The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2.

Proximity-dependent biotin labeling (BioID) may identify new targets for cancers driven by difficult-to-drug oncogenes such as Ras. Therefore, BioID was used with wild-type (WT) and oncogenic mutant (MT) H-, K-, and N-Ras, identifying known interactors, including Raf and PI3K, as well as a common set of 130 novel proteins proximal to all Ras isoforms. A CRISPR screen of these proteins for Ras dependence identified mTOR, which was also found proximal to MT Ras in human tumors. Oncogenic Ras directly bound two mTOR complex 2 (mTORC2) components, mTOR and MAPKAP1, to promote mTORC2 kinase activity at the plasma membrane. mTORC2 enabled the Ras pro-proliferative cell cycle transcriptional program, and perturbing the Ras-mTORC2 interaction impaired Ras-dependent neoplasia in vivo. Combining proximity-dependent proteomics with CRISPR screening identified a new set of functional Ras-associated proteins, defined mTORC2 as a new direct Ras effector, and offers a strategy for finding new proteins that cooperate with dominant oncogenes.

Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus B3-induced myocarditis and antiviral drug screening platform.

RATIONALE: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. OBJECTIVE: This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. METHODS AND RESULTS: hiPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferonß1, ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after interferonß1 treatment. CONCLUSIONS: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.

Cadherin-based intercellular adhesions organize epithelial cell-matrix traction forces.

Cell-cell and cell-matrix adhesions play essential roles in the function of tissues. There is growing evidence for the importance of cross talk between these two adhesion types, yet little is known about the impact of these interactions on the mechanical coupling of cells to the extracellular matrix (ECM). Here, we combine experiment and theory to reveal how intercellular adhesions modulate forces transmitted to the ECM. In the absence of cadherin-based adhesions, primary mouse keratinocytes within a colony appear to act independently, with significant traction forces extending throughout the colony. In contrast, with strong cadherin-based adhesions, keratinocytes in a cohesive colony localize traction forces to the colony periphery. Through genetic or antibody-mediated loss of cadherin expression or function, we show that cadherin-based adhesions are essential for this mechanical cooperativity. A minimal physical model in which cell-cell adhesions modulate the physical cohesion between contractile cells is sufficient to recreate the spatial rearrangement of traction forces observed experimentally with varying strength of cadherin-based adhesions. This work defines the importance of cadherin-based cell-cell adhesions in coordinating mechanical activity of epithelial cells and has implications for the mechanical regulation of epithelial tissues during development, homeostasis, and disease.

Patterning droplets with durotaxis.

Numerous cell types have shown a remarkable ability to detect and move along gradients in stiffness of an underlying substrate--a process known as durotaxis. The mechanisms underlying durotaxis are still unresolved, but generally believed to involve active sensing and locomotion. Here, we show that simple liquid droplets also undergo durotaxis. By modulating substrate stiffness, we obtain fine control of droplet position on soft, flat substrates. Unlike other control mechanisms, droplet durotaxis works without imposing chemical, thermal, electrical, or topographical gradients. We show that droplet durotaxis can be used to create large-scale droplet patterns and is potentially useful for many applications, such as microfluidics, thermal control, and microfabrication.

Universal deformation of soft substrates near a contact line and the direct measurement of solid surface stresses.

Droplets deform soft substrates near their contact lines. Using confocal microscopy, we measure the deformation of silicone gel substrates due to glycerol and fluorinated-oil droplets for a range of droplet radii and substrate thicknesses. For all droplets, the substrate deformation takes a universal shape close to the contact line that depends on liquid composition, but is independent of droplet size and substrate thickness. This shape is determined by a balance of interfacial tensions at the contact line and provides a novel method for direct determination of the surface stresses of soft substrates. Moreover, we measure the change in contact angle with droplet radius and show that Young's law fails for small droplets when their radii approach an elastocapillary length scale. For larger droplets the macroscopic contact angle is constant, consistent with Young's law.

Scaling of traction forces with the size of cohesive cell colonies.

To understand how the mechanical properties of tissues emerge from interactions of multiple cells, we measure traction stresses of cohesive colonies of 1-27 cells adherent to soft substrates. We find that traction stresses are generally localized at the periphery of the colony and the total traction force scales with the colony radius. For large colony sizes, the scaling appears to approach linear, suggesting the emergence of an apparent surface tension of the order of 10(-3) N/m. A simple model of the cell colony as a contractile elastic medium coupled to the substrate captures the spatial distribution of traction forces and the scaling of traction forces with the colony size.

Mutant collagen COL11A1 enhances cancerous invasion.

Collagens are the most abundant proteins in the body and comprise the basement membranes and stroma through which cancerous invasion occurs; however, a pro-neoplastic function for mutant collagens is undefined. Here we identify COL11A1 mutations in 66 of 100 cutaneous squamous cell carcinomas (cSCCs), the second most common U.S. cancer, concentrated in a triple helical region known to produce trans-dominant collagens. Analysis of COL11A1 and other collagen genes found that they are mutated across common epithelial malignancies. Knockout of mutant COL11A1 impairs cSCC tumorigenesis in vivo. Compared to otherwise genetically identical COL11A1 wild-type tissue, gene-edited mutant COL11A1 skin is characterized by induction of ß1 integrin targets and accelerated neoplastic invasion. In mosaic tissue, mutant COL11A1 cells enhanced invasion by neighboring wild-type cells. These results suggest that specific collagens are commonly mutated in cancer and that mutant collagens may accelerate this process.

KRAS regulation by small non-coding RNAs and SNARE proteins.

KRAS receives and relays signals at the plasma membrane (PM) where it transmits extracellular growth factor signals to downstream effectors. SNORD50A/B were recently found to bind KRAS and inhibit its tumorigenic action by unknown mechanisms. KRAS proximity protein labeling was therefore undertaken in SNORD50A/B wild-type and knockout cells, revealing that SNORD50A/B RNAs shape the composition of proteins proximal to KRAS, notably by inhibiting KRAS proximity to the SNARE vesicular transport proteins SNAP23, SNAP29, and VAMP3. To remain enriched on the PM, KRAS undergoes cycles of endocytosis, solubilization, and vesicular transport to the PM. Here we report that SNAREs are essential for the final step of this process, with KRAS localization to the PM facilitated by SNAREs but antagonized by SNORD50A/B. Antagonism between SNORD50A/B RNAs and specific SNARE proteins thus controls KRAS localization, signaling, and tumorigenesis, and disrupting SNARE-enabled KRAS function represents a potential therapeutic opportunity in KRAS-driven cancer.

Research Techniques Made Simple: Emerging Methods to Elucidate Protein Interactions through Spatial Proximity.

Interactions between proteins are essential for fundamental cellular processes, and the diversity of such interactions enables the vast variety of functions essential for life. A persistent goal in biological research is to develop assays that can faithfully capture different types of protein interactions to allow their study. A major step forward in this direction came with a family of methods that delineates spatial proximity of proteins as an indirect measure of protein-protein interaction. A variety of enzyme- and DNA ligation-based methods measure protein co-localization in space, capturing novel interactions that were previously too transient or low affinity to be identified. Here we review some of the methods that have been successfully used to measure spatially proximal protein-protein interactions.

The noncoding RNAs SNORD50A and SNORD50B bind K-Ras and are recurrently deleted in human cancer.

Small nucleolar RNAs (snoRNAs) are conserved noncoding RNAs best studied as ribonucleoprotein (RNP) guides in RNA modification. To explore their role in cancer, we compared 5,473 tumor-normal genome pairs to identify snoRNAs with frequent copy number loss. The SNORD50A-SNORD50B snoRNA locus was deleted in 10-40% of 12 common cancers, where its loss was associated with reduced survival. A human protein microarray screen identified direct SNORD50A and SNORD50B RNA binding to K-Ras. Loss of SNORD50A and SNORD50B increased the amount of GTP-bound, active K-Ras and hyperactivated Ras-ERK1/ERK2 signaling. Loss of these snoRNAs also increased binding by farnesyltransferase to K-Ras and increased K-Ras prenylation, suggesting that KRAS mutation might synergize with SNORD50A and SNORD50B loss in cancer. In agreement with this hypothesis, CRISPR-mediated deletion of SNORD50A and SNORD50B in KRAS-mutant tumor cells enhanced tumorigenesis, and SNORD50A and SNORD50B deletion and oncogenic KRAS mutation co-occurred significantly in multiple human tumor types. SNORD50A and SNORD50B snoRNAs thus directly bind and inhibit K-Ras and are recurrently deleted in human cancer.