bluuebunny/crossref_metadata_2025 huggingface dataset:
Frequency and orientation of pseudopod formation of Dictyostelium discoideum amebae chemotaxing in a spatial gradient: Further evidence for a temporal mechanism
Authors: Varnum‐Finney Barbara J. , Voss Edward , Soll David R.
Published: February 2005

Amebae of Dictyostelium discoideum normally chemotax to aggregation centers by assessing the direction of outwardly moving, nondissipating waves of the chemoattractant cAMP. However, D. discoideum amebae can also assess the direction of a relatively stable spatial gradient. We demonstrate that amebae migrating towards the “source” of a stable, spatial gradient move faster, extend fewer pseudopodia, and turn less frequently than amebae migrating away from the “source” in the same spatial gradient. In addition, amebae extend lateral pseudopods in a polarized fashion from the anterior half of the cell, and do so as frequently towards the source as away from the source. However, those formed towards the source more often produce a turn than those formed away from the source. These results suggest that there may be two decision‐making systems, one localized in the pseudopods, and one along the entire cell body; they support the suggestion that Dictyostelium amebae may employ a temporal mechanism to assess the direction of a spatial gradient of chemoattractant.
Characterization of the intermediate filament apparatus in skin fibroblasts from patients with giant axonal neuropathy: Effect of trypsin
Authors: Manetti Roberto , Ceccarini Constante , Guazzi Giancarlo , Federico Antonio , Tiezzi Antonio , Bugnoli Massimo , Ceccarini Elvira Costantino
Published: February 2005

Skin fibroblasts from two siblings with giant axonal neuropathy (GAN) were examined by both biochemical and immunocytochemical studies. The presence of intermediate filaments (IF) characteristic of these cells was affected by the growth conditions. Immediately after plating and during the following 24 hours the majority of the cells contained an IF “bundle”; however, after 4–6 days in culture only a minority of the cells retained this structure. We present evidence that trypsinization but not serum concentration is likely to influence the formation of the “bundle.” The results indicate that the formation of the “bundle” may result from a defective association or relationship between the cytoskeleton and the plasma membrane.
Motile mechanism of blue damselfish (Chrysiptera cyanea) iridophores
Authors: Oshima Noriko , Fujii Ryozo
Published: February 2005

Iridophores of the blue damselfish, Chrysiptera cyanea, responded to the sympathetic substance, norepinephrine by a shift towards longer wavelengths of the spectral peak of the light reflected by stacks of light‐reflecting platelets (“coloring response”). All antimitotic reagents tested, i.e., colchicine, vinblastine, and podophyllotoxin, inhibited the response reversibly, while an actin inhibitor, cytochalasin B, did not. Erythro‐9‐[3‐(2‐hydroxynonyl)]adenine (EHNA), a dynein ATPase inhibitor, also blocked the iridophore response effectively. These results indicate that the tubulin‐dynein system may be involved in the motility of iridophores, which is regarded as the simultaneous alteration of the distance between adjacent reflecting platelets within the cells.
Phosphorylation determines two distinct species of tau in the central nervous system
Authors: Papasozomenos Sozos Ch. , Binder Lester I.
Published: February 2005

The monoclonal antibody, Tau‐1, which had previously been used to localize tau to the axonal compartment in brain has been reutilized for light and electron microscopic immunohistochemistry following phosphatase treatment of tissue. We report here that a significant quantity of tau in the central nervous system is phosphorylated in situ at or near the Tau‐1 epitope, preventing the binding of the Tau‐1 antibody. Upon removal of this/these phosphate group(s), however, Tau‐1 was observed in the somatodendritic compartment of neurons as well as in axons. Furthermore, intense staining was also observed in astrocytes and in perineuronal glial cells. This immunoreactivity was present along the lengths of microtubules and on ribosomes (polysomes). Treatment of immunoblots of extracts of whole cerebral cortex with phosphatase confirmed the immunohistochemical results in that a 50–65% increase in Tau‐1 binding to the tau region of the blot was noted. Moreover, a novel monoclonal antibody, Tau‐2, was also used in these experiments. This antibody binds only to tau and localizes along microtubulesin axons, somata, dendrites, and astrocytes and on ribosomes (polysomes) without phosphatase pretreatment.
Structural and chemical characterization of isolated centrosomes
Authors: Bornens Michel , Paintrand Michel , Berges Josette , Marty Marie‐Chantal , Karsenti Eric
Published: February 2005

A procedure adapted from that described by Mitchison and Kirschner [Nature 312:232–237, 1984] was used to isolate centrosomes from human lymphoid cells. High yields of homogeneous centrosomes (60% of the theoretical total, assuming one centrosome per cell) were obtained. Centrosomes were isolated as pairs of centrioles, plus their associated pericentriolar material. Ultrastructural investigation revealed: 1) a link between both centrioles in a centrosome formed by the gathering in of a unique bundle of thin filaments surrounding each centriole; 2) a stereotypic organization of the pericentriolar material, including a rim of constant width at the proximal end of each centriole and a disc of nine satellite arms organized according to a ninefold symmetry at the distal end and; 3) an axial hub in the lumen of each centriole at the distal end surrounded by some ill‐defined material.

The total protein content was 2 to 3 × 10−2 pg per isolated centrosome, a figure that suggests that the preparations were close to homogeneity. The protein composition was complex but specific, showing proteins ranging from 180 to 300 kD, one prominent band at 130 kD, and a group of proteins between 50 and 65 kD. Actin was also present in centrosome preparations.

Functional studies demonstrated that the isolated centrosomes were competent to nucleate microtubules in vitro from purified tubulin in conditions in which spontaneous assembly could not occur. They were also very effective at inducing cleavage when microinjected into unfertilized Xenopus eggs.
Association of intermediate filaments with vinculin‐containing adhesion plaques of fibroblasts
Authors: Bershadsky Alexander D. , Tint Irena S. , Svitkina Tatjana M.
Published: February 2005

Double immunofluorescence staining of quail embryo fibroblasts with rabbit antibody to vinculin and mouse monoclonal antibody to vimentin revealed a coincidence between fluorescence patterns for cell‐substrate focal contacts and intermediate filaments. Most of the vinculin‐containing adhesion plaques coincided with the ends of vimentin‐positive fibrils.

This association was further corroborated by immunoelection microscopic observations of the cytoskeletons of quail and mouse fibroblasts using a platinum replica technique. The intermediate filaments were identified either by direct treatment with antivimentin IgM or by an indirect immunogold staining method.

Colcemid treatment of the cells caused a collapse of intermediate filaments and destroyed their association with focal contacts. During the early stages of the colcemid‐induced collapse of the intermediate filaments, single vimentin fibrils appeared to retain their association with focal contacts.

The possible role of the intermediate filaments in the formation and maintenance of focal contacts is discussed.
Binding and distribution of fluorescently labeled filamin in permeabilized and living cells
Authors: Mittal Balraj , Sanger Jean M. , Sanger Joseph W.
Published: February 2005

This study report the first development of a fluorescently labeled filamin. Smooth muscle was labeled with fluorscent dyes in order to study its interaction with stress fibers and myofibrils, both in living cells and in permeabilized cells. The labeled filamin bounds to the Z bands of isolated cross‐striated myofibrils and to the Z bands and intercalated discs in both permeabilized embryonic cardiac myocytes and in frozen sections of adult rat venticle. In permeabilized embryonic chick myotubes, filamin bound to early myotubes but was absent at later stages. In living embryonic chick myotubes, the fluorescently labeled filamin was incorporated into the Z bands of myofibirls during early and late stages of develoment but was absent during an intermediate stages. In living cardiac myocytes, filamin‐IAR was incorporated into nascent as well as fully formed sarcomeres throughout develoment. In permeabilized nonmuslce cells, labeled filamin bound to attachment plaques and foci of polygonal networks and to the dense bodies in stress fibers. The periodic bands of filamin in stress fibers had a longer spacing in fibroblasts than in epithelial cells. When injected into living cells, filamin was readily incorporated into stress fibers in a striated pattern. The fluorescent filamin bands were broader in injected cells, however, than they were in permeabilized cells. We have interpreted these results from living and permeabilized cells to mean that native filamin is distributed along the full lengh of the actin filaments in the stress fibers, with a higher concentration present in the dense bodies. A sarcomeric model is presented indicating the position of filamin with respect to other proteins in the stress fibers.
Structure and mass analysis of 12S and 19S dynein obtained from bull sperm flagella
Authors: Marchese‐Ragona Silvio P. , Gagnon Claude , White Daniel , Isles Marthe Belles , Johnson Kenneth A.
Published: February 2005

The Brookhaven scanning transmission electron microscpe (STEM) was used to elucidate the structures and masses of 12S and 19S dynein extracted from bull sperm flagella. The 12S particle was a single globular particle with an average mass of 311 ± 10 kdaltons. The 19S dynein particles consisted of two globular heads joined to a common base. The average mass of the 19S particle was 1.6 ± 0.04 × 106 daltons. Thus, with the exception of the larger mass, the bull sperm 19S dynein molecule resembles the two‐headed 21S dynein obtained from sea urchin sperm flagella and the 18S dynein obtained from Chlamydomonas with the possibility of a third head giving rise to the 12S particle. The structure, mass and polypeptide composition of bull sperm flagella dynein is compared with outer arm dyneins previously obtained from Chlamydomonas, Tetrahymena, and sea urchin sperm flagella.
The position of the microtubule‐organizing center relative to the nucleus is independent of the direction of cell migration in dictyostelium discoideum
Authors: Sameshima Masazumi , Imai Yoshihisa , Hashimoto Yohichi
Published: February 2005

Dictyostelium amoebae can migrate in several different modes. We tested for correlations of the direction of cell locomotion with the relative positions of the nucleus and microtubule‐organizing center (MTOC). Five cases were analyzed on electron micrographs with a microcomputer. Each mode of movement showed characteristic locations of the MTOC relative to the nucleus; however, they differed in the various cases. In randomly migrating interphase amoebae, the number of cells with the MTOC located behind the nucleus was twice as great as those with the MTOC located ahead of the nucleus. During chemotactic migration toward folic acid, cells with the MTOC behind the nucleus were more numerous, with a concomitant reduction of anterior MTOCs.

When amoebae aggregated on agar plates, a posterior location of the MTOC was most strikingly favored, whereas in cells aggregating under submerged conditions, the MTOC was indifferently anterior or posterior to the nucleus. (It may be significant that EDTA‐resistant cell‐cell adhesion was fully expressed in the former cells, but weaker in the latter.) Finally, in the case of chemotactically migrating cells from dissociated pseudoplasmodia, which adhere by means of other molecules, the MTOC was consistently ahead of the nucleus. Thus the MTOC shows no necessary preferential position anterior or posterior to the nucleus; its position, rather, correlates with the type of migration and perhaps with the nature of cell‐cell adhesion.
A monoclonal antibody to the Ca2+ ‐ATPase of cardiac sarcoplasmic reticulum cross‐reacts with slow type I but not with fast type II canine skeletal muscle fibers: An immunocytochemical and immunochemical study
Authors: Jorgensen Annelise O. , Arnold Wayne , Pepper David R. , Kahl Steven D. , Mandel Frederick , Campbell Kevin P.
Published: February 2005

Ca2+ ‐ATPase of the sarcoplasmic reticulum was localized in cryostat sections from three different adult canine skeletal muscles (gracilis, extensor carpi radialis, and superficial digitalis flexor) by immunofluorescence labeling with monoclonal antibodies to the Ca2+ ‐ATPase Type I (slow) myofibers were strongly labeled for the Ca2+ ‐ATPase with a monoclonal antibody (II D8) to the CA2+ ‐ATPase of canine cardiac sarcoplasmic reticulum; the type II (fast) myofibers were labeled at the level of the background with monoclonal antibody II D8. By contrast, type II (fast) myofibers were strongly labeled for Ca2+ ‐ATPase of rabbit skeletal sarcoplasmic reticulum. The subcellular distribution of the immunolabeling in type I (slow) myofibers with monoclonal antibody II D8 corresponded to that of the sarcoplasmic reticulum as previously determined by electron microscopy. The structural similarity between the canine cardiac Ca2+ ‐ATPase present in the sarcoplasmic reticulum of the canine slow skeletal muscle fibers was demonstrated by immunoblotting. Monoclonal antibody (II D8) to the cardiac Ca2+ ‐ATPase binds to only one protein band present in the extract from either cardiac or type I (slow) skeletal muscle tissue. By contrast, monoclonal antibody (II H11) to the skeletal type II (fast) Ca2+ ‐ATPase binds only one protein band in the extract from type II (fast) skeletal muscle tissue. These immunopositive proteins coelectrophoresed with the Ca2+ ‐ATPase of the canine cardiac sarcoplasmic reticulum and showed an apparent Mr of 115,000. It is concluded that the Ca2+ ‐ATPase of cardiac and type I (slow) skeletal sarcoplasmic reticulum have at least one epitope in common, which is not present on the Ca2+ ‐ATPase of sarcoplasmic reticulum in type II (fast) skeletal myofibers. It is possible that this site is related to the assumed necessity of the Ca2+ ‐ATPase of the sarcoplasmic reticulum in cardiac and type I (slow) skeletal myofibers to interact with phosphorylated phospholamban and thereby enhance the accumulation of Ca2+ in the lumen of the sarcoplasmic reticulum following β–adrenergic stimulation.
Analysis of bacterial flagellar rotation
Authors: Khan Shahid
Published: February 2005

Bacterial flagella have rotary motors at their base; embedded in the cytoplasmic membrane and powered by transmembrane ion gradients instead of ATP. Assays have been developed to measure the torque output of individual motors over a wide regime of load, to correlate the energizing proton flux with rotation speed and relate through genetic analysis motor structure to function. These assays promise substantial advances in understanding mechanochemical coupling in these motors. Here, I summarize the present status of our understanding of energy transduction in bacterial flagella and compare this with the case for muscle.
High‐performance optical filters for fluorescence analysis
Authors: Marcus David A.
Published: February 2005

Recent advances in thin film optical coating technology significantly improve the filters available for fluorescence spectroscopy. Bandpass and long‐ and shortpass filters with very sharply defined edges can provide from 10−5 to 10−6 blocking within 10–15 nm of the transmission region and are ideal for use as excitation and emission filters. A variety of nonpolarizing dichroic beamsplitters for use in epi‐illumination configurations or in multiple emission configurations provides optimum longpass, shortpass, band reflection, or bandpass spectral control. These dichroics, used with high‐performance bandpass, longpass, or shortpass filters, form matched sets that optimize the signal‐to‐noise ratio and system efficiency for fluorescence spectroscopic systems in single or multiple dye applications. Specially designed dichroic beamsplitters are used to reduce excitation filter overheating. Other dichroic beamsplitters efficiently separate two planes of polarization in a narrow wavelength band. Rejection band filters can be used to measure the fluorescent dye Indo 1 with very low emission signals.
In vitro reactivation of anaphase B in isolated spindles of the sea urchin egg
Authors: Rebhun Lionel I. , Palazzo Robert E.
Published: February 2005

Spindles may be isolated from sea urchin eggs so that some mitotic processes can be reactivated in vitro. The isolation media allow spindles to remain stable for days. Transfer of the spindles to reactivation media results in loss of birefringence and breakdown of the matrix within which the microtubules function. If, however, tubulin and either guanosine triphosphate or adenosine triphosphate are present in these media so that tubulin can cycle, the spindles do not break down but grow in size and birefringence and show some of the movements of in vivo spindles. The most prominent is that of anaphase B if the mitotic apparatuses (MAs) have been isolated at a time when anaphase was initiated. When isolated during metaphase, MAs either do not show chromosome movement or, if they do, it is a random movement which causes redistribution of the chromosomes on the spindle surface. In either case, such metaphase spindles grow in size and birefringence. Thus under the proper conditions, cycling microtubules can interact with the spindle matrix to induce chromosome movements which resemble those seen in in vivo cells in the case of anaphase B and show some aspects of anaphase A in at least half the spindles isolated at metaphase, although such movements are not coordinated to show a true anaphase movement.
Isolation and reactivation of highly‐coupled newt lung cilia
Authors: Hard Robert , Cypher Christopher , Schabtach Eric
Published: February 2005

The paired lungs of the newt, Taricha granulosa, are simple, unbranched sacs, 3.5–5.0 cm in length. The inner epithelium overlying the pulmonary vein is differentiated into a mucociliary tract that extends the entire length of the lung. Populations of single, demembranated ciliary axonemes, 12–13 μm in length, can be isolated by extracting whole lungs or primary cultures of the ciliated epithelium with Triton X‐100. The motile capabilities of the isolated axonemes are the highest yet obtained for any ciliary model. When exposed to a suitable reactivating medium containing Mg2+ and ATP, nearly 100% of the axonemes become motile. Uniform reactivation of high quality requires short extraction times, minimization of mechanical damage, and strict adherence to optimal conditions throughout the extraction, storage, and reactivation procedures. Significant deviations from either pH 7.0 or 0.12 M salt can lead to a rapid, irreversible decrease in the beat frequency of reactivated axonemes. Both DTT and EDTA serve to stabilize their motility. The isolated axonemes beat at 29.5 Hz in the presence of 1.75 mM ATP at 21°C, matching the beat frequencies measured for cultured cells at the same temperature. With 5 mM ATP, beat frequencies over 40 Hz are measured. Our results show that neither the plasma membrane, accessory structures, nor hydrodynamic coupling of cilia are required for this activity and imply that the lack of these factors is not responsible for the low motile capabilities of ciliary models isolated previously.
Fast axonal transport alterations in amyotrophic lateral sclerosis (ALS) and in parathyroid hormone (PTH)‐treated axons
Authors: Breuer Anthony C. , Atkinson Mark B.
Published: February 2005

Video‐enhanced contrast techniques have been used to study fast axonal transport of organelles in diseased and normal human axons. A broad perspective on the importance of axonal transport in the pathogenesis of human neurological disorders is presented and problems in dealing with human nerve summarized. Results from analysis of organelle traffic in axons from motor nerve in patients with amyotrophic lateral sclerosis (ALS) show: (1) higher mean speed of anterograde organelles, (2) lower mean speed of retrograde organelles, and (3) lower retrograde organelle traffic density. Hyperparathyroidism, another human clinical syndrome, can mimic ALS. The effect of parathyroid hormone (PTH) on axons in vitro is to increase the mean speed of both anterograde and retrograde organelle traffic. The dose response curve and time course of the PTH effect are delineated. Dihydropyridine calcium channel antagonists block the PTH effect, implicating extracellular calcium in the alteration of organelle traffic speed. The results are discussed in relation to neuronal function and the regulation of fast axonal transport.
Involvement of myxamoebal fragmin in the Ca2+‐induced reorganization of the microfilamentous cytoskeleton in flagellates of Physarum polycephalum
Authors: Uyeda Taro Q. P. , Hatano Sadashi , Furuya Masaki
Published: February 2005

When flagellates of Physarum polycephalum were treated with Triton X‐100 and more than 10−5 M Ca2+, the microfilamentous cytoskeleton disintegrated, as seen by staining with rhodamine‐phalloidin, and myxamoebal fragmin became associated with the Triton‐insoluble cytoskeleton as demonstrated by immunofluorescence microscopy and immunoblotting. The association of myxamoebal fragmin with the cytoskeleton was reversed by the subsequent addition of excess EGTA. When flagellates were permeabilized in the absence of Ca2+, myxamoebal fragmin did not associate with the cytoskeleton and diffused out of the cells. Subsequent treatment of these cells with Ca2+ was ineffective in inducing either the association of myxamoebal fragmin with the cytoskeleton or the disintegration of the microfilamentous cytoskeleton. However, treatment of these permeabilized flagellates with 10 μg/ml purified myxamoebal fragmin and 1 mM Ca2+ caused the disintegration of the microfilaments. Therefore, we conclude that myxamoebal fragmin participates in the Ca2+‐induced disintegration of the microfilamentous cytoskeleton in these permeabilized cells. Rapid cooling of flagellates caused the reversible association of myxamoebal fragmin with the Triton‐insoluble cytoskeleton in vivo. Thus myxamoebal fragmin may also participate in the reorganization of the microfilamentous cytoskeleton induced in vivo by the cold treatment.
Cell‐cycle modulation of MPM‐2‐specific spindle pole body phosphorylation in Aspergillus nidulans
Authors: Engle Dorothy B. , Doonan John H. , Morris N. Ronald
Published: February 2005

MPM‐2 is a monoclonal antibody that interacts with mitosis‐specific phosphorylated proteins in many different organisms. Immunocytochemistry of tissue culture cells has shown that MPM‐2 stains centrosomes, chromosomes, kinetochores, and spindles. In this paper, we demonstrate that MPM‐2 staining colocalizes with the spindle pole body (SPB) of Aspergillus nidulans and that SPB staining varies during the mitotic cycle. In an unsynchronized population, about one‐fourth to one‐third of the cells stain with MPM‐2 at the spindle plaques or SPBs. Nuclei in mitosis have two SPBs localized at the ends of the spindle, both of which stain with MPM‐2. To determine when MPM‐2 staining appears, we have examined the effects of temperature‐sensitive cell‐cycle mutations that block nuclear division in S or G2. Only a very small fraction of cells blocked in S‐phase stain with MPM‐2. In contrast, a large fraction of cells blocked in G2 stain brightly at the SPB. These data suggest that MPM‐2 reactivity of SPBs appears in G2. Moreover, the fact that cells blocked in G2 showed MPM‐2 staining but no spindles suggests that reactivity of SPBs occurs prior to mitosis but is not sufficient to trigger spindle formation. When G2‐blocked cells were downshifted to permissive temperature, they generated a mitotic spindle with an SPB at each end. Both SPBs stained with MPM‐2 in all of the mitotic cells.
Protease inhibitor and substrates block motility and microtubule sliding of sea urchin and carp spermatozoa
Authors: Cosson Marie‐Paule , Gagnon Claude
Published: February 2005

The effects of protease substrates and inhibitor, which have been previously shown to inhibit mammalian sperm motility (de Lamirande, E., and Gagnon, C. [1986] J. Cell Biol. 102:1378–1383), were investigated using reactivated sea urchin and carp spermatozoa as models of “9 + 2” flagella. Aprotinin in the 2 to 20 μM range interfered with sperm motility by reducing both the beat frequency and the percentage of motile spermatozoa. These inhibitory effects of aprotinin were reversible either by dilution or by the addition of high concentrations of MgATP to the incubation medium. Protease substrates with a lys–ester bond, such as N‐α‐benzyloxycarbonyl‐lys thiobenzyl ester (BLT), also affected motility, but in the 0.1 to 0.5 mM range. As with aprotinin, both the flagellar beat frequency and the percentage of motile spermatozoa were partially and completely decreased, respectively. Analysis of the beat frequencies as a function of MgATP concentration in the presence and absence of 6 μM aprotinin indicated that this protease inhibitor affects sperm motility by decreasing the maximal flagellar beat frequency rather than by altering the axoneme's apparent Km for MgATP. Furthermore, aprotinin concentrations that blocked flagellar reactivation completely inhibited the sliding of microtubules from trypsinized axonemes. Basic proteins or polypeptides of pI close to that of aprotinin (10.3) were also potent inhibitors of the reactivation of motility. However, the characteristics of their inhibition of flagellar beat frequencies and reversibility of their effects suggested that they might be acting on sites different from those sensitive to aprotinin. The inhibitory effects of protease inhibitor and substrates, as well as results of experiments showing the absolute requirement of an intact ester bond for the inhibitory action of protease substrates, suggest that the involvement of a protease in the reactivation of 9 + 2 flagellar beating might be considered as a possible mechanism to explain aprotinin and BLT actions.
Acrylamide treatment of PtKl cells causes dephosphorylation of keratin polypeptides
Authors: Eckert Barry S. , Yeagle Philip L.
Published: February 2005

Treatment of PtKl cells with 5 mM acrylamide for 4 hr results in alterations in the distribution of keratin filaments within the cells. This effect is reversible within 18 hr. Labeling of PtKl cells with 32P demonstrates that there are four phosphorylated keratins, having Mr of 56 k, 53 k, 45 k, and 40 k. Phosphate associated with these polypeptides appears to turn over with a t1/2 of 12 hr. Incubation of labeled cells in 5 mM acrylamide results in approximately 50% dephosphorylation of keratins within 2 hr, which is 3 times faster than normal turnover. Recovery of cells from acrylamide is accompanied by rephosphorylation of keratins within 18 hr. Analysis by 31P NMR spectroscopy shows that acrylamide treatments are accompanied by a transient decrease in soluble inorganic phosphate. This is followed by a rapid increase in Pi which gradually returns to normal levels. These studies show a strong correlation between phosphorylation of PtKl cell keratins and morphological response of keratin filaments to acrylamide. These observations suggest that normal distribution of keratin filaments may be, in part, mediated by protein phosphorylation.
Adhesion of cells to protein carpets: Do cells' feet have to be black?
Authors: Opas Michal
Published: February 2005

In most physiological situations, cell contact with a substratum is mediated by proteins of extracellular matrix. Therefore, an increasing number of cellsubstratum adhesion studies employ substrata covered with one or more proteins of extracellular matrix. To visualize the most adhesive cell structures, focal contacts and focal adhesions, the interference reflection microscopy has been widely used. It has been generally accepted that these strongly adhesive structures can be seen as black streaks in interference reflection microscopy. Calculations are presented herein, which although simplified, suggest that when cells are plated on protein‐covered substrata, their focal contacts may not always appear black in interference reflection microscopy.
Centripetal transport of cytoplasm, actin, and the cell surface in lamellipodia of fibroblasts
Authors: Fisher Gregory W. , Conrad Patricia A. , Debiasio Robbin L. , Taylor D. Lansing
Published: February 2005

Wound healing in Swiss 3T3 cultures was investigated with video‐enhanced contrast (VEC) microscopy. The formation of protrusions at the leading edge of cells along wounds was investigated in detail during the spreading stage, which usually lasted from 1 to 4 hr postwounding. Lamellipodia exhibited a continuous rearward, or centripetal, transport of a variety of cellular constituents at rates of ∼0.26 μm/sec from the leading edge. The lamellipodia were also the sites of lateral migration as well as extension and retraction of actin microspikes. Actin fibers oriented transversely to the direction of movement were also observed to transport centripetally at similar rates. These fibers may in part give rise to large actin fibers forming at the interface between the base of the lamellipodia and the lamellae. Beads 0.5 μm in diameter attached to the dorsal surfaces of lamellipodia also transported centripetally at rates of ∼0.21 μm/sec. Thus there is an apparent correlation between transport of a variety of structures within lamellipodia and with surface movements of lamellipodia.
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

The 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

This 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

Mitotic 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

To 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

To 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

Following 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

Actin 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

We 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

The 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

Microtubules 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.