Insulin-like growth factor binding protein-4 interacts with centrosomes and microtubules in primary astrocytes.
Insulin-like growth factor-1 and -2 (IGFs) are important for CNS development and have implications in pathological situations of the brain. Insulin-like growth factor binding protein-4 (IGFBP-4) regulates the biological effects of IGFs. We examined the expression of IGFBP-4 in primary rat and human astrocytes. IGFBP-4 mRNA was detectable by reverse transcription-polymerase chain reaction (RT-PCR) and protein expression was verified by Western blotting of cell lysates as well as conditioned culture medium. When astrocytes were immunostained for IGFBP-4 we detected an intracellular structure that did not derive from organelles involved in cellular trafficking. Ingestion of fluorescein isothiocyanate-labeled transferrin excluded detection of IGFBP-4 within vesicles of endosomal nature, in which the protein might have been incorporated. Double staining with gamma-tubulin demonstrated co-localization of IGFBP-4 with centrosomes of these cells. Treatment with nocodazole resulted in absence of IGFBP-4 signal on centrosomes, indicating a dependency on intact microtubules. Immunoelectron microscopy revealed IGFBP-4 localization not only at the centrioles but also a direct interaction with microtubules. There was no binding of IGFBP-4 to centrioles in primary rat oligodendrocytes, microglia or meningeal cells. The association of IGFBP-4 with centrioles and microtubules in astrocytes suggests an involvement of this molecule in microtubule functions of these cells.
Chesik
D
D
Department of Neurology, Academic Hospital Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands. d.chesnik@med.rug.nl
Wilczak
N
N
De Keyser
J
J
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D015153:Blotting, Western / Q000379:methods; D018385:Centrosome / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002540:Cerebral Cortex / Q000166:cytology; D016650:Fluorescein-5-isothiocyanate / Q000493:pharmacokinetics; D005455:Fluorescent Antibody Technique / Q000379:methods; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron / Q000379:methods; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D015739:Nocodazole / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014168:Transferrin / Q000494:pharmacology; D014404:Tubulin / Q000378:metabolism
D018974:Insulin-Like Growth Factor Binding Protein 4; D012333:RNA, Messenger; D014168:Transferrin; D014404:Tubulin; D016650:Fluorescein-5-isothiocyanate; D015739:Nocodazole