Characteristics of EYFP-actin and visualization of actin dynamics during ATP depletion and repletion.
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.
Herget-Rosenthal
S
S
Indiana Center for Biological Microscopy, Division of Nephrology, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Hosford
M
M
Kribben
A
A
Atkinson
S J
SJ
Sandoval
R M
RM
Molitoris
B A
BA
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D015151:Immunoblotting; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010590:Phalloidine / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022502:Stress Fibers / Q000378:metabolism; D013552:Swine
D000199:Actins; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D010590:Phalloidine; D000255:Adenosine Triphosphate