003396.
Cell polarization is a prerequisite for essential processes such as cell migration, proliferation or differentiation. The yeast Saccharomyces cerevisiae under control of the GTPase Cdc42 is able to polarize without the help of cytoskeletal structures and spatial cues through a pathway depending on its guanine nucleotide dissociation inhibitor (GDI) Rdi1. To develop a fundamental understanding of yeast polarization we establish a detailed mechanistic model of GDI-mediated polarization. We show that GDI-mediated polarization provides precise spatial and temporal control of Cdc42 signaling and give experimental evidence for our findings. Cell cycle induced changes of Cdc42 regulation enhance positive feedback loops of active Cdc42 production, and thereby allow simultaneous switch-like regulation of focused polarity and Cdc42 activation. This regulation drives the direct formation of a unique polarity cluster with characteristic narrowing dynamics, as opposed to the previously proposed competition between transient clusters. As the key components of the studied system are conserved among eukaryotes, we expect our findings also to apply to cell polarization in other organisms.
Author Summary
Cell polarization is a fundamental cellular process that defines a single orientation axis within prokaryotic or eukaryotic cells and is a prerequisite for developmental processes such as cell migration, proliferation or differentiation. In the yeast Saccharomyces cerevisiae cell polarization determines the position of a new growth or bud site. Although many studies have focused on identifying polarity regulators and their interactions, the fundamental mechanisms and features of cell polarity still remain controversial. Here, we develop a detailed mathematical model of diffusion-driven cell polarization, which we verify experimentally. We show that this polarization mechanism provides precise spatial and temporal control of signals, which determine the place of a new growth site. Changes induced by the cell cycle allow simultaneous switch-like regulation of polarization and activation of the GTPase Cdc42, the central polarity regulator which initiates formation of a new bud. This regulation drives direct formation of a unique Cdc42 cluster with characteristic narrowing dynamics and robustly narrow spatial focus. Hence, our analysis reveals fundamental design principles that allow cell polarization to reliably initiate developmental processes at a specific time and place. As the key components of the studied system are conserved among eukaryotes, we expect our findings also to apply to cell polarization in other organisms.
PMCID: PMC3861033  PMID: 24348237
2.  Erg Channel Is Critical in Controlling Cell Volume during Cell Cycle in Embryonic Stem Cells 
PLoS ONE  2013;8(8):e72409.
The cell cycle progression in mouse embryonic stem cells (mESCs) is controlled by ion fluxes that alter cell volume [1]. This suggests that ion fluxes might control dynamic changes in morphology over the cell cycle, such as rounding up of the cell at mitosis. However, specific channels regulating such dynamic changes and the possible interactions with actomyosin complex have not been clearly identified. Following RNAseq transcriptome analysis of cell cycle sorted mESCs, we found that expression of the K+ ion channel Erg1 peaked in G1 cell cycle phase, which was confirmed by immunostaining. Inhibition of Erg channel activity caused loss of G1 phase cells via non-apoptotic cell death. Cells first lost the ability of membrane blebbing, a typical feature of cultured embryonic stem cells. Continued Erg inhibition further increased cell volume and the cell eventually ruptured. In addition, atomic force measurements on live cells revealed a decreased cortical stiffness after treatment, suggesting alterations in actomyosin organization. When the intracellular osmotic pressure was experimentally decreased by hypertonic solution or block of K+ ion import via the Na, K-ATPase, cell viability was restored and cells acquired normal volume and blebbing activity. Our results suggest that Erg channels have a critical function in K+ ion homeostasis of mESCs over the cell cycle, and that cell death following Erg inhibition is a consequence of the inability to regulate cell volume.
PMCID: PMC3732234  PMID: 23936540
3.  Visualization of Endothelial Actin Cytoskeleton in the Mouse Retina 
PLoS ONE  2012;7(10):e47488.
Angiogenesis requires coordinated changes in cell shape of endothelial cells (ECs), orchestrated by the actin cytoskeleton. The mechanisms that regulate this rearrangement in vivo are poorly understood - largely because of the difficulty to visualize filamentous actin (F-actin) structures with sufficient resolution. Here, we use transgenic mice expressing Lifeact-EGFP to visualize F-actin in ECs. We show that in the retina, Lifeact-EGFP expression is largely restricted to ECs allowing detailed visualization of F-actin in ECs in situ. Lifeact-EGFP labels actin associated with cell-cell junctions, apical and basal membranes and highlights actin-based structures such as filopodia and stress fiber-like cytoplasmic bundles. We also show that in the skin and the skeletal muscle, Lifeact-EGFP is highly expressed in vascular mural cells (vMCs), enabling vMC imaging. In summary, our results indicate that the Lifeact-EGFP transgenic mouse in combination with the postnatal retinal angiogenic model constitutes an excellent system for vascular cell biology research. Our approach is ideally suited to address structural and mechanistic details of angiogenic processes, such as endothelial tip cell migration and fusion, EC polarization or lumen formation.
PMCID: PMC3480364  PMID: 23115648
4.  Cortical actin dynamics 
Bioarchitecture  2011;1(4):165-168.
The actin cytoskeleton plays essential roles in cell polarization and cell morphogenesis of the budding yeast Saccharomyces cerevisiae. Yeast cells utilize formin-generated actin cables as tracks for polarized transport, which forms the basis for a positive feedback loop driving Cdc42-dependent cell polarization. Previous studies on cable organization mostly focused on polarized actin cables in budded cells and their role as relatively static tracks for myosin-dependent organelle transport. Using quantitative live cell imaging, we have recently characterized the dynamics of cortical actin cables throughout the yeast cell cycle. Surprisingly, randomly oriented actin cables in G1 cells exhibited the highest level of dynamics, while cable dynamics was markedly slowed down upon cell polarization. We further demonstrated that the rapid dynamics of randomly oriented cables were driven by the formin Bni1 and Myosin V. Our data suggested a precise spatio-temporal regulation of the two yeast formins, as well as an unexpected mechanism of actin cable rearrangement through myosins. Here we discuss the immediate significance of these findings, which illustrates the importance of generating randomness for cellular organization.
PMCID: PMC3210520  PMID: 22069508
actin; formin; myosin; polarity; self organization
5.  Dynein Supports Motility of Endoplasmic Reticulum in the Fungus Ustilago maydisV⃞ 
Molecular Biology of the Cell  2002;13(3):965-977.
The endoplasmic reticulum (ER) of most vertebrate cells is spread out by kinesin-dependent transport along microtubules, whereas studies in Saccharomyces cerevisiae indicated that motility of fungal ER is an actin-based process. However, microtubules are of minor importance for organelle transport in yeast, but they are crucial for intracellular transport within numerous other fungi. Herein, we set out to elucidate the role of the tubulin cytoskeleton in ER organization and dynamics in the fungal pathogen Ustilago maydis. An ER-resident green fluorescent protein (GFP)-fusion protein localized to a peripheral network and the nuclear envelope. Tubules and patches within the network exhibited rapid dynein-driven motion along microtubules, whereas conventional kinesin did not participate in ER motility. Cortical ER organization was independent of microtubules or F-actin, but reformation of the network after experimental disruption was mediated by microtubules and dynein. In addition, a polar gradient of motile ER-GFP stained dots was detected that accumulated around the apical Golgi apparatus. Both the gradient and the Golgi apparatus were sensitive to brefeldin A or benomyl treatment, suggesting that the gradient represents microtubule-dependent vesicle trafficking between ER and Golgi. Our results demonstrate a role of cytoplasmic dynein and microtubules in motility, but not peripheral localization of the ER in U. maydis.
PMCID: PMC99612  PMID: 11907275
6.  Establishment of a robust single axis of cell polarity by coupling multiple positive feedback loops 
Nature Communications  2013;4:1807-.
Establishment of cell polarity—or symmetry breaking—relies on local accumulation of polarity regulators. Although simple positive feedback is sufficient to drive symmetry breaking, it is highly sensitive to stochastic fluctuations typical for living cells. Here, by integrating mathematical modelling with quantitative experimental validations, we show that in the yeast Saccharomyces cerevisiae a combination of actin- and guanine nucleotide dissociation inhibitor-dependent recycling of the central polarity regulator Cdc42 is needed to establish robust cell polarity at a single site during yeast budding. The guanine nucleotide dissociation inhibitor pathway consistently generates a single-polarization site, but requires Cdc42 to cycle rapidly between its active and inactive form, and is therefore sensitive to perturbations of the GTPase cycle. Conversely, actin-mediated recycling of Cdc42 induces robust symmetry breaking but cannot restrict polarization to a single site. Our results demonstrate how cells optimize symmetry breaking through coupling between multiple feedback loops.
A positive feedback loop which results in localized accumulation of the small GTPase Cdc42 generates cell polarity in budding yeast; however, such loops are inherently susceptible to noise. Here the authors demonstrate how two pathways that mediate Cdc42 recycling work together to ensure the robustness of symmetry breaking.
PMCID: PMC3674238  PMID: 23651995

Results 1-6 (6)