Inositol 1,4,5-trisphosphate (InsP3) is involved in the mobilization of Ca2+ from intracellular non-mitochondrial stores. In rat liver, it has been shown that the InsP3-binding site co-purifies with the plasma membrane. This suggests that in the liver the InsP3 receptor (InsP3R) associates with plasma membrane. We studied the subcellular distribution of the liver InsP3R by measuring the maximal binding capacity of [3H]InsP3 and using antibodies against the 14 C-terminal residues of the type 1 InsP3R. The antibodies recognized a large amount of an InsP3R protein of 260 kDa in a membrane fraction which is also enriched with [3H]InsP3-binding sites and with markers of the basal, the lateral and the bile-canalicular membrane and the plasma-membrane Ca2+ pump (PMCA). The fractions enriched in markers of the endoplasmic reticulum (ER) and the Ca2+ pump of the ER (SERCA2b) contained low levels of InsP3 receptors. The immunofluorescent labelling of cultured hepatocytes with anti-InsP3R antibodies indicated that the receptor is concentrated in the perinuclear area and in some regions near the plasma membrane. The fraction enriched with InsP3R is also contaminated with markers of the ER and with SERCA2b. It was exposed to alkaline medium (pH 10.5) to extract endogenous actin and membrane-associated proteins before being subfractionated by Percoll-gradient centrifugation. The alkaline treatment allowed partial separation of the markers of the ER from the markers of the plasma membrane. The InsP3R was recovered in the heavy subfraction, which was also enriched with markers for the ER and with the SERCA2b and contained low levels of markers of the plasma membrane. These data indicate that the InsP3R is neither localized on the plasma membrane itself nor homogeneously distributed on the ER membrane. This supports the view that part of the receptor is localized on a specialized sub-region of the ER which interacts with the plasma membrane.

Incorporation of Paramecium axonemal tubulin into lysed endosperm cells of the higher plant Haemanthus enabled us to identify sites of microtubule assembly. This exogenous Paramecium tubulin could be traced by specific antibodies that do not stain endogenous plant microtubules. Intracellular copolymerization of protozoan and higher plant tubulins gave rise to hybrid polymers that were visualized by immunofluorescence and by immunoelectron microscopy. The addition of exogenous tubulin revealed many free ends of endogenous microtubules that were competent to assemble ciliate tubulin. The functional roles of the nuclear surface and the equatorial region of the phragmoplast as plant microtubuleorganizing centers, which were revealed by the intense incorporation of exogenous tubulin, are-discussed. These data shed light on the present debate on higher plant microtubule organizing centers.Temporal and spatial regulation of microtubule (MT) assembly in acentriolar higher plant cells remains poorly understood, because of the lack of data from a functional assay of nucleation capacity that would unambiguously identify plant microtubule-organizing centers (MTOCs). Indirect information has been obtained through immunocytochemical and ultrastructural approaches. Human autoantibodies that stain pericentriolar nucleating material in animal cells (1, 2) decorate the periphery of the nucleus and the spindle poles of higher plant cells (3, 4). However, the significance of this cross-reactivity has been questioned (5). It has also been suggested that sites of MT anchoring, detected by antitubulin labeling (6-8), may correspond to nucleation centers since electron dense material resembling pericentriolar material is usually associated with them (9-12). Our specific aims were to obtain functional evidence that the nuclear envelope in higher plants acts as a MT nucleation center and to study the origin of phragmoplast MT assembly during anaphase-telophase.For this purpose we used a method that is derived from the analysis of MT dynamics and nucleation sites in animal cells in which a "reporter" tubulin is microinjected. The reporter tubulin can either be labeled tubulin (13,14) or tubulin from a distant organism for which a species-specific anti-tubulin antibody is available (15,16). In this context, we have developed an in vitro system of lysed Haemanthus endosperm cells (17) in which unmodified Paramecium axonemal (PA) tubulin is incorporated and then selectively labeled by its homologous antibody (15,18,19). Haemanthus endosperm cells are remarkably suited for investigating plant mitosis and cytoskeleton (6)(7)(8)20), and their lack of cell wall makes them ideal for studies involving permeabilization.In this report, we show (i) the intracellular copolymerization of ciliate and higher plant tubulins leading to hybrid MTs, (ii) the assembly of exogenous tubulin around the plant nucleus in interphase and telophase, and (iii) an intense incorporation of PA tubulin at the cell equator in the early stage of phragmoplast develop...

Abstract. MDCK cells form a polarized epithelium when they reach confluence in tissue culture. We have previously shown that concomitantly with the establishment of intercellular junctions, centrioles separate and microtubules lose their radial organization (Bacallao, R., C. Antony, C. Dotti, E. Karsenti, E. H. K. Stelzer, and K. Simons. 1989. J. Cell Biol. 109:2817-2832. Buendia, B., M. H. Brt, G. Griffiths, and E. Karsenti. 1990Karsenti. . 110:1123Karsenti. -1136. In this work, we have examined the pattern of microtubule nucleation before and after the establishment of intercellular contacts. We analyzed the elongation rate and stability of microtubules in single and confluent cells. This was achieved by microinjection of Paramecium axonemal tubulin and detection of the newly incorporated subunits by an antibody directed specifically against the Paramecium axonemal tubulin. The determination of newly nucleated microtubule localization has been made possible by the use of advanced doubleimmunofluorescence confocal microscopy. We have shown that in single cells, newly nucleated microtubules originate from several sites concentrated in a region localized close to the nucleus and not from a single spot that could correspond to a pair of centrioles. In confluent cells, newly nucleated microtubules were still more dispersed. The microtubule elongation rate of individual microtubules was not different in single and confluent cells (4/xm/min). However, in confluent cells, the population of long lived microtubules was strongly increased. In single or subconfluent cells most microtubules showed a tla of < 30 min, whereas in confluent monolayers, a large population of microtubules had a t~a of >2 h. These results, together with previous observations cited above, indicate that during the establishment of polarity in MDCK cells, microtubule reorganization involves both a relocalization of microtubule-nucleating activity and increased microtubule stabilization.