Glioblastoma ranks among the most aggressive and lethal of all human cancers. Functionally defi ned glioma stem cells (GSC) contribute to this poor prognosis by driving therapeutic resistance and maintaining cellular heterogeneity. To understand the molecular processes essential for GSC maintenance and tumorigenicity, we interrogated the superenhancer landscapes of primary glioblastoma specimens and in vitro GSCs. GSCs epigenetically upregulated ELOVL2, a key polyunsaturated fatty-acid synthesis enzyme. Targeting ELOVL2 inhibited glioblastoma cell growth and tumor initiation. ELOVL2 depletion altered cellular membrane phospholipid composition, disrupted membrane structural properties, and diminished EGFR signaling through control of fatty-acid elongation. In support of the translational potential of these fi ndings, dual targeting of polyunsaturated fatty-acid synthesis and EGFR signaling had a combinatorial cytotoxic effect on GSCs. SIGNIFICANCE : Glioblastoma remains a devastating disease despite extensive characterization. We profi led epigenomic landscapes of glioblastoma to pinpoint cell state-specifi c dependencies and therapeutic vulnerabilities. GSCs utilize polyunsaturated fatty-acid synthesis to support membrane architecture, inhibition of which impairs EGFR signaling and GSC proliferation. Combinatorial targeting of these networks represents a promising therapeutic strategy.

During prophase I of meiosis, chromosomes become organized as loop arrays around the proteinaceous chromosome axis. As homologous chromosomes physically pair and recombine, the chromosome axis is integrated into the tripartite synaptonemal complex (SC) as this structure’s lateral elements (LEs). While the components of the mammalian chromosome axis/LE—including meiosis-specific cohesin complexes, the axial element proteins SYCP3 and SYCP2, and the HORMA domain proteins HORMAD1 and HORMAD2—are known, the molecular organization of these components within the axis is poorly understood. Here, using expansion microscopy coupled with 2-color stochastic optical reconstruction microscopy (STORM) imaging (ExSTORM), we address these issues in mouse spermatocytes at a resolution of 10 to 20 nm. Our data show that SYCP3 and the SYCP2 C terminus, which are known to form filaments in vitro, form a compact core around which cohesin complexes, HORMADs, and the N terminus of SYCP2 are arrayed. Overall, our study provides a detailed structural view of the meiotic chromosome axis, a key organizational and regulatory component of meiotic chromosomes.

Genetically encoded Förster Resonance Energy Transfer (FRET)-based biosensors are powerful tools to illuminate spatiotemporal regulation of cell signaling in living cells, but the utility of the red spectrum for biosensing was limited due to a lack of bright and stable red fluorescent proteins. Here, we rationally improve the photophysical characteristics of the coralderived fluorescent protein TagRFP-T. We show that a new single-residue mutant, super-TagRFP (stagRFP) has nearly twice the molecular brightness of TagRFP-T and negligible photoactivation. stagRFP facilitates significant improvements on multiple green-red biosensors as a FRET acceptor and is an efficient FRET donor that supports red/far-red FRET biosensing. Capitalizing on the ability of stagRFP to couple with multiple FRET partners, we develop a novel multiplex method to examine the confluence of signaling activities from three kinases simultaneously in single living cells, providing evidence for a role of Src family kinases in regulating growth factor induced Akt and ERK activities.

The complex refractive index dispersion (CRID), which contains the information on the refractive index dispersion and extinction coefficient spectra, is an important optical parameter of biotissue. However, it is hard to perform the CRID measurement on biotissues due to their high scattering property. Continuous CRID measurement based on internal reflection (CCRIDM-IR) is introduced. By using a lab-made apparatus, internal reflectance spectra of biotissue samples at multiple incident angles were detected, from which the continuous CRIDs were calculated based on the Fresnel formula. Results showed that in 400-to 750-nm range, hemoglobin solution has complicated dispersion and extinction coefficient spectra, while other biotissues have normal dispersion properties, and their extinction coefficients do not vary much with different wavelengths. The normal dispersion can be accurately described by several coefficients of dispersion equations (Cauchy equation, Cornu equation, and Conrady equation). To our knowledge, this is the first time that the continuous CRID of scattering biotissue over a continuous spectral region is measured, and we hereby have proven that CCRIDM-IR is a good method for continuous CRID research of biotissue.

The serine/threonine protein kinase Akt regulates a wide range of cellular functions via phosphorylation of various substrates distributed throughout the cell, including at the plasma membrane and endomembrane compartments. Disruption of compartmentalized Akt signaling underlies the pathology of many diseases such as cancer and diabetes. However, the specific spatial organization of Akt activity and the underlying regulatory mechanisms, particularly the mechanism controlling its activity at the lysosome, are not clearly understood. We developed a highly sensitive excitation-ratiometric Akt activity reporter (ExRai-AktAR2), enabling the capture of minute changes in Akt activity dynamics at subcellular compartments. In conjunction with super-resolution expansion microscopy, we found that growth factor stimulation leads to increased colocalization of Akt with lysosomes and accumulation of lysosomal Akt activity. We further showed that 3-phosphoinositides (3-PIs) accumulate on the lysosomal surface, in a manner dependent on dynamin-mediated endocytosis. Importantly, lysosomal 3-PIs are needed for growth-factor-induced activities of Akt and mechanistic target of rapamycin complex 1 (mTORC1) on the lysosomal surface, as targeted depletion of 3-PIs has detrimental effects. Thus, 3-PIs, a class of critical lipid second messengers that are typically found in the plasma membrane, unexpectedly accumulate on the lysosomal membrane in response to growth factor stimulation, to direct the multifaceted kinase Akt to organize lysosome-specific signaling.

We present a novel scanning focused refractive-index microscopy (SFRIM) technique to obtain the refractive index (RI) profiles of objects. The method uses a focused laser as the light source, and combines the derivative total reflection method (DTRM), projection magnification, and scanning technique together. SFRIM is able to determine RIs with an accuracy of 0.002, and the central spatial resolution achieved is 1 µm, which is smaller than the size of the focal spot. The results of measurements carried out on cedar oil and a gradient-refractive-index (GRIN) lens agree well with theoretical expectations, verifying the accuracy of SFRIM. Furthermore, using SFRIM, to the best of our knowledge we have extracted for the first time the RI profile of a periodically modulated photosensitive gelatin sample. SFRIM is the first RI profile-resolved reflected light microscopy technique that can be applied to scattering and absorbing samples. SFRIM enables the possibility of performing RI profile measurements in a variety of applications, including optical waveguides, photosensitive materials and devices, photorefractive effect studies, and RI imaging in biomedical fields.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates growth, nutrient and energy status cues to control cell growth and metabolism. While mTORC1 activation at the lysosome is well characterized, it is not clear how this complex is regulated at other subcellular locations. Here, we combine location-selective kinase inhibition, live-cell imaging and biochemical assays to probe the regulation of growth factor-induced mTORC1 activity in the nucleus. Using a nuclear targeted Akt Substrate-based Tandem Occupancy Peptide Sponge (Akt-STOPS) that we developed for specific inhibition of Akt, a critical upstream kinase, we show that growth factor-stimulated nuclear mTORC1 activity requires nuclear Akt activity. Further mechanistic dissection suggests that nuclear Akt activity mediates growth factor-induced nuclear translocation of Raptor, a regulatory scaffolding component in mTORC1, and localization of Raptor to the nucleus results in nuclear mTORC1 activity in the absence of growth factor stimulation. Taken together, these results reveal a mode of regulation of mTORC1 that is distinct from its lysosomal activation, which controls mTORC1 activity in the nuclear compartment.

Traditionally, continuous refractive index dispersion (CRID) measurement of materials with scattering is hard to realize. In this paper, CRID measurement based on the derivative total reflection method (CRIDM-DTRM) is proposed to measure the CRID of both absorption and scattering materials. It effectively determined the CRID of K9 glass, concentrated milk, and 0.5% methyl red solution in the 400-750 nm range with the spectral resolution of about 0.259 nm. For the first time, CRID of a scattering material is measured. CRIDM-DTRM is a useful technique in the field of RID measurement, especially for biotissues and anomalous dispersion materials.