Binding of microtubules to pituitary secretory granules and secretory granule membranes.

Abstract

Microtubules assembled in vitro were bound to purified porcine pituitary secretory granules and to isolated granule membranes. The interaction between microtubules and whole secretory granules was demonstrated by alteration in the sedimentation properties of the microtubules. Incubation of secretory granules with mierotubules resulted in pelleting of microtubules which increased as a function of the number of granules added. Binding was quantitated by measurement of the tubulin remaining in the supernate after centrifugation. The interaction of secretory granules and microtubules was inhibited by nucleoside triphosphates and augmented by adenosine 5'-monophosphate and adenosine. When depolymerized protein from microtubules was incubated with secretory granules, the granules did not appear to bind the soluble tubulin dimer present in these preparations. However, the high molecular weight protein associated with microtubules was adsorbed by secretory granules during the binding process. Incubation of isolated secretory granule membranes with microtubules followed by centrifugation to density equilibrium in a discontinuous sucrose density gradient caused pelleting of the membranes, which otherwise banded higher in the gradient. The visible alteration in membrane sedimentation was confirmed by measurements of the membrane-associated magnesium-ATPase activity and by a shift in radioactivity in iodinated membrane preparations. Our data suggest a role for microtubules in the intracellular movement of secretory granules; this movement is perhaps brought about by dynein-like cross bridges which link the tubulin backbone and granule surface.In recent years, numerous studies have suggested that microtubule integrity is essential for normal secretion to occur (8, 11, 16, 22-24, 27, 28, 42, 43, 48). The bulk of the evidence for this proposition rests upon the ability of the microtubuledisruptive agents vinblastine and colchicine (49) to block the release of a variety of secretory products. Despite the general agreement that microtubules are necessary for secretion, their exact role is unclear.Those cells which release proteins, peptides, and neurotransmitters package the product in the Golgi apparatus within membrane-bounded vesicles or granules. Upon the appropriate stimulus, the granule membrane fuses with the cell membrane and exocytosis occurs. Movement of score-

Microtubule-associated proteins (MAPs) and the organization of actin filaments in vitro.

“…Some recent studies have shown that MAPS are necessary for the associations of secretary granule membranes and microtubules in vitro (32,38) and it also has been shown that a high molecular weight protein is associated with the bridge observed to link ciliary outer-doublet microtubules to the ciliary membrane in vivo (8). Other studies have shown that only a small portion of the MAP-2 molecule is necessary for the observed stimulation of microtubule assembly in vitro by MAPS (18,34,41).…”

A Molecular Description of Nerve Terminal Function

“…There is ample support biochemically for direct interactions between secretory vesicles and actin (293)(294)(295) and microtubules (296). The latter interactions may involve microtubules associated proteins (297).…”

Cellular control of renin secretion

“…Since a typical secretory process requires the movement of secretory granules from within the cell to the cellular plasma membrane, there is speculation that the cytosolic part of the cytoskeleton is involved in this process (Lacy et al 1968;Sherline et al 1977;Hoffstein and Weissmann 1978). The subplasmalemmal part of the cytoskeleton, however, is considered to have an inhibitory role in secretion (Burgoyne et al , 1988.…”