How cells integrate antagonistic receptor signaling events is enigmatic. Using superresolution optical microscopy, Lopes et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201608094 ) demonstrate the nanometer-scale molecular reorganization of antagonistic signaling receptors in macrophages, after engagement by the receptors of activating and inhibitory ligands. They propose that large-scale rearrangements of this type underpin decision-making by these cells.

Vertebrate Hedgehog (HH) signaling is controlled by several ligand-binding antagonists including Patched-1 (PTCH1), PTCH2, and HH-interacting protein 1 (HHIP1), whose collective action is essential for proper HH pathway activity. However, the molecular mechanisms used by these inhibitors remain poorly understood. In this paper, we investigated the mechanisms underlying HHIP1 antagonism of HH signaling. Strikingly, we found evidence that HHIP1 non–cell-autonomously inhibits HH-dependent neural progenitor patterning and proliferation. Furthermore, this non–cell-autonomous antagonism of HH signaling results from the secretion of HHIP1 that is modulated by cell type–specific interactions with heparan sulfate (HS). These interactions are mediated by an HS-binding motif in the cysteine-rich domain of HHIP1 that is required for its localization to the neuroepithelial basement membrane (BM) to effectively antagonize HH pathway function. Our data also suggest that endogenous, secreted HHIP1 localization to HS-containing BMs regulates HH ligand distribution. Overall, the secreted activity of HHIP1 represents a novel mechanism to regulate HH ligand localization and function during embryogenesis.

Somatic inactivation of the serine/threonine kinase gene STK11/LKB1/PAR-4 occurs in a variety of cancers, including ∼10% of melanoma. However, how the loss of LKB1 activity facilitates melanoma invasion and metastasis remains poorly understood. In LKB1-null cells derived from an autochthonous murine model of melanoma with activated Kras and Lkb1 loss and matched reconstituted controls, we have investigated the mechanism by which LKB1 loss increases melanoma invasive motility. Using a microfluidic gradient chamber system and time-lapse microscopy, in this paper, we uncover a new function for LKB1 as a directional migration sensor of gradients of extracellular matrix (haptotaxis) but not soluble growth factor cues (chemotaxis). Systematic perturbation of known LKB1 effectors demonstrated that this response does not require canonical adenosine monophosphate–activated protein kinase (AMPK) activity but instead requires the activity of the AMPK-related microtubule affinity-regulating kinase (MARK)/PAR-1 family kinases. Inhibition of the LKB1–MARK pathway facilitated invasive motility, suggesting that loss of the ability to sense inhibitory matrix cues may promote melanoma invasion.

Arp2/3-branched actin is critical for cytoskeletal dynamics and cell migration. However, perturbations and diseases affecting this network have phenotypes that cannot be fully explained by cell-autonomous effects. In this paper, we report nonautonomous effects of Arp2/3 depletion. We show that, upon Arp2/3 depletion, the expression of numerous genes encoding secreted factors, including chemokines, growth factors, and matrix metalloproteases, was increased, a signature resembling the senescence-associated secretory phenotype. These factors affected epidermal growth factor chemotaxis in a nonautonomous way, resolving the recent contradictions about the role of Arp2/3 in chemotaxis. We demonstrate that these genes were activated by nuclear factor κB via a CCM2–MEKK3 pathway that has been implicated in hyperosmotic stress signaling. Consistent with this, Arp2/3-depleted cells showed misregulation of volume control and reduced actin in the submembranous cortex. The defects in osmotic signaling in the Arp2/3-depleted cells can be rescued by hypoosmotic treatment. Thus, perturbations of Arp2/3 have nonautonomous effects that should be considered when evaluating experimental manipulations and diseases affecting the Arp2/3-actin cytoskeleton.

The protein kinase B-Raf is a critical component of the Ras/MAPK signaling pathway. An oncogenic B-Raf mutation that constitutively activates the kinase was identified in z50% of melanoma patients and in other cancers. A structure-guided drug discovery approach enabled the development of Zelboraf, a targeted inhibitor of oncogenic B-Raf. This drug has been used successfully in the clinic to treat metastatic melanoma patients harboring B-Raf mutations.

Ataxia telangiectasia mutated (ATM) plays a critical role in the cellular response to DNA damage. In response to DNA double-strand breaks (DSBs), ATM is autophosphorylated at serine 1981. Although this autophosphorylation is widely considered a sign of ATM activation, it is still not clear if autophosphorylation is required for ATM functions including localization to DSBs and activation of ATM kinase activity. In this study, we show that localization of ATM to DSBs is differentially regulated with the initial localization requiring the MRE11–RAD50–NBS1 complex and sustained retention requiring autophosphorylation of ATM at serine 1981. Autophosphorylated ATM interacts with MDC1 and the latter is required for the prolonged association of ATM to DSBs. Ablation of ATM autophosphorylation or knock-down of MDC1 protein affects the ability of ATM to phosphorylate downstream substrates and confer radioresistance. Together, these data suggest that autophosphorylation at serine 1981 stabilizes ATM at the sites of DSBs, and this is required for a proper DNA damage response.

Macrophage death in advanced atherosclerosis promotes necrosis and plaque destabilization. A likely cause of macrophage death is accumulation of free cholesterol (FC) in the ER, leading to activation of the unfolded protein response (UPR) and C/EBP homologous protein (CHOP)–induced apoptosis. Here we show that p38 MAPK signaling is necessary for CHOP induction and apoptosis. Additionally, two other signaling pathways must cooperate with p38-CHOP to effect apoptosis. One involves the type A scavenger receptor (SRA). As evidence, FC loading by non-SRA mechanisms activates p38 and CHOP, but not apoptosis unless the SRA is engaged. The other pathway involves c-Jun NH 2 -terminal kinase (JNK)2, which is activated by cholesterol trafficking to the ER, but is independent of CHOP. Thus, FC-induced apoptosis requires cholesterol trafficking to the ER, which triggers p38-CHOP and JNK2, and engagement of the SRA. These findings have important implications for understanding how the UPR, MAPKs, and the SRA might conspire to cause macrophage death, lesional necrosis, and plaque destabilization in advanced atherosclerotic lesions.

Tim23p (translocase of the inner membrane) is an essential import component located in the mitochondrial inner membrane. To determine how the Tim23 protein itself is transported into mitochondria, we used chemical cross-linking to identify proteins adjacent to Tim23p during its biogenesis. In the absence of an inner membrane potential, Tim23p is translocated across the mitochondrial outer membrane, but not inserted into the inner membrane. At this intermediate stage, we find that Tim23p forms cross-linked products with two distinct protein complexes of the intermembrane space, Tim8p–Tim13p and Tim9p–Tim10p. Tim9p and Tim10p cross-link to the COOH-terminal domain of the Tim23 protein, which carries all of the targeting signals for Tim23p. Therefore, our results suggest that the Tim9p–Tim10p complex plays a key role in Tim23p import. In contrast, Tim8p and Tim13p cross-link to the hydrophilic NH 2 -terminal segment of Tim23p, which does not carry essential import information and, thus, the role of Tim8p–Tim13p is unclear. Tim23p contains two matrix-facing, positively charged loops that are essential for its insertion into the inner membrane. The positive charges are not required for interaction with the Tim9p–Tim10p complex, but are essential for cross-linking of Tim23p to components of the inner membrane insertion machinery, including Tim54p, Tim22p, and Tim12p.

The zinc finger protein ZPR1 is present in the cytoplasm of quiescent mammalian cells and translocates to the nucleus upon treatment with mitogens, including epidermal growth factor (EGF). Homologues of ZPR1 were identified in yeast and mammals. These ZPR1 proteins bind to eukaryotic translation elongation factor-1α (eEF-1α). Studies of mammalian cells demonstrated that EGF treatment induces the interaction of ZPR1 with eEF-1α and the redistribution of both proteins to the nucleus. In the yeast Saccharomyces cerevisiae , genetic analysis demonstrated that ZPR1 is an essential gene. Deletion analysis demonstrated that the NH 2 -terminal region of ZPR1 is required for normal growth and that the COOH-terminal region was essential for viability in S . cerevisiae . The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation. Disruption of the binding of ZPR1 to eEF-1α by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth. Reconstitution of the ZPR1 interaction with eEF-1α restored normal growth. We conclude that ZPR1 is essential for cell viability and that its interaction with eEF-1α contributes to normal cellular proliferation.

Vasoactive effects of soluble matrix proteins and integrin-binding peptides on arterioles are mediated by α v β 3 and α 5 β 1 integrins. To examine the underlying mechanisms, we measured L-type Ca 2+ channel current in arteriolar smooth muscle cells in response to integrin ligands. Whole-cell, inward Ba 2+ currents were inhibited after application of soluble cyclic RGD peptide, vitronectin (VN), fibronectin (FN), either of two anti–β 3 integrin antibodies, or monovalent β 3 antibody. With VN or β 3 antibody coated onto microbeads and presented as an insoluble ligand, current was also inhibited. In contrast, beads coated with FN or α 5 antibody produced significant enhancement of current after bead attachment. Soluble α 5 antibody had no effect on current but blocked the increase in current evoked by FN-coated beads and enhanced current when applied in combination with an appropriate IgG. The data suggest that α v β 3 and α 5 β 1 integrins are differentially linked through intracellular signaling pathways to the L-type Ca 2+ channel and thereby alter control of Ca 2+ influx in vascular smooth muscle. This would account for the vasoactive effects of integrin ligands on arterioles and provide a potential mechanism for wound recognition during tissue injury.

SSH1, a newly identified member of the heat shock protein (hsp70) multigene family of the budding yeast Saccharomyces cerevisiae, encodes a protein localized to the mitochondrial matrix. Deletion of the SSH1 gene results in extremely slow growth at 23 degrees C or 30 degrees C, but nearly wild-type growth at 37 degrees C. The matrix of the mitochondria contains another hsp70, Ssc1, which is essential for growth and required for translocation of proteins into mitochondria. Unlike SSC1 mutants, an SSH1 mutant showed no detectable defects in import of several proteins from the cytosol to the matrix compared to wild type. Increased expression of Ssc1 partially suppressed the cold-sensitive growth defect of the SSH1 mutant, suggesting that when present in increased amounts, Ssc1 can at least partially carry out the normal functions of Ssh1. Spontaneous suppressors of the cold-sensitive phenotype of an SSH1 null mutant were obtained at a high frequency at 23 degrees C, and were all found to be respiration deficient. 15 of 16 suppressors that were analyzed lacked mitochondrial DNA, while the 16th had reduced amounts. We suggest that Ssh1 is required for normal mitochondrial DNA replication, and that disruption of this process in ssh1 cells results in a defect in mitochondrial function at low temperatures.

A subset of mRNAs, polyribosomes, and poly(A)-binding proteins copurify with microtubules from sea urchin embryos. Several lines of evidence indicate that the interaction of microtubules with ribosomes is specific: a distinct stalk-like structure appears to mediate their association; ribosomes bind to microtubules with a constant stoichiometry through several purification cycles; and the presence of ribosomes in these preparations depends on the presence of intact microtubules. Five specific mRNAs are enriched with the microtubule-bound ribosomes, indicating that translation of specific proteins may occur on the microtubule scaffolding in vivo.

Two variants of ankyrin have been distinguished in rat brain tissue using antibodies: a broadly distributed isoform (ankyrinB) that represents the major form of ankyrin in brain and another isoform with a restricted distribution (ankyrinR) that shares epitopes with erythrocyte ankyrin. The ankyrinR isoform was localized by immunofluorescence in cryosections of rat spinal cord gray matter and myelinated central and peripheral nerves to: (a) perikarya and initial axonal segments of neuron cells, (b) nodes of Ranvier of myelinated nerve with no detectable labeling in other areas of the myelinated axons, and (c) the axolemma of unmyelinated axons. Immunogold EM on ultrathin cryosections of myelinated nerve showed that ankyrinR was localized on the cytoplasmic face of the axolemma and was restricted to the nodal and, in some cases, paranodal area. The major isoform of ankyrin in brain (ankyrinB) displayed a broad distribution on glial and neuronal cells of the gray matter and a mainly glial distribution in central myelinated axons with no significant labeling on the axolemma. These results show that (a) ankyrin isoforms display a differential distribution on glial and neuronal cells of the nervous tissue; (b) an isoform of ankyrin codistributes with the voltage-dependent sodium channel in both myelinated and unmyelinated nerve fibers. Ankyrin interacts in vitro with the voltage-dependent sodium channel (Srinivasan, Y., L. Elmer, J. Davis, V. Bennett, and K. Angelides. 1988. Nature (Lond.). 333:177-180). A specific interaction of an isoform of ankyrin with the sodium channel thus may play an important role in the morphogenesis and/or maintenance of the node of Ranvier.