Deng, Ting et al. (2017) International Worm Meeting "Cell cycle-independent role of the pre-replication complex during anchor cell invasion."

Deng, Ting, Hajnal, Alex, Lattmann, Evelyn

[International Worm Meeting, 2017]

Anchor-cell (AC) invasion is serving as an excellent model of study fundamental aspects of cell invasion, a process occurring during normal development as well as tumor metastasis. By screening the C. elegans orthologs of genes up-regulated in invasive human melanoma cells, we have identified five pre-replication complex (pre-RC) components, mcm-7, cdc-6, cdt-1, orc-2 and orc-5, as regulators of AC invasion. The pre-RC is required for the licensing of replication origins during the G1 phase. Since the invading AC never replicates its DNA and remains arrested in G1, the similar penetrance of AC-specific and systemic mcm-7 RNAi suggest a cell-autonomous and cell cycle-independent function of MCM-7. An endogenous GFP::MCM-7 reporter shows a transient nuclear expression pattern in the AC prior to invasion, suggesting a function of the pre-RC in inducing the epithelial to mesenchymal transition of the AC. Accordingly, MCM-7 regulates expression of metalloproteinase zmp-1 in parallel with FOS-1A. To further study the function of the pre-RC during AC invasion, we have inserted FRT sites into the mcm-7 locus, allowing us to perform a conditional mcm-7 knock-out. We propose that components of the pre-RC have adopted a cell-cycle independent function in the AC to regulate cell invasion.

Fergin, Aleksandra et al. (2019) International Worm Meeting "The role of protein sumoylation during vulval morphogenesis and anchor cell invasion."

Hajnal, Alex, Lattmann, Evelyn, Fergin, Aleksandra, Lambert, Charlotte

[International Worm Meeting, 2019]

The sumoylation pathway is involved in a variety of processes in C. elegans including gonadal and vulval fate specification, cell cycle progression, maintenance of chromosome structure and chromosome segregation. In a targeted RNAi screen to identify genes regulating cell invasion, we have found that several components of the sumoylation pathway are required for normal anchor cell (AC) invasion, such as the E2 SUMO-conjugating enzyme ubc-9, the SUMO E3 ligase gei-17 or the SUMO ortholog smo-1. It is also known that many proteins are sumoylated during the epithelial to mesenchymal transition (EMT), highlighting the importance of the sumoylation pathway in cell invasion. Since many transcription factors crucial for normal development are modified by SUMO, studying the exact role of this posttranslational modification with respect to specific targets is a challenging question. Therefore, we have developed the tools to block sumoylation in a tissue-specific and temporally controlled manner to study how loss of sumoylation affects vulval development and AC invasion. For this purpose, we are using the auxin-inducible tissue-specific protein degradation system to down-regulate degron-tagged components of the sumoylation pathway (such as the E3 ligase gei-17) in the AC or vulval precursor cells (VPCs). To this aim, we generated animals expressing the modified Arabidopsis thaliana TIR1 protein (component of the E3 ubiquitin ligase complex) under the VPC specific bar-1 or the AC-specific cdh-3 promoters. The degradation of GEI-17 in these different somatic tissues resulted in defects in AC positioning, basement membrane breaching and utse formation. Moreover, VPC-specific inhibition of sumoylation perturbs vulval toroid formation. In further experiments we will examine specific transcription factors that require sumoylation for proper vulval morphogenesis and AC invasion.

Berger, Simon et al. (2017) International Worm Meeting "Long-term C. elegans Immobilization enables High Resolution Developmental Studies in vivo."

Aegarter-Wilmsen, Tinri, Lattmann, Evelyn, Hajnal, Alex, Casadevall i Solvas, Xavier, deMello, Andrew, Berger, Simon, Hengartner, Michael

[International Worm Meeting, 2017]

Owing to its small size, fecundity and genetical tractability C. elegans has become one of the most widely used model organisms in biology. One major challenge when working with C. elegans however, is its high mobility, making immobilization necessary for the study of most cellular and subcellular processes. Immobilization is usually achieved by simply placing the worms of interest on an agar pad, limiting motion through the pressure exerted on the animal and, if necessary, through addition of chemical tranquilizers. Unfortunately, this process has proven to be a major limitation when trying to follow developmental processes over extended periods of time, as it rapidly causes slowdown or arrest of many such processes. Here we present a novel set of microfluidic devices able to trap, feed and image C. elegans in a variety of developmental stages (L1 to adult), without any of the adverse effects encountered when using conventional immobilization methods. Our immobilization platform is based on a simple PDMS microfluidic device mounted on the back of a coverslip. Operation of the device is readily learned and the microfluidic device can easily be integrated with any type of microscope (inverted or upright), all imaging modalities commonly used (brightfield, epifluorescence and confocal microscopy), and any type of objective (high magnification and high numerical aperture). Thus requiring only small changes to existing microscope setups and microscopy protocols prior to adaptation, enabling the study of a great variety of developmental processes, interference free, in vivo. We demonstrate the platforms capabilities in several case studies. First, we studied long-term viability of adult C. elegans on-chip, readily achieving viabilities exceeding 100 hours while immobilized, all while observing normal feeding and egg laying rates. Optimal quality images of the adult gonad were then acquired, showcasing the platforms capabilities of following complex developmental processes over long time periods. Specifically we studied germ cell apoptosis, a process known to arrest on agar pad within 20 minutes, and factors involved in the cell fate decision by tracking 100 cells over the course of 12 hours. For the first time determining an apoptotic rate of 60% in vivo (n = 100). Secondly we demonstrate that crucial developmental process, e.g. anchor cell invasion and distal tip cell migration, occur normally on-chip in all larval stages assessed, and at rates comparable to on plate culture. Compared to development on agar pads, processes occurred 5-7 times faster and more reliable on-chip, with all worms undergoing normal development. This strongly suggests that our immobilization device has minimal negative effects on sensitive developmental processes, thus making it ideally suited for long-term studies of processes so far inaccessible, all while allowing the capture of high resolution images.