Spatiotemporal coordination of actin regulators generates invasive protrusions in cell-cell fusion.

Invasive membrane protrusions play a central role in a variety of cellular processes. Unlike filopodia, invasive protrusions are mechanically stiff and propelled by branched actin polymerization. However, how branched actin filaments are organized to create finger-like invasive protrusions is unclear.

Branched actin polymerization drives invasive protrusion formation to promote myoblast fusion during skeletal muscle regeneration.

Skeletal muscle regeneration is a multistep process involving the activation, proliferation, differentiation, and fusion of muscle stem cells, known as satellite cells. The fusion of satellite cell-derived mononucleated muscle cells (SCMs) is indispensable for the generation of multinucleated, contractile myofibers during muscle repair. However, the molecular and cellular mechanisms underlying SCM fusion during muscle regeneration remain poorly understood.

Digital droplet PCR analysis of organoids generated from mouse mammary tumors demonstrates proof-of-concept capture of tumor heterogeneity.

Breast cancer metastases exhibit many different genetic alterations, including copy number amplifications (CNA). CNA are genetic alterations that are increasingly becoming relevant to breast oncology clinical practice. Here we identify CNA in metastatic breast tumor samples using publicly available datasets and characterize their expression and function using a metastatic mouse model of breast cancer.

Projective light-sheet microscopy with flexible parameter selection.

Projection imaging accelerates volumetric interrogation in fluorescence microscopy, but for multi-cellular samples, the resulting images may lack contrast, as many structures and haze are summed up. Here, we demonstrate rapid projective light-sheet imaging with parameter selection (props) of imaging depth, position and viewing angle. This allows us to selectively image different sub-volumes of a sample, rapidly switch between them and exclude background fluorescence.

RNA Sensing and Innate Immunity Constitutes a Barrier for Interspecies Chimerism.

Interspecies chimera formation with human pluripotent stem cells (PSCs) holds great promise to generate humanized animal models and provide donor organs for transplant. However, the approach is currently limited by low levels of human cells ultimately represented in chimeric embryos. Different strategies have been developed to improve chimerism by genetically editing donor human PSCs.

Organoid generation from mouse mammary tumors captures the genetic heterogeneity of clinically relevant copy number alterations.

Breast cancer metastases exhibit many different genetic alterations, including copy number amplifications. Using publicly available datasets, we identify copy number amplifications in metastatic breast tumor samples and using our organoid-based metastasis assays, and we validate FGFR1 is amplified in collectively migrating organoids. Because the heterogeneity of breast tumors is increasingly becoming relevant to clinical practice, we demonstrate our organoid method captures genetic heterogeneity of individual tumors.

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix.

Multiphase coalescence mediates Hippo pathway activation.

The function of biomolecular condensates is often restricted by condensate dissolution. Whether condensates can be suppressed without condensate dissolution is unclear. Here, we show that upstream regulators of the Hippo signaling pathway form functionally antagonizing condensates, and their coalescence into a common phase provides a mode of counteracting the function of biomolecular condensates without condensate dissolution.

YAP induces an oncogenic transcriptional program through TET1-mediated epigenetic remodeling in liver growth and tumorigenesis.

Epigenetic remodeling is essential for oncogene-induced cellular transformation and malignancy. In contrast to histone post-translational modifications, how DNA methylation is remodeled by oncogenic signaling remains poorly understood. The oncoprotein YAP, a coactivator of the TEAD transcription factors mediating Hippo signaling, is widely activated in human cancers.

The cellular architecture and molecular determinants of the zebrafish fusogenic synapse.

Myoblast fusion is an indispensable process in skeletal muscle development and regeneration. Studies in Drosophila led to the discovery of the asymmetric fusogenic synapse, in which one cell invades its fusion partner with actin-propelled membrane protrusions to promote fusion. However, the timing and sites of vertebrate myoblast fusion remain elusive.

Dynamin regulates the dynamics and mechanical strength of the actin cytoskeleton as a multifilament actin-bundling protein.

The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure.

The fusogenic synapse at a glance.

Cell-cell fusion is a fundamental process underlying fertilization, development, regeneration and physiology of metazoans. It is a multi-step process involving cell recognition and adhesion, actin cytoskeletal rearrangements, fusogen engagement, lipid mixing and fusion pore formation, ultimately resulting in the integration of two fusion partners. Here, we focus on the asymmetric actin cytoskeletal rearrangements at the site of fusion, known as the fusogenic synapse, which was first discovered during myoblast fusion in embryos and later also found in mammalian muscle and non-muscle cells.

Myoblast Fusion: Invasion and Resistance for the Ultimate Union.

Cell-cell fusion is indispensable for creating life and building syncytial tissues and organs. Ever since the discovery of cell-cell fusion, how cells join together to form zygotes and multinucleated syncytia has remained a fundamental question in cell and developmental biology. In the past two decades, myoblast fusion has been used as a powerful genetic model to unravel mechanisms underlying cell-cell fusion in vivo.

Sex and the Single Fly: A Perspective on the Career of Bruce S. Baker.

Bruce Baker, a preeminent geneticist who made fundamental contributions to our understanding of the molecular genetic basis of sex differences, passed away July 1, 2018 at the age of 72. Members of Bruce's laboratory remember him as an intensely dedicated, rigorous, creative, deep-thinking, and fearless scientist. His trainees also remember his strong commitment to teaching students at every level.

Let curiosity lead you.

It is an incredible honor to receive the Woman in Cell Biology Mid-Career Award for Excellence in Research. My lab works on cell-cell fusion, an indispensable process in the conception, development, and physiology of multicellular organisms. In this essay, I reflect on my curiosity-led journey, which uncovered some unexpected mechanisms underlying cell-cell fusion.

Spectrin is a mechanoresponsive protein shaping fusogenic synapse architecture during myoblast fusion.

Spectrin is a membrane skeletal protein best known for its structural role in maintaining cell shape and protecting cells from mechanical damage. Here, we report that α/β-spectrin (β is also called karst) dynamically accumulates and dissolves at the fusogenic synapse between fusing Drosophila muscle cells, where an attacking fusion partner invades its receiving partner with actin-propelled protrusions to promote cell fusion. Using genetics, cell biology, biophysics and mathematical modelling, we demonstrate that spectrin exhibits a mechanosensitive accumulation in response to shear deformation, which is highly elevated at the fusogenic synapse.

Requirement of the fusogenic micropeptide myomixer for muscle formation in zebrafish.

Skeletal muscle formation requires fusion of mononucleated myoblasts to form multinucleated myofibers. The muscle-specific membrane proteins myomaker and myomixer cooperate to drive mammalian myoblast fusion. Whereas myomaker is highly conserved across diverse vertebrate species, myomixer is a micropeptide that shows relatively weak cross-species conservation.

Mechanisms of myoblast fusion during muscle development.

The development and regeneration of skeletal muscle require the fusion of mononucleated muscle cells to form multinucleated, contractile muscle fibers. Studies using a simple genetic model, Drosophila melanogaster, have discovered many evolutionarily conserved fusion-promoting factors in vivo. Recent work in zebrafish and mouse also identified several vertebrate-specific factors required for myoblast fusion.

Mechanical tension drives cell membrane fusion.

Membrane fusion is an energy-consuming process that requires tight juxtaposition of two lipid bilayers. Little is known about how cells overcome energy barriers to bring their membranes together for fusion. Previously, we have shown that cell-cell fusion is an asymmetric process in which an "attacking" cell drills finger-like protrusions into the "receiving" cell to promote cell fusion.

Actin-propelled invasive membrane protrusions promote fusogenic protein engagement during cell-cell fusion.

Cell-cell fusion is critical for the conception, development, and physiology of multicellular organisms. Although cellular fusogenic proteins and the actin cytoskeleton are implicated in cell-cell fusion, it remains unclear whether and how they coordinate to promote plasma membrane fusion. We reconstituted a high-efficiency, inducible cell fusion culture system in the normally nonfusing Drosophila S2R+ cells.

Group I PAKs function downstream of Rac to promote podosome invasion during myoblast fusion in vivo.

The p21-activated kinases (PAKs) play essential roles in diverse cellular processes and are required for cell proliferation, apoptosis, polarity establishment, migration, and cell shape changes. Here, we have identified a novel function for the group I PAKs in cell-cell fusion. We show that the two Drosophila group I PAKs, DPak3 and DPak1, have partially redundant functions in myoblast fusion in vivo, with DPak3 playing a major role.