ISSN:0886-1544    

DOI:10.1002/cm.970180202

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

年代:1991

数据来源: WILEY

摘要:

AbstractThe state of tubulin tyrosination in the fission yeastSchizosaccharomyces pombewas investigated using a combination of indirect immunofluorescence microscopy and Western blotting. Antibodies specific for the tyrosinated form of α‐tubulin stained all microtubule arrays in wild type cells and recognised the two α‐tubulin polypeptides in Western blots of cell extracts enriched for tubulin by DEAE‐Sephadex chromatography. Antisera that specifically recognised the detyrosinated, glu, form, on the other hand, gave consistently negative results, both in cells undergoing rapid exponential growth and in those allowed to accumulate in stationary phase. Neither the “ageing” of microtubules, by arresting cells at different points (late G1 or G2/M) in the cell division cycle, nor stabilising them, using D2O, lead to any detectable tubulin detyrosination. These results suggest thatS. pombelacks the carboxypeptidase that carries out the tubulin detyrosination reaction. This is the first report of an organism that possesses the correct C‐terminal α‐tubulin sequence yet fails to carry out this post‐translational modification. The implication of this novel finding for the biological role of these e

ISSN:0886-1544    

DOI:10.1002/cm.970180203

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

年代:1991

数据来源: WILEY

摘要:

AbstractTobacco BY‐2 suspension cultures were synchronized with aphidicolin in order to assess the relationship between microtubules (MTs), microfilaments (MFs), and the nuclear envelope (NE) at different stages of the cell cycle. Using immunofluorescence techniques, ordered MT arrays were found in the cortex in G1; few MTs are evident deeper in the cytoplasm or near the nucleus. However, MTs radiate from the surface of the nucleus during S and G2as the interphase cortical array is replaced by the preprophase band. Perinuclear fluorescence is also visible at the end of cytokinesis but does not overlap with new ordered cortical arrays early in G1. When isolated nuclei are examined, associated MTs are again evident in S and G2, but not in G1. Microfilaments are colocalized with the MTs in the radiating arrays, as ascertained by dual staining of cells with rhodamine phalloidin. Propyzamide treatment leads to the loss of MTs at all stages, while cytoplasmic and perinuclear MF networks persist. Conversely, cytochalasin D disrupts MFs, including those radiating from the nucleus during S and G2, without any apparent effect on MTs. The results cast doubt on a proposed role for the NE in the generation of cortical MTs in plants. A universal role for MFs in the deployment of MTs is also in questio

ISSN:0886-1544    

DOI:10.1002/cm.970180204

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

年代:1991

数据来源: WILEY

摘要:

AbstractA collaborative effort was initiated to resolve differences in two recent papers on the effects of cytochalasins in root cells. While both studies reported similar effects on interphase cells (i.e., replacement of microfilaments by many small specks and rods), Palevitz(Cell Motil. Cytoskeleton9:283‐298, 1988) maintained that cytochalasins B and D induce actin aggregation at the poles of dividingAlliumroot cells at a concentration of 10 μM with rhodamine phalloidin as a reporter probe, whereas McCurdy and Gunning(Cell Motil. Cytoskeleton15:76‐87, 1990) could not find these aggregates following antiactin immunocytochemistry inTriticumroots treated with CB at 50 μM. Employing identical methods and materials in the same laboratory, we found that CD induces polar actin aggregates in dividing cells of both species. However, the aggregates inTriticumare smaller and occur less frequently than those inAllium.A similar pattern is seen wi

ISSN:0886-1544    

DOI:10.1002/cm.970180205

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

年代:1991

数据来源: WILEY

摘要:

AbstractMonoclonal antibodies were raised against calmodulin purified fromDictyostelium discoideum.To increase its antigenicity, the calmodulin was conjugated to keyhole limpet hemocyanin; mice were immunized with the conjugate. Hybridomas producing antibodies against calmodulin were identified by screening culture supernatants with calmodulin coupled to bovine serum albumin. The specificity of antibodies from hybridoma culture supernatants was tested by Western blot ofDictyosteliumcell lysates. For this purpose, methods were developed that permitted sensitive detection of calmodulin bound to membranes. The key elements of the blotting protocol were use of PVDF membrane, transfer conducted in phosphate buffer, and glutaraldehyde fixation after transfer. These methods permitted detection of as little as 0.1 ng of calmodulin spotted directly onto the membrane, or 10 ng transferred from an SDS polyacrylamide gel. Ten calmodulin‐specific antibodies were identified; most of these reacted preferentially with the calcium‐containing form ofDictyosteliumcalmodulin. Several of the monoclonal antibodies cross‐reacted with calmodulin from bovine

ISSN:0886-1544    

DOI:10.1002/cm.970180206

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

年代:1991

数据来源: WILEY

摘要:

AbstractThe asymmetry of ATP‐reactivated flagellar bending waves of Triton‐demem‐brated sea urchin spermatozoa has been measured over a range of free Ca2+ion concentrations from 10−9to 10−4M. Detailed examination of the gradual response of asymmetry to Ca2+ion concentration over this wide range indicates the presence of two Ca2+sensors. A high‐affinity sensor operates at Ca2+concentrations near 10−7.5M. A lower‐affinity sensor operates at Ca2+concentrations above 10−6M, in the typical range for calmodulin‐mediated responses. Incubation of demembranated sperm flagella at high Ca2+concentrations to release calmodulin is required to enable these Ca2+responses to be observed. This treatment also causes a decrease in the apparent affinity of the flagella for cal‐modulin, as determined by measuring the increase in asymmetry in response to addition of exogenous calmodulin at

ISSN:0886-1544    

DOI:10.1002/cm.970180207

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

年代:1991

数据来源: WILEY

摘要:

AbstractA bioriented chromosome is tethered to opposite spindle poles during congression by bundles of kinetochore microtubules (kMts). At room temperature, kinetochore fibers are a dominant component of mitotic spindles of PtK2cells. PtK2cells at room temperature were injected with purified tubulin covalently bound to DTAF and congression movements of individual chromosomes were recorded in time lapse. Congression movements of bioriented chromosomes between the poles occur over distances of 4.5 μm or greater. DTAF‐tubulin injection had no effect on either the velocity or extent of these movements. Other cells were lysed, fixed, and the location of DTAF‐tubulin incorporation was detected from digitally processed images of indirect immunofluorescence of an antibody to DTAF. Microtubules were labeled with an anti‐beta tubulin antibody. At 2‐5 minutes after injection, concentrated DTAF‐tubulin staining was seen in the kinetochore fibers proximal to the kinetochores; a low concentration of DTAF‐tubulin staining occurred at various sites through the remaining length of the fibers toward the pole. Kinetochore fibers in the same cell displayed different lengths (0.2 to 4 μm) of concentrated DTAF‐tubulin incorporation proximal to the kinetochore, as did sister kinetochore fibers. Ten minutes after injection, the lengths of DTAF‐containing chromosomal fibers were greater than expected if incorporation resulted solely from the lengthening of kinetochore microtubules due to congression movements of the chromosomes. Besides incorporation as a result of chromosome movement, two other mechanisms might explain the length of the DTAF‐containing segments: (1) a poleward flux of tubulin subunits (Mitchison, 1989) or (2) capture of DTAF‐containing nonkin

ISSN:0886-1544    

DOI:10.1002/cm.970180208

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

年代:1991

数据来源: WILEY

摘要:

AbstractExamination of detergent‐extracted mouse eggs and embryos reveals the existence of two cytoskeletal networks. One network is the typical thin filament network observed in somatic cells while the other is composed of large planar elements. These latter cytoskeletal structures, with individual widths of 60.0±6.8 nm, alter their spatial organization in a developmental stage‐specific manner. The planar elements are composed of filaments with a diameter of 10 nm aligned side‐by‐side with these filaments exhibiting a linear periodicity of 20.0±1.6 nm. A biochemical fraction containing components of the planar elements has been prepared from different stages of development and disappearance of prominent polypep‐tides from this fraction correlates with the altered spatial organization of the planar elements. Ultrastructure and biochemistry of cytoskeletal planar elements in eggs and embryos of the mouse are comparable with cytoskeletal sheets of Syrian hamster eggs and embryos, suggesting these cytoskeletal components may have a functional role in mammalian embryogenesis. Because such structures have not been identified in eggs or embryos of species other than mammals, their function may be unique to mammalian em

ISSN:0886-1544    

DOI:10.1002/cm.970180209

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

年代:1991

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970180201

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

年代:1991

数据来源: WILEY