@article {69773, title = {Live imaging of the extracellular matrix with a glycan-binding fluorophore.}, journal = {Nat Methods}, year = {2025}, month = {2025 Feb 06}, abstract = {

All multicellular systems produce and dynamically regulate extracellular matrices (ECMs) that play essential roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small-molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, non-perturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

}, issn = {1548-7105}, doi = {10.1038/s41592-024-02590-2}, url = {https://doi.org/10.1038/s41592-024-02590-2}, author = {Fiore, Antonio and Yu, Guoqiang and Northey, Jason J and Patel, Ronak and Ravenscroft, Thomas A. and Ikegami, Richard and Kolkman, W and Pratik Kumar and Dilan, Tanya L and Ruetten, Virginia M.S. and Ahrens, Misha B and Shroff, Hari and Wang, Shaohe and Weaver, Valarie M and Pedram, Kayvon} } @article {69440, title = {Imaging the extracellular matrix in live tissues and organisms with a glycan-binding fluorophore}, journal = {bioRxiv}, year = {2024}, month = {2024 May 10}, abstract = {

All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

}, doi = {10.1101/2024.05.09.593460}, url = {https://www.biorxiv.org/content/early/2024/05/10/2024.05.09.593460}, author = {Fiore, Antonio and Yu, Guoqiang and Northey, Jason J. and Patel, Ronak and Ravenscroft, Thomas A. and Ikegami, Richard and Kolkman, Wiert and Kumar, Pratik and Grimm, Jonathan B. and Dilan, Tanya L. and Ruetten, Virginia M.S. and Ahrens, Misha B. and Shroff, Hari and Lavis, Luke D. and Wang, Shaohe and Weaver, Valerie M. and Pedram, Kayvon} } @article {69444, title = {The kinase ZYG-1 phosphorylates the cartwheel protein SAS-5 to drive centriole assembly in C. elegans}, journal = {EMBO Rep}, year = {2024}, month = {2024 May 14}, abstract = {

Centrioles organize centrosomes, the cell{\textquoteright}s primary microtubule-organizing centers (MTOCs). Centrioles double in number each cell cycle, and mis-regulation of this process is linked to diseases such as cancer and microcephaly. In C. elegans, centriole assembly is controlled by the Plk4 related-kinase ZYG-1, which recruits the SAS-5-SAS-6 complex. While the kinase activity of ZYG-1 is required for centriole assembly, how it functions has not been established. Here we report that ZYG-1 physically interacts with and phosphorylates SAS-5 on 17 conserved serine and threonine residues in vitro. Mutational scanning reveals that serine 10 and serines 331/338/340 are indispensable for proper centriole assembly. Embryos expressing SAS-5 exhibit centriole assembly failure, while those expressing SAS-5 possess extra centrioles. We show that in the absence of serine 10 phosphorylation, the SAS-5-SAS-6 complex is recruited to centrioles, but is not stably incorporated, possibly due to a failure to coordinately recruit the microtubule-binding protein SAS-4. Our work defines the critical role of phosphorylation during centriole assembly and reveals that ZYG-1 might play a role in preventing the formation of excess centrioles.

}, issn = {1469-3178}, doi = {10.1038/s44319-024-00157-y}, author = {Sankaralingam, Prabhu and Wang, Shaohe and Liu, Yan and Oegema, Karen F and O{\textquoteright}Connell, Kevin F} } @book {69492, title = {Salivary gland developmental mechanics}, series = {Current Topics in Developmental Biology}, year = {2024}, publisher = {Elsevier}, organization = {Elsevier}, abstract = {

The salivary gland undergoes branching morphogenesis to elaborate into a tree-like structure with numerous saliva-secreting acinar units, all joined by a hierarchical ductal system. The expansive epithelial surface generated by branching morphogenesis serves as the structural basis for the efficient production and delivery of saliva. Here, we elucidate the process of salivary gland morphogenesis, emphasizing the role of mechanics. Structurally, the developing salivary gland is characterized by a stratified epithelium tightly encased by the basement membrane, which is in turn surrounded by a mesenchyme consisting of a dense network of interstitial matrix and mesenchymal cells. Diverse cell types and extracellular matrices bestow this developing organ with organized, yet spatially varied mechanical properties. For instance, the surface epithelial sheet of the bud is highly fluidic due to its high cell motility and weak cell-cell adhesion, rendering it highly pliable. In contrast, the inner core of the bud is more rigid, characterized by reduced cell motility and strong cell-cell adhesion, which likely provide structural support for the tissue. The interactions between the surface epithelial sheet and the inner core give rise to budding morphogenesis. Furthermore, the basement membrane and the mesenchyme offer mechanical constraints that could play a pivotal role in determining the higher-order architecture of a fully mature salivary gland.

}, issn = {00702153}, doi = {10.1016/bs.ctdb.2024.05.002}, url = {https://doi.org/10.1016/bs.ctdb.2024.05.002}, author = {Morales, E. Angelo and Wang, Shaohe} } @article {68827, title = {Tissue Morphogenesis Through Dynamic Cell and Matrix Interactions.}, journal = {Annual Reviews Cell Developmental Biology}, year = {2023}, month = {2023 Jun 14}, abstract = {

Multicellular organisms generate tissues of diverse shapes and functions from cells and extracellular matrices. Their adhesion molecules mediate cell-cell and cell-matrix interactions, which not only play crucial roles in maintaining tissue integrity but also serve as key regulators of tissue morphogenesis. Cells constantly probe their environment to make decisions: They integrate chemical and mechanical information from the environment via diffusible ligand- or adhesion-based signaling to decide whether to release specific signaling molecules or enzymes, to divide or differentiate, to move away or stay, or even whether to live or die. These decisions in turn modify their environment, including the chemical nature and mechanical properties of the extracellular matrix. Tissue morphology is the physical manifestation of the remodeling of cells and matrices by their historical biochemical and biophysical landscapes. We review our understanding of matrix and adhesion molecules in tissue morphogenesis, with an emphasis on key physical interactions that drive morphogenesis. Expected final online publication date for the , Volume 39 is October 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

}, issn = {1530-8995}, doi = {10.1146/annurev-cellbio-020223-031019}, author = {Wu, Di and Yamada, Kenneth M and Wang, Shaohe} } @article {68751, title = {Polo-like kinase 1 independently controls microtubule-nucleating capacity and size of the centrosome}, journal = {Journal of Cell Biology}, volume = {220}, year = {2021}, month = {01/2021}, abstract = {

Centrosomes are composed of a centriolar core surrounded by a pericentriolar material (PCM) matrix that docks microtubule-nucleating γ-tubulin complexes. During mitotic entry, the PCM matrix increases in size and nucleating capacity in a process called centrosome maturation. Polo-like kinase 1 (PLK1) is recruited to centrosomes and phosphorylates PCM matrix proteins to drive their self-assembly, which leads to PCM expansion. Here, we show that in addition to controlling PCM expansion, PLK1 independently controls the generation of binding sites for γ-tubulin complexes on the PCM matrix. Selectively preventing the generation of PLK1-dependent γ-tubulin docking sites led to spindle defects and impaired chromosome segregation without affecting PCM expansion, highlighting the importance of phospho-regulated centrosomal γ-tubulin docking sites in spindle assembly. Inhibiting both γ-tubulin docking and PCM expansion by mutating substrate target sites recapitulated the effects of loss of centrosomal PLK1 on the ability of centrosomes to catalyze spindle assembly.

}, issn = {0021-9525}, doi = {10.1083/jcb.202009083}, url = {https://doi.org/10.1083/jcb.202009083}, author = {Ohta, Midori and Zhao, Zhiling and Wu, Di and Wang, Shaohe and Harrison, Jennifer L. and G{\'o}mez-Cavazos, J. Sebasti{\'a}n and Desai, Arshad and Oegema, Karen F.} } @article {68755, title = {Extracellular matrix dynamics in cell migration, invasion and tissue morphogenesis}, journal = {International Journal of Experimental Pathology}, volume = {100}, year = {2019}, month = {06/2019}, pages = {144-152}, abstract = {

Summary This review describes how direct visualization of the dynamic interactions of cells with different extracellular matrix microenvironments can provide novel insights into complex biological processes. Recent studies have moved characterization of cell migration and invasion from classical 2D culture systems into 1D and 3D model systems, revealing multiple differences in mechanisms of cell adhesion, migration and signalling{\textemdash}even though cells in 3D can still display prominent focal adhesions. Myosin II restrains cell migration speed in 2D culture but is often essential for effective 3D migration. 3D cell migration modes can switch between lamellipodial, lobopodial and/or amoeboid depending on the local matrix environment. For example, {\textquotedblleft}nuclear piston{\textquotedblright} migration can be switched off by local proteolysis, and proteolytic invadopodia can be induced by a high density of fibrillar matrix. Particularly, complex remodelling of both extracellular matrix and tissues occurs during morphogenesis. Extracellular matrix supports self-assembly of embryonic tissues, but it must also be locally actively remodelled. For example, surprisingly focal remodelling of the basement membrane occurs during branching morphogenesis{\textemdash}numerous tiny perforations generated by proteolysis and actomyosin contractility produce a microscopically porous, flexible basement membrane meshwork for tissue expansion. Cells extend highly active blebs or protrusions towards the surrounding mesenchyme through these perforations. Concurrently, the entire basement membrane undergoes translocation in a direction opposite to bud expansion. Underlying this slowly moving 2D basement membrane translocation are highly dynamic individual cell movements. We conclude this review by describing a variety of exciting research opportunities for discovering novel insights into cell-matrix interactions.

}, keywords = {3D culture, basement membrane remodelling, branching morphogenesis, cell migration, Extracellular Matrix, invasion}, doi = {https://doi.org/10.1111/iep.12329}, url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/iep.12329}, author = {Yamada, Kenneth M. and Collins, Joshua W. and Cruz Walma, David A. and Doyle, Andrew D. and Morales, Shaimar Gonzalez and Lu, Jiaoyang and Matsumoto, Kazue and Nazari, Shayan S. and Sekiguchi, Rei and Shinsato, Yoshinari and Wang, Shaohe} } @article {68757, title = {A high-content imaging approach to profile C. elegans embryonic development}, journal = {Development}, volume = {146}, year = {2019}, month = {04-2019}, abstract = {

The Caenorhabditis elegans embryo is an important model for analyzing mechanisms of cell fate specification and tissue morphogenesis. Sophisticated lineage-tracing approaches for analyzing embryogenesis have been developed but are labor intensive and do not naturally integrate morphogenetic readouts. To enable the rapid classification of developmental phenotypes, we developed a high-content method that employs two custom strains: a Germ Layer strain that expresses nuclear markers in the ectoderm, mesoderm and endoderm/pharynx; and a Morphogenesis strain that expresses markers labeling epidermal cell junctions and the neuronal cell surface. We describe a procedure that allows simultaneous live imaging of development in 80-100 embryos and provide a custom program that generates cropped, oriented image stacks of individual embryos to facilitate analysis. We demonstrate the utility of our method by perturbing 40 previously characterized developmental genes in variants of the two strains containing RNAi-sensitizing mutations. The resulting datasets yielded distinct, reproducible signature phenotypes for a broad spectrum of genes that are involved in cell fate specification and morphogenesis. In addition, our analysis provides new in vivo evidence for MBK-2 function in mesoderm fate specification and LET-381 function in elongation.

}, issn = {0950-1991}, doi = {10.1242/dev.174029}, url = {https://doi.org/10.1242/dev.174029}, author = {Wang, Shaohe and Ochoa, Stacy D. and Khaliullin, Renat N. and Gerson-Gurwitz, Adina and Hendel, Jeffrey M. and Zhao, Zhiling and Biggs, Ronald and Chisholm, Andrew D. and Desai, Arshad and Oegema, Karen and Green, Rebecca A.} } @article {68754, title = {A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development}, journal = {Journal of Visualized Experiments}, year = {2019}, month = {10/2019}, abstract = {

C. elegans\ is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80{\textendash}100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

}, doi = {10.3791/60362}, url = {https://www.jove.com/t/60362/a-semi-high-throughput-imaging-method-data-visualization-toolkit-tohttps://www.jove.com/t/60362/a-semi-high-throughput-imaging-method-data-visualization-toolkit-to}, author = {Khaliullin, Renat N. and Hendel, Jeffrey M. and Gerson-Gurwitz, Adina and Wang, Shaohe and Ochoa, Stacy D. and Zhao, Zhiling and Desai, Arshad and Oegema, Karen and Green, Rebecca A.} } @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 {68756, title = {Single Molecule RNA FISH (smFISH) in Whole-Mount Mouse Embryonic Organs}, journal = {Current Protocols in Cell Biology}, volume = {83}, year = {2018}, month = {11/2018}, pages = {e79}, abstract = {

Abstract Single molecule RNA fluorescence in situ hybridization (smFISH) has become the standard tool for high spatial resolution analysis of gene expression in the context of tissue organization. This article describes protocols to perform smFISH on whole-mount mouse embryonic organs, where tissue organization can be compared to RNA expression by co-immunostaining of known protein markers. An enzymatic labeling strategy is also introduced to produce low-cost smFISH probes. Important considerations and practical guidelines for imaging smFISH samples using fluorescence confocal microscopy are described. Finally, a suite of custom-written ImageJ macros is included with detailed instructions to enable semi-automated smFISH image analysis of both 2D and 3D images. {\textcopyright} 2018 by John Wiley \& Sons, Inc.

}, keywords = {co-immunostaining, enzymatic probe labeling, ImageJ macro quantification, smFISH}, doi = {https://doi.org/10.1002/cpcb.79}, url = {https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/cpcb.79}, author = {Wang, Shaohe} } @article {68762, title = {Centrioles initiate cilia assembly but are dispensable for maturation and maintenance in C. elegans}, journal = {Journal of Cell Biology}, volume = {216}, year = {2017}, month = {04/2017}, pages = {1659-1671}, abstract = {

Cilia are cellular projections that assemble on centriole-derived basal bodies. While cilia assembly is absolutely dependent on centrioles, it is not known to what extent they contribute to downstream events. The nematode C. elegans provides a unique opportunity to address this question, as centrioles do not persist at the base of mature cilia. Using fluorescence microscopy and electron tomography, we find that centrioles degenerate early during ciliogenesis. The transition zone and axoneme are not completely formed at this time, indicating that cilia maturation does not depend on intact centrioles. The hydrolethalus syndrome protein HYLS-1 is the only centriolar protein known to remain at the base of mature cilia and is required for intraflagellar transport trafficking. Surprisingly, targeted degradation of HYLS-1 after initiation of ciliogenesis does not affect ciliary structures. Taken together, our results indicate that while centrioles are essential to initiate cilia formation, they are dispensable for cilia maturation and maintenance.

}, issn = {0021-9525}, doi = {10.1083/jcb.201610070}, url = {https://doi.org/10.1083/jcb.201610070}, author = {Serwas, Daniel and Su, Tiffany Y. and Roessler, Max and Wang, Shaohe and Dammermann, Alexander} } @article {68763, title = {Patterned cell and matrix dynamics in branching morphogenesis}, journal = {Journal of Cell Biology}, volume = {216}, year = {2017}, month = {02/2017}, pages = {559-570}, abstract = {

Many embryonic organs undergo branching morphogenesis to maximize their functional epithelial surface area. Branching morphogenesis requires the coordinated interplay of multiple types of cells with the extracellular matrix (ECM). During branching morphogenesis, new branches form by {\textquotedblleft}budding{\textquotedblright} or {\textquotedblleft}clefting.{\textquotedblright} Cell migration, proliferation, rearrangement, deformation, and ECM dynamics have varied roles in driving budding versus clefting in different organs. Elongation of the newly formed branch and final maturation of the tip involve cellular mechanisms that include cell elongation, intercalation, convergent extension, proliferation, and differentiation. New methodologies such as high-resolution live imaging, tension sensors, and force-mapping techniques are providing exciting new opportunities for future research into branching morphogenesis.

}, issn = {0021-9525}, doi = {10.1083/jcb.201610048}, url = {https://doi.org/10.1083/jcb.201610048}, author = {Wang, Shaohe and Sekiguchi, Rei and Daley, William P. and Yamada, Kenneth M.} } @article {68761, title = {A toolkit for GFP-mediated tissue-specific protein degradation in C. elegans}, journal = {Development}, volume = {144}, year = {2017}, month = {07/2017}, pages = {2694-2701}, abstract = {

Proteins that are essential for embryo production, cell division and early embryonic events are frequently reused later in embryogenesis, during organismal development or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that uses expression of a fusion between a GFP-targeting nanobody and a SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or with rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues {\textendash} the epidermis, intestine, body wall muscle, ciliated sensory neurons and touch receptor neurons {\textendash} where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of C. elegans proteins.

}, issn = {0950-1991}, doi = {10.1242/dev.150094}, url = {https://doi.org/10.1242/dev.150094}, author = {Wang, Shaohe and Tang, Ngang Heok and Lara-Gonzalez, Pablo and Zhao, Zhiling and Cheerambathur, Dhanya K. and Prevo, Bram and Chisholm, Andrew D. and Desai, Arshad and Oegema, Karen} } @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} }