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Publication Open Access A bacteria-derived tail anchor localizes to peroxisomes in yeast and mammalian cells(Nature Publishing Group (NPG), 2018) Seferoğlu, Ayşe Bengisu; Department of Molecular Biology and Genetics; Dunn, Cory David; Keskin, Abdurrahman; Akdoğan, Emel; Lutfullahoglu-Bal, Guleycan; Department of Molecular Biology and Genetics; College of SciencesProkaryotes can provide new genetic information to eukaryotes by horizontal gene transfer (HGT), and such transfers are likely to have been particularly consequential in the era of eukaryogenesis. Since eukaryotes are highly compartmentalized, it is worthwhile to consider the mechanisms by which newly transferred proteins might reach diverse organellar destinations. Toward this goal, we have focused our attention upon the behavior of bacteria-derived tail anchors (TAs) expressed in the eukaryote Saccharomyces cerevisiae. In this study, we report that a predicted membrane-associated domain of the Escherichia coli YgiM protein is specifically trafficked to peroxisomes in budding yeast, can be found at a pre-peroxisomal compartment (PPC) upon disruption of peroxisomal biogenesis, and can functionally replace an endogenous, peroxisome-directed TA. Furthermore, the YgiM(TA) can localize to peroxisomes in mammalian cells. Since the YgiM(TA) plays no endogenous role in peroxisomal function or assembly, this domain is likely to serve as an excellent tool allowing further illumination of the mechanisms by which TAs can travel to peroxisomes. Moreover, our findings emphasize the ease with which bacteria-derived sequences might target to organelles in eukaryotic cells following HGT, and we discuss the importance of flexible recognition of organelle targeting information during and after eukaryogenesis.Publication Open Access Afrotropical montane birds experience upslope shifts and range contractions along a fragmented elevational gradient in response to global warming(Public Library of Science, 2021) Neate-Clegg, Montague H. C.; Stuart, Simon N.; Mtui, Devolent; Newmark, William D.; Department of Molecular Biology and Genetics; Şekercioğlu, Çağan Hakkı; Faculty Member; Department of Molecular Biology and Genetics; College of Sciences; 327589Global warming is predicted to result in upslope shifts in the elevational ranges of bird species in montane habitats. Yet few studies have examined changes over time in the elevational distribution of species along fragmented gradients in response to global warming. Here, we report on a resurvey of an understory bird community in the Usambara Mountains in Tanzania, along a forested elevational gradient that has been fragmented over the last 200 years. In 2019, we resurveyed seven sites, ranging in elevation from 360 m to 2110 m, that were originally surveyed between 1979 and 1981. We calculated differences in mean elevation and lower and upper range limits for 29 species between the two time periods and corrected for possible differences in elevation due to chance. Over four decades, we documented a significant mean upslope shift across species of 93 m. This shift was smaller than the 125 m expected shift due to local climate warming. Of the 29 focal species, 19 shifted upslope, eight downslope, and two remained unchanged. Mean upslope shifts in species were driven largely by contracting lower range limits which moved significantly upslope on average across species by 183 m, while upper range limits shifted non-significantly upslope by 72 m, leading to a mean range contraction of 114 m across species. Community composition of understory bird species also shifted over time, with current communities resembling communities found historically at lower elevations. Past forest fragmentation in combination with the limited gap-crossing ability of many tropical understory bird species are very likely important contributory factors to the observed asymmetrical shifts in lower and upper elevational range limits. Re-establishing forested linkages among the largest and closest forest fragments in the Eastern Arc Mountains are critical to permitting species to shift upslope and to reduce further elevational range contractions over time.Publication Open Access An LED-Based structured illumination microscope using a digital micromirror device and GPU accelerated image reconstruction(Public Library of Science, 2022) Aydın, Musa; Doğan, Buket; Department of Physics; Department of Electrical and Electronics Engineering; Department of Molecular Biology and Genetics; Kiraz, Alper; Karalar, Elif Nur Fırat; Morova, Berna; Uysallı, Yiğit; Özgönül, Ekin; Faculty Member; Researcher; PhD Student; PhD Student; Department of Physics; Department of Electrical and Electronics Engineering; Department of Molecular Biology and Genetics; Koç University Research Center for Translational Medicine (KUTTAM) / Koç Üniversitesi Translasyonel Tıp Araştırma Merkezi (KUTTAM); Graduate School of Sciences and Engineering; College of Engineering; School of Medicine; 22542; 206349; N/A; N/A; N/AWhen combined with computational approaches, fluorescence imaging becomes one of the most powerful tools in biomedical research. It is possible to achieve resolution figures beyond the diffraction limit, and improve the performance and flexibility of high-resolution imaging systems with techniques such as structured illumination microscopy (SIM) reconstruction. In this study, the hardware and software implementation of an LED-based superresolution imaging system using SIM employing GPU accelerated parallel image reconstruction is presented. The sample is illuminated with two-dimensional sinusoidal patterns with various orientations and lateral phase shifts generated using a digital micromirror device (DMD). SIM reconstruction is carried out in frequency space using parallel CUDA kernel functions. Furthermore, a general purpose toolbox for the parallel image reconstruction algorithm and an infrastructure that allows all users to perform parallel operations on images without developing any CUDA kernel code is presented. The developed image reconstruction algorithm was run separately on a CPU and a GPU. Two different SIM reconstruction algorithms have been developed for the CPU as mono-thread CPU algorithm and multi-thread OpenMP CPU algorithm. SIM reconstruction of 1024 × 1024 px images was achieved in 1.49 s using GPU computation, indicating an enhancement by*28 and*20 in computation time when compared with mono-thread CPU computation and multi-thread OpenMP CPU computation, respectively.Publication Open Access Biocompatible quantum funnels for neural photostimulation(American Chemical Society (ACS), 2019) N/A; Department of Chemical and Biological Engineering; N/A; Department of Electrical and Electronics Engineering; Department of Molecular Biology and Genetics; N/A; Jalali, Houman Bahmani; Doğru-Yüksel, Itır Bakış; Eren, Güncem Özgün; Nizamoğlu, Sedat; Karatüm, Onuralp; Melikov, Rustamzhon; Dikbaş, Uğur Meriç; Kavaklı, İbrahim Halil; Sadeghi, Sadra; Yıldız, Erdost; Ergün, Çağla; Şahin, Afsun; PhD Student; Faculty Member; PhD Student; Master Student; Faculty Member; PhD Student; PhD Student; Faculty Member; Department of Chemical and Biological Engineering; Department of Electrical and Electronics Engineering; Department of Molecular Biology and Genetics; Koç University Research Center for Translational Medicine (KUTTAM) / Koç Üniversitesi Translasyonel Tıp Araştırma Merkezi (KUTTAM); Graduate School of Sciences and Engineering; College of Engineering; College of Sciences; School of Medicine; N/A; N/A; N/A; 130295; N/A; N/A; N/A; 40319; N/A; N/A; N/A; 171267Neural photostimulation has high potential to understand the working principles of complex neural networks and develop novel therapeutic methods for neurological disorders. A key issue in the light-induced cell stimulation is the efficient conversion of light to bioelectrical stimuli. In photosynthetic systems developed in millions of years by nature, the absorbed energy by the photoabsorbers is transported via nonradiative energy transfer to the reaction centers. Inspired by these systems, neural interfaces based on biocompatible quantum funnels are developed that direct the photogenerated charge carriers toward the bionanojunction for effective photostimulation. Funnels are constructed with indium-based rainbow quantum dots that are assembled in a graded energy profile. Implementation of a quantum funnel enhances the generated photoelectrochemical current 215% per unit absorbance in comparison with ungraded energy profile in a wireless and free-standing mode and facilitates optical neuromodulation of a single cell. This study indicates that the control of charge transport at nanoscale can lead to unconventional and effective neural interfaces.Publication Open Access Comparative phosphoproteomic analysis reveals signaling networks regulating monopolar and bipolar cytokinesis(Nature Publishing Group (NPG), 2018) Department of Molecular Biology and Genetics; Karayel, Özge; Şanal, Erdem; Kagiali, Zeynep Cansu Üretmen; Köken, Ayşe Nur Polat; Faculty Member; Department of Molecular Biology and Genetics; Graduate School of Sciences and Engineering; N/A; N/A; N/A; N/A; 105301The successful completion of cytokinesis requires the coordinated activities of diverse cellular components including membranes, cytoskeletal elements and chromosomes that together form partly redundant pathways, depending on the cell type. The biochemical analysis of this process is challenging due to its dynamic and rapid nature. Here, we systematically compared monopolar and bipolar cytokinesis and demonstrated that monopolar cytokinesis is a good surrogate for cytokinesis and it is a well-suited system for global biochemical analysis in mammalian cells. Based on this, we established a phosphoproteomic signature of cytokinesis. More than 10,000 phosphorylation sites were systematically monitored; around 800 of those were up-regulated during cytokinesis. Reconstructing the kinase-substrate interaction network revealed 31 potentially active kinases during cytokinesis. The kinase-substrate network connects proteins between cytoskeleton, membrane and cell cycle machinery. We also found consensus motifs of phosphorylation sites that can serve as biochemical markers specific to cytokinesis. Beyond the kinase-substrate network, our reconstructed signaling network suggests that combination of sumoylation and phosphorylation may regulate monopolar cytokinesis specific signaling pathways. Our analysis provides a systematic approach to the comparison of different cytokinesis types to reveal alternative ways and a global overview, in which conserved genes work together and organize chromatin and cytoplasm during cytokinesis.Publication Open Access Diffraction data from aerosolized Coliphage PR772 virus particles imaged with the Linac Coherent Light Source(Nature Publishing Group (NPG), 2020) Li, H.; Nazari, R.; Abbey, B.; Alvarez, R.; Aquila, A.; Ayyer, K.; Barty, A.; Berntsen, P.; Bielecki, J.; Pietrini, A.; Bucher, M.; Carini, G.; Chapman, H. N.; Contreras, A.; Daurer, B. J.; Flűckiger, L.; Frank, M.; Hajdu, J.; Hantke, M. F.; Hogue, B. G.; Hosseinizadeh, A.; Hunter, M. S.; Jönsson, H. O.; Kirian, R. A.; Kurta, R. P.; Loh, D.; Maia, F. R. N. C.; Mancuso, A. P.; Morgan, A. J.; McFadden, M.; Muehlig, K.; Munke, A.; Reddy, H. K. N.; Nettelblad, C.; Ourmazd, A.; Rose, M.; Schwander, P.; Marvin, Seibert M.; Sellberg, J. A.; Sierra, R. G.; Sun, Z.; Svenda, M.; Vartanyants, I. A.; Walter, P.; Westphal, D.; Williams, G.; Xavier, P. L.; Yoon, C. H.; Zaare, S.; Department of Molecular Biology and Genetics; Demirci, Hasan; Faculty Member; Department of Molecular Biology and Genetics; College of Sciences; 307350Single Particle Imaging (SPI) with intense coherent X-ray pulses from X-ray free-electron lasers (XFELs) has the potential to produce molecular structures without the need for crystallization or freezing. Here we present a dataset of 285,944 diffraction patterns from aerosolized Coliphage PR772 virus particles injected into the femtosecond X-ray pulses of the Linac Coherent Light Source (LCLS). Additional exposures with background information are also deposited. The diffraction data were collected at the Atomic, Molecular and Optical Science Instrument (AMO) of the LCLS in 4 experimental beam times during a period of four years. The photon energy was either 1.2 or 1.7 keV and the pulse energy was between 2 and 4 mJ in a focal spot of about 1.3 μm x 1.7 μm full width at half maximum (FWHM). The X-ray laser pulses captured the particles in random orientations. The data offer insight into aerosolised virus particles in the gas phase, contain information relevant to improving experimental parameters, and provide a basis for developing algorithms for image analysis and reconstruction.Publication Open Access DNA damage sensitivity of SWI/SNF-deficient cells depends on TFIIH subunit p62/GTF2H1(Nature Publishing Group (NPG), 2018) Ribeiro-Silva, Cristina; Mesquita-Ribeiro, Raquel; Slyskova, Jana; Helfricht, Angela; Marteijn, Jurgen A.; Hoeijmakers, Jan H. J.; Lans, Hannes; Vermeulen, Wim; Department of Molecular Biology and Genetics; Aydın, Özge Zelal; Department of Molecular Biology and Genetics; Graduate School of Sciences and EngineeringMutations in SWI/SNF genes are amongst the most common across all human cancers, but efficient therapeutic approaches that exploit vulnerabilities caused by SWI/SNF mutations are currently lacking. Here, we show that the SWI/SNF ATPases BRM/SMARCA2 and BRG1/SMARCA4 promote the expression of p62/GTF2H1, a core subunit of the transcription factor IIH (TFIIH) complex. Inactivation of either ATPase subunit downregulates GTF2H1 and therefore compromises TFIIH stability and function in transcription and nucleotide excision repair (NER). We also demonstrate that cells with permanent BRM or BRG1 depletion have the ability to restore GTF2H1 expression. As a consequence, the sensitivity of SWI/SNF deficient cells to DNA damage induced by UV irradiation and cisplatin treatment depends on GTF2H1 levels. Together, our results expose GTF2H1 as a potential novel predictive marker of platinum drug sensitivity in SWI/SNF-deficient cancer cells.Publication Open Access Enzymatic spiroketal formation via oxidative rearrangement of pentangular polyketides(Nature Publishing Group (NPG), 2021) Frensch, Britta; Lechtenberg, Thorsten; Kather, Michel; Betschart, Martin; Kammerer, Bernd; Luedeke, Steffen; Mueller, Michael; Piel, Joern; Teufel, Robin; Department of Molecular Biology and Genetics; Yunt, Zeynep Sabahat; Teaching Faculty; Department of Molecular Biology and Genetics; College of Sciences; 116178The structural complexity and bioactivity of natural products often depend on enzymatic redox tailoring steps. This is exemplified by the generation of the bisbenzannulated [5,6]-spiroketal pharmacophore in the bacterial rubromycin family of aromatic polyketides, which exhibit a wide array of bioactivities such as the inhibition of HIV reverse transcriptase or DNA helicase. Here we elucidate the complex flavoenzyme-driven formation of the rubromycin pharmacophore that is markedly distinct from conventional (bio)synthetic strategies for spiroketal formation. Accordingly, a polycyclic aromatic precursor undergoes extensive enzymatic oxidative rearrangement catalyzed by two flavoprotein monooxygenases and a flavoprotein oxidase that ultimately results in a drastic distortion of the carbon skeleton. The one-pot in vitro reconstitution of the key enzymatic steps as well as the comprehensive characterization of reactive intermediates allow to unravel the intricate underlying reactions, during which four carbon-carbon bonds are broken and two CO2 become eliminated. This work provides detailed insight into perplexing redox tailoring enzymology that sets the stage for the (chemo)enzymatic production and bioengineering of bioactive spiroketal-containing polyketides.Rubromycin family of natural products belongs to aromatic polyketides with diverse bioactivities, but details of their biosynthesis are limited. Here, the authors report the complete in vitro reconstitution of enzymatic formation of the spiroketal moiety of rubromycin polyketides, driven by flavin-dependent enzymes, and characterize reaction intermediates.Publication Open Access Isolation of large dense-core vesicles from bovine adrenal medulla for functional studies(Nature Publishing Group (NPG), 2020) Preobraschenski, Julia; Ganzella, Marcelo; Jahn, Reinhard; Department of Molecular Biology and Genetics; Birinci, Yelda; Park, Yongsoo; Faculty Member; Department of Molecular Biology and Genetics; College of Sciences; 183492; 240759Large dense-core vesicles (LDCVs) contain a variety of neurotransmitters, proteins, and hormones such as biogenic amines and peptides, together with microRNAs (miRNAs). Isolation of LDCVs is essential for functional studies including vesicle fusion, vesicle acidification, monoamine transport, and the miRNAs stored in LDCVs. Although several methods were reported for purifying LDCVs, the final fractions are significantly contaminated by other organelles, compromising biochemical characterization. Here we isolated LDCVs (chromaffin granules) with high yield and purity from bovine adrenal medulla. The fractionation protocol combines differential and continuous sucrose gradient centrifugation, allowing for reducing major contaminants such as mitochondria. Purified LDCVs show robust acidification by the endogenous V-ATPase and undergo SNARE-mediated fusion with artificial membranes. Interestingly, LDCVs contain specific miRNAs such as miR-375 and miR-375 is stabilized by protein complex against RNase A. This protocol can be useful in research on the biological functions of LDCVs.Publication Open Access Mitochondrial dysfunction plus high-sugar diet provokes a metabolic crisis that inhibits growth(Public Library of Science, 2016) Kemppainen, Esko; George, Jack; Garipler, Görkem; Tuomela, Tea; Kiviranta, Essi; Soga, Tomoyoshi; Jacobs, Howard T.; Department of Molecular Biology and Genetics; Dunn, Cory David; Faculty Member; Department of Molecular Biology and Genetics; College of SciencesThe Drosophila mutant tko(25t) exhibits a deficiency ofmitochondrial protein synthesis, leading to a global insufficiency of respiration and oxidative phosphorylation. This entrains an organismal phenotype of developmental delay and sensitivity to seizures induced bymechanical stress. We found that the mutant phenotype is exacerbated in a dose-dependent fashion by high dietary sugar levels. tko(25t) larvae were found to exhibit severe metabolic abnormalities that were further accentuated by high-sugar diet. These include elevated pyruvate and lactate, decreased ATP and NADPH. Dietary pyruvate or lactate supplementation phenocopied the effects of high sugar. Based on tissue-specific rescue, the crucial tissue in which this metabolic crisis initiates is the gut. It is accompanied by down-regulation of the apparatus of cytosolic protein synthesis and secretion at both the RNA and post-translational levels, including a novel regulation of S6 kinase at the protein level.