Force-induced recruitment of cten along keratin network in epithelial cells.

The cytoskeleton provides structural integrity to cells and serves as a key component in mechanotransduction. Tensins are thought to provide a force-bearing linkage between integrins and the actin cytoskeleton; yet, direct evidence of tensin's role in mechanotransduction is lacking. We here report that local force application to epithelial cells using a micrometer-sized needle leads to rapid accumulation of cten (tensin 4), but not tensin 1, along a fibrous intracellular network. Surprisingly, cten-positive fibers are not actin fibers; instead, these fibers are keratin intermediate filaments. The dissociation of cten from tension-free keratin fibers depends on the duration of cell stretch, demonstrating that the external force favors maturation of cten-keratin network interactions over time and that keratin fibers retain remarkable structural memory of a cell's force-bearing state. These results establish the keratin network as an integral part of force-sensing elements recruiting distinct proteins like cten and suggest the existence of a mechanotransduction pathway via keratin network.

The Capicua C1 Domain is Required for Full Activity of the CIC::DUX4 Fusion Oncoprotein.

Rearrangements between genes can yield neomorphic fusions that drive oncogenesis. Fusion oncogenes are made up of fractional segments of the partner genes that comprise them, with each partner potentially contributing some of its own function to the nascent fusion oncoprotein. Clinically, fusion oncoproteins driving one diagnostic entity are typically clustered into a single molecular subset and are often treated a similar fashion. However, knowledge of where specific fusion breakpoints occur in partner genes, and the resulting retention of functional domains in the fusion, is an important determinant of fusion oncoprotein activity and may differ between patients. This study investigates this phenomena through the example of CIC::DUX4, a fusion between the transcriptional repressor capicua (CIC) and the double homeobox 4 gene (DUX4), which drives an aggressive subset of undifferentiated round cell sarcoma. Using a harmonized dataset of over 100 patient fusion breakpoints from the literature, we show that most bona fide CIC::DUX4 fusions retain the C1 domain, which is known to contribute to DNA binding by wild type CIC. Mechanistically, deletion or mutation of the C1 domain reduces, but does not eliminate, activation of CIC target genes by CIC::DUX4. We also find that expression of C1-deleted CIC::DUX4 is capable of exerting intermediate transformation-related phenotypes compared with those imparted by full-length CIC::DUX4, but was not sufficient for tumorigenesis in a subcutaneous mouse model. In summary, our results suggest a supercharging role for the C1 domain in the activity of CIC::DUX4.

Quantitative Comparison of Monomeric StayGold Variants Using Protein Nanocages in Living Cells.

To standardize comparison of fluorescent proteins and independently determine which monomeric StayGold variant is best for live microscopy, we analyzed fluorescent protein tagged I3-01 peptides that self-assemble into stable sixty subunit dodecahedrons inside live cells. We find mStayGold is 3-fold brighter and 3-fold more photostable compared with EGFP and superior to other monomeric variants in mammalian cytoplasm. In addition, analysis of intracellular nanocage diffusion confirms the monomeric nature of mStayGold.

Doublecortin reinforces microtubules to promote growth cone advance in soft environments.

Doublecortin (DCX) is a microtubule-associated protein critical for brain development. Although most highly expressed in the developing central nervous system, the molecular function of DCX in neuron morphogenesis remains unknown and controversial. We demonstrate that DCX function is intimately linked to its microtubule-binding activity. By using human induced pluripotent stem cell (hiPSC)- derived cortical i 3 Neurons genome engineered to express mEmerald-tagged DCX from the endogenous locus, we find that DCX-MT interactions become highly polarized very early during neuron morphogenesis. DCX becomes enriched only on straight microtubules in advancing growth cones with approximately 120 DCX molecules bound per micrometer of growth cone microtubule. At a similar saturation, microtubule-bound DCX molecules begin to impede lysosome transport, and thus can potentially control growth cone organelle entry. In addition, by comparing control, DCX-mEmerald and knockout DCX -/Y i 3 Neurons, we find that DCX stabilizes microtubules in the growth cone peripheral domain by reducing the microtubule catastrophe frequency and the depolymerization rate. DCX -/Y i 3 Neuron morphogenesis was inhibited in soft microenvironments that mimic the viscoelasticity of brain tissue and DCX -/Y neurites failed to grow toward brain-derived neurotrophic factor (BDNF) gradients. Together with high resolution traction force microscopy data, we propose a model in which DCX-decorated, rigid growth cone microtubules provide intracellular mechanical resistance to actomyosin generated contractile forces in soft physiological environments in which weak and transient adhesion-mediated forces in the growth cone periphery may be insufficient for productive growth cone advance. These data provide a new mechanistic understanding of how DCX mutations cause lissencephaly-spectrum brain malformations by impacting growth cone dynamics during neuron morphogenesis in physiological environments.

Proinflammatory immune cells disrupt angiogenesis and promote germinal matrix hemorrhage in prenatal human brain.

Germinal matrix hemorrhage (GMH) is a devastating neurodevelopmental condition affecting preterm infants, but why blood vessels in this brain region are vulnerable to rupture remains unknown. Here we show that microglia in prenatal mouse and human brain interact with nascent vasculature in an age-dependent manner and that ablation of these cells in mice reduces angiogenesis in the ganglionic eminences, which correspond to the human germinal matrix. Consistent with these findings, single-cell transcriptomics and flow cytometry show that distinct subsets of CD45+ cells from control preterm infants employ diverse signaling mechanisms to promote vascular network formation. In contrast, CD45+ cells from infants with GMH harbor activated neutrophils and monocytes that produce proinflammatory factors, including azurocidin 1, elastase and CXCL16, to disrupt vascular integrity and cause hemorrhage in ganglionic eminences. These results underscore the brain's innate immune cells in region-specific angiogenesis and how aberrant activation of these immune cells promotes GMH in preterm infants.

Notch1 cortical signaling regulates epithelial architecture and cell-cell adhesion.

Notch receptors control tissue morphogenic processes that involve coordinated changes in cell architecture and gene expression, but how a single receptor can produce these diverse biological outputs is unclear. Here we employ a 3D organotypic model of a ductal epithelium to reveal tissue morphogenic defects result from loss of Notch1, but not Notch1 transcriptional signaling. Instead, defects in duct morphogenesis are driven by dysregulated epithelial cell architecture and mitogenic signaling which result from loss of a transcription-independent Notch1 cortical signaling mechanism that ultimately functions to stabilize adherens junctions and cortical actin. We identify that Notch1 localization and cortical signaling are tied to apical-basal cell restructuring and discover a Notch1-FAM83H interaction underlies stabilization of adherens junctions and cortical actin. Together, these results offer new insights into Notch1 signaling and regulation, and advance a paradigm in which transcriptional and cell adhesive programs might be coordinated by a single receptor.

Notch1 cortical signaling regulates epithelial architecture and cell-cell adhesion.

Notch receptors control tissue morphogenic processes that involve coordinated changes in cell architecture and gene expression, but how a single receptor can produce these diverse biological outputs is unclear. Here, we employ a 3D model of a human ductal epithelium to reveal tissue morphogenic defects result from loss of Notch1, but not Notch1 transcriptional signaling. Instead, defects in duct morphogenesis are driven by dysregulated epithelial cell architecture and mitogenic signaling which result from the loss of a transcription-independent, Notch1 cortical signaling mechanism that ultimately functions to stabilize adherens junctions and cortical actin. We identify that Notch1 localization and cortical signaling are tied to apical-basal cell restructuring and discover that a Notch1-FAM83H interaction underlies control of epithelial adherens junctions and cortical actin. Together, these results offer new insights into Notch1 signaling and regulation and advance a paradigm in which transcriptional and cell adhesive programs might be coordinated by a single receptor.

Spatial proximity of proteins surrounding zyxin under force-bearing conditions.

Sensing physical forces is a critical first step in mechano-transduction of cells. Zyxin, a LIM domain-containing protein, is recruited to force-bearing actin filaments and is thought to repair and strengthen them. Yet, the precise force-induced protein interactions surrounding zyxin remain unclear. Using BioID analysis, we identified proximal proteins surrounding zyxin under normal and force-bearing conditions by label-free mass spectrometry analysis. Under force-bearing conditions, increased biotinylation of α-actinin 1, α-actinin 4, and AFAP1 were detected, and these proteins accumulated along force-bearing actin fibers independently from zyxin, albeit at a lower intensity than zyxin. VASP also accumulated along force-bearing actin fibers in a zyxin-dependent manner, but the biotinylation of VASP remained constant regardless of force, supporting the model of a free zyxin-VASP complex in the cytoplasm being corecruited to tensed actin fibers. In addition, ARHGAP42, a RhoA GAP, was also identified as a proximal protein of zyxin and colocalized with zyxin along contractile actin bundles. The overexpression of ARHGAP42 reduced the rate of small wound closure, a zyxin-dependent process. These results demonstrate that the application of proximal biotinylation can resolve the proximity and composition of protein complexes as a function of force, which had not been possible with traditional biochemical analysis.