Altered sensitivity of posttranslationally modified microtubules to methylmercury in differentiating embryonal carcinoma-derived neurons.
The effects of methylmercury (MeHg) on microtubules (MTs) in differentiating neurons derived from retinoic acid-induced embryonal carcinoma (EC) cells in culture were examined by immunofluorescence microscopy. Undifferentiated EC cells contained mostly kinetically labile tyrosinated (TYR) MTs which extended from the centrosome and a small population of stable acetylated (ACT) MTs usually (but not exclusively) associated with the centrosome. TYR MTs of undifferentiated cells underwent concentration- and time-dependent disassembly upon exposure to low concentrations of MeHg (1-2 microM), whereas ACT MTs were resistant to MeHg at low concentrations, with many remaining intact even in 5 microM MeHg. After 2 days in retinoic acid the appearance of short neuritic processes was indicative of early differentiation. TYR MTs predominated both in the short neurites and cell soma and remained susceptible to low concentrations of MeHg. ACT MTs also extended into the short neurites but were most often found in small bundles within the perikarya. ACT MTs remained more resistant to MeHg than TYR MTs. The neuron-specific tubulin isotype betaIII was first detected during the second day of differentiation. MTs labeled with antibodies to betaIII showed similar sensitivity to MeHg as TYR MTs, suggesting that MTs containing betaIII were largely tyrosinated. After 4 and 6 days of differentiation a greater number of betaIII-positive neurons were present with progressively longer and often branching neurites. TYR MTs were present in cell soma and neurites while ACT MTs were found almost exclusively in neurites. TYR MTs remained highly susceptible to MeHg (most notably in perikarya) in comparison to ACT MTs at all stages of differentiation; however, with increasing time in culture, even TYR MTs gained appreciable stability to MeHg. These data indicate that microtubules in developing neurons become progressively more resistant to disassembly by MeHg, suggesting that the most critical period of MT susceptibility occurs very early in development in vivo.
Graff
R D
RD
Joint Graduate Program in Toxicology, Rutgers College of Pharmacy, Piscataway, New Jersey 08854, USA.
Falconer
M M
MM
Brown
D L
DL
Reuhl
K R
KR
D000911:Antibodies, Monoclonal / Q000032:analysis; D018236:Carcinoma, Embryonal / Q000473:pathology*; D002454:Cell Differentiation / Q000187:drug effects*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008767:Methylmercury Compounds / Q000633:toxicity*; D008870:Microtubules / Q000737:chemistry / Q000187:drug effects* / Q000473:pathology; D009474:Neurons / Q000737:chemistry / Q000187:drug effects* / Q000473:pathology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D008767:Methylmercury Compounds