bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of alpha‐actinin from Acanthamoeba
Authors: Pollard Thomas D. , Tseng Peter C.‐H. , Rimm David L. , Bichell David P. , Williams Robley C. , Sinard John , Sato Masahiko
Published: February 2005

Characterization of a protein from Acanthamoeba that was originally called gelation protein [T.D. Pollard, J. Biol. Chem. 256:7666–7670, 1981] has shown that it resembles the actin filament cross‐linking protein, alpha‐actinin, found in other cells. It comprises about 1.5% of the total amoeba protein and can be purified by chromatography with a yield of 13%. The native protein has a molecular weight of 180,000 and consists of two polypeptides of 90,000 Da. The Stokes' radius is 8.5 nm, the intrinsic viscosity is 0.35 dl/dm, and the extinction coefficient at 280 nm is 1.8 × 105M−1·cm−1. Electron micrographs of shadowed specimens show that the molecule is a rod 48 nm long and 7 nm wide with globular domains at both ends and in the middle of the shaft. On gel electrophoresis in sodium dodecylsulfate the pure protein can run as bands with apparent molecular weights of 60,000, 90,000, 95,000, or 134,000 depending on the method of sample preparation. Rabbit antibodies to electrophoretically purified Acanthamoeba alpha‐actinin polypeptides react with all of these electrophoretic variants in samples of purified protein and cell extracts. By indirect fluorescent antibody staining of fixed amoebas, alpha‐actinin is distributed throughout the cytoplasmic matrix and concentrated in the hyaline cytoplasm of the cortex. The protein cross‐links actin filaments in the presence and absence of Ca++. It inhibits slightly the time course of the spontaneous polymerization of actin monomers but has no effect on the critical concentration for actin polymerization even though it increases the apparent rate of elongation to a small extent. Like some other cross‐linking proteins, amoeba alpha‐actinin inhibits the actin‐activated ATPase of muscle myosin subfragment‐1. Although Acanthamoeba alpha‐actinin resembles the alpha‐actinin from other cells in shape and ability to cross‐link actin filaments, antibodies to amoeba and smooth muscle alpha‐actinins do not cross react and there are substantial differences in the amino acid compositions and molecular dimensions.
Effect of microinjectecd calcium‐calmodulin on mitosis in PtK2 cells
Authors: Keith Charles H.
Published: February 2005

Calcium and calmodulin are believed in play a significant role in the regulation of mitosis, because they are both localized in the mitotic spindle and because they can potentiate microtubule depolymerization in the test tube and in the living cell. It has been hypothesized, specifically, that calcium‐saturated calmodulin drives the shortening of the kinetochore microtubules that must occur during prometaphase, when the chromosomes congress to the metaphase plate, and during anaphase A, when the half‐spindles shorten. We have examined the role of calmodulin in mitosis by observing the consequences of calmodulin microinjection on the progress of mitosis and morphology of the mitotic spindle in PtK2 cells. We have found that the injection of excess calcium‐saturated calmodulin during early prometaphase significantly prolongs the time required for the cell to go into anaphase, and that neither calcium‐depleted calmodulin nor buffer alone produce a similar perturbation. Calcium ion alone produces a similar but much smaller retardation of mitosis. Immunofluorescence and fluorescent analogue cytochemical studies of spindle morphology reveal that the immediate (<5‐min) effect of calcium‐saturated calmodulin on prometaphase spindles is a significant shortening of the kinetochore fibers and “interpolar” microtubules but not the astral microtubules. After this perturbation, however, the spindle quickly recovers its normal form. An equivalent transient shortening of the spindle fibers is seen following the injection of calcium chloride solutions but not after the injection of calciumdepleted calmodulin or buffer alone. Taken together, these observations suggest that calcium‐saturated calmodulin plays a significant role in the regulation of mitosis, and that this regulatory pathway involves more than spindle fiber shortening.
Caenorhabditis elegans spermatozoa assemble membrane proteins onto the surface at the tips of pseudopodial projections
Authors: Pavalko Fredrick M. , Roberts Thomas M.
Published: February 2005

The crawling movement of nematode sperm, like that of many other crawling metazoan cells, is accompanied by movement of membrane components from the leading edge of the cell rearward. We used colloidal gold conjugates of monoclonal antibodies (CGP‐ABY) to membrane proteins on Caenorhabditis elegans sperm to examine this surface movement by electron microscopy. Antibody binding sites on fixed sperm are distributed uniformly over the cell surface. However, blocking these sites on live sperm with unlabelled antibody or removing them with protease and then pulse‐labelling the cell with CGP‐ABY revealed that new antigen is assembled onto the surface at the tips of the stubby projections that stud the pseudopod surface. These proteins then move rearward rapidly so that the pseudopod surface pool of antigen is replaced within 2 min. The same pattern of surface movement was observed when live cells were labelled with CGP‐ABY and then washed with buffer before fixation. Bound CGP‐ABY was cleared first from the tips of the projections and subsequently from the entire pseudopod surface. These gold particles accumulated at the base of the pseudopod without moving onto the cell body or being internalized. We did, however, detect a pool of antigen in the pseudopod cytoplasm that may be available for assembly onto the pseudopod surface. We propose that the localized assembly of new membrane and its subsequent rearward movement may play an important role in sperm locomotion.
Stress fiber and cleavage furrow formation in living cells microinjected with fluorescently labeled α‐actinin
Authors: Sanger Jean M. , Mittal Balraj , Pochapin Mark B. , Sanger Joseph W.
Published: February 2005

α‐Actinins, isolated from muscle and nonmuscle sources and labeled with various fluorescent dyes, were microinjected into living PtK2 cells during interphase to observe the reformation of stress fibers following cell division. Fluorescently labeled ovalbumin and bovine serum albumin were also injected as control proteins. α‐Actinin was incorporated into stress fibers within 5 minutes after injection and remained present in the fibers for up to 11 days. The pattern of incorporation was the same regardless of whether the α‐actinin was isolated from muscle or nonmuscle tissues or whether it was labeled with fluorescein, Lucifer Yellow, or rhodamine dyes. In contrast, neither labeled ovalbumin nor bovine serum albumin were incorporated into stress fibers. When the injected cells entered prophase, all stress fibers disassembled, resulting in a distribution of the fluorescent α‐actinin throughout the cytoplasm. During cytokinesis, the fluorescent α‐actinin was concentrated in the broad area between the separated chromosomes and along the edge of the cell in the cleavage area. Within 10 minutes after the completion of cleavage, the first fluorescent stress fibers reformed parallel to the spreading edges of the daughter cells and in close association with the midbody with a concomitant loss of α‐actinin in the former cleavage furrow. Additional fibers formed adjacent to these first stress fibers. In some cases, new stress fibers formed between two existing stress fibers and some stress fibers moved up to 4 μm apart from one another in the course of 2 hours. Thus, fluorescent α‐actinin, injected into living cells, undergoes the same cyclical changes in distribution as endogenous α‐actinin during the cell cycle: from stress fibers to cleavage furrow and back to stress fibers.
Evidence that MAP‐2 may be involved in pigment granule transport in squirrel fish erythrophores
Authors: Stearns Mark E. , Binder Lester I.
Published: February 2005

We have demonstrated the presence of MAP‐2 in squirrel fish erythrophores using SDS‐PAGE, immunobolt, and immunoprecipitation techniques. The monoclonal antibodies used (AP‐9, ‐13, ‐14) were raised against distinct antigenic sites on Chinese hamster brain MAP‐2. Immunoprecipitation studies demonstrated that all three antibodies bind a 300 K protein found in crude cell extracts and in partially purified MAP fractions isolated from erythrophores of the squirrel fish Holocentrus rufus. Immunofluorescent studies confirmed that the 300 K protein was present in cultured erythrophores. Studies of cells induced to aggregate and disperse their pigment granules revealed that the 300 K protein comigrated with the pigment, suggesting that the 300 K protein may constitute part of the “α‐cytomatrix” involved in pigment translocations.
Mitotic inhibition and chromosome displacement induced by estradiol in chinese hamster cells
Authors: Wheeler W. J. , Hsu T. C. , Tousson A. , Brinkley B. R.
Published: February 2005

We tested diethylstilbestrol (DES) and 17 β‐estradiol as mitotic arrestants to determine their effects on chromosome distribution, spindle microtubules, and the cytoplasmic microtubule complex (CMTC) in the Chinese hamster strain Don. Cytological experiments assessed micronuclei induction, chromosome displacement, and anaphase recovery Indirect immunofluorescence microscopy with antibody to tubulin and electron microscopy were used to illustrate effects on microtubules. Both DES and estradiol were potent inhibitors of mitosis when applied to cells in vitro. Estradiol induced micronuclei at a greater frequency than did DES. Estradiol‐arrested metaphases often contained misaligned chromosomes despite the presence of a bipolar spindle and an equatorial plate. Equatorial plates were not observed in DES‐arrested cells. Cells recovered quickly from estradiol exposure upon removal of the steroid. The frequency of abnormal metaphases and abnormal anaphases declined as the recovery period increased. Microtubule experiments showed that DES inhibited spindle assembly and disassembled the CMTC, whereas estradiol, at similar concentrations, arrested mitosis in a manner that allowed spindle assembly. A definite effect on the CMTC by estradiol could not be determined. However, changes in cell morphology were observed. In the presence of estradiol, centrosomes organized microtubules that joined with kinet‐ochores of chromosomes at the equatorial plate as well as with those of misaligned chromosomes. Misaligned chromosomes appeared predominantly at polar regions of mitotic cells. Following drug removal, the pole‐oriented chromosomes reoriented at the equatorial plate. The unique arresting properties of estradiol may prove useful in studies of chromosome migration and segregation during mitosis.
Cytoskeletal and myonemal structures of dinoflagellates are made of 2–3‐nm filaments
Authors: Cachon J. , Cachon M. , Boillot A. , Brown D. L.
Published: February 2005

An important cortical fibrillar cytoskeleton is described in some unarmoured dinoflagellates (Gymnodinium, Gyrodinium). The filaments which constitute this cortical layer are 2–3 nm in diameter, tightly packed, entangled, and resistant to denaturing agents. Issuing from them are other bundles of filaments which appear to be involved in the contraction of the cell, and for this reason they are termed “myonemal.”

In highly contractile unarmoured dinoflagellates (Leptophyllus) the cytoskeleton is also made of 2–3‐nm filaments, but they are regularly arrayed and more directly involved in contraction and thus correspond also to myonemal structures.

Both structures—cytoskeletal and myonemal—are made of 2–3‐nm filaments.
3T3 cell motility in the temperature range 33°C to 39°C
Authors: Thurston G. , Palcic B.
Published: February 2005

The phenomena of mammalian cell motility in tissue culture is an integrated function of many cellular components. As such, cell motility is very sensitive to external stimuli and perturbation. In this article we report the effect of temperature in the range 33°C to 39°C on cell motility. For this 3T3 cells were plated in plastic tissue culture flasks. A large number of individual cells (60 per experiment) were tracked as a function of time by means of an automated device, the Cell Analyzer. The data show a peak in the average cell speed in the range 36.5°C to 38.5°C, falling off sharply at lower and higher temperatures. The average rate of cell motility closely correlates to the average cell proliferation rate in the range 33°C to 39°C.
Microtubule cycle in Chlamydomonas reinhardtii: An Immunofluorescence study
Authors: Doonan John H. , Grief Christopher
Published: February 2005

Monoclonal antibodies to yeast tubulin have been used to investigate the distribution of microtubules throughout the cell cycle of the green alga Chlamydomonas reinhardtii. By indirect immunofluorescence microscopy we detect all the classes of microtubule‐containing structures previously described from ultrastructural investigations but we can now demonstrate the complex spatial and temporal relationships between the different microtubule arrays. During interphase a cortical cytoplasmic microtubule system emanates from the base of the flagellar microtubules. These microtubules become reorganised on entry into mitosis, being largely disassembled and replaced by the spindle and “metaphase band” microtubules. The “metaphase band” is shown to be not one array but two distinct sets of microtubules, each linking a spindle pole with a region of the cell cortex near the spindle equator. It persists until late telophase, when it is replaced by cortical and cleavage furrow microtubules.
Isolation and initial biochemical characterisation of caps of two major rat thymocyte glycoprotens: Evidence for the involvement of a 205 K Con A binding protein and cytoskeletal components in capping
Authors: Tuner Christopher E. , Shotton David M.
Published: February 2005

Two major rat thymocyte surface glycoproteins, the leucocyte‐common (L‐C) antigen and the leucocyte sialoglycoprotein (LSGP), were induced to cap independently, using the specific monoclonal antibodies OX‐1 and W3/13, respectively, and an appropriate fluorescently labeled second antibody layer. The caps were subsequently isolated from detergent extracted cells by a procedure involving gentle shearing.

TRITC‐phalloidin staining of the isolated caps demonstrated the presence of F‐actin within these structures, and lectin‐affinity staining after fractionation on SDS polyacrylamide gels revealed the presence of a concanavalin A (Con A) binding protein of relative molecular weight (Mr) 205,000, gp205, in both the L‐C antigen and LSGP caps, but absent from the detergent‐insoluble residue isolated from unchallenged cells. These results suggest that gp205 may be involved in the association of cross‐linked glycoproteins with the cytoskeleton during capping.
Topographical relationship between the axonemal arrangement and the bend direction in starfish sperm flagella
Authors: Mohri Hideo , Mohri Toshiko , Okuno Makoto
Published: February 2005

Since starfish spermatozoa have spherical heads, it is not easy to determine the topographical relationship of the axoneme to the directions of the flagellar bends, the principal, and the reverse bends as defined by Gibbons and Gibbons [J. Cell. Biol. 1972, 63:970–985]. The demembranated spermatozoa are known to take the quiescent “cane” shape with a sharp principal bend at the proximal region of the flagellum in the presence of high concentration of Ca2+. When such spermatozoa were placed on a grid for electron microscopy, fixed with osmic acid vapor, washed with distilled water, and negatively stained with urany1 acetate, the head of the spermatozoon was disrupted and dispersed disclosing the proximal centriole at at the proximal end of the flagellum. The proximal centriole was always found on the concave side of the “cane” ‐shaped flagella. Electron microscopy of the serial thin sections of intact and demembranated spermatozoa revealed that the doublet microtobules numbers 5 and 6 were contained in the convex edge of the principal bend.
Platelet‐derived growth factor‐induced alterations in vinculin distribution in porcine vascular smooth muscle cells
Authors: Herman B. , Roe M. W. , Harris C. , Wray B. , Clemmons D.
Published: February 2005

Exposure of porcine vascular smooth muscle cells to platelet‐derived growth factor (PDGF; 18–180 ng/ml) but not epidermal growth factor (EGF; 30ng/ml), somatomedin C (SmC; 30 ng/ml), or insulin (10 μM), results in a rapid, reversible, time‐ and concentration‐dependent disapperance of vinculin staining in adhesion plaques; actin‐containing stress fibers also become disrupted following exposure of cells to PDGF. Disapperance of vinculin staining from adhesion plaques is also caused by 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA; 200–400 nM), though the time course of the disapperance of vinculin staining under these conditions takes longer than in cells exposed to PDGF. The PDGF‐induced removal of vinculin from adhesion plaques was inhibited in a concentration‐dependent fashion by 8‐(N, N‐diethylamin) octy1–3,4,5‐trimethoxybenzoate (TMA‐8; 0.25–4 μM) and leupepetin (2–300 μM), and by n‐α‐rosyl‐L‐lysine chloromethylketone (TLCK; 100 μM) and trifluoperazine (TFP; 2.5 μM). Addition of PDGF to vascular smooth muscle cells caused a rapid, tranient increase in cytosolic free calcium, from a basal resting level of 146 ± 6.9 nM (SEM, n=62) to 414 ± 34 nM (SEM, n=22) as determined using the calcium‐sensitive indicator Fura‐2 and Digitized Video Microscopy. This increase in cellular calcium preceded the disappearance of vinculin from adhesion plaques and was partially blocked by pretreatment of cells with TMB‐8 but not leupeption. This rise in cytosolic free calcium was found to occur in ∼ 80% of the sample population and dispalyed both spatial and temporal subcellular heterogeneity. Exposure of cells to TPA (100 nM) did not result in a change in cytosolic free calcium. Both PDGF (20 ng/ml) and TPA (100 nM) caused cytosolic alkalinization which occurred after PDGF‐induced disruption of vinculin from adhesion plaques, as determined using the pH‐sensitive indicator BCECF and Digitized Video Microscopy. PDGF stimulated DNA synthesis and vinculin disruption in a similar dose‐dependent fashion. Both could be inhibited by leupeptin or TMB‐8. These results suggest that 1) exposure of vascular smooth muscle cells to PDGF is associated with the disruption of vinculin from adhesion plaques, 2) PDGF‐induced vinculin disruption is regulated by an increase in cytosolic calcium (but not cytosolic alkalinization), and involves proteolysis; 3) activation of protein kinase C also causes vinculin removal from adhesion plaques but by a calcium‐independent mechanism, and 4) the cellular response to PDGF‐stimulated increases in cytosolic free calcium is heterogeneous. Our data also suggest that cytosolic vinculin distribution is a sensitive indicator of the response of vascular smooth muslce cells to PDGF.
Stress fiber reformation after ATP depletion
Authors: Glascott Peter A. , McSorley Karen M. , Mittal Balraj , Sanger Jean M. , Sanger Joseph W.
Published: February 2005

Flurescently labeled heavy mermoyosin, alpha‐actinin, and vinculin were used to localize actin, and vinculin, respectively, in permeabilized and living cells during the process of stress fiber reassembly, which occurred when cells were removed from ATP‐depleting medium (20 mM sodium azide and 10 mM 2‐deoxyglucose). In 80% of the cells recovering from ATP depletion, small, scattered plaques containing actin, alpha‐actinin, and vinculin were replaced by long, thin, periodic fibers within 5 minutes of removal of the inhibitors. These nascent stress fibers grew broader as recovery progressed, until they attained the thickness of stress fibers in control cells. In the other 20% of the cells, the scattered plaques aggregated within 5 minutes of reversal, and almost all the actin, alpha‐actinin, and vinculin in the cell became localized in one perinuclear aggregate, with a diameter of approximaterly 15–25 μm. As recovery progressed, all aggregates resembled rings, with diameters that increased at about 0.5 μm/minute and grew to as large as 70 μm in some giant cells. As the size of the rings increased, fibers radiated outward from them and sometimes spanned the diamater of te rings. The shape of the cells did not change during this time. By 1 hour after reversal, the rings were no longer present and all cells had networks of stress fibers. Indirect immunofluorescence techniques used to localize tubulin and vimentin indicated that microtubules and intermediate filaments were not constituents of the rings, and the rings were not closely apposed to the substrate, judging from reflection contrast optics. The rapid rearrangement of attachment plaques into a perinuclear aggregate that spreads radially in the cytoplasm occurs at the same speed as fibroblast and chromosomal movement, but is unlike other types of intracytoplasmic motility.
Invited review: Fine (2–5‐nm) filaments: New types of cytoskeletal structures
Authors: Roberts Thomas M.
Published: February 2005

Over the past 30 years filaments 2–5 nm in diameter have been foun in a number of different types of eukaryotic cells. As a group, these fine filaments lack the similarity of composition and function that characterize the three major classes of cytoskeleta elements—microfilaments, microtubules, and intermediate filaments. Six different proteins that form fine filaments have been identified; proposed functions for these fibers range from cell motility to cytoarchitecture. Recent studies, however, have revealed filaments with similar compositions and/or functions in otherwise different cells, sugesting that the fine filaments may eventually fit into a limited number of subgroups.
Cell type‐specific association between two types of spectrin and two types of intermediate filaments
Authors: Langley Robert C. , Cohen Carl M.
Published: February 2005

We have demonstrated a differential association between two types of spectrin, from erythrocytes and brain, with two types of intermediate filaments, vimentin filaments and neurofilaments. Electron microscopy showed that erythrocyte spectrin promoted the binding of vimentin filaments to red cell inside‐out vesicles via lateral associations with the filaments. In vitro binding studies showed that the association of spectrin with vimentin filaments was apparently saturable, increased with temperature, and could be prevented by heat denaturation of the spectrin. Comparisons were made between erythrocyte and brain spectrin binding to both vimentin filaments and neurofilaments. We found that vimentin filaments bound more erythrocyte spectrin than brain spectrin, while neurofilaments bound more brain spectrin than erythrocyte spectrin. Our results show that both erythroid and nonerythroid spectrins are capable of binding to intermediate filaments and that such association may be characterized by differential affinities of the various types of spectrin with the several classes of intermediate filaments present in cells. Our results also suggest a role for both erythroid and nonerythroid spectrins in mediating the association of intermediate filaments with plasma membranes or other cytoskeletal elements.
“Pull” and “push” in neurite elongation: Observations on the effects of different concentrations of cytochalasin B and taxol
Authors: Letourneau Paul C. , Shattuck Terri A. , Ressler Alice H.
Published: February 2005

Neurite elongation involves two distinct cytoskeletal functions the “push” of anterograde transport of the cytoskeleton and associated organelles to the neurite tip, and the “pull” exerted by protrusion and generation of tensions in the growth cone. We investigated the roles of these two activities in neurite elongation via the drugs taxol and cytochalasin B (CB), which act on the key cytoskeletal components, microtubules and actin filaments, respectively. When neurons are treated with concentrations of CB below 0.2 μg/ml, neurite elongation, growth cone protrusion, and neurite tension are all inhibited in a similar concentration dependent manner. Protrusive activity and tensions are absent at CB concentrations above 0.3 μg/ml, yet neurite elongation continues at a plateau level. Thus, “pull” does modulate, but it is not required for neurite elongation. Surprisingly, the inhibitory effects of taxol on neurite elongation are removed by the addition of CB at levels that substantially disrupt the actin filaments of neurites. The neurites extended by taxol‐CB neurons are unbranched and curiously unattached to the substratum. When CB is added to taxol‐treated neurons, neurite extension begins rapidly, even if protein synthesis is severely reduced. We propose that taxol inhibits microtubule transport in neurites, and this inhibition of “push” is reversed by the disruptive effects of CB on the cytoplasmic matrix, allowing taxol‐induced microtubule bundles to be transported distally.
Vimentin dynamics during the mitogenic stimulation of mouse splenic lymphocytes
Authors: Paulin‐Levasseur Micheline , Brown David L.
Published: February 2005

We have used double immunofluorescence and electron microscopy to examine the distribution of tubulin and vimentin during the stimulation of mouse splenic lymphocytes by the mitogen concanavalin A. In unstimulated cells, vimentin forms a filamentous network partially coincident with the radial pattern of microtubules. In stimulated cells, the numbers of microtubules assembled from the centrosome. When these cells enter mitosis, vimentin is arranged into a filamentous cage enclosing the mitotic apparatus. During cytokinesis, the polar centrosomes are observed at a position adjacent to the midbody and vimentin is detected as an aggregate, similar to that seen prior to mitosis, close to the centrosome in each daughter cell. Using several agents, such as colchicine, colcemid, nocodazole, and taxol, which affect microtubule assembly, we have observed that the vimentin system, although closely related spatially to the microtubule complex in lymphocytes, can still reorganize independently as these cells progress through in the cell cycle. Throughout mitogenic stimulation in the continued presence of taxol, microtubules are reorganized into a few thick bundles while the vimentin system undergoes a sequence of rearragements similar to those observed during normal stimulation. These data suggest that vimentin dynamics may be important in the progression of lymphocytes through the cell cycle in response to mitogen.
Analysis of the flagellar bending waves of ejaculated ram sperm
Authors: Chevrier C. , Dacheux J. L.
Published: February 2005

The variability of flagellar movement, illustrated by the highly heterogenous nature of the ejaculated sperm population of the ram, was analyzed by the use of a stroboscopic technique and an adapted microphotographic 24 × 36 camera system. The multiple‐moving‐exposures (MME) records give very distinct successive sequences of the flagellar beats and are particularly suitable for the analysis of bend development and propagation along the tail. With this technique, the parameters of the flagellar bending waves of ejaculated ram sperm have been determined. Most of the sperm have planar flagellar beatings; few are rolling under the conditions of observation. The trajectories of the gametes are mostly linear; nevertheless, some have circular paths. The analysis of bending has been focused on two examples for which the difference in the progressiveness ratio was maximum. The circular pathways for ram spermatozoa are linked to an asymmetry between principal and reverse bend probably induced by differences in wave propagation evidenced along the flagellum. A typical sperm flagellar movement may be related either to the conditions of the observations or to some differences in the maturation process of the sperm.
Acetylated and detyrosinated α‐tubulins are co‐localized in stable microtubules in rat meningeal fibroblasts
Authors: Cambray‐Deakin Martin A. , Burgoyne Robert D.
Published: February 2005

We have examined the distribution of acetylated α‐tubulin using immunofluorescence microscopy in fibroblastic cells of rat brain meaninges. Meningeal fibroblasts showed heterogenous staining patterns with a monoclonal antibody against acetylated α‐tubulin ranging from staining of primary cilia or microtubule‐organising centers (MTOCs) alone to extensive microtubule networks. Staining with a broad spectrum anti‐α‐tubulin monoclonal indicated that all cells possessed cytoplasmic microtubule networks. From double‐labeling experiments using an antibody against acetylated α‐tubulin (6–11B‐1) and antibodies against either tyrosinated or detyrosinated α‐tubulin, it was found that acetylated α‐tubulin and tyrosinated α‐tubulin were often segregated to different microtubules. The microtubules containing acetylated but not tyrosinated α‐tubulin were cold stable. Therefore, it appeared that in general meningeal cells possessed two subset of microtubules: One subset contained detyrosinated and acetylated α‐tubulin and was cold stable, and the other contained tyrosinated α‐tubulin and was cold labile. These results are consistent with the idea that acetylation and detyrosination of α‐tubulin are involved in the specification of stable microtubules.
Flagellar movement of intact and demembranated, reactivated ram spermatozoa
Authors: Ishijima Sumio , Witman George B.
Published: February 2005

The flagellar movement of intact ejaculated ram sperm, and of demembranated models reactivated with ATP, has been studied using high‐speed, high‐resolution video microscopy.

Intact sperm attached to the coverslip by their heads had an average beat frequency of 20.9 Hz and an average wave amplitude of 20.2 μm. There was little difference in the beat frequency or waveform of these sperm and sperm swimming freely near the coverslip or captured by their heads with a micropipette and held far from the coverslip, inducationg that the flagellar waveform of ram sperm is relatively resistant to distorition as a result of immobilization of the head or proximity to a surface. The beat envelope was nearly planar as determined by observations of free‐swimming sperm and sperm captured by their head and oriented so they were beating either parallel or perpendicular to the plane of focus.

The effect of various conditions for demembranation and reactivation of the sperm were examined. Treatment of sperm with 0.2 % Triton X‐100 removed most of their plasma membrane. Under optimal conditions, nearly 100 % of the demembranted sperm reactivated at MgATP2− concentrations ranging from ∼4 μM to ∼20 mM. From ∼ 1 mM to ∼ 10 mM MgATP2−, their beat pattern closely resembled that of intact sperm; beat frequency depended on MgATP2− concentration. Percent motility was maximal between pH 7.5 and 8.0 and decreased sharply below pH 7.0 and avove pH 8.5. The addition of 50 μM cAMP to the reactivation medium had no effect on percent motility or the beat pattern and did not accelerate the initiation of movement.
Unfertilized sea urchin eggs contain a discrete cortical shell of actin that is subdivided into two organizational states
Authors: Spudich Annamma , Wrenn Joan T. , Wessells Norman K.
Published: February 2005

Changes in the distribution and organizational state of actin in the cortex of echinoderm eggs are believed to be important events following fertilization. To examine the initial distribution and form of actin in unfertilized eggs, we have adapted immunogold‐labeling procedures for use with eggs of Strongylocentrotus purpuratus. Using these procedures, as well as fluorescence microscopy, we have revealed a discrete 1‐μm‐thick concentrated shell of actin in the unfertilized egg cortex. This actin is located in the short surface projections of unfertilized eggs and around the cortical granules in a manner that suggests it is associated with the cortical granule surface. The actin in the short surface projections appears to be organized into filaments. However, most if not all of the actin surrounding the cortical granules is organized in a form that does not bind phalloidin, even though it is accessible to actin antibody. The lack of phalloidin binding is consistent with either the presence of nonfilamentous actin associated with the cortical granules or the masking of actin‐filament phalloidin‐binding sites by some cellular actin‐binding component. In addition to the concentrated shell of actin found in the cortex, actin was also found to be concentrated in the nuclei of unfertilized eggs.
Polymorphonuclear leukocyte locomotion is insensitive to lowered cytoplasmic calcium levels
Authors: Zigmond Sally H. , Slonczewski Joan L. , Wilde Mary W. , Carson Monica
Published: February 2005

Chemotactic factors stimulate the rate of locomotion of polymorphonuclear leukocytes (PMNs). To investigate the importance of cytoplasmic calcium we have examined the ability of the chemotactic peptide N‐formylnorleucyl eucylphenalanine (FNLLP) to stimulate the locomotion of PMNs whose cytoplasmic calcium levels were reduced by incubation in EGTA or in EGTA plus the calcium ionophores, ionomycin or A23187. Locomotion was assayed by migration through micropore filters and by time‐lapse videomicroscopy. Cells in EGTA exhibited similar or slightly reduced rates of locomotion compared to cells in Hanks' balanced salt solution (HBSS). The peptide dose dependence for the stimulation of locomotion was similar in medium containing calcium or EGTA. The presence of 1 μM ionophore plus EGTA had no effect on the stimulation of locomotion by peptide. The presence of ionophores (1 μM) plus external calcium inhibited locomotion.
Dynamics of a fluorescent calmodulin analog in the mammalian mitotic spindle at metaphase
Authors: Stemple Derek L. , Sweet Stuart C. , Welsh Michael J. , Richard McIntosh J.
Published: February 2005

We have compared the exchange kinetics of fluorescein‐labeled calmodulin and tubulin in the spindles of living mitotic cells at metaphase. Cultured mammalian cells in early stages of mitosis were microinjected with labeled calmodulin or tubulin and returned to an incubator to allow equilibration of the fluorescent protein with the endogenous protein pools. Calmodulin becomes concentrated in the mitotic spindle, and treatments with inhibitors of tubulin assembly show that this concentration is dependent on the presence of microtubules. The steady‐state exchange rates of both tubulin and calmodulin were measured by an analysis of fluorescence redistribution after photobleaching (FRAP), using cells preequilibrated to either 26 ± 2°C or 36 ± 2°C. A pulse of laser light focused to a 5‐μm diameter column was used to destroy the fluorescence at one pole of a metaphase mitotic spindle. Ratios of fluorescence intensity from the two half‐spindles and from the two polar regions were calculated for each image in a post‐bleach time series to determine the rates and extents of FRAP. For tubulin, we confirm earlier observations concerning the temperature dependence of the extent of FRAP, but our data do not show a significant temperature dependence for the rate of FRAP. We hypothesize that the reduced extent of tubulin FRAP at the lower temperatures is a result of microtubules that are stable to depolymerization at 26°C and are thus less likely to exchange subunits. Calmodulin's FRAP, however, does not exhibit any of the temperature dependence observed with fluorescent tubulin. At 26 ± 2°C calmodulin exchanges rapidly with the relatively stable population of microtubules, suggesting that calmodulin is bound, either directly or indirectly, to microtubule walls.
Wave of free calcium at fertilization in the sea urchin egg visualized with fura‐2
Authors: Hafner Mathias , Petzelt Christian , Nobiling Rainer , Pawley James B. , Kramp Douglas , Schatten Gerald
Published: February 2005

A wave front of increased free calcium traversing the egg at fertilization is demonstrated in the sea urchin Lytechinus pictus. The use of the fluorescent calcium chelator fura‐2 in combination with low‐light‐level TV microscopy and image processing allows the visualization of the Ca2+ wave front with high spatial and temporal resolution. Such a wave is demonstrated as increased fluorescence after an excitation of 340‐nm wavelength and as the reciprocal image in form of a reduced fluorescence when excited at 380 nm. The band‐like appearance of the wave resembles the Ca2+ wave described for larger eggs of other species. In a dispermic egg the high resolution of the system used allows us to recognize two waves of Ca2+ originating from the respective points of sperm entry.
Determination of the average shape of flagellar bends: A gradient curvature model
Authors: Eshel Dan , Brokaw Charles J.
Published: February 2005

Data obtained by manual digitization of photographs of flagellar bending waves have been analyzed by determining size parameters for the bends by least‐squares fitting of a model waveform. These parameters were then used to normalize the data so that the average shape of the bends could be determined. Best fits were obtained with a model waveform derived from the constant curvature waveforms used previously but with provision for a linear change in curvature across the central region of the bend–the gradient curvature model (GCM). The central regions of the GCM bending waves are separated by transition regions with length determined by a parameter called the truncation factor (FT). Fitting the GCM to sine‐generated bending waves give optimal fit when FT = 0.34. Fitting the GCM to four different samples of flagellar bending waves gave best fits with values of FT ranging from 0.17 for ATP‐reactivated Lytechinus spermatozoa beating at approximately 10 Hz to 0.32 for live spermatozoa of Arbacia. The difference between the Arbacia waveforms and a sine‐generated waveform is therefore very small, but a sine‐generated waveform lacks the degree of freedom represented by FT that is required to fit other waveforms optimally.

The residual differences between the waveform data and optimal GCM waveforms were averaged and found to be small. In most cases, the curvature in the central region of the optimal GCM decreased in magnitude towards the tip of the flagellum; however, this slope was highly variable and sometimes positive. Significant variations in both this slope and FT were found in individual bends as they propagated along a flagellum.
The cytoskeletal microtubular system of some naked dinoflagellates
Authors: Brown D. L. , Cachon J. , Cachon M. , Boillot A.
Published: February 2005

The cytoskeletal microtubule system has been studied in six species of unarmoured Dinoflagellates using immunofluorescence and electron microscopy. Several structures have been detected and described: (1) a subpellicular layer of microtubules, constituting the microtubular cytoskeleton, running singly or in bundles from the anterior part of the cell to the posterior; (2) a feeding apparatus, containing a ribbon of microtubules, which corresponds to a small peduncle in some species and is simply represented by a cytostome in some other species; and (3) the longitudinal flagellum that runs in a long intracytoplasmic pocket before becoming free at the extremity of the sulcus. A thorough study of the organization of the microtubular structures in a wide spectrum of Dinoflagellates is a prerequisite for understanding the evolutionary history of the group.
Nanometer‐scale measurements using video light microscopy
Authors: Schnapp B. J. , Gelles J. , Sheetz M. P.
Published: February 2005

Video and digital image processing have been used to amplify the contrast of light microscopic images, making it possible to observe in real time the diffraction images of cell structures 10 times smaller than the Raleigh resolution limit of 0.2 μm. In this paper we discuss how quantitative analysis of diffraction images can be used to extract information about motion or structure at the nanometer level. This issue is considered in the context of a method for tracking the motion of kinesin‐coated beads on microtubules with 1–2 nm precision (Gelles et al.: Nature 331:450–453, 1988).
Actin polymerization and pseudopod extension during amoeboid chemotaxis
Authors: Condeelis J. , Hall Anne , Bresnick Anne , Warren Vivien , Hock Rick , Bennett Holly , Ogihara Satoshi
Published: February 2005

Amoebae of the cellular slime mold Dictyostelium discoideum are an excellent model system for the study of amoeboid chemotaxis. These cells can be studied as a homogeneous population whose response to chemotactic stimulation is sufficiently synchronous to permit the correlation of the changes in cell shape and biochemical events during chemotaxis. Having demonstrated this synchrony of response, we show that actin polymerization occurs in two stages during stimulation with chemoattractants. The assembly of F‐actin that peaks between 40 and 60 sec after the onset of stimulation is temporally correlated with the growth of new pseudopods. F‐actin, which is assembled by 60 sec after stimulation begins, is localized in the new pseudopods that are extended at this time. Both stages of actin polymerization during chemotactic stimulation involve polymerization at the barbed ends of actin filaments based on the cytochalasin sensitivity of this response. We present a hypothesis in which actin polymerization is one of the major driving forces for pseudopod extension during chemotaxis. The predictions of this model, that localized regulation of actin nucleation activity and actin filament cross‐linking must occur, are discussed in the context of current models for signal transduction and of recent information regarding the types of actin‐binding proteins that are present in the cell cortex.
Dynamic organization of Physarum plasmodium
Authors: Kamiya N. , Allen R. D. , Yoshimoto Y.
Published: February 2005

Birefringent fibrils (BRFs) with a positive sign composed of bundles of F‐actin were found throughout the Physarum plasmodium with the mode of existence differing regionally. In the zone behind the leading edge of an advancing plasmodium, where cytoplasmic sol and gel were still not well differentiated, more BRFs came to the foreground when the endoplasm flowed backward (emptying phase), and a substantial portion disappeared when the endoplasm flowed forward (filling phase), except for nodes, from which BRFs were reorganized in the early emptying phase of each cycle. BRFs found in the wall of the streaming channel in the posterior network and the branched vein section ran in parallel to or helically around the channel. They were much more stable and maintained strong birefringence irrespective of the direction of the cytoplasmic flow. When the fan‐like expanse ceased moving forward, the BRFs no longer appeared and disappeared cyclically but persisted in the area which had previously been the front. We concluded that the site of the active contraction‐relaxation rhythm in an advancing plasmodium with antero‐posterior polarity is restricted to its frontal zone and that the rest of the plasmodium is in a state of “tonus” which continuously imparts a certain level of hydrostatic pressure to the interior. The meaning of the tonus and the mechanics of tensile force production in the plasmodium are discussed in terms of a working hypothesis arrived at from the phase relationship between isometric and isotonic contraction waves.
A precursor of the focal contact in cultured fibroblasts
Authors: Izzard Colin S.
Published: February 2005

Evidence for a structural precursor of the focal contact in cultured fibroblasts and continuing studies on the development of the precursor and contact are discussed. The structural precursor consists of an F‐actin‐rich, rib‐like fiber within the motile lamellipodium. The focal contact forms beneath the fiber, part of which is retained at the contact as the initial adhesion plaque. Therefore, F‐actin is present at the contact from the beginning. Vinculin accumulates at the plaque during a 90‐second period after the contact forms. A novel feature of the distribution of talin has been found. The protein is present along the distal margin of the lamellipodium, where it is further concentrated as a series of nodes at the tips of each precursor and between precursors. This distribution of talin is independent of that which develops at the plaque after the contact forms. The structural development of the precursor has been followed with AVEC‐DIC optics. The process begins with the development of fine oblique fibers from small structural nodes at the margin of the lamellipodium, and continues with the fusion of the nodes at the margin and inward coalescence of the fibers. It is suggested that talin may function as a cross‐linking protein in the convergence of actin filaments at the membrane, while other actin‐bundling proteins participate in the inward coalescence of the filaments to form fibers. The F‐actin core of the precursor could provide a structural framework against which differences at the external surface of the membrane develop prior to contact formation.
Micromanipulation studies of the mitotic apparatus in sand dollar eggs
Authors: Hiramoto Yukio , Nakano Yoshitaro
Published: February 2005

Mechanical properties of the mitotic spindle and the effects of various operations of the mitotic apparatus on the chromosome movement and spindle elongation were investigated in fertilized eggs and blastomeres of the sand dollar, Clypeaster japonicus. On the basis of results with mechanical stretching and compression of the spindle with a pair of microneedles and the behavior of an oil drop microinjected into the spindle, it was concluded that the equatorial region of the spindle is mechanically weaker than the half‐spindle region. Anaphase chromosome movement occurred in the spindle from which an aster had been removed or separated with its polar end and in the spindle in which the interzonal region had been removed. This fact indicates that chromosomes move poleward in anaphase by forces generated near the kinetochores in the half‐spindle. Because of the effects of separation or removal of an aster from the spindle on the spindle elongation in anaphase and the behavior of the aster, it was concluded that the spindle elongation in anaphase is caused by pulling forces generated by asters attached to the ends of the spindle.
Dynein as a microtubule translocator in ciliary motility: Current studies of arm structure and activity pattern
Authors: Satir Peter
Published: February 2005

The dynein arms of ciliary doublet microtubules cause adjacent axonemal doublets to slide apart with fixed polarity. This suggests that there is a unique mechanochemistry to the dynein arm with unidirectional force generation in all active arms and also that not all arms are active at once during a ciliary beat. Negative stain and thin‐section images of arms in axonemes treated with β, γ methylene adenosine triphosphate (AMP‐PCP) show a consistent subunit construction where the globular head of the arm interacts with subfiber B of doublet N + 1. This interpretation differs from that provided by freeze etch and STEM interpretations of in situ arm construction and has implications for the mechanochemical cycle of the arm. A computer model of the arms in relation to other axonemal structures has been constructed to test these interpretations. Attachment of the head of the arm to subfiber B is directly demonstrable in splayed axonemes in AMP‐PCP. About half of the doublets in an axoneme show such attachments, while half do not. This might imply that about half the doublets in an axoneme are active at any given instant and can be identified as such. This information may be useful in probing questions of how active arms differ biochemically from inactive arms and of how microtubule translocators in general become active.