摘要:

AbstractA physical model developed to explain microtubule sliding patterns in the trypsintreated ciliary axoneme has been extended to investigate the generation of bending moments by microtubules sliding in an axoneme in which the dublets are anchored at one end. With sliding restricted, a bending moment is developed by the polarized shearing interaction between neighbouring doublets, effected by the activity of dynein arms on doublet N pushing N + 1 in a tipward ( + ) direction. In arrested axonemes in which arms on several contiguous doublets are active, the bending moment causes splitting of the 9 + 2 microtubule array into two or more sets of doublets. In the absence of special constraints, splitting depends only on breaking the circumferential interdoublet links most distorted by the bending moment. The analysis, which permits assignment of arm activity to specific microtubules in each of the observed patterns of splitting, indicates that the axoneme will split between doublet N and N + 1 if arms on doublet N are inactive and arms on either N + 1 or N−1 are active. To produce the observed major splits, dynein arms on the microtubules of roughly one‐half of the axoneme are predicted to be active, in a manner consistent with the switch‐point hypothesis of ciliary motion. Electron microscopic examination indicates that virtually every set of doublets in the split axonemes retains its cylindrical form. Maintenance of cylindrical symmetry can be ascribed to the mechanical properties of the unbroken links, which may resist both tensile and compressive stress, and to active dynein arms. © 1994 Wiley‐L

ISSN:0886-1544    

DOI:10.1002/cm.970270402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThis study considers the relationship between two structural forms of the 22S dynein arm ofTetrahymena thermophila:the bouquet and the compact arm. The compact arm differs from the bouquet and from other proposed forms (e.g., the “toadstool”) in that the globular domains are situated transversely across the interdoublet gap with one globular subunit, the head, proximal to the adjacent doublet microtubule. The other models place all three globular domains proximal to the neighboring doublet microtubule. When sliding of an isolated axoneme is induced, at least 57% of total attached arms on exposed doublets are in the compact form within dimensions of 24 × 24 × 12 nm, and only about 2% of the arms are bouquets. Toadstools are incompatible with the images seen. Bouquets are not found in regions of the doublet protected by a neighboring doublet. When axonemes with exposed doublets are treated with 0.5 M KCl for 30 min, the compct arms and the dynein heavy (H)‐chains disappear, while isolated bouquets and dynein H‐chains appear in the medium, suggesting that the compact arms give rise to the bouquets as they are solubilized. The bouquet is the predominant form of isolated 22S dynein molecules, which are found in two apparently enantiomorphic forms, within dimensions 45 × 39 × 13 nm; bouquets attached to doublets have dimensions similar to those of isolated bouquets. Computer modeling indicates that in an intact standard‐diameter axoneme, these dimensions are incompatible with the interdoublet volume available for an arm; the bouquet therefore represents an unfolded compact arm. A plausible sequence of changes can be modeled to illustrate the conversion of an attached compact arm to an attached and then free bouquet. The toadstool is probably an artifact that arises after unfolding. Consistent with the conformational difference, H‐chains of attached compact arms differ from those of isolated bouquets in their susceptibility to limited proteolysis. These results suggest that the compact arm, rather than the unfolded bouquet or the toadstool, is the functional form of the outer arm in the intact axoneme. © 1994

ISSN:0886-1544    

DOI:10.1002/cm.970270403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractManipulation of the single conventional myosin heavy chain (mhc) gene inDictyostelium discoideum(Dd) has delineated an essential role for the filament‐forming, or light meromyosin (LMM) domain of the myosin molecule in cyto‐kinesis, development, and in the capping of cell surface receptors (see Spudich:Cell Regulation1:1–11, 1989; Egelhoff et al.:Journal of Cell Biology, 112:677–688, 1991a). In order to assess the functional relationship between sarcomeric and cytoplasmic myosins, a chimeric gene encoding theDdmyosin head and subfragment 2 fused to rat β cardiac LMM was transfected into both wild‐type andDdmhc null cells. Chimeric myosin was organized into dense cortical patches in the cytoplasm of both wild‐type andDdmhc null cells. Although null cells expressing chimeric mhc at ∼10% ofDdmhc levels were unable to grow in shaking suspension or to complete development, chimeric myosin was able to rescue capping of cell surface receptors, to associate with filamentous actin, and to localize to the correct subcellular position during aggregation. Deletion of 29 amino acids in the rod corresponding to a previously defined filament assembly competent region eliminated the cortical patches and the posterior localization during chemotaxis. Taken together, these observations suggest that sarcomeric and cytoplasmic myosin rods are functionally interchangeable in several aspects of nonmuscle motility. © 1994

ISSN:0886-1544    

DOI:10.1002/cm.970270404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractHuman erythroleukemia (HEL) cells grow in suspension, but after treatment with nM PMA the cells adhere and spread on glass or fibronectin [Jarvinen et al., 1987:Eur. J. Cell Biol.44:238–246]. We observed an early (20–30 min) stage of spreading in which F‐actin was organized into peripheral arcs near the spreading margin and vinculin was localised to the cell's periphery at the ends of these arcs. By 1 h the cells were well spread with straight actin bundles many of which ended at more central sites terminating on patches containing vinculin and talin; thus the cells assemble typical stress fibers but do not appear to polarize. The cells also spread on RGD polymer. DiC8 (1,2‐dioctanoyl‐sn‐glycerol, C8:0, Sigma Chemical Co., St. Louis, MO) induced spreading but only if DAG kinase inhibitor and A‐23187 were also present; in their absence cells adhered but did not spread. Spreading was ∼85% inhibited by 100 nM staurosporine. PKC‐β was shown to be present in the cells by immunoblotting. In cells spread for 1 h with PMA, F‐actin increased to 180% of control levels as measured by RP binding and the actin sequestering complex of G‐actin‐thymosin β4decreased significantly.To determine whether the F‐actin increase required adhesion, we inhibited cell attachment to the substratum by adding RGDS, by coating glass surfaces with hemoglobin, or by a combined treatment. Under these conditions PMA‐treated suspended cells still increased their F‐actin to 126–137% of controls, a significant increase over control levels. Staurosporine inhibited F‐actin increases under all the conditions studied.Permeabilized cell suspensions, incubated with rhodamine labelled G‐actin, incorporated the labelled actin along cell membranes at a low level. A few minutes preincubation with either diC8 plus DAG kinase inhibitor or with PMA strongly increased the incorporation. This increased incorporation was reduced to below control levels by either staurosporine (100 nM) or cytochalasin D (1 μM).We conclude that both suspended and spreading HEL cells can be stimulated to polymerize actin by a mechanism dependent on PKC or a PKC‐like molecule. In suspended cells, the polymerization occurs along the membrane. When cells spread, F‐actin increased to a significantly greater extent. This second step could involve additional polymerization, perhaps at the observed adhesion sites, decreased turnover of the actin bundles, or a combined effect

ISSN:0886-1544    

DOI:10.1002/cm.970270405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractMicrotubular networks are extensively developped in many ciliate species. In several of them, we investigate the occurrence of the post‐translational glutamylation of tubulin [Eddé et al., 1990:Science247:82–85; Eddé et al., 1991:J. Cell. Biochem.46:134–142] using as a probe for such modified tubulin, the monoclonal antibody GT335 [Wolff et al., 1992:Eur. J. Cell Biol.59:425–432]. Results obtained inParameciumstrongly suggest that both axonemal and cytoplasmic tubulin are glutamylated. As in the vertebrate brain tubulin so far tested, the GT335 epitope is located at the carboxy‐terminal fragment of cytoplasmic tubulin removed by subtilisin treatment. Immunoblotting and immunofluorescence experiments reveal that, unlike tubulin acetylation, glutamylation is not restricted to cold‐resistant microtubules. In addition, immunofluorescence studies performed on dividing cells show that glutamylation takes place soon after the polymerization of microtubules.Finally, glutamylated tubulin is also detected in the ciliate speciesEuplotes, Tetrahymena, andParaurostyla.Together with results obtained on flagellate species, this suggests that tubulin glutamylation came out early in the course of eukaryotic evolution and has been widely exploited in various cellular strategies. © 1994 W

ISSN:0886-1544    

DOI:10.1002/cm.970270406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractTo determine which proteins were associated with and intrinsic to the marginal band (MB) of microtubules (MTs), we studied protein components of MBs isolated from nucleated erythrocytes by differential detergent solubilization of the membrane skeleton (MS). MBs isolated from dogfish erythrocytes contained major proteins in the tubulin Mrrange. A high molecular weight protein of ∼290 kD that bound antibody to syncolin and to heat‐stable brain MAPs was present in the whole cytoskeleton. However, most of it was solubilized by the MB isolation medium, together with the MS. Dogfish erythrocyte cytoskeletons and isolated MBs were examined with polyclonal and monoclonal antibodies against mammalian brain tau and chicken erythrocyte tau. As shown by immunofluorescence and immunoblotting, these antibodies bound to proteins in the 50 to 67 kD range, located along the length of isolated MBs. Two‐dimensional SDS‐PAGE revealed isolated MB proteins of pI ∼6.8 in the same molecular weight range, as well as α‐ and β‐tubulin with pI ∼5.4. Subtilisin or high‐salt treatment of isolated MBs resulted in unbundling of MTs, indicating involvement of MAPs. MBs isolated from chicken erythrocyte cytoskeletons also contained tau as shown by antimammalian brain tau immunofluorescence. Both chicken and dogfish isolated MBs also bound phalloidin, but the binding was usually discontinuous and, for any given MB, matched the pattern of anti‐syncolin binding. Both syncolin and F‐actin were part of the MS remnant remaining after MT disassembly, supporting their assignment to a specialized MS region at the MB/MS interface. In contrast, tau protein appears to be intrinsic to the MB, where it may have an MT stabilizing and bundling function.

ISSN:0886-1544    

DOI:10.1002/cm.970270407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970270408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970270401

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY