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Article: A Role for Cross-linking Proteins in Actin Filament Network Organization and Force Generation.

Hill, Jennifer M / Cai, Songlin / Carver, Michael D / Drubin, David G

bioRxiv : the preprint server for biology

2024

Abstract: The high turgor pressure across the plasma membrane of yeasts creates a requirement for substantial force production by actin polymerization and myosin motor activity for clathrin-mediated endocytosis (CME). Endocytic internalization is severely impeded ... ...

Abstract	The high turgor pressure across the plasma membrane of yeasts creates a requirement for substantial force production by actin polymerization and myosin motor activity for clathrin-mediated endocytosis (CME). Endocytic internalization is severely impeded in the absence of fimbrin, an actin filament crosslinking protein called Sac6 in budding yeast. Here, we combine live-cell imaging and mathematical modeling to gain new insights into the role of actin filament crosslinking proteins in force generation. Genetic manipulation showed that CME sites with more crosslinking proteins are more effective at internalization under high load. Simulations of an experimentally constrained, agent- based mathematical model recapitulate the result that endocytic networks with more double-bound fimbrin molecules internalize the plasma membrane against elevated turgor pressure more effectively. Networks with large numbers of crosslinks also have more growing actin filament barbed ends at the plasma membrane, where the addition of new actin monomers contributes to force generation and vesicle internalization. Our results provide a richer understanding of the crucial role played by actin filament crosslinking proteins during actin network force generation, highlighting the contribution of these proteins to the self-organization of the actin filament network and force generation under increased load.
Language	English
Publishing date	2024-04-20
Publishing country	United States
Document type	Preprint
DOI	10.1101/2024.04.19.590161
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Article: Endocytic myosin-1 is a force-insensitive, power-generating motor.

Pedersen, Ross Ta / Snoberger, Aaron / Pyrpassopoulos, Serapion / Safer, Daniel / Drubin, David G / Ostap, E Michael

bioRxiv : the preprint server for biology

2023

Abstract: Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical properties of the relevant motors have not been investigated. Myosins have diverse ... ...

Abstract	Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical properties of the relevant motors have not been investigated. Myosins have diverse mechanochemical activities, ranging from powerful contractility against mechanical loads to force-sensitive anchoring. To better understand the essential molecular contribution of myosin to endocytosis, we studied the in vitro force-dependent kinetics of the Summary: Pedersen, Snoberger et al. measure the force-sensitivity of the yeast endocytic the myosin-1 called Myo5 and find that it is more likely to generate power than to serve as a force-sensitive anchor in cells. Implications for Myo5's role in clathrin-mediated endocytosis are discussed.
Language	English
Publishing date	2023-06-30
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.03.21.533689
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Article ; Online: Endocytic myosin-1 is a force-insensitive, power-generating motor.

Pedersen, Ross T A / Snoberger, Aaron / Pyrpassopoulos, Serapion / Safer, Daniel / Drubin, David G / Ostap, E Michael

The Journal of cell biology

2023 Volume 222, Issue 10

Abstract	Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical properties of the relevant motors have not been investigated. Myosins have diverse mechanochemical activities, ranging from powerful contractility against mechanical loads to force-sensitive anchoring. To better understand the essential molecular contribution of myosin to endocytosis, we studied the in vitro force-dependent kinetics of the Saccharomyces cerevisiae endocytic type I myosin called Myo5, a motor whose role in clathrin-mediated endocytosis has been meticulously studied in vivo. We report that Myo5 is a low-duty-ratio motor that is activated ∼10-fold by phosphorylation and that its working stroke and actin-detachment kinetics are relatively force-insensitive. Strikingly, the in vitro mechanochemistry of Myo5 is more like that of cardiac myosin than that of slow anchoring myosin-1s found on endosomal membranes. We, therefore, propose that Myo5 generates power to augment actin assembly-based forces during endocytosis in cells.
MeSH term(s)	Actins ; Clathrin ; Myosin Type I/genetics ; Myosins/genetics ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae Proteins/genetics
Chemical Substances	Actins ; Clathrin ; Myosin Type I (EC 3.6.1.-) ; Myosins (EC 3.6.4.1) ; MYO5 protein, S cerevisiae ; Saccharomyces cerevisiae Proteins
Language	English
Publishing date	2023-08-07
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	218154-x
ISSN	1540-8140 ; 0021-9525
ISSN (online)	1540-8140
ISSN	0021-9525
DOI	10.1083/jcb.202303095
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Article ; Online: Reconstitution of kinetochore motility and microtubule dynamics reveals a role for a kinesin-8 in establishing end-on attachments.

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

eLife

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
MeSH term(s)	Kinesins ; Kinetochores/metabolism ; Microtubule-Associated Proteins/metabolism ; Microtubules/metabolism ; Mitosis ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae Proteins/metabolism
Chemical Substances	KIP3 protein, S cerevisiae ; Microtubule-Associated Proteins ; Saccharomyces cerevisiae Proteins ; Kinesins (EC 3.6.4.4)
Language	English
Publishing date	2022-07-05
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.78450
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Article ; Online: Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis.

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

eLife

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.
MeSH term(s)	Actin Cytoskeleton/chemistry ; Actin Cytoskeleton/metabolism ; Actin-Related Protein 2-3 Complex/chemistry ; Actin-Related Protein 2-3 Complex/metabolism ; Actins/chemistry ; Actins/metabolism ; Biomechanical Phenomena/physiology ; Cell Line ; Cell Membrane/chemistry ; Cell Membrane/metabolism ; Clathrin/chemistry ; Clathrin/metabolism ; Endocytosis/physiology ; Humans ; Induced Pluripotent Stem Cells
Chemical Substances	Actin-Related Protein 2-3 Complex ; Actins ; Clathrin
Language	English
Publishing date	2020-01-17
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.49840
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Article ; Online: Mechanistic insights into actin force generation during vesicle formation from cryo-electron tomography.

Serwas, Daniel / Akamatsu, Matthew / Moayed, Amir / Vegesna, Karthik / Vasan, Ritvik / Hill, Jennifer M / Schöneberg, Johannes / Davies, Karen M / Rangamani, Padmini / Drubin, David G

Developmental cell

2022 Volume 57, Issue 9, Page(s) 1132–1145.e5

Abstract: Actin assembly provides force for a multitude of cellular processes. Compared to actin-assembly-based force production during cell migration, relatively little is understood about how actin assembly generates pulling forces for vesicle formation. Here, ... ...

Abstract	Actin assembly provides force for a multitude of cellular processes. Compared to actin-assembly-based force production during cell migration, relatively little is understood about how actin assembly generates pulling forces for vesicle formation. Here, cryo-electron tomography identified actin filament number, organization, and orientation during clathrin-mediated endocytosis in human SK-MEL-2 cells, showing that force generation is robust despite variance in network organization. Actin dynamics simulations incorporating a measured branch angle indicate that sufficient force to drive membrane internalization is generated through polymerization and that assembly is triggered from ∼4 founding "mother" filaments, consistent with tomography data. Hip1R actin filament anchoring points are present along the entire endocytic invagination, where simulations show that it is key to pulling force generation, and along the neck, where it targets filament growth and makes internalization more robust. Actin organization described here allowed direct translation of structure to mechanism with broad implications for other actin-driven processes.
MeSH term(s)	Actin Cytoskeleton/metabolism ; Actins/metabolism ; Clathrin/metabolism ; Cytoskeleton/metabolism ; Electron Microscope Tomography ; Endocytosis ; Humans
Chemical Substances	Actins ; Clathrin
Language	English
Publishing date	2022-05-02
Publishing country	United States
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2054967-2
ISSN	1878-1551 ; 1534-5807
ISSN (online)	1878-1551
ISSN	1534-5807
DOI	10.1016/j.devcel.2022.04.012
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Zs.A 5742: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (2.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: Direct comparison of clathrin-mediated endocytosis in budding and fission yeast reveals conserved and evolvable features.

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

eLife

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
MeSH term(s)	Clathrin/metabolism ; Endocytosis ; Fungal Proteins/metabolism ; Intravital Microscopy ; Saccharomyces cerevisiae/metabolism ; Schizosaccharomyces/metabolism
Chemical Substances	Clathrin ; Fungal Proteins
Language	English
Publishing date	2019-12-12
Publishing country	England
Document type	Comparative Study ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.50749
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Article ; Online: Tagging endogenous loci for live-cell fluorescence imaging and molecule counting using ZFNs, TALENs, and Cas9.

Dambournet, D / Hong, S H / Grassart, A / Drubin, D G

Methods in enzymology

2014 Volume 546, Page(s) 139–160

Abstract: The programmable ZFN, TALEN, and Cas9 nucleases allow genome editing of any cell line or organism. In this chapter, we describe methods to create gene fusions at endogenous loci in mammalian cells to express fluorescent fusions of proteins of interest at ...

Abstract	The programmable ZFN, TALEN, and Cas9 nucleases allow genome editing of any cell line or organism. In this chapter, we describe methods to create gene fusions at endogenous loci in mammalian cells to express fluorescent fusions of proteins of interest at endogenous levels. The donor DNA, which includes the sequence encoding a fluorescent protein, is provided to the cell to repair a double-strand break induced by a nuclease. The engineered donor sequence is integrated by homology-directed repair into the genome in frame with the coding region of the gene of interest, resulting in expression of a fusion protein at physiological levels. We further describe techniques to study protein dynamics and numbers using the genome-edited cell lines. In contrast to cell lines stably overexpressing fusion proteins from modified cDNAs, genes encoding fluorescent proteins are targeted to the endogenous genetic locus, avoiding perturbation of alternative splicing and expression levels.
MeSH term(s)	Animals ; DNA/genetics ; Electroporation ; Endonucleases/metabolism ; Gene Targeting ; Genetic Engineering/methods ; Genetic Loci ; Humans ; Luminescent Proteins/analysis ; Luminescent Proteins/genetics ; Microscopy, Fluorescence ; Plasmids/genetics ; Recombinant Fusion Proteins/analysis ; Recombinant Fusion Proteins/genetics
Chemical Substances	Luminescent Proteins ; Recombinant Fusion Proteins ; DNA (9007-49-2) ; Endonucleases (EC 3.1.-)
Language	English
Publishing date	2014
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	1557-7988 ; 0076-6879
ISSN (online)	1557-7988
ISSN	0076-6879
DOI	10.1016/B978-0-12-801185-0.00007-6
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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 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
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Article ; Online: 4D cell biology: big data image analytics and lattice light-sheet imaging reveal dynamics of clathrin-mediated endocytosis in stem cell-derived intestinal organoids.

Schöneberg, Johannes / Dambournet, Daphné / Liu, Tsung-Li / Forster, Ryan / Hockemeyer, Dirk / Betzig, Eric / Drubin, David G

Molecular biology of the cell

2018 Volume 29, Issue 24, Page(s) 2959–2968

Abstract: New methods in stem cell 3D organoid tissue culture, advanced imaging, and big data image analytics now allow tissue-scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and ... ...

Abstract	New methods in stem cell 3D organoid tissue culture, advanced imaging, and big data image analytics now allow tissue-scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high-content imaging data. We expressed fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome-edited human embryonic stem cells, which were differentiated into hESC-derived intestinal epithelial organoids. Lattice light-sheet imaging with adaptive optics (AO-LLSM) allowed us to image large volumes of these organoids (70 × 60 × 40 µm xyz) at 5.7 s/frame. We developed an open-source data analysis package termed pyLattice to process the resulting large (∼60 Gb) movie data sets and to track clathrin-mediated endocytosis (CME) events. CME tracks could be recorded from ∼35 cells at a time, resulting in ∼4000 processed tracks per movie. On the basis of their localization in the organoid, we classified CME tracks into apical, lateral, and basal events and found that CME dynamics is similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO-LLSM makes possible quantitative high temporal and spatial resolution analysis of subcellular events within tissues.
MeSH term(s)	Animals ; Big Data ; Cell Culture Techniques/methods ; Cell Differentiation/physiology ; Clathrin/metabolism ; Clathrin-Coated Vesicles/metabolism ; Dynamin II/metabolism ; Endocytosis/physiology ; Human Embryonic Stem Cells/cytology ; Human Embryonic Stem Cells/metabolism ; Humans ; Image Processing, Computer-Assisted/methods ; Intestinal Mucosa/cytology ; Intestinal Mucosa/metabolism ; Intestines/cytology ; Mice ; Organoids/cytology ; Organoids/diagnostic imaging ; Organoids/metabolism
Chemical Substances	Clathrin ; Dynamin II (EC 3.6.5.5)
Language	English
Publishing date	2018-09-06
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1098979-1
ISSN	1939-4586 ; 1059-1524
ISSN (online)	1939-4586
ISSN	1059-1524
DOI	10.1091/mbc.E18-06-0375
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Zs.A 2853: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (2.OG) ab Jg. 2022: Lesesaal (EG)
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