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

Am J Physiol Cell Physiol. 2001 Dec;281(6):C1858-70. doi: 10.1152/ajpcell.2001.281.6.C1858.


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-PK(10) 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-PK(10) 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. Homogeneous 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-PK(10) 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.

MeSH terms

  • Actins / genetics
  • Actins / metabolism*
  • Adenosine Triphosphate / metabolism*
  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Cell Line
  • Cytoskeleton / metabolism*
  • Cytoskeleton / ultrastructure
  • Immunoblotting
  • Kidney Tubules, Proximal / cytology
  • Kidney Tubules, Proximal / metabolism*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Microscopy, Fluorescence
  • Phalloidine / chemistry
  • Phalloidine / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Stress Fibers / metabolism
  • Swine


  • Actins
  • Bacterial Proteins
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • yellow fluorescent protein, Bacteria
  • Phalloidine
  • Adenosine Triphosphate