Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p-and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/ disassembly.

BackgroundMany high-throughput genomic experiments, such as Synthetic Genetic Array and yeast two-hybrid, use colony growth on solid media as a screen metric. These experiments routinely generate over 100,000 data points, making data analysis a time consuming and painstaking process. Here we describe ScreenMill, a new software suite that automates image analysis and simplifies data review and analysis for high-throughput biological experiments.ResultsThe ScreenMill, software suite includes three software tools or "engines": an open source Colony Measurement Engine (CM Engine) to quantitate colony growth data from plate images, a web-based Data Review Engine (DR Engine) to validate and analyze quantitative screen data, and a web-based Statistics Visualization Engine (SV Engine) to visualize screen data with statistical information overlaid. The methods and software described here can be applied to any screen in which growth is measured by colony size. In addition, the DR Engine and SV Engine can be used to visualize and analyze other types of quantitative high-throughput data.ConclusionsScreenMill automates quantification, analysis and visualization of high-throughput screen data. The algorithms implemented in ScreenMill are transparent allowing users to be confident about the results ScreenMill produces. Taken together, the tools of ScreenMill offer biologists a simple and flexible way of analyzing their data, without requiring programming skills.

Most human somatic cells do not express telomerase. Consequently, with each cell division their telomeres progressively shorten until replicative senescence is induced. Approximately 15% of human cancers maintain their telomeres using telomerase-independent, recombination-based mechanisms collectively termed Alternative Lengthening of Telomeres (ALT). In the yeast Saccharomyces cerevisiae, ALT cells are referred to as “survivors”. One type of survivor (type II) resembles human ALT cells in that both are defined by the amplification of telomeric repeats. We analyzed recombination-mediated telomere extension events at individual telomeres in telomerase-negative yeast during type II survivor formation and find that long telomeres are preferentially extended. Furthermore, we find that senescent cells with long telomeres are more efficient at bypassing senescence via the type II pathway. We speculate that telomere length may be important in determining whether cancer cells utilize telomerase or ALT to bypass replicative senescence.

The potential impacts of sea level rise (SLR) on 95% of the land areas of the Florida Keys were estimated through analysis of a digital elevation model (DEM) derived from airborne light detection and ranging (LiDAR) measurements in a geographic information system. The topographic detail of the LiDAR DEM allowed projections of land, population, and property inundation in 0.15 m increments across a broad range of SLR scenarios for the next century. The results showed that a 0.6 m SLR by 2100 would inundate about 70% of the total land surface, but smaller percentages of the population (17%) and real property (12%). A 1.5 m rise in sea level during the same period would inundate 91% of the land surface, 71% of the population and 68% of property in the study area. Comparison of inundation dynamics indicates that the Lower Florida Keys are more susceptible to SLR than the Upper Florida Keys. The inundation dynamics exhibit non-linear behavior and demonstrate tipping points in inundation processes beyond which the inundation of land, population, and property speeds up. Acceleration of SLR will amplify the nonlinear inundation, causing tipping points to be reached sooner.

Srs2 is a 3’ to 5’ DNA helicase that regulates many aspects of DNA metabolism in Saccharomyces cerevisiae. It is best known for its ability to counteract homologous recombination by dismantling Rad51 filaments, but is also involved in checkpoint activation, adaptation and recovery, and in resolution of late recombination intermediates. To further address its biological roles and uncover new genetic interactions, we examined the consequences of overexpressing SRS2 as well as two helicase-dead mutants, srs2-K41A and srs2-K41R, in the collection of 4827 yeast haploid deletion mutants. We identified 274 genes affecting a large variety of cellular functions that are required for cell growth when SRS2 or its mutants are overexpressed. Further analysis of these interactions reveals that Srs2 acts independently of its helicase function at replication forks likely through its recruitment by the sumoylated PCNA replication clamp. This helicase-independent function is responsible for the negative interactions with DNA metabolism genes and for the toxicity of SRS2 overexpression in many of the diverse cellular pathways revealed in our screens.