bluuebunny/crossref_metadata_2025 huggingface dataset:
Preferential dendritic localization of pericentriolar material in hippocampal pyramidal neurons in culture
Authors: Ferreira Adriana , Palazzo Robert E. , Rebhun Lionel I.
Published: February 2005

Centrosomes are unique cytoplasmic structures which serve as microtubule organizing centers (MTOC). In most animal cells centrosomes consist of one or more pair of centrioles surrounded by electron dense amorphous pericentriolar material (PCM) responsible for nucleation of microtubules. In the present study we analyzed the pattern of induction and localization of proteins of the PCM at different stages of neuronal development in cell cultures prepared from the embryonic hippocampus. For this purpose we used a human polyclonal antibody that recognizes two proteins of the PCM (100 kd and 60 kd, respectively). The results indicate that in mature neurons, pericentriolar immunoreactive material is preferentially localized in dendritic processes, and that throughout the course of neurite development and differentiation it is systematically excluded from the neuron's axon. Western blot analysis showed that during neuronal development in situ, there is an increase in he immunoreactivity for both proteins recognized by this antibody. In contrast, in hippocampal pyramidal neurons that develop in culture, there is an increase in the 60 kd polypeptide, while the 100 kd one is not detected after 7 days in vitro. © 1993 Wiley‐Liss, Inc.
Dynamics of organelles in the mitotic spindles of living cells: Membrane and microtubule interactions
Authors: Waterman‐Storer Clare M. , Sanger Joseph W. , Sanger Jean M.
Published: February 2005

The distribution and dynamics of the membranous organelles in two cell types were investigated during cell division. Live cells (either PtK2 or LLC‐PK1) labeled with the vital dye 3,3′‐dihexyloxacarbocyanine iodide [DiOC6(3)] were observed via serial optical sectioning with the laser‐scanning confocal microscope. Z‐series of labeled, dividing cells were collected every 1–2 minutes throughout mitosis, beginning at prophase and extending to the spreading of the daughter cells. Membrane distribution began to change from the onset of prophase in both cell types. When the mitotic spindle formed in prometaphase, fine tubular membranes, similar to those extending out to the edges of interphase cells aligned along the kinetochore spindle fibers. The lacy polygonal network typical of interphase cells persisted beneath the spindle, and a membrane network was also associated with the dorsal layer of the cell. As PtK2 cells reached metaphse, their spindles were nearly devoid of membrane staining, whereas the spindles of LLC‐PK1 cells contained many tubular and small vesicular membranous structures. X–Z series of the LLC‐PK1 metaphase spindle revealed a small cone of membranes that was separated from the rest of the cytoplasm by kinetochore MTs. In both cell types, as chromosome separation proceeded, the interzone remained nearly devoid of membranes until the onset of anaphase B. At this time the elongating interzonal microtubules were closely associated with the polygonal network of endoplasmic reticulum. Cytokinesis caused a compression, and then an exclusion of organelles from the midbody. Immunofluorescence staining with anti‐tubulin antibodies suggested that spindle membranes were associated with microtubules throughout mitosis. In addition, taxol induced a dense and extensive collection of small vesicles to collect at the spindle poles of both cell types. Nocodazole treatment induced a distinct loss of organization of the membranous components of the spindles. Together these results suggest that microtubules organize the membrane distribution in mitotic cells, and that this organization may vary in different cell types depending on the quantity of microtubules within the spindle. © 1993 Wiley‐Liss, Inc.
Probing actin incorporation into myofibrils using Asp11 and His73 actin mutants
Authors: Xia Dong , Peng Bonnie , Sesok Deborah A. , Peng Isaac
Published: February 2005

We used a cell free system [Bouché et al.: J. Cell Biol. 107:587–596, 1988] to study the incorporation of actin into myofibrils. We used α‐skeletal muscle actin and actins with substitutions of either His73 [Solomon and Rubenstein: J. Biol. Chem. 262:11382, 1987], or Asp11 [Solomon et al.: J. Biol. Chem. 263:19662, 1988]. Actins were translated in reticulocyte lysate and incubated with myofibrils. The incorporated wild type actin could be cross‐linked into dimers using N,N′‐1,4‐phenylenebismaleimide (PBM), indicating that the incorporated actin is actually inserted into the thin filaments of the myofibril. The His73 mutants incorporated to the same extent as wild type actin and was also cross‐linked with PBM. Although some of the Asp11 mutants co‐assembled with carrier actin, only 1–3% of the Asp11 mutant actins incorporated after 2 min and did not increase after 2 hr. Roughly 17% of wild type actin incorporated after 2 min and 31% after 2 hr. ATP increased the release of wild type actin from myofibrils, but did not increase the release of Asp11 mutants. We suggest that (1) the incorporation of wild type and His73 mutant actins was due to a physiological process whereas association of Asp11 mutants with myofibrils was non‐specific. (2) the incorporation of wild type actin involved a rapid initial phase, followed by a slower phase, and (3) since some of the Asp11 mutants can co‐assemble with wild type actin, the ability to self‐assemble was not sufficient for incorporation into myofibrils. Thus, incorporation probably includes interaction between actin and a thin filament associated protein. We also showed that incorporation occurred at actin concentrations which would cause disassembly of F‐actin. Since the myofibrils did not show large scale disassembly but incorporated actin, filament stability and monomer incorporation are likely to be mediated by actin associated proteins of the myofibril. © 1993 Wiley‐Liss, Inc.
Coactosin, a 17 kDA F‐actin binding protein from Dictyostelium discoideum
Authors: de Hostos E. L. , Bradtke B. , Lottspeich F. , Gerisch G.
Published: February 2005

A 17 kDa protein, designated as coactosin, has been purified from an actinmyosin complex reconstituted in vitro from a soluble fraction of Dictyostelium discoideum cells. The protein binds to F‐actin in vitro without significantly altering its viscosity. Immunoblots labeled with monoclonal antibodies indicate that part of the protein is associated with the detergent‐insoluble cytoskeleton. cDNA clones comprising the entire coding region of coactosin have been isolated from an expression library. The cDNA‐derived amino‐acid sequence reveals similarities of coactosin to the drebrins identified in neurons and to actin‐binding proteins from other organisms, including yeast ABP1p, and yeast and vertebrate cofilins. © 1993 Wiley‐Liss, Inc.
Sarcomeric myosin heavy chain expressed in nonmuscle cells forms thick filaments in the presence of substoichiometric amounts of light chains
Authors: Vikstrom Karen L. , Rovner Art S. , Saez Claudia G. , Bravo‐Zehnder Marcela , Straceski Anthony J. , Leinwand Leslie A.
Published: February 2005

Central to the function of myosin is its ability to assemble into thick filaments which interact precisely and specifically with other myofibrillar proteins. We have established a novel experimental system for studying myofibrillogenesis using transient transfections of COS cells, a monkey kidney cell line. We have expressed both full‐length rat α cardiac myosin heavy chain (MHC) and a truncated heavy meromyosin‐like α MHC (sHMM) and shown that immunoreactive MHC proteins of the expected sizes were detected in lysates of transfected cells. Surprisingly, the full‐length MHC formed large spindle‐shaped structures throughout the cytoplasm of transfected cells as determined by immunofluorescence microscopy. The structures were not found in cells expressing the sHMM construct, indicating that their formation required an MHC rod. The spindle‐shaped structures ranged in length from approximately 1 μm to over 20 μm in length and were birefringent suggesting that they are ordered arrays of thick filaments. This was confirmed by electron microscopic analysis of the transfected cells which revealed arrays of filamentous structures approximately 12 nm in diameter at their widest point. In addition, the vast majority of transfected MHC did not associate with the endogenous nonmuscle myosin light chains, demonstrating that myosin thick filaments can form in the absence of stoichiometric amounts of myosin light chains. © 1993 Wiley‐Liss, Inc.
In vitro functional characterization of bacterially expressed human fibroblast tropomyosin isoforms and their chimeric mutants
Authors: Novy Robert E. , Sellers James R. , Liu Li‐Fei , Lin Jim Jung‐Ching
Published: February 2005

At least eight tropomyosin isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsmα) are expressed from four distinct genes in human fibroblasts. In order to elucidate isoform properties, we have subcloned hTM3 and hTM5 full‐length cDNAs, as well as their chimeric cDNAs into the bacterial expression pET8C system. Bacterially expressed tropomyosin isoforms (called PEThTM3, PEThTM5. PEThTM5/3, and PEThTM3/5) were purified and characterized. Under optimal binding conditions, the binding of PEThTM5 isoform to F‐actin was stronger than the PEThTM3 isoform. However, analysis of actin‐binding by the McGhee and von Hippel equation revealed that PEThTM3 exhibits higher cooperativity in binding than PEThTM5 does. Furthermore, the chimera PEThTM5/3 which possessed the N‐terminal fragment of hTM5 fused to the C‐terminal fragment of hTM3 had even stronger actin binding ability. The reverse chimera PEThTM3/5 which possessed the N‐terminal fragment of hTM3 fused to the C‐terminal fragment of hTM5 demonstrated greatly reduced affinity to actin filaments. In addition, both chimeras had different KCl requirements for optimal binding to F‐actin than their parental tropomyosins. A bacterially made C‐terminal fragment of human fibroblast caldesmon (PETCaD39) and native chicken gizzard caldesmon were both able to enhance the actin‐binding of these bacterially expressed tropomyosins. However, PETCaD39′s enhancement of binding to F‐actin was greater for PEThTM5 than PEThTM3. Under 30 mM KCl and 4 mM MgCl2, the low Mr isoform PEThTM5 appeared to be able to amplify the actin‐activated HMM ATPase activity by 4.7 fold, while the high Mr isoform PEThTM3 stimulated the activity only 1.5 fold. The higher enhancement of ATPase activity by PEThTM5 than by PEThTM3 suggested that the low Mr isoform hTM5 may be more involved in modulating nonmuscle cell motility than hTM3. These results further suggested that different isoforms of tropomyosin might have finite differences in their specific functions (e.g., cytoskeletal vs. motile) inside the cell. © 1993 Wiley‐Liss, Inc.
Effects of cytochalasin and colcemid on cortical flow in coelomocytes
Authors: Edds Kenneth T.
Published: February 2005

Sea urchin coelomocytes naturally flatten on a substratum into a discoid morphology and display striking, centripetally directed cortical flow along the radii of the cell when viewed with time lapse, video enhanced microscopy. The rate of cortical flow averaged 4.5 μm/min in the peripheral most 10 μm of cytoplasm but slows considerably in the perinuclear region. Cytochalasin B causes: (1) the flow to stop, (2) the buildup of an actin filament‐rich peripheral ridge of cytoskeletal material, (3) the centrifugal dissolution of a portion of the actin cytoskeleton, and (4) the contraction of other portions of the cytoskeleton into foci. Cytochalasin D (CD), on the other hand, causes the flowing actin meshwork to become severed from the edge of the cell and allows it to be drawn at least part way in towards the nucleus. A smaller peripheral ridge of actin filament buildup is also seen with CD. Colcemid induces another striking change in the cytoskeleton. The centripetal progression of the actin is not stopped by colcemid, but shortly after leaving the periphery of the cell, the linear elements within the flow become reoriented into arcs. The long axis of the arcs is roughly parallel with the cell's edge. The effects of all three drugs are reversible. The results are discussed in light of other systems and potential mechanisms for cortical flow. © 1993 Wiley‐Liss, Inc.
Contractile protein dynamics of myofibrils in paired adult rat cardiomyocytes
Authors: Imanaka‐Yoshida Kyoko , Sanger Jean M. , Sanger Joseph W.
Published: February 2005

The purpose of this study was to determine how quickly contractile proteins are incorporated into the myofibrils of freshly isolated cardiomyocytes and to determine whether there are regions of the cells that are more dynamic than others in their ability to incorporate the proteins. Paired cardiomyocytes joined at intercalated discs and single cells were isolated from adult rats, and microinjected 3 hours later with fluorescently labeied actin, alpha‐actinin, myosin light chains, and vinculin. The cells were fixed and permeabilized at various period, 5 seconds and longer, after microinjection. Actin became incorporated throughout the I‐Bands in as short a time as 5 seconds. The free edges of the cells, which were formerly intercalated discs, exhibited concentrations of actin greater than that incorporated in the I‐Bands. This extra concentration of actin was not detected, however, at intact intercalated discs connecting paired cells. Alpha‐actinin was incorporated immediately into Z‐Bands and intercalated discs. Vinculin, also, was localized at the Z‐Bands and at intercalated discs, but in contrast to alpha‐actinin, there was a higher concentration of vinculin in the region of the intact intercalated discs. Both alpha‐actinin and vinculin were concentrated at the free ends of the cells that were formerly parts of intercalated discs. Myosin light chains were observed to incorporate into the A‐Bands in periods as short as 5 seconds. These results suggest that the myofibrils of adult cardiomyocytes may be capable of rapid isoform transitions along the length of the myofibrils. The rapid accumulation of fluorescent actin, alpha‐actinin, and vinculin in membrane sites that were previously parts of intercalated discs, may reflect the response to locomotory activity that is initiated in these areas as cells spread in culture. A similar response after an injury in the intact heart could allow repair to occur. © 1993 Wiley‐Liss, Inc.
Effects of thymosin β4 and thymosin β10 on actin structures in living cells
Authors: Yu Fu‐Xin , Yin Helen L. , Morrison‐Bogorad Marcelle , Lin Sheng‐Cai
Published: February 2005

The β‐thymosins are a family of small proteins originally isolated from the thymus. Recently, two of the major mammalian isoforms, thymosin β4 (Tβ4) and thymosin β10 (Tβ10), are identified as significant actin monomer sequestering proteins which may be involved in regulating actin filament assembly. To study the cellular function of β‐thymosins, we have used isoform‐specific antibodies to determine their concentration and intracellular distribution, and examined the effects of inducing overexpression of Tβ4 and Tβ10 on actin filament structures. Immunofluorescence labeling of peritoneal macrophages showed that both β‐thymosins are uniformly distributed within the cytoplasm. cDNA‐mediated overexpression of β‐thymosins in CV1 fibroblasts induced extensive loss of phalloidinstained actin stress fibers. Stress fibers in the cell center were more susceptible than those at the periphery. There was a decrease in the number of focal adhesions, as evidenced by a decrease in discrete vinculin staining and an increase in diffuse vinculin fluorescence. The majority of the transfected cells had normal shape in spite of extensive loss of actin filaments. Occasionally, cells overexpressing β‐thymosin were observed to divide. In these cells, β‐thymosin was excluded from the midbody which contains an actin filament‐rich contractile ring. Our results indicate that Tβ4 and Tβ10 are functionally very similar and both are effective regulators of a large subset of actin filaments in living cells. © 1994 Wiley‐Liss, Inc.
Calmyonemin: A 23 kDa analogue of algal centrin occurring in contractile myonemes of Eudiplodinium maggii (ciliate)
Authors: David Christine , Viguès Bernard
Published: February 2005

Myonemes are bundles of thin filaments (3‐6 nm in diameter) which mediate calcium‐induced contraction of the whole or only parts of the cell body in a number of protists. In Eudiplodinium maggii, a rumen ciliate which lacks a uniform ciliation of the cell body, myonemes converge toward the bases of apical ciliary zones that can be retracted under stress conditions, entailing immobilization of the cell. An mAB (A69) has been produced that identifies a calciumbinding protein by immunoblot, immunoprecipitation experiments and specifically labels the myonemes in immunoelectron microscopy. Solubility properties, apparent molecular weight (23 kDa) and isoelectric point (4.9) of the myonemal protein, are similar to the values reported for the calcium‐modulated contractile protein centrin. Western‐blot analysis indicates that the 23 kDa protein crossreacts antigenically with anti‐centrin antibodies. In addition, the 23 kDa protein displays calcium‐induced changes in both electrophoretic and chromatographic behaviour, and contains calcium‐binding domains that conform to the EF‐hand structure, as known for centrin. Based on these observations, we conclude that a calcium‐binding protein with major similarities to centrin occurs in the myonemes of E. maggii. We postulate that this protein plays an essential role in myonememediated retraction of the ciliature. © 1994 Wiley‐Liss, Inc.
ATP‐induced sliding of microtubules on tracks of 22S dynein molecules aligned with the same polarity
Authors: Mimori Y. , Miki‐Nomura T.
Published: February 2005

Chlamydomonas and Tetrahymena axonemal dyneins have previously been found to bind to porcine brain microtubules to produce a microtubule‐dynein complex. At appropriate microtubule:dynein concentration, microtubules in the complex became covered to saturation by dynein arms of the same polarity and at a spacing of 24 nm [Haimo et al., 1979; Haimo and Fenton, 1988; Haimo, 1989; Porter and Johnson, 1983a].

In the present study, two different types of microtubule‐dynein complexes (α‐and β‐complexes) were prepared from Tetrahymena ciliary 22S dynein and porcine brain tubulin. The characteristics of the adenosine triphosphate (ATP)‐induced extrusion of microtubules from these complexes were analyzed, as a simple and direct in vitro assay for the ATP‐induced extrusion of single microtubules. The α‐complex prepared by adding dynein to microtubules showed an interrupted sliding movement, which would stop and start several times following the addition of ATP. In the β‐complex, prepared by adding dynein bound to DEAE‐tubulin to pre‐assembled microtubules, microtubules became covered with dynein molecules whose orientation and binding were uniform with respect to microtubule polarity. The microtubules in the β‐complex extruded at 12 μm/second following the addition of ATP. Dark‐field and electron microscopy indicated that the extruded microtubules had undergone sliding on a dynein‐track that had become detached from the complexes and had been absorbed onto the surface of the glass slide. At higher light intensity under a dark‐field microscope, the dynein‐track was seen to be composed of rows of dynein molecules arranged densely. The orientation of dynein molecules in rows appeared to be uniform considering the images of bound dynein in the β‐complex under electron microscope. The higher sliding velocity of the microtubules on these dynein‐tracks compared to that seen on slides coated at random with dynein [Vale and Toyoshima, 1988, 1989], may be due to more efficient force generation by this dense arrangement of dynein molecules with the same polarity on the tracks. © 1994 Wiley‐Liss, Inc.
Cellular infrared detector appears to be contained in the centrosome
Authors: Albrecht‐Buehler Guenter
Published: February 2005

Previous experiments have suggested that 3T3 cells were able to extend pseudopodia toward latex particles up to 60 μm away from the cell body if the particles were irradiated by an infrared beam in the range of 700–900 nm [Albrecht‐Buehler, 1991: J. Cell Biol. 114:493–502]. The present article reports that this response of cells to infrared light can be inhibited if the cell center is simultaneously irradiated with a beam of the same light. In marked contrast, the cells responded normally to the presence of infrared light scattering particles if the second beam irradiated other parts of the cell body. The results imply that the cellular mechanism of infrared detection is located at the cell center. The infrared sensing mechanism remains intact in enucleated cells and in cells which were incubated in monensin to vesiculate their Golgi apparatus and inhibit their Golgi functions. Accordingly, it is proposed that the centrosome which contains the centrioles is the only remaining candidate in the cell center for a cellular detection device for the direction of infrared signal sources. The results support an earlier suggestion that centrioles may be such detection devices [Albrecht‐Buehler, 1981: Cell Motil. Cytoskeleton 1:237–245]. © 1994 Wiley‐Liss, Inc.
Structural and geometrical constraints on the outer dynein arm in situ
Authors: Barkalow Kurt , Avolio Jock , Holwill Michael E. J. , Hamasaki Toshikazu , Satir Peter
Published: February 2005

This study considers the relationship between two structural forms of the 22S dynein arm of Tetrahymena thermophila: the bouquet and the compact arm. The compact arm differs from the bouquet and from other proposed forms (e.g., the “toadstool”) in that the globular domains are situated transversely across the interdoublet gap with one globular subunit, the head, proximal to the adjacent doublet microtubule. The other models place all three globular domains proximal to the neighboring doublet microtubule. When sliding of an isolated axoneme is induced, at least 57% of total attached arms on exposed doublets are in the compact form within dimensions of 24 × 24 × 12 nm, and only about 2% of the arms are bouquets. Toadstools are incompatible with the images seen. Bouquets are not found in regions of the doublet protected by a neighboring doublet. When axonemes with exposed doublets are treated with 0.5 M KCl for 30 min, the compct arms and the dynein heavy (H)‐chains disappear, while isolated bouquets and dynein H‐chains appear in the medium, suggesting that the compact arms give rise to the bouquets as they are solubilized. The bouquet is the predominant form of isolated 22S dynein molecules, which are found in two apparently enantiomorphic forms, within dimensions 45 × 39 × 13 nm; bouquets attached to doublets have dimensions similar to those of isolated bouquets. Computer modeling indicates that in an intact standard‐diameter axoneme, these dimensions are incompatible with the interdoublet volume available for an arm; the bouquet therefore represents an unfolded compact arm. A plausible sequence of changes can be modeled to illustrate the conversion of an attached compact arm to an attached and then free bouquet. The toadstool is probably an artifact that arises after unfolding. Consistent with the conformational difference, H‐chains of attached compact arms differ from those of isolated bouquets in their susceptibility to limited proteolysis. These results suggest that the compact arm, rather than the unfolded bouquet or the toadstool, is the functional form of the outer arm in the intact axoneme. © 1994 Wiley‐Liss, Inc.
Microtubule centers and the interphase microtubule cytoskeleton in amoebae of the cellular slime molds (mycetozoans) Acytostelium leptosomum and Protostelium mycophaga
Authors: Guhl Bruno , Roos Urs‐Peter
Published: February 2005

We investigated the microtubule (MT) cytoskeleton and microtubule centers (MTC) in undifferentiated amoebae by indirect immunofluorescence with six monoclonal antitubulin antibodies, and by transmission electron microscopy and immunogold ultracytochemistry. Interphase amoebae of both species contain a distinct cytoplasmic complex of MTs, which is more elaborate in Protostelium mycophaga. In Acytostelium leptosomum amoebae a single MTC is attached to each interphase nucleus at its pointed end, as in the other dictyostelid cellular slime molds Dictyostelium discoideum and Polysphondylium violaceum. Ultrastructurally, MTCs of A. leptosomum also resemble those of these two species: They consist of an electron‐opaque core shaped like a stout rod, which is embedded, together with nodules, in a fuzzy matrix. The nodules are the points of origin of the MTs. In most amoebae of P. mycophaga there are two MTCs on opposite sides of and close to the nucleus, but many amoebae also contain a variable number of MTCs that are remote from the nucleus. Nucleus‐associated and “remote” MTCs are structurally identical. They consist of a ring‐shaped core with inner and outer diameters of ca. 130 nm and 340 nm. A plug sits in the ring, and satellites are connected to the core by fine fibrils. The satellites are the points of origin of MTs. New MTCs are apparently formed during mitosis, the parent MTC probably serving as a template for the genesis of a new ring. The results support the notion that phylogenetically related organisms have similarly constructed MTCs and that these are dissimilar in less closely related organisms. © 1994 Wiley‐Liss, Inc.
pH‐Dependent solubility and assembly of microtubules in bovine brain extracts
Authors: Tiwari Suresh C. , Suprenant Kathy A.
Published: February 2005

Alkaline pH favors the assembly of microtubules (MTs) in marine egg extracts [Suprenant and Marsh, 1987: J. Cell Sci. 184:167‐180; Suprenant, 1989: Exp. Cell Res. 184:167‐180; 1991: Cell Motil. Cytoskeleton 19:207‐220] and mammalian brain extracts [Tiwari and Suprenant, 1993: Anal. Biochem. 215:96‐103], even though the assembly of purified microtubule protein (MTP) from both of these sources is favored at slightly acidic pH. The present investigation examines whether alkaline pH has a direct or indirect effect on MT nucleation and growth in soluble brain extracts. Cell‐free extracts were prepared from bovine cerebral cortex, and a nucleated assembly assay was used to demonstrate that MT assembly in brain extracts is favored at slightly acidic pH. The increase in MT mass found at alkaline pH is due to an increase in the solubility of tubulin not an increase in the extent of assembly On average, 47.7 ± 11.3% of the total tubulin is soluble at pH 7.2, while only 30.9 ± 8.9% of the tubulin is soluble at pH 6.8. A model is proposed that indicates how microtubule proteins from both mammalian brain and marine eggs may be associated with pH‐dependent factors. © 1994 Wiley‐Liss, Inc.
Orientation, assembly, and stability of microtubule bundles induced by a fragment of tau protein
Authors: Brandt Roland , Lee Gloria
Published: February 2005

The neuronal microtubule‐associated protein tau has been implicated in the development of axonal morphology including the organization of microtubules into a uniformly oriented array of microtubules commonly referred to as “bundle.” Determination of the functional organization of tau has revealed that regions of tau protein which flank the microtubule‐binding domain affect the bundling of microtubules in vitro with a microtubule‐binding fragment of tau being most effective [Brandt and Lee, 1993: J. Biol. Chem. 268:3414‐3419]. In order to study the relation of microtubule bundles that form in vitro to those observed in the axon, we determined the orientation of individual microtubules in bundles and the effects of bundling on microtubule assembly and stability in cell‐free assembly reactions. Here we report that bundles induced by a microtubule‐binding fragment of tau contain randomly oriented microtubules as determined by using the difference in growth rates at microtubule plus and minus ends. We demonstrate that in vitro bundling increases microtubule growth (about 30%), stabilizes microtubules against dilution‐ and cold‐induced disassembly, and allows microtubule nucleation despite the absence of a tau region which has previously been shown to be required for tau‐dependent microtubule nucleation. We conclude that conditions that stabilize microtubules can lead to bundle formation and allow microtubule assembly by a mechanism different from that employed by microtubule‐associated proteins. The data also support the view that additional mechanisms besides the action of tau and tubulin exist in order to organize microtubules in the axon. © 1994 Wiley‐Liss, Inc.
Role of tropomyosin, α‐actinin, and actin binding protein 280 in stabilizing triton insoluble f‐actin in basal and chemotactic factor activated neutrophils
Authors: Watts Raymond G. , Howard Thomas H.
Published: February 2005

F‐actin is a major component of the neutrophil (PMN) cytoskeleton. In basal PMNs, F‐actin exists in two structurally and functionally distinct pools: Triton insoluble F‐actin (TIF)–cold insensitive, not depolymerizable by dilution, and distributed in pseudopods and submembranous locations; and Triton soluble F‐actin (TSF)–unstable in cold, diffusely distributed, and gelsolin enriched. The element(s) conferring these unique properties to the Triton insoluble F‐actin pool are unknown, but logically include distinct actin regulatory proteins. To study the morphologic and functional determinants of the Triton insoluble F‐actin pool, the distribution and quantity of three candidate regulatory proteins, α‐actinin, tropomyosin (TM), and actin binding protein (ABP‐280), were compared in F‐actin (Triton insoluble and Triton soluble) and G‐actin pools isolated from basal and chemotactic factor activated human PMNs in suspension, using immunoblots and ionic extraction. F‐actin content was measured by NBDphallacidin binding and gel scans. The results show that: (1) α‐actinin, actin binding protein 280, and tropomyosin are localized to TIF and excluded from TSF; (2) TM, α‐actinin, and ABP 280 are required to stabilize fractions of Triton insoluble F‐actin in PMNs; and (3) chemotactic factor activation results in release of a fraction of TM from the Triton insoluble F‐actin pool in temporal association with F‐actin polymerization in the Triton insoluble F‐actin pool. Shifts in ABP 280 or α‐actinin do not occur. The results suggest that TM, α‐actinin, and ABP 280 provide structure to TIF and that TM release from TIF is involved in chemotactic factor induced actin polymerization in PMNs. © 1994 Wiley‐Liss, Inc.
Clustered acetylcholine receptors have two levels of organization in Xenopus muscle cells
Authors: Luther Paul W. , Samuelsson Steven J. , Bloch Robert J. , Pumplin David W.
Published: February 2005

We studied the organization of acetylcholine receptor (AChR) clusters by shearing cultured Xenopus muscle cells with a stream of buffer, and preparing rotary replicas of the exposed cytoplasmic surface of the sarcolemma. AChR clusters contained numerous particles that protruded from the sarcolemma and formed an irregular array composed of discrete aggregates. AChR were located within these particle aggregates, as shown by comparison of the replicas to labeling by fluorescent α‐bungarotoxin, and by immunogold cytochemistry with antibodies specific for the receptor. The aggregates were cross‐linked by a dense network of 7 nm filaments that replicated with the banded pattern characteristic of actin microfilaments. The organization of receptors into the small aggregates was independent of the organization of these aggregates into clusters, as alkaline extraction removed the microfilament network and disrupted the irregular array of particle aggregates, but did not disperse individual receptors from the aggregates. We conclude that two levels of interactions organize AChR clusters in Xenopus muscle cells: short‐range interactions that assemble individual AChR into small aggregates, and long‐range interactions, perhaps mediated by actin microfilaments, that anchor the aggregates into larger clusters. © 1994 Wiley‐Liss, Inc.
A conserved region in the tail domain of vimentin is involved in its assembly into intermediate filaments
Authors: Makarova Irina , Carpenter David , Khan Sohaib , Ip Wallace
Published: February 2005

Although the head and rod domains of intermediate filament (IF) proteins are known to play significant roles in filament assembly, the role of the tail domain in this function is unclear and the available information supports contradictory conclusions. We examined this question by comparing transfection of the same cDNA constructs, encoding vimentins with modified tail domains, into cell lines that do and do not contain endogenous IF proteins. By this approach, we were able to distinguish between the ability of a mutant IF protein to initiate assembly de novo, from that of incorporating into existing filament networks. Vimentins with modifications at or near a highly conserved tripeptide, arg‐asp‐gly (RDG), of the tail domain incorporated into existing IF networks in vimentin‐expressing (vim+) cells, but were assembly‐incompetent in cells that did not express IF proteins (vim−). The failure of the RDG mutant vimentins to assemble into filament arrays in vim‐ cells was reversible by re‐introducing a wild‐type vimentin cDNA, whereupon both wild‐type and mutant vimentins coassembled into one and the same IF network. We conclude that the function of the tail domain of type III IF proteins, and possibly of keratins K8 and K18, in IF assembly is distinct from those of other domains; a region encompassing the RDG tripeptide appears to be important in the assembly process. © 1994 Wiley‐Liss, Inc.
Coordinated expression of five tropomyosin isoforms and β‐actin in astrocytes treated with dibutyryl cAMP and cytochalasin D
Authors: Ferrier Richard , Had Laurence , Rabié Alain , Faivre‐Sarrailh Catherine
Published: February 2005

Cytochalasin D and dBcAMP cause cultured astrocytes to change from flat cells to retrated process‐bearing cells. F‐actin was present throughout cells stimulated with dBcAMP for 16 h, whereas cytochalasin D caused F‐actin to form massive aggregates at the tips of the cell processes. The two drugs differently regulated the expression of both β‐actin and tropomyosin genes in astrocytes cultured in the presence or absence of serum: dBcAMP caused down‐regulation and cytochalasin D caused up‐regulation. Northern blot analyses indicated that: (1) serum deprivation halved the concentration of all tropomyosin transcripts (TM‐1, TM‐2, TM‐4, TMBr‐1, TMBr‐2). Serum induced TM‐4 via transcriptional activation, independent of protein synthesis, (2) dBcAMP induced down‐regulation of β‐actin (–50%) and tropomyosin transcripts (–35 to 52%) even in the presence of serum. The concentration of profilin mRNA decreased in dBcAMP‐reactive astrocytes (‐46%). The decrease in β‐actin mRNA concentration was not blocked by cycloheximide, whereas down‐regulation of tropomyosin transcripts was completely reversed when protein synthesis was inhibited, and (3) cytochalasin D induced an increase in the concentration of tropomyosin transcripts (+ 69 to 185%) which was cumulative with serum stimulation. Cytochalasin D induction of both β‐actin and TM‐4 operated through transcriptional activation, independent of protein synthesis.

The production of all tropomyosin transcripts examined here were strictly coordinated with β‐actin expression in serum‐, dBcAMP‐ and cytochalasin D‐treated astrocytes. This indicates that the differential expression of tropomyosin isoforms occurring during astrocyte maturation is due to more complex regulation than that involved in serum‐ or cAMP‐stimulated astrocytes. © 1994 Wiley‐Liss, Inc.
Dynamic instability of microtubules from cold‐living fishes
Authors: Billger Martin , Wallin Margareta , Williams Robley C. , Detrich H. William
Published: February 2005

The dynamic instability of microtubules free of microtubule‐associated proteins from two genera of cold‐living fishes was measured, by means of video‐enhanced differential interference‐contrast microscopy, at temperatures near those of their habitats. Brain microtubules were isolated from the boreal Atlantic cod (Gadus morhua; habitat temperature ∽ 2–15°C) and from two austral Antarctic rockcods (Notothenia gibberifrons and N. coriiceps neglecta; habitat temperature ∽ −1.8 to + 2°C). Critical concentrations for polymerization of the fish tubulins were in the neighborhood of 1 mg/ml, consistent with high interdimer affinities. Rates of elongation and frequencies of growth‐to‐shortening transitions (“catastrophes”) for fish microtubules were significantly smaller than those for mammalian microtubules. Slow dynamics is therefore an intrinsic property of these fish tubulins, presumably reflecting their adaptation to low temperatures. Two‐dimensional electrophoresis showed striking differences between the isoform compositions of the cod and the rockcod tubulins, which suggests that the cold‐adapted microtubule phenotypes of northern and southern fishes may have arisen independently. © 1994 Wiley‐Liss, Inc.
cAMP‐induced morphological changes in an immortalized schwann cell line: A prelude to differentiation?
Authors: De Deyne Patrick G. , De Vries George H. , Bigbee John W.
Published: February 2005

Schwann cells (SC), the myelinating cells of the peripheral nervous system, show a remarkable capacity to switch from a differentiated state to a proliferative state both during development and peripheral nerve regeneration. In order to better understand the regulatory mechanisms involved with this change we are studying a Schwann cell line transfected with the SV‐40 large T gene (TSC). Serum‐free medium combined with elevating intra‐cellular cAMP levels produced a slower proliferating TSC whose morphology changed from pleiomorphic to process bearing, reminiscent of primary SC in culture. This change was abrogated by colcemid but was unaltered by cytochalasin D, indicating a major role for microtubules. Ultrastructural studies demonstrated numerous microtubules in the cellular extensions which correlated with strong immunocytochemical staining for tubulin in the processes. Analysis of cytoskeletal fractions from the treated cells revealed a greater proportion of tubulin in the polymerized state compared with untreated cells which closely resembled the distribution in primary SC. The cytoskeletal changes observed in the TSC as a result of elevating the intra‐cellular cAMP levels may reflect the earliest cellular changes in the induction of myelination. © 1994 Wiley‐Liss, Inc.
Kinesin‐related polypeptide is associated with vesicles from Corylus avellana pollen
Authors: Liu Guo‐Qin , Cai Giampiero , Casino Cecilia Del , Tiezzi Antonio , Cresti Mauro
Published: February 2005

A 100‐kDa polypeptide with microtubule‐interacting properties was identified in a Golgi vesicle‐enriched fraction from Corylus avellana pollen. The k71s23 antibody (directed to the kinesin heavy chain from bovine brain) [Tiezzi et al., 1992: Cell Motil. Cytoskeleton 21:132–137] localized the polypeptide on the external surface of membrane‐bounded organelles. Some 100‐kDa‐containing vesicles co‐pelleted with microtubules (polymerized from purified bovine brain tubulin) either in presence or absence of 5 mM AMPPNP, but they could be released by 10 mM ATP or 0.5 M KCl. The pollen microtubule‐interacting protein, salt‐extracted from membranes and partially purified by gel filtration, exhibited an ATPase activity (16.2 nmolPi/mg/min) which could be stimulated about 2‐fold (32.5 nmolPi/mg/min) by addition of bovine brain microtubules. We suppose that the 100‐kDa polypeptide is part of a molecular complex showing properties of the kinesin class. © 1994 Wiley‐Liss, Inc.
Localization of NuMA protein isoforms in the nuclear matrix of mammalian cells
Authors: Zeng Changqing , He Dacheng , Brinkley B. R.
Published: February 2005

Using a monoclonal antibody 2D3 generated against a kinetochore‐enriched human chromosome preparation, we identified a high molecular mass protein with nuclear staining in interphase and polar staining of the pericentriolar region in the mitotic spindle. Initially termed centrophilin, this protein associates with the minus‐ends of spindle microtubules (MT) and appears to be important in spindle organization [Tousson et al., 1991: J. Cell Biol. 112:427–440]. Comparison of a partial cDNA sequence obtained for centrophilin with the full length cDNA sequence of nuclear mitotic apparatus protein (NuMA) [Compton et al., 1992: J. Cell Biol. 116:1395–1408; Yang et al., 1992: J. Cell Biol. 116:1303–1317] has indicated that NuMA and centrophilin are the same protein. Using a polyclonal NuMA antibody, we have provided further evidence that NuMA exists as iso‐forms as shown by peptide mapping and immunoblots. Sequential fractionation experiments along with immunofluorescence, immunoblotting, and EM immunogold labeling have demonstrated that NuMA isoforms are novel components of nuclear core filaments. Thus, NuMA, a long coiled‐coil protein, appears to have dual functions in interphase and mitosis during the cell cycle. In interphase, NuMA likely plays a structural role in the nucleoskeleton that may be important in nuclear organization and functions, whereas in mitosis, NuMA appears to be associated with spindle MT organization and chromosome positioning. © 1994 Wiley‐Liss, Inc.
Substructure of cytoplasmic dense bodies and changes in distribution of desmin and α‐actinin in developing smooth muscle cells
Authors: Chou Rong‐Ghi R. , Stromer Marvin H. , Robson Richard M. , Huiatt Ted W.
Published: February 2005

The substructure of assembling cytoplasmic dense bodies (CDBs) and changes in the distribution of desmin and α‐actinin during development of smooth muscle were studied in gizzard samples from 10‐ and 16‐day embryos and from 1‐ and 7‐day post‐hatch chickens. CDBs in these cells lack the density of CDBs in mature or adult smooth muscle cells and, thus, allow observations of the changes inside CDBs. The random filament orientation seen in younger embryonic cells is first modified to include relatively small patches of IFs that are somewhat straighter and are approaching a side‐by‐side arrangement. As development proceeds, the IFs in these arrays become straighter, are parallel over longer lengths of the IFs and later acquire the density characteristic of mature CDBs. Anti‐desmin labeling in embryonic 10‐ and 16‐day cells showed that desmin intermediate filaments (IFs) were located in the myofilament compartment but were concentrated in or near assembling CDBs. Anti‐desmin labeling shifted to the perimeter of CDBs after hatching. Cross sections, longitudinal sections, and stereo pairs all show that IF profiles are present inside unlabeled assembling CDBs. Anti‐α‐actinin labeling was directly on CDBs and was often associated with the cross‐connecting filaments (CCFs) (average diameter of 2–3nm) inside CDBs. We propose, based on these data, that desmin IFs, α‐actinin‐containing CCFs, and actin filaments are the principal components of the substructure of assembling CDBs. We also present a proposed model for CDB assembly. © 1994 Wiley‐Liss, Inc.
Gain setting in Chlamydomonas reinhardtii: Mechanism of phototaxis and the role of the photophobic response
Authors: Zacks David N. , Spudich John L.
Published: February 2005

The unicellular green alga Chlamydomonas reinhardtii maintains sensitivity of its phototaxis response (alignment of swimming direction along the axis of a light beam) over several orders of magnitude of light intensities. It is widely accepted that the rotation of the swimming cell provides temporal comparisons of light intensities via periodic contrast generated by its asymmetrically positioned refractile eyespot organelle. The cells also exhibit a second behavioral response to light called the photophobic (or stop) response, which is a brief cessation of swimming caused by a temporal change in light intensity. The cells are desensitized to photophobic stimuli by light exposure. Through comparative measurements of both responses, we explain the behavioral basis of the large dynamic range of phototaxis in terms of precise desensitization of the photophobic response. The basis of the explanation is that the flagellar beat changes which cause phototactic orientation are the residual of the photophobic response after desensitization (i.e., “mini‐photophobic” reactions which cause brief reorienting motions without a full stop). This interpretation predicts quantitatively the dependence of the extent of desensitization on light intensity and the dependence of onset and maintenance of phototaxis on extent of desensitization. These predictions are tested and confirmed in this report. © 1994 Wiley‐Liss, Inc.
Different reactivity with monoclonal anti‐tubulin antibodies between native and fixed mitotic microtubules in sea urchin eggs
Authors: Oka Mikako T. , Arai Takao , Hamaguchi Yukihisa
Published: February 2005

The effect on fixation on the reactivities of mitotic microtubules with monoclonal anti‐tubulin antibodies was investigated by the indirect immunofluorescence procedure. All of the seven antibodies used intensely stained mitotic microtubules in sea urchin eggs lysed and fixed with methanol at −20°C, whereas only two of them stained the stabilized microtubules in the lysed eggs before the fixation. The other five did not stain the mitotic microtubules even after microtubule components other than tubulin were removed by treating the lysed eggs with 0.4 M KCl solution containing taxol. These results exclude the possibility that the fixation affects proteins, which interact with microtubules including microtubule‐associated proteins (MAPs) and interfere with the binding of monoclonal antibodies with tubulin, and strongly suggest that the fixation directly affects the three‐dimensional conformation of tubulin Furthermore, microinjection of these antibodies indicated the results as follows [combining the results reported previously; Oka et al., 1990: Cell Struct. Funct. 15: 373–378]: The antibodies which stained mitotic microtubules stabilized in the lysed eggs induced disassembly of native mitotic microtubules in the living eggs, but those which did not stain the stabilized microtubules did not disassemble the native microtubules. From these results, it is suggested that the monoclonal antibodies which stain microtubules in the eggs lysed but not fixed are useful for microinjection experiments. © 1994 Wiley‐Liss, Inc.
Switching of the dominant calcium sequestering protein during skeletal muscle differentiation
Authors: Koyabu Sukenari , Imanaka‐Yoshida Kyoko , Ioshii Sérgio O. , Nakano Takeshi , Yoshida Toshimichi
Published: February 2005

A major Ca2+‐storing protein in endoplasmic reticulum (ER) of non‐muscle cells is calreticulin (CR), which is considered to be functionally homologous to calsequestrin. Calsequestrin is a Ca2+‐binding protein in sarcoplasmic reticulum (SR) of striated muscle, which stores Ca2+ during muscle relaxation. In order to investigate the expression and distribution of calsequestrin and calreticulin during skeletal muscle differentiation, cultured chick embryonic skeletal muscles were observed by immunofluorescence using anti‐calsequestrin, anti‐calreticulin, antidesmin, and anti‐sarcomeric myosin antibodies and rhodamine‐phalloidin. Within 6 hours in culture, myoblasts started to express desmin. Desmin‐positive cells demonstrated the reticular staining of calreticulin, as did desmin‐negative cells. Around fusion, calsequestrin and sarcomeric myosin started to appear in desmin‐positive cells. The expression of calsequestrin slightly preceded that of sarcomeric myosin. As the myotubes matured, the fluorescent dots of calsequestrin increased and spread to the cell periphery along the myofibrils, while the reticular pattern of calreticulin gradually disappeared. Double labeling showed that calsequestrin colocalized with calreticulin. In mature myotubes, anti‐calsequestrin staining demonstrated many dots along myofibrils, whereas calreticulin was barely seen except at the perinuclear region. These results suggest that the expression of calsequestrin and calreticulin are switched during skeletal muscle differentiation. © 1994 Wiley‐Liss, Inc.