bluuebunny/crossref_metadata_2025 huggingface dataset:
Probing actin polymerization by intermolecular cross-linking.
Authors: Millonig R , Salvo H , Aebi U
Published: May 2004

We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an apparent molecular mass of 86 kD by SDS-PAGE [LD]) is attenuated, while an actin filament dimer ("upper" dimer with an apparent molecular mass of 115 kD by SDS-PAGE [UD] as characterized [Elzinga, M., and J. J. Phelan. 1984. Proc. Natl. Acad. Sci. USA. 81:6599-6602]) is formed. This shift from LD to UD occurs concomitant with formation of filaments as assayed by N-(1-pyrenyl)iodoacetamide fluorescence enhancement and electron microscopy. Isolated cross-linked LD does not form filaments, while isolated cross-linked UD will assemble into filaments indistinguishable from those polymerized from unmodified G-actin under typical filament-forming conditions. The presence of cross-linked LD does not effect the kinetics of polymerization of actin monomer, whereas cross-linked UD shortens the "lag phase" of the polymerization reaction in a concentration-dependent fashion. Several converging lines of evidence suggest that, although accounting for a significant oligomeric species formed during early polymerization, the LD is incompatible with the helical symmetry defining the mature actin filament; however, it could represent the interfilament dimer found in paracrystalline arrays or filament bundles. Furthermore, the LD is compatible with the unit cell structure and symmetry common to various types of crystalline actin arrays (Aebi, U., W. E. Fowler, G. Isenberg, T. D. Pollard, and P. R. Smith. 1981. J. Cell Biol. 91:340-351) and might represent the major structural state in which a mutant beta-actin (Leavitt, J., G. Bushar, T. Kakunaga, H. Hamada, T. Hirakawa, D. Goldman, and C. Merril. 1982. Cell. 28:259-268) is arrested under polymerizing conditions.
Epidermal growth factor controls smooth muscle alpha-isoactin expression in BC3H1 cells.
Authors: Wang Y C , Rubenstein P A
Published: May 2004

We have examined the effects of epidermal growth factor (EGF), platelet-derived growth factor, and insulin on the differentiation of a mouse vascular smooth muscle-like cell line, the BC3H1 cells. On the basis of cell morphology and smooth muscle alpha-isoactin synthesis, we demonstrate that EGF at physiological concentrations prevents the differentiation of these cells, whereas platelet-derived growth factor has no apparent effect. The induction of alpha-isoactin synthesis by serum deprivation is inhibited by EGF in a dose-dependent manner with a half-maximal effect at 3-5 ng/ml and a maximal inhibition at approximately 30 ng/ml. Northern analysis also shows that EGF blocks the accumulation of alpha-isoactin mRNA normally observed during cell differentiation. Addition of EGF to differentiated cells results in a repression of alpha-isoactin synthesis, a stimulation of beta- and gamma-isoactin synthesis, and the stabilization of the nonmuscle isoactins. The synthesis of creatine phosphokinase, a muscle-specific noncontractile protein, is also regulated by EGF in a similar fashion. Modulation by EGF of alpha-isoactin expression is not affected by aphidicolin and is therefore independent of its mitogenic effect on these cells. Insulin is not required for observation of the EGF-dependent effects but instead seems to promote differentiation. Our results show that EGF can replace serum in controlling the differentiation of BC3H1 cells.
A role for gangliosides in astroglial cell differentiation in vitro.
Authors: Facci L , Skaper S D , Favaron M , Leon A
Published: May 2004

Rat cerebral astroglial cells in culture display specific morphological and biochemical behaviors in response to exogenously added gangliosides. To examine a potential function for endogenous gangliosides in the processes of astroglial cell differentiation, we have used the B subunit of cholera toxin as a ganglioside-specific probe. The B subunit, which is multivalent and binds specifically to GM1 ganglioside on the cell surface, induced a classical star-shaped (stellate) morphology in the astroglial cells and inhibited DNA synthesis in a dose-dependent manner. The morphological response was massive and complete within 2 h, with an ED50 of 0.8 nM, and appeared to depend on the direct interaction of the B subunit with GM1 on the cell surface. A B subunit-evoked inhibition of DNA synthesis and cell division (ED50 = 0.2 nM) was observed when the cells were stimulated with defined mitogens, such as epidermal growth factor and basic fibroblast growth factor. Maximal inhibition approached 80% within 24 h. The effects of the B subunit were unrelated to increases in cAMP. These observations, taken together with previous studies, demonstrate that both endogenously occurring plasma membrane gangliosides and exogenously supplied gangliosides can influence the differentiative state (as judged by morphological and growth behaviors) of astroglial cells in vitro.
Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation.
Authors: Martin D P , Schmidt R E , DiStefano P S , Lowry O H , Carter J G , Johnson E M
Published: May 2004

We have developed an experimental paradigm to study the mechanism by which nerve growth factor (NGF) allows the survival of sympathetic neurons. Dissociated sympathetic neurons from embryonic day-21 rats were grown in vitro for 7 d in the presence of NGF. Neurons were then deprived of trophic support by adding anti-NGF antiserum, causing them to die between 24 and 48 h later. Ultrastructural changes included disruption of neurites, followed by cell body changes characterized by an accumulation of lipid droplets, changes in the nuclear membrane, and dilation of the rough endoplasmic reticulum. No primary alterations of mitochondria or lysosomes were observed. The death of NGF-deprived neurons was characterized biochemically by assessing [35S]methionine incorporation into TCA precipitable protein and by measuring the release of the cytosolic enzyme adenylate kinase into the culture medium. Methionine incorporation began to decrease approximately 18 h post-deprivation and was maximally depressed by 36 h. Adenylate kinase began to appear in the culture medium approximately 30 h after deprivation, reaching a maximum by 54 h. The death of NGF-deprived neurons was entirely prevented by inhibiting protein or RNA synthesis. Cycloheximide, puromycin, anisomycin, actinomycin-D, and dichlorobenzimidazole riboside all prevented neuronal death subsequent to NGF deprivation as assessed by the above morphologic and biochemical criteria. The fact that sympathetic neurons must synthesize protein and RNA to die when deprived of NGF indicates that NGF, and presumably other neurotrophic factors, maintains neuronal survival by suppressing an endogenous, active death program.
Effects of heat shock on the expression of thrombospondin by endothelial cells in culture.
Authors: Ketis N V , Lawler J , Hoover R L , Karnovsky M J
Published: May 2004

Heat-shock proteins from confluent primary cultures of bovine aortic endothelial cells were analyzed by SDS-polyacrylamide gels. In addition to the increased synthesis of the classical heat-shock proteins, there is an increase of a 180,000-mol wt polypeptide in the growth media of heat-shocked cells. Immunoprecipitation with specific antiserum indicates that the 180,000-mol wt polypeptide is thrombospondin. Assay of mRNA levels coding for thrombospondin after brief hyperthermic treatment (45 degrees C, 10 min), followed by a recovery of 2 h at 37 degrees C, results in a twofold increase in mRNA abundance. In contrast, the activation level of the 71,000-mol wt heat-shock protein mRNA occurs at an earlier time than for thrombospondin mRNA. Immunofluorescence microscopy was used to study the intracellular and extracellular distribution of thrombospondin. Thrombospondin is localized to a prominent pattern of granules of intracellular fluorescence in a perinuclear distribution in cells not exposed to heat. Upon heat treatment, the pattern of granules of intracellular fluorescence appears more pronounced, and the fluorescence appears to be clustered more about the nucleus. There are at least three pools of extracellular forms of thrombospondin: (a) the fine fibrillar extracellular matrix thrombospondin; (b) the punctate granular thrombospondin; and (c) the thrombospondin found in the conditioned medium not associated with the extracellular matrix. When bovine aortic endothelial cells are exposed to heat, the extracellular matrix staining of a fibrillar nature is noticeably decreased, with an increase in the number and degree of fluorescence of focal areas where the punctate granule thrombospondin structures are highly localized. No gross morphological changes in extracellular matrix staining of fibronectin was noted. However, the intermediate filament network was very sensitive and collapsed around the nucleus after heat shock. We conclude that the expression of thrombospondin is heat-shock stimulated.
Differential effects of transforming growth factor-beta on the synthesis of extracellular matrix proteins by normal fetal rat calvarial bone cell populations.
Authors: Wrana J L , Maeno M , Hawrylyshyn B , Yao K L , Domenicucci C , Sodek J
Published: May 2004

To determine the effects of transforming growth factor-beta (TGF-beta) on the different cell types that exist in bone, cell populations (I-IV), progressively enriched in osteoblastic cells relative to fibroblastic cells, were prepared from fetal rat calvaria using timed collagenase digestions. TGF-beta did not induce anchorage-independent growth of these cells, nor was anchorage-dependent growth stimulated in most populations studied, despite a two- to threefold increase in the synthesis of cellular proteins. In all populations the synthesis of secreted proteins increased 2-3.5-fold. In particular, collagen, fibronectin, and plasminogen activator inhibitor synthesis was stimulated. However, different degrees of stimulation of individual proteins were observed both within and between cell populations. A marked preferential stimulation of plasminogen activator inhibitor was observed in each population, together with a slight preferential stimulation of collagen; the effect on collagen expression being directed primarily at type I collagen. In contrast, the synthesis of SPARC (secreted protein acidic rich in cysteine/osteonectin was stimulated approximately two-fold by TGF-beta, but only in fibroblastic populations. Collectively, these results demonstrate that TGF-beta stimulates matrix production by bone cells and, through differential effects on individual matrix components, may also influence the nature of the matrix formed by different bone cell populations. In the presence of TGF-beta, osteoblastic cells lost their polygonal morphology and alkaline phosphatase activity was decreased, reflecting a suppression of osteoblastic features. The differential effects of TGF-beta on bone cell populations are likely to be important in bone remodeling and fracture repair.
Modulation of matrix adhesive responses of human neuroblastoma cells by neighboring sequences in the fibronectins.
Authors: Mugnai G , Lewandowska K , Carnemolla B , Zardi L , Culp L A
Published: May 2004

Attachment and neurite extension have been measured when Platt or La-N1 human neuroblastoma cells respond to tissue culture substrata coated with a panel of complementary fragments from the individual chains of human plasma (pFN) or cellular fibronectins (cFN) purified from thermolysin digests. A 110-kD fragment (f110), which contains the Arg-Gly-Asp-Ser sequence (RGDS)-dependent cell-binding domain but no heparin-binding domains and whose sequences are shared in common by both the alpha- and beta-subunits of pFN, facilitated attachment of cells that approached the level observed with either intact pFN or the heparan sulfate-binding platelet factor-4 (PF4). This attachment on f110 was resistant to RGDS-containing peptide in the medium. Neurite outgrowth was also maximal on f110, and half of these neurites were also resistant to soluble RGDS peptide. Treatment of cells with glycosaminoglycan lyases failed to alter these responses on f110. Therefore, there is a second "cell-binding" domain in the sequences represented by f110 that is not RGDS- or heparan sulfate-dependent and that facilitates stable attachment and some neurite outgrowth; this domain appears to be conformation-dependent. Comparisons were also made between two larger fragments generated from the two subunits of pFN-f145 from the alpha-subunit and f155 from the beta-subunit--both of which contain the RGDS-dependent cell-binding domain and the COOH-terminal heparin-binding domain but which differ in the former's containing some IIICS sequence at its COOH terminus and the latter's having an additional type III homology unit. Heparin-binding fragments (with no RGDS activity) of f29 and f38, derived from f145 or f155 of pFN, respectively, and having the same differences in sequence, were also compared with f44 + 47 having the "extra domain" characteristic of cFN. Attachment on f145 was slightly sensitive to soluble RGDS peptide; attachment on f155 was much more sensitive. There were also differences in the percentage of cells with neurites on f145 vs. f155 but neurites on either fragment were completely sensitive to RGDS peptide. Mixing of f29, f38, or PF4 with f110 could not reconstitute the activities demonstrated in f145 or f155, demonstrating that covalently linked sequences are critical in modulating these responses. However, mixing of f44 + 47 from cFN with f110 from pFN increased the sensitivity to RGDS peptide.(ABSTRACT TRUNCATED AT 400 WORDS)
Adhesion of glycosaminoglycan-deficient chinese hamster ovary cell mutants to fibronectin substrata.
Authors: LeBaron R G , Esko J D , Woods A , Johansson S , Höök M
Published: May 2004

We have examined the role of cell surface glycosaminoglycans in fibronectin-mediated cell adhesion by analyzing the adhesive properties of Chinese hamster ovary cell mutants deficient in glycosaminoglycans. The results of our study suggest that the absence of glycosaminoglycans does not affect the initial attachment and subsequent spreading of these cells on substrata composed of intact fibronectin or a fibronectin fragment containing the primary cell-binding domain. However, in contrast to wild-type cells, the glycosaminoglycan-deficient cells did not attach to substrate composed of a heparin-binding fibronectin fragment. Furthermore, the wild-type but not the glycosaminoglycan-deficient cells formed F-actin-containing stress fibers and focal adhesions on substrata composed of intact fibronectin. We propose, therefore, that cell surface proteoglycan(s) participate in the transmembrane linking of intracellular cytoskeletal components to extracellular matrix components which occurs in focal adhesions.
Mouse mammary epithelial cells produce basement membrane and cell surface heparan sulfate proteoglycans containing distinct core proteins.
Authors: Jalkanen M , Rapraeger A , Bernfield M
Published: May 2004

Cultured mouse mammary (NMuMG) cells produce heparan sulfate-rich proteoglycans that are found at the cell surface, in the culture medium, and beneath the monolayer. The cell surface proteoglycan consists of a lipophilic membrane-associated domain and an extracellular domain, or ectodomain, that contains both heparan and chondroitin sulfate chains. During culture, the cells release into the medium a soluble proteoglycan that is indistinguishable from the ectodomain released from the cells by trypsin treatment. This medium ectodomain was isolated, purified, and used as an antigen to prepare an affinity-purified serum antibody from rabbits. The antibody recognizes polypeptide determinants on the core protein of the ectodomain of the cell surface proteoglycan. The reactivity of this antibody was compared with that of a serum antibody (BM-1) directed against the low density basement membrane proteoglycan of the Englebarth-Holm-Swarm tumor (Hassell, J. R., W. C. Leyshon, S. R. Ledbetter, B. Tyree, S. Suzuki, M. Kato, K. Kimata, and H. Kleinman. 1985. J. Biol. Chem. 250:8098-8105). The BM-1 antibody recognized a large, low density heparan sulfate-rich proteoglycan in the cells and in the basal extracellular materials beneath the monolayer where it accumulated in patchy deposits. The affinity-purified anti-ectodomain antibody recognized the cell surface proteoglycan on the cells, where it is seen on apical cell surfaces in subconfluent cultures and in fine filamentous arrays at the basal cell surface in confluent cultures, but detected no proteoglycan in the basal extracellular materials beneath the monolayer. The amino acid composition of the purified medium ectodomain was substantially different from that reported for the basement membrane proteoglycan. Thus, NMuMG cells produce at least two heparan sulfate-rich proteoglycans that contain distinct core proteins, a cell surface proteoglycan, and a basement membrane proteoglycan. In newborn mouse skin, these proteoglycans localize to distinct sites; the basement membrane proteoglycan is seen solely at the dermal-epidermal boundary and the cell surface proteoglycan is seen solely at the surfaces of keratinocytes in the basal, spinous, and granular cell layers. These results suggest that although heparan sulfate-rich proteoglycans may have similar glycosaminoglycan chains, they are sorted by the epithelial cells to different sites on the basis of differences in their core proteins.
Collagen type I and type V are present in the same fibril in the avian corneal stroma.
Authors: Birk D E , Fitch J M , Babiarz J P , Linsenmayer T F
Published: May 2004

The distribution, supramolecular form, and arrangement of collagen types I and V in the chicken embryo corneal stroma were studied using electron microscopy, collagen type-specific monoclonal antibodies, and a preembedding immunogold method. Double-label immunoelectron microscopy with colloidal gold-tagged monoclonal antibodies was used to simultaneously localize collagen type I and type V within the chick corneal stroma. The results definitively demonstrate, for the first time, that both collagens are codistributed within the same fibril. Type I collagen was localized to striated fibrils throughout the corneal stroma homogeneously. Type V collagen could be localized only after pretreatment of the tissue to partially disrupt collagen fibril structure. After such pretreatments the type V collagen was found in regions where fibrils were partially dissociated and not in regions where fibril structure was intact. When pretreated tissues were double labeled with antibodies against types I and V collagen coupled to different size gold particles, the two collagens colocalized in areas where fibril structure was partially disrupted. Antibodies against type IV collagen were used as a control and were nonreactive with fibrils. These results indicate that collagen types I and V are assembled together within single fibrils in the corneal stroma such that the interaction of these collagen types within heterotypic fibrils masks the epitopes on the type V collagen molecule. One consequence of the formation of such heterotypic fibrils may be the regulation of corneal fibril diameter, a condition essential for corneal transparency.
In vivo system for characterizing clonal variation and tissue-specific gene regulatory factors based on function.
Authors: Hardeman E C , Minty A , Benton-Vosman P , Kedes L , Blau H M
Published: May 2004

The inducibility of stably transfected alpha-cardiac actin genes differs among L cell clones. We examined the ability of muscle-specific factors to induce the expression of the human muscle alpha-cardiac actin gene promoter when stably transfected into mouse fibroblast L cells. This promoter is transcriptionally active in L cells at a low level, 2-5% of that in transfected muscle cells. Upon fusion with muscle cells to form heterokaryons, expression of the transfected alpha-cardiac actin gene promoter can be induced. However, induction is observed with only 10% of transfected L cell clones and the magnitude of this induction varies between 5- and 50-fold. These properties of the transfected L cell appear to be stably inherited. Our results are consistent with the hypothesis that muscle cells contain factors capable of increasing the transcription of the transfected gene, but that differences among L cell clones, possibly in the site of integration in the genome, determine the extent to which the gene can respond. By fusion into heterokaryons, transfectants with responsive genes can be identified. Such clones should prove useful in determining the basis for clonal variation. In addition, they provide an in vivo system for isolating functionally active tissue-specific transcription factors and the genes that encode them.
Prepro-carboxypeptidase Y and a truncated form of pre-invertase, but not full-length pre-invertase, can be posttranslationally translocated across microsomal vesicle membranes from Saccharomyces cerevisiae.
Authors: Hansen W , Walter P
Published: May 2004

We have determined that prepro-carboxypeptidase Y and a truncated form of pre-invertase can be translocated across the yeast microsomal membrane post-translationally in a homologous in vitro system. The yeast secretory protein prepro-alpha-factor which was previously shown to be an efficient posttranslational translocation substrate is therefore not unique in this regard, but rather the yeast ER protein translocation machinery is generally capable of accepting substrates from a ribosome-free, soluble pool. However, within our detection limits, full-length pre-invertase could not be translocated posttranslationally, but was translocated co-translationally. This indicates that not every fully synthesized pre-protein can use this pathway, presumably because normal or aberrant folding characteristics can interfere with translocation competence.
Targeting of the hepatitis B virus precore protein to the endoplasmic reticulum membrane: after signal peptide cleavage translocation can be aborted and the product released into the cytoplasm.
Authors: Garcia P D , Ou J H , Rutter W J , Walter P
Published: May 2004

The major hepatitis B virus (HBV) core protein is a viral structural protein involved in nucleic acid binding. Its coding sequence contains an extension of 29 codons (the "precore" region) at the amino terminus of the protein which is present in a fraction of the viral transcripts. This region is evolutionarily conserved among mammalian and avian HBVs, suggesting it has functional importance, although at least for duck HBV it has been shown to be nonessential for replication of infectious virions. Using in vitro assays for protein translocation across the endoplasmic reticulum membrane, we found that the precore region of the HBV genome encodes a signal sequence. This signal sequence was recognized by signal recognition particle, which targeted the nascent precore protein to the endoplasmic reticulum membrane with efficiencies comparable to those of other mammalian secretory proteins. A 19-amino acid signal peptide was removed by signal peptidase on the lumenal side of the microsomal membrane, generating a protein similar to the HBV major core protein, but containing 10 additional amino acids from the precore region at its amino terminus. Surprisingly, we found that 70-80% of this signal peptidase-cleaved product was localized on the cytoplasmic side of the microsomal vesicles and was not associated with the membranes. We conclude that translocation was aborted by an unknown mechanism, then the protein disengaged from the translocation machinery and was released back into the cytoplasm. Thus, a cytoplasmically disposed protein was created whose amino terminus resulted from signal peptidase cleavage. The remaining 20-30% appeared to be completely translocated into the lumen of the microsomes. A deletion mutant lacking the carboxy-terminal nucleic acid binding domain of the precore protein was similarly partitioned between the lumen of the microsomes and the cytoplasmic compartment, indicating that this highly charged domain is not responsible for the aborted translocation. We discuss the implications of our findings for the protein translocation process and suggest a possible role in the virus life cycle.
Characterization of the thermotolerant cell. II. Effects on the intracellular distribution of heat-shock protein 70, intermediate filaments, and small nuclear ribonucleoprotein complexes.
Authors: Welch W J , Mizzen L A
Published: May 2004

Here we further characterize a number of properties inherent to the thermotolerant cell. In the preceding paper, we showed that the acquisition of the thermotolerant state (by a prior induction of the heat-shock proteins) renders cells translationally tolerant to a subsequent severe heat-shock treatment and thereby results in faster kinetics of both the synthesis and subsequent repression of the stress proteins. Because of the apparent integral role of the 70-kD stress proteins in the acquisition of tolerance, we compared the intracellular distribution of these proteins in both tolerant and nontolerant cells before and after a severe 45 degrees C/30-min shock. In both HeLa and rat embryo fibroblasts, the synthesis and migration of the major stress-induced 72-kD protein into the nucleolus and its subsequent exit was markedly faster in the tolerant cells as compared with the nontolerant cells. Migration of preexisting 72-kD into the nucleolus was shown to be dependent upon heat-shock treatment and independent of active heat-shock protein synthesis. Using both microinjection and immunological techniques, we observed that the constitutive and abundant 73-kD stress protein similarly showed a redistribution from the cytoplasm and nucleus into the nucleolus as a function of heat-shock treatment. We show also that other lesions that occur in cells after heat shock can be prevented or at least minimized if the cells are first made tolerant. Specifically, the heat-induced collapse of the intermediate filament cytoskeleton did not occur in cells rendered thermotolerant. Similarly, the disruption of intranuclear staining patterns of the small nuclear ribonucleoprotein complexes after heat-shock treatment was less apparent in tolerant cells exposed to a subsequent heat-shock treatment.
Light-induced structural changes of cytoskeleton in squid photoreceptor microvilli detected by rapid-freeze method.
Authors: Tsukita S , Tsukita S , Matsumoto G
Published: May 2004

The cytoskeleton in squid photoreceptor microvilli was studied by freeze-substitution electron microscopy combined with rapid freezing using liquid helium, under dark-adapted and light-illuminated conditions. In the dark-adapted microvilli, actin filaments were regularly associated with granular structures on their surface; these granular structures were cross-linked to the rhodopsin-bearing plasma membranes through slender strands. Upon exposure to light, the granular components detached from the actin filaments, which then appeared to be fragmented and/or depolymerized. These observations have led us to conclude that light stimulation triggers the breakdown of the microvillar actin filament complex in squid photoreceptor cells. The results are discussed with special reference to the physiological role of actin filaments in photoreception.
A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase.
Authors: Hirano T , Hiraoka Y , Yanagida M
Published: May 2004

A temperature-sensitive mutant nuc2-663 of the fission yeast Schizosaccharomyces pombe specifically blocks mitotic spindle elongation at restrictive temperature so that nuclei in arrested cells contain a short uniform spindle (approximately 3-micron long), which runs through a metaphase plate-like structure consisting of three condensed chromosomes. In the wild-type or in the mutant cells at permissive temperature, the spindle is fully extended approximately 15-micron long in anaphase. The nuc2' gene was cloned in a 2.4-kb genomic DNA fragment by transformation, and its complete nucleotide sequence was determined. Its coding region predicts a 665-residues internally repeating protein (76.250 mol wt). By immunoblots using anti-sera raised against lacZ-nuc2+ fused proteins, a polypeptide (designated p67; 67,000 mol wt) encoded by nuc2+ is detected in the wild-type S. pombe extracts; the amount of p67 is greatly increased when multi-copy or high-expression plasmids carrying the nuc2+ gene are introduced into the S. pombe cells. Cellular fractionation and Percoll gradient centrifugation combined with immunoblotting show that p67 cofractionates with nuclei and is enriched in resistant structure that is insoluble in 2 M NaCl, 25 mM lithium 3,5'-diiodosalicylate, and 1% Triton but is soluble in 8 M urea. In nuc2 mutant cells, however, soluble p76, perhaps an unprocessed precursor, accumulates in addition to insoluble p67. The role of nuc2+ gene may be to interconnect nuclear and cytoskeletal functions in chromosome separation.
Microtubule dynamics and chromosome motion visualized in living anaphase cells.
Authors: Gorbsky G J , Sammak P J , Borisy G G
Published: May 2004

Chromosome segregation in most animal cells is brought about through two events: the movement of the chromosomes to the poles (anaphase A) and the movement of the poles away from each other (anaphase B). Essential to an understanding of the mechanism of mitosis is information on the relative movements of components of the spindle and identification of sites of subunit loss from shortening microtubules. Through use of tubulin derivatized with X-rhodamine, photobleaching, and digital imaging microscopy of living cells, we directly determined the relative movements of poles, chromosomes, and a marked domain on kinetochore fibers during anaphase. During chromosome movement and pole-pole separation, the marked domain did not move significantly with respect to the near pole. Therefore, the kinetochore microtubules were shortened by the loss of subunits at the kinetochore, although a small amount of subunit loss elsewhere was not excluded. In anaphase A, chromosomes moved on kinetochore microtubules that remained stationary with respect to the near pole. In anaphase B, the kinetochore fiber microtubules accompanied the near pole in its movement away from the opposite pole. These results eliminate models of anaphase in which microtubules are thought to be traction elements that are drawn to and depolymerized at the pole. Our results are compatible with models of anaphase in which the kinetochore fiber microtubules remain anchored at the pole and in which microtubule dynamics are centered at the kinetochore.
Localization and mobility of gelsolin in cells.
Authors: Cooper J A , Loftus D J , Frieden C , Bryan J , Elson E L
Published: May 2004

To investigate the physiologic role of gelsolin in cells, we have studied the location and mobility of gelsolin in a mouse fibroblast cell line (C3H). Gelsolin was localized by immunofluorescence of fixed and permeabilized cells and by fluorescent analog cytochemistry of living cells and cells that were fixed and/or permeabilized. Overall, the images show that in living cells gelsolin has a diffuse cytoplasmic distribution, but in fixed cells a minor fraction is associated with regions of the cell that are rich in actin filaments. The latter fraction is more prominent after permeabilization of the fixed cells because some diffuse gelsolin is not fixed and is therefore lost during permeabilization, confirmed by immunoblots. To determine quantitatively whether gelsolin is bound to actin filaments in living cells, we measured the mobility of microinjected fluorescent gelsolin by fluorescence photobleaching recovery. Gelsolin is fully mobile with a diffusion coefficient similar to that of control proteins. As a positive control, fluorescent phalloidin, which binds actin filaments, is totally immobile. These results are supported by immunoblots on cells permeabilized with detergent. All the endogenous gelsolin is extracted, and the half-time for the extraction is approximately 5 s, which is about the rate predicted for diffusion. Therefore, gelsolin is not tightly bound to actin filaments in cells. The most likely interpretation of the difference between living and fixed cells is that fixation traps a fraction of gelsolin that is associated with actin filaments in short-lived complexes.
Molecular characterization and expression of the stratification-related cytokeratins 4 and 15.
Authors: Leube R E , Bader B L , Bosch F X , Zimbelmann R , Achtstaetter T , Franke W W
Published: May 2004

A number of human cytokeratins are expressed during the development of stratified epithelia from one-layered polar epithelia and continue to be expressed in several adult epithelial tissues. For studies of the regulation of the synthesis of stratification-related cytokeratins in internal tissues, we have prepared cDNA and genomic clones encoding cytokeratin 4, as a representative of the basic (type II) cytokeratin subfamily and cytokeratin 15, as representative of the acidic (type I) subfamily, and determined their nucleotide sequences. The specific expression of mRNAs encoding these two polypeptides in certain stratified tissues and cultured cell lines is demonstrated by Northern blot hybridization. Hybridization in situ with antisense riboprobes and/or synthetic oligonucleotides shows the presence of cytokeratin 15 mRNA in all layers of esophagus, whereas cytokeratin 4 mRNA tends to be suprabasally enriched, although to degrees varying in different regions. We conclude that the expression of the genes encoding these stratification-related cytokeratins starts already in the basal cell layer and does not depend on vertical differentiation and detachment from the basal lamina. Our results also show that simple epithelial and stratification-related cytokeratins can be coexpressed in basal cell layers of certain stratified epithelia such as esophagus. Implications of these findings for epithelial differentiation and the formation of squamous cell carcinomas are discussed.
Differential effects of laminin, intact type IV collagen, and specific domains of type IV collagen on endothelial cell adhesion and migration.
Authors: Herbst T J , McCarthy J B , Tsilibary E C , Furcht L T
Published: May 2004

Laminin and type IV collagen were compared for the ability to promote aortic endothelial cell adhesion and directed migration in vitro. Substratum-adsorbed IV promoted aortic endothelial cell adhesion in a concentration dependent fashion attaining a maximum level 141-fold greater than controls within 30 min. Aortic endothelial cell adhesion to type IV collagen was not inhibited by high levels (10(-3) M) of arginyl-glycyl-aspartyl-serine. In contrast, adhesion of aortic endothelial cells on laminin was slower, attaining only 53% of the adhesion observed on type IV collagen by 90 min. Type IV collagen when added to the lower well of a Boyden chamber stimulated the directional migration of aortic endothelial cells in a concentration dependent manner with a maximal response 6.9-fold over control levels, whereas aortic endothelial cells did not migrate in response to laminin at any concentration (.01-2.0 X 10(-7) M). Triple helix-rich fragments of type IV collagen were nearly as active as intact type IV collagen in stimulating both adhesion and migration whereas the carboxy terminal globular domain was less active at promoting adhesion (36% of the adhesion promoted by intact type IV collagen) or migration. Importantly, aortic endothelial cells also migrate to substratum adsorbed gradients of type IV collagen suggesting that the mechanism of migration is haptotactic in nature. These results demonstrate that the aortic endothelial cell adhesion and migration is preferentially promoted by type IV collagen compared with laminin, and has a complex molecular basis which may be important in angiogenesis and large vessel repair.
Protein kinase C mediates platelet-derived growth factor-induced tyrosine phosphorylation of p42.
Authors: Kazlauskas A , Cooper J A
Published: May 2004

One of the early events after stimulation of Swiss 3T3 cells with either platelet-derived growth factor (PDGF), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), diacylglycerol, or several other mitogens is the near stoichiometric phosphorylation at tyrosine and serine of a scarce cytoplasmic protein (p42). TPA and diacylglycerol are known to directly stimulate the activity of a protein-serine/threonine kinase, protein kinase C (PKC). PDGF and several other mitogens stimulate tyrosine kinases directly and PKC indirectly. We have therefore examined the involvement of PKC in p42 tyrosine phosphorylation in Swiss 3T3 cells. Firstly, six agents which stimulated phosphorylation of p42 also stimulated phosphorylation of a known PKC substrate, an 80,000-Mr protein (p80). Secondly, in PKC-deficient cells (cells in which PKC activity was reduced to undetectable levels by prolonged exposure to TPA), PDGF-induced p42 phosphorylation was reduced three- to fourfold. Phosphoamino acid analysis of phosphorylated p42 from PDGF-stimulated PKC-deficient cells revealed primarily phosphoserine and only a trace of phosphotyrosine, suggesting that the reduction in PDGF-stimulated tyrosine phosphorylation of p42 resulting from PKC deficiency is greater than three- to fourfold. Finally, comparison of antiphosphotyrosine immunoprecipitates of PKC-deficient versus naive cells revealed that most other PDGF-induced tyrosine phosphorylation events were quite similar. These data suggest that mitogens such as PDGF, which directly stimulate phosphorylation of some proteins at tyrosine, induce p42 tyrosine phosphorylation via a cascade of events involving PKC.
The function of the nuclear envelope in nuclear protein accumulation.
Authors: Zimmer F J , Dreyer C , Hausen P
Published: May 2004

The mechanism by which proteins accumulate in the cell nucleus is not yet known. Two alternative mechanisms are discussed here: (a) selective unidirectional entry of karyophilic proteins through the nuclear pores, and (b) free diffusion of all proteins through the nuclear pores and specific binding of nuclear proteins to nondiffusible components of the nucleoplasm. We present experiments designed to distinguish between these alternatives. After mechanical injury of the Xenopus oocyte nuclear envelope, nuclear proteins were detected in the cytoplasm by immunohistochemical methods. In a second approach, nuclei from X. borealis oocytes were isolated under oil, the nuclear envelopes were removed, and the pure nucleoplasm was injected into the vegetal pole of X. laevis oocytes. With immunohistochemical methods, it was found that each of five nuclear proteins rapidly diffuses out of the injected nucleoplasm into the surrounding cytoplasm. The subsequent transport and accumulation in the intact host nucleus could be shown for the nuclear protein N1 with the aid of a species-specific mAb that reacts only with X. borealis N1. Purified and iodinated nucleoplasmin was injected into the cytoplasm of Xenopus oocytes and its uptake into the nucleus was studied by biochemical methods.
Amphibian oocyte maturation induced by extracts of Physarum polycephalum in mitosis.
Authors: Adlakha R C , Shipley G L , Zhao J Y , Jones K B , Wright D A , Rao P N , Sauer H W
Published: May 2004

The orderly progression of eukaryotic cells from interphase to mitosis requires the close coordination of various nuclear and cytoplasmic events. Studies from our laboratory and others on animal cells indicate that two activities, one present mainly in mitotic cells and the other exclusively in G1-phase cells, play a pivotal role in the regulation of initiation and completion of mitosis, respectively. The purpose of this study was to investigate whether these activities are expressed in the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony. Extracts were prepared from plasmodia in various phases of the cell cycle and tested for their ability to induce germinal vesicle breakdown and chromosome condensation after microinjection into Xenopus laevis oocytes. We found that extract of cells at 10-20 min before metaphase consistently induced germinal vesicle breakdown in oocytes. Preliminary characterization, including purification on a DNA-cellulose affinity column, indicated that the mitotic factors from Physarum were functionally very similar to HeLa mitotic factors. We also identified a number of mitosis-specific antigens in extracts from Physarum plasmodia, similar to those of HeLa cells, using the mitosis-specific monoclonal antibodies MPM-2 and MPM-7. Interestingly, we also observed an activity in Physarum at 45 min after metaphase (i.e., in early S phase since it has no G1) that is usually present in HeLa cells only during the G1 phase of the cell cycle. These are the first studies to show that maturation-promoting factor activity is present in Physarum during mitosis and is replaced by the G1 factor (or anti-maturation-promoting factor) activity in a postmitotic stage. A comparative study of these factors in this slime mold and in mammalian cells would be extremely valuable in further understanding their function in the regulation of eukaryotic cell cycle and their evolutionary relationship to one another.
Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast.
Authors: Payne G S , Baker D , van Tuinen E , Schekman R
Published: May 2004

Clathrin heavy chain-deficient mutants (chcl) of Saccharomyces cerevisiae are viable but exhibit compromised growth rates. To investigate the role of clathrin in intercompartmental protein transport, two pathways have been monitored in chcl cells: transport of newly synthesized vacuolar proteins to the vacuole and receptor-mediated uptake of mating pheromone. Newly synthesized precursors of the vacuolar protease carboxypeptidase Y (CPY) were converted to mature CPY with similar kinetics in mutant and wild-type cells. chcl cells did not aberrantly secrete CPY and immunolocalization techniques revealed most of the CPY in chcl cells within morphologically identifiable vacuolar structures. Receptor-mediated internalization of the mating pheromone alpha-factor occurred in chcl cells at 36-50% wild-type levels. The mutant cells were fully competent to respond to pheromone-induced cell-cycle arrest. These results argue that in yeast, clathrin may not play an essential role either in vacuolar protein sorting and delivery or in receptor-mediated endocytosis of alpha-factor. Alternative mechanisms ordinarily may execute these pathways, or be activated in clathrin-deficient cells.
Glucocorticoid-regulated localization of cell surface glycoproteins in rat hepatoma cells is mediated within the Golgi complex.
Authors: Haffar O K , Aponte G W , Bravo D A , John N J , Hess R T , Firestone G L
Published: May 2004

Glucocorticoid hormones regulate the post-translational maturation and sorting of cell surface and extracellular mouse mammary tumor virus (MMTV) glycoproteins in M1.54 cells, a stably infected rat hepatoma cell line. Exposure to monensin significantly reduced the proteolytic maturation and externalization of viral glycoproteins resulting in a stable cellular accumulation of a single 70,000-Mr glycosylated polyprotein (designated gp70). Cell surface- and intracellular-specific immunoprecipitations of monensin-treated cells revealed that gp70 can be localized to the cell surface only in the presence of 1 microM dexamethasone, while in uninduced cells gp70 is irreversibly sequestered in an intracellular compartment. Analysis of oligosaccharide processing kinetics demonstrated that gp70 acquired resistance to endoglycosidase H with a half-time of 65 min in the presence or absence of hormone. In contrast, gp70 was inefficiently galactosylated after a 60-min lag in uninduced cells while rapidly acquiring this carbohydrate modification in the presence of dexamethasone. Furthermore, in the absence or presence of monensin, MMTV glycoproteins failed to be galactosylated in hormone-induced CR4 cells, a complement-selected sorting variant defective in the glucocorticoid-regulated compartmentalization of viral glycoproteins to the cell surface. Since dexamethasone had no apparent global effects on organelle morphology or production of total cell surface-galactosylated species, we conclude that glucocorticoids induce the localization of cell surface MMTV glycoproteins by regulating a highly selective step within the Golgi apparatus after the acquisition of endoglycosidase H-resistant oligosaccharide side chains but before or at the site of galactose attachment.
Integration of a small integral membrane protein, M2, of influenza virus into the endoplasmic reticulum: analysis of the internal signal-anchor domain of a protein with an ectoplasmic NH2 terminus.
Authors: Hull J D , Gilmore R , Lamb R A
Published: May 2004

The M2 protein of influenza A virus is a small integral membrane protein of 97 residues that is expressed on the surface of virus-infected cells. M2 has an unusual structure as it lacks a cleavable signal sequence yet contains an ectoplasmic amino-terminal domain of 23 residues, a 19 residue hydrophobic transmembrane spanning segment, and a cytoplasmic carboxyl-terminal domain of 55 residues. Oligonucleotide-mediated deletion mutagenesis was used to construct a series of M2 mutants lacking portions of the hydrophobic segment. Membrane integration of the M2 protein was examined by in vitro translation of synthetic mRNA transcripts prepared using bacteriophage T7 RNA polymerase. After membrane integration, M2 was resistant to alkaline extraction and was converted to an Mr approximately equal to 7,000 membrane-protected fragment after digestion with trypsin. In vitro integration of M2 requires the cotranslational presence of the signal recognition particle. Deletion of as few as two residues from the hydrophobic segment of M2 markedly decreases the efficiency of membrane integration, whereas deletion of six residues completely eliminates integration. M2 proteins containing deletions that eliminate stable membrane anchoring are apparently not recognized by signal recognition particles, as these polypeptides remain sensitive to protease digestion, indicating that in addition they do not have a functional signal sequence. These data thus indicate that the signal sequence that initiates membrane integration of M2 resides within the transmembrane spanning segment of the polypeptide.