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Membrane fusion was initiated at on average 5. To investigate whether this discrepancy is related to the different virus strains used, we analyzed the infectious properties of both viruses on HeLa cells expressing dominant-negative Rab5S34N and Rab7T22N. In agreement with the above results, viral infectivity of S1 was severely impaired in cells expressing dominant-negative Rab7, whereas the infectious properties of NGC were unaffected under the conditions of the experiments Figure S1.

To investigate the requirement for Rab7 of S1 infectivity in more detail, we performed single-particle tracking experiments in cells transiently expressing the dominant-negative Rab7T22N mutant [42] , [43]. Discussion Despite the medical importance of DENV, little information is available about the infectious cell entry pathway of the virus.

In this study, we investigated the cell entry process of single DENV particles in real-time by simultaneous tracking of fluorescently labeled DENV particles and endocytic structures in cells. This approach allowed us to obtain mechanistic and kinetic insights into the route of internalization and endocytic trafficking behavior of individual DENV particles in living cells.

Previous electron-microscopy studies suggested that DENV penetrates both mammalian and insect cells by direct fusion with the plasma membrane [12] , [13]. In contrast, this report shows that DENV enters cells via clathrin-mediated endocytosis and fuses from within late endosomes.

Furthermore, treatment of cells with chlorpromazine as well as expression of a dominant-negative Eps15 mutant significantly suppressed the number of DENV-infected cells. It is not clear what the explanation is for the discrepancy, but it might be related to the methodology that was used to investigate the cell entry process of the virus. The conclusion that DENV utilizes clathrin-mediated endocytosis for internalization is in agreement with recent observations of Acosta and co-workers [14].

Real-time imaging studies showed that macromolecules either induce de novo formation of clathrin-coated pits or are recruited to pre-existing clathrin-coated pits [31] , [44] , [45]. For example, influenza virus particles land on the cell surface and induce de novo formation of clathrin-coated pits at the site of binding [30]. This study indicates that DENV particles first diffuse along the cell surface before they encounter pre-existing clathrin-coated pits.

After the virus associates with the pit, the clathrin signal around the virus particle increases, which implies maturation of the clathrin-coated pit and formation of a clathrin-coated vesicle. Thereafter, the clathrin signal rapidly disappears again, typically within a time scale of a few seconds.

This behavior is similar to that of reoviruses, which have been shown to stabilize and induce maturation of pre-existing clathrin-coated pits [31]. Recently, several modes of endosome maturation have been described. Rink et al. Vonderheit et al.

The SFV particles are sequestered into this Rab7 domain, which pinches off as a Rab7-positive late endosome, leaving a Rab5-positive endosome behind [37]. We observed both modes of endosome maturation. Occasionally, we observed that DENV particles sequestered into a distinct Rab7 domain, similar to the behavior observed from endosomes containing SFV [37].

DENV particles predominantly fused from within Rab7-positive endosomes. Furthermore, the membrane fusion activity was significantly impaired in cells expressing dominant-negative forms of Rab7, which indicates that progression of DENV to Rab7-positive endosomes is important for its infectious entry.

In contrast, Krishnan et al. A direct comparison between both virus strains revealed that viral infectivity of S1 was severely impaired in cells expressing dominant-negative Rab7, whereas the infectivity of NGC was unaffected. These results suggest that both virus strains have distinct entry characteristics. Young, University of Queensland, Australia. The different pH-dependent properties of these virus strains may therefore reflect the distinct requirements for functional endocytic trafficking in cells.

Future experiments should reveal whether the pH threshold determines in which organelle membrane fusion occurs. DENV particles reside on average for 5. This result is surprising considering that TBEV efficiently fuses with liposomes in a model system in a time scale of seconds after low-pH exposure [46]. Pre-exposure of TBEV to low pH for 10—20 seconds in the absence of liposomes completely abolishes the membrane fusion activity of the virus [46]. Similar results were obtained for WNV unpublished results, J.

Portella , M. Vendruscolo , D. Frenkel , T. Schlick , and M. Orozco, Chromatin unfolding by epigenetic modifications explained by dramatic impairment of internucleosome interactions: a multiscale computational study, J. Ozer, A. Luque, and T. Schlick, The chromatin fiber: multiscale problems and approaches, Curr.

Kim, M. Zahran, and T. Ozer, R. Collepardo-Guevara, and T. Beard, L. Perera, D. Shock, T. Kim, T. Schlick, and S. Wilson, Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide, Nature, , , DOI Kim, Z. Zheng, S. Elmetwaly and T. Luque, R. Collepardo-Guevara, S. Grigoryev and T. Schlick, Dynamic condensation of linker histone C-terminal domain regulates chromatin structure, Nucleic Acids Res.

Laing, S. Elmetwaly, S. Jung, J. Curuksu, and T. Li, B. Jung and T. Barbati, K. Arora, J. Bogdanovic and T. Jung, N. Kim, S. Elmetwaly, M. Li, and T. Schlick, Insights into chromatin fibre structure by in vitro and in silico single-molecule stretching experiments, Biochem.

Jung, and T. Kim, L. Kim, N. Fuhr and T. Collepardo-Guevara and T. Schlick, Crucial role of dynamic linker histone binding and divalent ions for DNA accessibility and gene regulation revealed by mesoscale modeling of oligonucleosomes, Nucleic Acids Research, DOI Schlick, K.

Arora, W. Beard, and S. Kim and T. Li, C. Gridley, J. Jaeger, J. Sweasy, and T. Schlick ed. Schlick, J. Hayes, and S. Quarta, K. Sin and T. Laing, D. Wen, J. Wang and T. Izzo, N. Laing and T. Yunlang and T. Gan and T. Schlick, R. Collepardo-Guevara, L. Halvorsen, S. Jung and X. Foley, V.

Padow and T. Perisic, R. Matter, Kim, J. Izzo, S. Elmetwaly, H. Acids Res. Schlick and O. Quarta, N. Izzo, and T. Arora and T. Arya, S. Correll, C. Woodcock and T. USA, Fish, Editor, Oxford University Press Foley and T. B, 39 : Schlick, Monte Carlo, harmonic approximation, and coarse-graining approaches for enhanced sampling of biomolecular structure, F Biology Reports, Arya and T.

A, 16, Sampoli Benitez, K. Arora, L. Balistreri, and T. Bebenek, M. Garcia-Diaz, M. Foley, L. Pedersen, T. Schlick, and T. Xin, G. Quarta, H. Gan, and T. Shin, S. Kim, H. Phys, Radhakrishnan and T. Radhakrishnan, K. Arora, Y. Wang, W. Wang, S. Reddy, W. Sponer and F. Foley, K. Arora, and T. Arya, Q. Zhang, and T. Zhang and T. Wang, K. Laserson, H. Leimkuhler, C. Chipot, R. Elber, A. Laaksonen, A. Mark, T. Schlick, C. Schuette, R.

Gevertz, H. Sun, Q. B Pasquali, H. Fera, N. Shiffeldrim, J. Zorn, U. Zhang, S. Broyde, and T. Royal Soc. Yang, W. Wilson, S. Zorn, H. Gan, N. Shiffeldrim, and T. Shiffeldrim, H. Yang, K. Gan, D. Fera, J. Zorn, M. Tang, N. Shiffeldrim, U. Laserson, N. USA Huang, Q. Gan, S. Pasquali, and T. Wilson, B. Roux, S. Qian and T. Batcho, D.

Case, and T. Qian, D. Strahs, and T. Beard and T. Schlick, D. Beard, J. Huang, D. Strahs, and X. Special Issue on Computational Chemistry 2: Schlick, Inertial Stochastic Dynamics: I. Skeel, A. Brunger, L. Kale, J. Hermans, K.

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Single molecule imaging and in recent years the emergence of super-resolution microscopy have opened new doors to visualize the dynamic assembly and disassembly of macromolecular complexes in living cells. Importantly, we have taken a highly quantitative biophysical approach in studying these biological processes, going beyond qualitative descriptions towards precise quantitative models.

For example, we have made important strides in quantifying the stoichiometry of macromolecular assemblies such as nucleosomes at nanoscale resolution Durisic et al, J. Nat Commun. PMID: Zanacchi, C. Manzo, R.

Magrassi, N. Garbacik, M. Cella Zanacchi, C. Manzo, A. Sandoval Alvarez, N, Derr,M. Lakadamyali,Nature Methods, doi Durisic, L. Cuervo, A. Borbely, M. Lakadamyali, Nature Methods, 11, Determined a novel nanoscale organization of chromatin:An important goal of the Lakadamyali lab is to reconcile the epigenomic and microscopic views of chromatin organization and determine how chromatin structure regulates gene function.

Nuclear organization of the chromatin fiber spans many length-scales and while the nanoscale level organization plays a key role in regulating gene expression, this organization is impossible to visualize using conventional microscopy methods. Lakadamyali lab has been using and further developing quantitative super-resolution microscopy methods to overcome this limitation.

We visualized and estimated the number of nucleosomes along the chromatin fiber of different cells at nanoscale resolution. Remarkably, the median number of nucleosomes and their packing density inside clutches highly correlated with cellular state, such that clutch size correlates with gene expression and pluripotency grade of iPSCs. Additionally, nanoscale chromatin organization is aberrantly remodeled in disease states.

Heo, S. Thakur, X. Chen, C. Loebel, B. Xia, R. McBeath, J. Burdick, V. Shenoy, R. Mauck, M. Lakadamyali, Nature Biomedical Engineering, in press b. Lerner, P. Gomez-Garcia, R. McCarthy, Z. Liu, M. Lakadamyali, K. Otterstrom, A. Garcia, C. Vicario, P. Gomez-Garcia, M. Cosma, M. Lakadamyali, Nucleic Acids Research, doi: Ricci, C. Manzo, M. Garcia-Parajo, M. Developed cutting-edge methods for studying intracellular trafficking: One of the overarching goals of Lakadamyali lab is to determine how molecular motors coordinate to transport vesicles and organelles in the complex cellular environment.

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Then she realized the possibilities. In protein immunolabeling, quantification is tricky. Multiple antibodies can bind to one protein, and other proteins can be missed altogether. Although she and her team have used in vitro assembled arrays of histone proteins and specific DNA sequences to accurately count nucleosomes, she sought a more generalizable method. DNA origami can be used with antibodies that recognize green fluorescent protein GFP to calibrate the efficiency of antibody labeling and to quantify protein copy numbers.

Precise stoichiometry can help labs understand how protein numbers affect protein function and, for example, learn about the coordination and tug-of-war between motors involved in intracellular trafficking. Lakadamyali studies intracellular transport and wants to quantify and understand the motor protein clusters she sees that likely move lysosomes along microtubules.

The cluster could be multiple antibodies attached to one protein or multiple fluorophores on each antibody, or each fluorophore could be actually blinking multiple times. She wants to clarify this with her new method, and hopes others will use the method for their puzzles of interest.

In her lab, Lakadamyali enjoys being there for her students and postdocs for big-picture discussions as well as the small day-to-day technical details and troubleshooting. Good ideas can be challenging but she will not give up easily on a project. She has lived in Austin, Boston and Barcelona and is now in Philadelphia.

A DNA origami platform for quantifying protein copy number in super-resolution Nat. Contributions to Science Developed versatile methods for quantitative, multiplexed imaging of molecular complexes using super-resolution microscopy:Proteins in cells assemble into nanoscale complexes in order to carry out a specific function.

Changes in nanoscale organization of protein complexes, for example a change from monomeric to oligomeric stoichiometry, often triggers disease states. However, visualizing many proteins simultaneously and quantifying protein copy number with high spatial resolution is highly challenging. Super-resolution methods hold promise for overcoming this hurdle, however, the complex photophysics of fluorophores limits both multi-color imaging capabilities and the ability to extract quantitative information.

Lakadamyali lab has been developing new methods to overcome these challenges. To address the limitations in simultaneous, high-throughput, multi-color super-resolution imaging, we recently combined DNA-Paint with excitation multiplexing to demonstrate the initial proof of concept for fm-DNA-Paint. Further, to address the challenges with quantification of protein copy number at the nanoscale level, we built calibration standards based on DNA origami.

Overall, these methods begin to overcome some of the main challenges associated to super-resolution microscopy making it a multiplexed and quantitative tool. Zanacchi, C. Manzo, R. Magrassi, N. Garbacik, M. Cella Zanacchi, C. Manzo, A. Sandoval Alvarez, N, Derr,M. Lakadamyali,Nature Methods, doi Durisic, L. Cuervo, A. Borbely, M. Lakadamyali, Nature Methods, 11, Determined a novel nanoscale organization of chromatin:An important goal of the Lakadamyali lab is to reconcile the epigenomic and microscopic views of chromatin organization and determine how chromatin structure regulates gene function.

Nuclear organization of the chromatin fiber spans many length-scales and while the nanoscale level organization plays a key role in regulating gene expression, this organization is impossible to visualize using conventional microscopy methods. Lakadamyali lab has been using and further developing quantitative super-resolution microscopy methods to overcome this limitation.

We visualized and estimated the number of nucleosomes along the chromatin fiber of different cells at nanoscale resolution. Remarkably, the median number of nucleosomes and their packing density inside clutches highly correlated with cellular state, such that clutch size correlates with gene expression and pluripotency grade of iPSCs.

Additionally, nanoscale chromatin organization is aberrantly remodeled in disease states. Heo, S. Thakur, X. Chen, C. Loebel, B. Xia, R. McBeath, J. Burdick, V. Shenoy, R. Mauck, M. Lakadamyali, Nature Biomedical Engineering, in press b. Lerner, P. Gomez-Garcia, R.

McCarthy, Z. Liu, M. Lakadamyali, K. Otterstrom, A. Garcia, C.

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