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  1. Article ; Online: Reconstitution of kinetochore motility and microtubule dynamics reveals a role for a kinesin-8 in establishing end-on attachments

    Julia R Torvi / Jonathan Wong / Daniel Serwas / Amir Moayed / David G Drubin / Georjana Barnes

    eLife, Vol

    2022  Volume 11

    Abstract: During mitosis, individual microtubules make attachments to chromosomes via a specialized protein complex called the kinetochore to faithfully segregate the chromosomes to daughter cells. Translocation of kinetochores on the lateral surface of the ... ...

    Abstract During mitosis, individual microtubules make attachments to chromosomes via a specialized protein complex called the kinetochore to faithfully segregate the chromosomes to daughter cells. Translocation of kinetochores on the lateral surface of the microtubule has been proposed to contribute to high fidelity chromosome capture and alignment at the mitotic midzone, but has been difficult to observe in vivo because of spatial and temporal constraints. To overcome these barriers, we used total internal reflection fluorescence (TIRF) microscopy to track the interactions between microtubules, kinetochore proteins, and other microtubule-associated proteins in lysates from metaphase-arrested Saccharomyces cerevisiae. TIRF microscopy and cryo-correlative light microscopy and electron tomography indicated that we successfully reconstituted interactions between intact kinetochores and microtubules. These kinetochores translocate on the lateral microtubule surface toward the microtubule plus end and transition to end-on attachment, whereupon microtubule depolymerization commences. The directional kinetochore movement is dependent on the highly processive kinesin-8, Kip3. We propose that Kip3 facilitates stable kinetochore attachment to microtubule plus ends through its abilities to move the kinetochore laterally on the surface of the microtubule and to regulate microtubule plus end dynamics.
    Keywords mitosis ; kinetochore ; Kip3 ; kinesin-8 ; microtubules ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 572
    Language English
    Publishing date 2022-07-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Branched actin networks are organized for asymmetric force production during clathrin-mediated endocytosis in mammalian cells

    Meiyan Jin / Cyna Shirazinejad / Bowen Wang / Amy Yan / Johannes Schöneberg / Srigokul Upadhyayula / Ke Xu / David G. Drubin

    Nature Communications, Vol 13, Iss 1, Pp 1-

    2022  Volume 12

    Abstract: Drubin et al. use three different advanced imaging approaches to show that actin assembles preferentially at stalled clathrin-mediated endocytosis sites, where the actin pulls vesicles into the cell asymmetrically, as a bottle opener pulls off a cap. ...

    Abstract Drubin et al. use three different advanced imaging approaches to show that actin assembles preferentially at stalled clathrin-mediated endocytosis sites, where the actin pulls vesicles into the cell asymmetrically, as a bottle opener pulls off a cap.
    Keywords Science ; Q
    Language English
    Publishing date 2022-06-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis

    Matthew Akamatsu / Ritvik Vasan / Daniel Serwas / Michael A Ferrin / Padmini Rangamani / David G Drubin

    eLife, Vol

    2020  Volume 9

    Abstract: Force generation by actin assembly shapes cellular membranes. An experimentally constrained multiscale model shows that a minimal branched actin network is sufficient to internalize endocytic pits against membrane tension. Around 200 activated Arp2/3 ... ...

    Abstract Force generation by actin assembly shapes cellular membranes. An experimentally constrained multiscale model shows that a minimal branched actin network is sufficient to internalize endocytic pits against membrane tension. Around 200 activated Arp2/3 complexes are required for robust internalization. A newly developed molecule-counting method determined that ~200 Arp2/3 complexes assemble at sites of clathrin-mediated endocytosis in human cells. Simulations predict that actin self-organizes into a radial branched array with growing ends oriented toward the base of the pit. Long actin filaments bend between attachment sites in the coat and the base of the pit. Elastic energy stored in bent filaments, whose presence was confirmed by cryo-electron tomography, contributes to endocytic internalization. Elevated membrane tension directs more growing filaments toward the base of the pit, increasing actin nucleation and bending for increased force production. Thus, spatially constrained actin filament assembly utilizes an adaptive mechanism enabling endocytosis under varying physical constraints.
    Keywords actin mechanics ; clathrin-mediated endocytosis ; multiscale modeling ; quantitative microscopy ; human induced pluripotent stem cells ; cryo-electron tomography ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 612
    Language English
    Publishing date 2020-01-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Direct comparison of clathrin-mediated endocytosis in budding and fission yeast reveals conserved and evolvable features

    Yidi Sun / Johannes Schöneberg / Xuyan Chen / Tommy Jiang / Charlotte Kaplan / Ke Xu / Thomas D Pollard / David G Drubin

    eLife, Vol

    2019  Volume 8

    Abstract: Conserved proteins drive clathrin-mediated endocytosis (CME), which from yeast to humans involves a burst of actin assembly. To gain mechanistic insights into this process, we performed a side-by-side quantitative comparison of CME in two distantly ... ...

    Abstract Conserved proteins drive clathrin-mediated endocytosis (CME), which from yeast to humans involves a burst of actin assembly. To gain mechanistic insights into this process, we performed a side-by-side quantitative comparison of CME in two distantly related yeast species. Though endocytic protein abundance in S. pombe and S. cerevisiae is more similar than previously thought, membrane invagination speed and depth are two-fold greater in fission yeast. In both yeasts, accumulation of ~70 WASp molecules activates the Arp2/3 complex to drive membrane invagination. In contrast to budding yeast, WASp-mediated actin nucleation plays an essential role in fission yeast endocytosis. Genetics and live-cell imaging revealed core CME spatiodynamic similarities between the two yeasts, although the assembly of two zones of actin filaments is specific for fission yeast and not essential for CME. These studies identified conserved CME mechanisms and species-specific adaptations with broad implications that are expected to extend from yeast to humans.
    Keywords clathrin-mediated endocytosis ; protein counting ; budding and fission yeast ; force generation ; N-WASp and Type I myosin ; actin patch ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 570
    Language English
    Publishing date 2019-12-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: Machine-Learning-Based Analysis in Genome-Edited Cells Reveals the Efficiency of Clathrin-Mediated Endocytosis

    Sun Hae Hong / Christa L. Cortesio / David G. Drubin

    Cell Reports, Vol 12, Iss 12, Pp 2121-

    2015  Volume 2130

    Abstract: Cells internalize various molecules through clathrin-mediated endocytosis (CME). Previous live-cell imaging studies suggested that CME is inefficient, with about half of the events terminated. These CME efficiency estimates may have been confounded by ... ...

    Abstract Cells internalize various molecules through clathrin-mediated endocytosis (CME). Previous live-cell imaging studies suggested that CME is inefficient, with about half of the events terminated. These CME efficiency estimates may have been confounded by overexpression of fluorescently tagged proteins and inability to filter out false CME sites. Here, we employed genome editing and machine learning to identify and analyze authentic CME sites. We examined CME dynamics in cells that express fluorescent fusions of two defining CME proteins, AP2 and clathrin. Support vector machine classifiers were built to identify and analyze authentic CME sites. From inception until disappearance, authentic CME sites contain both AP2 and clathrin, have the same degree of limited mobility, continue to accumulate AP2 and clathrin over lifetimes >∼20 s, and almost always form vesicles as assessed by dynamin2 recruitment. Sites that contain only clathrin or AP2 show distinct dynamics, suggesting they are not part of the CME pathway.
    Keywords Biology (General) ; QH301-705.5
    Language English
    Publishing date 2015-09-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: Switch-like Arp2/3 activation upon WASP and WIP recruitment to an apparent threshold level by multivalent linker proteins in vivo

    Yidi Sun / Nicole T Leong / Tommy Jiang / Astou Tangara / Xavier Darzacq / David G Drubin

    eLife, Vol

    2017  Volume 6

    Abstract: Actin-related protein 2/3 (Arp2/3) complex activation by nucleation promoting factors (NPFs) such as WASP, plays an important role in many actin-mediated cellular processes. In yeast, Arp2/3-mediated actin filament assembly drives endocytic membrane ... ...

    Abstract Actin-related protein 2/3 (Arp2/3) complex activation by nucleation promoting factors (NPFs) such as WASP, plays an important role in many actin-mediated cellular processes. In yeast, Arp2/3-mediated actin filament assembly drives endocytic membrane invagination and vesicle scission. Here we used genetics and quantitative live-cell imaging to probe the mechanisms that concentrate NPFs at endocytic sites, and to investigate how NPFs regulate actin assembly onset. Our results demonstrate that SH3 (Src homology 3) domain-PRM (proline-rich motif) interactions involving multivalent linker proteins play central roles in concentrating NPFs at endocytic sites. Quantitative imaging suggested that productive actin assembly initiation is tightly coupled to accumulation of threshold levels of WASP and WIP, but not to recruitment kinetics or release of autoinhibition. These studies provide evidence that WASP and WIP play central roles in establishment of a robust multivalent SH3 domain-PRM network in vivo, giving actin assembly onset at endocytic sites a switch-like behavior.
    Keywords WASP (yeast Las17) ; WIP (yeast Vrp1) ; Intersectin (Pan1-End3-Sla1) ; clathrin-mediated endocytosis ; multivalent PRM-SH3 domain interactions ; phase seperation ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Language English
    Publishing date 2017-08-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  7. Article ; Online: Simultaneous co-localized super-resolution fluorescence microscopy and atomic force microscopy

    Ana I. Gómez-Varela / Dimitar R. Stamov / Adelaide Miranda / Rosana Alves / Cláudia Barata-Antunes / Daphné Dambournet / David G. Drubin / Sandra Paiva / Pieter A. A. De Beule

    Scientific Reports, Vol 10, Iss 1, Pp 1-

    combined SIM and AFM platform for the life sciences

    2020  Volume 10

    Abstract: Abstract Correlating data from different microscopy techniques holds the potential to discover new facets of signaling events in cellular biology. Here we report for the first time a hardware set-up capable of achieving simultaneous co-localized imaging ... ...

    Abstract Abstract Correlating data from different microscopy techniques holds the potential to discover new facets of signaling events in cellular biology. Here we report for the first time a hardware set-up capable of achieving simultaneous co-localized imaging of spatially correlated far-field super-resolution fluorescence microscopy and atomic force microscopy, a feat only obtained until now by fluorescence microscopy set-ups with spatial resolution restricted by the Abbe diffraction limit. We detail system integration and demonstrate system performance using sub-resolution fluorescent beads and applied to a test sample consisting of human bone osteosarcoma epithelial cells, with plasma membrane transporter 1 (MCT1) tagged with an enhanced green fluorescent protein (EGFP) at the N-terminal.
    Keywords Medicine ; R ; Science ; Q
    Language English
    Publishing date 2020-01-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  8. Article ; Online: Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast

    Yansong Miao / Xuemei Han / Liangzhen Zheng / Ying Xie / Yuguang Mu / John R. Yates / David G. Drubin

    Nature Communications, Vol 7, Iss 1, Pp 1-

    2016  Volume 12

    Abstract: Metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. Here the authors identify fimbrin as one of the main metaphase Cdk1 targets for cell cycle regulation of actin cable assembly in budding yeast. ...

    Abstract Metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. Here the authors identify fimbrin as one of the main metaphase Cdk1 targets for cell cycle regulation of actin cable assembly in budding yeast.
    Keywords Science ; Q
    Language English
    Publishing date 2016-04-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  9. Article ; Online: The mechanochemistry of endocytosis.

    Jian Liu / Yidi Sun / David G Drubin / George F Oster

    PLoS Biology, Vol 7, Iss 9, p e

    2009  Volume 1000204

    Abstract: Endocytic vesicle formation is a complex process that couples sequential protein recruitment and lipid modifications with dramatic shape transformations of the plasma membrane. Although individual molecular players have been studied intensively, how they ...

    Abstract Endocytic vesicle formation is a complex process that couples sequential protein recruitment and lipid modifications with dramatic shape transformations of the plasma membrane. Although individual molecular players have been studied intensively, how they all fit into a coherent picture of endocytosis remains unclear. That is, how the proper temporal and spatial coordination of endocytic events is achieved and what drives vesicle scission are not known. Drawing upon detailed knowledge from experiments in yeast, we develop the first integrated mechanochemical model that quantitatively recapitulates the temporal and spatial progression of endocytic events leading to vesicle scission. The central idea is that membrane curvature is coupled to the accompanying biochemical reactions. This coupling ensures that the process is robust and culminates in an interfacial force that pinches off the vesicle. Calculated phase diagrams reproduce endocytic mutant phenotypes observed in experiments and predict unique testable endocytic phenotypes in yeast and mammalian cells. The combination of experiments and theory in this work suggest a unified mechanism for endocytic vesicle formation across eukaryotes.
    Keywords Biology (General) ; QH301-705.5
    Subject code 612
    Language English
    Publishing date 2009-09-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  10. Article ; Online: Lsb1 is a negative regulator of las17 dependent actin polymerization involved in endocytosis.

    Matthias Spiess / Johan-Owen de Craene / Alphée Michelot / Bruno Rinaldi / Aline Huber / David G Drubin / Barbara Winsor / Sylvie Friant

    PLoS ONE, Vol 8, Iss 4, p e

    2013  Volume 61147

    Abstract: The spatial and temporal regulation of actin polymerization is crucial for various cellular processes. Members of the Wiskott-Aldrich syndrome protein (WASP) family activate the Arp2/3-complex leading to actin polymerization. The yeast Saccharomyces ... ...

    Abstract The spatial and temporal regulation of actin polymerization is crucial for various cellular processes. Members of the Wiskott-Aldrich syndrome protein (WASP) family activate the Arp2/3-complex leading to actin polymerization. The yeast Saccharomyces cerevisiae contains only one WASP homolog, Las17, that requires additional factors for its regulation. Lsb1 and Lsb2/Pin3 are two yeast homologous proteins bearing an SH3 domain that were identified as Las17-binding proteins. Lsb2/Pin3 that promotes prion induction was suggested to link this prion formation to the actin cytoskeleton. However, the cellular role of Lsb1 and the molecular function of both Lsb1 and Lsb2 remain unknown. In this study, we show that Lsb1 and/or Lsb2 full-length proteins inhibit Las17-mediated actin polymerization in vitro, Lsb2 being a less potent inhibitor of Las17 activity compared to Lsb1. Addition of Lsb1 or Lsb2 to the corresponding full-length Lsb1/2 further inhibits Las17 activity. Lsb1 and Lsb2 form homo- and hetero-oligomeric complexes suggesting that these two proteins could regulate Las17 activity via dimerization or cooperative binding. In vivo, overexpressed Lsb1 and Lsb2 proteins cluster Las17-CFP in few cytoplasmic punctate structures that are also positive for other Arp2/3-dependent actin polymerization effectors like Sla1 or Abp1. But, only Lsb1 overexpression blocks the internalization step of receptor-mediated endocytosis. This shows a specific function of Lsb1 in endocytosis.
    Keywords Medicine ; R ; Science ; Q
    Subject code 570
    Language English
    Publishing date 2013-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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