bluuebunny/crossref_metadata_2025 huggingface dataset:
Flagellar photoresponses of Chlamydomonas cells held on micropipettes: I. Change in flagellar beat frequency
Authors: Rüffer Ursula , Nultsch Wilhelm
Published: February 2005

Chlamydomonas 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

Phototaxis 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

Definition 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.
Light chains of sea urchin kinesin identified by immunoadsorption
Authors: Johnson Catharine S. , Buster Dan , Scholey Jonathan M.
Published: February 2005

Previous studies with monoclonal antibodies indicate that sea urchin kinesin contains two heavy chains arranged in parallel such that their N‐terminal ends fold into globular mechanochemical heads attached to a thin stalk ending in a bipartitetail [Scholey et al. 1989]. In the present, complementary study, we have used the monoclonal antikinesin. SUK4, to probe the quaternary structure of sea urchin (Strongylocentrotus purpuratus) kinesin. Kinesin prepared from sea urchin cyto‐sol sedimented at 9.6 S on sucrose density gradients and consisted of 130‐kd heavy chains plus an 84‐kd/78 kd doublet (1 mol heavy chain: 1 mol doublet determined by gel densitomctry). Low levels of 110‐kd and 90‐kd polypeptides were sometimes present as well. The 84‐kd/78 kd polypeptides are thought to be light chains because they were precipitated from the kinesin preparation at a stoichiometry of one mol doublet per 1 mol heavy chain using SUK4‐Sepharose immunoaffinity resins. The 110‐kd and 90‐kd peptides, by contrast, were removed using this immunoadsorption method. SUK4‐Sepharose immunoaffinity chromatography was also used to purify the 130‐kd heavy chain and 84‐kd/78‐kd doublet (1 mol heavy chain: 1 mol doublet) directly from sea urchin egg cytosolic extracts, and from a MAP (microtubule‐associated protein) fraction eluted by ATP from microtubules prepared in the presence of AMPPNP but not from microtubules prepared in ATP. The finding that sea urchin kinesin contains equi‐molar quantities of heavy und light chains, together with the aforementioned data on kinesin morphology, suggests that native sea urchin kinesin is a tetramer assembled from two light chains and two heavy chains.
Identification and characterization of a novel mammalian intermediate filament‐associated protein
Authors: Brown Kevin D. , Binder Lester I.
Published: February 2005

A novel monoclonal antibody, designated M1.4, recognizes the high molecular weight microtubule‐associated protein MAPIA (ca. Mr 380 kD) in both bovine and rat brain. In HeLa cells, however, M1.4 binds to a 240 kD polypeptide on immunoblots and co‐localizes with both vimentin and cytokeratin filaments using double‐label immunofluorescence microscopy. Immunoelectron microscopy indicates that the 240 kD polypeptide localizes along bundled intermediate filaments in a periodic manner. Two‐dimensional electrophoretic analysis indicates that the 240 kD polypeptide has a basic pI of 7.7. When HeLa cell intermediate filaments are isolated using standard non‐ionic detergent/high‐salt conditions the 240 kD polypeptide does not sediment with the intermediate filaments, unlike the established intermediate filament‐associated protein plectin. Immunoblot analysis with M1.4 shows the 240 kD polypeptide is expressed in a number of mammalian cell lines. Additionally, double‐label immunofluorescence shows the 240 kD polypeptide to associate with vimentin filaments in African Green Monkey kidney (CV‐1) and JC neuroblastoma cells. Due to its unique biochemical and biological characteristics, the 240 kD polypeptide is clearly a novel intermediate filament‐associated protein for which we have proposed the designation gyronemin (Gr.gyros: around: nemin: filament).
Neurite elongation is blocked if microtubule polymerization is inhibited in PC12 cells
Authors: Keith Charles H.
Published: February 2005

We have injected process‐bearing PC12 cells with colchicine‐tubulin mixed with either fluorescein‐dextran or a rhodamine‐labelled tubulin analogue to determine the role of microtubule polymerization in neurite elongation. Colchicine‐tubulin is a specific, substoichiometric poison of microtubule assembly. We have shown that colchicine‐tubulin does not cause existing PC12 microtubules to disassemble, and yet can inhibit the assembly of rhodamine‐tubulin injected along with it. In population s'udies of neurite outgrowth in injected and uninjected cells, we find that colchicine‐tubulin substantially inhibits neurite extension from injected cells over a wide variety of concentrations. In acute time‐course studies of injected cells, we find that colchicine‐tubulin does not block neurite outgrowth until the injectate reaches the neurite tip. Thereafter, however, it blocks process elongation completely. Thus we can conclude that microtubule polymerization in the region of the growth cone is an important element in neurite elongation. While polymerization at the cell body may be important in supplying subunits to the distal neurite, it does not play a direct role in process extension.
Stoichiometry of estramustine phosphate binding to MAP2 measured by the disassembly of chick brain MAP2: Tubulin microtubules
Authors: Burns Roy G.
Published: February 2005

The concentration of estramustine phosphate required to inhibit the assembly or to induce the disassembly of chick brain MAP2:tubulin microtubules is markedly dependent upon the microtubule protein concentration. Analysis of this relationship shows that estramustine phosphate and tubulin compete for common MAP2 sites, that MAP2 can bind 5–6 moles‐mole−1 estramustine phosphate, and that the Kd of these sites is ≏ 20 μM estramustine phosphate. It is proposed that two molecules of estramustine phosphate interact with each of the three tubulin‐binding sites of MAP2 and inhibit the MAP2:tubulin interaction by neutralising two highly conserved basic residues.
Computerized analysis of flagellar motility by digitization and fitting of film images with straight segments of equal length
Authors: Brokaw Charles J.
Published: February 2005

Methods are described for computerized analysis of digitized images obtained by scanning photomicrographs of swimming sperm flagella. After storing a series of image frames in computer memory, the entire series is analyzed automatically. For each sperm image, the sperm head is located to obtain a starting point for analysis of the flagellum. This location is obtained by minimizing image intensity along a model of the sperm head outline. The flagellum is modelled by a series of straight segments of equal length: 0.5 or 1 μm. The angles between these segments are adjusted to give minimum image intensity along the line of the model as well as minimizing smoothing functions. Extensions to analyze a series of images in each frame, and to measure the positions of beads attached to the flagellar microtubules, are also described.
Identification of surface components of mammalian respiratory tract cilia
Authors: Hastie Annette T. , Krantz Mori J. , Colizzo Frank P.
Published: February 2005

Cilia isolation methods were modified to retain respiratory tract ciliary membranes and to identify accessible surface components. Prior to isolation of cilia, halves of cow tracheae were treated with the extended spacer arm analog of N‐hydroxy succinimido‐biotin (NHS‐LC‐biotin) to label accessible membrane constituents. Mechanical disruption of the epithelium and substitution of CHAPS for Triton X‐100 provided a good yield of cilia with membranes and with minimal contamination. Subsequent extraction of these cilia with Triton X‐100 solubilized the membranes and released soluble matrix proteins. Proteins of membrane + matrix and axoneme fractions were analyzed after electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The major biotin‐labeled components in the membrane + matrix fraction were 105, 98, and 92 kd, were glycosylated, and remained with reconstituted, pelleted membrane vesicles along with the major non‐biotinylated protein at 51 kd. Other membrane+matrix proteins at 126 and 76 kd bound streptavidin even from noniabeled trachea, but remained soluble. Several biotin‐labeled proteins distinct from those in the membrane fraction remained with Triton X‐100‐extracted axonemes. Streptavidin‐colloidal‐gold (SAG) particles appeared to bind randomly along the length of cilia. The peripheral join between A and B microtubules was a predominant nonspecific location of SAG on axonemes. Axonemes with biotin label also bound significant numbers of SAG to outer dynein arms, confirming the streptavidin reaction with separated proteins on transfers. These results suggest close association of the membrane with the axoneme in respiratory tract cilia and a membrane composition somewhat different from protozoan cilia.
Alpha‐actinin and actin in the outer retina: A double immunoelectron microscopic study
Authors: Arikawa Kentaro , Williams David S.
Published: February 2005

Actin has many diverse functions in the outer retina. To help elucidate its organization in this area, we have investigated the extent of its association with the actin cross‐linking protein alpha‐actinin. Ultrathin sections of chicken retina were double‐immunolabelled with monospecific antibodies against actin and alpha‐actinin. The highest relative amount of alpha‐actinin to actin label was measured in the adherens junctions between the individual retinal pigmented epithelial (RPE) cells and between the photoreceptor and Mueller cells; in the photoreceptor myoid; and in the RPE basal microvilli. The lowest amount was in the Mueller cell microvilli, the RPE apical processes, and in the photoreceptor ellipsoid. It is likely that the areas containing the highest ratio of alpha‐actinin to actin labelling are where the actin filaments are most highly cross‐linked into bundles and linked to the plasma membrane by alpha‐actinin. Actin filaments terminate in these areas, and, except for the myoid region, they are involved in cell‐cell or cell‐substrate adherens junctions.
Cell division in Dictyostelium with special emphasis on actomyosin organization in cytokinesis
Authors: Fukui Yoshio , Inoué Shinya
Published: February 2005

This study focuses on the dynamic reorganization of actin and myosin (“conventional” myosin, or myosin‐II) during cytokinesis in D. discoideum. This is the first study identifying the birefringence of the spindle microtubules as well as three sets of microfilamentous structure in Dictyostelium. The change of organization in these fibrillar structures was followed in real‐time with video microscopy, using a Universal Polarizing Microscope equipped with polarized‐light (POL) and differential interference contrast (DIC) optics combined with digital image processing. High‐frequency mitotic cells were obtained by semi‐synchronous culture, and high‐resolution observations were made by utilizing the agar‐overlay method (Yumura et al.: Journal of Cell Biology 99:894–899, 1984). The molecular identity of the birefringent structures was determined by fluorescence microscopy. Through‐focus observations were performed with an axial resolution of 0.3 μm depth of field.

The actomyosin fibrils show a dramatic reorganization throughout mitosis. The fibrils at the leading lamellipodia disappear, and there is a striking assembly of the cortical actomyosin in pro‐metaphase, which is accompanied by a decrease in cell volume. The cortical actomyosin gradually increases through anaphase. After late anaphase, very active polar lamellipodia, with an average life of less than 1 minute, are formed. We confirmed that the polar lamellipodia include actin, but not myosin‐II. At the cleavage furrow, the microfilaments form two distinctive structures: circular contractile ring at the equator, and a cortical filament array parallel to the polar axis. Myosin is localized in the contractile ring, but not associated with the axial array of F‐actin. Actomyosin in the contractile ring gradually transforms into cortical network at the posterior region of daughter cells. The constriction of the furrow is accompanied by a drastic efflux of water as evidenced by highly active contractile vacuole formation and turbulent motion of minute vesicles connected to the furrow. This study demonstrates the presence of a new microfilament structure, as well as the dynamic property of the contractile ring, and sheds new light on the contractile mechanisms underlying cytoki‐nesis.
Production and specificity of monoclonal antibodies against calmodulin from dictyostelium discoideum
Authors: Hulen Durvis , Baron Andre , Salisbury Jeffrey , Clarke Margaret
Published: February 2005

Monoclonal antibodies were raised against calmodulin purified from Dictyostelium discoideum. To increase its antigenicity, the calmodulin was conjugated to keyhole limpet hemocyanin; mice were immunized with the conjugate. Hybridomas producing antibodies against calmodulin were identified by screening culture supernatants with calmodulin coupled to bovine serum albumin. The specificity of antibodies from hybridoma culture supernatants was tested by Western blot of Dictyostelium cell lysates. For this purpose, methods were developed that permitted sensitive detection of calmodulin bound to membranes. The key elements of the blotting protocol were use of PVDF membrane, transfer conducted in phosphate buffer, and glutaraldehyde fixation after transfer. These methods permitted detection of as little as 0.1 ng of calmodulin spotted directly onto the membrane, or 10 ng transferred from an SDS polyacrylamide gel. Ten calmodulin‐specific antibodies were identified; most of these reacted preferentially with the calcium‐containing form of Dictyostelium calmodulin. Several of the monoclonal antibodies cross‐reacted with calmodulin from bovine brain.
Calcium sensors in sea urchin sperm flagella
Authors: Brokaw Charles J.
Published: February 2005

The asymmetry of ATP‐reactivated flagellar bending waves of Triton‐demem‐brated sea urchin spermatozoa has been measured over a range of free Ca2+ ion concentrations from 10−9 to 10−4 M. Detailed examination of the gradual response of asymmetry to Ca2+ ion concentration over this wide range indicates the presence of two Ca2+ sensors. A high‐affinity sensor operates at Ca2+ concentrations near 10−7.5 M. A lower‐affinity sensor operates at Ca2+ concentrations above 10−6 M, in the typical range for calmodulin‐mediated responses. Incubation of demembranated sperm flagella at high Ca2+ concentrations to release calmodulin is required to enable these Ca2+ responses to be observed. This treatment also causes a decrease in the apparent affinity of the flagella for cal‐modulin, as determined by measuring the increase in asymmetry in response to addition of exogenous calmodulin at low Ca2+ concentration.
Chromosome fiber dynamics and congression oscillations in metaphase PtK2 Cells at 23°C
Authors: Wise Dwayne , Cassimeris Lynne , Rieder Conly L. , Wadsworth Patricia , Salmon Edward D.
Published: February 2005

A bioriented chromosome is tethered to opposite spindle poles during congression by bundles of kinetochore microtubules (kMts). At room temperature, kinetochore fibers are a dominant component of mitotic spindles of PtK2 cells. PtK2 cells at room temperature were injected with purified tubulin covalently bound to DTAF and congression movements of individual chromosomes were recorded in time lapse. Congression movements of bioriented chromosomes between the poles occur over distances of 4.5 μm or greater. DTAF‐tubulin injection had no effect on either the velocity or extent of these movements. Other cells were lysed, fixed, and the location of DTAF‐tubulin incorporation was detected from digitally processed images of indirect immunofluorescence of an antibody to DTAF. Microtubules were labeled with an anti‐beta tubulin antibody. At 2‐5 minutes after injection, concentrated DTAF‐tubulin staining was seen in the kinetochore fibers proximal to the kinetochores; a low concentration of DTAF‐tubulin staining occurred at various sites through the remaining length of the fibers toward the pole. Kinetochore fibers in the same cell displayed different lengths (0.2 to 4 μm) of concentrated DTAF‐tubulin incorporation proximal to the kinetochore, as did sister kinetochore fibers. Ten minutes after injection, the lengths of DTAF‐containing chromosomal fibers were greater than expected if incorporation resulted solely from the lengthening of kinetochore microtubules due to congression movements of the chromosomes. Besides incorporation as a result of chromosome movement, two other mechanisms might explain the length of the DTAF‐containing segments: (1) a poleward flux of tubulin subunits (Mitchison, 1989) or (2) capture of DTAF‐containing nonkinetochore microtubules.
A monoclonal antibody recognizing a common antigen on Drosophila embryos and human fibroblasts
Authors: Callaini Giuliano , Riparbelli Maria Giovanna
Published: February 2005

We used a monoclonal antibody specific for vimentin from human fibroblasts to stain whole mounts of Drosophila embryos. In immunofluorescence observations this antibody cross‐reacts with an antigenic determinant localized throughout mitosis at the nuclear boundary. Double fluorescence observations with the Rb188 antibody that specifically recognizes a centrosomal protein of the Drosophila embryo [Whitfield et al., 1988] showed that the anti‐vimentin antibody cross‐reacts with an antigen localized in the centrosomal region.
Discrete subcellular localization of a cytoplasmic and a mitochondrial isozyme of creatine kinase in intestinal epithelial cells
Authors: Keller Thomas C. S. , Gordon Phillip V.
Published: February 2005

Two isozymes of creatine kinase have been purified differentially from mitochondrial and cytoplasmic subfractions of intestinal epithelial cells. These intestinal epithelial cell creatine kinases were indistinguishable from the cytoplasmic (B‐CK) and mitochondrial (Mi‐CK) creatine kinase isozymes of brain when compared by SDS‐PAGE, cellulose polyacetate electrophoresis, and peptide mapping. In intestinal epithelial cells, immunolocalization of the Mi‐CK isozyme indicates that it is associated with long, thin mitochondria, which are excluded from the brush border at the apical end of each cell. In contrast, immunolocalization of the B‐CK isozyme indicates that it is concentrated distinctly in the brush border terminal web domain. Although absent from the microvilli, B‐CK also is distributed diffusely throughout the cytoplasm. Terminal web localization of B‐CK was maintained in glycerol‐permeabilized cells and in isolated brush borders, indicating that B‐CK binds to the brush border structure. The abundance and localization of the mitochondrial and cytoplasmic creatine kinase isozymes suggest that they are part of a system that temporally and/or spatially buffers dynamic energy requirements of intestinal epithelial cells.
Unidirectional microtubule assembly in cell‐free extracts of Spisula solidissima oocytes is regulated by subtle changes in pH
Authors: Suprenant Kathy A.
Published: February 2005

Most, if not all, microtubules in vivo grow unidirectionally from a nucleation site such as the centrosome. This organized growth of microtubules can generate and maintain the radially symmetrical array of interphase microtubules as well as the bipolar mitotic apparatus. To investigate the regulation of polarized microtubule growth, we have prepared a cell‐free extract from surf clam oocytes that exhibits unidirectional microtubule assembly. Immunofluorescence microscopy was used to visualize the net assembly of microtubules onto the fast (plus)‐ and slow (minus)‐ growing ends of isolated ciliary axonemes. All detectable microtubule growth in these cytoplasmic extracts occurred at the plus (+) ends and the extent of (+) end growth was regulated by subtle changes in pH. Microtubule assembly in these crude extracts was highly favored at pH 7.3, the pH of the post‐fertilization cytoplasm. In contrast, when tubulin was purified from these oocyte extracts, integral components were lost, and microtubule growth became predominantly bidirectional and was favored at acidic pH. These results indicate that cytoplasmic factors may inhibit bidirectional growth in vivo and that temporal or local changes in cytoplasmic pH may influence microtubule assembly during the cell cycle.
Synchronous triggering of trout sperm is followed by an invariable set sequence of movement parameters whatever the incubation medium
Authors: Cosson Marie‐Paule , Cosson Jacky , Billard Roland
Published: February 2005

The movement of live trout spermatozoa is very brief (25 sec at 20°C) and conditions have been developed to get synchronous initiation of sperm motility which allowed quantification of the major parameters of sperm movement during the motility phase.

Recorded flagellar beat frequencies decreased steadily from values of 55 Hz at the beginning to 20 Hz at the end of the motility phase. Sperm forward velocities followed a similar pattern from 250 to 20 μm.sec−1 in the same conditions and the diameters of sperm trajectories were reduced from 370 to 40 μm. Thus none of the characteristics of sperm movement was constant during the motile phase which ended abruptly by a straightening of the flagella.

The decrease in flagellar beat frequencies and sperm velocities are much greater than what could be extrapolated from the decrease of intracellular ATP (Christen R. et al: Eur. J. Biochem, 166:667–671, 1987) or from measurements of ATP‐dependence of reactivated sperm velocities (Okuno M. and Morisawa N.: In Biological Functions of Microtubules and Related Structures. New York: Academic Press, pp. 151–162, 1982). Therefore, the cessation of flagellar beating at 25 sec is not directly the result of the low concentration of intracellular ATP.

The decrease in the diameters of sperm trajectories which occurred during the first part of the motility phase was correlated with [Ca]i measurements (Cosson M.P. et al, Cell Motil. Cytoskeleton, 14:424–434, 1989). The effect of Ca2+ at the axonemal level does not indicates that Ca2+ influx is previous to flagellar beating but rather suggests a classical Ca2+ regulation of the flagellar assymetry.

The short duration of the motility phase and the characteristics of sperm movement were very similar in various conditions (high external K+, low pH media) where increased external Ca2+ or divalent ions were shown to overcome K+ and H+ inhibition of sperm motility, both conditions which have been shown to depolarize the plasma membrane potential (Gatti J.L. et al: J. Cell Physiol., 143:546–554, 1990).

The present study of the parameters of sperm movement suggests that once motility is initiated, a defined set of axonemal events will take place whatever the external conditions.
Examination of the calcium‐modulated protein S100α and its target proteins in adult and developing skeletal muscle
Authors: Zimmer Danna B.
Published: February 2005

In this study radioimmunoassay, immunohistochemistry, Northern blot analysis, and a gel overlay technique have been used to examine the level, subcellular distribution, and potential target proteins of the S100 family of calcium‐modulated proteins in adult and developing rat skeletal muscles. Adult rat muscles contained high levels of S100 proteins but the particular form present was dependent on the muscle type: cardiac muscle contained exclusively S100α, slow‐twitch skeletal muscle fibers contained predominantly S100α, vascular smooth muscle contained both S100α and S100β, and fast‐twitch skeletal muscle fibers contained low but detectable levels of S100α and S100β. While the distribution of S100 mRNAs paralled the protein distribution in all muscles there was no direct correlation between the mRNA and protein levels in different muscle types, suggesting that S100 protein expression is differentially regulated in different muscle types. Immunohistochemical analysis of the cellular distribution of S100 proteins in adult skeletal muscles revealed that S100α staining was associated with muscle cells, while S100β staining was associated with nonmuscle cells. Radioimmunoassays of developing rat skeletal muscles demonstrated that all developing muscles contained low levels of S100α at postnatal day 1 and that as development proceeded the S100α levels increased. In contrast to adult muscle, S100α expression as confined to fast‐twitch fibers in developing skeletal muscle until postnatal day 21. At postnatal day 1, developing contractile elements were S100α positive, but no staining periodicity was detectable. At postnatal day 21, S100α exhibited the same subcellular localization as seen in the adult: colocalization with the A‐band and/or longitudinal sarcoplasmic reticulum. Comparison of the S100α‐binding protein profiles in fast‐ and slow‐twitch fibers of various species revealed few, if any, species‐ or fiber type‐specific S100 binding proteins. Isolated sarcoplasmic reticulum fractions and myo fibrils contained multiple S100α‐hinding proteins. The colocalization of S100α and S100α‐binding proteins with the contractile apparatus and sarcoplasmic reticulum suggest that S100α may regulate excitation and/or contraction in slow‐twitch fibers.
Buffer conditions and non‐tubulin factors critically affect the microtubule dynamic instability of sea urchin egg tubulin
Authors: Simon John R. , Parsons Stephen F. , Salmon E. D.
Published: February 2005

The dynamic instability of individual microtubules (Mts) in cytoplasmic extracts or assembled from highly purified sea urchin egg tubulin was examined using video‐enhanced, differential‐interference contrast (VE‐DIC) light microscopy. Extract Mts (endogenous tubulin = 12.1 μM) displayed only plus‐ended growth. The elongation velocity was 7.8 μm/min for an average duration of 1.3 min before switching (catastrophe) to rapid shortening, which occurred at 13.0 μm/ min for an average duration of 0.5 min before switching (rescue) back to the elongation phase. These parameters are typical of interphase Mt dynamic instability. Surprisingly, Mts assembled from purified urchin egg tubulin in standard buffers were less dynamic that those reported for purified brain tubulin or Mts in the extract. Buffer parameters were changed in an attempt to mimic the extract Mt results. The pH buffer itself, Hepes or Pipes, drastically altered Mt dynamics but could not achieve high elongation velocity with high catastrophe frequencies. Calcium at 1 μM had negligible effects, while increasing pH from 6.9 to 7.2 stimulated elongation velocity. Finally, Mt dynamics of purified egg tubulin (11.9 μM) were assayed in ultrafiltiates (MW cut‐off <30 kD) of the cytoplasmic extracts. Mts elongated slowly at 1.2 μm/min for 26 min before a catastrophe and rapid shortening at 11.8 μm/min. Rescue was less frequent than unfiltered extracts, minus‐ended growth was observed, and self‐assembly occurred at slightly higher tubulin concentrations. Therefore, the egg extracts and cytoplasm must contain non‐buffer factors which stimulate elongation velocity by 6.5‐fold without self‐assembly, increase catastrophe frequency by 20‐fold, and block minus‐ended growth.
Functional coupling to brush border creatine kinase imparts a selective energetic advantage to contractile ring myosin in intestinal epithelial cells
Authors: Gordon Phillip V. , Keller Thomas C. S.
Published: February 2005

The B‐CK isozyme of cytoplasme creatine kinase is localized distinctly in the terminal web region of the intestinal epithelial cell brush border (Keller and Gordon: Cell Motil. Cytoskeletoa 19:169–179, 1991). Experiments were performed to determine whether this CK is energetically coupled to the myosin II that is present in the circumferential ring and interrootlet structural domains of the brush border terminal web. In isolated brush borders, ATP‐dependent circumferential ring contraction and interrootlet myosin solubilization were supported either by an exogenous PEP‐pyruvate kinase‐based ATP‐regeneration system (PEP‐PK) or by the addition of phosphocreatine to the endogenous B‐CK‐based ATP‐regeneration system (PCr‐B‐CK). Addition of an exogenous hexokinase‐glucose ATP‐hydrolysis system (HK‐G) effectively blocked both contraction and myosin solubilization in the PEP‐PK assay. In contrast, HK‐G had no significant effect on PCr‐B‐CK‐supported brush border contraction, although it did inhibit interrootlet myosin solubilization. Thus, when high‐energy phosphate is supplied as phos‐phocreatine, brush border B‐CK imparts to the circumferential ring myosin a selective energetic advantage over other ATPases. These results suggest that myosin and B‐CK are functionally coupled in the brush border circumferential ring, where they might comprise one end of an energy circuit that supplies energy for contraction, but that colocalizaton of CK with myosin in the brush border interrootlet domain is insufficient to establish functional coupling.
Cytochalasins induce actin polymerization in human leukocytes
Authors: Rao K. Murali Krishna , Padmanabhan Jaya , Cohen Harvey J.
Published: February 2005

We studied the effect of cytochalasins (B, D, and E) on the F‐actin content in human neutrophils and lymphocytes using NBD‐phallacidin labeling followed by flow cytometry. All three cytochalasins induced a concentration‐ and time‐dependent increase in the F‐actin content in both cell types. The order of potency was cytochalasin D > E > B. The increase in F‐actin content was accompanied by a decrease in the G‐actin content as measured by DNase I inhibition assay. These observations suggest that in intact cells cytochalasins may function differently compared to purified and semipurified systems, and their effects may be modified through other actin‐binding or sequestering proteins. 2‐deoxyglucose (20 mM) caused a decrease in the basal F‐actin content and significantly reduced the change induced by the cytochalasins. These results suggest that the state of actin in intact cells is regulated by cytosolic ATP levels, primarily by the integrity of the glycolytic pathway. Based on these observations, we conclude that the mechanism of action of cytochalasins in intact cells is more complex than current models suggest.
An immunoreactive homolog of mammalian kinesin in Nicotiana tabacum pollen tubes
Authors: Tiezzi Antonio , Moscatelli Alessandra , Cai Giampiero , Bartalesi Alessandro , Cresti Mauro
Published: February 2005

A cytoskeletal apparatus is involved in the movement of vesicles, organelles, and gametes in the pollen tube. The function of microfilaments has been defined quite precisely, but the role of microtubules needs to be further clarified. On the basis of immunological and biochemical investigations, we have identified a polypep‐tide showing common properties with kinesin, a microtubule‐based motor mainly described in nonplant tissues, in the pollen tube of Nicotiana tabacum. Like mammalian kinesin, the kinesin‐immunoreactive homolog from Nicotiana tabacum pollen tubes binds to mammalian microtubules in an AMP‐PNP dependent manner. The kinesin‐like component is likely to be involved in the movement of vesicular material in the growing pollen tube.
Isolation and characterization of a unique 15 kilodalton trypanosome subpellicular microtubule‐associated protein
Authors: Balaban N. , Goldman R.
Published: February 2005

A protein of 15 kDa (p15) was isolated from Trypanosoma brucei subpellicular microtubules by tubulin affinity chromatography. The protein bound tubulin specifically both in its native form and after SDS‐PAGE in tubulin overlay experiments. p15 promoted both the in vitro polymerization of purified calf brain tubulin and the bundling of preformed mammalian microtubules. Immunolabeling identified p15 at multiple sites along microtubule polymers comprising calf brain tubulin and p15 as well as on the subpellicular microtubules of cryosectioned trypanosomes. Antibodies directed against p15 did not cross react with mammalian microtubules. It is suggested that p15 is a trypanosome‐specific microtubule‐associated protein (MAP) that contributes to the unique organization of the sub‐pellicular microtubules.
Characterization of tetramethylrhodaminyl‐phalloidin binding to cellular F‐actin
Authors: Cano Manuel L. , Cassimeris Lynne , Joyce Michael , Zigmond Sally H.
Published: February 2005

Fluorescent derivatives of phallcidin are widely used to measure filamentous actin (F‐actin) levels and to stabilize F‐actin. We have characterized the kinetics and affinity of binding of tetramethylrhodaminy (TRITC)‐phalloidin to rabbit skeletal muscle F‐actin and to F‐actin in lysates of rabbit polymorphonuclear leukocytes (PMNs). We have defined conditions where TRITC‐phalloidin can be used to inhibit F‐actin depolymerization and to quantify F‐actin without prior fixation. By equi librium measurements, the affinity of TRITC‐phalloidin binding to rabbit skeletal muscle F‐actin (pyrene labeled) or to PMN lysate F‐actin was 1–4 × 10−7 M. In both cases, the stoichiometry of binding was approximately 1:1. Kinetic measurements of TRITC‐phalloidin binding to PMN lysate F‐actin resulted in an association rate constant of 420 ± 120 M−1 sec−1 and a dissociation rate constant of 8.3 ± 0.9 ± 10−5 sec−1. The affinity calculated from the kinetic measurements. (2 ± 1 × 10−7 M) agreed well with that obtained by equilibrium measurements. The rate with which 0.6 μM TRITC‐phalloidin inhibited 0.1 μM pyrenyl F‐actin depolymerization (90% inhibition in 10 sec) was much faster than the rate of binding to pyrenyl F‐actin (<1% bound in 10 sec), suggesting that phalloidin binds to filament ends more rapidly than to the rest of the filament. We show that TRITC‐phalloidin can be used to measure F‐actin levels in cell lysates when G‐actin is also present (i.e., in cell lysates at high concentrations) if DNase I is included to prevent phalloidin‐induced polymerization.
Reactivation of outer‐arm‐depleted lung axonemes: Evidence for functional differences between inner and outer dynein arms in situ
Authors: Hard Robert , Blaustein Kathy , Scarcello Lisa
Published: February 2005

Demembranated axonemes isolated from newt lung ciliated cells show a complex beat frequency response to varying [MgATP] and temperature [Hard and Cypher, 1992, Cell Motil. Cytoskeleton 21:187–198]. The present study was undertaken to ascertain whether the beat frequency of outer‐arm‐depleted newt lung axonemes is controlled in a manner similar to that of intact axonemes. Populations of demembranated ciliary axonemes were isolated by Triton X‐100 extraction of lungs from the newt, Taricha granulosa. Aliquots of the demembranated axonemes were further treated with solutions containing high salt (0.375 M KCl) and 1.25 mM MgATP. This treatment resulted in the selective removal of outer dynein arms and a concomitant decrease in beat frequency to a stable level, 33–35% of control values. The effects of pH, salt concentration, nucleotides, and temperature on the beat frequency of reactivated outer‐arm‐depleted axonemes were ascertained and compared with those of intact axonemes. Some reactivation properties, such as nucleotide specificity, the effect of pH on beat frequency and the threshold [MgATP] required for reactivation (approximately 5 μM) were similar to those observed for intact axonemes. Other properties, such as the relationship between beat frequency and varying [MgATP] or salt concentration, differed both qualitatively and quantitatively from those of control axonemes, as did their response to temperature over the range, 5°–32°C. The nature of the results obtained with temperature and MgATP suggests that inner and outer dynein arms are not functionally equivalent in situ.
Actin in the ciliated protozoan Climacostomum virens: Purification by DNAse I affinity chromatography, electrophoretic characterization, and immunological analysis
Authors: Fahrni José F.
Published: February 2005

The anti‐actin monoclonal antibody (mab) JLA20 (Lin: Proc. Natl. Acad. Sci. U.S.A. 78:2335–2339, 1981) labels a 43 kD protein on Western blots of Climacostomum cell extracts; this protein does not react with an anti‐α‐smooth muscle actin mab (Skalli et al.: J. Cell Biol. 103:2787–2796, 1986) nor with an anti‐α‐sarcomeric actin mab (Skalli et al.: Am. J. Pathol. 130:515–531, 1988). This protein binds to DNAse I and can be purified by DNAse I affinity chromatography. The affinity‐purified actin also reacts with mab JLA20. Two‐dimensional gel analysis reveals that Climacostomum actin focuses as three spots which are more basic than the mammalian actin isoforms. After addition of KC1, the affinity‐purified actin polymerizes into filaments as shown by electron microscopy after negative staining. © 1992 Wiley‐Liss, Inc.
Polar organizers in monoplastidic mitosis of hepatics (Bryophyta)
Authors: Brown Roy C. , Lemmon Betty E.
Published: February 2005

Archesporial cells of the hepaticMonoclea gottscheiLindb. undergo a series of monoplastidic mitotic divisions prior to enlarging into sporocytes. Interphase cells have a nuclear‐based endoplasmic microtubule system that is predominantly aligned in the long axis of the cell and lack a hoop‐like cortical system. No preprophase bands (PPBs) are formed. Prior to mitosis, the single plastid divides and daughter plastids move to the incipient spindle poles as is typical of monoplastidic cell division. However, the plastids do not serve as microtubule organizing centers (MTOCs) as they do in both mitosis and meiosis of hornworts and lycopsids, and in meiosis of mosses. Rather, microtubules of the developing spindle emanate from distinct polar organizers (POs) arising just outside the nuclear envelope as in polyplastidic mitosis in other hepatics. The POs, which appear to arise de novo on opposite ends of the nucleus during preprophase, consist of vesicles, endoplasmic reticulum, and radiating microtubules. The developmental and evolutionary significance of distinct POs, plastid MTOCs, and the diffuse MTOCs of higher plants is discussed. © 1992 Wiley‐Liss, Inc.
Actin dynamics during the cell cycle in Chlamydomonas reinhardtii
Authors: Harper John D. I. , McCurdy David W. , Sanders Mark A. , Salisbury Jeffrey L. , John Peter C. L.
Published: February 2005

We have used two monoclonal antibodies to demonstrate the presence and localization of actin in interphase and mitotic vegetative cells of the green alga Chlamydomonas reinhardtii. Commercially available monoclonal antibodies raised against smooth muscle actin (Lessard: Cell Motil. Cytoskeleton 10:349–362, 1988; Lin: Proc. Natl. Acad. Sci. USA 78:2335–2339, 1981) identify Chlamydomonasactin as a ∼43,000‐Mr protein by Western immunoblot procedures. In an earlier study, Detmers and coworkers (Cell Motil. 5:415–430, 1985) first identified Chlamydomonas actin using NBD‐phallacidin and an antibody raised against Dictyostelium actin; they demonstrated that F‐actin is localized in the fertilization tubule of mating gametes. Here, we show by immunofluorescence that vegetative Chlamydomonas cells have an array of actin that surrounds the nucleus in interphase cells and undergoes dramatic reorganization during mitosis and cytokinesis. This includes the following: reorganization of actin to the ante‐ rior of the cell during preprophase; the formation of a cruciate actin band in prophase; reorganization to a single anterior actin band in metaphase; rearrange‐ ment forming a focus of actin anterior to the metaphase plate; reextension of the actin band in anaphase; presence of actin in the forming cleavage furrow during telophase and cytokinesis; and finally reestablishment of the interphase actin array. The studies presented here do not allow us to discriminate between G and F‐actin. None the less, our observations, demonstrating dynamic reorganization of actin during the cell cycle, suggest a role for actin that may include the movement of basal bodies toward the spindle poles in mitosis and the formation of the cleavage furrow during cytokinesis. © 1992 Wiley‐Liss, Inc.
Association of the β isoform of protein kinase C with vimentin filaments
Authors: Spudich Annamma , Meyer Tobias , Stryer Lubert
Published: February 2005

Protein kinase C (PKC) isoforms are key mediators in hormone, growth factor, and neurotransmitter triggered pathways of cell activation (Nishizuka: Science 233:305–312, 1986; Nature 334:661–665, 1988). Stimulation of kinase activity by diacylglycerol and calcium often leads to translocation of PKC from the cytosol to a particulate fraction (Kraft and Anderson: Nature 301:621–623, 1983). The β isoform of PKC is translocated and degraded much more rapidly than the β isoform in phorbolester‐stimulated rat basophilic leukemia (RBL) cells (Huang et al.: J. Biol. Chem. 264:4238–4243, 1989). We report here immunofluorescence evidence that the distributions of PKC α and β are strikingly different in antigen‐activated RBL cells. PKC β associates with perinuclear filaments and filaments that extend from the perinuclear area to the cell periphery whereas PKC β concentrates in regions of the cell periphery. This distribution of PKC β is distinctly different from that of actin filaments and microtubules as determined by phalloidin staining and by anti‐tubulin antibody labeling. In contrast, the staining patterns obtained with antibodies to PKC β and to the intermediate filament protein vimentin are almost identical, indicating that PKC β associates with vimentin filaments. These bundles of 100 Å filaments may provide docking sites for interactions of PKC β with its substrates and thus confer specificity to the actions of this isoform. © 1992 Wiley‐Liss, Inc.
Three microtubule‐organizing centers are required for ascus growth and sporulation in the fungus Sordaria macrospora
Authors: Thompson‐Coffe Catherine , Zickler Denise
Published: February 2005

The microtubule system of the Sordaria macrospora ascus was examined by antitubulin immunofluorescence, without the removal of the cell wall. The complex cytoskeleton revealed three possible microtubule‐organizing centers (MTOCs): the spindle pole body (SPB), the nuclear envelope, and an apical organizing center. MPM‐2, a mitotic phosphoprotein antibody which reacts with MTOCs, stained the apical center in a developmentally specific manner, and the nuclear envelope and SPB in a cell cycle‐dependent fashion. Nocodazole was used in both high (10–15 μg/ml) and low (0.5 μg/ml) concentrations to depolymerize the networks and reveal their points of origin and recovery. The apical center was active from prophase I to the end of first meiosis. The nuclear envelope was the site of microtubule nucleation in early prophase and at the telophase/interphase transition, while SPBs were active in both nuclear division and sporulation.

Mutant strains deficient in sporulation and with aberrant morphology were analyzed by antitubulin and MPM‐2 immunofluorescence. Shape mutants showed abnormal or absent apical organizing centers and abnormal cortical microtubule patterns, indicating a possible role for the cortical network in the establishment and maintenance of ascus shape. © 1992 Wiley‐Liss, Inc.