SummaryLocalized Gurken (Grk) translation specifies the anterior-posterior and dorsal-ventral axes of the developing Drosophila oocyte; spindle-class females lay ventralized eggs resulting from inefficient grk translation. This phenotype is thought to result from inhibition of the Vasa RNA helicase. In a screen for modifiers of the eggshell phenotype in spn-B flies, we identified a mutation in the lnk gene. We show that lnk mutations restore Grk expression but do not suppress the persistence of double-strand breaks nor other spn-B phenotypes. This suppression does not affect Egfr directly, but rather overcomes the translational block of grk messages seen in spindle mutants. Lnk was recently identified as a component of the insulin/insulin-like growth factor signaling (IIS) and TOR pathway. Interestingly, direct inhibition of TOR with rapamycin in spn-B or vas mutant mothers can also suppress the ventralized eggshell phenotype. When dietary protein is inadequate, reduced IIS-TOR activity inhibits cap-dependent translation by promoting the activity of the translation inhibitor eIF4E-binding protein (4EBP). We hypothesize that reduced TOR activity promotes grk translation independent of the canonical Vasa-and cap-dependent mechanism. This model might explain how flies can maintain the translation of developmentally important transcripts during periods of nutrient limitation when bulk cap-dependent translation is repressed.

Mechanical forces are integral to a wide range of cellular processes including migration, differentiation and tissue morphogenesis; however, it has proved challenging to directly measure strain at high spatial resolution and with minimal tissue perturbation. Here, we fabricated, calibrated, and tested a fibronectin (FN)-based nanomechanical biosensor (NMBS) that can be applied to cells and tissues to measure the magnitude, direction, and dynamics of strain from subcellular to tissue length-scales. The NMBS is a fluorescently-labeled, ultrathin square lattice FN mesh with spatial resolution tailored by adjusting the width and spacing of the lattice fibers from 2-100 µm. Time-lapse 3D confocal imaging of the NMBS demonstrated strain tracking in 2D and 3D following mechanical deformation of known materials and was validated with finite element modeling. Imaging and 3D analysis of the NMBS applied to single cells, cell monolayers, and Drosophila ovarioles demonstrated the ability to dynamically track microscopic tensile and compressive strains in various biological applications with minimal tissue perturbation. This fabrication and analysis platform serves as a novel tool for studying cells, tissues, and more complex systems where forces guide structure and function.

Mechanical forces are integral to cellular migration, differentiation and tissue morphogenesis; however, it has proved challenging to directly measure strain at high spatial resolution with minimal perturbation in living sytems. Here, we fabricate, calibrate, and test a fibronectin (FN)-based nanomechanical biosensor (NMBS) that can be applied to the surface of cells and tissues to measure the magnitude, direction, and strain dynamics from subcellular to tissue length-scales. The NMBS is a fluorescently-labeled, ultra-thin FN lattice-mesh with spatial resolution tailored by adjusting the width and spacing of the lattice from 2–100 µm. Time-lapse 3D confocal imaging of the NMBS demonstrates 2D and 3D surface strain tracking during mechanical deformation of known materials and is validated with finite element modeling. Analysis of the NMBS applied to single cells, cell monolayers, and Drosophila ovarioles highlights the NMBS’s ability to dynamically track microscopic tensile and compressive strains across diverse biological systems where forces guide structure and function.

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Wnt signaling generates patterns in all embryos, from flies to humans, and controls cell fate, proliferation and metabolic homeostasis. Inappropriate Wnt pathway activation results in diseases, including colorectal cancer. The adenomatous polyposis coli (APC) tumor suppressor gene encodes a multifunctional protein that is an essential regulator of Wnt signaling and cytoskeletal organization. Although progress has been made in defining the role of APC in a normal cellular context, there are still significant gaps in our understanding of APC-dependent cellular function and dysfunction. We expanded the APC-associated protein network using a combination of genetics and a proteomic technique called twodimensional difference gel electrophoresis (2D-DIGE). We show that loss of Drosophila Apc2 causes protein isoform changes reflecting misregulation of post-translational modifications (PTMs), which are not dependent on β-catenin transcriptional activity. Mass spectrometry revealed that proteins involved in metabolic and biosynthetic pathways, protein synthesis and degradation, and cell signaling are affected by Apc2 loss. We demonstrate that changes in phosphorylation partially account for the altered PTMs in APC mutants, suggesting that APC mutants affect other types of PTM. Finally, through this approach Aminopeptidase P was identified as a new regulator of β-catenin abundance in Drosophila embryos. This study provides new perspectives on the cellular effects of APC that might lead to a deeper understanding of its role in development.

Cancer recurrence and metastasis involves many biological interactions, such as genetic, transcription, environmental, endocrine signaling, and metabolism. These interactions add a complex understanding of cancer recurrence and metastatic progression, delaying the advancement in therapeutic opportunities. We highlight the recent advances on the molecular complexities of endocrine-related cancers, focusing on breast and prostate cancer, and briefly review how endocrine signaling and metabolic programs can influence transcriptional complexes for metastasis competence. Nuclear receptors and transcriptional coregulators function as molecular nodes for the crosstalk between endocrine signaling and metabolism that alter downstream gene expression important for tumor progression and metastasis. This exciting regulatory axis may provide insights to the development of cancer therapeutics important for these desensitized endocrine-dependent cancers.

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This protocol details the step-by-step fabrication process to create an extracellular matrix (ECM) protein-based nanomechanical biosensor (NMBS) via an adaptation of the surface-initiated assembly (SIA) procedure1. The NMBS is a fluorescently labeled, ultra-thin fibronectin lattice-mesh with spatial resolution tailored by adjusting the width and spacing of the lattice from 2-100 µm. By applying the NMBS to the surface of cells and tissues one can directly measure deformation from subcellular to tissue length-scales. The procedure outlined here covers all aspects of NMBS construction from pattern design, photolithography for mold creation, casting of polydimethylsiloxane (PDMS) silicone stamps, conjugation of fluorescent dye to fibronectin, microcontact printing of fibronectin, creation of gelatin carriers2, transfer of the NMBS to gelatin carriers, and the use of gelatin carriers for application of the NMBS onto cells and tissue. The protocol can be broken down into three phases: material preparation (2 days), NMBS patterning (4 hours), and transfer to cell or tissue (1 hour). Material preparation and NMBS patterning can be performed ahead of time and the patterned NMBS can be stored for up to 1 year prior to use.