Explore the vast range of titles available.

Tumor-initiating cells (TICs) share features and regulatory pathways with normal stem cells, yet how the stem cell niche contributes to tumorigenesis remains unclear. Here, we identify CXCR4 + macrophages as a niche population enriched in normal mammary ducts, where they promote the regenerative activity of basal cells in response to luminal cell-derived CXCL12. CXCL12 triggers AKT-mediated stabilization of β-catenin, which induces Wnt ligands and pro-migratory genes, enabling intraductal macrophage infiltration and supporting regenerative activity of basal cells. Notably, these same CXCR4 + niche macrophages regulate the tumor-initiating activity of various breast cancer subtypes by enhancing TIC survival and tumor-forming capacity, while promoting early immune evasion through regulatory T cell induction. Furthermore, a CXCR4 + niche macrophage gene signature correlates with poor prognosis in human breast cancer. These findings highlight the pivotal role of the CXCL12-CXCR4 axis in orchestrating interactions between niche macrophages, mammary epithelial cells, and immune cells, thereby establishing a supportive niche for both normal tissue regeneration and mammary tumor initiation. Mammary gland resident macrophages are known to be crucial components of the mammary stem cell niche. Here, the authors show that CXCR4 + macrophages form a niche that regulates the tumor-initiating activity of breast cancer cells and induces early immune evasion through the recruitment of regulatory T cells.

Keystone species or ecological engineers are vital to the health of an ecosystem; however, often, their low abundance or biomass present challenges for their discovery, identification, visualization and selection. We report the development of fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB), a fixation-free protocol that is applicable to archaea and bacteria. The FISH-TAMB method differs from existing FISH methods by the absence of fixatives or surfactants in buffers, the fast hybridization time of as short as 15 min at target cells’ growth temperature, and the omission of washing steps. Polyarginine cell-penetrating peptides are employed to deliver molecular beacons (MBs) across prokaryotic cell walls and membranes, fluorescently labeling cells when MBs hybridize to target mRNA sequences. Here, the detailed protocol of the preparation and application of FISH-TAMB is presented. To demonstrate FISH-TAMB’s ability to label intracellular mRNA targets, differentiate transcriptional states, detect active and rare taxa, and keep cell viability, labeling experiments were performed that targeted the messenger RNA (mRNA) of methyl-coenzyme M reductase A (mcrA) expressed in (1) Escherichia coli containing a plasmid with a partial mcrA gene of the methanogen Methanosarcina barkeri (E. coli mcrA⁺); (2) M. barkeri; and (3) an anaerobic methanotrophic (ANME) enrichment from a deep continental borehole. Although FISH-TAMB was initially envisioned for mRNA of any functional gene of interest without a requirement of prior knowledge of 16S ribosomal RNA (rRNA)-based taxonomy, FISH-TAMB has the potential for multiplexing and going beyond mRNA and thus is a versatile addition to the molecular ecologist’s toolkit, with potentially widespread application in the field of environmental microbiology.

The complexity of intracellular signalling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid–liquid phase separation provide a distinct mechanism for the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGFβ and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters casein kinase 2—a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis. Esposito et al. show that TGF-β-induced DACT1 forms biomolecular condensates that sequester CK2 to repress Wnt signalling and modulate bone metastasis in cancer.

The complexity of intracellular signalling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid-liquid phase separation provide a distinct mechanism for the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGF[beta] and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters casein kinase 2–a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis.

Chemotaxis and phagocytosis are two distinct mechanisms that occur as a result of extracellular activation, inducing polarized cytoskeletal reorganization. Phagocytosis is defined as binding and internalization of a particle greater than 0.5 μm in diameter. The binding induces the propagation of a trans-membrane signal that is relayed to the cytosolic region of the cell. This signal results in localized actin polymerization at the site of internalization. Here we report the first attempt at introducing a live, viable substrate (bacteria) expressing a red fluorescent protein as a marker that allows for the plate fluorometric and flow cytometric determination of uptake and degradation rates in D. discoideum. We now have an assay that allows for the reproducible quantification of these rates as well as image analysis of the localized molecular mechanisms involved. Chemotaxis is the process whereby recognition of an external gradient induces polarized actin polymerization resulting in persistent and directional cell body translocation. Binding of chemoattractant ligand activates a signal transduction cascade that promotes the formation of a pseudopod at the “front” of the cell and subsequent retraction of the midbody anteriorly, resulting in “forward” cell motility. The development of an under-agarose folate chemotaxis assay in which cells must (a) recognize a chemoattractant gradient, and (b) deform the agarose upwards while flattening themselves as they transition under the agarose, allows us to analyze this process. We have found that in the restricted environment of this under-agarose assay, cells lacking the motor function of myosin II are able to chemotax normally, whereas cells proposed to be lacking the cross-linking function of myosin II are severely limited in their motile characteristics. These results have been determined via computerized measurement of cell motility and image analysis of GFP fusion proteins. We hypothesize the cross-linking function of myosin II is critical for cell motility in a restrictive environment. Phagocytosis and chemotaxis are specialized mechanisms of cellular polarity. I propose the hypothesis that both of these mechanisms are types of polarized cytoskeletal reorganization that are the result of the induction of specific types of receptors.

High-throughput sequencing and cellular imaging have expanded our knowledge of microbial diversity and expression of cellular activity. However, it remains challenging to characterize low-abundance, slow-growing microorganisms that play key roles in biogeochemical cycling. With the goal of isolating transcriptionally active cells of these microorganisms from environmental samples, we developed fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB) to label living prokaryotic cells. FISH-TAMB utilizes polyarginine cell-penetrating peptides to deliver molecular beacons across cell walls and membranes. Target cells are fluorescently labeled via hybridization between molecular beacons and messenger RNA of targeted functional genes. FISH-TAMB's target specificity and deliverance into both bacterial and archaeal cells were demonstrated by labeling intracellular methyl-coenzyme M reductase A (mcrA) transcripts expressed by Escherichia coli mcrA+, Methanosarcina barkeri, and a methanogenic enrichment of deep continental fracture fluid. Growth curve analysis supported sustained cellular viability following FISH-TAMB treatment. Flow cytometry and confocal microscopy detected labeled single cells and single cells in aggregates with unlabeled cells. As FISH-TAMB is amenable to target any functional gene of interest, when coupled with cell sorting, imaging, and sequencing techniques, FISH-TAMB will enable characterization of key uncharacterized rare biosphere microorganisms and of the syntrophically activated metabolic pathways between physically associated microorganisms.