A comparison of techniques for localizing actin and tubulin in hyphae of Saprolegnia ferax.
We have evaluated protocols for immunofluorescence (IF) staining of the potentially interacting actin filaments (F-actin) and microtubules in hyphae of Saprolegnia ferax, using rhodamine-phalloidin (RP) and freeze-substitution electron microscopy (FSEM), respectively, as standards for their distribution. Saprolegnia has four distinguishable cortical F-actin populations with characteristic organizations and RP- and actin-IF-staining affinities, all of which could be labeled with both probes after some protocols. Other protocols stained only some of the populations. Cortical F-actin was always more reproducibly and sharply stained with RP than IF, indicating that the former is the probe of choice for F-actin in these cells. Although no single IF protocol revealed all of the F-actin and microtubule populations, showing the potential need to optimize protocols for specific antibodies, simultaneous localization was readily achieved by dual labeling with RP and tubulin IF. Tubulin IF patterns differed from FSEM: mitotic spindles were revealed but not the more abundant prophase microtubule arrays, and the cytoplasmic microtubules were subapically displaced and bundled into long cables. These cables, which apparently linked nuclei, indicate a previously undetected involvement in nuclear spacing. The tubulin antibody successfully used for IF failed to recognize any proteins in immunoblots, indicating that immunoblots may not always be a useful indicator of success with IF.
Kaminskyj
S G
SG
Department of Biology, York University, North York, Ontario, Canada.
Heath
I B
IB
D000199:Actins / Q000302:isolation & purification*; D003599:Cytoskeleton / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D017110:Freeze Substitution; D006651:Histocytochemistry / Q000379:methods*; D008854:Microscopy, Electron; D009868:Oomycetes / Q000737:chemistry* / Q000648:ultrastructure; D010590:Phalloidine; D012235:Rhodamines; D013194:Staining and Labeling; D014404:Tubulin / Q000302:isolation & purification*
D000199:Actins; D012235:Rhodamines; D014404:Tubulin; D010590:Phalloidine