The neutrophil protein CD177 is a novel PDPN receptor that regulates human cancer-associated fibroblast physiology

The cancer-associated fibroblast (CAF) marker podoplanin (PDPN) is generally correlated with poor clinical outcomes in cancer patients and thus represents a promising therapeutic target. Despite its biomedical relevance, basic aspects of PDPN biology such as its cellular functions and cell surface ligands remain poorly uncharacterized, thus challenging drug development. Here, we utilize a high throughput platform to elucidate the PDPN cell surface interactome, and uncover the neutrophil protein CD177 as a new binding partner. Quantitative proteomics analysis of the CAF phosphoproteome reveals a role for PDPN in cell signaling, growth and actomyosin contractility, among other processes. Moreover, cellular assays demonstrate that CD177 is a functional antagonist, recapitulating the phenotype observed in PDPN-deficient CAFs. In sum, starting from the unbiased elucidation of the PDPN co-receptome, our work provides insights into PDPN functions and reveals the PDPN/CD177 axis as a possible modulator of fibroblast physiology in the tumor microenvironment.

Optogenetic relaxation of actomyosin contractility uncovers mechanistic roles of cortical tension during cytokinesis

Actomyosin contractility generated cooperatively by nonmuscle myosin II and actin filaments plays essential roles in a wide range of biological processes, such as cell motility, cytokinesis, and tissue morphogenesis. However, subcellular dynamics of actomyosin contractility underlying such processes remains elusive. Here, we demonstrate an optogenetic method to induce relaxation of actomyosin contractility at the subcellular level. The system, named OptoMYPT, combines a protein phosphatase 1c (PP1c)-binding domain of MYPT1 with an optogenetic dimerizer, so that it allows light-dependent recruitment of endogenous PP1c to the plasma membrane. Blue-light illumination is sufficient to induce dephosphorylation of myosin regulatory light chains and a decrease in actomyosin contractile force in mammalian cells and Xenopus embryos. The OptoMYPT system is further employed to understand the mechanics of actomyosin-based cortical tension and contractile ring tension during cytokinesis. We find that the relaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, revealing that the cortical tension substantially antagonizes constriction of the cleavage furrow. Based on these results, the OptoMYPT system provides opportunities to understand cellular and tissue mechanics.

Anti-Malignant Effect of Tensile Loading to Adherens Junctions in Cutaneous Squamous Cell Carcinoma Cells

Actomyosin contractility regulates various cellular processes including proliferation and differentiation while dysregulation of actomyosin activity contributes to cancer development and progression. Previously, we have reported that actomyosin-generated tension at adherens junctions is required for cell density-dependent inhibition of proliferation of normal skin keratinocytes. However, it remains unclear how actomyosin contractility affects the hyperproliferation ability of cutaneous squamous cell carcinoma (cSCC) cells. In this study, we find that actomyosin activity is impaired in cSCC cells both in vitro and in vivo. External application of tensile loads to adherens junctions by sustained mechanical stretch attenuates the proliferation of cSCC cells, which depends on intact adherens junctions. Forced activation of actomyosin of cSCC cells also inhibits their proliferation in a cell-cell contact-dependent manner. Furthermore, the cell cycle arrest induced by tensile loading to adherens junctions is accompanied by epidermal differentiation in cSCC cells. Our results show that the degree of malignant properties of cSCC cells can be reduced by applying tensile loads to adherens junctions, which implies that the mechanical status of adherens junctions may serve as a novel therapeutic target for cSCC.

Optogenetic dissection of the roles of actomyosin in the mechanics underlying tissue fluidity

Rapid epithelial tissue flows are essential to building and shaping developing embryos. However, it is not well understood how the mechanical properties of tissues and the forces driving them to flow are jointly regulated to accommodate rapid tissue remodeling. To dissect the roles of actomyosin in the mechanics of epithelial tissue flows, here we use two optogenetic tools, optoGEF and optoGAP, to manipulate Rho/Rho-kinase signaling and actomyosin contractility in the germband epithelium, which flows via convergent extension during Drosophila body axis elongation. The ability to perturb actomyosin across the tissue allows us to analyze the effects of actomyosin on cell rearrangements, tissue tensions, and tissue mechanical properties. We find that either optogenetic activation or deactivation of Rho1 signaling and actomyosin contractility at the apical surface of the germband disrupts cell rearrangements and tissue-level flows. By probing mechanical tensions in the tissue using laser ablation and predicting tissue mechanical properties from cell packings, we find that actomyosin influences both the anisotropic forces driving tissue flow and the mechanical properties of the tissue resisting flow, leading to complex relationships between actomyosin activity and tissue-level flow. Moreover, our results indicate that changes in the distribution of medial and junctional myosin in the different perturbations alter tissue tension and cell packings in distinct ways, revealing how junctional and medial myosin have differential roles in promoting and orienting cell rearrangements to tune tissue flows in developing embryos.

The cytosolic role of EZH2-IMPDH2 complex in melanoma progression and metastasis via GTP regulation

Although conventional EZH2 enzymatic inhibitors are effective in various tumors, we demonstrated that B-Raf mutant melanoma cells do not respond effectively to both GSK126 and EPZ6438 when used in physiological levels in vitro. In addition, the EZH2 knockdown phenotype (lowered tumorigenesis and metastasis) was rescued by both wild-type EZH2 and methyl-transferase-deficient H689A mutant and cytosolic nuclear localization signal (NLS) deletion-mutant EZH2 overexpression in vitro and in vivo. This clearly indicates a methyl-transferase-independent role of cytosolic EZH2 in melanoma cell tumorigenicity and metastasis. To identify potential methyltransferase-independent mechanisms of EZH2 in melanoma, we performed Liquid Chromatography-Mass Spectrometry (LC-MS) on EZH2 immunoprecipitates from multiple melanoma cell lines and human PDXs. We identified an interacting protein called inosine monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme in de-novo GTP synthesis. Biochemical studies showed that N-terminal EED-binding domain of cytosolic EZH2 interacts with CBS domain of IMPDH2 in a PRC2- and methylation-independent manner. EZH2 silencing reduces cellular GTP levels by impeding IMPDH2 tetramerization, stability and its cytosolic localization. On the other hand, guanosine, which replenishes GTP, stabilized ribosomal biogenesis and actomyosin contractility and thereby, promoted invasive and clonogenic cell states even in EZH2 silenced cells. In human melanoma clinical samples, high cytosolic EZH2 and IMPDH2 expressions are directly correlated with the nucleolar enlargement in the metastatic melanomas. In addition, IMPDH2 silencing reduces EZH2 overexpression induced proliferation and invasion phenotype that is reversed later by guanosine addition. In addition, EZH2-IMPDH2 complex was also validated across a range of cancers. These results point to a methyltransferase-independent but GTP-dependent non-canonical mechanism of EZH2 regulation in various cancers. Sappanone A (SA), that is shown to inhibit IMPDH2/EZH2 interaction and thereby IMPDH2 tetrametization, is anti-tumorigenic across a range of cancers including melanoma, but not in normal melanocytes or bone marrow progenitor cells. In summary, EZH2 contributes to melanoma tumorigenicity and invasion by upregulating ribosomal biogenesis and actomyosin contractility via IMPDH2-induced GTP synthesis.

Plexin-B2 orchestrates collective stem cell dynamics via actomyosin contractility, cytoskeletal tension and adhesion

During morphogenesis, molecular mechanisms that orchestrate biomechanical dynamics across cells remain unclear. Here, we show a role of guidance receptor Plexin-B2 in organizing actomyosin network and adhesion complexes during multicellular development of human embryonic stem cells and neuroprogenitor cells. Plexin-B2 manipulations affect actomyosin contractility, leading to changes in cell stiffness and cytoskeletal tension, as well as cell-cell and cell-matrix adhesion. We have delineated the functional domains of Plexin-B2, RAP1/2 effectors, and the signaling association with ERK1/2, calcium activation, and YAP mechanosensor, thus providing a mechanistic link between Plexin-B2-mediated cytoskeletal tension and stem cell physiology. Plexin-B2-deficient stem cells exhibit premature lineage commitment, and a balanced level of Plexin-B2 activity is critical for maintaining cytoarchitectural integrity of the developing neuroepithelium, as modeled in cerebral organoids. Our studies thus establish a significant function of Plexin-B2 in orchestrating cytoskeletal tension and cell-cell/cell-matrix adhesion, therefore solidifying the importance of collective cell mechanics in governing stem cell physiology and tissue morphogenesis.

Actin capping protein regulates actomyosin contractility to maintain germline architecture in C. elegans

Actin dynamics play an important role in the morphogenesis of cells and tissues, yet the control of actin filament growth takes place at the molecular level. A challenge in the field is to link the molecular function of actin regulators with their physiological function. Here, we report the in vivo role of the actin capping protein CAP-1 in the C. elegans germline. We show that CAP-1 is associated with actomyosin structures in the cortex and rachis, where it keeps the level of contractility in check. A 60% reduction in the level of CAP-1 leads to a 2-fold increase in F-actin and non-muscle myosin II and only a 30% increase in Arp2/3. CAP-1 depletion leads to severe structural defects in the syncytial germline and oocytes, which can be rescued by reducing myosin activity. Thus, we uncover a physiological role for actin capping protein in maintaining C. elegans fertility by regulating the level of actomyosin contractility.

A unique form of collective epithelial migration is crucial for tissue fusion in the secondary palate and can overcome loss of epithelial apoptosis

Tissue fusion is an oft-employed process in morphogenesis which often requires the removal of the epithelia intervening multiple distinct primordia to form one continuous structure. In the mammalian secondary palate, a midline epithelial seam (MES) forms between two palatal shelves and must be removed to allow mesenchymal confluence. Abundant apoptosis and cell extrusion in this epithelial seam support their importance in its removal. However, by genetically disrupting the intrinsic apoptotic regulators BAX and BAK within the MES, we find a complete loss of cell death and cell extrusion, but successful removal of the MES, indicating that developmental compensation enables fusion. Novel static and live imaging approaches reveal that the MES is removed through a unique form of collective epithelial cell migration in which epithelial trails and islands stream through the mesenchyme to reach the oral and nasal epithelial surfaces. These epithelial trails and islands begin to express periderm markers while retaining expression of the basal epithelial marker ΔNp63, suggesting their migration to the oral and nasal surface is concomitant with their differentiation to an epithelial intermediate. Live imaging reveals anisotropic actomyosin contractility within epithelial trails that drives their peristaltic movement, and genetic loss of non-muscle myosin IIA-mediated actomyosin contractility results in dispersion of epithelial collectives and dramatic failure of normal MES migration. These findings demonstrate redundancy between cellular mechanisms of morphogenesis and reveal a crucial role for a unique form of collective epithelial migration during tissue fusion.