Showing 1 - 6 of 6 results
Mps1 releases Mad1 from nuclear pores to ensure a robust mitotic checkpoint and accurate chromosome segregation.
The strength of the Spindle Assembly Checkpoint (SAC) depends on the amount of the Mad1-C-Mad2 heterotetramer at kinetochores but also on its binding to Megator/Tpr at nuclear pore complexes (NPCs) during interphase. However, the molecular underpinnings controlling the spatiotemporal redistribution of Mad1-C-Mad2 as cells progress into mitosis remain elusive. Here, we show that Mps1-mediated phosphorylation of Megator/Tpr abolishes its interaction with Mad1 in vitro and in Drosophila cells. Timely activation of Mps1 during prophase triggers Mad1 release from NPCs, which we find to be required for competent kinetochore recruitment of Mad1-C-Mad2 and robust checkpoint response. Importantly, preventing Mad1 binding to Megator/Tpr rescues the fidelity of chromosome segregation and aneuploidy in larval neuroblasts of Drosophila mps1-null mutants. Our findings demonstrate that the subcellular localization of Mad1 is stringently coordinated with cell cycle progression by kinetochore-extrinsic activity of Mps1. This ensures that both NPCs in interphase and kinetochores in mitosis can generate anaphase inhibitors to efficiently preserve genomic stability.
Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in Drosophila S2 Cells.
Drosophila Schneider 2 (S2) cells are a simple and powerful system commonly used in cell biology because they are well suited for high resolution microscopy and RNAi-mediated depletion. However, understanding dynamic processes, such as cell division, also requires methodology to interfere with protein function with high spatiotemporal control. In this research study, we report the adaptation of an optogenetic tool to Drosophila S2 cells. Light-activated reversible inhibition by assembled trap (LARIAT) relies on the rapid light-dependent heterodimerization between cryptochrome 2 (CRY2) and cryptochrome-interacting bHLH 1 (CIB1) to form large protein clusters. An anti-green fluorescent protein (GFP) nanobody fused with CRY2 allows this method to quickly trap any GFP-tagged protein in these light-induced protein clusters. We evaluated clustering kinetics in response to light for different LARIAT modules, and showed the ability of GFP-LARIAT to inactivate the mitotic protein Mps1 and to disrupt the membrane localization of the polarity regulator Lethal Giant Larvae (Lgl). Moreover, we validated light-induced co-clustering assays to assess protein-protein interactions in S2 cells. In conclusion, GFP-based LARIAT is a versatile tool to answer different biological questions, since it enables probing of dynamic processes and protein-protein interactions with high spatiotemporal resolution in Drosophila S2 cells.
Control of microtubule dynamics using an optogenetic microtubule plus end-F-actin cross-linker.
We developed a novel optogenetic tool, SxIP-improved light-inducible dimer (iLID), to facilitate the reversible recruitment of factors to microtubule (MT) plus ends in an end-binding protein-dependent manner using blue light. We show that SxIP-iLID can track MT plus ends and recruit tgRFP-SspB upon blue light activation. We used this system to investigate the effects of cross-linking MT plus ends and F-actin in Drosophila melanogaster S2 cells to gain insight into spectraplakin function and mechanism. We show that SxIP-iLID can be used to temporally recruit an F-actin binding domain to MT plus ends and cross-link the MT and F-actin networks. Cross-linking decreases MT growth velocities and generates a peripheral MT exclusion zone. SxIP-iLID facilitates the general recruitment of specific factors to MT plus ends with temporal control enabling researchers to systematically regulate MT plus end dynamics and probe MT plus end function in many biological processes.
The Spatiotemporal Limits of Developmental Erk Signaling.
Animal development is characterized by signaling events that occur at precise locations and times within the embryo, but determining when and where such precision is needed for proper embryogenesis has been a long-standing challenge. Here we address this question for extracellular signal regulated kinase (Erk) signaling, a key developmental patterning cue. We describe an optogenetic system for activating Erk with high spatiotemporal precision in vivo. Implementing this system in Drosophila, we find that embryogenesis is remarkably robust to ectopic Erk signaling, except from 1 to 4 hr post-fertilization, when perturbing the spatial extent of Erk pathway activation leads to dramatic disruptions of patterning and morphogenesis. Later in development, the effects of ectopic signaling are buffered, at least in part, by combinatorial mechanisms. Our approach can be used to systematically probe the differential contributions of the Ras/Erk pathway and concurrent signals, leading to a more quantitative understanding of developmental signaling.
Optogenetic Control of Gene Expression in Drosophila.
To study the molecular mechanism of complex biological systems, it is important to be able to artificially manipulate gene expression in desired target sites with high precision. Based on the light dependent binding of cryptochrome 2 and a cryptochrome interacting bHLH protein, we developed a split lexA transcriptional activation system for use in Drosophila that allows regulation of gene expression in vivo using blue light or two-photon excitation. We show that this system offers high spatiotemporal resolution by inducing gene expression in tissues at various developmental stages. In combination with two-photon excitation, gene expression can be manipulated at precise sites in embryos, potentially offering an important tool with which to examine developmental processes.
Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo.
The small GTPase Rac induces actin polymerization, membrane ruffling and focal contact formation in cultured single cells but can either repress or stimulate motility in epithelial cells depending on the conditions. The role of Rac in collective epithelial cell movements in vivo, which are important for both morphogenesis and metastasis, is therefore difficult to predict. Recently, photoactivatable analogues of Rac (PA-Rac) have been developed, allowing rapid and reversible activation or inactivation of Rac using light. In cultured single cells, light-activated Rac leads to focal membrane ruffling, protrusion and migration. Here we show that focal activation of Rac is also sufficient to polarize an entire group of cells in vivo, specifically the border cells of the Drosophila ovary. Moreover, activation or inactivation of Rac in one cell of the cluster caused a dramatic response in the other cells, suggesting that the cells sense direction as a group according to relative levels of Rac activity. Communication between cells of the cluster required Jun amino-terminal kinase (JNK) but not guidance receptor signalling. These studies further show that photoactivatable proteins are effective tools in vivo.