A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATP-dependent manner: a new chaperone role is proposed.
In the present study, double immunofluorescence and immunoblot analysis have been used to show that centrosomes, isolated from Paracentrotus lividus sea urchin embryos at the first mitotic metaphase, contain the constitutive chaperone, heat-shock protein (HSP) 70. More specifically, we demonstrate that centrosomes contain only the HSP70-d isoform, which is one of the four isoforms identified in P. lividus. We also provide evidence that p34(cell division control kinase-2) and t complex polypeptide-1 (TCP-1) alpha, a subunit of the TCP-1 complex, are localized on the centrosomes. Furthermore, inhibition of TCP-1 in vivo, via microinjecting an anti-(TCP-1 alpha) antibody into P. lividus eggs before fertilization, either impaired mitosis or induced severe malformations in more than 50% of embryos. In addition, we have isolated the whole mitotic apparatus and shown that HSP70 localizes on the fibres of spindles and asters, and binds them in an ATP-dependent manner. These observations suggest that HSP70 has a chaperone role in assisting the TCP-1 complex in tubulin folding, when localized on centrosomes, and during the assembling and disassembling of the mitotic apparatus, when localized on the fibres of spindles and asters.
Agueli
C
C
Dipartimento di Biologia Cellulare e dello Sviluppo A. Monroy, Università di Palermo, Viale delle Scienze, 90128 Palermo, Italy.
Geraci
F
F
Giudice
G
G
Chimenti
L
L
Cascino
D
D
Sconzo
G
G
D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002458:Cell Fractionation; D018385:Centrosome / Q000737:chemistry; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000378:metabolism; D046508:Culture Techniques; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D019085:Fluorescent Antibody Technique, Direct; D019084:Fluorescent Antibody Technique, Indirect; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D008677:Metaphase / Q000502:physiology*; D020033:Protein Isoforms / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D012617:Sea Urchins / Q000166:cytology* / Q000196:embryology / Q000378:metabolism / Q000502:physiology*; D008941:Spindle Apparatus / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D014404:Tubulin / Q000378:metabolism
D018840:HSP70 Heat-Shock Proteins; D020033:Protein Isoforms; D014404:Tubulin; D000255:Adenosine Triphosphate; D016203:CDC2 Protein Kinase; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins