Improved procedures for electron microscopic visualization of the cytoskeleton of cultured cells.
We have developed an improved electron microscopic procedure appropriate for correlative light and electron microscopy of the cytoskeleton. The procedure is based on detergent extraction, chemical fixation, critical point drying, and platinum/carbon coating of cultured cells and the improvements consist of modifications which are minor individually but collectively of substantial impact. They are: inclusion of polyethylene glycol into the extraction medium; cell lysis at room temperature; fixation by sequential application of glutaraldehyde, tannic acid, and uranyl acetate; horizontal position of specimens during dehydration and drying; and uranyl acetate treatment during dehydration. As a result, we have obtained a greatly improved quality of electron microscopic images together with a high consistency of results. Long and straight actin filaments were clearly seen in stress fibers and newly formed lamellipodia. Their polarity was distinctly revealed by decoration with myosin subfragment 1. Depletion of actin from cytoskeletons by gelsolin treatment allowed for better visualization of myosin, intermediate filaments, and microtubules. Intermediate filaments exposed by this treatment exhibited numerous side projections in a hitherto unreported millipede-like appearance. The suggested procedure was compatible with immunogold labeling as demonstrated with an antibody to tubulin. Correlative light and electron microscopy of cells microinjected with a fluorescent derivative of myosin II was reliable and efficient, producing a close resemblance between the two kinds of images.
Svitkina
T M
TM
A. N. Belozersky Institute of Physical and Chemical Biology, Moscow State University, Russia.
Verkhovsky
A B
AB
Borisy
G G
GG
D016475:3T3 Cells / Q000648:ultrastructure; D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000199:Actins / Q000648:ultrastructure; D000818:Animals; D002244:Carbon; D002478:Cells, Cultured / Q000648:ultrastructure*; D002522:Chlorocebus aethiops; D003599:Cytoskeleton / Q000648:ultrastructure*; D003890:Desiccation / Q000295:instrumentation / Q000379:methods; D003902:Detergents; D005347:Fibroblasts / Q000648:ultrastructure; D005456:Fluorescent Dyes; D018260:Gelsolin; D005976:Glutaral; D047348:Hydrolyzable Tannins; D007150:Immunohistochemistry; D007382:Intermediate Filaments / Q000648:ultrastructure; D007668:Kidney; D051379:Mice; D008845:Microinjections; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000648:ultrastructure; D009218:Myosins / Q000648:ultrastructure; D009942:Organometallic Compounds; D010984:Platinum; D011092:Polyethylene Glycols; D051381:Rats; D016628:Replica Techniques; D012235:Rhodamines; D016626:Shadowing Technique, Histology; D013048:Specimen Handling; D016707:Tissue Fixation; D014404:Tubulin / Q000276:immunology
D000199:Actins; D003902:Detergents; D005456:Fluorescent Dyes; D018260:Gelsolin; D047348:Hydrolyzable Tannins; D009942:Organometallic Compounds; D012235:Rhodamines; D014404:Tubulin; C005460:uranyl acetate; D011092:Polyethylene Glycols; D010984:Platinum; C005358:tetramethylrhodamine; D002244:Carbon; D009218:Myosins; D005976:Glutaral