Cytoskeletal architecture in mouse lung epithelial cells is regulated by protein-kinase C-alpha and calpain II.

Brief exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a uniformly flattened population of mouse lung epithelial cells to become more heterogeneous; some cells rounded up, and others detached to overlap with flatter cells. Actin stress fiber organization was disrupted, and F-actin accumulated in lemellipodia. Vinculin dissociated from the focal adhesion plaques to diffuse throughout the cytoplasm. Inhibition of protein kinase C (PKC) activity blocked these effects of TPA. After 8 h of TPA exposure, actin filaments reassembled and vinculin again localized to the cell periphery. Calpain inhibition attenuated the decrease of PKC-alpha protein and PKC activity from the membrane fraction, and prevented the redistribution of cytoskeletal elements. Talin immunostaining was widespread throughout control cells but was localized to the periphery 8 h after treatment with TPA or with inhibitors of PKC and calpain. Both vinculin and talin concentrations increased with prolonged TPA treatment. PKC-zeta and calpain II were not appreciably affected by TPA exposure. Translocation of PKC-alpha to the membrane, followed by its calpain-induced downmodulation, is apparently required for the reversible pattern of cytoskeletal changes caused by TPA.

Blot overlay identification of microtubule-binding peptides from bovine brain.

Microtubule (MT)-binding peptides have been detected in homogenates of bovine brain tissue utilizing a blot overlay assay. Blots were prepared by the electrophoretic transfer to nitrocellulose of proteins separated on polyacrylamide gels. These blots were incubated with taxol stabilized MTs or tubulin, rinsed, and then fixed by air drying. About 17 soluble MT-associated proteins (MAPs) were identified by immunodetection of bound tubulin, including MAP2, kinesin, and tau. The interaction of MTs with these peptides appears to be specific, since MT binding can be displaced by a fluorescent tubulin analog, is competitively inhibited by the addition of exogenous brain MAPs, is decreased by raising the salt concentration, and is diminished by sodium dodecyl sulfate (SDS) denaturation. Only one protein (150 kDa) appears to have an interaction with MTs that is stable in high salt. The specificity of the binding on blots is further illustrated by the interaction of MTs with the MT-binding domains of MAP2 (32-35 kDa fragments) and kinesin (64 kDa fragment). Specific MT-binding peptides or domains can thus be isolated and characterized with this method, which requires little protein and is suitable for use with proteins that are either soluble or insoluble under physiological conditions.

Microtubule-associated proteins from Antarctic fishes.

Microtubules and presumptive microtubule-associated proteins (MAPs) were isolated from the brain tissues of four Antarctic fishes (Notothenia gibberifrons, N. coriiceps neglecta, Chaenocephalus aceratus, and a Chionodraco sp.) by means of a taxol-dependent, microtubule-affinity procedure (cf. Vallee: Journal of Cell Biology 92:435-442, 1982). MAPs from these fishes were similar to each other in electrophoretic pattern. Prominent in each preparation were proteins in the molecular weight ranges 410,000-430,000, 220,000-280,000, 140,000-155,000, 85,000-95,000, 40,000-45,000, and 32,000-34,000. The surfaces of MAP-rich microtubules were decorated by numerous filamentous projections. Exposure to elevated ionic strength released the MAPs from the microtubules and also removed the filamentous projections. Addition of fish MAPs to subcritical concentrations of fish tubulins at 0-5 degrees C induced the assembly of microtubules. Both the rate and the extent of this assembly increased with increasing concentrations of the MAPs. Sedimentation revealed that approximately six proteins, with apparent molecular weights between 60,000 and 300,000, became incorporated into the microtubule polymer. Bovine MAPs promoted microtubule formation by fish tubulin at 2-5 degrees C, and proteins corresponding to MAPs 1 and 2 co-sedimented with the polymer. MAPs from C. aceratus also enhanced the polymerization of bovine tubulin at 33 degrees C, but the microtubules depolymerized at 0 degrees C. We conclude that MAPs are part of the microtubules of Antarctic fishes, that these proteins promote microtubule assembly in much the same way as mammalian MAPs, and that they do not possess special capacities to promote microtubule assembly at low temperatures or to prevent cold-induced microtubule depolymerization.

Cell cycle-dependent changes in the dynamics of MAP 2 and MAP 4 in cultured cells.

To examine the behavior of microtubule-associated proteins (MAPs) in living cells, MAP 4 and MAP 2 have been derivatized with 6-iodoacetamido-fluorescein, and the distribution of microinjected MAP has been analyzed using a low light level video system and fluorescence redistribution after photobleaching. Within 1 min following microinjection of fluoresceinated MAP 4 or MAP 2, fluorescent microtubule arrays were visible in interphase or mitotic PtK1 cells. After cold treatment of fluorescent MAP 2-containing cells (3 h, 4 degrees C), microtubule fluorescence disappeared, and the only fluorescence above background was located at the centrosomes; microtubule patterns returned upon warming. Loss of microtubule immunofluorescence after nocodozole treatment was similar in MAP-injected and control cells, suggesting that injected fluorescein-labeled MAP 2 did not stabilize microtubules. The dynamics of the MAPs were examined further by FRAP. FRAP analysis of interphase cells demonstrated that MAP 2 redistributed with half-times slightly longer (60 +/- 25 s) than those for MAP 4 (44 +/- 20 s), but both types of MAPs bound to microtubules in vivo exchanged with soluble MAPs at rates exceeding the rate of tubulin turnover. These data imply that microtubules in interphase cells are assembled with constantly exchanging populations of MAP. Metaphase cells at 37 degrees C or 26 degrees C showed similar mean redistribution half-times for both MAP 2 and MAP 4; these were 3-4 fold faster than the interphase rates (MAP 2, t1/2 = 14 +/- 6 s; MAP 4, t1/2 = 17 +/- 5 s). The extent of recovery of spindle fluorescence in MAP-injected cells was to 84-94% at either 26 or 37 degrees C. Although most metaphase tubulin, like the MAPs, turns over rapidly and completely under physiologic conditions, published work shows either reduced rates or extents of turnover at 26 degrees C, suggesting that the fast mitotic MAP exchange is not simply because of fast tubulin turnover. Exchange of MAP 4 bound to telophase midbodies occurred with dynamics comparable to those seen in metaphase spindles (t1/2 = approximately 27 s) whereas midbody tubulin exchange was slow (greater than 300 s). These data demonstrate that the rate of MAP exchange on microtubules is a function of time in the cell cycle.

Localization of kinesin in cultured cells.

Kinesin was isolated from bovine brain and used to elicit polyclonal antibodies in rabbits. The specificities of the resulting antibodies were evaluated by immunoblotting. Antibodies purified from these sera by their affinity for brain kinesin react with a polypeptide of approximately 120 kD in extracts from bovine brain, PtK1 cells, and mouse neuroblastoma cells. They bind to a pair of polypeptides of approximately 120 kD present in crude kinesin prepared from Xenopus eggs and with a single polypeptide of approximately 115 kD in extracts from Drosophila embryos. Antibodies raised against kinesin prepared from fruit fly embryos (by W. M. Saxton, Indiana University, Bloomington, IN) and from neural tissues of the squid (by M. P. Sheetz, Washington University, St. Louis, MO) cross react with the mammalian, the fly, and the frog polypeptides. Kinesin antigen was localized in cultured cells by indirect immunofluorescence. PtK1 cells in interphase showed dim background staining of cytoplasmic membranous components and bright staining of a small, fibrous, juxtanuclear structure. Double staining with antibodies to microtubules showed that the fibrous object was usually located near the centrosome. On the basis of shape, size, and location, we identify the kinesin-positive structure as a primary cilium. PtK1 cells in mitosis are stained at their poles during all stages of division. The structure stained is approximately spherical, but wisps of faint fluorescence also extend into the body of the spindle. Antibodies to squid or fruit fly kinesin produce identical patterns in PtK1 cells. Controls with preimmune and preabsorbed sera show that the centrosome staining is not due simply to the common tendency of rabbit antisera to stain this structure. Similar centrosome and spindle pole staining was visible when antibodies to bovine brain or squid kinesin were applied to the A6 cell line (kidney epithelial cells from Xenopus laevis). Some possible functions of kinesin localized at the spindle poles are discussed.