Fluorescent tagging of endogenous IRS2 with an auxin-dependent degron to assess dynamic intracellular localization and function.

Insulin Receptor Substrate 2 (IRS2) is a signaling adaptor protein for the insulin (IR) and Insulin-like Growth Factor-1 (IGF-1R) receptors. In breast cancer, IRS2 contributes to both the initiation of primary tumor growth and the establishment of secondary metastases through regulation of cancer stem cell (CSC) function and invasion. However, how IRS2 mediates its diverse functions is not well understood. We used CRISPR/Cas9-mediated gene editing to modify endogenous IRS2 to study the expression, localization, and function of this adaptor protein. A cassette containing an auxin-inducible degradation (AID) sequence, 3x-FLAG tag, and mNeon-green was introduced at the N-terminus of the IRS2 protein to provide rapid and reversible control of IRS2 protein degradation and analysis of endogenous IRS2 expression and localization. Live fluorescence imaging of these cells revealed that IRS2 shuttles between the cytoplasm and nucleus in response to growth regulatory signals in a PI3K-dependent manner. Inhibition of nuclear export or deletion of a putative nuclear export sequence in the C-terminal tail promotes nuclear retention of IRS2, implicating nuclear export in the mechanism by which IRS2 intracellular localization is regulated. Moreover, the acute induction of IRS2 degradation reduces tumor cell invasion, demonstrating the potential for therapeutic targeting of this adaptor protein. Our data highlight the value of our model of endogenously tagged IRS2 as a tool to study IRS2 localization and function.

Transcriptional suppression of sphingolipid catabolism controls pathogen resistance in C. elegans.

Sphingolipids are required for diverse biological functions and are degraded by specific catabolic enzymes. However, the mechanisms that regulate sphingolipid catabolism are not known. Here we characterize a transcriptional axis that regulates sphingolipid breakdown to control resistance against bacterial infection. From an RNAi screen for transcriptional regulators of pathogen resistance in the nematode C. elegans, we identified the nuclear hormone receptor nhr-66, a ligand-gated transcription factor homologous to human hepatocyte nuclear factor 4. Tandem chromatin immunoprecipitation-sequencing and RNA sequencing experiments revealed that NHR-66 is a transcriptional repressor, which directly targets sphingolipid catabolism genes. Transcriptional de-repression of two sphingolipid catabolic enzymes in nhr-66 loss-of-function mutants drives the breakdown of sphingolipids, which enhances host susceptibility to infection with the bacterial pathogen Pseudomonas aeruginosa. These data define transcriptional control of sphingolipid catabolism in the regulation of cellular sphingolipids, a process that is necessary for pathogen resistance.

Xenopus embryos show a compensatory response following perturbation of the Notch signaling pathway.

As an essential feature of development, robustness ensures that embryos attain a consistent phenotype despite genetic and environmental variation. The growing number of examples demonstrating that embryos can mount a compensatory response to germline mutations in key developmental genes has heightened interest in the phenomenon of embryonic robustness. While considerable progress has been made in elucidating genetic compensation in response to germline mutations, the diversity, mechanisms, and limitations of embryonic robustness remain unclear. In this work, we have examined whether Xenopus laevis embryos are able to compensate for perturbations of the Notch signaling pathway induced by RNA injection constructs that either upregulate or inhibit this signaling pathway. Consistent with earlier studies, we found that at neurula stages, hyperactivation of the Notch pathway inhibited neural differentiation while inhibition of Notch signaling increases premature differentiation as assayed by neural beta tubulin expression. However, surprisingly, by hatching stages, embryos begin to compensate for these perturbations, and by swimming tadpole stages most embryos exhibited normal neuronal gene expression. Using cell proliferation and TUNEL assays, we show that the compensatory response is, in part, mediated by modulating levels of cell proliferation and apoptosis. This work provides an additional model for addressing the mechanisms of embryonic robustness and of genetic compensation.

Cutting Edge: IL-1R1 Mediates Host Resistance to Mycobacterium tuberculosis by Trans-Protection of Infected Cells.

IL-1R1 deficiency in mice causes severe susceptibility to Mycobacterium tuberculosis Mice and macrophage cultures lacking IL-1R1 display increased bacterial growth, suggesting that phagocytes may require IL-1R1-dependent antimicrobial signals to limit intracellular M. tuberculosis replication directly. However, the myeloid-cell-intrinsic versus -extrinsic requirements for IL-1R1 to control M. tuberculosis infection in mice have not been directly addressed. Using single-cell analysis of infected cells, competitive mixed bone marrow chimeras, and IL-1R1 conditional mutant mice, we show in this article that IL-1R1 expression by pulmonary phagocytes is uncoupled from their ability to control intracellular M. tuberculosis growth. Importantly, IL-1R1-dependent control was provided to infected cells in trans by both nonhematopoietic and hematopoietic cells. Thus, IL-1R1-mediated host resistance to M. tuberculosis infection does not involve mechanisms of cell-autonomous antimicrobicidal effector functions in phagocytes but requires the cooperation between infected cells and other cells of hematopoietic or nonhematopoietic origin to promote bacterial containment and control of infection.

The Insulin-like Growth Factor Signaling Pathway in Breast Cancer: An Elusive Therapeutic Target.

In this review, we provide an overview of the role of the insulin-like growth factor (IGF) signaling pathway in breast cancer and discuss its potential as a therapeutic target. The IGF pathway ligands, IGF-1 and IGF-2, and their receptors, primarily IGF-1R, are important for normal mammary gland biology, and dysregulation of their expression and function drives breast cancer risk and progression through activation of downstream signaling effectors, often in a subtype-dependent manner. The IGF signaling pathway has also been implicated in resistance to current therapeutic strategies, including ER and HER2 targeting drugs. Unfortunately, efforts to target IGF signaling for the treatment of breast cancer have been unsuccessful, due to a number of factors, most significantly the adverse effects of disrupting IGF signaling on normal glucose metabolism. We highlight here the recent discoveries that provide enthusiasm for continuing efforts to target IGF signaling for the treatment of breast cancer patients.

The insulin and IGF signaling pathway sustains breast cancer stem cells by IRS2/PI3K-mediated regulation of MYC.

Despite the strong association of the insulin/insulin-like growth factor (IGF) signaling (IIS) pathway with tumor initiation, recurrence, and metastasis, the mechanism by which this pathway regulates cancer progression is not well understood. Here, we report that IIS supports breast cancer stem cell (CSC) self-renewal in an IRS2-phosphatidylinositol 3-kinase (PI3K)-dependent manner that involves the activation and stabilization of MYC. IRS2-PI3K signaling enhances MYC expression through the inhibition of GSK3ß activity and suppression of MYC phosphorylation on threonine 58, thus reducing proteasome-mediated degradation of MYC and sustaining active pS62-MYC function. A stable T58A-Myc mutant rescues CSC function in Irs2-/- cells, supporting the role of this MYC stabilization in IRS2-dependent CSC regulation. These findings establish a mechanistic connection between the IIS pathway and MYC and highlight a role for IRS2-dependent signaling in breast cancer progression.

Rapid GPR183-mediated recruitment of eosinophils to the lung after Mycobacterium tuberculosis infection.

Influx of eosinophils into the lungs is typically associated with type II responses during allergy and fungal and parasitic infections. However, we previously reported that eosinophils accumulate in lung lesions during type I inflammatory responses to Mycobacterium tuberculosis (Mtb) in humans, macaques, and mice, in which they support host resistance. Here we show eosinophils migrate into the lungs of macaques and mice as early as one week after Mtb exposure. In mice this influx is CCR3 independent and instead requires cell-intrinsic expression of the oxysterol receptor GPR183, which is highly expressed on human and macaque eosinophils. Murine eosinophils interact directly with bacilli-laden alveolar macrophages, which upregulate the oxysterol-synthesizing enzyme Ch25h, and eosinophil recruitment is impaired in Ch25h-deficient mice. Our findings show that eosinophils are among the earliest cells from circulation to sense and respond to Mtb infection of alveolar macrophages and reveal a role for GPR183 in the migration of eosinophils into lung tissue.

Subcellular patterns of SPE-6 localization reveal unexpected complexities in Caenorhabditis elegans sperm activation and sperm function.

To acquire and maintain directed cell motility, Caenorhabditis elegans sperm must undergo extensive, regulated cellular remodeling, in the absence of new transcription or translation. To regulate sperm function, nematode sperm employ large numbers of protein kinases and phosphatases, including SPE-6, a member of C. elegans' highly expanded casein kinase 1 superfamily. SPE-6 functions during multiple steps of spermatogenesis, including functioning as a "brake" to prevent premature sperm activation in the absence of normal extracellular signals. Here, we describe the subcellular localization patterns of SPE-6 during wild-type C. elegans sperm development and in various sperm activation mutants. While other members of the sperm activation pathway associate with the plasma membrane or localize to the sperm's membranous organelles, SPE-6 surrounds the chromatin mass of unactivated sperm. During sperm activation by either of two semiautonomous signaling pathways, SPE-6 redistributes to the front, central region of the sperm's pseudopod. When disrupted by reduction-of-function alleles, SPE-6 protein is either diminished in a temperature-sensitive manner (hc187) or is mislocalized in a stage-specific manner (hc163). During the multistep process of sperm activation, SPE-6 is released from its perinuclear location after the spike stage in a process that does not require the fusion of membranous organelles with the plasma membrane. After activation, spermatozoa exhibit variable proportions of perinuclear and pseudopod-localized SPE-6, depending on their location within the female reproductive tract. These findings provide new insights regarding SPE-6's role in sperm activation and suggest that extracellular signals during sperm migration may further modulate SPE-6 localization and function.

Eosinophils are part of the granulocyte response in tuberculosis and promote host resistance in mice.

Host resistance to Mycobacterium tuberculosis (Mtb) infection requires the activities of multiple leukocyte subsets, yet the roles of the different innate effector cells during tuberculosis are incompletely understood. Here we uncover an unexpected association between eosinophils and Mtb infection. In humans, eosinophils are decreased in the blood but enriched in resected human tuberculosis lung lesions and autopsy granulomas. An influx of eosinophils is also evident in infected zebrafish, mice, and nonhuman primate granulomas, where they are functionally activated and degranulate. Importantly, using complementary genetic models of eosinophil deficiency, we demonstrate that in mice, eosinophils are required for optimal pulmonary bacterial control and host survival after Mtb infection. Collectively, our findings uncover an unexpected recruitment of eosinophils to the infected lung tissue and a protective role for these cells in the control of Mtb infection in mice.

Lamin A and microtubules collaborate to maintain nuclear morphology.

Lamin A (LA) is a critical structural component of the nuclear lamina. Mutations within the LA gene (LMNA) lead to several human disorders, most striking of which is Hutchinson-Gilford Progeria Syndrome (HGPS), a premature aging disorder. HGPS cells are best characterized by an abnormal nuclear morphology known as nuclear blebbing, which arises due to the accumulation of progerin, a dominant mutant form of LA. The microtubule (MT) network is known to mediate changes in nuclear morphology in the context of specific events such as mitosis, cell polarization, nucleus positioning and cellular migration. What is less understood is the role of the microtubule network in determining nuclear morphology during interphase. In this study, we elucidate the role of the cytoskeleton in regulation and misregulation of nuclear morphology through perturbations of both the lamina and the microtubule network. We found that LA knockout cells exhibit a crescent shape morphology associated with the microtubule-organizing center. Furthermore, this crescent shape ameliorates upon treatment with MT drugs, Nocodazole or Taxol. Expression of progerin, in LA knockout cells also rescues the crescent shape, although the response to Nocodazole or Taxol treatment is altered in comparison to cells expressing LA. Together these results describe a collaborative effort between LA and the MT network to maintain nuclear morphology.