摘要:

AbstractThe phenotypes induced by the expression of neuronal microtubule‐associated proteins (MAPs) in Sf9 cells have provided data on the in situ function of these proteins. Both MAP2 and tau can induce long processes in Sf9 cells, and the processes contain bundles of microtubules. In both cases the microtubules are aligned with their plus ends distal. Tau expression usually induces a single process that is unbranched and of uniform caliber. Processes can form even when the cells are grown in suspension. Microtubules do not extend all the way to the tip; instead the terminal region contains an actin‐rich meshwork. Taxol treatment of Sf9 cells also induces the assembly of microtubules into bundles but does not induce process formation in Sf9 cells. Therefore the in vitro properties of tau as a molecule capable of assembling, stabilizing, and bundling microtubules do not fully account for the in vivo ability of tau alone to transduce microtubule assembly into a change in cell shape. The morphological features of the processes induced by MAP2 differ in highly informative ways. © 1994 Wiley‐Lis

ISSN:0886-1544    

DOI:10.1002/cm.970280302

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

年代:1994

数据来源: WILEY

摘要:

AbstractObservations that were interpreted to provide evidence for equivalent functions of all axonemal dyneins should be reinterpreted, and models based on this assumption should be abandoned. In the future, attempts to understand the mechanisms for flagellar bending, oscillation, and bend propagation should start from the assumption that each type of axonemal dynein may have a specific function. At least three distinct functions can now be identified: bend initiation, maintenance of the angle of propagating bends, and generation of power to overcome viscous resistances. Only the last of these three functions is an outer arm dynein function. © 1994 Wiley‐Liss, I

ISSN:0886-1544    

DOI:10.1002/cm.970280303

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

年代:1994

数据来源: WILEY

摘要:

AbstractCytoplasmic dynein is the putative motor protein for retrograde organelle transport along microtubules in cells and, thus, must be capable of binding to organelle membranes. Such an attachment may occur via receptor proteins or through a direct interaction of dynein with the membrane phospholipids. We show here that cytoplasmic dynein‐synaptic membrane binding does not require a receptor protein and that this binding is mediated by an electrostatic interaction with acidic phospholipids. The properties of cytoplasmic dynein binding to NaOH‐extracted synaptic membranes are not significantly affected when those membranes are treated with trypsin to digest endogenous integral membrane proteins. Moreover, purified cytoplasmic dynein is capable of binding to liposomes composed of pure phospholipids. Dynein binds to liposomes with a profile remarkably similar to that of dynein binding to native membranes. Dynein‐liposome binding is dependent upon the presence of acidic phospholipids and is disrupted by NaCl. Thus, these studies suggest that electrostatic interactions can effect dynein‐membrane binding. © 1994 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970280304

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

年代:1994

数据来源: WILEY

摘要:

AbstractMicrotubule‐membrane cross‐linkers in motile and nonmotile cilia are supramolecular structures, held together by strong interactions between the constituent molecules. We have characterized these interactions in the photoreceptor connecting cilium, where cross‐linkers co‐fractionate and maintain their in situ location after Triton X‐100 extraction of axonemes. In bovine photoreceptor cells, the transmembrane assemblage that is cross‐linked to the connecting cilium axoneme contains three high molecular mass glycoconjugates of 425, 600, and 700 kDa (Horst et al., 1987). The relative amounts of the three glycoconjugates, as judged from band intensity in electrophoretograms, depend strongly on sample treatment prior to electrophoresis. The electrophoretic pattern was reproducible after several weeks of storage of the axoneme fraction in extraction buffer containing 50% sucrose. Removal of sucrose from the buffer by dialysis eliminated the 600 kDa and 700 kDa, and decreased the detected amount of the 425 kDa glycoconjugate. When samples were incubated in Laemmli sample buffer at increasing temperatures (23°, 60°, 95°C), a gradual reduction in the intensity of the three bands was observed. The quantitative reduction of high molecular mass glycoconjugates was accompanied by the appearance of novel protein species of lower molecular mass, as detected by lectin and antibody overlays of axonemal transblots. These results suggest that the previously characterized cross‐linker glycoconjugates are complex, SDS‐resistant multi‐molecular conglomerates. We have further used fluorescent lectins to monitor the presence of glycoconjugates on whole‐mounted axonemes, in conditions aimed to selectively solubilize the cross‐linkers. The cross‐linker complexes could not be dissociated from the axoneme by incubation with buffers containing 1 M of either Na2SO4or NaI. The results indicate that the connecting cilium‐specific cross‐linker complexes are bound via high‐affinity interactions to both axoneme and overlying plasma

ISSN:0886-1544    

DOI:10.1002/cm.970280305

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

年代:1994

数据来源: WILEY

摘要:

AbstractIt was recently shown that, in addition to the well‐established microtubule‐dependent mechanism, fast transport of organelles in squid giant axons also occurs in the presence of actin filaments [Kuznetsov et al., 1992,Nature356:722‐725]. The objectives of this study were to obtain direct evidence of axoplasmic organelle movement on actin filaments and to demonstrate that these organelles are able to move on skeletal muscle actin filaments. Organelles and actin filaments were visualized by video‐enhanced contrast differential interference contrast (AVEC‐DIC) microscopy and by video intensified fluorescence microscopy. Actin filaments, prepared by polymerization of monomeric actin purified from rabbit skeletal muscle, were stabilized with rhodamine‐phalloidin and adsorbed to cover slips. When axoplasm was extruded on these cover slips in the buffer containing cytochalasin B that prevents the formation of endogenous axonal actin filaments, organelles were observed to move at the fast transport rate. Also, axoplasmic organelles were observed to move on bundles of actin filaments that were of sufficient thickness to be detected directly by AVEC‐DIC microscopy. The range of average velocities of movement on the muscle actin filaments was not statistically different from that on axonal filaments. The level of motile activity (number of organelles moving/min/field) on the exogenous filaments was less than on endogenous filaments probably due to the entanglement of filaments on the cover slip surface. We also found that calmodulin (CaM) increased the level of motile activity of organelles on actin filaments. In addition, CaM stimulated the movement of elongated membranous organelles that appeared to be tubular elements of smooth endoplasmic reticulum or extensions of prelysosomes. These studies provide the first direct evidence that organelles from higher animal cells such as neurons move on biochemically defined actin filaments. © 1994 W

ISSN:0886-1544    

DOI:10.1002/cm.970280306

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

年代:1994

数据来源: WILEY

摘要:

AbstractWe have used degenerate oligonucleotides, derived from the amino acid sequence of transgelin peptides [Shapland et al., 1993:J. Cell Biol.121:1065‐1073], to isolate and sequence overlapping cDNA clones encoding this actin gelling protein. Primers with 5′ restriction enzyme sites directed against the N and C terminal amino acids present in these clones were then used to amplify and clone the entire transgelin coding region from reverse transcribed rat small intestine cDNA (RT‐PCR). These studies have shown that transgelin is the product of a single gene which is conserved between yeast,Drosophila, molluscs, and humans. Transgelin is expressed as a single message that is regulated at the level of transcription in SV40 transformed 3T3 cells. Our data have shown that transgelin and several other proteins of unknown function, SM22α [Pearlstone et al., 1987:J. Biol. Chem.262:5985‐5991], mouse p27 [Almendral et al., 1989:Exp. Cell Res.181:518‐530], and human WS3‐10 [Thweatt et al., 1992:Biochem. Biophys. Res. Commun.187:1‐7], share extensive homology. More limited regions of homology shared between transgelin and other proteins such as rat NP25 (unpublished), chicken calponins α and β [Takahashi and Nadal‐Ginard, 1991:J. Biol. Chem.266:13284‐13288], andDrosophilamp20 [Ayme‐Southgate et al., 1989:J. Cell Biol.108:521‐531] suggest that all of these proteins may be classified as members of a new transgelin multigene family

ISSN:0886-1544    

DOI:10.1002/cm.970280307

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

年代:1994

数据来源: WILEY

摘要:

AbstractInsect Sf9 cells usually elaborate a highly characteristic single process when infected with a baculovirus encoding recombinant human tau. The processes are unbranched, of uniform caliber, and contain bundles of microtubules. Because taxol treatment alone does not induce process outgrowth in these cells, it is believed that tau confers properties on microtubules that permits the conversion of microtubule assembly into the formation of processes. Here we have analyzed the reorganization of both actin filaments and microtubules during process initiation. A zone of organelle exclusion representing the focal reorganization of actin at one pole of the cell anticipated process emergence. A relationship between actin organization and process emergence was also suggested by a shift from single to multiple process formation after treatment with cytochalasin D. The rate of process elongation doubled after cytochalasin treatment of tau‐expressing cells. The increase in rate was due to the inhibition of the growth arrest phases which occur in the absence of cytochalasin. In contrast, Sf9 cells treated with cytochalasin after more than 20 h of tau expression were relatively resistant to the drug's effects. We conclude that actin and microtubules are specifically reorganized during tau‐induced process outgrowth and that a dynamic relationship between actin filaments and microtubules effects process formation. © 1994 Wiley‐Lis

ISSN:0886-1544    

DOI:10.1002/cm.970280308

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

年代:1994

数据来源: WILEY

摘要:

AbstractAlthough the head and rod domains of intermediate filament (IF) proteins are known to play significant roles in filament assembly, the role of the tail domain in this function is unclear and the available information supports contradictory conclusions. We examined this question by comparing transfection of the same cDNA constructs, encoding vimentins with modified tail domains, into cell lines that do and do not contain endogenous IF proteins. By this approach, we were able to distinguish between the ability of a mutant IF protein to initiate assembly de novo, from that of incorporating into existing filament networks. Vimentins with modifications at or near a highly conserved tripeptide, arg‐asp‐gly (RDG), of the tail domain incorporated into existing IF networks in vimentin‐expressing (vim+) cells, but were assembly‐incompetent in cells that did not express IF proteins (vim−). The failure of the RDG mutant vimentins to assemble into filament arrays in vim‐cells was reversible by re‐introducing a wild‐type vimentin cDNA, whereupon both wild‐type and mutant vimentins coassembled into one and the same IF network. We conclude that the function of the tail domain of type III IF proteins, and possibly of keratins K8 and K18, in IF assembly is distinct from those of other domains; a region encompassing the RDG tripeptide appears to be important in the assembly process. © 19

ISSN:0886-1544    

DOI:10.1002/cm.970280309

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.970280301

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

年代:1994

数据来源: WILEY