bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heterogeneity of microtubule organizing center components as revealed by monoclonal antibodies to mammalian centrosomes and to nucleus‐associated bodies from Dictyostelium
Authors: Sellitto Caterina , Kimble Mary , Kuriyama Ryoko 
Published: February 2005

AbstractThe molecular composition of two morphologically distinct microtubule‐organizing centers (MTOCs) was compared by probing with monoclonal antibodies raised against (i) nucleus‐associated bodies (NABs) isolated in a complex with nuclei from the cellular slime mold Dictyostelium discoideum and (ii) mammalian mitotic spindles isolated from Chinese hamster ovary (CHO) cells. The staining patterns observed by immunofluorescence microscopy in whole CHO cells and Dictyostelium amoebae showed that the distribution of thirteen MTOC antigens is heterogeneous. Not all antibodies recognized the MTOC in both interphase and mitosis. Most of the anti‐MTOC antibodies cross‐reacted with other cellular organelles such as nuclei, Golgi apparatus‐like aggregates and cytoskeletal elements. Two antibodies, CHO3 and AX3, recognized phosphorylated epitopes present in both mammalian centrosomes and Dictyostelium NABs. On immunoblots, most of the antibodies showed multiple bands, often of high molecular weight, indicating that the antigenic determinants are shared among different molecules. One antibody inhibited the regrowth of microtubules onto centrosomes in vitro after addition of exogenous tubulin to detergent‐lysed CHO cells on coverslips; this antibody binds to an antigen(s) that might be essential for the microtubule‐nucleating activity of centrosomes. These observations demonstrate that molecular components in different MTOCs exhibit a variety of distinct subcellular localizations and functional properties, and that some antigenic molecules have been conserved among morphologically distinct MTOCs. © 1992 Wiley‐Liss, Inc.
An immunofluorescence study of the effects of ultraviolet radiation on the organization of microfilaments, keratin intermediate filaments, and microtubules in human keratinocytes
Authors: Zamansky Glen B. , Nguyen Uyensa , Chou Lih‐Nan 
Published: February 2005

AbstractIndirect immunofluorescence microscopy has been used to investigate the ultraviolet (UV) radiation induced disruption of the organization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. Following irradiation, concurrent changes in the organization of the three major cytoskeletal components were observed in cells incubated under low Ca2+ (0.15 mM) conditions. UV irradiation induced a dose‐dependent condensation of keratin filaments into the perinuclear region. This collapse of the keratin network was accompanied by the reorganization of microfilaments into rings and a restricted distribution of microtubules, responses normally elicited by exposure to high Ca2+ (1.05 mM) medium. The UV induced alteration of the keratin network appears to disrupt the interactions between keratin and actin, permitting the reorganization of actin filaments in the absence of Ca2+ stimulation.In addition to the perinuclear condensation of keratin filaments, UV irradiation inhibits the Ca2+ induced formation of keratin alignments at the membrane of apposed cells if UV treatment precedes exposure to high Ca2+ medium. Incubation of keratinocytes in high Ca2+ medium for 24 hours prior to irradiation results in the stabilization of membrane associated keratin alignments and a reduced susceptibility of cytoplasmic keratin filaments to UV induced disruption. Unlike results from investigations with isogenic skin fibroblasts, no UV induced disassembly of microtubules was discernible in irradiated human keratinocytes. © 1992 Wiley‐Liss, Inc.
Evolution of the flagellar waveform of ram spermatozoa in relation to the degree of epididymal maturation
Authors: Chevrier C. , Dacheux J.‐L. 
Published: February 2005

AbstractMotility and flagellar movement of ram spermatozoa along the epididymis were analysed in vitro. From the caput to the cauda of the epididymis, the percentage of motile and progressive spermatozoa increases. No flagellar bending was observed in spermatozoa from the testis or the epididymal anterior caput. When spermatozoa reached the distal caput of the epididymis, a static curvature, associated with an initiation of the flagellar beating, appeared on the flagella. This curvature normally disappeared during epididymal transit. Its disappearance was associated with an increase in the flagellar beat efficiency. Our results suggest that the initiation of motility is related to two mechanisms involving: (1) the presence of a transient static curvature, and (2) the establishment of a symmetric regular beating of the flagellum. © 1992 Wiley‐Liss, Inc.
Timing of mitotic chromosome loss caused by the ncd mutation of Drosophila melanogaster
Authors: Nelson Catherine R. , Szauter Paul 
Published: February 2005

AbstractWe studied the timing of mitotic loss of maternally and paternally derived chromosomes among the progeny of Drosophila melanogaster females homozygous for an amorphic mutation in ncd, a gene encoding a kinesin‐like protein. In order to determine the division at which chromosome loss occurs, we estimated the fraction of XO nuclei resulting from X chromosome loss by scoring the phenotype of 47 adult cuticular landmarks in 160 XX–XO mosaics (gynandromorphs) derived from maternal X chromosome loss, and 33 gynandromorphs derived from paternal X chromosome loss. The results show that while most of the mitotic loss of maternally derived chromosomes occurs at the first cleavage division, the mitotic loss of paternally derived chromosomes occurs only at the second and later divisions. This means that paternally derived chromosomes are immune from the effects of ncd prior to karyogamy, which occurs after the first cleavage division. We discuss the implications of these results for the function of the ncd gene product and for other kinesin‐like proteins in Drosophila. © 1992 Wiley‐Liss, Inc.
Activation of maternal centrosomes in unfertilized sea urchin eggs
Authors: Schatten Heide , Walter Marika , Biessmann Harald , Schatten Gerald 
Published: February 2005

AbstractCentrosomes are undetectable in unfertilized sea urchin eggs, and normally the sperm introduces the cell's microtubule‐organizing center (MTOC) at fertilization. However, artificial activation or parthenogenesis triggers microtubule assembly in the unfertilized egg, and this study explores the reappearance and behavior of the maternal centrosome. During activation with A23187 or ammonia, microtubules appear first at the cortex; centrosomal antigen is detected diffusely throughout the entire cytoplasm. Later, the centrosome becomes more distinct and organizes a radial microtubule shell, and eventually a compact centrosome at the egg center organizes a monaster. In these activated eggs, centrosomes undergo cycles of compaction and decompaction in synchrony with the chromatin, which also undergoes cycles of condensation and decondensation. Parthenogenetic activation with heavy water (50% D2O) or the microtubule‐stabilizing drug taxol (10 μM) induces numerous centrosomal foci in the unfertilized sea urchin egg. Within 15 min after incubation in D2O, numerous fine centrosomal foci are detected, and they organize a connected network of numerous asters which fill the entire egg. Taxol induces over 100 centrosomal foci by 15 min after treatment, which organize a corresponding number of asters. The centrosomal material in either D2O‐ or taxol‐treated eggs aggregates with time to form fewer but denser foci, resulting in fewer and larger asters. Fertilization of eggs pretreated with either D2O or taxol shows that the paternal centrosome is dominant over the maternal centrosome. The centrosomal material gradually becomes associated with the enlarged sperm aster. These experiments demonstrate that maternal centrosomal material is present in the unfertilized egg, likely as dispersed undetectable material, which can be activated without paternal contributions. At fertilization, paternal centrosomes become dominant over the maternal centrosomal material. © 1992 Wiley‐Liss, Inc.
Behavior of Dictyostelium amoebae is regulated primarily by the temporal dynamic of the natural cAMP wave
Authors: Wessels Deborah , Murray John , Soll David R. 
Published: February 2005

AbstractThe instantaneous velocity plots of Dictyostelium discoideum amoebae responding to natural waves and simulated temporal waves of cAMP with periods of 7 min are highly similar. This similarity has been used to deduce the dynamics of a natural wave crossing an amoeba, and the behavior of amoebae has been characterized during the different phases of a natural wave with a computer‐assisted dynamic image analyzing system. During the first ∼150 sec of the front of a natural wave, cells move persistently toward the aggregation center, with high instantaneous velocity and a decreased frequency of lateral pseudopod formation. During the last 30 sec of the front of the wave and the first 30 sec of the back of the wave, there is a “freeze” in cell shape and a dramatic depression in cell motility, pseudopod formation, and intracellular particle movement. During the last 180 sec of the back of the wave, there is a rebound in pseudopod formation, but it is random in direction and leads to no net cellular translocation. The data suggest that all of the behavior of a cell but orientation during the translocation phase is mediated by the temporal dynamics of the wave. The data also suggest that orientation toward the aggregation center occurs early in the front of the wave and that, once oriented, cells move in a blind fashion during the translocation phase. © 1992 Wiley‐Liss, Inc.
Expression of desmin cDNA in PtK2 cells results in assembly of desmin filaments from multiple sites throughout the cytoplasm
Authors: Mittal Balraj , Danowski Barbara A. , Sanger Jean M. , Sanger Joseph W. 
Published: February 2005

AbstractThe assembly of intermediate filaments into a cytoplasmic network was studied by microinjecting into the nuclei and cytoplasms of PtK2 cells, plasmids that contained a full length desmin cDNA and an RSV promoter. Immunofluorescence was used to monitor the expression of desmin and its integration into the cells' vimentin intermediate filament network. We found that the expressed desmin co‐localized with filaments of vimentin just as it does when fluorescently labelled desmin is microinjected into the cytoplasm of PtK2 cells. As early as two hours after microinjection of the plasmids, small discrete dots and short fragments of desmin could be detected throughout the cytoplasm of the cells. This initial distribution of desmin was superimposed on the filamentous pattern of vimentin in the cells. At 8 hours after microinjection of the plasmids, some of the desmin was present in long filaments that were coincident with vimentin filaments. By 18 hours, most of the desmin was in a filamentous network co‐localizing with vimentin. There was no indication that desmin assembly began in the perinuclear region and proceeded toward the cell periphery. In some cells, excessively high levels of desmin were expressed. In these cases, overexpression led to clumping of desmin filaments as well as to an accumulation of diffusely distributed desmin protein in the center of the cells. This effect was apparent at approximately 18 hours after introduction of the plasmid. The native vimentin filaments in such cells were also aggregated around the nucleus, colocalizing with desmin. The microtubule networks in all injected cells appeared normal; microtubules were extended in typical arrays out to the periphery of the cells. © 1992 Wiley‐Liss, Inc.
Characterization of the retraction response of goldfish retinal ganglion cell axons induced by monoclonal antibody 8A2 in vitro
Authors: Finnegan Sarah G. , Lemmon Vance P. , Koenig Edward 
Published: February 2005

AbstractRetraction similar to that occurring spontaneously in senescent axonal fields of goldfish regenerating ganglion cell axons is reliably induced by monoclonal antibody (mAb) 8A2. The retraction response is characterized by transformation of the growth cone into a nodular motile mass, which undergoes retrograde translocation in conjunction with the contiguous column of axoplasm, generating evacuated distal strands. The growth cone‐to‐motile mass transformation involves a reorganization of F‐actin. In addition, the reorganization of F‐actin is a necessary antecedent for retrograde bulk translocation of axoplasm. Contractile tension contributes to compaction within the motile mass, while that within the column of distal axoplasm is oriented longitudinally and appears to contribute to bulk movement. As a derivative of the growth cone, the motile mass exhibits protrusive activities and a capacity to translocate independently when microtubules are partially disrupted. Apparent compressive forces cause buckling of microtubules in the adjacent segment which appear as elbow‐like protrusions. Cytochalasin D blocks mAb 8A2 induced retraction and immediately arrests retrograde translocation when it is in progress; however, neither nocodazole nor taxol blocks retraction. Phalloidin and immunofluorescence double labeling of retracted axons reveals that myosin 11, MLCK, and calmodulin co‐localize with dense F‐actin structures within the motile mass. These results suggest that microtubules play a subordinate, passive role, and that actomyosin interactions mediate the formation of the motile mass and the retraction response. Finally, axons grown on laminin exhibit a more robust retraction response than those grown on polylysine, implicating membrane‐cytoskeletal interactions as modulating factors. © 1992 Wiley‐Liss, Inc.
PDGF and neomycin induce similar changes in the actin cytoskeleton in human fibroblasts
Authors: Hedberg Karin M. , Bengtsson Torbjörn , Safiejko‐Mroczka Barbara , Bell Paul B. , Lindroth Margaretha 
Published: February 2005

AbstractThe addition of platelet‐derived growth factor (PDGF) to serum‐starved fibroblasts induces increased motility, formation of lamellipodia, increased ruffling activity, and actin ring structures associated with dorsal ruffles. Involvement of the phosphatidylinositol cycle (PI‐cycle) in these morphological changes was investigated by observing the effects of neomycin, an inhibitor of the PI‐cycle, on cultured human foreskin fibroblasts. The role of actin in the changes was investigated by using cytochalasin D (CD). Actin in detergent‐extracted cells was labelled with TRITC‐phalloidin and examined with fluorescence microscopy. Using PDGF and neomycin simultaneously potentiated lamellipodia formation, ruffling activity, as well as the number of cells with actin rings. Furthermore, neomycin by itself induced morphological changes similar to those induced by PDGF. Quantitation of actin rings showed dose and time dependency for PDGF and neomycin respectively, with a maximal number of cells containing rings after 15 min of exposure to either 3.5 mM neomycin or 10 ng PDGF/ml. Comparing the two substances, PDGF induced ring formation in a greater number of cells. These processes were inhibited by the presence of CD. PDGF‐ and neomycin‐induced changes in the actin cytoskeleton were also observed in human embryonic lung fibroblasts, human glial cells, and embryonic mouse fibroblasts, all of which are known to express PDGF‐receptors. In conclusion, the present study indicates that an increased turnover of the PI‐cycle is not essential for the changes in actin organization induced by PDGF. © 1993 Wiley‐Liss, Inc.
Myosin‐I in mammalian liver
Authors: Coluccio Lynne M. , Conaty Cathleen 
Published: February 2005

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 single cell type. © 1993 Wiley‐Liss, Inc.
Primary and secondary chick heart fibroblasts: Fast and slow‐moving cells show no significant difference in microtubule dynamics
Authors: Brown Denise A. , Warn Richard M. 
Published: February 2005

AbstractHighly motile chick heart fibroblasts in primary culture (1° CHFs) gradually convert into much slower‐moving secondary (2°) cells. The polarized movement of the latter, but not the former, cell type has been found to be dependent on an intact microtubule (MT) network [Middleton et al., 1989, J. Cell Sci. 94:25–32]. To investigate the comparative stability of the MT networks of 1°s and 2°s, turnover was investigated by microinjection of biotin‐labeled brain tubulin to act as a reporter. MTs in both cell types were found to be very dynamic, with the MT networks effectively disassembled by about 30 min in 1° CHFs and 60 min in 2° CHFs, with mainly MT fragments remaining beyond these times. All MTs and fragments were found to have turned over by 1 h in 1° CHFs and 80 min in 2°s. Because 2° CHFs were found to be on average six times larger than 1°s, the difference in MT turnover time was considered largely due to the size difference. For both 1° and 2° cells, the more slowly turning over MTs were generally curly and perinuclear in distribution, resembling stable MTs in other systems, but they appeared significantly earlier in CHFs. However, no discrete subpopulations of slower turning over MTs were found to be associated with either the leading edges or the processes of either cell type. In addition, no major differences were identified in the patterns of modified α‐tubulin along the MTs or of MT cold or drug stability. It is concluded that MTs do not have a direct structural or skeletal function in maintaining a polarized 2° CHF cell shape, but rather play an ancillary role. © 1993 Wiley‐Liss, Inc.
Elastic properties of titin filaments demonstrated using a “freeze‐break” technique
Authors: Trombitás Károly , Pollack Gerald H. , Wright John , Wang Kuan 
Published: February 2005

AbstractA “freeze‐break” technique (Trombitás, K.: Acta Biochim. Biophys. Hung. 6:419–427, 1971) and immunoelectron microscopy were used to study the elastic properties of titin filaments. Small bundles of freshly prepared rabbit psoas muscle fibers were quickly frozen and broken under liquid nitrogen to fracture sarcomeres in planes perpendicular to the filament axis, in each of various regions along the sarcomere. The still‐frozen specimens were thawed during fixation to allow elastic filaments to retract. The broken specimens were then labelled with monoclonal anti‐titin antibodies against an unique epitope in the I‐band.The titin epitopes were normally positioned symmetrically about the Z‐line. However, in sarcomeres broken at the A‐I junction, the epitopes no longer remained symmetrical: the titin filaments in the broken half‐sarcomere retracted, independently of the thin filaments, forming a dense band just near the Z‐line. The retracted density apparently did not reach the Z‐line; retraction stopped at the level of the so‐called N1‐line. In sarcomeres broken at the Z‐line level, the titin filaments retracted in the opposite direction. In this case the titin epitope retracted all the way to the ends of the thick filaments.It appears then that titin molecules form elastic filaments that are independent of thin filaments in most of the I‐band. Near the Z‐line, however, the titin filaments either have an inelastic domain or associate firmly with the thin filaments at the N1‐line level. © 1993 Wiley‐Liss, Inc.
Actin filament structure probed with monoclonal antibodies
Authors: Moncman Carole L. , Peng Isaac , Winkelmann Donald A. 
Published: February 2005

AbstractThe interaction of two monoclonal antibodies (mAbs) with actin has been characterized to map the epitopes defined by these mAbs and to determine the accessibility of these sites in the actin filament (F‐actin). Both mAbs react specifically with actin in radioimmunoassays and Western blot assays, and by immunoprecipitation. The location of the epitopes within the primary structure of actin has been determined using limited proteolysis of actin and Western blots, or using immunoprecipitation of truncated actin fragments synthesized in a cell free translation assay. Both mAbs bind to the C‐terminal fragment of actin (residues 68–375) produced by chymotrypsin cleavage. One epitope is further localized to a 9.9 kD peptide corresponding to residues 5–93. Therefore, the epitope defined by this mAb (2G11.4) lies between residues Lys68 and Glu93 of actin. The location of the other epitope was determined by immunoprecipitation of actin fragments synthesized in vitro. Removal of residues 356–365 from the C‐terminus of actin completely abolished the binding of mAb 4E3.adl. Therefore, this mAb defines an epitope that involves residues between Trp356 and Ala365. The accessibility of these epitopes in native F‐actin was determined with solution binding assays and characterized by immunoelectron microscopy. Monoclonal antibody 4E3.adl binds strongly to filaments, resulting in bundling or decoration of F‐actin depending on the valency of the mAb, and indicating that the epitope is readily accessible in F‐actin. In contrast, mAb 2G11.4 disrupts F‐actin structure, resulting in the formation of an amorphous immunoprecipitate. These results place constraints on models of actin filament structure. © 1993 Wiley‐Liss, Inc.