bluuebunny/crossref_metadata_2025 huggingface dataset:
Two-dimensional structure of the light-harvesting chlorophyll a/b complex by cryoelectron microscopy.
Authors: Lyon M K , Unwin P N
Published: May 2004

The light-harvesting chlorophyll a/b complex (LHC-II) found in green plants has at least three functions: it absorbs light energy for transfer to the reaction centers, it is involved in keeping the photosynthetic membranes stacked, and it regulates energy distribution between the two photosystems. We have developed a procedure to produce large vesicles consisting almost exclusively of two-dimensional crystalline domains of LHC-II in which LHC-II is biochemically and structurally intact, as shown by SDS-PAGE, response to cations, and 77K fluorescence excitation spectra. The vesicles were examined by cryoelectron microscopy and analyzed, in projection, to a resolution of 17 A. Their surface lattice consists of trimers arranged in interlocking circles; the two-sided plane group is p321 (unit cell dimension, a = 124 A) with two, oppositely facing trimers/unit cell. Individual trimers consist of matter arranged in a ring, around a central cavity, an appearance similar to that obtained in some conditions using negative stain (Li, J., 1985. Proc. Natl. Acad. Sci. USA. 82:386-390). The monomer (approximately 45 x 20 A) is seen as two domains of slightly different size at this resolution. The thickness of single layers is approximately 48 A, measured from edge-on views of the frozen vesicles. Based on these dimensions, the molecular mass of the monomer is approximately 30 kD. Therefore, each monomer appears to be composed of a single polypeptide and its associated pigments.
Accelerated sliding of pollen tube organelles along Characeae actin bundles regulated by Ca2+.
Authors: Kohno T , Shimmen T
Published: May 2004

Pollen tubes show active cytoplasmic streaming. We isolated organelles from pollen tubes and tested their ability to slide along actin bundles in characean cell models. Here, we show that sliding of organelles was ATP-dependent and that motility was lost after N-ethylmaleimide or heat treatment of organelles. On the other hand, cytoplasmic streaming in pollen tube was inhibited by either N-ethylmaleimide or heat treatment. These results strongly indicate that cytoplasmic streaming in pollen tubes is supported by the "actomyosin"-ATP system. The velocity of organelle movement along characean actin bundles was much higher than that of the native streaming in pollen tubes. We suggested that pollen tube "myosin" has a capacity to move at a velocity of the same order of magnitude as that of characean myosin. Moreover, the motility was high at Ca2+ concentrations lower than 0.18 microM (pCa 6.8) but was inhibited at concentration higher than 4.5 microM (pCa 5.4). In conclusion, cytoplasmic streaming in pollen tubes is suggested to be regulated by Ca2+ through "myosin" inactivation.
The mechanism of cytoplasmic streaming in characean algal cells: sliding of endoplasmic reticulum along actin filaments.
Authors: Kachar B , Reese T S
Published: May 2004

Electron microscopy of directly frozen giant cells of characean algae shows a continuous, tridimensional network of anastomosing tubes and cisternae of rough endoplasmic reticulum which pervade the streaming region of their cytoplasm. Portions of this endoplasmic reticulum contact the parallel bundles of actin filaments at the interface with the stationary cortical cytoplasm. Mitochondria, glycosomes, and other small cytoplasmic organelles enmeshed in the endoplasmic reticulum network display Brownian motion while streaming. The binding and sliding of endoplasmic reticulum membranes along actin cables can also be directly visualized after the cytoplasm of these cells is dissociated in a buffer containing ATP. The shear forces produced at the interface with the dissociated actin cables move large aggregates of endoplasmic reticulum and other organelles. The combination of fast-freezing electron microscopy and video microscopy of living cells and dissociated cytoplasm demonstrates that the cytoplasmic streaming depends on endoplasmic reticulum membranes sliding along the stationary actin cables. Thus, the continuous network of endoplasmic reticulum provides a means of exerting motive forces on cytoplasm deep inside the cell distant from the cortical actin cables where the motive force is generated.
Regulation of microtubule protein levels during cellular morphogenesis in nerve growth factor-treated PC12 cells.
Authors: Drubin D , Kobayashi S , Kellogg D , Kirschner M
Published: May 2004

Nerve growth factor induces neurite process formation in pheochromacytoma (PC12) cells and causes the parallel increase in levels of the microtubule-associated proteins, tau and MAP1, as well as increases in tubulin levels. Mechanisms to insure balanced accumulation of microtubule proteins and make their levels highly responsive to nerve growth factor were investigated. The effects on tau, MAP1, and tubulin are due to changes in protein synthesis rates, which for tau and tubulin we could show are due in part to changes in the mRNA levels. Whereas tubulin shows feedback regulation to modulate synthesis up or down, tau protein synthesis is not affected in a straightforward way by microtubule polymerization and depolymerization. The degradation of tau, MAP1, and both tubulin polypeptides, however, are stimulated by microtubule depolymerization caused by colchicine, or nerve growth factor removal. Combined feedback on synthesis and stability make tubulin levels highly responsive to assembly states. In addition, the linkage of tau and MAP1 turnover with the state of microtubule polymerization amplifies any change in their rate of synthesis, since tau and MAP1 promote microtubule polymerization. This linkage lends itself to rapid changes in the state of the system in response to nerve growth factor.
Organization of microtubules in centrosome-free cytoplasm.
Authors: McNiven M A , Porter K R
Published: May 2004

Many different cell types possess microtubule patterns which appear to be polarized and oriented, in part, by cytoplasmic factors not directly associated with a centrosome. Recently, we demonstrated that cytoplasmic extensions ("arms") of teleost melanophores will reorganize their microtubule population outward from their centers after surgical isolation (McNiven, M. A., M. Wang, and K. R. Porter. 1984. Cell. 37:753-765). In the study reported here, we examine microtubule dynamics within the centrosome-free fragments and find that, after severing, microtubule reorganization is initiated at the proximal (cut) end of an arm and migrates distally with the aggregated pigment mass until it becomes permanently positioned at the middle of the arm. Computer-aided image analysis demonstrates that this middle position is located at the arm centroid, implicating the action of a cytoplasmic gel in this process. Morphological studies of arms devoid of pigment reveal that microtubules do not emanate from a single site or structure within the centroid area, but from a more generalized region. Taken together, these findings suggest that factors distributed throughout cytoplasm participate in microtubule assembly and organization.
Differential regulation of Paramecium ciliary motility by cAMP and cGMP.
Authors: Bonini N M , Nelson D L
Published: May 2004

cAMP and cGMP had distinct effects on the regulation of ciliary motility in Paramecium. Using detergent-permeabilized cells reactivated to swim with MgATP, we observed effects of cyclic nucleotides and interactions with Ca2+ on the swimming speed and direction of reactivated cells. Both cAMP and cGMP increased forward swimming speed two- to threefold with similar half-maximal concentrations near 0.5 microM. The two cyclic nucleotides, however, had different effects in antagonism with the Ca2+ response of backward swimming and on the handedness of the helical swimming paths of reactivated cells. These results suggest that cAMP and cGMP differentially regulate the direction of the ciliary power stroke.
Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm.
Authors: Tash J S , Krinks M , Patel J , Means R L , Klee C B , Means A R
Published: May 2004

Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca2+ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KCl from isolated flagella from dog, pig, and sea urchin sperm. All salt-extractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.
Expression of simple epithelial type cytokeratins in stratified epithelia as detected by immunolocalization and hybridization in situ.
Authors: Bosch F X , Leube R E , Achtstätter T , Moll R , Franke W W
Published: May 2004

Multi-layered ("stratified") epithelia differ from one-layered ("simple") polar epithelia by various architectural and functional properties as well as by their cytoskeletal complements, notably a set of cytokeratins characteristic of stratified tissue. The simple epithelial cytokeratins 8 and 18 have so far not been detected in any stratified epithelium. Using specific monoclonal antibodies we have noted, in several but not all samples of stratified epithelia, including esophagus, tongue, exocervix, and vagina, positive immunocytochemical reactions for cytokeratins 8, 18, and 19 which in some regions were selective for the basal cell layer(s) but extended into suprabasal layers in others. In situ hybridization with different probes (riboprobes, synthetic oligonucleotides) for mRNAs of cytokeratin 8 on esophageal epithelium has shown, in extended regions, relatively strong reactivity for cytokeratin 8 mRNA in the basal cell layer. In contrast, probes to cytokeratin 18 have shown much weaker hybridization which, however, was rather evenly spread over basal and suprabasal strata. These results, which emphasize the importance of in situ hybridization in studies of gene expression in complex tissues, show that the genes encoding simple epithelial cytokeratins can be expressed in stratified epithelia. This suggests that continual expression of genes coding for simple epithelial cytokeratins is compatible with the formation of squamous stratified tissues and can occur, at least in basal cell layers, simultaneously with the synthesis of certain stratification-related cytokeratins. We also emphasize differences of expression and immunoreactivity of these cytokeratins between different samples and in different regions of the same stratified epithelium and discuss the results in relation to changes of cytokeratin expression during fetal development of stratified epithelia, in response to environmental factors and during the formation of squamous cell carcinomas.
Localization of phospholipase A2 in normal and ras-transformed cells.
Authors: Bar-Sagi D , Suhan J P , McCormick F , Feramisco J R
Published: May 2004

The cellular localization of phospholipase A2 (PLA2) was examined in normal and ras-transformed rat fibroblasts using immunohistochemical techniques. Polyclonal antibodies were generated against porcine pancreatic PLA2 and were affinity purified for use in this study. The antibodies detected a 16-kD band on immunoblots of total cellular proteins from fibroblasts. In cell-free assays of phospholipase A2 activity, the purified antibodies inhibited the bulk of the enzyme activity whereas control IgG preparations had no effect. Immunofluorescence microscopy indicated that PLA2 was diffusely distributed throughout the cell. Increased concentration of PLA2 was detected under membrane ruffles in normal and ras-transformed cells. Specific immunofluorescence staining was also detected on the outer surface of the normal cells. Immunoelectron microscopy demonstrated the increased accumulation of PLA2 in membrane ruffles and also revealed the presence of the enzyme in microvilli and its association with intracellular vesicles. Ultrastructural localization of PLA2 and the ras oncogene protein, using a double immunogold labeling technique, indicated a spatial proximity between PLA2 and ras proteins in the ruffles of ras-transformed cells. The possible role of PLA2 in the structural rearrangements that underlie membrane ruffling is discussed.
The control of chick myoblast fusion by ion channels operated by prostaglandins and acetylcholine.
Authors: Entwistle A , Zalin R J , Bevan S , Warner A E
Published: May 2004

Chick myoblast fusion in culture was investigated using prostanoid synthesis inhibitors to delay spontaneous fusion. During this delay myoblast fusion could be induced by prostaglandin E1 (PGE1), by raising extracellular potassium and by addition of carbachol. Carbachol-induced fusion, but not PGE-induced fusion, was prevented by the acetylcholine receptor blocker alpha-bungarotoxin. Fusion induced by any of these agents was prevented by the Ca channel blockers lanthanum and D600. The threshold for potassium-induced fusion was 7-8 mM; maximal fusion occurred at 16-20 mM. Low extracellular potassium inhibited spontaneous fusion. Intracellular potassium in fusion competent myoblasts was 101 m-moles/l cell. Calcium flux measurements demonstrated that high potassium increased calcium permeability in fusion-competent myoblasts. A 30-s exposure to high potassium or PGE1 was sufficient to initiate myoblast fusion. Anion-exchange inhibitors (SITS and DIDS) delayed spontaneous myoblast fusion and blocked fusion induced by PGE1 but not carbachol. Blocking the acetylcholine receptor shifted the dose-response relation for PGE-induced fusion to higher concentrations. PGE1-induced fusion required chloride ions; carbachol-induced fusion required sodium ions. Provided calcium channels were available, potassium always induced fusion. We conclude that myoblasts possess at least three, independent pathways, each of which can initiate myoblast fusion and that the PGE-activated pathway and the acetylcholine receptor-activated pathway act synergistically. We suggest that fusion competent myoblasts have a high resting membrane potential and that fusion is controlled by depolarization initiated directly (potassium), by an increase in permeability to chloride ions (PGE), or by activation of the acetylcholine receptor (carbachol); depolarization triggers a rise in calcium permeability. The consequent increase in intracellular calcium initiates myoblast fusion.
Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and myelin-associated glycoprotein) in regenerating adult mouse sciatic nerve.
Authors: Martini R , Schachner M
Published: May 2004

The localization of the neural cell adhesion molecules L1, N-CAM, and the myelin-associated glycoprotein was studied by pre- and postembedding staining procedures at the light and electron microscopic levels in transected and crushed adult mouse sciatic nerve. During the first 2-6 d after transection, myelinated and nonmyelinated axons degenerated in the distal part of the proximal stump close to the transection site and over the entire length of the distal part of the transected nerve. During this time, regrowing axons were seen only in the proximal, but not in the distal nerve stump. In most cases L1 and N-CAM remained detectable at cell contacts between nonmyelinating Schwann cells and degenerating axons as long as these were still morphologically intact. Similarly, myelin-associated glycoprotein remained detectable in the periaxonal area of the degenerating myelinated axons. During and after degeneration of axons, nonmyelinating Schwann cells formed slender processes which were L1 and N-CAM positive. They resembled small-diameter axons but could be unequivocally identified as Schwann cells by chronical denervation. Unlike the nonmyelinating Schwann cells, only few myelinating ones expressed L1 and N-CAM. At the cut ends of the nerve stumps a cap developed (more at the proximal than at the distal stump) that contained S-100-negative and fibronectin-positive fibroblast-like cells. Most of these cells were N-CAM positive but always L1 negative. Growth cones and regrowing axons expressed N-CAM and L1 at contact sites with these cells. Regrowing axons of small diameter were L1 and N-CAM positive where they made contact with each other or with Schwann cells, while large-diameter axons were only poorly antigen positive or completely negative. 14 d after transection, when regrowing axons were seen in the distal part of the transected nerve, regrowing axons made L1- and N-CAM-positive contacts with Schwann cells. When contacting basement membrane, axons were rarely found to express L1 and N-CAM. Most, if not all, Schwann cells associated with degenerating myelin expressed L1 and N-CAM. In crushed nerves, the immunostaining pattern was essentially the same as in the cut nerve. During formation of myelin, the sequence of adhesion molecule expression was the same as during development: L1 disappeared and N-CAM was reduced on myelinating Schwann cells and axons after the Schwann cell process had turned approximately 1.5 loops around the axon. Myelin-associated glycoprotein then appeared both periaxonally and on the turning loops of Schwann cells in the uncompacted myelin.(ABSTRACT TRUNCATED AT 400 WORDS)
Tyrosine phosphorylated proteins in different tissues during chick embryo development.
Authors: Maher P A , Pasquale E B
Published: May 2004

A high affinity polyclonal antibody specific for phosphotyrosyl residues has been used in immunoblotting experiments to survey developing embryonic chicken tissues for the presence and characteristics of tyrosine phosphorylated proteins. Proteins phosphorylated on tyrosine were found to be present in all the embryonic tissues examined, including heart, thigh, gizzard, intestine, lung, liver, kidney, brain, and lens, from 7 to 21 d of development in ovo, but were greatly reduced or absent in the same tissues taken from adult chickens. A limited number of major tyrosine phosphorylated proteins were seen in all the tissues examined and they ranged in molecular mass from 35 to 220 kD. Most of the tissues contained proteins phosphorylated on tyrosine with apparent molecular masses of 120, 70, 60, and 35 kD, suggesting that the substrates of tyrosine protein kinases in different tissues may be related proteins. One-dimensional peptide mapping of the 120- and 70-kD protein bands indicated a close structural relationship among the phosphotyrosine-containing proteins of 120 kD, and similarly among those of 70 kD, from the different tissues.
Phosphotyrosine-modified proteins are concentrated at the membranes of epithelial and endothelial cells during tissue development in chick embryos.
Authors: Takata K , Singer S J
Published: May 2004

We have used high affinity polyclonal antibodies specific for phosphotyrosine (PTyr) residues to examine the localization in various chick embryonic tissues in situ of PTyr-modified proteins by immunocytochemical methods. During the period from 9 to 21 d of development, most tissues exhibit elevated levels of PTyr-modified proteins as determined by immunoblotting experiments of tissue extracts with the anti-PTyr antibodies (Maher, P. A., and E. B. Pasquale. 1988. J. Cell Biol. 106:1747-1755). By immunofluorescence labeling of semithin frozen sections, the highest concentrations of PTyr immunolabeling in all of the embryonic tissues examined were localized to the membranes of the epithelial and endothelial cells with other cells showing no detectable labeling. These results were confirmed by immunoelectron microscopic labeling, which showed particularly high concentrations of PTyr-modified proteins close to the membranes at the apical junctions. The corresponding adult tissues showed no labeling. It is proposed that these results reflect the molecular basis for the functional plasticity of epithelial and endothelial cell junctions during embryonic development.
Interaction between pathogenic amebas and fibronectin: substrate degradation and changes in cytoskeleton organization.
Authors: Talamás-Rohana P , Meza I
Published: May 2004

Invasion of human tissues by the parasitic protozoan Entamoeba histolytica is a multistep process involving, as a first step, the recognition of surface molecules on target tissues by the amebas or trophozoites. This initial contact is followed by the release of proteolytic and other activities that lyse target cells and degrade the extracellular matrix. In other parasitic diseases, as well as in certain cancers, the interaction of invasive organisms or cells with fibronectin (FN) through specific receptors has been shown to be the initial step in target cell recognition. Interaction with FN triggers the release of proteolytic activities necessary for the effector cell migration and invasion. Here, we describe the specific interaction of Entamoeba histolytica trophozoites with FN, and identify a 37-kD membrane peptide as the putative receptor for FN. The interaction between the parasite and FN leads to a response reaction that includes the secretion of proteases that degrade the bound FN and the rearrangement of amebic actin into "adhesion plates" at sites of contact with FN-coated surfaces. The kinetics of the interaction was determined by measuring the binding of soluble 125I-FN to the trophozoites and visualization of the bound protein using specific antibodies. Degradation of FN was measured by gel electrophoresis and the release of radioactivity into the incubation medium. Focal degradation of FN was visualized as black spots under the trophozoites at contact sites with fluorescent FN. We conclude that the interaction of E. histolytica with FN occurs through a specific surface receptor. The interaction promotes amebic cytoskeleton changes and release of proteases from the parasite. The binding and degradation of extracellular matrix components may facilitate the migration and penetration of amebas into tissues, causing the lesions seen in human hosts.
A novel marker glycoprotein for the microvillus membrane of surface colonocytes of rat large intestine and its presence in small-intestinal crypt cells.
Authors: Gorr S U , Stieger B , Fransen J A , Kedinger M , Marxer A , Hauri H P
Published: May 2004

Murine mAbs were produced against purified microvillus membranes of rat colonocytes in order to establish a marker protein for this membrane. The majority of antibodies binding to the colonic microvillus membrane recognized a single protein with a mean apparent Mr of 120 kD in both proximal and distal colon samples. The antigen is membrane bound as probed by phase-partitioning studies using Triton X-114 and by the sodium carbonate extraction procedure and is extensively glycosylated as assessed by endoglycosidase F digestion. Localization studies in adult rats by light and electron microscopy revealed the microvillus membrane of surface colonocytes as the principal site of the immunoreaction. The antigen was not detectable in kidney or liver by immunoprecipitation but was present in the small intestine, where it was predominantly confined to the apical membrane of crypt cells and much less to the microvillus membrane of differentiated enterocytes. During fetal development, the antigen appears first in the colon at day 15 and 1-2 d later in the small intestine. In both segments, it initially covers the whole luminal surface but an adult-like localization pattern develops soon after birth. The antibodies were also used to develop a radiometric assay for the quantification of the antigen in subcellular fractions of colonocytes in order to assess the validity of a previously developed method for the purification of colonic brush-border membranes (Stieger, B., A. Marxer, and H.P. Hauri. 1986. J. Membr. Biol. 91:19-31.). The results suggest that we have identified a valuable marker glycoprotein for the colonic microvillus membrane, which in adult rats may also serve as a marker for early differentiation of enterocyte progenitor cells in small-intestinal crypt cells.
Direct demonstration of actin filament annealing in vitro.
Authors: Murphy D B , Gray R O , Grasser W A , Pollard T D
Published: May 2004

Direct electron microscopic examination confirms that short actin filaments rapidly anneal end-to-end in vitro, leading over time to an increase in filament length at steady state. During annealing of mixtures of native unlabeled filaments and glutaraldehyde-fixed filaments labeled with myosin subfragment-1, the structural polarity within heteropolymers is conserved absolutely. Annealing does not appear to require either ATP hydrolysis or the presence of exogenous actin monomers, suggesting that joining occurs through the direct association of filament ends. During recovery from sonication the initial rate of annealing is consistent with a second-order reaction involving the collision of two filament ends with an apparent annealing rate constant of 10(7) M-1s-1. This rapid phase lasts less than 10 s and is followed by a slow phase lasting minutes to hours. Annealing is calculated to contribute minimally to filament elongation during the initial stages of self-assembly. However, the rapid rate of annealing of sonicated fixed filaments observed in vitro suggests that it may be an efficient mechanism for repairing breaks in filaments and that annealing together with polymer-severing mechanisms may contribute significantly to the dynamics and function of actin filaments in vivo.
Characterization of 83-kilodalton nonmuscle caldesmon from cultured rat cells: stimulation of actin binding of nonmuscle tropomyosin and periodic localization along microfilaments like tropomyosin.
Authors: Yamashiro-Matsumura S , Matsumura F
Published: May 2004

Nonmuscle caldesmon purified from cultured rat cells shows a molecular weight of 83,000 on SDS gels, Stokes radius of 60.5 A, and sedimentation coefficient (S20,w) of 3.5 in the presence of reducing agents. These values give a native molecular weight of 87,000 and a frictional ratio of 2.04, suggesting that the molecule is a monomeric, asymmetric protein. In the absence of reducing agents, the protein is self-associated, through disulfide bonds, into oligomers with a molecular weight of 230,000 on SDS gels. These S-S oligomers appear to be responsible for the actin-bundling activity of nonmuscle caldesmon in the absence of reducing agents. Actin binding is saturated at a molar ratio of one 83-kD protein to six actins with an apparent binding constant of 5 X 10(6) M-1. Because of 83-kD nonmuscle caldesmon and tropomyosin are colocalized in stress fibers of cultured cells, we have examined effects of 83-kD protein on the actin binding of cultured cell tropomyosin. Of five isoforms of cultured rat cell tropomyosin, tropomyosin isoforms with high molecular weight values (40,000 and 36,500) show higher affinity to actin than do tropomyosin isoforms with low molecular weight values (32,400 and 32,000) (Matsumura, F., and S. Yamashiro-Matsumura. 1986. J. Biol. Chem. 260:13851-13859). At physiological concentration of KCl (100 mM), 83-kD nonmuscle caldesmon stimulates binding of low molecular weight tropomyosins to actin and increases the apparent binding constant (Ka from 4.4 X 10(5) to 1.5 X 10(6) M-1. In contrast, 83-kD protein has slight stimulation of actin binding of high molecular weight tropomyosins because high molecular weight tropomyosins bind to actin strongly in this condition. As the binding of 83-kD protein to actin is regulated by calcium/calmodulin, 83-kD protein regulates the binding of low molecular weight tropomyosins to actin in a calcium/calmodulin-dependent way. Using monoclonal antibodies to visualize nonmuscle caldesmon along microfilaments or actin filaments reconstituted with purified 83-kD protein, we demonstrate that 83-kD nonmuscle caldesmon is localized periodically along microfilaments or actin filaments with similar periodicity (36 +/- 4 nm) as tropomyosin. These results suggest that 83-kD protein plays an important role in the organization of microfilaments, as well as the control of the motility, through the regulation of the binding of tropomyosin to actin.
Diverse effects of beta-tubulin mutations on microtubule formation and function.
Authors: Huffaker T C , Thomas J H , Botstein D
Published: May 2004

We have used in vitro mutagenesis and gene replacement to construct five new cold-sensitive mutations in TUB2, the sole gene encoding beta-tubulin in the yeast Saccharomyces cerevisiae. These and one previously isolated tub2 mutant display diverse phenotypes that have allowed us to define the functions of yeast microtubules in vivo. At the restrictive temperature, all of the tub2 mutations inhibit chromosome segregation and block the mitotic cell cycle. However, different microtubule arrays are present in these arrested cells depending on the tub2 allele. One mutant (tub2-401) contains no detectable microtubules, two (tub2-403 and tub2-405) contain greatly diminished levels of both nuclear and cytoplasmic microtubules, one (tub2-104) contains predominantly nuclear microtubules, one (tub2-402) contains predominantly cytoplasmic microtubules, and one (tub2-404) contains prominent nuclear and cytoplasmic microtubule arrays. Using these mutants we demonstrate here that cytoplasmic microtubules are necessary for nuclear migration during the mitotic cell cycle and for nuclear migration and fusion during conjugation; only those mutants that possess cytoplasmic microtubules are able to perform these functions. We also show that microtubules are not required for secretory vesicle transport in yeast; bud growth and invertase secretion occur in cells which contain no microtubules.
Generation of antisera that discriminate among mammalian alpha-tubulins: introduction of specialized isotypes into cultured cells results in their coassembly without disruption of normal microtubule function.
Authors: Gu W , Lewis S A , Cowan N J
Published: May 2004

To assay the functional significance of the multiple but closely related alpha-tubulin polypeptides that are expressed in mammalian cells, we generated three specific immune sera, each of which uniquely recognizes a distinct alpha-tubulin isotype. All three isotypes are expressed in a tissue-restricted manner: one (M alpha 3/7) only in mature testis, one (M alpha 4) mainly in muscle and brain, and the third (M alpha 6) in several tissues at a very low level. A fourth specific antiserum was also generated that distinguishes between the tyrosinated and nontyrosinated form of a single alpha-tubulin isotype. Because individual tubulin isotypes cannot be purified biochemically, these sera were raised using cloned fusion proteins purified from host Escherichia coli cells. To suppress the immune response to shared epitopes, animals were first rendered tolerant to fusion proteins encoding all but one of the known mammalian alpha-tubulin isotypes. Subsequent challenge with the remaining fusion protein then resulted in the elicitation of an immune response to unique epitopes. Three criteria were used to establish the specificity of the resulting sera: (a) their ability to discriminate among cloned fusion proteins representing all the known mammalian alpha-tubulin isotypes; (b) their ability to uniquely detect alpha-tubulin in whole extracts of tissues; and (c) their capacity to stain microtubules in fixed preparations of cells transfected with sequences encoding the corresponding isotype. The transfection experiments served to demonstrate (a) the coassembly of M alpha 3/7, M alpha 4, and M alpha 6 into both interphase and spindle microtubules in HeLa cells and NIH 3T3 cells, and (b) that the M alpha 4 isotype, which is unique among mammalian alpha-tubulins in that it lacks an encoded carboxy-terminal tyrosine residue, behaves like other alpha-tubulin isotypes with respect to the cycle of tyrosination/detyrosination that occurs in most cultured cells.
The alpha-tubulin gene family expressed during cell differentiation in Naegleria gruberi.
Authors: Lai E Y , Remillard S P , Fulton C
Published: May 2004

Genes that direct the programmed synthesis of flagellar alpha-tubulin during the differentiation of Naegleria gruberi from amebae to flagellates have been cloned, and found to be novel with respect to gene organization, sequence, and conservation. The flagellar alpha-tubulin gene family is represented in the genome by about eight homologous DNA segments that are exceptionally similar and yet are neither identical nor arrayed in a short tandem repeat. The coding regions of three of these genes have been sequenced, two from cDNA clones and one from an intronless genomic gene. These three genes encode an identical alpha-tubulin that is conserved relative to the alpha-tubulins of other organisms except at the carboxyl terminus, where the protein is elongated by two residues and ends in a terminal glutamine instead of the canonical tyrosine. In spite of the protein conservation, the Naegleria DNA sequence has diverged markedly from the alpha-tubulin genes of other organisms, a counterexample to the idea that tubulin genes are conserved. alpha-Tubulin mRNA homologous to this gene family has not been detected in amebae. This mRNA increases markedly in abundance during the first hour of differentiation, and then decreases even more rapidly with a half-life of approximately 8 min. The abundance of physical alpha-tubulin mRNA rises and subsequently falls in parallel with the abundance of translatable flagellar tubulin mRNA and with the in vivo rate of flagellar tubulin synthesis, which indicates that flagellar tubulin synthesis is directly regulated by the relative rates of transcription and mRNA degradation.
Mitosis-inducing factors are present in a latent form during interphase in the Xenopus embryo.
Authors: Dunphy W G , Newport J W
Published: May 2004

During the conversion to the mitotic state, higher eukaryotic cells activate a cascade of reactions which result in the disintegration of the nuclear envelope, the condensation of the DNA into chromosomes, and the reorganization of the cytoskeleton. In Xenopus, the induction of the mitotic state appears to be under the control of a cytoplasmic factor(s) known as mitosis-promoting factor or MPF. We have developed a rapid and highly sensitive version of an in vitro assay for MPF. The assay uses reconstituted nuclei in interphase cytoplasm from activated Xenopus eggs. The MPF-induced conversion from interphase to mitosis is conveniently monitored by the visual observation of the loss of the nuclear envelope from the substrate nuclei. At near saturating concentrations of MPF, nuclear breakdown requires 20-30 min. Preincubation experiments have revealed that the action of MPF requires only a few minutes and that the disassembly process itself takes up the remainder of the incubation period. Using this cell-free system, we have investigated the observation that protein synthesis is required for the progression through each successive mitotic cycle in the developing Xenopus embryo. A simple explanation for this finding would be that MPF is degraded after each mitosis and then resynthesized before the next mitotic cycle. However, using in vitro reactivation experiments, we have found that MPF is present in a latent, inactive form during interphase. These results suggest that the block in the cell cycle induced by inhibitors of protein synthesis is due to the lack of production of an activator of MPF.
Distribution of extrajunctional acetylcholine receptors on a vertebrate muscle: evaluated by using a scanning electron microscope autoradiographic procedure.
Authors: Salpeter M M , Marchaterre M , Harris R
Published: May 2004

A scanning electron microscope (SEM) autoradiographic technique was calibrated and used to determine the site density of acetylcholine receptors within 250 micron of the neuromuscular junction in innervated as well as 3- and 10-d denervated sternomastoid muscle of the mouse. In all these groups sharp gradients of receptor site density are seen around the endplates in the first 2-7 micron, continuing less sharply to between 25 and 50 micron. Beyond 50 micron (to 250 micron) a spatial density gradient is present 3 d after denervation, but none exist by 10 d. These results suggest that the postdenervation steady-state extrajunctional receptor site density is reached sooner near the junction than away from the junction. The usefulness of SEM autoradiography to study the expression and distribution of membrane molecules at high resolution is demonstrated.
Molecular basis of growth cone adhesion: anchoring of adheron-containing filaments at adhesive loci.
Authors: Tsui H C , Schubert D , Klein W L
Published: May 2004

Adhesive contacts made by filopodia of neuronal growth cones are essential for proper neurite elongation and may have a role in the formation of synaptic junctions. Previously we described the appearance of filamentous materials extending from growth cone surfaces that seem to be associated with the strongly adhesive behavior of filopodia (Tsui, H.-C., K. L. Lankford, and W. L. Klein. 1985. Proc. Natl. Acad. Sci. USA. 82:8256-8260). Here, we have used immunogold labeling to determine whether known adhesive molecules might be localized at points of adhesion and possibly be constituents of the filamentous material. Antibodies to an adhesive molecule (neural cell adhesion molecule [N-CAM]) and to an adhesive macromolecular complex of proteins and proteoglycans (adheron) were localized at the EM level in whole mounts of cultured avian retina cells. Labeling of fixed cells showed that N-CAM and adheron molecules were both present on growth cones and on filopodia. However, filamentous materials extending from the cell surface were labeled with anti-adheron but not with anti-N-CAM. If cells were labeled before fixation, patches of anti-N-CAM labeling occurred in random areas over the growth cones, but adheron antibodies concentrated at points of apparent adhesion. Particularly dense clustering of anti-adheron occurred at individual filopodial tips and at points of contact between pairs of filopodia. The different patterns of labeling imply that N-CAMS do not associate with the main antigenic components of adheron on the membrane surface. Most importantly, the data indicate the N-CAMs were mobile in the membrane but that constituents of adherons were anchored at adhesive loci. An appealing hypothesis is that molecules found in adheron preparations have an important role in establishing the adhesive junctions formed by growth cone filopodia.
Dexamethasone-dependent inhibition of differentiation of C2 myoblasts bearing steroid-inducible N-ras oncogenes.
Authors: Gossett L A , Zhang W , Olson E N
Published: May 2004

ras proteins are localized to the plasma membrane where they are postulated to interact with growth factor receptors and other proximal elements in intracellular cascades triggered by growth factors. The molecular events associated with terminal differentiation of certain skeletal myoblasts are inhibited by specific polypeptide growth factors and by constitutive expression of transforming ras oncogenes. To determine whether the inhibitory effects of ras on myogenic differentiation were reversible and to investigate whether muscle-specific genes remained susceptible to ras-dependent repression in terminally differentiated myotubes, the murine myoblast cell line, C2, was transfected with a plasmid containing a mutationally activated human N-ras oncogene under transcriptional control of the steroid-sensitive promoter of the mouse mammary tumor virus long terminal repeat. Addition of dexamethasone to myoblasts bearing steroid-inducible ras oncogenes prevented myotube formation and induction of muscle creatine kinase and acetylcholine receptors. Inhibition of differentiation by dexamethasone occurred in a dose-dependent manner and was a titratable function of ras expression. In the presence of dexamethasone, myoblasts bearing steroid-inducible ras genes retained their dependence on exogenous growth factors to divide and exhibited contact inhibition of growth at confluent densities, indicating that the inhibitory effects of ras on differentiation were independent of cell proliferation. Removal of dexamethasone from N-ras-transfected myoblasts led to fusion and induction of muscle-specific gene products in a manner indistinguishable from control C2 cells. Examination of the effects of culture media conditioned by ras-transfected myoblasts on differentiation of normal C2 cells yielded no evidence for inhibition of differentiation via an autocrine mechanism. In contrast to the ability of N-ras to prevent up-regulation of muscle-specific gene products in myoblasts, induction of N-ras in terminally differentiated myotubes failed to extinguish muscle-specific gene expression. Together, these results suggest that oncogenic ras proteins reversibly activate an intracellular cascade that prevents establishment of the differentiated phenotype. The inability of ras to extinguish muscle-specific gene expression in terminally differentiated myotubes also suggests that ras may interfere with an early step in the pathway of myoblasts toward the differentiated state.
Yeast vacuoles fragment when microtubules are disrupted.
Authors: Guthrie B A , Wickner W
Published: May 2004

To identify whether microtubules are involved in the maintenance of vacuolar morphology, we treated Saccharomyces cerevisiae with nocodazole and methyl benzimidazole-2-yl-carbamate, drugs which inhibit the polymerization of microtubules. Treated cells arrest with a single large bud in the G2/prophase portion of the cell cycle. Labeling the vacuole with either quinacrine or FITC-dextran revealed vacuole fragmentation that was not found in untreated cells or in cells arrested in G2 by unrelated means. A drug-resistant mutant in beta tubulin does not show vacuolar fragmentation when treated with drug. We propose that microtubules are involved in the regulation of vacuole morphology.
Purification and characterization of a basal body-associated Ca2+-binding protein.
Authors: Huang B , Watterson D M , Lee V D , Schibler M J
Published: May 2004

Isolated basal body complexes from the unicellular alga, Chlamydomonas reinhardtii were found to contain a low molecular mass acidic polypeptide, distinct from calmodulin, but with biochemical features in common with members of the calmodulin family of calcium-binding proteins. These common characteristics included a relative low molecular mass of 20 kD, an experimentally determined acidic pI of 5.3, an altered electrophoretic mobility in SDS-polyacrylamide gels in the presence of added calcium, and a calcium-dependent binding to the hydrophobic ligand phenyl-Sepharose which allowed its purification by affinity chromatography. The relatedness of the basal body-associated 20-kD calcium-binding protein (CaBP) to calmodulin was confirmed by amino acid compositional analysis and partial peptide sequencing of the isolated protein. A rabbit antibody specific for the 20-kD CaBP was raised and used to determine by indirect immunofluorescence the cellular localization of the protein in Chlamydomonas cells. In interphase cells the antibody stained intensely the region between the paired basal bodies, two fibers extending between the basal bodies and the underlying nucleus, and an array of longitudinal filaments surrounding the nucleus. The two basal body-nuclear connecting fibers were identified in thin-section electron micrographs to be narrow striated fiber roots. In mitotic cells the 20-kD CaBP was specifically associated with the poles of the mitotic spindle at the sites of the duplicated basal body complexes.
Molecular cloning of cDNA for caltractin, a basal body-associated Ca2+-binding protein: homology in its protein sequence with calmodulin and the yeast CDC31 gene product.
Authors: Huang B , Mengersen A , Lee V D
Published: May 2004

An extended synthetic oligonucleotide (59-mer) was used to isolate a Chlamydomonas cDNA containing the entire coding region for a novel basal body-associated 20-kD calcium-binding protein (CaBP). DNA and RNA blot analysis indicate that the 20-kD CaBP is encoded by a single copy gene from which is derived an approximately 1.1-kb-long transcript. The deduced amino acid sequence for the protein shows a linear relatedness with calmodulin from Chlamydomonas and other organisms (45-48% identity). The primary protein sequence of the 20-kD CaBP and its predicted secondary structure suggests that the protein is likely to contain four homologous calcium-binding domains that conform to the helix-loop-helix (or EF hand) structure found in calmodulin and related calcium-modulated proteins. The major difference between the protein and calmodulin is an amino-terminal domain of 21 amino acids present on the 20-kD CaBP. In addition to its relatedness to calmodulin, the Chlamydomonas 20-kD CaBP shows a strong sequence identity (50%) with the yeast Saccharomyces cerevisiae CDC31 gene product required for spindle pole body duplication. The association of these sequence-related calcium-binding proteins to microtubule-organizing centers of divergent structure suggests a potential conserved function for the proteins.
Mutations that encode partially functional beta 2 tubulin subunits have different effects on structurally different microtubule arrays.
Authors: Fuller M T , Caulton J H , Hutchens J A , Kaufman T C , Raff E C
Published: May 2004

The testis-specific beta 2 tubulin of Drosophila is required for assembly and function of at least three architecturally different microtubule arrays (Kemphues et al., 1982). Two recessive male-sterile mutations in the B2t locus that encode partially functional, stable, variant forms of beta 2 tubulin cause defects in only certain microtubule-based processes during spermatogenesis. These mutations could thus identify aspects of beta tubulin primary structure critical for function only in specific microtubule arrays. In males carrying the B2t6 mutation, meiotic chromosome segregation and nuclear shaping are normal and flagellar axonemes are formed, but there is a subtle defect in axoneme structure; the outer doublet microtubules fill in with a central core normally seen only in the central pair and accessory microtubules. In homozygous B2t7 males, chromosome movement is usually normal during meiosis but cytokinesis often fails, cytoplasmic microtubules are assembled and nuclear shaping appears to be normal, but the flagellar axoneme lacks structural integrity. In contrast, the B2t8 allele affects a general property of tubulin, the ability to form normal side-to-side association of protofilaments (Fuller et al., 1987), and causes defects in meiosis, axoneme assembly and nuclear shaping. Certain combinations of these beta 2 tubulin mutations show interallelic complementation; in B2t6/B2t8 males functional sperm are produced and both variant subunits are incorporated into mature sperm, in the absence of wild-type beta 2 tubulin. Comparison of the phenotypes of the three partially functional beta 2 tubulin alleles reveals some aspects of tubulin primary structure more important for function in specific subsets of microtubule arrays, and other aspects required for the construction of microtubules in general.