Muscle contraction relies on the interaction of myosin motors with F-actin, which is regulated through a translocation of tropomyosin by the troponin complex in response to Ca 2+ . The current model of muscle regulation holds that at relaxing (low-Ca 2+ ) conditions tropomyosin blocks myosin binding sites on F-actin, whereas at activating (high-Ca 2+ ) conditions tropomyosin translocation only partially exposes myosin binding sites on F-actin so that binding of rigor myosin is required to fully activate the thin filament (TF). Here we used a single-particle approach to helical reconstruction of frozen hydrated native cardiac TFs under relaxing and activating conditions to reveal the azimuthal movement of the tropomyosin on the surface of the native cardiac TF upon Ca 2+ activation. We demonstrate that at either relaxing or activating conditions tropomyosin is not constrained in one structural state, but rather is distributed between three structural positions on the surface of the TF. We show that two of these tropomyosin positions restrain actomyosin interactions, whereas in the third position, which is significantly enhanced at high Ca 2+ , tropomyosin does not block myosin binding sites on F-actin. Our data provide a structural framework for the enhanced activation of the cardiac TF over the skeletal TF by Ca 2+ and lead to a mechanistic model for the regulation of the cardiac TF.thin filament | cardiac muscle regulation | cryoelectron microscopy

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shown that tension applied to the filament accelerates its elongation and that, in the absence of actin monomers, formins were able to track depolymerizing filaments in spite of an opposing tension of a few picoNewtons (Jegou et al., Nat. Commun. 2013). Here, with similar single filament experiments using microfluidics, we investigate the impact of tension and protein concentration on another key feature of formins, processivity. We show that tension drastically enhances the detachment of the formin from the filament barbed end, both in the elongation and the depolymerization regime. Moreover, we find that tension and Capping Protein act in synergy to rapidly detach the filament from the formin. We also characterize the impact of actin and profilin concentrations on formin processivity.

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Hyperactivated K-RAS oncoproteins are known to drive cell proliferation, tumor growth, and metastasis. Oncogene addiction is often associated with increased ER and mitochondrial stress due to excessive reactive oxygen and nitrogen species (ROS & NROS) production. RING finger E3 ubiquitin ligase, Seven in Absentia homolog 2 (Siah2) is a highly conserved, critical signaling module downstream of the EGFR/HER2/K-RAS pathway. SIAH2 function is required for proper K-RAS signal transduction and has been shown to target key regulators of oxidative stress for ubiquitination and proteasome degradation. However, the mechanisms by which SIAH2 regulates cellular stress and protects against programmed cell death in oncogenic K-RAS-addicted cancer cells are not well understood. Since SIAH2 is upregulated in proliferating tumor cells, we hypothesized that SIAH2-deficient cancer cells may be compromised in their ability to adapt to increased cellular and oxidative stress induced by oncogenic K-RAS pathway blockade. In this study, SIAH2 knockdown was carried out in cancer cells carrying oncogenic K-RAS mutations (A549, MDA-MB-231 and MiaPaCa cells). Triplicates of fresh cell lysates from SIAH2 proficient and SIAH2 deficient cells were then interrogated for altered expression of 19 signaling molecules with known functions in modulating stress response and apoptosis using the PathScan® Stress and Apoptosis Signaling Antibody Array kit. Differences in the protein expression were quantified and validated by Western blots, immunofluorescence (IF) and flow cytometry. We found that oxidative stress was markedly increased, evidenced by mitochondrial aggregations and dysfunction in a subset K-RAS-driven cancer cells, as we blocked SIAH2 function downstream of the oncogenic K-RAS signaling pathway. SIAH2 deficiency in breast cancer (MDA-MB-231) and non-small cell lung adenocarcinoma (A549) cells resulted in decreased ERK1/2 and AKT (S473) phosphorylation, increased ROS production, compromised mitochondria integrity and function, increased Caspase 3 and 7 activity, and PARP 1 cleavage that led to massive cell death observed in these cancer cells. In contrast, the prominent cell death was not observed in SIAH2 deficient pancreatic cancer (MiaPaCa) cells that expressed high levels of Survivin, which is an anti-apoptosis protein. The anti-SIAH2 therapy is highly effective in inducing oxidative stress, altered mitochondrial bioenergetics, and apoptosis in a subset of invasive and metastatic cancer lines expressing insufficient levels of Survivin. Our results suggest that, anti-SIAH2 strategy reveals novel oncogenic K-RAS-dependent cancer cell vulnerability in stress response that can be exploited in future for anti-SIAH2 therapy alone, or in combination with other anti-proliferative treatment to effectively combat KRAS-dependent cancers. Citation Format: Monicah M. Njogu, Caroline Lee, Jamie Eisner, Minglei Bian, Robert Vansciver, Amy H. Tang. Increased cellular stress and apoptosis induced by anti-SIAH2 therapy in human cancer cells with oncogenic K-RAS addiction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3498.