GDI-mediated cell polarization in yeast provides precise spatial and temporal control of Cdc42 signaling.

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.

Cortical actin dynamics driven by formins and myosin V.

Cell morphogenesis requires complex and rapid reorganization of the actin cytoskeleton. The budding yeast Saccharomyces cerevisiae is an invaluable model system for studying molecular mechanisms driving actin dynamics. Actin cables in yeast are formin-generated linear actin arrays that serve as tracks for directed intracellular transport by type V myosins. Cables are constantly reorganized throughout the cell cycle but the molecular basis for such dynamics remains poorly understood. By combining total internal reflection microscopy, quantitative image analyses and genetic manipulations we identify kinetically distinct subpopulations of cables that are differentially driven by formins and myosin. Bni1 drives elongation of randomly oriented actin cables in unpolarized cells, whereas both formins Bnr1 and Bni1 mediate slower polymerization of cables in polarized cells. Type V myosin Myo2 surprisingly acts as a motor for translational cable motility along the cell cortex. During polarization, cells change from fast to slow cable dynamics through spatio-temporal regulation of Bni1, Bnr1 and Myo2. In summary, we identify molecular mechanisms for the regulation of cable dynamics and suggest that fast actin reorganization is necessary for fidelity of cell polarization.

Activation of Imd pathway in hemocyte confers infection resistance through humoral response in Drosophila.

Upon microbial invasion the innate immune system of Drosophila melanogaster mounts a response that comes in two distinct but complimentary forms, humoral and cellular. A screen to find genes capable of conferring resistance to the Gram-positive Staphylococcus aureus upon ectopic expression in immune response tissues uncovered imd gene. This resistance was not dependent on cellular defenses but rather likely a result of upregulation of the humoral response through increased expression of antimicrobial peptides, including a Toll pathway reporter gene drosomycin. Taken together it appears that Imd pathway is capable of playing a role in resistance to the Gram-positive S. aureus, counter to notions of traditional roles of the Imd pathway thought largely to responsible for resistance to Gram-negative bacteria.

Establishment of a robust single axis of cell polarity by coupling multiple positive feedback loops.

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.

Phosphatidylserine promotes polar Cdc42 localization.

The establishment and maintenance of cell polarity requires targeted recruitment of polarity regulators to the plasma membrane. Phosphatidylserine is now shown to have a key role in polarization of yeast cells and the localization of the central polarity regulator Cdc42.

Molecular and genetic analysis of condensin function in vertebrate cells.

We engineered mutants into residues of SMC2 to dissect the role of ATPase function in the condensin complex. These residues are predicted to be involved in ATP binding or hydrolysis and in the Q-loop, which is thought to act as a mediator of conformational changes induced by substrate binding. All the engineered ATPase mutations resulted in lethality when introduced into SMC2 null cells. We found that ATP binding, but not hydrolysis, is essential to allow stable condensin association with chromosomes. How SMC proteins bind and interact with DNA is still a major question. Cohesin may form a ring structure that topologically encircles DNA. We examined whether condensin behaves in an analogous way to its cohesin counterpart, and we have generated a cleavable form of biologically active condensin with PreScission protease sites engineered into the SMC2 protein. This has allowed us to demonstrate that topological integrity of the SMC2-SMC4 heterodimer is not necessary for the stability of the condensin complex in vitro or for its stable association with mitotic chromosomes. Thus, despite their similar molecular organization, condensin and cohesin exhibit fundamental differences in their structure and function.