@article {68884, title = {Ten-fold Robust Expansion Microscopy.}, journal = {Bio-Protocol}, volume = {13}, year = {2023}, month = {2023 Jun 20}, pages = {e4698}, abstract = {

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. By systematic exploration of the ExM recipe space, we developed a novel ExM method termed Ten-fold Robust Expansion Microscopy (TREx) that, as the original ExM method, requires no specialized equipment or procedures. TREx enables ten-fold expansion of both thick mouse brain tissue sections and cultured human cells, can be handled easily, and enables high-resolution subcellular imaging with a single expansion step. Furthermore, TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.

}, issn = {2331-8325}, doi = {10.21769/BioProtoc.4698}, author = {Damstra, Hugo G J and Mohar, Boaz and Eddison, Mark and Akhmanova, Anna and Kapitein, Lukas C and Tillberg, Paul W} } @article {68759, title = {CYK-4 functions independently of its centralspindlin partner ZEN-4 to cellularize oocytes in germline syncytia}, journal = {eLife}, volume = {7}, year = {2018}, month = {07/2018}, pages = {e36919}, abstract = {

Throughout metazoans, germ cells undergo incomplete cytokinesis to form syncytia connected by intercellular bridges. Gamete formation ultimately requires bridge closure, yet how bridges are reactivated to close is not known. The most conserved bridge component is centralspindlin, a complex of the Rho family GTPase-activating protein (GAP) CYK-4/MgcRacGAP and the microtubule motor ZEN-4/kinesin-6. Here, we show that oocyte production by the syncytial \textitCaenorhabditis elegans germline requires CYK-4 but not ZEN-4, which contrasts with cytokinesis, where both are essential. Longitudinal imaging after conditional inactivation revealed that CYK-4 activity is important for oocyte cellularization, but not for the cytokinesis-like events that generate syncytial compartments. CYK-4{\textquoteright}s lipid-binding C1 domain and the GTPase-binding interface of its GAP domain were both required to target CYK-4 to intercellular bridges and to cellularize oocytes. These results suggest that the conserved C1-GAP region of CYK-4 constitutes a targeting module required for closure of intercellular bridges in germline syncytia.

}, keywords = {centralspindlin, germline syncytia, intercellular bridge, Kinesin-6, MgcRacGAP, ring canal}, issn = {2050-084X}, doi = {10.7554/eLife.36919}, url = {https://doi.org/10.7554/eLife.36919}, author = {Lee, Kian-Yong and Green, Rebecca A and Gutierrez, Edgar and Gomez-Cavazos, J Sebastian and Kolotuev, Irina and Wang, Shaohe and Desai, Arshad and Groisman, Alex and Oegema, Karen}, editor = {Balasubramanian, Mohan K and Akhmanova, Anna} } @article {68766, title = {NOCA-1 functions with γ-tubulin and in parallel to Patronin to assemble non-centrosomal microtubule arrays in \textitC. elegans}, journal = {eLife}, volume = {4}, year = {2015}, month = {09/2015}, pages = {e08649}, abstract = {

Non-centrosomal microtubule arrays assemble in differentiated tissues to perform mechanical and transport-based functions. In this study, we identify \textitCaenorhabditis elegans NOCA-1 as a protein with homology to vertebrate ninein. NOCA-1 contributes to the assembly of non-centrosomal microtubule arrays in multiple tissues. In the larval epidermis, NOCA-1 functions redundantly with the minus end protection factor Patronin/PTRN-1 to assemble a circumferential microtubule array essential for worm growth and morphogenesis. Controlled degradation of a γ-tubulin complex subunit in this tissue revealed that γ-tubulin acts with NOCA-1 in parallel to Patronin/PTRN-1. In the germline, NOCA-1 and γ-tubulin co-localize at the cell surface, and inhibiting either leads to a microtubule assembly defect. γ-tubulin targets independently of NOCA-1, but NOCA-1 targeting requires γ-tubulin when a non-essential putatively palmitoylated cysteine is mutated. These results show that NOCA-1 acts with γ-tubulin to assemble non-centrosomal arrays in multiple tissues and highlight functional overlap between the ninein and Patronin protein families.

}, keywords = {ninein, NOCA-1, non-centrosomal microtubule array, Patronin, PTRN-1}, issn = {2050-084X}, doi = {10.7554/eLife.08649}, url = {https://doi.org/10.7554/eLife.08649}, author = {Wang, Shaohe and Wu, Di and Quintin, Sophie and Green, Rebecca A and Cheerambathur, Dhanya K and Ochoa, Stacy D and Desai, Arshad and Oegema, Karen}, editor = {Akhmanova, Anna} }