bluuebunny/crossref_metadata_2025_split huggingface dataset:
Correlation between the distribution of smooth muscle or non muscle myosins and α‐smooth muscle actin in normal and pathological soft tissues
Authors: Benzonana Gilbert , Skalli Omar , Gabbiani Giulio 
Published: February 2005

AbstractThe distribution of smooth muscle (SM) and non muscle myosins was compared with that of α‐SM actin in various normal and pathological tissues and in cultured cells by means of indirect immunofluorescence using a monoclonal antibody specific for α‐SM actin [anti‐αsm‐1, Skalli et al., 1986b] and two polyclonal antibodies raised against bovine aortic myosin (ABAM) and human platelet myosin (AHPM), respectively.In normal tissues ABAM stained vascular and parenchymal smooth muscle cells (SMC), myoepithelial cells and myoid cells of the testis in a pattern similar to that reported by other authors with antisera raised against non vascular SM myosin. Cells stained with ABAM were always positive for anti‐αsm‐1. In human and experimental atheromatous plaques, most cells were positive for AHPM; a variable proportion was also stained for ABAM plus anti‐αsm‐1. Myofibroblasts from rat granulation tissue, Dupuytren's nodule and stroma from breast carcinoma were constantly positive for AHPM and negative for ABAM; however, myofibroblasts from Dupuytren's nodule and breast carcinoma were anti‐αsm‐1 positive. Early primary cultures of rat aortic SMC were positive for ABAM and anti‐αsm‐1 and became negative for ABAM and positive for AHPM after a few days in culture. They remained positive for AHPM and anti‐αsm‐1 after passages; the staining of AHPM and anti‐αsm‐1 appeared to be colocalized along the same stress fibers.These results may be relevant for the understanding of SMC function and adaptation, and show that in non malignant SMC proliferation, α‐SM actin represents a more general marker of SM origin than SM myosin.
In vitro phosphorylation of Paramecium axonemes and permeabilized cells
Authors: Hamasaki Toshikazu , Murtaugh Timothy J. , Satir Birgit H. , Satir Peter 
Published: February 2005

AbstractThis study seeks to identity phosphoproteins in axonemes from Paramecium letraurelia whose phosphorylation responses to adenosine 3′,5′‐cyclic monophosphate (cAMP) and Ca2+ parallel responses induced by these agents in ciliary behavior in this cell. In purified rxonemes, over 15 bands ranging from Mr >300 kDa to 19 kDa on SDS‐PAGE incorporate 32P from adenosine 5′‐γ‐[32P]tri‐phosphate (γ‐32P‐ATP) at pCa 7 in the absence of cAMP. A major band whose label turns over rapidly was identified at Mr 43 kDa. In the presence of 5 μM cAMP, more than eight bands, but not the Mr 43 kDa band, were labeled additionally or enhanced their labeling. These phosphoproteins and their kinases are structural components of the axoneme. Overall, some of the same major bands are labeled in the presence of cAMP in Triton X‐100‐permeabilized paramecia that retain their behavioral responses and in axonemes mechanically isolated from these cells. In particular, two major bands have been identified whose phosphorylation is greatly enhanced by cAMP at low concentrations: (1) a 29 kDa polypeptide whose cAMP‐dependent phosphorylation is diminished at pCa 4 compared with pCa 7 and (2) a 65 kDa polypeptide whose phosphorylation is pCa insensitive. These polypeptides meet minimal criteria for signal‐sensitive regulators of motility parameters in the Paramecium axoneme.
Distribution of phosphorylated spindle‐associated proteins in the diatom Stephanopyxis turris
Authors: Wordeman L. , Davis F. M. , Rao P. N. , Cande W. Z. 
Published: February 2005

AbstractMitotic spindles isolated from the diatom Stephanopyxis turris become thiophosphorylated in the presence of ATPγS at specific locations within the mitotic apparatus, resulting in a stimulation of ATP‐dependent spindle elongation in vitro. Here, using indirect immunofluorescence, we compare the staining pattern of an antibody against thiophosphorylated proteins to that of MPM‐2, an antibody against mitosis‐specific phosphoproteins, in isolated spindles. Both antibodies label spindle poles, kinetochores, and the midzone. Neither antibody exhibits reduced labeling in salt‐extracted spindles, although prior salt extraction inhibits thiophosphorylation in ATPγS. Furthermore, both antibodies recognize a 205 kd band on immunoblots of spindle extracts. Microtubule‐organizing centers and mitotic spindles label brightly with the MPM‐2 antibody in intact cells. These results show that functional mitotic spindles isolated from S. turris are phosphorylated both in vivo and in vitro. We discuss the possible role of phosphorylated cytoskeletal proteins in the control of mitotic spindle function.
Microinjection into Acanthamoeba castellanii of monoclonal antibodies to myosin‐II slows but does not stop cell locomotion
Authors: Sinard John H. , Pollard Thomas D. 
Published: February 2005

AbstractTo study the in vivo role of myosin‐II in Acanthamoeba castellanii, motile cells were microinjected with monoclonal antibodies raised against the myosin‐II heavy chain. All injected cells underwent a transient shock response. It was found that although injection of buffer alone or of an endogenous Acanthamoeba protein decreased the motility of injected cells from 7 μm/min to ∼3 μm/min, injection of monoclonal antibodies specific for myosin‐II decreased motility further to ∼0.8 μm/min. This effect was seen whether or not the monoclonal antibody to myosin‐II inhibited the actomyosin‐II MgATPase activity in vitro. Levels of antibody far in excess of endogenous myosin‐II concentrations could not completely block amoeboid movement. The morphology of moving antimyosin‐II‐injected cells was unusual, suggesting a greater defect in the ability to retract the trailing edge of the cell rather than to extend the leading edge. Endosomes frequently disappeared from injected cells, and although buffer‐injected cells rapidly recovered visible endosomes (50% recovery at 5 min), endosomes were not seen in antimyosin‐II‐injected cells until, on the average, ∼50 min after injection. Injection of a nonspecific antibody or of a nonspecific exogenous protein (ovalbumin) also decreased the mobility of the injected cells beyond that of buffer‐injected cells (to ∼1 μm/min). These cells tended to recover endosomes more rapidly (∼25 min) than cells injected with antimyosin‐II monoclonal antibodies. The inability of antibodies to myosin‐II to inhibit completely any of the movements studied suggests that although myosin‐II probably plays a role in these motilities, the cell either routinely uses or can draw upon another cytoplasmic motor to maintain locomotion, organelle movement, contractile vacuole activity, and endocytosis.
Calmodulin is associated with microtubules forming in PTK1 cells upon release from nocodazole treatment
Authors: Sweet Stuart C. , Rogers Charles M. , Welsh Michael J. 
Published: February 2005

AbstractTo investigate the association of calmodulin (CaM) with microtubules (MTs) in the mitotic apparatus (MA), the distributions of CaM and tubulin were examined in cells in which the normal spindle organization had been altered. A fluorescent CaM conjugate with tetramethylrhodamine isothiocyanate (CaM‐TRITC) and a dichlorotriazinyl aminofluorescein conjugate with tubulin (tubulin‐DTAF) were injected into cells that had been treated with the MT inhibitor nocodazole. With moderate nocodazole concentration (0.3 μg/ml, 37°C, 4 h) in live cells, CaM‐TRITC and tubulin‐DTAF concentrated identically on or near the centrosomes and kinetochores. In serial sections of these cells, small MT segments were observed by transmission electron microscopy (TEM) in the regions where fluorescent protein had concentrated. When a higher drug concentration was used (3.0 μg/ml, 37°C, 4 h), no regions of CaM‐TRITC or tubulin‐DTAF localization were observed, and no MTs were observed when serial sections were examined by TEM. However, following release from the high‐concentration nocodazole block, CaM‐TRITC colocalized with newly formed MTs at the kinetochores and centrosomes. Later in the recovery period, when chromosome‐to‐pole fibers had formed, CaM association with kinetochores diminished, ultimately attaining its normal pole‐proximal association with kinetochore MTs in cells that progressed through mitosis. We interpret these observations as supporting the hypothesis that in the MA, CaM attains a physical association with kinetochore MTs and suggest that CaM‐associated MTs may be inherently more stable.
Myosin regulation and calcium transients in fibroblast shape change, attachment, and patching
Authors: Rees David A. , Charlton Jillian , Ataliotis Paris , Woods Anne , Stones Amanda J. , Bayley Susan A. 
Published: February 2005

AbstractFollowing our study in Balb/c 3T3 cells and other cultured fibroblasts of the changes in myosin light chain phosphorylation associated with alterations in cell shape, attachment, and receptor patching, we have now determined the corresponding changes in cytoskeletal myosin distribution, and in the cellular calcium concentration, since this might, in part, mediate such responses.Immunofluorescence microscopy showed that myosin assembly into ordered forms such as actomyosin bundles and myosin sheath almost always correlated with previously shown high phosphorylation levels of myosin regulatory light chain, whereas diffuse distributions usually correlated with low or undetectable levels. An exception was observed in treatment to alter cellular cAMP levels when, in a biphasic response, assembly was correlated inversely with the phosphorylation states shown previously.Fluorescent indicators for intracellular calcium concentration, [Ca++]i, showed that myosin disassembly by trypsin or EGTA acting externally on the cells was preceded by a transient increase in [Ca++]i. For EGTA this was associated with transient recruitment of myosin into dorsal sheath structure as well as the transient enhancement of phosphorylation shown earlier. Blockage of EGTA‐induced disassembly could be achieved by azide, which also caused an immediate increase in [Ca++]i and inhibited its subsequent decline. Trypsin‐induced dephosphorylation did not appear to involve an eventual reduction of [Ca++]i. Therefore, in many but not all of the systems studied, correlated changes were observed in myosin assembly, [Ca++]i, and the myosin phosphorylation levels shown earlier.
Analysis of cell division using fluorescently labeled actin and myosin in living PtK2 cells
Authors: Sanger Jean M. , Mittal Balraj , Dome Jeffrey S. , Sanger Joseph W. 
Published: February 2005

AbstractActin and the light chains of myosin were labeled with fluorescent dyes and injected into interphase PtK2 cells in order to study the changes in distribution of actin and myosin that occurred when the injected cells subsequently entered mitosis and divided. The first changes occurred when stress fibers in prophase cells began to disassemble. During this process, which began in the center of the cell, individual fibers shortened, and in a few fibers, adjacent bands of fluorescent myosin could be seen to move closer together. In most cells, stress fiber disassembly was complete by metaphase, resulting in a diffuse distribution of the fluorescent proteins throughout the cytoplasm with the greatest concentration present in the mitotic spindle. The first evidence of actin and myosin concentration in a cleavage ring occurred at late anaphase, just before furrowing could be detected. Initially, the intensity of fluorescence and the width of the fluorescent ring increased as the ring constricted. In cells with asymmetrically positioned mitotic spindles, both protein concentration and furrowing were first evident in the cortical regions closest to the equator of the mitotic spindle. As cytokinesis progressed in such asymmetrically dividing cells, fluorescent actin and myosin appeared at the opposite side of the cell just before furrowing activity could be seen there. At the end of cytokinesis, myosin and actin were concentrated beneath the membrane of the midbody and subsequently became organized in two rings at either end of the midbody.
Cell migration into neural tube lumen provides evidence for the “fixed cortex” theory of cell motility
Authors: Bilozur Michael E. , Hay Elizabeth D. 
Published: February 2005

AbstractWe present a model of cell motility based on emigration of neural crest cells into the neural tube lumen under in vitro conditions (10% fetal calf serum or YIGSR) that inhibit their normal emigration from the base of the neuroepithelium into surrounding extracellular matrix (ECM). Ultrastructural observations reveal that cells lining the lumen are joined by zonulae adherentes (ZA), which are points of strong intercellular attachment, and thereby serve as markers for fixed regions of plasmalemma and cortical actin. Three major observations of the relationship of cells to the ZA support the “fixed cortex” model of mesenchymal cell migration. First, cells extend apical cel processes past the ZA into the lumen. To do this, they must make new apical plasmalemma and actin corrtex that the endoplasm slides into. Second, elongated cells are observed in the lumen that are still attached via ZA to the neuroepithelium. This indicates that all of the endoplasm finally slides past the ZA. Third, numerous cytoplasmic pieces, often attached to each other and to the neuroepithelium via ZA, are found at the site where cells appear to have detached from the epithelium after entering the lumen. Since the ZA is fixed in location, the endoplasm must have slid past it into newly manufactured anterior cortex and plasmalemma, with the trailing end of the cell finally snapping off. The “fixed cortex” theory of cell migration agrees with existing data in that it predicts the polarized insertion of new plasmalemma and actin at the leading end of the cell, but it differs significantly from existing theories of mesenchymal cell migration in that it states that the cell surface remains firmly attached to the substratum while the myosin‐rich endoplasm slides past it.
Correlated distribution of actin, myosin, and microtubules at the leading edge of migrating swiss 3T3 fibroblasts
Authors: Conrad Patricia A. , Nederlof Michel A. , Herman Ira M. , Taylor D. Lansing 
Published: February 2005

AbstractThe formation of lamellipodia in migrating cells involves dynamic processes that occur in a cyclic manner as the leading edge of a cell slowly advances. We used video‐enhanced contrast microscopy (VEC) to monitor the motile behavior of cells to classify protrusions into the temporal stages of initial and established protrusions (Fisher et al.: Cell Motility and the Cytoskeleton 11:235–247, 1988), and to monitor the fixation of cells. Multiple parameter fluorescence imaging methods (DeBiasio et al.: Journal of Cell Biology 105:1613–1622, 1987; Waggoner et al.: Methods in Cell Biology, Vol. 30, Part B, pp. 449–478, 1989) were then used to determine and to map accurately the distributions of actin, myosin and microtubules in specific types of protrusions. Initial protrustions exhibited no substructure as evidenced by VEC and actin was diffusely arranged, while myosin and microtubules were absent. Newly established protrusions contained diffuse actin as well as actin in microspikes. There was a delay in the appearance of myosin into established protrusions relative to the presence of actin. Microtubules were found in established protrusions after myosin was detected, and they were oriented parallel to the direction of migration. Actin and myosin were also localized in fibers transverse to the direction of migration at the base of initial and established protrusions. Image analysis was used to quantify the orientation of actin fibers relative to the leading edge of motile cells. The combined use of VEC, multiple parameter immunofluorescence, and image analysis should have a major impact on defining complex relationships within cells.
Structural plugs at microtubule ends may regulate polymer dynamics in vitro
Authors: Azhar Salman , Murphy Douglas B. 
Published: February 2005

AbstractMicrotubules contain in their lumens distinct structures (plugs) that influence their dynamic behavior in vitro. As observed by electron microscopy, plugs are stainoccluding structures 10–30 nm in length that occur along the lengths and at the ends of microtubules. Plugs occur at a frequency of 20–40% at the ends of microtubules assembled from cycled microtubule protein containing MAPs. While the composition of plugs is not known, preliminary evidence suggests that they are accretions of tubulin, that they are labile, and that they are more common in preparations containing MAPs. When polymers are induced to depolymerize by endwise subuit dissociation, the frequency of plugged microtubule ends increases transiently, suggesting that plugs temporarily stabilize microtubules. The functional significance of plugs may be that they prevent the sudden complete loss of microtubules through catastrophic disassembly. It is possible that plugs, by slowing the rate of disassembly, enable a polymer to add GTP‐tubulin subunits, thereby forming a stabilizing GTP‐cap. These observations suggest that plugs may stabilize polymers and account for the frequent transitions from shortening to growing phases that characterize dynamic instability.
Flagellar photoresponses of Chlamydomonas cells held on micropipettes: I. Change in flagellar beat frequency
Authors: Rüffer Ursula , Nultsch Wilhelm 
Published: February 2005

AbstractChlamydomonas cells immobilized on micropipettes were filmed at high‐speed (500 f/s) while photostimulated either by one continuous light stimulus or by pulsed light in the frequency range of cell rotation (1 or 2 Hz). Two kinds of photophobic step‐up and two kinds of photophobic step‐down beat frequency changes without a reversal of flagellar beat were observed after frame‐by‐frame evaluation of 141 records. So far, a single step‐up response, the “shock” response, has been considered the only photophobic response. However, the present results show that the cells always responded to step‐up as well as to step‐down light stimuli. Either a decrease of beat frequency by step‐up was combined with an increase by step‐down (type I), or an increase by step‐up was combined with a decrease by step‐down (type II). Whether type I or type II was observed depended on the preirradiation of the cells. All four responses are blue‐light responses with a lag‐time of ∼40 ms. Nothing can be said about the photoreceptor site. Regardless, these responses cannot be the basic mechanism for phototaxis, as assumed till now, because the flagella remain synchronized during the flagella beat frequency changes. Even if they are uncoupled before and after stimulation, both flagella respond in the same sense, i.e., either both increase or both decrease their beat frequency. The behavioral relevance of these responses for Chlamydomonas is not yet clear.
Adaptation of Chlamydomonas phototaxis: I. A light‐scattering apparatus for measuring the phototactic rate of microorganisms with high time resolution
Authors: Uhl Rainer , Hegemann Peter 
Published: February 2005

AbstractPhototaxis of the unicellular alga Chlamydomonas was studied with subsecond time resolution by using a newly developed taxigraph. The taxigraph determines the cell density in a particular volume element of a cuvette by measuring the amount of scattered light originating from the cells in this region. When the cell density is kept below 106/ml, a linear relationship exists between the scattered photon irradiance and the number of scattering particles. Time‐dependent scattering changes can be used to determine direction and extent of phototactic activity as well as the time course of various adaptation processes. This communication describes design and performance of the taxigraph in detail and compares results obtained from Chlamydomonas cell populations with those obtained from single‐cell analysis by using a computer‐aided motion analysis system.The high time resolution of the taxigraph permits the study of rapid adaptational processes. Chlamydomonas strain 806 cells, which have been reported to show exclusively negative phototaxis, were found to turn transiently towards the light upon a rapid change in irradiance, before eventually moving away from it. The duration of the initial positive phototactic response was critically dependent on the magnitude of the irradiance increment. Adaptation to a step‐up stimulus was consistently faster than to a step‐down stimulation. The statistical nature of the switch from positive to negative phototaxis is demonstrated by single‐cell observation.Different adaptation levels were characterised by stimulus‐response curves, either in the presence of a constant background or following a defined delay after long previous irradiation. To describe the observed behaviour the existence of two adaptation processes, occurring on a vastly different time scale, must be anticipated: a rapid (seconds) background adaptation and a slow desensitisation in steady light which is completed in 30‐40 min.
Kinetic analysis of chemotactic peptide‐induced actin polymerization in neutrophils
Authors: Wang Danher , Berry Keith , Howard Thomas H. 
Published: February 2005

AbstractDefinition of the kinetics of ligand‐activated actin polymerization in the neutrophil is important for ultimately understanding the mechanisms utilized for regulation of actin polymerization in this non‐muscle cell. To better define the kinetics of formyl peptide (fMLP) ‐induced actin polymerization in neutrophils we determined F‐actin content at 5 second intervals after activation of human neutrophils with a range (10−11−10−9M) of fMLP concentrations. The state of actin polymerization was monitored by quantifying F‐actin content with NBD phallacidin binding in both flow cytometric and extraction assays. Results demonstrate three successive kinetic periods of fMLP‐induced actin polymerization in neutrophils, a lag period, a 5 second period when rate of polymerization is maximal, and a period of declining rate of actin polymerization as F‐actin content approaches a maximum. The duration of the lag period, the maximum rate of polymerization, and the maximum extent of polymerization all depend upon the fMLP concentration. The lag period varies from 0 to 12 seconds and is followed in 5–10 seconds by a 5 second burst of actin polymerization when the rate is as great as 9% increase in F‐actin content per second. After the 5 second burst of polymerization, the rate of polymerization rapidly declines. The study defines three distinct kinetic periods of fMLP‐induced actin polymerization during which important rate‐limiting biochemical events occur. The mechanistic and motile implications of kinetic periods are discussed.