bluuebunny/crossref_metadata_2025 huggingface dataset:
DiOC6 staining reveals organelle structure and dynamics in living yeast cells
Authors: Koning Ann J. , Lum Pek Yee , Williams Jennifer M. , Wright Robin
Published: February 2005

When present at low concentrations, the fluorescent lipophilic dye, DiOC6, stains mitochondria in living yeast cells [Pringle et al.: Methods in Cell Biol. 31:357–435, 1989; Weisman et al.: Proc. Natl. Acad. Sci. U.S.A. 87:1076–1080, 1990]. However, we found that the nuclear envelope and endoplasmic reticulum were specifically stained if the dye concentration was increased or if certain respiratory‐deficient yeast strains were examined. The quality of nuclear envelope staining with DiOC6 was sufficiently sensitive to reveal alterations in the nuclear envelope known as karmellae. These membranes were previously apparent only by electron microscopy. At the high dye concentrations required to stain the nuclear envelope, wild‐type cells could no longer grow on non‐fermentable carbon sources. In spite of this effect on mitochondrial function, the presence of high dye concentration did not adversely affect cell viability or general growth characteristics when strains were grown under standard conditions on glucose. Consequently, time‐lapse confocal microscopy was used to examine organelle dynamics in living yeast cells stained with DiOC6. These in vivo observations correlated very well with previous electron microscopic studies, including analyses of mitochondria, karmellae, and mitosis. For example, cycles of mitochondrial fusion and division, as well as the changes in nuclear shape and position that occur during mitosis, were readily imaged in time‐lapse studies of living DiOC6‐stained cells. This technique also revealed new aspects of nuclear disposition and interactions with other organelles. For example, the nucleus and vacuole appeared to form a structurally coupled unit that could undergo coordinated movements. Furthermore, unlike the general view that nuclear movements occur only in association with division, the nucleus/vacuole underwent dramatic migrations around the cell periphery as cells exited from stationary phase. In addition to the large migrations or rotations of the nucleus/vacuole, DiOC6 staining also revealed more subtle dynamics, including the forces of the spindle on the nuclear envelope during mitosis. This technique should have broad application in analyses of yeast cell structure and function. © 1993 Wiley‐Liss, Inc.
Characterization of spontaneous and action potential‐induced calcium transients in developing myotubes in vitro
Authors: Flucher Bernhard E. , Andrews S. Brain
Published: February 2005

We have investigated the onset and maturation of action potential‐ and calcium‐induced calcium release from the sarcoplasmic reticulum during the differentiation of excitation‐contraction coupling in skeletal muscle. Microfluorometry and video imaging of cultured myotubes loaded with the fluorescent calcium indicator fluo‐3 revealed the dynamics, time course, and physiological properties of calcium transients as well as their changes during development. Spontaneous and stimulated contractions in well‐differntiated myotubes are accompanied by brief (200–500 ms) increases in the concentration of free cytoplasmic calcium. These transients are modulated by sub‐threshold concentrations of caffeine, resulting in a plateau of elevated calcium. Two novel types of calcium transients were observed in non‐contracting myotubes. (1) Fast localized transients (FLTs) are radially restricted focal release events that occur spontaneously within the myoplasm at various densities and frequencies. (2) Upon addition of caffeine, propagating calcium waves are generated (35–70 μm/s velocity), which are accompanied by contractures. Aside from caffeine sensitivity, calcium waves and contractionrelated sustained release events are similar in amplitude and duration, as well as in their inactivation and refractory properties. Thus, these transients may represent calcium‐induced calcium release in quiescent and active myotubes, respectively. Following one calcium‐induced calcium release event, myotubes become refractory to new calcium‐induced transients; however, action potential‐induced transients and FLTs are not blocked. This suggests that these transients occur by distinct release mechanisms and that dual modes of calcium release exist prior to the coupling of calcium release to excitation. © 1993 Wiley‐Liss, Inc.
Tipping of flagellar agglutinins by gametes of Chlamydomonas reinhardtii
Authors: Goodenough Ursula W.
Published: February 2005

The migration of cross‐linked agglutinins to the gametic flagellar tips (tipping) is a hallnmark of the Chlamydomonas mating reaction. In this study, an assay was developed to analyze the kinetics and biological requirements for the tipping response: isolated flagella from mt− gametes of C. reinhardtii were allowed to agglutinate to the immotile flagella of pf‐18 mt+ gametes, and their migration to the tips was monitored by phase microscopy. The tipping process is shown to require both adhesion and elevated levels of cAMP. The cAMP may activate tipping motors directly. In addition, cAMP stimulates the recruitment of agglutinins to flagellar surface to replace those inactivated by adhesion. These results are compared with previous studies on the tipping of flagellar surface proteins cross‐linked by soluble ligands, and an integrated model is presented. © 1993 Wiley‐Liss, Inc.
Fimbrin localized to an insoluble cytoskeletal fraction is constitutively phosphorylated on its headpiece domain in adherent macrophages
Authors: Messier Jeanne M. , Shaw Leslie M. , Chafel Mark , Matsudaira Paul , Mercurio Arthur M.
Published: February 2005

The actin‐bundling protein fimbrin is homologous to l‐plastin, a 65kD phosphoprotein expressed in leukocytes and transformed cells [de Arruda et al., J. Cell Biol. 111, 1069–1080]. Because fimbrin is present in cell adhesion sites, we studied the phosphorylation state of fimbrin and its distribution in macrophages sequentially extracted with Triton‐X‐100 (soluble fraction), Tween 40‐deoxy‐cholate (cytoskeletal fraction), and SDS (insoluble cytoskeletal fraction). The approximate distribution of fimbrin and actin among these fractions was found to be: 65% fimbrin/55% actin in the soluble fraction, 30% fimbrin/20% actin in the cytoskeletal fraction, and 5% fimbrin/25% actin in the insoluble cytoskeletal fraction. PMA did not alter this distribution. Fluorescence microscopy of acetone‐extracted macrophages showed that actin is concentrated in podosomes at the substratum interface and is diffusely distributed throughout the remainder of the cell. Fimbrin colocalizes with actin in podosomes and also exhibits a punctate distribution in the cytoplasm that overlaps with actin. In Tween 40/DOC‐extracted cells, podosomes remain, and fimbrin also exhibits a punctate distribution along actin filaments. Metabolic 32PO4 labeling revealed that fimbrin is constitutively phosphorylated and that phosphorylated fimbrin is concentrated in the insoluble cytoskeletal fraction. PMA increased the relative levels of fimbrin phosphorylation twofold but did not alter the pattern of fimbrin fluorescence or the distribution of phosphorylated fimbrin. Limited trypsin digestion and phosphoamino acid analysis demonstrated that phosphorylation occurs specifically on serine residues within the 10kD headpiece domain of fimbrin. Phosphorylation of the headpiece domain could regulate the actin binding and bundling properties of fimbrin, or it could regulate the interaction of fimbrin with other proteins. © 1993 Wiley‐Liss, Inc.
Human fibroblast tropomyosin isoforms: Characterization of cDNA clones and analysis of tropomyosin isoform expression in human tissues and in normal and transformed cells
Authors: Novy Robert E. , Lin Jenny Li‐Chun , Lin Ching‐Shwun , Lin Jim Jung‐Ching
Published: February 2005

A tropomyosin‐specific oligonucleotide probe (REN29) designed to hybridize to all known human tropomyosin isoforms was used to study tropomyosin mRNA levels in normal and transformed human cells. At least four different sizes of RNAs were detected in normal human fibroblast KD cells by Northern blot analysis. The major bands of 1.1 kb RNA for hTM1 and 3.0 kb RNA for hTM4 were decreased substantially in various transformed cell lines. One of the minor RNA bands (2.0 kb for hTM2 and hTM3) appeared to be absent in a human pancreatic carcinoma cell line. The level of the other minor RNA band (2.5 kb for hTM5) was found to be unchanged or slightly decreased in transformed cells. This differential expression of tropomyosin isoforms at the RNA level was not totally in agreement with the difference in the protein amounts found in normal and transformed cells, suggesting that translational control may also play an important role in the expression of some tropomyosin isoforms. The REN29 probe was further used to screen γgt10 and γgt11 cDNA libraries, which were constructed from poly(A)+ RNAs of human fibroblast cell lines HuT‐14 and WI‐38, respectively. In addition to cDNA clones encoding known isoforms, we obtained three classes of new cDNA clones that encode two low Mr isoforms (hTM5a and hTM5b), and a high Mr isoform (hTMsmα). Sequence comparison revealed that hTM5a and hTM5b are alternatively spliced products derived from the same gene that encodes hTM2 and hTM3. Northern blot analysis and amino acid sequence comparison suggested that the hTMsmα represents a smooth muscle tropomyosin which is also expressed in human fibroblasts. The exon specific for, and common to, hTM5a and hTM5b was found to be highly expressed in small intestine. However, there was no detectable expression of this exon in stomach and skeletal muscle. The difference in tissue‐specific expression suggests that different isoforms may perform distinct functions in different tissues. © 1993 Wiley‐Liss, Inc.
Expression of four myosin heavy chain isoforms with development in mouse uterus
Authors: Eddinger Thomas J. , Wolf Jane A.
Published: February 2005

In smooth muscle tissue, two or three isoforms of myosin heavy chain (MHC) have been reported (SM1, SM2, and/or NM). In mouse uterus tissue, four bands in the region of the MHC's can be resolved on high resolution SDS polyacrylamide gels. Western blots using smooth muscle (SM) MHC‐specific and nonmuscle (NM) MHC‐specific polyclonal antibodies show the upper two bands in the MHC region are SM isoforms, whereas the lower two bands are NM isoforms. One‐dimensional peptide maps of these four bands show each to have a unique pattern of polypeptide fragments following α‐chymotrypsin digestion. Developmental expression of myosin heavy chains (MHC) in mouse uterus, aorta, bladder, and stomach (6 ages, 10–150 days) was determined using tissue homogenates. In the uterus, both SM MHC's show an increase in relative content with increasing age, whereas the NM MHC's show a decrease. The mouse aorta shows a significant increase in the SM MHC's and a significant decrease in the NM MHC from day 10 to day 30, which is similar to data reported for the rat aorta. Whereas both the bladder and stomach contain relatively small amounts of NM MHC's (∼ 10% or less), these quantities do show decreases with development. The SM1:SM2 ratio for the uterus remains high (3.4 at 150 days) through development; the aorta, bladder, and stomach also start out high, but tend toward 1.0 in the 150‐day animals. The presence of four MHC isoforms in the uterus with unique developmental regulation of expression is consistent with hypotheses of unique functional roles for these isoforms. © 1993 Wiley‐Liss, Inc.
Biochemical and immunochemical identification of a microtubule‐binding protein from bovine pancreas
Authors: Michalik Liliane , Neuville Pascal , Vanier Marie‐Thérèse , Launay Jean‐François
Published: February 2005

We have identified a 67 kDa heat‐stable protein among the proteins which bind specifically to brain microtubules immobilized on a chromatographic support. Its relationship to tubulin and to the cytoskeleton using polyclonal antibodies has been studied. This 67 kDa protein is present in cytoskeleton and microtubule preparations from pancreas. This heat‐stable microtubule‐associated protein (MAP) copolymerized with phosphocellulose purified brain tubulin. The 67 kDa polypeptide was immunoreactive to antibodies against the 210 kDa MAP from HeLa cells; it also reacted with antibodies against an oligopeptide whose sequence corresponded to the second repeat of mouse brain tau. © 1993 Wiley‐Liss, Inc.
PKC mediates 12(S)‐HETE‐induced cytoskeletal rearrangement in B16A melanoma cells
Authors: Timar J. , Tang D. , Bazaz R. , Haddad M. M. , Kimler V. A. , Taylor J. D. , Honn K. V.
Published: February 2005

The fatty acid 12(S)‐HETE may be a new second messenger capable of activating PKC. In tumor cells 12(S)‐HETE stimulates cytoskeleton‐dependent cellular responses such as adhesion and spreading. Analysis of 12(S)‐HETE effects on B16a melanoma cell cytoskeleton revealed reversible rearrangement of microtubules, microfilaments, the actin‐binding proteins, vinculin, myosin heavy (MHC) and light chains (MLC), as well as bundling of vimentin intermediate filaments. The alterations in microfilaments and intermediate filaments occurred very rapidly, i.e., 5 min after exposure of tumor cells to 12(S)‐HETE. The 12(S)‐HETE‐induced cytoskeletal alterations were accompanied by centrifugal organelle‐translocation. Interestingly, MLC exhibited clear association with the cytoplasmic organelles. Biochemical analysis of the 12(S)‐HETE effect indicated a PKC‐mediated reversible hyperphosphorylation of MLC, vimentin, and a 130 kD cytoskeletal‐associated protein. Optimal effects were obtained after 5 min treatment with 12(S)‐HETE at 0.1 μM concentration. 12(S)‐HETE pretreatment induced tumor cell spreading on a fibronectin matrix which required the intactness of all three major cytoskeletal components. The spreading process was dependent upon the activity of PKC. Our data suggest that 12(S)‐HETE is a physiological stimulant of PKC. Further, it induces rearrangement of the cytoskeleton of tumor cells in interphase resulting in the stimulation of cytoskeleton‐dependent cell activity such as spreading. © 1993 Wiley‐Liss, Inc.
Inhibition of microtubule sliding by Ni2+ and Cd2+: Evidence for a differential response of certain microtubule pairs within the bovine sperm axoneme
Authors: Kanous Kathleen S. , Casey Christina , Lindemann Charles B.
Published: February 2005

Bovine sperm, extracted with 0.1% Triton X‐100, frozen at −20°C for 48–120 hours, and thawed, disintegrated by microtubule sliding when 1 mM MgATP was added. Microtubules and outer dense fibers (ODFs) were usually extruded in groups or “bundles.” A total of 44.5% of the cells extruded two distinct bundles, one from each side of the connecting piece, exhibiting opposite curvatures. Only one bundle was observed in 46.2% of the cells, and 9.2% showed no signs of sliding. Transmission electron microscopy (T.E.M.) showed one group consisting of the 4,5‐6,7 elements, with the 9,1,2 elements on the other side of the axoneme making up the other bundle. T.E.M. revealed that when only one side of the axoneme had extruded elements, they were always from the 4,5‐6,7 group. The remainder of the axoneme (8,9,1,2,3 and the central pair) was left relatively intact, suggesting a difference in the sliding response of the nine pairs of axonemal microtubules. These results indicate a predisposition for sliding between elements 7 and 8 over that between doublets 2 and 3, perhaps due to a disparity in activation thresholds. Also, both Ni2+ and Cd2+ appear to selectively block activation of 2–3 interdoublet sliding.

Incubation with 0.25 mM Ni2+ prior to adding MgATP modified the percentages of sliding patterns: 8.6% demonstrated two‐sided extrusion, 58.2% showed one‐sided, and 33.2% had no extruded bundles. Again, when half the axoneme was missing, it was always the 4,5‐6,7 group. Ten micromolar Cd2+ altered the sliding pattern similarly to Ni2+, with 28% two‐sided extrusion, 55.9% one‐sided extrusion and 16.1% with no extruded bundles.

Either pretreatment regimen impeded extrusion of the 9,1,2 group in a high percentage of cells, compared to untreated cells. This specific inhibition of the 9,1,2 side by Ni2+ or Cd2+ is especially significant since Ni2+ also inhibits spontaneous wave initiation in bull sperm (Lindemann et al.: Journal of Cell Biology 87:420–426, 1980), and both Ni2+ and Cd2+ reportedly block the flagellar Ca2+‐response in rat sperm (Lindemann and Goltz: Cell Motility and the Cytoskeleton 10:420–431, 1988; Lindemann et al.: Cell Motility and the Cytoskeleton 20:316–324, 1991). © 1993 Wiley‐Liss, Inc.
Method of cell handling affects leakiness of cell surface labeling and detection of intracellular keratins
Authors: Riopel Carrie L. , Butt Isha , Omary M. Bishr
Published: February 2005

Keratins are a subgroup of cytoskeletal intermediate filament proteins found in most epithelial cells. Some reports have suggested that keratins may be found on the cell surface as well as their well‐accepted cytoskeletal location. A major part of the evidence in the interpretation of cell surface expression of keratins is cell surface radioiodination. Here we show that lactoperoxidase‐catalyzed iodination of colonic and breast tissue culture cells results in radiolabeling of the keratins when cells are manipulated. No labeling of keratins is detected when cells are labeled directly on the tissue culture dish. A similar result was obtained when intact cells were biotinylated using water‐soluble sulfo–NHS–biotin. Partitioning of the keratins to a soluble and an insoluble pool after “cell surface” 125I‐labeling showed that both pools became iodinated. Indirect immunofluorescence showed that binding of a panel of anti‐keratin antibodies to intact epithelial cells occurs only on the cells that are more adherent, which are the cells that require longer manipulation to remove from the tissue culture dish. Taken together, our results indicate that the reported expression of cell surface keratins in some cells likely reflects intracellular keratins. In addition, the method of epithelial cell handling can dramatically alter the leakiness of cell surface iodination techniques. © 1993 Wiley‐Liss, Inc.
Neurofilaments move apart freely when released from the circumferential constraint of the axonal plasma membrane
Authors: Brown Anthony , Lasek Raymond J.
Published: February 2005

Squid giant axons were used to obtain axonal cytoskeletons that had been separated from the confines of their plasma membranes. To remove the plasma membrane, axoplasm was extruded from the giant axon directly into an artificial axoplasm solution (AAS). This procedure produces a smooth axoplasmic cylinder in which neurofilaments (NFs) are the most prevalent cytological elements. The NFs scatter light strongly and thus dark‐field light microscopy can be used to quantify the volume occupied by these polymers. Measurements of the widths of the dark‐field images of the axoplasmic cylinders showed that the cross‐sectional area of the NF population increased by 60–110% (n = 8) between 1–100 min after plasma membrane removal, and then continued to increase more slowly for many hours. After 1,000 min, the cross‐sectional area was 75–160% (n = 8) larger than at 1 min. These light microscopic measurements of axoplasm suggest that the NF population disperses to occupy a continuously increasing volume after removal of the plasma membrane and immersion in AAS. This inference was confirmed by quantitative ultrastructural studies of NFs in axoplasmic cross‐sections, which demonstrated that the spacing between the NFs increased between 1–1,000 min after plasma membrane removal. Comparison of the NF density distribution after 1,000 min with a theoretical distribution calculated using the Poisson theorem indicated that the NFs dispersed randomly. These studies on NFs in isolated axoplasm suggest that ordinary thermal forces of Brownian motion are sufficient to move axonal NFs apart independently and thereby to disperse them. We propose that, in the intact axon, the dispersive movements of the NFs spread the NF cytoskeleton radially and expansively to fill out the cylindrical space contained by the axonal plasma membrane and its surrounding connective tissue elements. © 1993 Wiley‐Liss, Inc.
Role of phosphorylation in keratin and vimentin filament integrity in cultured thyroid epithelial cells
Authors: Deery William J.
Published: February 2005

Cytokeratin and vimentin intermediate filaments (IFs) possess relatively stable polymeric properties which can be affected by phosphorylation. The present study, using cultures of thyroid epithelial cells, shows by indirect immunofluorescence that these cells contain both keratin tonofilament and vimentin IF complexes. Immunoblots of Triton X‐100 insoluble cytoskeletal fractions show vimentin, and ∼52 kDa type II and 40/38 kDa type I keratins. Under “basal” conditions, following prelabeling of cells with [32PO4], vimentin is not significantly phosphorylated, while both type II and I keratins are phosphorylated. Treatment of cells for 20 min with 1 mM dbcAMP or 0.4 μM 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), to stimulate protein kinase A and C, respectively, has no effect on either the phosphorylation state or cytoplasmic filament integrity of vimentin. However, while dbcAMP also does not affect keratin filaments, TPA increases both type II and I phosphorylation ∼3‐fold, and concomitantly disrupts tonofilament complexes associated with the nucleus, cytoplasm, and desmosomes. TPA‐treated cells also show dramatic shape changes and protrusive activity. Tryptic peptide mappings show phosphorylations of at least 6 and ∼2 additional sites for type II and I keratins, respectively, vs. [32P]‐peptides from control cells. Treatment of [32PO4]‐labeled cells with 0.4 μM calyculin A to inhibit types 1 and 2A phosphatase activity causes hyperphosphorylation of both vimentin and keratin, disruption of IF complexes, and actomyosin/cell contraction within 20 min. Quantitatively, ∼50% of the type II/I keratin hyperphosphorylations are at some sites apparently also phosphorylated after TPA treatment. Thus, in these cells, IFs are specifically and differentially affected and regulated by the activity of several kinases. © 1993 Wiley‐Liss, Inc.
Assembly dynamics of actin in adherent human neutrophils
Authors: Wang Jia‐Sheng , Pavlotsky Nelli , Tauber Alfred I. , Zaner Ken S.
Published: February 2005

We have extended our previous studies of adherent neutrophils and compared actin depolymerization and intracellular calcium changes induced by adherence to laminin and fibronectin. In order to accurately assess cellular actin changes, F‐actin depolymerization in the cell lysates must be inhibited. We found that phalloidin or 3.7% formaldehyde treatment effectively inhibited the depolymerization of F‐actin fragments following cell lysis. Formaldehyde and phalloidin treatment reduced G‐actin levels 75–80% in suspended cells, 35–73% in cells adherent for 1 min, and about 50% for cells adherent for 3 min. When the actin was fixed, there were highly significant differences in G‐actin levels between the suspended and adherent cells as compared with unfixed cells. Adhesion to both laminin and fibronectin initiated a rapid rise in G‐actin with a corresponding decrease in F‐actin. However, the changes were more pronounced in cells adherent to laminin. The peak of depolymerization occurred by 1 min and, thereafter, G‐actin decreased and F‐actin increased reaching a steady state at 5 min. Adhesion to both laminin‐ and fibronectin‐coated surfaces was accompanied by an increase of [Ca2+]i with a peak at 3 min, followed by a decrease from 3–5 min and a steady state attained between 5 and 10 min. The rise of [Ca2+]i in laminin‐adherent cells was about twice that in fibronectin‐adherent cells at 3 min (P < 0.02). Pertussis toxin, H‐7, and staurosporin treatments did not alter the dynamic changes of actin in adherent cells, suggesting that these metabolic events are transduced by a G‐protein and Protein Kinase C independent mechanism. The results support the hypothesis that a transient mobilization of F‐actin to a monomeric pool, which then serves as a source for further repolymerization, is induced by adherence of neutrophils to extracellular matrix proteins. © 1993 Wiley‐Liss, Inc.
Dithiothreitol prevents membrane fusion but not centrosome or microtubule organization during the first cell cycles in sea urchins
Authors: Schatten Heide
Published: February 2005

Dithiothreitol (DTT), a disulfide reducing agent, inhibits the fusion of male and female pronuclei within the activated cytoplasm of sea urchin eggs. The migrations of the pronuclei are not affected by DTT, indicating that microtubule function is not impaired. Centrosomal antigens are detected in the sperm aster and in all subsequent microtubule‐based configurations. Nuclear membranes never fuse and the chromatin of male and female pronuclei never mix in the DTT‐treated cells. During prophase, when nuclear envelopes break down to undergo mitosis, both sets of chromosomes undergo condensation cycles independent from each other. Both pronuclei initially stain for centrosomal material and surrounding microtubules. With time, the female's centrosomal material as well as the microtubules disappear while the male forms a bipolar spindle. Interestingly, one pole of the paternal mitotic apparatus communicates with the separate maternal chromatin, forming a half spindle which moves the egg‐derived chromatin towards its pole. At the time for cell division, the individual karyomeres are not able to fuse their nuclear membranes to reconstitute the blastomere nuclei. When DTT is applied at prometaphase of the first cell cycle, the chromosome cycle continues until next metaphase. Centrosomes also continue their cycle and undergo somewhat atypical splitting during the time for second telophase. Division furrows are initiated but aborted. These results support the hypothesis that disulfide groups are required for membrane fusion of the pronuclei, for membrane fusion of the karyomeres, and for the completion of the division furrow to achieve successful cell division. © 1994 Wiley‐Liss, Inc.
Microtubules with altered assembly kinetics have a decreased rate of kinesin‐based transport
Authors: Redenbach Darlene M. , Richburg John H. , Boekelheide Kim
Published: February 2005

Microtubules treated with the γ‐diketone 2,5‐hexanedione (2,5‐HD) have altered assembly behavior characterized by precocious nucleation and rapid elongation. By measuring the rate of microtubule transport, we have examined the potential functional significance of this 2,5‐HD‐induced microtubule modification. 2,5‐HD‐treated microtubules were transported at only 70% of the rate of control microtubules in a simple kinesin‐based motility assay on glass coverslips using video and computer enhanced differential interference contrast microscopy. Since 2,5‐HD is capable of forming both pyrrole adducts and crosslinks with tubulin, the contributions of pyrrole formation and crosslinking to slowed microtubule transport were determined. 3‐Acetyl‐2,5‐hexanedione (AcHD), a pyrrole forming, non‐crosslinking congener of 2,5‐HD which does not alter microtubule assembly, did not produce slowed microtubule transport as occurs with 2,5‐HD. However, glutaraldehyde, a pyrrole‐independent crosslinking agent which alters microtubule assembly in the same way as 2,5‐HD, slowed microtubule transport. These results indicate that a 2,5‐HD‐induced microtubule modification, possibly a crosslink‐related conformational change, produces both an alteration in the kinetics of assembly and an alteration in the microtubule‐motor interaction. © 1994 Wiley‐Liss, Inc.
Control of flagellar bending: A new agenda based on dynein diversity
Authors: Brokaw Charles J.
Published: February 2005

Observations that were interpreted to provide evidence for equivalent functions of all axonemal dyneins should be reinterpreted, and models based on this assumption should be abandoned. In the future, attempts to understand the mechanisms for flagellar bending, oscillation, and bend propagation should start from the assumption that each type of axonemal dynein may have a specific function. At least three distinct functions can now be identified: bend initiation, maintenance of the angle of propagating bends, and generation of power to overcome viscous resistances. Only the last of these three functions is an outer arm dynein function. © 1994 Wiley‐Liss, Inc.
Cytoplasmic dynein binds to phospholipid vesicles
Authors: Lacey Merri Lynn , Haimo Leah T.
Published: February 2005

Cytoplasmic dynein is the putative motor protein for retrograde organelle transport along microtubules in cells and, thus, must be capable of binding to organelle membranes. Such an attachment may occur via receptor proteins or through a direct interaction of dynein with the membrane phospholipids. We show here that cytoplasmic dynein‐synaptic membrane binding does not require a receptor protein and that this binding is mediated by an electrostatic interaction with acidic phospholipids. The properties of cytoplasmic dynein binding to NaOH‐extracted synaptic membranes are not significantly affected when those membranes are treated with trypsin to digest endogenous integral membrane proteins. Moreover, purified cytoplasmic dynein is capable of binding to liposomes composed of pure phospholipids. Dynein binds to liposomes with a profile remarkably similar to that of dynein binding to native membranes. Dynein‐liposome binding is dependent upon the presence of acidic phospholipids and is disrupted by NaCl. Thus, these studies suggest that electrostatic interactions can effect dynein‐membrane binding. © 1994 Wiley‐Liss, Inc.
Metachronal activity of cultured mucociliary epithelium under normal and stimulated conditions
Authors: Gheber Larisa , Priel Zvi
Published: February 2005

In the present work we measured in real time the metachronism and degree of correlation between beating cilia from cultured mucociliary epithelium. The method is based on simultaneous measurement of ciliary beat frequency, phase shifts, and correlation factors in two directions: parallel and perpendicular to the effective stroke direction (ESD). From the phase shifts the lengths of wave components, and consequently the metachronal wavelength and direction, were evaluated.

On active ciliary areas of cultured frog esophagus under normal conditions, a relatively high degree of correlation is observed, but cilia are more correlated in direction parallel to ESD which is also the direction of the mucus propulsion. The length of the wave component parallel to ESD is more than twice as large as that of the perpendicular component. The metachronal wavelength was found to be in the range of 5–9 μm, and the direction of the wave propagation was in the range of 90°–125° clockwise to the ESD.

When ciliary beat frequency was rapidly increased by extracellular ATP or acetylcholine, only minor effects were observed on the degree of correlation between beating cilia. The length of the wave component parallel to ESD showed the most dramatic effect increasing up to tenfold. The perpendicular to ESD component was not affected by the stimulation. Consequently, the metachronism became more laeoplectic with the angle between the ESD and the wave directions decreasing by 10°–30°, and the metachronal wavelength remained unaltered. © 1994 Wiley‐Liss, Inc.
A model of flagellar and ciliary functioning which uses the forces transverse to the axoneme as the regulator of dynein activation
Authors: Lindemann Charles B.
Published: February 2005

Ciliary and flagellar motion is driven by the dynein‐tubulin interaction between adjacent doublets of the axoneme, and the resulting sliding displacements are converted into axonemal bends that are propagated. When the axoneme is bent in the normal beating plane, force develops across the axoneme in the plane of the bend. This transverse force (t‐force) has maximal effect on the interdoublet spacing of outer doublets 2–4 on one side of the axoneme and doublets 7–9 on the opposite side. Episodes of sliding originates as the t‐force brings these doublets into closer proximity (allowing dynein bridges to form) and are terminated when these doublets are separated from each other by the t‐force. A second factor, the adhesive force of the dynein‐tubulin attachments (bridges), also acts to pull neighboring doublets closer together. This force resists termination of a sliding episode once initiated, and acts locally to give the population of dynein bridges a type of excitability. In other words, as bridges form, the probability of nearby bridges attaching is increased by a positive feedback exerted through the interdoublet spacing. A conceptual working hypothesis explaining the behavior of cilia and flagella is proposed based on the above concepts. Additionally, the feasibility of this proposed mechanism is demonstrated using a computer simulation. The simulation uses a Monte Carlo‐type algorithm for dynein attachment and adhesive force, together with a geometric evaluation of the t‐force on the key microtubule pairs. This model successfully develops spontaneous oscillations from any starting configuration (including a straight position). It is compatible with the physical dimensions, mechanical properties and bridge forces measured in real cilia and flagella. In operation, it exhibits many of the observed actions of cilia and flagella, most notably wave propagation and the ability to produce both cilia‐like and flagella‐like waveforms. © 1994 Wiley‐Liss, Inc.
Extrusion of rotating microtubules on the dynein‐track from a microtubule‐dynein γ‐complex
Authors: Mimori Y. , Miki‐Noumura T.
Published: February 2005

Applying a new in vitro motility assay system for microtubules and 22S dynein, we recently reported on an ATP‐induced extrusion of microtubules from microtubule‐dynein α‐ and β‐complexes [Mimori and Miki‐Noumura, 1994:Cell Motil. Cytoskeleton 27:180–191]. In the present study, we prepared a γ‐complex by copolymerizing porcine brain tubulin and Tetrahymena ciliary 22S dynein, and examined the ATP‐induced microtubule movement from the γ‐complex. The extrusion process appeared quite similar to that of the β‐complex. The sliding velocity was 18.39 ± 2.20 m̈m/sec, which was a value comparable to that of trypsin‐digested flagellar axonemes [Yano and Miki‐Noumura, 1980:J. Cell Sci. 44:169–186]. Higher velocity may be due to a densely arranged dynein‐track with the same polarity, which was detached from the γ‐complex and absorbed in rows on a glass surface of the slide. Sometimes a free‐floating microtubule in the perfusion chamber was observed riding and sliding on the dynein‐track remaining on the slide after extrusion.

Unexpectedly, we found that when the front part of the microtubule was fixed to a glass surface, a continuous sliding microtubule at the rear part on the dyneintrack often transformed into a left‐handed helix, and subsequently a twisted helix with several turns. The helix formation may be due to some rigidity in the microtubule and a right‐handed torque component in the sliding force of 22S dynein. The addition of ATP may release some distortion accumulated in the complex structure during copolymerization of tubulin and 22S dynein, inducing reverse rotation of the microtubule. © 1995 Wiley‐Liss, Inc.
Listeria monocytogenes intracellular migration: Inhibition by profilin, vitamin D‐binding protein and DNase I
Authors: Sanger Jean M. , Mittal Balraj , Southwick Frederick S. , Sanger Joseph W.
Published: February 2005

Infection of host cells by Listeria monocytogenes results in the recruitment of cytoplasmic actin into a tail‐like appendage that projects from one end of the bacterium. Each filamentous actin tail progressively lengthenes, providing the force which drives the bacterium in a forward direction through the cytoplasm and later results in Listeria cell‐to‐cell spread. Host cell actin monomers are incorporated into the filamentous actin tail at a discrete site, the bacterial‐actin tail interface. We have studied the consequences of microinjecting three different actin monomer‐binding proteins on the actin tail assembly and Listeria intracellular movement. Introduction of high concentrations of profilin (estimated injected intracellular concentration 11–22 m̈M) into infected PtK2 cells causes a marked slowing of actin tail elongation and bacterial migration. Lower intracellular concentrations of two other injected higher affinity monomer‐sequenstering proteins, Vitamin D‐binding protein (DBP; 1–2 m̈M) and DNase I (6–7 m̈M) completely block bacterial‐induced actin assembly and bacterial migration. The onset of inhibition by each protein is gradual (10–20 min) indicating that the mechanisms by which these proteins interfere with Listeria‐induced actin assembly are likely to be complex. To exclude the possibility that Listeria recruits preformed actin filaments to generate the tails and that these monomer‐binding proteins act by depolymerizing such performed actin filaments, living infected cells have been injected with fluorescently labeled phalloidin (3 m̈M). Although the stress fibers are labeled, no fluorescent phalloidin is found in the tails of the moving bacteria. These results demonstrate that Listeria‐induced actin assembly in PtK2 cells is the result of assembly of actin monomers into new filaments and that Listeria's ability to recruit polymerization competent monomeric actin is very sensitive to the introduction of exogenous actin monomer‐binding proteins. © 1995 Wiley‐Liss, Inc.
Cytoskeletal domains in the activated platelet
Authors: Bearer E. L.
Published: February 2005

Platelets circulate in the blood as discoid cells which, when activated, change shape by polymerizing actin into various structures, such as filopodia and stress fibers. In order to understand this process, it is necessary to determine how many other proteins are involved. As a first step in defining the full complement of actin‐binding proteins in platelets, filamentous (F)‐actin affinity chromatography was used. This approach identified >30 different proteins from ADP‐activated human blood platelets which represented 4% of soluble protein. Although a number of these proteins are previously identified platelet actin‐binding proteins, many others appeared to be novel. Fourteen different polyclonal antibodies were raised against these apparently novel proteins and used to sort them into nine categories based on their molecular weights and on their location in the sarcomere of striated muscle, in fibroblasts and in spreading platelets. Ninety‐three percent of these proteins (13 of 14 proteins tested) were found to be associated with actin‐rich structures in vivo.

Four distinct actin filament structures were found to form during the initial 15 min of activation on glass: filopodia, lamellipodia, a contractile ring encircling degranulating granules, and thick bundles of filaments resembling stress fibers. Actin‐binding proteins not localized in the discoid cell became highly concentrated in one or another of these actin‐based structures during spreading, such that each structure contains a different complement of proteins. These results present crucial information about the complexity of the platelet cytoskeleton, demonstrating that four different actin‐based structures form during the first 15 min of surface activation, and that there remain many as yet uncharacterized proteins awaiting further investigation that are differentially involved in this process. © 1995 Wiley‐Liss, Inc.
Identification and molecular characterization of a yeast myosin I
Authors: Goodson Holly V. , Spudich James A.
Published: February 2005

The family of myosin motors is comprised of numerous classes distributed among a diverse set of organisms and cell types. We have identified an unconventional myosin gene (MYO3) in the yeast Saccharomyces cerevisiae and show that it is member of a subclass of unconventional myosin proteins originally found only in the amoeboid organisms Dictyostelium and Acanthamoeba. Identification of this protein in these genetically and morphologically divergent organisms suggests that it will be ubiquitous in eukaryotes and that it has a role in the basic functions of the eukaryotic cell. We have constructed a strain of yeast missing 99% of the MYO3 coding sequence. This mutation has no observable phenotypic effect, placing MYO3 into a growing class of yeast genes which are dispensable under laboratory conditions, perhaps due to genetic redundancy. Alignment of MYO3 with other unconventional myosins shows that it shares with a subset of them a previously unrecognized region of homology in the tail; this region falls within a domain identified as important for mediating nonspecific electrostatic interactions with membranes. The existence of this region suggests that it may be involved in mediating specific protein‐protein interactions, possibly helping to localize this myosin to specific membranes or membrane regions. In addition, we show that “classic” myosin I proteins share a region of hyper‐proline‐richness 10 amino acids before the SH3 domain. Proline‐rich regions have recently been implicated as SH3 binding sites, which suggests that this region might be involved with regulating or in other ways interacting with SH3 domains. © 1995 Wiley‐Liss, Inc.
Myosin heavy chain isoform expression in human myometrium: Presence of an embryonic nonmuscle isoform in leiomyomas and in cultured cells
Authors: Cavaillé F. , Fournier T. , Dallot E. , Dhellemes C. , Ferré F.
Published: February 2005

We had previously found no myosin heavy chain (MHC) changes in expression during pregnancy in human myometrium. In the present work, we compared the MHC pattern of expression in normal human myometrium, pregnant and non‐pregnant, to that in benign tumors of the uterine musculature and in cultured myometrial cells. We used a high‐resolution gel electrophoretic system and monoclonal antibodies directed against smooth muscle and nonmuscle MHCs. Smooth muscle MHCs (SM1, 204 kDa, and SM2, 200 kDa, MHCs) and a nonmuscle MHC of 196 kDa (NM MHC) were detected in pregnant and nonpregnant human myometrium. Pregnant myometrium was found to differ from nonpregnant myometrium by its slightly lower content in NM MHC, whereas the ration of SM1/SM2 was equivalent. In leiomyomas and in cultured cells grown from human myometrium explants, SM1, SM2, and NM MHCs were also expressed. In addition, a nonmuscle MHC of 198/200 kDa (SMemb MHC), which was present in a fetal human uterus but not in adult normal tissue, was observed in leiomyomas and in cultured cells. Expression of SM1 and SM2 MHCs was variable in the different leiomyomas studied. In cultured cells, SM1 and SM2 MHC content was low, but it was enhanced by suppression of serum after cell confluency. Present results confirm that pregnancy‐associated smooth muscle cell hypertrophy is not accompanied by major changes in MHCs. In contrast, cell culturing and cell hyperplasia leading to leiomyoma formation induce substantial modifications in MHCs, including the occurrence of a second type of nonmuscle MHC. © 1995 Wiley‐Liss, Inc.
Antibody against ahosphorylated proteins (MPM‐2) recognizes mitotic microtubules in endosperm cells of higher plant haemanthus
Authors: Smirnova E. A. , Cox D. L. , Bajer A. S.
Published: February 2005

In diverse cell types, monoclonal antibody MPM‐2 recognizes a class of phosphorylated proteins related to microtubule organizing centers and abundant during mitosis. We have used this antibody in an attempt to identify the spatial and temporal localization of putative microtubule organizing centers in endosperm cells of the higher plant Haemanthus. Our results show that MPM‐2 recognized epitope is present in interphase cells and enriched in mitotic cells. In interphase the antibody usually stains cytoplasmic granules. During the interphase‐prophase transition immunoreactive material appears in the nucleus, at the nuclear envelope, and in association with microtubules. Concomitantly, we observed an increase of immunoreactivity of the cytoplasm. During mitosis the phosphorproteins recognized by MPM‐2 are detected in the cytoplasm, in association with microtubules of the spindle, the phragmoplast, and in the newly‐formed cell plate. After completion of mitosis, only the cell plate and cytoplasmic granules are MPM‐2 positive. Extraction of the cells with Triton X‐100 prior to fixation removes staining of the cytoplasm by MPM‐2. The detergent resistant immunoreactive material remains associated with surrounding the nucleus microtubules of the prophase spindle, the core of kinetochore fibers, and the phragmoplast. In the phragmoplast, however, segments of microtubules which are distal to the cell plate are depleted of MPM‐2.

These data demonstrate that microtubule arrays of endosperm cells are phosphorylated during mitosis. Thus, similar to animal cells, interphase and mitotic microtubules of higher plants have different properties. Additionally, the localization of detergent resistant MPM‐2 antigen points to the difference in microtubule nucleation/organization between higher plant and animal cells.
Focal adhesion proteins associated with apical stress fibers of human fibroblasts
Authors: Katoh Kazuo , Masuda Michitaka , Kano Yumiko , Fujiwara Keigi , Jinguji Yoichi
Published: February 2005

Human fibroblasts stained with fluorescently labeled phalloidin revealed many stress fibers within the apical cytoplasm in addition to those located along the basal plasma membrane and associated with focal adhesions. The staining patterns of these apical stress fibers with fluorescent phalloidin, anti‐α‐actinin, and anti‐myosin were identical to those of the basal stress fibers, suggesting the same macromolecular organization for both types f stress fibers. There were two types of apical stress fibers that clearly interacted with the apical plasma membrane, those extending between the basal and the apical plasma membrane and those having both ends on the basal membrane forming arches whose top interacted with the apical plasma membrane. By electron microscopy, we observed that apical stress fibers were associated with the apical plasma membrane via electron‐dense plaques reminiscent of the focal adhesion. Since several proteins have been specifically localized to the focal adhesion site, we examined whether they were also present at the apical stress fiber‐membrane association site by using immunocy‐tochemical methods and image reconstruction techniques. We found that vinculin, talin, paxillin, a fibronectin receptor protein, several integrin subunits including β1, fibronectin, and proteins with phosphorylated tyrosine were also components of the apical plaque. These observations indicate that apical stress fibers are attached to the plasma membrane by using principally the same molecular assembly as the focal adhesion associated with the basal stress fiber. We suggest that the complex molecular organization of the focal adhesion is not demanded by cell adhesion, but rather it is needed for anchoring stress fibers to the plasma membrane. Apical plaques did not stain with the anti‐integrin αv subunit or anti‐focal adhesion associated kinase (FAK), although these antibodies stained focal adhesions. These results suggest that the apical stress fiber‐membrane contact has some important functions different from those of the focal adhesion.