Observing and tracking single small ribosomal subunits in vivo

Landvogt, Lisa; Ruland, Jan A.; Montellese, Christian; Siebrasse, Jan Peter; Kutay, Ulrike; Kubitscheck, Ulrich

doi:10.1016/j.ymeth.2018.09.001

Cited by 8 publications

(7 citation statements)

References 24 publications

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“…Tracking of the single particles revealed a maximal nuclear diffusion coefficient of 1.7± 0.1 µm 2 /s (see Methods). This value was smaller than the maximal diffusion coefficient that we recently determined for single pre-40S particles, 2.3 ± 0.3 µm 2 /s 25 , and significantly smaller than the maximal diffusion coefficient of free, unbound proteins in cell nuclei, >10 µm 2 /s 32 .…”

Section: Tracking Of Single Pre-60s Particles At Single Npcscontrasting

confidence: 72%

“…However, pre-40S and pre-60S particles represent large RNP particles that can be labeled by a comparable approach as we have used previously for transport experiments on mRNA particles 24 . Only recently we succeeded in labeling the pre-40S particles using DIM2 (Pno1) coupled C-terminally to a SnapTag 25 . Our approach enabled us to observe pre-40S particles inside cell nuclei at the single particle level.…”

Section: Introductionmentioning

confidence: 99%

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Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time

Ruland

Krueger

Doerner

et al. 2021

Preprint

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Ribosomal subunit biogenesis within mammalian cells initiates in the nucleolus with the assembly of a 90S precursor particle, which is subsequently split into the pre-40S and pre-60S subunits. During further processing steps, pre-ribosomal subunits are loaded with export receptors, which enables their passage through the pore complexes (NPCs) into the cytoplasm. Here export factors are released and both subunits can form a mature ribosome. Ribosomal biogenesis has been studied in great detail by biochemical, genetic and electron microscopic approaches, however, until now live cell data on the in vivo kinetics are still missing. We analysed export kinetics of the large ribosomal subunit (pre-60S particle) through single NPCs in living human cells. To assess the in vivo dynamics of this process, we established a stable cell line co-expressing Halo-tagged eIF6 and GFP-fused NTF2 to simultaneously label ribosomal 60S subunits (eIF6) and NPCs (NTF2). By combining single molecule tracking and super resolution confocal microscopy in a highly customized microscopic setup, we visualized the dynamics of single pre-60S particles during the interaction with and export through single NPCs. In this way we obtained unprecedented insights into this key cellular process. Our results revealed that for export events, maximum particle accumulation is found in the centre of the pore, while unsuccessful export terminates within the nuclear basket. The export process takes place with a single rate limiting step and an export dwell time of ~24 milliseconds. Only about 1/3 of attempted export events were successful. Given the molecular mass of the pre-60S particles our results show that the mass flux through a single NPC can reach up to ~125 MDa s-1 in vivo.

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Section: Tracking Of Single Pre-60s Particles At Single Npcscontrasting

confidence: 72%

Section: Introductionmentioning

confidence: 99%

Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time

Ruland

Krueger

Doerner

et al. 2021

Preprint

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“…The same instrument was employed for fluorescence microscopy based on HILO illumination 64 by reducing the incidence angle of the laser beam in the back focal plane of the objective lens compared to that for TIRF excitation 65 . Illumination using HILO reduced fluorescence excitation to a sheet-like axial section near the coverslip surface resulting in suppression of fluorescence outside the illuminated section.…”

Section: Microscopy Of Bacteriamentioning

confidence: 99%

Ca2+-Daptomycin targets cell wall biosynthesis by forming a tripartite complex with undecaprenyl-coupled intermediates and membrane lipids

et al. 2020

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The lipopeptide daptomycin is used as an antibiotic to treat severe infections with grampositive pathogens, such as methicillin resistant Staphylococcus aureus (MRSA) and drugresistant enterococci. Its precise mechanism of action is incompletely understood, and a specific molecular target has not been identified. Here we show that Ca 2+-daptomycin specifically interacts with undecaprenyl-coupled cell envelope precursors in the presence of the anionic phospholipid phosphatidylglycerol, forming a tripartite complex. We use microbiological and biochemical assays, in combination with fluorescence and optical sectioning microscopy of intact staphylococcal cells and model membrane systems. Binding primarily occurs at the staphylococcal septum and interrupts cell wall biosynthesis. This is followed by delocalisation of components of the peptidoglycan biosynthesis machinery and massive membrane rearrangements, which may account for the pleiotropic cellular events previously reported. The identification of carrier-bound cell wall precursors as specific targets explains the specificity of daptomycin for bacterial cells. Our work reconciles apparently inconsistent previous results, and supports a concise model for the mode of action of daptomycin.

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“… [ 17 ] 28S rRNA C.tentans 2.87 (60%) 0.41 (40%) n.a. [ 21 ] 40S subunit HeLa 2.3 (28%) b 0.31 (39%) b 0.054 (33%) b [ 22 ] 2.3 (7%) c 0.31 (28%) c 0.054 (65%) c 60S subunit HeLa 1.7 n.a. n.a.…”

Section: Transport To the Nuclear Pore Complexesmentioning

confidence: 99%

“…This approach has been successfully used to perform transport experiments on mRNA particles [ 24 ]. Recently, Landvogt et al [ 22 ] succeeded in labeling pre-40S particles using PNO1 (Partner of NOb1, Defective in DNA Methylation 2; DIM2 in yeast [ 25 , 26 ]) coupled C-terminally to a SnapTag. They created a stable cell line carrying an inducible SNAP-PNO1 fusion protein and observed the pre-40S subunits in live cells.…”

Section: Transport To the Nuclear Pore Complexesmentioning

confidence: 99%

Pre-ribosomal particles from nucleoli to cytoplasm

Kubitscheck,

Siebrasse

2024

Nucleus

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Observing and tracking single small ribosomal subunits in vivo

Cited by 8 publications

References 24 publications

Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time

Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time

Ca2+-Daptomycin targets cell wall biosynthesis by forming a tripartite complex with undecaprenyl-coupled intermediates and membrane lipids

Pre-ribosomal particles from nucleoli to cytoplasm

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