bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of solation-contraction coupling in regulating stress fiber dynamics in nonmuscle cells.
Authors: Kolega J , Janson L W , Taylor D L
Published: May 2004

Serum-deprived Swiss 3T3 fibroblasts constitutively form stress fibers at their edges. These fibers move centripetally towards the perinuclear region where they disassemble. Serum stimulation causes shortening of fibers in a manner suggesting active actin-myosin-based contraction (Giuliano, K.A. and D.L. Taylor. 1990. Cell Motil. and Cytoskeleton. 16:14-21). To elucidated the role of actin-based gel structure in these movements, we examined the effects of disrupting actin organization with cytochalasin. Serum-deprived fibroblasts were microinjected with rhodamine analogs of actin or myosin II and fiber dynamics were monitored with a multimode light microscope workstation using video-enhanced contrast and fluorescence modes. When cells were perfused with greater than or equal to 3 microM cytochalasin B or 0.5 microM cytochalasin D, formation and transport of stress fibers were reversibly inhibited, and rapid and immediate shortening of existing fibers was induced. Quantification of actin and myosin II fluorescence associated with individual shortening fibers demonstrated that fluorescence per length of fiber increased for both components, suggesting sliding filament contraction. However, there was also a net loss of both actin and myosin II from fibers as they shortened, indicating a self-destructive process. Loss of material from fibers coupled with increased overlap of actin and myosin II remaining in the fibers suggested that contraction could be induced not only by increasing the force exerted by contractile motors, but also by decreasing gel structure through partial solation. Finally, cytochalasin accelerated contraction of actin-myosin-based gels reconstituted from purified proteins in the absence of myosin-based regulation, further supporting solation-contraction coupling as a possible mechanism for modulating cytoplasmic contractility (Taylor, D.L. and M. Fechheimer. 1982. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 299:185-197).
Immunolocalization of myosin I heavy chain kinase in Acanthamoeba castellanii and binding of purified kinase to isolated plasma membranes.
Authors: Kulesza-Lipka D , Baines I C , Brzeska H , Korn E D
Published: May 2004

The actin-activated Mg(2+)-ATPase activities of Acanthamoeba myosins I are known to be maximally expressed only when a single threonine (myosin IA) or serine (myosins IB and IC) is phosphorylated by myosin I heavy chain kinase. The purified kinase is highly activated by autophosphorylation and the rate of autophosphorylation is greatly enhanced by the presence of acidic phospholipids. In this paper, we show by immunofluorescence and immunoelectron microscopy of permeabilized cells that myosin I heavy chain kinase is highly concentrated, but not exclusively, at the plasma membrane. Judged by their electrophoretic mobilities, kinase associated with purified plasma membranes may differ from the cytoplasmic kinase, possibly in the extent of its phosphorylation. Purified kinase binds to highly purified plasma membranes with an apparent KD of approximately 17 nM and a capacity of approximately 0.8 nmol/mg of plasma membrane protein, values that are similar to the affinity and capacity of plasma membranes for myosins I. Binding of kinase to membranes is inhibited by elevated ionic strength and by extensive autophosphorylation but not by substrate-level concentrations of ATP. Membrane-bound kinase autophosphorylates to a lesser extent than free kinase and does not dissociate from the membranes after autophosphorylation. The co-localization of myosin I heavy chain kinase and myosin I at the plasma membrane is of interest in relation to the possible functions of myosin I especially as phospholipids increase kinase activity.
A human centrosomal protein is immunologically related to basal body-associated proteins from lower eucaryotes and is involved in the nucleation of microtubules.
Authors: Moudjou M , Paintrand M , Vigues B , Bornens M
Published: May 2004

Isolation of centrosomes from human cells has revealed a proteic pattern which is both complex and specific. As the most prominent structural element of centrosomes in animal cells, the centriole which is present as two copies, is a highly conserved structure, we have attempted to identify centrosomal proteins on the basis of immunocross-reaction with proteins identified in basal bodies from lower eucaryotes. We report that two antibodies, one raised against the Ca(+)-binding protein centrin (Salisbury, J. L., A. T. Baron, B. Surek, and M. Melkonian. 1984. J. Cell Biol. 99:962-970) and the other directed against a 230-kD protein isolated from the infraciliary cytoskeletal lattice of the protozoan Polyplastron m., decorate the centrosome of human cultured cells, and identify one of the major centrosomal components revealed as a doublet of 62/64 kD. Moreover the nucleation reaction of microtubules, which can be efficiently produced on isolated centrosomes, is blocked by the antibodies, a result which strongly implicates the 62/64-kD protein in this centrosomal activity. We also show that the 62/64-kD protein remains insoluble in conditions (0.5 M KI or 8 M urea) which are capable of extracting most of the centrosomal proteins. Immunocytochemical localization by EM of isolated centrosomes revealed the association of this 62/64-kD doublet with the intercentriolar link and the pericentriolar lattice. Our results suggest that conservation of structure in the centrosome from divergent organisms could be matched by conservation of proteins and activity, evidence for the maintenance of a specific function, which could involve Ca2+, associated with the microtubule organizing centers.
Metastatic behavior of human melanoma cell lines in nude mice correlates with urokinase-type plasminogen activator, its type-1 inhibitor, and urokinase-mediated matrix degradation.
Authors: Quax P H , van Muijen G N , Weening-Verhoeff E J , Lund L R , Danø K , Ruiter D J , Verheijen J H
Published: May 2004

Five out of six human melanoma cell lines tested were able to degrade in vitro a smooth muscle cell extracellular matrix in a plasmin-dependent way. In three of these five cell lines, this process was mediated by tissue-type plasminogen activator (t-PA) and in the other two cell lines by urokinase-type plasminogen activator (u-PA). All melanoma cell lines produced t-PA mRNA and protein, whereas only the two cell lines showing u-PA-mediated matrix degradation produced u-PA mRNA and protein. These latter cell lines also produced plasminogen activator inhibitor type-1 (PAI-1) and type-2 (PAI-2) mRNA and protein. u-PA receptor (u-PA-R) mRNA and binding of radiolabeled u-PA was found in all melanoma cell lines. The metastatic capacity of these cell lines was studied in nude mice. All cell lines were able to develop primary tumors at the subcutaneous inoculation site. The production of plasminogen activators, their inhibitors and urokinase receptor by subcutaneous tumors corresponded with the production by the parental cell lines in vitro. The two u-PA and PAI-1 producing cell lines showed the highest frequency to form spontaneous lung metastases after subcutaneous inoculation, whereas five of the six cell lines formed lung colonies after intravenous inoculation. In conclusion, u-PA mediated matrix degradation in vitro and production of u-PA and PAI-1 by human melanoma cell lines correlated with their ability to form spontaneous lung metastasis in nude mice. No correlation was found with the ability to form lung colonies after intravenous injection. These findings suggest a role for u-PA and PAI-1 in a relatively early stage of melanoma metastasis.
The complement binding-like domains of the murine homing receptor facilitate lectin activity.
Authors: Watson S R , Imai Y , Fennie C , Geoffrey J , Singer M , Rosen S D , Lasky L A
Published: May 2004

The leukocyte homing receptor (HR), the endothelial leukocyte adhesion molecule, and gmp140/platelet activation-dependent granule membrane protein are members of a family of adhesion molecules, termed the lectin cell adhesion molecules (LEC-CAMS) which are unified by a multi-domain structure containing a lectin motif, an epidermal growth factor-like (egf) motif, and variable numbers of a complement binding-like (CB) motif. Previous data have indicated a predominant role for the lectin motif in cell adhesion directed by the LEC-CAMS, although the egf-like domain of the HR may also play a potential role in cell binding. While the role(s) of the CB domains in the LEC-CAMS is currently not understood, they have been hypothesized to act as rigid spacers or stalks for lectin and perhaps, egf domain presentation. In this paper, we analyze the functional characteristics of murine HR-IgG chimeras containing the lectin, lectin plus egf, and lectin plus egf plus CB domains. The Mel 14 mAb, an adhesion blocking antibody which recognizes a conformational determinant in the N-terminus of the HR lectin domain, shows a significantly decreased affinity for a HR construct which lacks the CB motifs, consistent with the possibility that the CB domains are involved with lectin domain structure. In agreement with this conjecture, HR mutants lacking the CB domains show a profound decrease in lectin-specific interaction with the carbohydrate polyphosphomannan ester, suggesting that the changes in Mel 14 affinity for the lectin domain are reflected in lectin functionality. Various assays investigating the interactions between the HR deletion mutants and the peripheral lymph node high endothelium, including cell blocking, immunohistochemical staining, and radioactively labeled ligand binding, all showed that removal of the CB domains results in a lack of HR adhesive function. These results imply that the CB domains of the HR, and, by analogy, the other members of the LEC-CAM family, may play important structural roles involving induction of lectin domain conformation and resultant functionality.
Calcium transients during Fc receptor-mediated and nonspecific phagocytosis by murine peritoneal macrophages.
Authors: Hishikawa T , Cheung J Y , Yelamarty R V , Knutson D W
Published: May 2004

Studies with populations of macrophages have produced conflicting results concerning the possibility that the concentration of intracellular ionized calcium [( Ca2+]i) may act as an important mediator for phagocytosis. Since asynchronous changes in [Ca2+]i in individual cells undergoing phagocytosis may be averaged to undetectability in population studies, we studied single adhering murine macrophages using fura-2 and our previously described digital imaging system. The proportion of macrophages phagocytosing IgG-coated latex beads was greater than for uncoated beads (percent phagocytosing cells: 71 +/- 7 vs. 27 +/- 7, P less than 0.01). Phagocytosis of IgG-coated and uncoated beads was always associated with a calcium transient that preceded the initiation of phagocytosis. No calcium transients were detected in cells that bound but did not phagocytose beads. Four major differences between Fc receptor-mediated and nonspecific phagocytosis were detected: (a) the duration of calcium transients was longer for nonspecific phagocytosis compared with Fc receptor-mediated phagocytosis (69.9 +/- 10.2 vs. 48.7 +/- 4.7 s, P less than 0.05) and the magnitude of calcium transients was less for nonspecific phagocytosis (178 +/- 43 vs. 349 +/- 53 nM, P less than 0.05); (b) removal of extracellular calcium abolished the calcium transients associated with nonspecific phagocytosis but had no effect on those associated with receptor-mediated phagocytosis; (c) in the absence of extracellular calcium, buffering intracellular calcium with a chelator reduced Fc receptor-mediated phagocytosis but had no additive inhibitory effect on nonspecific phagocytosis; and (d) inhibition of protein kinase C (PKC) with staurosporine inhibited nonspecific phagocytosis but had no effect on receptor-mediated phagocytosis. Our observations suggest that despite both types of phagocytosis being associated with intracellular calcium transients, the role played by intracellular calcium in the signaling pathways may differ for Fc receptor-mediated and nonspecific phagocytosis by elicited murine macrophages.
The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle.
Authors: Byers T J , Kunkel L M , Watkins S C
Published: May 2004

We use a highly specific and sensitive antibody to further characterize the distribution of dystrophin in skeletal, cardiac, and smooth muscle. No evidence for localization other than at the cell surface is apparent in skeletal muscle and no 427-kD dystrophin labeling was detected in sciatic nerve. An elevated concentration of dystrophin appears at the myotendinous junction and the neuromuscular junction, labeling in the latter being more intense specifically in the troughs of the synaptic folds. In cardiac muscle the distribution of dystrophin is limited to the surface plasma membrane but is notably absent from the membrane that overlays adherens junctions of the intercalated disks. In smooth muscle, the plasma membrane labeling is considerably less abundant than in cardiac or skeletal muscle and is found in areas of membrane underlain by membranous vesicles. As in cardiac muscle, smooth muscle dystrophin seems to be excluded from membrane above densities that mark adherens junctions. Dystrophin appears as a doublet on Western blots of skeletal and cardiac muscle, and as a single band of lower abundance in smooth muscle that corresponds most closely in molecular weight to the upper band of the striated muscle doublet. The lower band of the doublet in striated muscle appears to lack a portion of the carboxyl terminus and may represent a dystrophin isoform. Isoform differences and the presence of dystrophin on different specialized membrane surfaces imply multiple functional roles for the dystrophin protein.
The structural basis for the intrinsic disorder of the actin filament: the "lateral slipping" model.
Authors: Bremer A , Millonig R C , Sütterlin R , Engel A , Pollard T D , Aebi U
Published: May 2004

Three-dimensional (3-D) helical reconstructions computed from electron micrographs of negatively stained dispersed F-actin filaments invariably revealed two uninterrupted columns of mass forming the "backbone" of the double-helical filament. The contact between neighboring subunits along the thus defined two long-pitch helical strands was spatially conserved and of high mass density, while the intersubunit contact between them was of lower mass density and varied among reconstructions. In contrast, phalloidinstabilized F-actin filaments displayed higher and spatially more conserved mass density between the two long-pitch helical strands, suggesting that this bicyclic hepta-peptide toxin strengthens the intersubunit contact between the two strands. Consistent with this distinct intersubunit bonding pattern, the two long-pitch helical strands of unstabilized filaments were sometimes observed separated from each other over a distance of two to six subunits, suggesting that the intrastrand intersubunit contact is also physically stronger than the interstrand contact. The resolution of the filament reconstructions, extending to 2.5 nm axially and radially, enabled us to reproducibly "cut out" the F-actin subunit which measured 5.5 nm axially by 6.0 nm tangentially by 3.2 nm radially. The subunit is distinctly polar with a massive "base" pointing towards the "barbed" end of the filament, and a slender "tip" defining its "pointed" end (i.e., relative to the "arrowhead" pattern revealed after stoichiometric decoration of the filaments with myosin subfragment 1). Concavities running approximately parallel to the filament axis both on the inner and outer face of the subunit define a distinct cleft separating the subunit into two domains of similar size: an inner domain confined to radii less than or equal to 2.5-nm forms the uninterrupted backbone of the two long-pitch helical strands, and an outer domain placed at radii of 2-5-nm protrudes radially and thus predominantly contributes to the outer part of the massive base. Quantitative evaluation of successive crossover spacings along individual F-actin filaments revealed the deviations from the mean repeat to be compensatory, i.e., short crossovers frequently followed long ones and vice versa. The variable crossover spacings and diameter of the F-actin filament together with the local unraveling of the two long-pitch helical strands are explained in terms of varying amounts of compensatory "lateral slipping" of the two strands past each other roughly perpendicular to the filament axis. This intrinsic disorder of the actin filament may enable the actin moiety to play a more active role in actin-myosin-based force generation than merely act as a rigid passive cable as has hitherto been assumed.
Synthetic peptides from the carboxy-terminal globular domain of the A chain of laminin: their ability to promote cell adhesion and neurite outgrowth, and interact with heparin and the beta 1 integrin subunit.
Authors: Skubitz A P , Letourneau P C , Wayner E , Furcht L T
Published: May 2004

The large carboxy-terminal globular domain (G domain; residues 2,110-3,060) of the A chain of murine-derived laminin has been shown to promote heparin binding, cell adhesion, and neurite outgrowth. This study was conducted to define the potential sequence(s) originating from the G domain of laminin with any of these functional activities. A series of peptides were synthesized from the G domain, termed GD peptides, each approximately 20 amino acids long and containing multiple positively charged amino acids. In direct 3H-heparin binding assays, peptides GD-1 and GD-2 bound high levels of 3H-heparin, while peptides GD-3 and GD-4 bound lower levels of 3H-heparin, and GD-5 bound essentially no 3H-heparin. The binding of 3H-heparin to peptides GD-1 and GD-2 appeared to be of high affinity, since significant binding of 3H-heparin to these two peptides was still observed even when the NaCl concentration was raised to 1.0 M. Four of the peptides, GD-1, GD-2, GD-3, and GD-4, directly promoted the adhesion and spreading of HT-1080 human fibrosarcoma cells as well as the outgrowth of neurites from chick spinal cord and dorsal root ganglia neurons. In addition, solutions of these peptides or antibodies generated against these peptides inhibited laminin-mediated HT-1080 cell adhesion. Antibodies against the beta 1 integrin subunit inhibited HT-1080 cell adhesion and neurite outgrowth on surfaces adsorbed with peptides GD-3 and GD-4. Therefore, laminin appears to have multiple, independent sequences in the G domain that serve a similar cell adhesion promoting function for different cell types. Furthermore, these results suggest that the sequences comprising peptides GD-3 and GD-4 use an integrin as a receptor, of which the beta 1 integrin subunit is a component for these various cell types.
Mini-collagens in hydra nematocytes.
Authors: Kurz E M , Holstein T W , Petri B M , Engel J , David C N
Published: May 2004

We have isolated and characterized four collagen-related c-DNA clones (N-COL 1, N-COL 2, N-COL 3, N-COL 4) that are highly expressed in developing nematocytes in hydra. All four c-DNAs as well as their corresponding transcripts are small in size (600-1,000 bp). The deduced amino acid sequences show that they contain a central region consisting of 14 to 16 Gly-X-Y triplets. This region is flanked amino-terminal by a stretch of 14-23 proline residues and carboxy-terminal by a stretch of 6-9 prolines. At the NH2- and COOH-termini are repeated patterns of cysteine residues that are highly conserved between the molecules. A model is proposed which consists of a central stable collagen triple helix of 12-14 nm length from which three 9-22 nm long polyproline II type helices emerge at both ends. Disulfide linkage between cysteine-rich segments in these helices could lead to the formation of oligomeric network structures. Electrophoretic characterization of nematocyst extracts allows resolution of small proline-rich polypeptides that correspond in size to the cloned sequences.
Evidence from lateral mobility studies for dynamic interactions of a mutant influenza hemagglutinin with coated pits.
Authors: Fire E , Zwart D E , Roth M G , Henis Y I
Published: May 2004

Replacement of cysteine at position 543 by tyrosine in the influenza virus hemagglutinin (HA) protein enables the endocytosis of the mutant protein (Tyr 543) through coated pits (Lazarovits, J., and M. G. Roth. 1988. Cell. 53:743-752). To investigate the interactions between Tyr 543 and the clathrin coats in the plasma membrane of live cells, we performed fluorescence photobleaching recovery measurements comparing the lateral mobilities of Tyr 543 (which enters coated pits) and wild-type HA (HA wt, which is excluded from coated pits), following their expression in CV-1 cells by SV-40 vectors. While both proteins exhibited the same high mobile fractions, the lateral diffusion rate of Tyr 543 was significantly slower than that of HA wt. Incubation of the cells in a sucrose-containing hypertonic medium, a treatment that disperses the membrane-associated coated pits, resulted in similar lateral mobilities for Tyr 543 and HA wt. These findings indicate that the lateral motion of Tyr 543 (but not of HA wt) is inhibited by transient interactions with coated pits (which are essentially immobile on the time scale of the lateral mobility measurements). Acidification of the cytoplasm by prepulsing the cells with NH4Cl (a treatment that arrests the pinching-off of coated vesicles from the plasma membrane and alters the clathrin lattice morphology) led to immobilization of a significant part of the Tyr 543 molecules, presumably due to their entrapment in coated pits for the entire duration of the lateral mobility measurement. Furthermore, in both untreated and cytosol-acidified cells, the restrictions on Tyr 543 mobility were less pronounced in the cold, suggesting that the mobility-restricting interactions are temperature dependent and become weaker at low temperatures. From these studies we conclude the following. (a) Lateral mobility measurements are capable of detecting interactions of transmembrane proteins with coated pits in intact cells. (b) The interactions of Tyr 543 with coated pits are dynamic, involving multiple entries of Tyr 543 molecules into and out of coated pits. (c) Alterations in the clathrin lattice structure can modulate the above interactions.
Localization of the DMDL gene-encoded dystrophin-related protein using a panel of nineteen monoclonal antibodies: presence at neuromuscular junctions, in the sarcolemma of dystrophic skeletal muscle, in vascular and other smooth muscles, and in proliferating brain cell lines.
Authors: Nguyen T M , Ellis J M , Love D R , Davies K E , Gatter K C , Dickson G , Morris G E
Published: May 2004

mAbs have been raised against different epitopes on the protein product of the DMDL gene, which is an autosomal homologue of the X-linked DMD gene for dystrophin. These antibodies provide direct evidence that DMDL protein is localized near acetylcholine receptors at neuromuscular junctions in normal and mdx mouse intercostal muscle. The primary location in tissues other than skeletal muscle is smooth muscle, especially in the vascular system, which may account for the wide tissue distribution previously demonstrated by Western blotting. The DMDL protein was undetectable in the nonjunctional sarcolemma of normal human muscle, but was observed in nonjunctional sarcolemma of Duchenne muscular dystrophy patients, where dystrophin itself is absent or greatly reduced. The expression of DMDL protein is not restricted to smooth and skeletal muscle, however, since relatively large amounts are present in transformed brain cell lines of both glial and Schwann cell origin. This contrasts with the low levels of DMDL protein in adult brain tissue.
Surface relocation of alpha 6 beta 4 integrins and assembly of hemidesmosomes in an in vitro model of wound healing.
Authors: Kurpakus M A , Quaranta V , Jones J C
Published: May 2004

A transmembrane extracellular matrix receptor of the integrin family, alpha 6 beta 4, is a component of the hemidesmosome, an adhesion complex of importance in epithelial cell-connective tissue attachment (Stepp, M. A., S. Spurr-Michaud, A. Tisdale, J. Elwell, and I. K. Gipson. 1990. Proc. Natl. Acad. Sci. USA. 87:8970-8974; Jones, J. C. R., M. A. Kurpakus, H. M. Cooper, and V. Quaranta. 1991. Cell Regulation. 2:427-438). Cytosolic components of hemidesmosomes include bullous pemphigoid (BP) antigens while extracellular components include a 125-kD component of anchoring filaments (CAF) and collagen type VII-containing anchoring fibrils. We have monitored the incorporation of the alpha 6 beta 4 integrins into forming hemidesmosomes in an in vitro wound-healing explant model. In epithelial cells recently migrated from the edges of unwounded sites over bare connective tissue, alpha 6 beta 4 first appears along the entire cell surface. At this stage, these cells contain little or no cytosolic hemidesmosomal components, at least as detectable by immunofluorescence using BP autoantibodies, whereas they are already positive for laminin and CAF. At a later stage, as cells become positive for cytosolic hemidesmosome components such as BP antigens as well as collagen type VII, alpha 6 beta 4 becomes concentrated along the basal pole of the epithelial cell where it abuts the connective tissue of the explant. Polyclonal antibodies to beta 4 do not interfere with the migration of epithelial cells in the explant. However, they prevent assembly of hemidesmosomal complexes and inhibit expression of collagen type VII in cells that have migrated over wound areas. In addition, they induce disruption of established hemidesmosomes in nonmigrating cells of the unwounded area of the explant. Monoclonal antibodies to alpha 6 have a more dramatic effect, since they completely detach epithelial cells in the unwounded area of the explant. Antibodies to CAF also detach epithelial cells in unwounded areas, apparently by inducing separation between epithelium and connective tissue at the lamina lucida of the basement membrane zone. These results suggest a model whereby polarization of alpha 6 beta 4 to the basal surface of the cells, perhaps induced by a putative anchoring filament-associated ligand, triggers assembly of hemidesmosome plaques.
A mutation in the signal recognition particle 7S RNA of the yeast Yarrowia lipolytica preferentially affects synthesis of the alkaline extracellular protease: in vivo evidence for translational arrest.
Authors: Yaver D S , Matoba S , Ogrydziak D M
Published: May 2004

Replacement of the signal recognition particle (SRP) 7S gene (SCR1) on a replicating plasmid with scr1-1 (G to A at 129 and A to T at 131 in the consensus sequence -GNAR- in the loop of domain III) resulted in temperature sensitivity for growth of cells in which both chromosomal SRP 7S RNA genes were deleted. Pulse-chase immunoprecipitation experiments were done after a shift to non-permissive temperature using the major secreted protein the alkaline extracellular protease (AEP) as a reporter molecule. No untranslocated AEP precursor was detected in a strain with scr1-1 on a plasmid, but the amount of the largest AEP precursor (55 kD) immunoprecipitated as a percentage of total protein synthesized was reduced 68% compared to an isogenic strain with SCR1 on the plasmid. The possibility that an untranslocated precursor was synthesized but not detected because of instability was largely eliminated by detection of a 53-kD untranslocated precursor of a mutated AEP (P17M; methionine replaced proline in the second position of the pro-peptide) which chased to the 55-kD translocated AEP precursor. Thus, SRP has a role in the biosynthesis of AEP. Possibly, the scr1-1 mutation does not affect signal recognition or translational arrest but instead results in maintenance of translational arrest of AEP synthesis. The results also suggest that AEP can be translocated in vivo either co-translationally in which SRP is at least involved in biosynthesis or posttranslationally without SRP involvement.
Phosphorylation of conserved serine residues does not regulate the ability of mosxe protein kinase to induce oocyte maturation or function as cytostatic factor.
Authors: Freeman R S , Meyer A N , Li J , Donoghue D J
Published: May 2004

Expression of the mosxe protein kinase is required for the normal meiotic maturation of Xenopus oocytes and overexpression induces maturation in the absence of other stimuli. In addition, mosxe functions as a component of cytostatic factor (CSF), an activity responsible for arrest of the mature egg at metaphase II. After microinjection of Xenopus oocytes with in vitro synthesized RNA encoding either wild-type mosxe or kinase-inactive mosxe(R90), both proteins are phosphorylated exclusively on serine residues and exhibit essentially identical chymotryptic maps. Since the phosphorylated kinase-inactive mosxe(R90) protein was recovered from resting oocytes that have not yet begun to translate endogenous mosxe, this indicates that the major phosphopeptides of mosxe(R90) are phosphorylated by a preexisting protein kinase present in resting oocytes, and are not the result of autophosphorylation. The results presented here also indicate that the mosxe protein does not undergo significant phosphorylation at unique sites during oocyte maturation. If the biological activity of mosxe were regulated by phosphorylation, a site of regulatory phosphorylation would most likely be conserved among mos proteins of different species. Site-directed mutagenesis was used to construct 13 individual serine----alanine mutations at conserved residues (3, 16, 18, 25, 26, 57, 71, 76, 102, 105, 127, 211, and 258). These 13 mutants were analyzed for their abilities to induce oocyte maturation and to function as CSF. Results obtained with the mosxe(A105) mutant revealed that serine-105 is required for both maturation induction and CSF activity, even though serine-105 does not represent a major site of phosphorylation. All of the remaining serine----alanine mosxe mutants induced oocyte maturation and exhibited CSF activity comparable with the wild type. These results demonstrate that none of the conserved serines examined in this study function as regulatory phosphorylation sites for these biological activities. Peptide mapping of the remaining mosxe mutants identified serine-3 as a major phosphorylation site in vivo, which is contained within the chymotryptic peptide MPSPIPVERF.
Thin-section ratiometric Ca2+ images obtained by optical sectioning of fura-2 loaded mast cells.
Authors: Monck J R , Oberhauser A F , Keating T J , Fernandez J M
Published: May 2004

The availability of the ratiometric Ca2+ indicator dyes, fura-2, and indo-1, and advances in digital imaging and computer technology have made it possible to detect Ca2+ changes in single cells with high temporal and spatial resolution. However, the optical properties of the conventional epifluorescence microscope do not produce a perfect image of the specimen. Instead, the observed image is a spatial low pass filtered version of the object and is contaminated with out of focus information. As a result, the image has reduced contrast and an increased depth of field. This problem is especially important for measurements of localized Ca2+ concentrations. One solution to this problem is to use a scanning confocal microscope which only detects in focus information, but this approach has several disadvantages for low light fluorescence measurements in living cells. An alternative approach is to use digital image processing and a deblurring algorithm to remove the out of focus information by using a knowledge of the point spread function of the microscope. All of these algorithms require a stack of two-dimensional images taken at different focal planes, although the "nearest neighbor deblurring" algorithm only requires one image above and below the image plane. We have used a modification of this scheme to construct a simple inverse filter, which extracts optical sections comparable to those of the nearest neighbors scheme, but without the need for adjacent image sections. We have used this "no neighbors" processing scheme to deblur images of fura-2-loaded mast cells from beige mice and generate high resolution ratiometric Ca2+ images of thin sections through the cell. The shallow depth of field of these images is demonstrated by taking pairs of images at different focal planes, 0.5-microns apart. The secretory granules, which exclude the fura-2, appear in focus in all sections and distinct changes in their size and shape can be seen in adjacent sections. In addition, we show, with the aid of model objects, how the combination of inverse filtering and ratiometric imaging corrects for some of the inherent limitations of using an inverse filter and can be used for quantitative measurements of localized Ca2+ gradients. With this technique, we can observe Ca2+ transients in narrow regions of cytosol between the secretory granules and plasma membrane that can be less than 0.5-microns wide. Moreover, these Ca2+ increases can be seen to coincide with the swelling of the secretory granules that follows exocytotic fusion.
Induction of proliferation or hypertrophy of chondrocytes in serum-free culture: the role of insulin-like growth factor-I, insulin, or thyroxine.
Authors: Böhme K , Conscience-Egli M , Tschan T , Winterhalter K H , Bruckner P
Published: May 2004

In bone forming cartilage in vivo, cells undergo terminal differentiation, whereas most of the cells in normal articular cartilage do not. Chondrocyte hypertrophy can be induced also in vitro by diffusible signals. We have identified growth factors or hormones acting individually on 17-d chick embryo sternal chondrocytes cultured in agarose gels under strictly serum-free conditions. Insulin-like growth factor I or insulin triggered the first steps of chondrocyte maturation, i.e., cell proliferation and increased matrix deposition while the chondrocytic phenotype was maintained. However, cells did not progress to the hypertrophic stage. Proliferation and stimulated collagen production was preceded by a lag period, indicating that synthesis of other components was required before cells became responsive to insulin-like growth factor I or insulin. Very small amounts of FBS exerted effects similar to those of insulin-like growth factor I or insulin. However, FBS could act directly and elicited hypertrophy when constituting greater than 1% of the culture media. Basic FGF has been claimed to be the most potent chondrocyte mitogen, but had negligible effects under serum-free conditions. The same is true for PDGF, a major serum-mitogen. Under the direction of thyroxine, cells did not proliferate but became typical hypertrophic chondrocytes, extensively synthesizing collagen X and alkaline phosphatase.
Remodeling the cell surface distribution of membrane proteins during the development of epithelial cell polarity.
Authors: Wollner D A , Krzeminski K A , Nelson W J
Published: May 2004

The development of polarized epithelial cells from unpolarized precursor cells follows induction of cell-cell contacts and requires resorting of proteins into different membrane domains. We show that in MDCK cells the distributions of two membrane proteins, Dg-1 and E-cadherin, become restricted to the basal-lateral membrane domain within 8 h of cell-cell contact. During this time, however, 60-80% of newly synthesized Dg-1 and E-cadherin is delivered directly to the forming apical membrane and then rapidly removed, while the remainder is delivered to the basal-lateral membrane and has a longer residence time. Direct delivery of greater than 95% of these proteins from the Golgi complex to the basal-lateral membrane occurs greater than 48 h later. In contrast, we show that two apical proteins are efficiently delivered and restricted to the apical cell surface within 2 h after cell-cell contact. These results provide insight into mechanisms involved in the development of epithelial cell surface polarity, and the establishment of protein sorting pathways in polarized cells.
Analysis of the actin-binding domain of alpha-actinin by mutagenesis and demonstration that dystrophin contains a functionally homologous domain.
Authors: Hemmings L , Kuhlman P A , Critchley D R
Published: May 2004

To define the actin-binding site within the NH2-terminal domain (residues 1-245) of chick smooth muscle alpha-actinin, we expressed a series of alpha-actinin deletion mutants in monkey Cos cells. Mutant alpha-actinins in which residues 2-19, 217-242, and 196-242 were deleted still retained the ability to target to actin filaments and filament ends, suggesting that the actin-binding site is located within residues 20-195. When a truncated alpha-actinin (residues 1-290) was expressed in Cos cells, the protein localized exclusively to filament ends. This activity was retained by a deletion mutant lacking residues 196-242, confirming that these are not essential for actin binding. The actin-binding site in alpha-actinin was further defined by expressing both wild-type and mutant actin-binding domains as fusion proteins in E. coli. Analysis of the ability of such proteins to bind to F-actin in vitro showed that the binding site was located between residues 108 and 189. Using both in vivo and in vitro assays, we have also shown that the sequence KTFT, which is conserved in several members of the alpha-actinin family of actin-binding proteins (residues 36-39 in the chick smooth muscle protein) is not essential for actin binding. Finally, we have established that the NH2-terminal domain of dystrophin is functionally as well as structurally homologous to that in alpha-actinin. Thus, a chimeric protein containing the NH2-terminal region of dystrophin (residues 1-233) fused to alpha-actinin residues 244-888 localized to actin-containing structures when expressed in Cos cells. Furthermore, an E. coli-expressed fusion protein containing dystrophin residues 1-233 was able to bind to F-actin in vitro.
The activity of cAMP-dependent protein kinase is required at a posttranslational level for induction of voltage-dependent sodium channels by peptide growth factors in PC12 cells.
Authors: Ginty D D , Fanger G R , Wagner J A , Maue R A
Published: May 2004

The synthesis and expression of voltage-dependent sodium (Na) channels is a crucial aspect of neuronal differentiation because of the central role these ion channels play in the generation of action potentials and the transfer of information in the nervous system. We have used rat pheochromocytoma (PC12) cell lines deficient in cAMP-dependent protein kinase (PKA) activity to examine the role of PKA in the induction of Na channel expression by nerve growth factor (NGF) and basic FGF (bFGF). In the parental PC12 cell line both NGF and bFGF elicit an increase in the density of functional Na channels, as determined from whole-cell patch clamp recordings. This increase does not occur in two PC12 cell lines deficient in both isozymes of PKA (PKAI and PKAII), and is strongly reduced in a third line deficient in PKAII, but not PKAI. Despite the inability of the neurotrophic factors to induce functional Na channel expression in the PKA-deficient cells, Northern blot hybridization studies and saxitoxin binding assays of intact cells indicate that NGF and bFGF are still capable of eliciting increases in both Na channel mRNA and Na channel protein in the membrane. Thus, PKA activity appears to be necessary at a posttranslational step in the synthesis and expression of functional Na channels, and thereby plays an important role in determining neuronal excitability.
Mechanisms of cell shape change: the cytomechanics of cellular response to chemical environment and mechanical loading
Authors: Adams DS
Published: May 2004

Processes such as cell locomotion and morphogenesis depend on both the generation of force by cytoskeletal elements and the response of the cell to the resulting mechanical loads. Many widely accepted theoretical models of processes involving cell shape change are based on untested hypotheses about the interaction of these two components of cell shape change. I have quantified the mechanical responses of cytoplasm to various chemical environments and mechanical loading regimes to understand better the mechanisms of cell shape change and to address the validity of these models. Measurements of cell mechanical properties were made with strands of cytoplasm submerged in media containing detergent to permeabilize the plasma membrane, thus allowing control over intracellular milieu. Experiments were performed with equipment that generated sinusoidally varying length changes of isolated strands of cytoplasm from Physarum polycephalum. Results indicate that stiffness, elasticity, and viscosity of cytoplasm all increase with increasing concentration of Ca2+, Mg2+, and ATP, and decrease with increasing magnitude and rate of deformation. These results specifically challenge assumptions underlying mathematical models of morphogenetic events such as epithelial folding and cell division, and further suggest that gelation may depend on both actin cross-linking and actin polymerization.
Balbiani ring hnRNP substructure visualized by selective staining and electron spectroscopic imaging.
Authors: Olins A L , Olins D E , Bazett-Jones D P
Published: May 2004

The Balbiani Rings (BR) in the polytene chromosomes of Chironomus salivary glands are intense sites of transcription. The nascent RNPs fold during transcription into 40-50-nm granules, containing in the mature transcript approximately 37-kb RNA. Using a new nucleic acid specific stain, osmium ammine B on Lowicryl sections, in combination with electron energy filtered imaging of sections containing BR granules, we demonstrate a RNA-rich particulate substructure (10-nm particle diameter; 10-12 particles per BR granule). Elemental imaging supports that these particles are enriched in phosphorus. The possible relationship of these RNA-rich particles to ribonucleosomes is discussed, as well as models for their arrangement in the mature BR granules.
Neutrophil migration across monolayers of cytokine-prestimulated endothelial cells: a role for platelet-activating factor and IL-8.
Authors: Kuijpers T W , Hakkert B C , Hart M H , Roos D
Published: May 2004

In a previous study we observed that neutrophils respond with a rapid rise in [Ca2+]i during adherence to cytokine-activated endothelial cells (EC), caused by EC membrane-associated platelet-activating factor (PAF). In the present study, we investigated whether this form of PAF was important in neutrophil adherence and migration across monolayers of rIL-1 beta- or rTNF alpha-prestimulated EC. PAF receptor antagonists prevented neutrophil migration across cytokine-pretreated EC by approximately 60% (P less than 0.005) without interfering with the process of adherence. The antagonists WEB 2086 and L-652,731 had no effect on neutrophil migration across resting EC induced by formylmethionyl-leucyl-phenylalanine (FMLP). A murine anti-IL-8 antiserum was found to also partially inhibit the neutrophil transmigration across cytokine-activated EC. When the anti-IL-8 antiserum was used in combination with a PAF receptor antagonist, neutrophil migration across cytokine-pretreated monolayers of EC was completely prevented. During transmigration, LAM-1 and CD44 on the neutrophils were down-modulated; both WEB 2086 and anti-IL-8 antiserum partially prevented this down-modulation caused by cytokine-prestimulated EC. Our results indicate that human neutrophils are activated and guided by EC-associated PAF and EC-derived IL-8 during the in vitro diapedesis in between cytokine-stimulated EC.
In vitro microtubule-nucleating activity of spindle pole bodies in fission yeast Schizosaccharomyces pombe: cell cycle-dependent activation in xenopus cell-free extracts
Authors: Masuda H , Sevik M , Cande WZ
Published: May 2004

The spindle pole body (SPB) is the equivalent of the centrosome in fission yeast. In vivo it nucleates microtubules (MTs) during mitosis, but, unlike animal centrosomes, does not act as a microtubule organizing center (MTOC) during interphase. We have studied the MT-nucleating activity of SPBs in vitro and have found that SPBs in permeabilized cells retain in vivo characteristics. SPBs in cells permeabilized during mitosis can nucleate MTs, and are recognized by two antibodies: anti-gamma-tubulin and MPM-2 which recognizes phosphoepitopes. SPBs in cells permeabilized during interphase cannot nucleate MTs and are only recognized by anti-gamma-tubulin. Interphase SPBs which cannot nucleate can be converted to a nucleation competent state by incubation in cytostatic factor (CSF)-arrested Xenopus egg extracts. After incubation, they are recognized by MPM-2, and can nucleate MTs. The conversion does not occur in Xenopus interphase extract, but occurs in Xenopus interphase extract driven into mitosis by preincubation with exogenous cyclin B. The conversion is ATP dependent and inhibited by protein kinase inhibitors and alkaline phosphatase. Purified, active, cdc2 kinase/cyclin B complex in itself is not effective for activation of MT nucleation, although some interphase SPBs are now stained with MPM-2. These results suggest that the ability of SPBs in vitro to nucleate MTs after exposure to CSF-arrested extracts is activated through a downstream pathway which is regulated by cdc2 kinase.
Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase
Authors: Shamu CE , Murray AW
Published: May 2004

We have produced metaphase spindles and induced them to enter anaphase in vitro. Sperm nuclei were added to frog egg extracts, allowed to replicate their DNA, and driven into metaphase by the addition of cytoplasm containing active maturation promoting factor (MPF) and cytostatic factor (CSF), an activity that stabilizes MPF. Addition of calcium induces the inactivation of MPF, sister chromatid separation and anaphase chromosome movement. DNA topoisomerase II inhibitors prevent chromosome segregation at anaphase, demonstrating that the chromatids are catenated at metaphase and that decatenation occurs at the start of anaphase. Topoisomerase II activity towards exogenous substrates does not increase at the metaphase to anaphase transition, showing that chromosome separation at anaphase is not triggered by a bulk activation of topoisomerase II.
Local expression and exocytosis of viral glycoproteins in multinucleated muscle cells
Authors: Metsikkö K , Hentunen T , Väänänen K
Published: May 2004

We have analyzed the distribution of enveloped viral infections in multinucleated L6 muscle cells. A temperature-sensitive vesicular stomatitis virus (mutant VSV ts045) was utilized at the nonpermissive temperature (39 degrees C). As expected, the glycoprotein (G protein) of this mutant was restricted to the ER when the multinucleated cells were maintained at 39 degrees C. We demonstrate that this G protein remained localized when the infection was performed at low dose. By 4 h after infection the G protein patches spanned an average of 220 microns. The localization was independent of nuclear positions, showing that the ER was a peripheric structure. Thus, the infection did not recognize nuclear domains characteristic of nuclearly encoded proteins. After release of the 39 degrees C block, transport through a perinuclear compartment into a restricted surface domain lying above the internal G protein patch occurred. Accordingly, the transport pathway was locally restricted. After a 16-h infection the G protein spanned 420 microns, while the matrix protein occupied 700-800 microns of the myotube length. Double infection of multinucleated L6 muscle cells with Semliki Forest virus and VSV at high multiplicities showed that the glycoprotein of each virus occupied intracellular domains which were devoid of the other respective glycoprotein. Taken together, these findings indicate that the viral glycoproteins did not range far from their site of synthesis within the ER or other intracellular membrane compartments in these large cells. This result also suggests that relocation of viral RNA synthesis occurred slowly.