bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the mRNA levels of nimA, a gene required for the G2-M transition in Aspergillus nidulans.
Authors: Osmani S A , May G S , Morris N R
Published: May 2004

The temperature-sensitive cell cycle mutation nimA5 causes nuclei of Aspergillus nidulans to be blocked in late G2 at restrictive temperature. Under these conditions the spindle pole body divides but does not separate and the mitotic index drops to zero. If nimA5 is blocked for more than one doubling time and then shifted from restrictive to permissive temperature, nuclei immediately enter mitosis, the mitotic spindle forms, and the chromosomes condense (Oakley, B. R., and N. R. Morris, 1983, J. Cell Biol., 96:1155-8). We have cloned the wild-type nimA gene by DNA-mediated complementation of the nimA5 mutant phenotype and have characterized nimA mRNA expression by Northern blot analysis. The transcript is 3.6 kb in length and is under tight nuclear cycle regulation. In synchronously dividing cells, the levels of nimA mRNA become elevated as cells enter mitosis and drop sharply as cells progress through mitosis. Cells blocked in S-phase with hydroxyurea have very low levels of nimA mRNA. Cells blocked in mitosis, either by the antimitotic agent benomyl or by the cell cycle mutation bimE7, maintain elevated levels of the nimA transcript. These data demonstrate not only that nimA is required for entry into mitosis, but because the transcript is normally expressed cyclically and is under tight cell cycle control, they suggest that nimA may play a regulatory role in the initiation of mitosis.
Cloning and characterization of mammalian myosin regulatory light chain (RLC) cDNA: the RLC gene is expressed in smooth, sarcomeric, and nonmuscle tissues.
Authors: Taubman M B , Grant J W , Nadal-Ginard B
Published: May 2004

The 20-kD regulatory light chain (RLC) plays a central role in the regulation of smooth muscle contraction. Little is known about the structure or expression of smooth muscle myosin light chain (MLC) genes. A cDNA library was constructed in the expression vector, lambda gt-11, with mRNA derived from cultured rat aortic smooth muscle cells. Using antibody generated against tracheal smooth muscle myosin, three cDNA clones encoding a RLC were isolated, one of which, SmRLC-2, represents a full-length transcript of the RLC mRNA. The derived amino acid sequence shows 94.2% homology with the chicken gizzard RLC, and 70 and 52% homology with the rat skeletal and cardiac muscle MLC-2 proteins, respectively. Thus, the gene encoding the putative smooth muscle RLC appears to have originated by duplication of the same ancestor that gave rise to the sarcomeric MLC-2 genes. Contrary to the stringent tissue-specific expression of sarcomeric MLC-2 genes, RNA blot hybridization and S1 nuclease mapping demonstrates that the putative smooth muscle RLC gene is expressed in smooth, sarcomeric, and nonmuscle tissues at significant levels. Primer extension analysis suggests that the same promoter region is used in these different tissues. Thus the putative smooth muscle RLC gene appears to be a gene that is constitutively expressed in a large variety of cells and has a differentiated function in smooth muscle.
Actin in the preprophase band of Allium cepa.
Authors: Palevitz B A
Published: May 2004

F-actin has been identified in the preprophase band of Allium cepa. Cells attached to subbed slides were obtained from formaldehyde-fixed root tips digested in EGTA and Cellulysin. The air-dried cells were extracted in Triton X-100, treated with rhodamine-phalloidin, rinsed briefly in PBS, and viewed in the fluorescence microscope. Interphase cells contain a network of actin fibers that extends into all areas of the cytoplasm. During preprophase, the network is replaced by a band of fibers aligned in the position of the preprophase band. Colocalization of F-actin with rhodamine-phalloidin and microtubules with tubulin immunocytochemistry confirms that the two bands are coincident. The actin appears to comprise a thin layer of fibers next to the plasmalemma. Like the microtubule preprophase band, the actin band narrows as preprophase progresses and disappears by midprophase. Fluorescent actin bands are not seen in fixed cells pretreated with excess unlabeled phalloidin before staining. They are also absent in roots exposed to cytochalasins B and D before fixation, but preprophase band microtubules at all stages of aggregation are still present. Colchicine treatment leads to the loss of both preprophase band microtubules and actin. The possible function of preprophase band actin is discussed.
Modulation of fodrin (membrane skeleton) stability by cell-cell contact in Madin-Darby canine kidney epithelial cells.
Authors: Nelson W J , Veshnock P J
Published: May 2004

During growth of Madin-Darby canine kidney (MDCK) epithelial cells, there is a dramatic change in the stability, biophysical properties, and distribution of the membrane skeleton (fodrin) which coincides temporally and spatially with the development of the polarized distribution of the Na+, K+-ATPase, a marker protein of the basolateral domain of the plasma membrane. These changes occur maximally upon the formation of a continuous monolayer of cells, indicating that extensive cell-cell contact may play an important role in the organization of polarized MDCK cells (Nelson, W. J., and P. J. Veshnock, 1986, J. Cell Biol., 103:1751-1766). To directly analyze the role of cell-cell contact in these events, we have used an assay in which the organization of fodrin and membrane proteins is analyzed in confluent monolayers of MDCK cells in the absence or presence of cell-cell contact by adjusting the concentration Ca++ in the growth medium. Our results on the stability and solubility properties of fodrin reported here show directly that there is a positive correlation between cell-cell contact and increased stability and insolubility of fodrin. Furthermore, we show that fodrin can be recruited from an unstable pool of protein to a stable pool during induction of cell-cell contact; significantly, the stabilization of fodrin is not affected by the addition of cyclohexamide, indicating that proteins normally synthesized during the induction of cell-cell contact are not required. Together these results indicate that cell-cell contact may play an important role in the development of polarity in MDCK cells by initiating the formation of a stable, insoluble matrix of fodrin with preexisting (membrane) proteins at the cell periphery. This matrix may function subsequently to trap proteins targeted to the membrane, resulting in the maintenance of membrane domains.
"Buttonin," a unique button-shaped microtubule-associated protein (75 kD) that decorates spindle microtubule surface hexagonally.
Authors: Hirokawa N , Hisanaga S
Published: May 2004

A 75-kD protein was purified from sea urchin egg microtubule proteins through gel filtration. It enhanced the polymerization of porcine brain tubulin, but was not heat-stable and did not bind to calmodulin in the presence of calcium as demonstrated by calmodulin affinity column chromatography. Rotary shadowing of the freeze-etched 75-kD protein adsorbed on mica revealed the protein to be a spherical molecule (approximately 9 nm in diameter). Quick-freeze deep-etch electron microscopy revealed that the surface of microtubules polymerized with 75-kD protein was entirely covered with hexagonally packed, round, button-like structures that were quite uniform in shape and size (approximately 9 nm) and similar to the buttons observed on microtubules of mitotic spindles in vivo or microtubules isolated from mitotic spindles. Judging from calibration studies of molecular mass by gel filtration, the 75-kD protein probably exists in a dimeric form (approximately 150 kD) in its native condition. The stoichiometry of tubulin (dimer) versus 75-kD protein (dimer) in the polymerized pellet was 3-3.4:1. Hence, we concluded that the 75-kD protein was a unique microtubule-associated protein that formed the microtubule button in vivo and in vitro. We propose to name this protein "buttonin".
Cross-reactivity of antibodies specific for flagellar tektin and intermediate filament subunits.
Authors: Chang X J , Piperno G
Published: May 2004

Monoclonal antibodies specific for each of the flagellar tektins were prepared and used to determine whether structures similar to tektin filaments are present in cells lacking cilia or flagella. This analysis was performed by double-label immunofluorescence microscopy of several cell lines and by immunoblots of protein fractions. Two of the four anti-tektin antibodies, the antibodies 3-7-1 and 3-10-1, which bind different epitopes of the C-tektin, label 3T3, HeLa, PtK2, and BHK-21 cells as well as myotubes. The antibody 3-7-1 stains intermediate filament structures in the cells and binds vimentin or desmin in preparations of cytoskeletal proteins; whereas the antibody 3-10-1 stains nuclear envelopes in the cells and binds lamin A and C in preparations of cytoskeletal proteins or nuclear lamina. Structural similarities between the C-tektin and intermediate filament proteins probably are extended to more than two epitopes because polyclonal antibodies anti-vimentin and anti-desmin bind to C-tektin. These polyclonal antibodies also bind to A-tektin. The cross-reaction of monoclonal and polyclonal antibodies binding to epitopes in tektin and intermediate filament components and the existence of a high content of alpha-helical structure in the tektin subunits (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22) indicate that tektin and intermediate filaments are homologous in several parts of their structure.
Colocalization of microtubule-associated protein 1A and microtubule-associated protein 2 on neuronal microtubules in situ revealed with double-label immunoelectron microscopy.
Authors: Shiomura Y , Hirokawa N
Published: May 2004

Microtubule-associated protein 1A (MAP1A) and microtubule-associated protein 2 (MAP2) were shown to be colocalized on the same microtubules (MTs) within neuronal cytoskeletons by double-label immunoelectron microscopy. To investigate the electron microscopic disposition of MAP1A and MAP2 and their relationship to MTs in vivo, and to determine whether there are different subsets of MTs which specifically bind either MAP1 or MAP2, we employed a double-label immunogold procedure on rat cerebella using mouse monoclonal antibody against rat brain MAP1A and affinity-purified rabbit polyclonal antibody against rat brain MAP2. MAP1A and MAP2 were identified with secondary antibodies coupled to 10- and 5-nm gold particles, respectively. In Purkinje cell dendrites, both 10- and 5-nm gold particles were observed to be studded on the fuzzy structures attached to the same MTs. Many such structures connected MTs to each other. There was no particular MT which bound either MAP1A or MAP2 alone. Furthermore, there seemed to be no specific regions on MTs where either MAP1A or MAP2 was specifically attached. Hence, we conclude that MAP1A and MAP2 are colocalized on MTs in dendrites and assume that MAP1A and MAP2 have some interrelationship in vivo and that their interactions are responsible for forming the network of cross-bridges between MTs and MTs in neuronal cytoskeletons.
Visualization of neural cell adhesion molecule by electron microscopy.
Authors: Hall A K , Rutishauser U
Published: May 2004

The 130- and 160-kD polypeptide forms of the neural cell adhesion molecule (NCAM) were analyzed by electron microscopy after low angle rotary shadowing and freeze replication. Individual NCAM molecules appeared as uniformly thick rods, with a distinct bend or hinge region near their middle. Aggregates were also present, containing two to six rods in a pinwheel-like configuration without measurable overlap between rods. The 130- and 160-kD NCAM forms had lengths of 38 and 51 nm, respectively, with a difference in arm length distal to the bend, but not toward the center of the pinwheel. Although enzymatic removal of the polysialic acid moiety on NCAM did not alter the appearance of individual molecules, it did increase the average number of arms per aggregate. Monoclonal antibodies that recognize defined regions of the NCAM polypeptide were used to provide landmarks on the observed molecular figures. Two antibodies specific for cytoplasmic epitopes near the COOH terminus were clustered at the distal tip of aggregated arms. Two other antibodies that react with epitopes near the NH2 terminus and the middle of the molecule bound to sites more centrally located on the pinwheel structure. Together, these results suggest that the observed aggregates represent an association of molecules near their NH2-terminal homophilic binding site, and have led to several predictions about the nature of an NCAM-mediated cell-cell bond.
The novel carbohydrate epitope L3 is shared by some neural cell adhesion molecules.
Authors: Kücherer A , Faissner A , Schachner M
Published: May 2004

The monoclonal L3 antibody reacts with an N-glycosidically linked carbohydrate structure on at least nine glycoproteins of adult mouse brain. Three out of the L3 epitope-carrying glycoproteins could be identified as the neural cell adhesion molecules L1 and myelin-associated glycoprotein, and the novel adhesion molecule on glia. Expression of the L3 carbohydrate epitope is regulated independently of the protein backbone of these three glycoproteins. Based on the observation that out of three functionally characterized L3 epitope-carrying glycoproteins three fulfill the operational definition of an adhesion molecule, we would like to suggest that they form a new family of adhesion molecules that is distinct from the L2/HNK-1 carbohydrate epitope family of neural cell adhesion molecules. Interestingly, some members in each family appear to be unique to one family while other members belong to the two families.
Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide.
Authors: Brenneman D E , Neale E A , Foster G A , d'Autremont S W , Westbrook G L
Published: May 2004

The developmental regulation of neuronal survival by vasoactive intestinal peptide (VIP) was investigated in dissociated spinal cord-dorsal root ganglion (SC-DRG) cultures. Previous studies demonstrated that VIP increased neuronal survival in SC-DRG cultures when synaptic transmission was blocked with tetrodotoxin (TTX). This effect was further investigated to determine if VIP acted directly on neurons or via nonneuronal cells. For these studies, SC-DRG cells were cultured under conditions designed to provide preparations enriched for a particular cell type: astrocyte-enriched background cell (BG) cultures, meningeal fibroblast cultures, standard mixed neuron-nonneuron (STD) cultures, and neuron-enriched (N) cultures. Addition of 0.1 nM VIP to TTX-treated STD cultures for 5 d prevented the TTX-mediated death and the death that occurred naturally during development in culture, whereas the same treatment on N cultures did not prevent neuronal cell death. Conditioned medium from VIP-stimulated BG cultures prevented neuronal cell death when added to the medium (10% of total volume) of N cultures treated with TTX. The same amount of conditioned medium from BG cultures that were not treated with VIP had no protective action on N cultures. Conditioned medium from N or meningeal fibroblast cultures, either with or without VIP treatment, did not prevent TTX-mediated cell death in N test cultures. These data indicate that VIP increases the availability of neurotrophic survival-promoting substances derived from nonneuronal cultures, the most likely source being astroglial cells. This study suggests that VIP has a role in mediating a neuron-glia-neuron interaction that influences the trophic regulation of neuronal survival.
Long-term, heterologous down-regulation of the epidermal growth factor receptor in PC12 cells by nerve growth factor.
Authors: Lazarovici P , Dickens G , Kuzuya H , Guroff G
Published: May 2004

Cells of the rat pheochromocytoma clone PC12 possess receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF), thus enabling the study of the interaction of these receptors in the regulation of proliferation and differentiation. Treatment of the cells with NGF induces a progressive and nearly total decrease in the specific binding of EGF beginning after 12 h and completed within 4 d. Three different measures of receptor show that the decreased binding capacity represents, in fact, a decreased amount of receptor: (a) affinity labeling of PC12 cell membranes by cross-linking of receptor-bound 125I-EGF showed a 60-90% decrease in the labeling of 170- and 150-kD receptor bands in cells treated with NGF for 1-4 d; (b) EGF-dependent phosphorylation of a src-related synthetic peptide or EGF receptor autophosphorylation with membranes from NGF-differentiated cells showed a decrease of 80 and 90% in the tyrosine kinase activity for the exogenous substrate and for receptor autophosphorylation, respectively; (c) analysis of 35S-labeled glycoproteins isolated by wheat germ agglutinin-Sepharose chromatography from detergent extracts of PC12 membranes showed a 70-90% decrease in the 170-kD band in NGF-differentiated cells. These findings permit the hypothesis that long-term heterologous down-regulation of EGF receptors by NGF in PC12 cells is mediated by an alteration in EGF receptor synthesis. It is suggested that this heterologous down-regulation is part of the mechanism by which differentiating cells become insensitive to mitogens.
Biosynthesis and processing of platelet GPIIb-IIIa in human megakaryocytes.
Authors: Duperray A , Berthier R , Chagnon E , Ryckewaert J J , Ginsberg M , Plow E , Marguerie G
Published: May 2004

Platelet membrane glycoprotein IIb-IIIa forms a calcium-dependent heterodimer and constitutes the fibrinogen receptor on stimulated platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIIa is a single chain protein. These proteins are synthesized in the bone marrow by megakaryocytes, but the study of their synthesis has been hampered by the difficulty in obtaining enriched population of megakaryocytes in large numbers. To examine the biosynthesis and processing of GPIIb-IIIa, purified human megakaryocytes were isolated from liquid cultures of cryopreserved leukocytes stem cell concentrates from patients with chronic myelogenous leukemia. Immunoprecipitation of [35S]methionine pulse-chase-labeled cell extracts by antibodies specific for the alpha or beta subunits of GPIIb indicated that GPIIb was derived from a precursor of Mr 130,000 that contains the alpha and beta subunits. This precursor was converted to GPIIb with a half-life of 4-5 h. No precursor form of GPIIIa was detected. The glycosylation of GPIIb-IIIa was examined in megakaryocytes by metabolic labeling in the presence of tunicamycin, monensin, or treatment with endoglycosidase H. The polypeptide backbones of the GPIIb and the GPIIIa have molecular masses of 120 and 90 kD, respectively. High-mannose oligosaccharides are added to these polypeptide backbones co-translationally. The GPIIb precursor is then processed with conversion of high-mannose to complex type carbohydrates yielding the mature subunits GPIIb alpha (Mr 116,000) and GPIIb beta (Mr 25,000). No posttranslational processing of GPIIIa was detected.
The influenza hemagglutinin insertion signal is not cleaved and does not halt translocation when presented to the endoplasmic reticulum membrane as part of a translocating polypeptide.
Authors: Finidori J , Rizzolo L , Gonzalez A , Kreibich G , Adesnik M , Sabatini D D
Published: May 2004

The co-translational insertion of polypeptides into endoplasmic reticulum membranes may be initiated by cleavable amino-terminal insertion signals, as well as by permanent insertion signals located at the amino-terminus or in the interior of a polypeptide. To determine whether the location of an insertion signal within a polypeptide affects its function, possibly by affecting its capacity to achieve a loop disposition during its insertion into the membrane, we have investigated the functional properties of relocated insertion signals within chimeric polypeptides. An artificial gene encoding a polypeptide (THA-HA), consisting of the luminal domain of the influenza hemagglutinin preceded by its amino-terminal signal sequence and linked at its carboxy-terminus to an intact prehemagglutinin polypeptide, was constructed and expressed in in vitro translation systems containing microsomal membranes. As expected, the amino-terminal signal initiated co-translational insertion of the hybrid polypeptide into the membranes. The second, identical, interiorized signal, however, was not recognized by the signal peptidase and was translocated across the membrane. The failure of the interiorized signal to be cleaved may be attributed to the fact that it enters the membrane as part of a translocating polypeptide and therefore cannot achieve the loop configuration that is thought to be adopted by signals that initiate insertion. The finding that the interiorized signal did not halt translocation of downstream sequences, even though it contains a hydrophobic region and must enter the membrane in the same configuration as natural stop-transfer signals, indicates that the HA insertion signal lacks essential elements of halt transfer signals that makes the latter effective membrane-anchoring domains. When the amino-terminal insertion signal of the THA-HA chimera was deleted, the interior signal was incapable of mediating insertion, probably because of steric hindrance by the folded preceding portions of the chimera. Several chimeras were constructed in which the interiorized signal was preceded by polypeptide segments of various lengths. A signal preceded by a segment of 111 amino acids was also incapable of initiating insertion, but insertion took place normally when the segment preceding the signal was only 11-amino acids long.(ABSTRACT TRUNCATED AT 400 WORDS)
Is movement of mannose 6-phosphate-specific receptor triggered by binding of lysosomal enzymes?
Authors: Braulke T , Gartung C , Hasilik A , von Figura K
Published: May 2004

Mannose 6-phosphate-specific receptors with an apparent molecular mass of 215,000 are present in fibroblasts at the cell surface and in intracellular membranes. The cell surface receptors mediate endocytosis of exogenous lysosomal enzymes and exchange with the intracellular receptors, which function in the sorting of endogenous lysosomal enzymes. In the present study, several methods independent of receptor ligands were designed in order to examine the exchange of receptors under conditions where receptor-ligand complexes do not dissociate (weak bases and monensin) or where receptor-ligand complexes are not formed due to absence of endogenous ligands as a result of inhibition of protein synthesis. Weak bases and monensin reduce the concentration of receptors at the cell surface by 20-30% and free cell surface receptors were replaced by occupied receptors. The latter continued to be exchanged with internal ligand-occupied receptors and the rates of the exchange were similar to the control values. The exchange of receptors between the cell surface and internal membranes was also not affected when the receptor ligands were depleted from the transport compartments by treating the cells with cycloheximide for up to 10 h. We conclude from these results that movement of mannose 6-phosphate-specific receptors along the endocytosis and sorting pathways is constitutive and not triggered by binding or dissociation of ligands.
Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways.
Authors: Lemansky P , Hasilik A , von Figura K , Helmy S , Fishman J , Fine R E , Kedersha N L , Rome L H
Published: May 2004

The molecular forms of two lysosomal enzymes, cathepsin C and cathepsin D, have been examined in lysosomes and coated vesicles (CVs) of rat liver. In addition, the relative proportion of these lysosomal enzymes residing in functionally distinct CV subpopulations was quantitated. CVs contained newly synthesized precursor forms of the enzymes in contrast to lysosomes where only the mature forms were detected. Exocytic and endocytic CV subpopulations were prepared by two completely different protocols. One procedure, a density shift method, uses cholinesterase to alter the density of CVs derived from exocytic or endocytic pathways. The other relies on electrophoretic heterogeneity to accomplish the CV subfractionation. Subpopulations of CVs prepared by either procedure showed similar results, when examined for their relative proportion of cathepsin C and cathepsin D precursors. Within the starting CV preparation, exocytic CVs contained approximately 80-90% of the total steady-state levels of these enzymes while the level in the endocytic population was approximately 10-13%. The implications of these findings are discussed with regard to lysosome trafficking.
The three-dimensional structure of the Na,K-ATPase from electron microscopy.
Authors: Mohraz M , Simpson M V , Smith P R
Published: May 2004

The structure of Na,K-ATPase has been studied by electron microscopy and image reconstruction. A three-dimensional structure of this enzyme has been obtained to an overall resolution of 2.5 nm using data from specimens of negatively stained dimer sheets tilted through a range of angles +/- 60 degrees. The reconstruction shows a complex mass distribution consisting of ribbons of paired molecules extending approximately 6.0 nm from the cytoplasmic side of the membrane. The molecular envelope consists of a massive "body" with "lobe" and "arm" structures projecting from it. The body has a columnar shape and is tilted with respect to the plane of the membrane. The region of interaction responsible for dimer formation is located between two bodies and is clearly visible in the reconstruction. It has been identified as a segment in the amino-terminal portion of the alpha subunit. The arms that interconnect the ribbons are located close to the membrane and are most probably formed by the beta subunits.
Two distinct attachment sites for vimentin along the plasma membrane and the nuclear envelope in avian erythrocytes: a basis for a vectorial assembly of intermediate filaments.
Authors: Georgatos S D , Blobel G
Published: May 2004

In vitro binding studies with isolated bovine lens vimentin and avian erythrocyte membranes reveal the existence of two functionally distinct sets of intermediate filament attachment sites. One population of such receptors is located along the nuclear envelope and comprises polypeptides recognizing the carboxy-terminal tail domain of vimentin. Vimentin associates with these nuclear surface receptors in a cooperative manner and forms extensive 10-nm filaments in a concentration-dependent fashion. Conversely, the plasma membrane contains binding sites that interact in a noncooperative, saturable fashion with vimentin, recognizing its amino-terminal head domain. The functional dichotomy of the vimentin-binding sites under in vitro conditions may reflect a vectorial assembly process whereby 10-nm filaments, although structurally apolar, acquire polar features brought about by the differential attachment to specific receptors arranged along the plasma membrane and the nuclear envelope.
Lamin B constitutes an intermediate filament attachment site at the nuclear envelope.
Authors: Georgatos S D , Blobel G
Published: May 2004

We found that urea extraction of turkey erythrocyte nuclear envelopes abolished their ability to bind exogenous 125I-vimentin, while, at the same time, it removed the nuclear lamins from the membranes. After purification of the lamins from such urea extracts, a specific binding between isolated vimentin and lamin B, or a lamin A + B hetero-oligomer, was detected by affinity chromatography. Similar analysis revealed that the 6.6-kD vimentin tail piece was involved in this interaction. By other approaches (quantitative immunoprecipitation, rate zonal sedimentation, turbidometric assays) a substoichiometric lamin B-vimentin binding was determined under in vitro conditions. It was also observed that anti-lamin B antibodies but not other sera (anti-lamin A, anti-ankyrin, preimmune) were able to block 70% of the binding of 125I-vimentin to native, vimentin-depleted, nuclear envelopes. These data, which were confirmed by using rat liver nuclear lamins, indicate that intermediate filaments may be anchored directly to the nuclear lamina, providing a continuous network connecting the plasma membrane skeleton with the karyoskeleton of eukaryotic cells.
A single histone acetyltransferase from Tetrahymena macronuclei catalyzes deposition-related acetylation of free histones and transcription-related acetylation of nucleosomal histones.
Authors: Chicoine L G , Richman R , Cook R G , Gorovsky M A , Allis C D
Published: May 2004

A salt-extracted histone acetyltransferase activity from Tetrahymena macronuclei acetylates mostly histone H3 and H4 when free histones are used as substrate. Free histone H4 is acetylated first at position 11 (monoacetylated) or positions 11 and 4 (diacetylated). This activity strongly resembles in vivo, deposition-related acetylation of newly synthesized histones. When acetylase-free mononucleosomes are used as substrate, all four core histones are acetylated by the same extract, and H4 is acetylated first at position 7 (monoacetylated) or positions 7 and 4 (diacetylated). In this respect, the activity of the extract is indistinguishable from postsynthetic, transcription-related histone acetylation that occurs in vivo or in isolated nuclei. Heat inactivation curves with both substrates are indistinguishable, and free histones compete with chromatin for limiting amounts of enzyme activity. These results argue strongly that two distinct, biologically important histone acetylations, one deposition related and one transcription related, are carried out by a single acetyltransferase.
Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway.
Authors: Rhodes C J , Halban P A
Published: May 2004

The pancreatic B cell has been used as a model to compare the release of newly synthesized prohormone/hormone with that of stored hormone. Secretion of newly synthesized proinsulin/insulin (labeled with [3H]leucine during a 5-min pulse) and stored total immunoreactive insulin was monitored from isolated rat pancreatic islets at basal and stimulatory glucose concentrations over 180 min. By 180 min, 15% of the islet content of stored insulin was released at 16.7 mM glucose compared with 2% at 2.8 mM glucose. After a 30-min lag period, release of newly synthesized (labeled) proinsulin and insulin was detected; from 60 min onwards this release was stimulated up to 11-fold by 16.7 mM glucose. At 180 min, 60% of the initial islet content of labeled proinsulin was released at 16.7 mM glucose and 6% at 2.8 mM glucose. Specific radioactivity of the released newly synthesized hormone relative to that of material in islets indicated its preferential release. A similar degree of isotopic enrichment of released, labeled products was observed at both glucose concentrations. Quantitative HPLC analysis of labeled products indicated that glucose had no effect on intracellular proinsulin to insulin conversion; release of both newly synthesized proinsulin and insulin was sensitive to glucose stimulation; 90% of the newly synthesized hormone was released as insulin; and only 0.5% of proinsulin was rapidly released (between 30 and 60 min) in a glucose-independent fashion. It is thus concluded that the major portion of released hormone, whether old or new, processed or unprocessed, is directed through the regulated pathway, and therefore the small (less than 1%) amount released via a constitutive pathway cannot explain the preferential release of newly formed products from the B cell.
Further evidence for importance of the subunit combination of silk fibroin in its efficient secretion from the posterior silk gland cells.
Authors: Takei F , Kikuchi Y , Kikuchi A , Mizuno S , Shimura K
Published: May 2004

A locus responsible for the Nd-s mutation of the silkworm, Bombyx mori, has been mapped very close to or within the fibroin light (L) chain gene on the 14th chromosome (Takei, F., K. Kimura, S. Mizuno, T. Yamamoto, and K. Shimura, 1984, Jpn. J. Genet., 59:307-313). A strain of B. mori carrying the homozygous Nd-sD mutation (Nd-sD/Nd-sD; Nd-sD is allelic to Nd-s) secretes less than 0.3% of fibroin into the lumen of the posterior silk gland compared with a strain carrying the homozygous wild-type alleles (+/+). The small amount of fibroin that is secreted in the Nd-sD/Nd-sD strain consists of the heavy (H) chain only and lacks the L chain, although the L chain mRNA and the proteins that are cross-reactable with the anti-L chain serum are present in the posterior silk gland cells. In the hybrid silkworm, Nd-sD/+, the H chain derived from either the Nd-sD or + allele forms disulfide linkage with the L chain derived from the + allele and these fibroins are secreted into the lumen with an equal efficiency, but the L chain derived from the Nd-sD allele remains in the cell unbound to the H chain. Some evidence suggesting structural abnormality of the L chain derived from the Nd-sD allele is presented. These results, together with the previous results on the effect of the H chain gene-linked Nd(2) mutation (Takei, F., F. Oyama, K. Kimura, A. Hyodo, S. Mizuno, and K. Shimura, 1984, J. Cell Biol., 99:2005-2010), strongly suggest that the H-L subunit combination of silk fibroin is important for its efficient secretion.
Ordered transmembrane and extracellular structure in squid photoreceptor microvilli.
Authors: Saibil H , Hewat E
Published: May 2004

Invertebrate retinas contain hexagonal arrays of microvilli that form the honeycomb structure of rhabdome photoreceptors. The largest and most crystalline rhabdomes are found in the squid retina, and we have taken advantage of their unique properties to derive a model for the electron density distribution in microvillar membranes using low angle X-ray diffraction combined with correlation averaging of electron microscope images. The model electron density map, calculated to a resolution of approximately 35 A, shows an unusually high protein content in the membranes. This may be associated with a dense meshwork of membrane junctions between neighboring microvilli as revealed by electron microscope image analysis. Membrane pair contacts are resolved as two or more strands of density crossing the membranes. The microvilli are also linked together by Y-shaped junctions at their three-way contacts. These two sorts of junctions link the membranes into a three-dimensional array and partition them into a mosaic of deformable and rigid domains. This arrangement maintains a remarkable degree of long-range order in squid rhabdomes, and may be responsible for the alignment of rhodopsin molecules. The structural order observed is necessary for these photoreceptors to achieve their high sensitivity to the plane of polarized light. Rhodopsin constitutes about one-half the microvillar protein. The remaining proteins, which can be divided into approximately equal detergent-soluble and insoluble fractions, could account for the composition of the new structures described.
Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor.
Authors: McGraw T E , Greenfield L , Maxfield F R
Published: May 2004

Transferrin (Tf) receptor-variant Chinese hamster ovary cells have been isolated by selection for resistance to two Tf-toxin conjugates. The hybrid toxins contain Tf covalently linked to ricin A chain or a genetically engineered diphtheria toxin fragment. The Tf-receptor-variant (TRV) cells do not have detectable cell-surface Tf receptor; they do not bind fluorescein-Tf or 125I-Tf. TRV cells are at least 100-fold more resistant to the Tf-diphtheria toxin conjugate than are the parent cells. The TRV cells have retained sensitivity to native diphtheria toxin, indicating that the increased resistance to the conjugate is correlated with the loss of Tf binding. The endocytosis of fluorescein-labeled alpha 2-macroglobulin is normal in TRV cells, demonstrating that the defect does not pleiotropically affect endocytosis. Since these cells lack endogenous Tf receptor activity, they are ideally suited for studies of the functional expression of normal or altered Tf receptors introduced into the cells by cDNA transfection. One advantage of this system is that Tf binding and uptake can be used to monitor the behavior of the transfected receptor. A cDNA clone of the human Tf receptor has been transfected into TRV cells. In the stably expressing transfectants, the behavior of the human receptor is very similar to that of the endogenous Chinese hamster ovary cell Tf receptor. Tf binds to cell surface receptors, and is internalized into the para-Golgi region of the cell. Iron is released from Tf, and the apo-Tf and its receptor are recycled back to the cell surface. Thus, the TRV cells can be used to study the behavior of genetically altered Tf receptors in the absence of interfering effects from endogenous receptors.
The endosomal concentration of a mannose 6-phosphate receptor is unchanged in the absence of ligand synthesis.
Authors: Pfeffer S R
Published: May 2004

The cation-independent mannose-6-phosphate (Man-6-P) receptor is involved in the targeting of newly synthesized lysosomal hydrolases. To investigate the intracellular distribution of this receptor, a conjugate of lactoperoxidase coupled to asialoorosomucoid was used to catalyze its iodination within the endosomes of human hepatoma (HepG2) cells. The 215-kD, cation-independent Man-6-P receptor was iodinated by this procedure as shown by pentamannosyl-6-phosphate-Sepharose affinity chromatography and by immunoprecipitation of labeled cell extracts. The amount of this receptor detected in endosomes was found to be unchanged after inhibition of protein synthesis with cycloheximide. If the Man-6-P receptor accumulates in the Golgi apparatus in the absence of lysosomal hydrolase synthesis, it should have been correspondingly depleted from endosomes after a period of cycloheximide treatment, because these pools of receptor are in rapid equilibrium. Therefore, these data suggest that newly synthesized ligands are not required for the transport of the cation-independent Man-6-P receptor from the Golgi apparatus to endosomes.
Import of the carboxy-terminal portion of acyl-CoA oxidase into peroxisomes of Candida tropicalis.
Authors: Small G M , Lazarow P B
Published: May 2004

We report the sequence of a cDNA clone that codes for the carboxy-terminal portion of the peroxisomal protein, acyl-CoA oxidase, from the yeast, Candida tropicalis. This is a newly identified acyl-CoA oxidase sequence, most likely a second allele of POX4. The cDNA clone was expressed by in vitro transcription followed by translation. The major product, a 43-kD protein, associated with isolated peroxisomes in an in vitro import assay. More than half of the peroxisome-associated protein was protected from added protease, implying that it was internalized within the organelle. These findings indicate that there is sufficient information in the carboxy-terminal portion of the protein to target it to peroxisomes.
Postpolymerization detyrosination of alpha-tubulin: a mechanism for subcellular differentiation of microtubules.
Authors: Gundersen G G , Khawaja S , Bulinski J C
Published: May 2004

Tyrosinated (Tyr) and detyrosinated (Glu) alpha-tubulin, species interconverted by posttranslational modification, are largely segregated in separate populations of microtubules in interphase cultured cells. We sought to understand how distinct Tyr and Glu microtubules are generated in vivo, by examining time-dependent alterations in Tyr and Glu tubulin levels (by immunoblots probed with antibodies specific for each species) and distributions (by immunofluorescence) after microtubule regrowth and stabilization. When microtubules were allowed to regrow after complete depolymerization by microtubule antagonists, Glu microtubules reappeared with a delay of approximately 25 min after the complete array of Tyr microtubules had regrown. In these experiments, Tyr tubulin immunofluorescence first appeared as an aster of distinct microtubules, while Glu tubulin staining first appeared as a grainy pattern that was not altered by detergent extraction, suggesting that Glu microtubules were created by detyrosination of Tyr microtubules. Treatments with taxol, azide, or vinblastine, to stabilize polymeric tubulin, all resulted in time-dependent increases in polymeric Glu tubulin levels, further supporting the hypothesis of postpolymerization detyrosination. Analysis of monomer and polymer fractions during microtubule regrowth and in microtubule stabilization experiments were also consistent with postpolymerization detyrosination; in each case, Glu polymer levels increased in the absence of detectable Glu monomer. The low level of Glu monomer in untreated or nocodazole-treated cells (we estimate that Glu tubulin comprises less than 2% of the monomer pool) also suggested that Glu tubulin entering the monomer pool is efficiently retyrosinated. Taken together these results demonstrate that microtubules are polymerized from Tyr tubulin and are then rapidly converted to Glu microtubules. When Glu microtubules depolymerize, the resulting Glu monomer is retyrosinated. This cycle generates structurally, and perhaps functionally, distinct microtubules.
Sequential phosphorylation of chartin microtubule-associated proteins is regulated by the presence of microtubules.
Authors: Aletta J M , Greene L A
Published: May 2004

Chartins are a unique class of three families of microtubule-associated proteins, each consisting of several isoforms possessing varying degrees of phosphorylation. The most highly phosphorylated chartin isoforms are highly enriched in neuronal cell fractions containing microtubules and there is evidence that their phosphorylation may play a role in promoting neurite outgrowth. The present work describes the relationship between the phosphorylation state of chartins and the presence of intact microtubules in long-term cultures of NGF-treated, neurite-bearing PC12 cells. Cultures were depleted of microtubules by exposure to high concentrations of depolymerizing agents for 2-24 h. Radiolabeling of cellular proteins with [32P]orthophosphate or [35S]methionine revealed that both the ongoing and steady-state phosphorylation of chartins is markedly altered under these conditions. Two-dimensional isoelectric focusing by SDS-PAGE of whole cell extracts demonstrated that the more acidic, highly phosphorylated isoforms are diminished with a concomitant increase in the more basic, less phosphorylated isoforms. These phosphorylation changes were relatively specific for the chartins and were not observed for phosphorylated MAP 1.2, phospho-beta-tubulin, or most other phosphoproteins. Thus, the phosphorylation state of chartins, but not of other phosphoproteins, is regulated by the presence of native microtubules. Despite depolymerization of microtubules, neurites remained extended for at least 24 h. Neurite elongation, however, was arrested. Microtubules, therefore, may be required for extension, but not for short-term maintenance of well-established neurites. Taxol, which promotes tubule assembly and stability, does not, conversely, drive phosphorylation of the chartins. Instead, taxol appeared to decrease the turnover of phosphate in microtubule-associated, acidic chartin isoforms. These data suggest several models as to how chartin phosphorylation is regulated in neurite-bearing cells and indicate that phosphorylation of cytoplasmic and microtubule-associated chartins occurs via different mechanisms.
Structural relationships of actin, myosin, and tropomyosin revealed by cryo-electron microscopy.
Authors: Milligan R A , Flicker P F
Published: May 2004

We have calculated three-dimensional maps from images of myosin subfragment-1 (S1)-decorated thin filaments and S1-decorated actin filaments preserved in frozen solution. By averaging many data sets we obtained highly reproducible maps that can be interpreted simply to provide a model for the native structure of decorated filaments. From our results we have made the following conclusions. The bulk of the actin monomer is approximately 65 X 40 X 40 A and is composed of two domains. In the filaments the monomers are strongly connected along the genetic helix with weaker connections following the long pitch helix. The long axis of the monomer lies roughly perpendicular to the filament axis. The myosin head (S1) approaches the actin filament tangentially and binds to a single actin, the major interaction being with the outermost domain of actin. In the map the longest chord of S1 is approximately 130 A. The region of S1 closest to actin is of high density, whereas the part furthest away is poorly defined and may be disordered. By comparing maps from decorated thin filaments with those from decorated actin, we demonstrate that tropomyosin is bound to the inner domain of actin just in front of the myosin binding site at a radius of approximately 40 A. A small change in the azimuthal position of tropomyosin, as has been suggested by others to occur during Ca2+-mediated regulation in vertebrate striated muscle, appears to be insufficient to eclipse totally the major site of interaction between actin and myosin.
Hemodynamics and the vascular endothelial cytoskeleton.
Authors: Herman I M , Brant A M , Warty V S , Bonaccorso J , Klein E C , Kormos R L , Borovetz H S
Published: May 2004

Although there is considerable evidence to suggest that hemodynamics play an important role in vascular disease processes, the exact mechanisms are unknown. With this in mind, we have designed a pulsatile perfusion apparatus which reproducibly delivers pulsatile hemodynamics upon freshly excised canine carotid arteries in vitro. Quantifiable simulations included normotension with normal or lowered flow rates (120/80 mmHg, 120 and 40 ml/min), normotension with lowered or elevated transmural pressures (40-170 mmHg), and elevated pulse pressure (120 and 80 mmHg) with normal (150 ml/min) or elevated rates of flow (300 and 270 ml/min). Arterial biomechanical stresses and cellular behaviors were characterized biochemically and morphologically under all these stimulations which continued for 2-24 h. We found that increased pulse pressure alone had little effect on the total amount of radiolabeled [4-14C]cholesterol present within the medial compartment. However, normotension when coupled with altered transmural pressure yielded a three- to fourfold increase. Combinations of increased pulse pressure and flow potentiated cholesterol uptake by a factor of 10 when compared with normotension control values. Simulations that enhanced carotid arterial cholesterol uptake also influenced the endothelial cytoskeletal array of actin. Stress fibers were not present within the carotid endothelial cells of either the sham controls or the normotension and increased pulse pressure (normal flow) simulations. Endothelial cells lining carotids exposed to elevations in flow or those present within vessels perfused as per simulation b above assembled stress fibers (x = 4 and 10 per cell, respectively) within the time course of these studies. When endothelial cells were subjected to hemodynamic conditions that potentiated maximally cholesterol transport, no diffuse or stress fiber staining could be seen, but the cortical array of actin was intact. These results suggest that those biomechanical stresses that alter endothelial permeability and intimal integrity may do so via cytoskeletal actin signaling.