摘要:

AbstractThe development and dynamics of the phragmoplast cytoskeleton have been analyzed in living stamen hair cells ofTradescantia.Microtubules and actin microfilaments have been identified by microinjecting either carboxyfluorescein labeled brain tubulin or rhodamine phalloidin. Examination with the confocal laser scanning microscope has permitted sequential imaging of the fluorescent cytoskeletal elements in single living cells progressing through division. Phragmoplast microtubules initially emerge through the lateral coalescence of preexisting interzone microtubules. As cytokinesis progresses, these tightly clustered microtubules shorten in length and expand centrifugally toward the cell periphery. By contrast, the phragmoplast microfilaments appear to arise de novo in late anaphase in close association with the proximal surfaces of the reconstituting daughter nuclei. The microfilaments are oriented parallel to the microtubules but conspicuously do not occupy the equatorial region where microtubules interdigitate and where the cell plate vesicles aggregate and fuse. As development proceeds the microfilaments shorten in length and expand in girth, similar to microtubules, although they remain excluded from the cell plate region. In terminal phases of cell plate formation, microtubules degrade first in the central regions of the phragmoplast and later toward the edges, whereas microfilaments break down more uniformly throughout the phragmoplast. © 1993 Wiley‐Liss, I

ISSN:0886-1544    

DOI:10.1002/cm.970240302

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe cytoplasmic microtubules of the cold‐adapted Antarctic fishes, unlike those of homeotherms and temperate poikilotherms, assemble and function at body temperatures in the range −1.8 to +2°C. To determine whether alterations to the primary sequence of β tubulin may contribute to enhancement of microtubule assembly at cold temperatures, we have cloned and sequenced a 1.8‐kilobase neural β‐chain cDNA, Ncnβ1, from an Antarctic rockcod,Notothenia coriiceps neglecta. Based on nucleotide sequence homology, Ncnβ1 probably corresponds to a class‐II β‐tubulin gene. The 446‐residue β chain encoded by Ncnβ1 is closely related (sequence homology ∼95%) both to the neural class‐I/II isotypes and to the neural/testicular class‐IV variants of higher vertebrates, but the sequence of its carboxy‐terminal isotype‐defining region (residues 431–446) has diverged markedly (≥ 25% change relative to the I/II/IV referents). Furthermore, the NcnβsZ1 polypeptide contains six unique amino‐acid substitutions (five conservative, one nonconservative) not found in other vertebrate brain isotypes, and the carboxyterminal region possesses a unique tyrosine inserted at position 442. We conclude that Ncnβ1 encodes a class‐II β tubulin that contains sequence modifications, located largely in its interdimer contact domain, that may contribute to cold adaptation of mi

ISSN:0886-1544    

DOI:10.1002/cm.970240303

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

年代:1993

数据来源: WILEY

摘要:

AbstractWe have developed a method for producing sea urchin egg cytoplasmic extracts which support substantial microtubule‐associated motility, particularly minus end‐directed motility characteristic of cytoplasmic dynein. Particles translocated along microtubules and axonemes predominantly in the minus end direction; microtubules and axonemes glided across the coverslip surface only in the plus end direction (as expected for a minus‐end directed motor bound to the coverslip surface); and microtubules crosslinked into bundles in an antiparallel orientation. Velocities of particle and microtubule translocation were in the range of 0.5–1.8 μm/sec. Vanadate at 10 μM inhibited all gliding of the microtubules and axonemes, yet bidirectional particle transport persisted. Vanadate at concentrations of 25 μM and higher inhibited nearly all microtubule‐based motility in the preparation and produced parallel bundling of the microtubules. Motility was slowed but not stopped in the presence of 5 mM AMP‐PNP.Usually when a particle bound to a microtubule wall, it moved to the microtubule minus end. These particles often remained attached to the minus end. When a microtubule plus end in the shortening phase of dynamic instability reached a stationary particle on the microtubule, sometimes normal minus enddirected motility was activated, or at other times the particle remained attached to the shortening plus end. © 1993

ISSN:0886-1544    

DOI:10.1002/cm.970240304

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

年代:1993

数据来源: WILEY

摘要:

AbstractProfilin is a small G‐actin binding protein implicated in sequestering actin monomers in vivo. We have quantitated profilin and actin expression in human hepatoma HepG‐2 cells and in two mouse myogenic cell lines, BC3H1 and C2C12, to determine whether the expression of profilin and the expression of nonmuscle isoactin or total actin are co‐regulated. During differentiation of both muscle cell types, profilin and nonmuscle actin expression decrease in a coordinate manner as shown by measurements of steady state mRNA and newly synthesized protein. In human hepatoma HepG‐2 cells, the twofold increase in actin synthesis observed after 24 hours of exposure to cytochalasin D did not result in an increase in profilin synthesis. Thus, profilin and actin expression are not coregulated in all cells. To determine if there is sufficient profilin to sequester a large portion of cellular G‐actin, we measured total profilin and G‐actin levels in the three cell types. In each case, profilin accounted for less than 10% of the total G‐actin on a molar basis. Thus, profilin is not responsible for total G‐actin sequestration in these cells. Finally, using poly‐L‐proline affinity chromatography, we showed that, in the cell types tested, less than 20% of the poly‐L‐proline purified profilin existed as a complex with G‐actin. The profilin in these cells may be interacting with cellular components other than acti

ISSN:0886-1544    

DOI:10.1002/cm.970240305

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

年代:1993

数据来源: WILEY

摘要:

AbstractMyosin‐I refers to a class of proteins with a molecular weight of approximately 110‐kDa, which have characteristics of conventional myosin but are unable to form filaments. Previous studies have implicated myosin‐I in motile cellular processes including cell migration and phagocytosis. Although the first example of myosin‐I in higher eukaryotes was the intestinal 110K‐calmodulin complex, which forms in microvilli the lateral links connecting the core bundle of actin filaments to the membrane, myosin‐I has now been shown to be a component of rat kidney and to be present in bovine adrenal gland and brain. We have now purified and characterized two polypeptides from rat liver which have several characteristics of the intestinal 110K‐calmodulin complex. Both liver polypeptides are solubilized with ATP and co‐elute on gel filtration with calmodulin. The polypeptides, of 110‐kDa and 130‐kDa, bind calmodulin in 1 mM EGTA. Both polypeptides bind to F‐actin in an ATP reversible fashion, and crosslink actin filaments. The purified polypeptides possess an actin‐activated Mg2+‐ATPase activity typical of brush border myosin‐I. A polyclonal antiserum directed against the chicken intestinal 110‐kDa polypeptide recognizes both rat liver polypeptides, whereas another serum recognizes the 130‐kDa but not the 110‐kDa rat liver polypeptide. Controlled proteolysis of the purified polypeptides with α‐chymotrypsin indicates that the two polypeptides are distinct but related. Immunofluorescence microscopy on isolated hepatocytes shows distribution of myosin‐I to be vesicular, distributed throughout the cytoplasm, but more concentrated near the nucleus. These data contribute new evidence by several functional criteria that multiple myosin‐I molecules are present in higher organisms and may coexist in a

ISSN:0886-1544    

DOI:10.1002/cm.970240306

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe transition from metaphase to anaphase was examined in primary oocytes of the Mediterranean mealmoth,Ephestia kuehniella.Isolated spindles were analysed using phase‐contrast microscopy and antitubulin immunofluorescence. Metaphase I bivalents in female Lepidoptera contain material derived from the synaptonemal complexes, the elimination chromatin, which is located between the homologs. Upon the onset of anaphase I, the homologs detach from the elimination chromatin. The present observations revealed that prior to the start of anaphase A, the homologs move centripetally, i.e., toward the central spindle axis, while the elimination chromatin remains stationary. This type of movement is not directly comparable with conventional chromosome movements such as prometaphase congression, anaphase A, and anaphase B, since it occurs at right angles to the preferential orientation of the spindle MTs. A MT‐based mechanism may nevertheless account for the centripetal shift of homologs in meiosis I of the moth: bundling of kinetochore MTs of different chromosomes could transfer the chromosomes toward the spindle axis. Since the homologs move along the poleward surfaces of the plate of elimination chromatin, a MT‐independent force producer could exist as well at the interface between the chromosomes and the plate of elimination chromatin. © 1993 Wiley‐L

ISSN:0886-1544    

DOI:10.1002/cm.970240307

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

年代:1993

数据来源: WILEY

摘要:

AbstractFluorescent brain tubulin, injected into living cells of the green algaNitella pseudoflabellataand the higher plantTradescantia virginiana, incorporates into the cortical microtubules, allowing these structures to be observed. With confocal laser scanning microscopy, clear images of microtubules were recorded and changes in microtubule patterns documented. After injection, fluorescent lengths of microtubules appeared within a few minutes and their number and length increased rapidly to a “steady state” over the first 15 min. In many instances, fluorescent microtubules could still be detected several hours after injection. In the cells examined, microtubules are arranged as an array of separate units only occasionally displaying close association or accurate co‐alignment with neighboring microtubules. In what we perceive to be the steady state condition, some microtubules remain relatively static, while others undergo rapid changes in length or small translocations. We also document what appears to be bidirectional microtubule elongation during postdepolymerization assembly. © 1993 Wiley‐L

ISSN:0886-1544    

DOI:10.1002/cm.970240308

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

年代:1993

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970240301

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

年代:1993

数据来源: WILEY