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  1. Article ; Online: Toward a standard model for autophagosome biogenesis.

    Cook, Annan S I / Hurley, James H

    The Journal of cell biology

    2023  Volume 222, Issue 7

    Abstract: Two papers in this issue resolve a long-standing obstacle to a "standard model" for autophagosome biogenesis in mammals. The first, Olivas et al. (2023. J. Cell Biol. https://doi.org/10.1083/jcb.202208088), uses biochemistry to confirm that the lipid ... ...

    Abstract Two papers in this issue resolve a long-standing obstacle to a "standard model" for autophagosome biogenesis in mammals. The first, Olivas et al. (2023. J. Cell Biol. https://doi.org/10.1083/jcb.202208088), uses biochemistry to confirm that the lipid scramblase ATG9A is a bona fide autophagosome component, while the second, Broadbent et al. (2023. J. Cell Biol. https://doi.org/10.1083/jcb.202210078), uses particle tracking to show that the dynamics of autophagy proteins are consistent with the concept.
    MeSH term(s) Animals ; Autophagosomes ; Autophagy ; Macroautophagy ; Mammals ; Autophagy-Related Proteins
    Chemical Substances Autophagy-Related Proteins
    Language English
    Publishing date 2023-06-05
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Comment
    ZDB-ID 218154-x
    ISSN 1540-8140 ; 0021-9525
    ISSN (online) 1540-8140
    ISSN 0021-9525
    DOI 10.1083/jcb.202304011
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end.

    Cleary, Joseph M / Kim, Tae / Cook, Annan S I / McCormick, Lauren A / Hancock, William O / Rice, Luke M

    eLife

    2022  Volume 11

    Abstract: Microtubule polymerization dynamics result from the biochemical interactions of αβ-tubulin with the polymer end, but a quantitative understanding has been challenging to establish. We used interference reflection microscopy to make improved measurements ... ...

    Abstract Microtubule polymerization dynamics result from the biochemical interactions of αβ-tubulin with the polymer end, but a quantitative understanding has been challenging to establish. We used interference reflection microscopy to make improved measurements of microtubule growth rates and growth fluctuations in the presence and absence of GTP hydrolysis. In the absence of GTP hydrolysis, microtubules grew steadily with very low fluctuations. These data were best described by a computational model implementing slow assembly kinetics, such that the rate of microtubule elongation is primarily limited by the rate of αβ-tubulin associations. With GTPase present, microtubules displayed substantially larger growth fluctuations than expected based on the no GTPase measurements. Our modeling showed that these larger fluctuations occurred because exposure of GDP-tubulin on the microtubule end transiently 'poisoned' growth, yielding a wider range of growth rates compared to GTP only conditions. Our experiments and modeling point to slow association kinetics (strong longitudinal interactions), such that drugs and regulatory proteins that alter microtubule dynamics could do so by modulating either the association or dissociation rate of tubulin from the microtubule tip. By causing slower growth, exposure of GDP-tubulin at the growing microtubule end may be an important early event determining catastrophe.
    MeSH term(s) Guanosine Diphosphate/metabolism ; Guanosine Triphosphate/metabolism ; Kinetics ; Microtubules/metabolism ; Tubulin/metabolism
    Chemical Substances Tubulin ; Guanosine Diphosphate (146-91-8) ; Guanosine Triphosphate (86-01-1)
    Language English
    Publishing date 2022-04-14
    Publishing country England
    Document type Journal Article
    ZDB-ID 2687154-3
    ISSN 2050-084X ; 2050-084X
    ISSN (online) 2050-084X
    ISSN 2050-084X
    DOI 10.7554/eLife.75931
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Insights into Kinesin-1 Stepping from Simulations and Tracking of Gold Nanoparticle-Labeled Motors.

    Mickolajczyk, Keith J / Cook, Annan S I / Jevtha, Janak P / Fricks, John / Hancock, William O

    Biophysical journal

    2019  Volume 117, Issue 2, Page(s) 331–345

    Abstract: High-resolution tracking of gold nanoparticle-labeled proteins has emerged as a powerful technique for measuring the structural kinetics of processive enzymes and other biomacromolecules. These techniques use point spread function (PSF) fitting methods ... ...

    Abstract High-resolution tracking of gold nanoparticle-labeled proteins has emerged as a powerful technique for measuring the structural kinetics of processive enzymes and other biomacromolecules. These techniques use point spread function (PSF) fitting methods borrowed from single-molecule fluorescence imaging to determine molecular positions below the diffraction limit. However, compared to fluorescence, gold nanoparticle tracking experiments are performed at significantly higher frame rates and utilize much larger probes. In the current work, we use Brownian dynamics simulations of nanoparticle-labeled proteins to investigate the regimes in which the fundamental assumptions of PSF fitting hold and where they begin to break down. We find that because gold nanoparticles undergo tethered diffusion around their anchor point, PSF fitting cannot be extended to arbitrarily fast frame rates. Instead, camera exposure times that allow the nanoparticle to fully populate its stationary positional distribution achieve a spatial averaging that increases fitting precision. We furthermore find that changes in the rotational freedom of the tagged protein can lead to artifactual translations in the fitted particle position. Finally, we apply these lessons to dissect a standing controversy in the kinesin field over the structure of a dimer in the ATP waiting state. Combining new experiments with simulations, we determine that the rear kinesin head in the ATP waiting state is unbound but not displaced from its previous microtubule binding site and that apparent differences in separately published reports were simply due to differences in the gold nanoparticle attachment position. Our results highlight the importance of gold conjugation decisions and imaging parameters to high-resolution tracking results and will serve as a useful guide for the design of future gold nanoparticle tracking experiments.
    MeSH term(s) Adenosine Triphosphate/chemistry ; Animals ; Binding Sites ; Computer Simulation ; Drosophila ; Gold/chemistry ; Kinesin/chemistry ; Metal Nanoparticles/chemistry ; Molecular Motor Proteins/chemistry ; Photons ; Rotation ; Staining and Labeling
    Chemical Substances Molecular Motor Proteins ; Gold (7440-57-5) ; Adenosine Triphosphate (8L70Q75FXE) ; Kinesin (EC 3.6.4.4)
    Language English
    Publishing date 2019-06-20
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 218078-9
    ISSN 1542-0086 ; 0006-3495
    ISSN (online) 1542-0086
    ISSN 0006-3495
    DOI 10.1016/j.bpj.2019.06.010
    Database MEDical Literature Analysis and Retrieval System OnLINE

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