Anjon Audhya et al. (2006) Development & Evolution Meeting "A novel mitotic function for rab-5 in structural reorganization of the endoplasmic reticulum and nuclear envelope disassembly"

Karen Oegema, Anjon Audhya

[Development & Evolution Meeting, 2006]

In many cells, mitotic entry is accompanied by a dramatic restructuring of the endoplasmic reticulum (ER). Despite its prevalence, both the molecular mechanics and functional significance of this structural transition remain poorly understood. To investigate the mitotic reorganization of the ER, we combine imaging-based assays with RNA interference during the first mitotic division of the C. elegans embryo. Timelapse imaging of embryos expressing GFP-labeled ER markers reveals that restructuring occurs in two steps with distinct molecular requirements: (1) reticulation to form a highly tubular network, and (2) consolidation of ER into larger sheet-like structures. Interestingly, we show that the early endosomal Rab type GTPase RAB-5 is required for sheet formation, but not for reorganization of the ER into a reticular network. We also identify a pair of functionally redundant ER components whose depletion similarly blocks sheet formation but not reticulation. Interestingly, blocking sheet formation by either depletion of RAB-5 or simultaneous depletion of the pair of ER-localized proteins results in a pronounced defect in nuclear envelope disassembly. In contrast, preventing nuclear envelope disassembly by other means does not perturb reorganization of the ER. Cumulatively, these results suggest that structural reorganization of the ER during mitosis is required for efficient disassembly of the nuclear envelope.

Anjon Audhya et al. (2007) International Worm Meeting "A screen to identify factors necessary for plasma membrane remodeling during cellularization and cell division."

Karen Oegema, Anjon Audhya, Rebecca Green

[International Worm Meeting, 2007]

Membrane trafficking between different cellular compartments is required in all eukaryotic cells to maintain cellular integrity, communicate with the extracellular environment, and facilitate cell division. To identify components of the membrane transport machinery that contribute to cell division, we initiated an RNAi-based screen to identify genes required for cellularization in the C. elegans gonad (an event analogous to cytokinesis). To execute this screen, we have constructed a transgenic worm strain co-expressing an RFP fusion with histone H2B and a GFP fusion with a specific PH domain, to simultaneously label the DNA and plasma membrane. Using this strain, we can rapidly screen through RNA-treated hermaphrodites to identify genes whose inhibition results in the formation of polyploid oocytes or defects in the organization of membrane partitions in the gonad. Initial studies have shown that loss of particular proteins known to function in membrane transport, such as the Rab-type GTPase Rab11, leads to the production of oocytes containing multiple nuclei, indicating a defect in cellularization. Using data from a number of independent RNAi-based genome-wide screens, we have identified the set of non-redundant genes essential for embryo production. Individual depletion of proteins encoded by these genes has recently been completed, allowing an initial assessment of those important for cellularization. Using both fluorescence and biochemical-based secondary screening approaches, new regulators of membrane transport have been identified and their further characterization is underway.

Anjon Audhya et al. (2005) International Worm Meeting "The Scd6p family protein CAR-1 is a component of a complex of RNA-binding proteins required for cytokinesis in C. elegans"

Ian MacLeod, Anjon Audhya, Francie Hyndman, Amy Maddox, Karen Oegema

[International Worm Meeting, 2005]

Cytokinesis completes cell division by partitioning the intracellular constituents of one cell to form two topologically distinct daughter cells. Here we describe the characterization of CAR-1, a predicted RNA binding protein whose depletion results in a specific defect in cytokinesis. Consistent with a role in RNA metabolism, CAR-1 localizes to germline-specific RNA-containing P-granules and co-purifies with the essential RNA helicase CGH-1, which controls its localization. The atypical Sm domain of CAR-1, which is predicted to mediate an association with RNA, is dispensable for CAR-1 localization but critical for its function. The failure of cytokinesis in CAR-1 depleted embryos likely results from a pronounced defect in the structure of the anaphase spindle, which normally interacts with the cortex to promote the completion of cytokinesis. In CAR-1 depleted embryos, inter-zonal microtubule bundles that recruit Aurora B kinase and the kinesin ZEN-4 fail to form. Depletion of CGH-1 results in sterility, but partially depleted worms produce embryos that exhibit a nearly identical phenotype to that resulting from depletion of CAR-1. Cumulatively, these results suggest that CAR-1 and CGH-1 function together to regulate RNAs important for anaphase spindle structure and point to a connection between RNA metabolism and cytokinesis.

Shao, Xiangqiang et al. (2013) International Worm Meeting "An In Vivo Analysis of Critical Functional Domains of a-catenin in C. elegans."

Shao, Xiangqiang, Hardin, Jeff, Simske, Jeffrey, Audhya, Anjon

[International Worm Meeting, 2013]

Stable intercellular adhesions are critical for normal embryonic development in metazoans, during which tissue integrity is maintained as cells undergo morphogenetic movements. The adherens junction, a key cell-cell adhesion structure, contains a highly conserved cadherin-catenin complex (CCC). a-catenin is an important regulator that connects the CCC to F-actin at adherens junctions. C. elegans provides a good model to study the function of a-catenin in vivo, since hmp-1 is the sole a-catenin homolog. Given the potential role of a-catenin as a physical linker between CCC and the actin cytoskeleton, we hypothesized that recruiting a-catenin to junctions irrespective of its normal binding partners is sufficient for its function. To test this, we fused full length HMP-1 with the junctional transmembrane protein VAB-9 (VAB-9::HMP-1::GFP). This fusion protein localizes correctly to junctions in embryos, and is able to rescue the hmp-1 strong loss-of-function mutant allele zu242. HMP-1 is normally recruited to junctions via HMP-2/b-catenin. The presumed HMP-2/b-catenin binding site of HMP-1 is located in the N-terminus of HMP-1. To test if this binding site has additional functional roles, we fused a truncated form of HMP-1 lacking the HMP-2/b-catenin binding site with VAB-9 (VAB-9::HMP-1(315-927)::GFP). This fusion protein also localizes correctly to junctions; however, it only partially rescues zu242 embryos. Our results suggest that hmp-1 is a stable physical linker between the cadherin-catenin complex and F-actin, while its N-terminal interaction with hmp-2 may indeed be required for robust linkage. The N terminus of HMP-1 may do so by recruiting additional binding partners that strengthen the connection to F-actin at the junction. In order to find potential binding partners for hmp-1, we performed co-immunoprecipitation followed by mass spectrometry and are currently testing the candidates to determine functional roles for these proteins.

Green, Rebecca A. et al. (2009) International Worm Meeting "A High-Content Screen to Functionally Classify Proteins Required for Cell Viability and Division."

Desai, Arshad, Audhya, Anjon, Green, Rebecca A., Oegema, Karen

[International Worm Meeting, 2009]

Systems biology analyzes the role of molecular assemblies within functional networks. The success of this approach relies upon the availability of systematic and functional information about the components of these molecular assemblies. RNA-mediated interference (RNAi) is currently the method of choice for linking genes to their cellular functions. In addition to relatively ''low content'' single reporter assays, RNAi has been used to perform high-content microscopy-based assays, integrating spatial and/or temporal information. This approach has been used to characterize the set of genes required for cell viability and division in C. elegans. Of the 20,000 genes, ~10% are required for embryo production or viability; these 2000 genes were previously screened by filming the first two embryonic divisions following individual RNAi depletion. This screen provided high quality data that allowed for the functional classification of ~400 genes, however it did not provide high quality functional information on the ~560 genes whose inhibition blocks embryo production (the sterile collection). Here, we functionally characterize the sterile collection by performing a second high-content screen. We use two-color fluorescence confocal microscopy to examine gonad structure in anesthetized worms after individual depletion of each of the 560 sterile gene products. The gonad-morphology data was analyzed by binary scoring for 94 potential defects. A clustering algorithm was used to group genes with similar phenotypic profiles, and phenotypes within the major clusters were then re-examined by eye for accuracy and identification of subclasses within each broad phenotypic group. Our live-imaging data, annotation and analysis will be integrated with the data from the prior DIC imaging based screen of the embryonic lethal collection in an online database that will be made available upon publication. Our analysis placed genes into ~20 broad classes that could be partitioned into ~100 different phenotypic sub-classes, which typically corresponded to the subunits of a specific protein complex. In addition to characterizing the 390 genes in the sterile collection, for which there was some prior functional information, our screen has placed the ~50 unknown and 120 previously uncharacterized genes in the sterile collection into functional groups. Cumulatively, our data doubles the number of genes for which we have high quality systematic functional information and provides an important platform for systems biology based analysis of the pathways contributing to embryo production and development.

Green, Rebecca A. et al. (2013) International Worm Meeting "The midbody ring, not the midbody microtubules, dictates abscission patterning in vivo."

Desai, Arshad, Audhya, Anjon, Green, Rebecca A., Lewellyn, Lindsay, Oegema, Karen, Mayers, Jonathan

[International Worm Meeting, 2013]

During cytokinesis, the cell must transition from constriction phase into abscission phase, by patterning the abscission components at the appropriate spatiotemporal location. Two structural features within the intercellular bridge are poised to serve as guidance cues to promote this process - the midbody microtubules and the midbody ring. It has been widely anticipated that the midbody microtubules are required-since key abscission determinants localize to this structure. In this study, we find that the midbody microtubules are dispensable for abscission in vivo and provide the first detailed analysis of the steps that occure during abscission phase in an intact organism. We define three temporally distinct and assayable abscission steps in vivo in the C. elegans embryo: cytoplasmic isolation, membrane shedding onset and midbody/midbody ring release; ESCRT assemblies are required only for the final step. We find that midbody microtubules are not required for any step, and that key abscission determinants are recruited to the abscission site in the absence of midbody microtubules. Furthermore, we show that the midbody ring component, the septins, is important for cytoplasmic isolation and is essential for midbody release. These results suggest that the midbody ring orchestrates the constriction to abscission phase transition in vivo.

Li, Zao et al. (2015) International Worm Meeting "Necrotic Cells Actively Attract Phagocytes through the Collaborative Action of Two Distinct PS-exposure Mechanisms."

Li, Zao, Nagaoka, Yuji, Zhou, Zheng, Morino, Eri, Venegas, Victor, Audhya, Anjon, Raghavan, Prashant, Nakanishi, Yoshinobu

[International Worm Meeting, 2015]

Necrosis is the premature death of cells caused by cell injury and genetic alternations and is closely related to human diseases including neuron degeneration, stroke, and cancer. Like apoptotic cells, necrotic cell also need to be swiftly removed from the organisms to prevent inflammatory and autoimmune responses. However, unlike apoptosis, the programmed cell death, necrosis is a caspase-independent cellular event and necrotic cells show morphologically distinct features from the button-like apoptotic cells. In the nematode Caenorhabditis elegans, gain-of-function mutations in certain ion channel subunits result in necrotic-like cell death of six touch neurons. Necrotic touch neurons are subsequently engulfed and degraded inside engulfing cells. However, it is unclear of how necrotic cells are recognized by phagocytes. Phosphatidylserine (PS) is an important apoptotic cell surface signal that attracts engulfing cells. Using ectopically expressed MFG-E8, a high-affinity PS-binding protein, we observed that PS was actively present on the surface of necrotic touch neurons. In addition, we found that CED-1, the phagocytic receptor could also recognize necrotic cells by associating with PS. We further found CED-7, the worm homolog of mouse ABC1 transporter, was necessary for PS-exposure on necrotic cell surfaces. In addition to CED-7, ANOH-1, the C. elegans homolog of the mammalian Ca2+-dependent phospholipid scramblase TMEM16F, plays an independent role in promoting PS exposure on necrotic cells. The combined activities from CED-7 and ANOH-1 ensure sufficient exposure of PS on necrotic cells to attract their phagocytes. Our work indicates that cells killed by different mechanisms (necrosis or apoptosis) expose a common "eat me" signal to attract their phagocytic receptor(s); furthermore, unlike what was previously believed, necrotic cells actively present PS on their outer surfaces through two distinct molecular mechanisms rather than leaking out PS passively.

Elliott J. Hagedorn et al. (2007) International Worm Meeting "Time-lapse visualization of anchor cell invasion in C. elegans."

David R. Sherwood, Elliott J. Hagedorn

[International Worm Meeting, 2007]

A lack of in vivo models has hampered insight into the mechanisms driving cell-invasive behavior. The behavior of a single uterine cell in Caenorhabditis elegans called the anchor cell (AC) provides such an in vivo model, allowing for easy visualization and genetic manipulation. During normal development in the C. elegans hermaphrodite, the AC invades through the underlying juxtaposed uterine and ventral epidermal basement membranes (BM) to establish the initial uterine-vulval contact. In addition to the genetic tractability and ease of visualization, the consistency of this event, occurring at the same time in every wild-type hermaphrodite, makes for a powerful model of cell invasion. We have established an in vivo system to visualize anchor cell invasion as it occurs in real time using a laminin-GFP fusion (LAM-1::GFP, Kao and Wadsworth 2006, Dev Biol. 290(1): 211-219) or a hemicentin-GFP fusion (GFP-hemicentin, Vogel and Hedgecock 2001, Development 128(6): 883-894), both markers of the BM, in combination with a PH::mCherry (PH domain from PLC?, a gift from Anjon Audhya) driven by an AC-specific promoter, labeling the invasive membrane (Sherwood et al. 2005, Cell 121(6): 951-962). To investigate the real-time dynamics of invasion, we have begun to characterize AC invasion in wild-type animals. Prior to invasion there is a deposition of GFP-hemicentin and an apparent increase of LAM-1::GFP in the uterine BM directly below the AC, suggesting that the AC deposits or recruits additional BM components, prior to migration through it. Invasion appears to initiate with a single filopod extending through a tiny hole in the BM. Once this filopodial process reaches through the BM, possibly contacting vulval cells below, it begins to widen, often fanning out on the ventral side of the BM. This widening of the AC extension seems to be coincident with widening of the hole in the BM, as visualized by LAM-1::GFP. The initial characterization of wild-type invasion has brought about several questions. 1) How is the initial filopod generated, and how does it penetrate through the BM? 2) What dictates the precise location of the initial protrusive filopod? 3) What happens to the BM under the AC as the filopod widens? 4) Do other BM components similarly increase under the AC, how are they recruited there, and what function does their increase serve? It is our hope that through a detailed cell-biological analysis, in combination with genetic screens and mutant analysis, that we will gain insight into these questions and lead to a better understanding of the mechanisms that endow invasive cells with the ability to traverse basement membranes.

Green, Rebecca A. et al. (2011) International Worm Meeting "A High-Resolution C. elegans Essential Gene Network Based on Phenotypic Profiling of a ComplexTissue."

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.

Hideaki Hiraki et al. (2007) International Worm Meeting "Semi-automatic system for the creation of cell shape models in C. elegans embryogenesis."

Yuji KOHARA, Hideaki Hiraki

[International Worm Meeting, 2007]

Cell to cell interactions play critical roles in early embryogenesis, therefore, it is very important to have information about the arrangements of cells, cell shapes and the contact among them. We have been developing a computer system to create cell shape models from a time series of confocal microscopic images of the embryo whose plasma membrane is labeled with a vital fluorescent dye or a fluorescent protein. In this system, cell shapes are automatically calculated by a seeded region growing algorithm from a 3D image and a set of seed point coordinates. Manual editing of the seed coordinates is required and is assisted by its graphical user interface. We applied this system on the "dub" data set in WORM 4D CDs kindly provided by Bill Mohler, and obtained cell shape models of 24-200 cell stages successfully for the most part. This time, in collaboration with Jon Audhya, we performed live recording of OD58, the strain expressing the fusion of GFP with pleckstrin homology domain which is targeted to the plasma membrane. We successfully obtained data sets of 1-24 cell stages. We also developed a post-processing system to remove the bumps of cell shapes derived from image noises by implementing an active balloon model under gradient vector flow. As a result, the phenotype of par-2 RNAi embryos could be evaluated in terms of cell volumes and cell-to-cell contacting areas. We are improving the system more, and plan to apply this system to compare the cellular arrangements and the cell-to-cell contacts among mutant embryos and the embryos from other species closely related to C. elegans.