A functional role for intrinsic disorder in the tau-tubulin complex.

Tau is an intrinsically disordered protein with an important role in maintaining the dynamic instability of neuronal microtubules. Despite intensive study, a detailed understanding of the functional mechanism of tau is lacking. Here, we address this deficiency by using intramolecular single-molecule Förster Resonance Energy Transfer (smFRET) to characterize the conformational ensemble of tau bound to soluble tubulin heterodimers. Tau adopts an open conformation on binding tubulin, in which the long-range contacts between both termini and the microtubule binding region that characterize its compact solution structure are diminished. Moreover, the individual repeats within the microtubule binding region that directly interface with tubulin expand to accommodate tubulin binding, despite a lack of extension in the overall dimensions of this region. These results suggest that the disordered nature of tau provides the significant flexibility required to allow for local changes in conformation while preserving global features. The tubulin-associated conformational ensemble is distinct from its aggregation-prone one, highlighting differences between functional and dysfunctional states of tau. Using constraints derived from our measurements, we construct a model of tubulin-bound tau, which draws attention to the importance of the role of tau's conformational plasticity in function.

Association of Patient Characteristics and Tumor Genomics With Clinical Outcomes Among Patients With Non-Small Cell Lung Cancer Using a Clinicogenomic Database.

Importance: Data sets linking comprehensive genomic profiling (CGP) to clinical outcomes may accelerate precision medicine. Objective: To assess whether a database that combines EHR-derived clinical data with CGP can identify and extend associations in non-small cell lung cancer (NSCLC). Design, Setting, and Participants: Clinical data from EHRs were linked with CGP results for 28 998 patients from 275 US oncology practices. Among 4064 patients with NSCLC, exploratory associations between tumor genomics and patient characteristics with clinical outcomes were conducted, with data obtained between January 1, 2011, and January 1, 2018. Exposures: Tumor CGP, including presence of a driver alteration (a pathogenic or likely pathogenic alteration in a gene shown to drive tumor growth); tumor mutation burden (TMB), defined as the number of mutations per megabase; and clinical characteristics gathered from EHRs. Main Outcomes and Measures: Overall survival (OS), time receiving therapy, maximal therapy response (as documented by the treating physician in the EHR), and clinical benefit rate (fraction of patients with stable disease, partial response, or complete response) to therapy. Results: Among 4064 patients with NSCLC (median age, 66.0 years; 51.9% female), 3183 (78.3%) had a history of smoking, 3153 (77.6%) had nonsquamous cancer, and 871 (21.4%) had an alteration in EGFR, ALK, or ROS1 (701 [17.2%] with EGFR, 128 [3.1%] with ALK, and 42 [1.0%] with ROS1 alterations). There were 1946 deaths in 7 years. For patients with a driver alteration, improved OS was observed among those treated with (n = 575) vs not treated with (n = 560) targeted therapies (median, 18.6 months [95% CI, 15.2-21.7] vs 11.4 months [95% CI, 9.7-12.5] from advanced diagnosis; P < .001). TMB (in mutations/Mb) was significantly higher among smokers vs nonsmokers (8.7 [IQR, 4.4-14.8] vs 2.6 [IQR, 1.7-5.2]; P < .001) and significantly lower among patients with vs without an alteration in EGFR (3.5 [IQR, 1.76-6.1] vs 7.8 [IQR, 3.5-13.9]; P < .001), ALK (2.1 [IQR, 0.9-4.0] vs 7.0 [IQR, 3.5-13.0]; P < .001), RET (4.6 [IQR, 1.7-8.7] vs 7.0 [IQR, 2.6-13.0]; P = .004), or ROS1 (4.0 [IQR, 1.2-9.6] vs 7.0 [IQR, 2.6-13.0]; P = .03). In patients treated with anti-PD-1/PD-L1 therapies (n = 1290, 31.7%), TMB of 20 or more was significantly associated with improved OS from therapy initiation (16.8 months [95% CI, 11.6-24.9] vs 8.5 months [95% CI, 7.6-9.7]; P < .001), longer time receiving therapy (7.8 months [95% CI, 5.5-11.1] vs 3.3 months [95% CI, 2.8-3.7]; P < .001), and increased clinical benefit rate (80.7% vs 56.7%; P < .001) vs TMB less than 20. Conclusions and Relevance: Among patients with NSCLC included in a longitudinal database of clinical data linked to CGP results from routine care, exploratory analyses replicated previously described associations between clinical and genomic characteristics, between driver mutations and response to targeted therapy, and between TMB and response to immunotherapy. These findings demonstrate the feasibility of creating a clinicogenomic database derived from routine clinical experience and provide support for further research and discovery evaluating this approach in oncology.

Quantification of protein delivery in live cells using fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a quantitative single-molecule method that measures the concentration and rate of diffusion of fluorophore-tagged molecules, both large and small, in vitro and within live cells, and even within discrete cellular compartments. FCS is exceptionally well-suited to directly quantify the efficiency of intracellular protein delivery-specifically, how well different "cell-penetrating" proteins and peptides guide proteinaceous materials into the cytosol and nuclei of live mammalian cells. This article provides an overview of the procedures necessary to execute robust FCS experiments and evaluate endosomal escape efficiencies: preparation of fluorophore-tagged proteins, incubation with mammalian cells and preparation of FCS samples, setup and execution of an FCS experiment, and a detailed discussion of and custom MATLAB® script for analyzing the resulting autocorrelation curves in the context of appropriate diffusion models.