Characteristics of EYFP-actin and visualization of actin dynamics during ATP depletion and repletion

Stefan Herget-Rosenthal, Melanie Hosford, Andreas Kribben, Simon J. Atkinson, Ruben M. Sandoval, Bruce Molitoris

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Disruption of the actin cytoskeleton in proximal tubule cells is a key pathophysiological factor in acute renal failure. To investigate dynamic alterations of the actin cytoskeleton in live proximal tubule cells, LLC-PK10 cells were transfected with an enhanced yellow fluorescence protein (EYFP)-actin construct, and a clone with stable EYFP-actin expression was established. Confluent live cells were studied by confocal microscopy under physiological conditions or during ATP depletion of up to 60 min. Immunoblots of stable transfected LLC-PK10 cells confirmed the presence of EYFP-actin, accounting for 5% of total actin. EYFP-actin predominantly incorporated in stress fibers, i.e., cortical and microvillar actin as shown by excellent colocalization with Texas red phalloidin. Homogenous cytosolic distribution of EYFP-actin indicated colocalization with G-actin as well. Beyond previous findings, we observed differential subcellular disassembly of F-actin structures: Stress fibers tagged with EYFP-actin underwent rapid and complete disruption, whereas cortical and microvillar actin disassembled at slower rates. In parallel, ATP depletion induced the formation of perinuclear EYFP-actin aggregates that colocalized with F-actin. During ATP depletion the G-actin fraction of EYFP-actin substantially decreased while endogenous and EYFP-F-actin increased. During intracellular ATP repletion, after 30 min of ATP depletion, there was a high degree of agreement between F-actin formation from EYFP-actin and endogenous actin. Our data indicate that EYFP-actin did not alter the characteristics of the endogenous actin cytoskeleton or the morphology of LLC-PK10 cells. Furthermore, EYFP-actin is a suitable probe to study the spatial and temporal dynamics of actin cytoskeleton alterations in live proximal tubule cells during ATP depletion and ATP repletion.

Original languageEnglish
JournalAmerican Journal of Physiology - Cell Physiology
Volume281
Issue number6 50-6
StatePublished - 2001

Fingerprint

Actins
Visualization
Adenosine Triphosphate
Fluorescence
Proteins
Actin Cytoskeleton
Stress Fibers
Phalloidine
Acute Kidney Injury
Confocal Microscopy
Fibers
Confocal microscopy

Keywords

  • Actin cytoskeleton
  • Enhanced yellow fluorescent protein
  • Green fluorescent protein
  • Live imaging
  • Renal proximal tubule cell

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology
  • Physiology (medical)

Cite this

Characteristics of EYFP-actin and visualization of actin dynamics during ATP depletion and repletion. / Herget-Rosenthal, Stefan; Hosford, Melanie; Kribben, Andreas; Atkinson, Simon J.; Sandoval, Ruben M.; Molitoris, Bruce.

In: American Journal of Physiology - Cell Physiology, Vol. 281, No. 6 50-6, 2001.

Research output: Contribution to journalArticle

Herget-Rosenthal, Stefan ; Hosford, Melanie ; Kribben, Andreas ; Atkinson, Simon J. ; Sandoval, Ruben M. ; Molitoris, Bruce. / Characteristics of EYFP-actin and visualization of actin dynamics during ATP depletion and repletion. In: American Journal of Physiology - Cell Physiology. 2001 ; Vol. 281, No. 6 50-6.
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AU - Herget-Rosenthal, Stefan

AU - Hosford, Melanie

AU - Kribben, Andreas

AU - Atkinson, Simon J.

AU - Sandoval, Ruben M.

AU - Molitoris, Bruce

PY - 2001

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N2 - Disruption of the actin cytoskeleton in proximal tubule cells is a key pathophysiological factor in acute renal failure. To investigate dynamic alterations of the actin cytoskeleton in live proximal tubule cells, LLC-PK10 cells were transfected with an enhanced yellow fluorescence protein (EYFP)-actin construct, and a clone with stable EYFP-actin expression was established. Confluent live cells were studied by confocal microscopy under physiological conditions or during ATP depletion of up to 60 min. Immunoblots of stable transfected LLC-PK10 cells confirmed the presence of EYFP-actin, accounting for 5% of total actin. EYFP-actin predominantly incorporated in stress fibers, i.e., cortical and microvillar actin as shown by excellent colocalization with Texas red phalloidin. Homogenous cytosolic distribution of EYFP-actin indicated colocalization with G-actin as well. Beyond previous findings, we observed differential subcellular disassembly of F-actin structures: Stress fibers tagged with EYFP-actin underwent rapid and complete disruption, whereas cortical and microvillar actin disassembled at slower rates. In parallel, ATP depletion induced the formation of perinuclear EYFP-actin aggregates that colocalized with F-actin. During ATP depletion the G-actin fraction of EYFP-actin substantially decreased while endogenous and EYFP-F-actin increased. During intracellular ATP repletion, after 30 min of ATP depletion, there was a high degree of agreement between F-actin formation from EYFP-actin and endogenous actin. Our data indicate that EYFP-actin did not alter the characteristics of the endogenous actin cytoskeleton or the morphology of LLC-PK10 cells. Furthermore, EYFP-actin is a suitable probe to study the spatial and temporal dynamics of actin cytoskeleton alterations in live proximal tubule cells during ATP depletion and ATP repletion.

AB - Disruption of the actin cytoskeleton in proximal tubule cells is a key pathophysiological factor in acute renal failure. To investigate dynamic alterations of the actin cytoskeleton in live proximal tubule cells, LLC-PK10 cells were transfected with an enhanced yellow fluorescence protein (EYFP)-actin construct, and a clone with stable EYFP-actin expression was established. Confluent live cells were studied by confocal microscopy under physiological conditions or during ATP depletion of up to 60 min. Immunoblots of stable transfected LLC-PK10 cells confirmed the presence of EYFP-actin, accounting for 5% of total actin. EYFP-actin predominantly incorporated in stress fibers, i.e., cortical and microvillar actin as shown by excellent colocalization with Texas red phalloidin. Homogenous cytosolic distribution of EYFP-actin indicated colocalization with G-actin as well. Beyond previous findings, we observed differential subcellular disassembly of F-actin structures: Stress fibers tagged with EYFP-actin underwent rapid and complete disruption, whereas cortical and microvillar actin disassembled at slower rates. In parallel, ATP depletion induced the formation of perinuclear EYFP-actin aggregates that colocalized with F-actin. During ATP depletion the G-actin fraction of EYFP-actin substantially decreased while endogenous and EYFP-F-actin increased. During intracellular ATP repletion, after 30 min of ATP depletion, there was a high degree of agreement between F-actin formation from EYFP-actin and endogenous actin. Our data indicate that EYFP-actin did not alter the characteristics of the endogenous actin cytoskeleton or the morphology of LLC-PK10 cells. Furthermore, EYFP-actin is a suitable probe to study the spatial and temporal dynamics of actin cytoskeleton alterations in live proximal tubule cells during ATP depletion and ATP repletion.

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KW - Enhanced yellow fluorescent protein

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KW - Live imaging

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