|Budget Amount *help
¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1994 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1993 : ¥1,100,000 (Direct Cost : ¥1,100,000)
At the aggregation stage of Dictyostelium discoideum, the signal transduction system depending on cAMP becomes active. We recently have found that cross-bride structures are formed between the outer nuclear membrane and cytoplasmic microtubules (MTs) at this stage. To search molecules composing these structures, microtubule-associated proteins (MAPs) of D.discoideum were examined.Although there were no bands specific to aggregating cells, several common components, 42,52,55,59,72,75,95,220 and 280 kDa bands, were detected on SDS-PAGE.Each component was analyzed with western blotting and their partial amino acid sequences also were examined.
(1) The 55 kDa band is originated from depolymerized porcine tubulin.
(2) The 42 and 95 kDa are actin and an actin-cross linking protein, alpha actinin, respectively. Partial amino acid sequence of the 75 kDa component is similar to HSC70, actin-capping protein.
(3) MTs fraction incubated with Dictyostelium cell sup has been examined by immuno-negative staining. Signals for anti-actin antibody are found on structures attached to the terminal end of MTs. If this is the+end, this type of binding of actin to MTs is interested from a point of view of the relation of MTs and actin systems under the cytoplasmic membranes.
(4) Partial amino acid sequence of the 280 kDa band coincides with human cytokeratin 9. Since this band reproducibly is found and molecular weight of the keratin is 64 kDa, this band seems not to be originated simply from contaminations of human debris during experiments.
(5) Little is known about other bands. It will be possible to determine molecules composing cross-bridge structures through the examination of intracellular distribution using antibody against each band separated on the gels