Jordan, Shawn, Laband, Kimberley, Shirasu-Hiza, Mimi, Davies, Tim, Canman, Julie, Chand, Vandana, Dumont, Julien
[
International Worm Meeting,
2013]
We have developed novel technology for rapid temperature shift experiments using a custom-built machine we call "The Therminator". We use the Therminator to dissect the precise temporal requirement for proteins in cytokinesis, or the physical division of one cell into two. Cytokinesis requires a stereotypical and highly synchronized series of events within only 5-10 minutes in the first mitotic division of the C. elegans embryo. Because many of the proteins required for division also play crucial roles during oogenesis and development, they are difficult to study by traditional genetic approaches. Combining the Therminator with fast-acting (20s), temperature-sensitive (ts) cytokinesis-defective mutants allows us to bypass developmental requirements and identify the precise temporal window during which specific proteins contribute to cytokinesis. We set out to examine the temporal requirements of five key proteins or protein complexes known to play a role in cell division including the myosin-II motor (NMY-2), a formin family actin filament nucleator (CYK-1), a regulator of Rho GTPase signaling (CYK-4), a midzone microtubule organizer (ZEN-4), and the master regulatory kinase Aurora-B (AIR-2). All of these proteins are assumed to function throughout cytokinesis; however, only myosin-II (Liu et al., Dev. Biol. 2010) has been functionally studied at high temporal resolution. Unexpectedly, we find that each protein exhibits a unique temporal profile of functional requirement. These results challenge several current models of the cellular mechanisms underlying cytokinesis. Furthermore, these data illustrate the power of fast-acting ts mutants to dissect the temporal requirement for protein function during a complex cellular event such as cytokinesis.
Schedl, Tim, Audhya, Anjon, Niessen, Sherry, Desai, Arshad, Swathi, Arur, Laband, Kimberley, Piano, Fabio, Mayers, Jonathan, Green, Rebecca A., Wang, Shaohe, Fridolfsson, Heidi, Gunsalus, Kristin, Schulman, Monty, Oegema, Karen, Kao, Huey-Ling, Starr, Daniel, Schloissnig, Siegfried, Hyman, Anthony
[
International Worm Meeting,
2011]
High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach- profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for 106 uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes- pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.