bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but not of their activator MAP kinase kinase (p45mapkk) in fibroblasts
Authors: Lenormand P , Sardet C , Pagès G , L'Allemain G , Brunet A , Pouysségur J
Published: May 2004

Mitogen-activated protein kinases (p42mapk and p44mapk) are serine/threonine kinases that are activated rapidly in cells stimulated with various extracellular signals. This activation is mediated via MAP kinase kinase (p45mapkk), a dual specificity kinase which phosphorylates two key regulatory threonine and tyrosine residues of MAP kinases. We reported previously that the persistent phase of MAP kinase activation is essential for mitogenically stimulated cells to pass the "restriction point" of the cell cycle. Here, using specific polyclonal antibodies and transfection of epitope-tagged recombinant MAP kinases we demonstrate that these signaling protein kinases undergo distinct spatio-temporal localization in growth factor-stimulated cells. In G0-arrested hamster fibroblasts the activator p45mapkk and MAP kinases (p42mapk, p44mapk) are mainly cytoplasmic. Subsequent to mitogenic stimulation by serum or alpha-thrombin both MAP kinase isoforms translocate into the nucleus. This translocation is rapid (seen in 15 min), persistent (at least during the entire G1 period up to 6 h), reversible (by removal of the mitogenic stimulus) and apparently 'coupled' to the mitogenic potential; it does not occur in response to nonmitogenic agents such as alpha-thrombin-receptor synthetic peptides and phorbol esters that fail to activate MAP kinases persistently. When p42mapk and p44mapk are expressed stably at high levels, they are found in the nucleus of resting cells; this nuclear localization is also apparent with kinase-deficient mutants (p44mapk T192A or Y194F). In marked contrast the p45mapkk activator remains cytoplasmic even during prolonged growth factor stimulation and even after high expression levels achieved by transfection. We propose that the rapid and persistent nuclear transfer of p42mapk and p44mapk during the entire G0-G1 period is crucial for the function of these kinases in mediating the growth response.
Serum-induced translocation of mitogen-activated protein kinase to the cell surface ruffling membrane and the nucleus
Authors: Gonzalez FA , Seth A , Raden DL , Bowman DS , Fay FS , Davis RJ
Published: May 2004

The mitogen-activated protein (MAP) kinase signal transduction pathway represents an important mechanism by which growth factors regulate cell function. Targets of the MAP kinase pathway are located within several cellular compartments. Signal transduction therefore requires the localization of MAP kinase in each sub-cellular compartment that contains physiologically relevant substrates. Here, we show that serum treatment causes the translocation of two human MAP kinase isoforms, p40mapk and p41mapk, from the cytosol into the nucleus. In addition, we report that p41mapk (but not p40mapk) is localized at the cell surface ruffling membrane in serum-treated cells. To investigate whether the protein kinase activity of MAP kinase is required for serum-induced redistribution within the cell, we constructed mutated kinase-negative forms of p40mapk and p41mapk. The kinase-negative MAP kinases were not observed to localize to the cell surface ruffling membrane. In contrast, the kinase-negative MAP kinases were observed to be translocated to the nucleus. Intrinsic MAP kinase activity is therefore required only for localization at the cell surface and is not required for transport into the nucleus. Together, these data demonstrate that the pattern of serum-induced redistribution of p40mapk is different from p41mapk. Thus, in addition to common targets of signal transduction, it is possible that these MAP kinase isoforms may differentially regulate targets located in distinct sub-cellular compartments.
The conversion of mouse skin squamous cell carcinomas to spindle cell carcinomas is a recessive event
Authors: Stoler AB , Stenback F , Balmain A
Published: May 2004

Squamous carcinomas of both human and rodent origin can undergo a transition to a more invasive, metastatic phenotype involving reorganization of the cytoskeleton, loss of cell adhesion molecules such as E-cadherin and acquisition of a fibroblastoid or spindle cell morphology. We have developed a series of cell lines from mouse skin tumors which represent different stages of carcinogenesis, including benign papillomas, and clonally related squamous and spindle carcinomas derived from the same primary tumor. Some spindle cells continue to express keratins, but with a poorly organized keratin filament network, whereas in others no keratin expression is detectable. All of the spindle cells lack expression of the cell adhesion molecule E-cadherin and the desmosomal component desmoplakin. Loss of these cell surface proteins therefore appears to precede the destabilization of the keratin network. At the genetic level, it is not known whether such changes involve activation of dominantly acting oncogenes or loss of a suppressor function which controls epithelial differentiation. To examine this question, we have carried out a series of fusion experiments between a highly malignant mouse skin spindle cell carcinoma and cell lines derived from premalignant or malignant mouse skin tumors, including both squamous and spindle carcinoma variants. The results show that the spindle cell phenotype as determined by cell morphology and lack of expression of keratin, E-cadherin, and desmoplakin proteins, is recessive in all hybrids with squamous cells. The hybrids expressed all of these differentiation markers, and showed suppression of tumorigenicity to a variable level dependent upon the tumorigenic properties of the less malignant fusion partner. Our results suggest that acquisition of the spindle cell phenotype involves functional loss of a gene(s) which controls epithelial differentiation.
Integrin-mediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium channels
Authors: Arcangeli A , Becchetti A , Mannini A , Mugnai G , De Filippi P , Tarone G , Del Bene MR , Barletta E , Wanke E , Olivotto M
Published: May 2004

Electrical signals elicited by integrin interaction with ECM components and their role in neurite outgrowth were studied in two clones (N1 and N7) isolated from 41A3 murine neuroblastoma cell line. Although the two clones similarly adhered to fibronectin (FN) and vitronectin (VN), this adhesion induced neurite outgrowth in N1 but not in N7 cells. Patch clamp recordings in whole cell configuration showed that, upon adhesion to FN or VN but not to platelet factor 4 (PF4), N1 cells undergo a marked (approximately equal to 20 mV) hyperpolarization of the resting potential (Vrest) that occurred within the first 20 min after cell contact with ECM, and persisted for approximately 1 h before reverting to the time zero values. This hyperpolarization was totally absent in N7 cells. A detailed analysis of the molecular mechanisms involved in N1 and N7 cell adhesion to ECM substrata was performed by using antibodies raised against the FN receptor and synthetic peptides variously competing with the FN or VN binding to integrin receptor (GRGDSP and GRGESP). Antibodies, as well as GRGDSP, abolished adhesion of N1 and N7 clones to FN and VN, revealing a similar implication of integrins in the adhesion of these clones to the ECM proteins. However, these anti-adhesive treatments, while ineffective on Vrest of N7 cells, abolished in N1 cells the FN- or VN-induced hyperpolarization and neurite outgrowth, that appeared therefore strictly associated and integrin-mediated phenomena. The nature of this association was deepened through a comparative analysis of the integrin profiles and the ion channels of N1 and N7 cells. The integrin immunoprecipitation profile resulted very similarly in the two clones, with only minor differences concerning the alpha V containing complexes. Both clones possessed Ca2+ and K+ delayed rectifier (KDR) channels, while only N1 cells were endowed with inward rectifier K+ (KIR) channels. The latter governed the Vrest, and, unlike KDR channels, were blocked by Ba2+ and Cs+. By moving patched cells in contact with FN-coated beads, it was shown that KIR channel activation was responsible for the FN-mediated hyperpolarization of Vrest. Treatment with Pertuxis toxin (PTX) abolished this hyperpolarization and neurite outgrowth, indicating that a G protein is interposed between integrins and KIR channels and that the activation of these channels is required for neuritogenesis. In fact, the block of KIR channels by Cs+ abolished both hyperpolarization and neurite outgrowth, provided that the cation was supplied during the first two hours after N1 cell contact with FN.(ABSTRACT TRUNCATED AT 400 WORDS)
Reversible effects of nuclear membrane permeabilization on DNA replication: evidence for a positive licensing factor
Authors: Coverley D , Downes CS , Romanowski P , Laskey RA
Published: May 2004

We have investigated the mechanism which prevents reinitiation of DNA replication within a single cell cycle by exploiting the observation that intact G2 HeLa nuclei do not replicate in Xenopus egg extract, unless their nuclear membranes are first permeabilized (Leno et al., 1992). We have asked if nuclear membrane permeabilization allows escape of a negative inhibitor from the replicated nucleus or entry of a positive activator as proposed in the licensing factor hypothesis of Blow and Laskey (1988). We have distinguished these possibilities by repairing permeabilized nuclear membranes after allowing soluble factors to escape. Membrane repair of G2 nuclei reverses the effects of permeabilization arguing that escape of diffusible inhibitors is not sufficient to allow replication, but that entry of diffusible activators is required. Membrane repair has no significant effect on G1 nuclei. Pre-incubation of permeable G2 nuclei in the soluble fraction of egg extract before membrane repair allows semiconservative DNA replication of these nuclei when incubated in complete extract. Addition of the same fraction after membrane repair has no effect. Our results provide direct evidence for a positively acting "licensing" activity which is excluded form the interphase nucleus by the nuclear membrane. Nuclear membrane permeabilization and repair can be used as an assay for licensing activity which could lead to its purification and subsequent analysis of its action within the nucleus.
Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells
Authors: Xu H , Shields D
Published: May 2004

Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence from several laboratories has implicated either the TGN or immature secretory granules as the site of prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin (proSRIF) (Stoller, T. J., and D. Shields. J. Cell Biol. 1988. 107:2087-2095). By incubating these cells at 20 degrees C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident; processing resumed upon shifting the cells back to 37 degrees C. After the 20 degrees C block, the cells were mechanically permeabilized and pro-SRIF processing determined. Cleavage of proSRIF to the mature hormone was approximately 35-50% efficient, required incubation at 37 degrees C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATP-dependent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCP, a protonophore, or by preincubating the permeabilized cells with low concentrations of N-ethylmaleimide, an inhibitor of vacuolar-type ATP-dependent proton pumps. These data suggest that: (a) proSRIF cleavage is initiated in the TGN, and (b) this reaction requires an acidic pH which is facilitated by a Golgi-associated vacuolar-type ATPase. A characteristic feature of polypeptide hormone-producing cells is their ability to store the mature hormone in dense core secretory granules. To investigate the mechanism of protein sorting to secretory granules, the budding of nascent secretory vesicles from the TGN was determined. No vesicle formation occurred at 20 degrees C; in contrast, at 37 degrees C, the budding of secretory vesicles was approximately 40% efficient and was dependent on ATP, GTP, and cytosolic factors. Vesicle formation was inhibited by GTP gamma S suggesting a role for GTP-binding proteins in this process. Vesicle budding was dependent on cytosolic factors that were tightly membrane associated and could be removed only by treating the permeabilized cells with high salt. After high salt treatment, vesicle formation was dependent on added cytosol or the dialyzed salt extract. The formation of nascent secretory vesicles contrasts with prosomatostatin processing which required only ATP for efficient cleavage. Our results demonstrate that prohormone cleavage which is initiated in the TGN, precedes vesicle formation and that processing can be uncoupled from the generation of nascent secretory vesicles.
Microtubule independent vesiculation of Golgi membranes and the reassembly of vesicles into Golgi stacks
Authors: Veit B , Yucel JK , Malhotra V
Published: May 2004

We have recently shown that ilimaquinone (IQ) causes the breakdown of Golgi membranes into small vesicles (VGMs for vesiculated Golgi membranes) and inhibits vesicular protein transport between successive Golgi cisternae (Takizawa et al., 1993). While other intracellular organelles, intermediate filaments, and actin filaments are not affected, we have found that cytoplasmic microtubules are depolymerized by IQ treatment of NRK cells. We provide evidence that IQ breaks down Golgi membranes regardless of the state of cytoplasmic microtubules. This is evident from our findings that Golgi membranes break down with IQ treatment in the presence of taxol stabilized microtubules. Moreover, in cells where the microtubules are first depolymerized by microtubule disrupting agents which cause the Golgi stacks to separate from one another and scatter throughout the cytoplasm, treatment with IQ causes further breakdown of these Golgi stacks into VGMs. Thus, IQ breaks down Golgi membranes independently of its effect on cytoplasmic microtubules. Upon removal of IQ from NRK cells, both microtubules and Golgi membranes reassemble. The reassembly of Golgi membranes, however, takes place in two sequential steps: the first is a microtubule independent process in which the VGMs fuse together to form stacks of Golgi cisternae. This step is followed by a microtubule-dependent process by which the Golgi stacks are carried to their perinuclear location in the cell. In addition, we have found that IQ has no effect on the structural organization of Golgi membranes at 16 degrees C. However, VGMs generated by IQ are capable of fusing and assembling into stacks of Golgi cisternae at 16 degrees C. This is in contrast to the cells recovering from BFA treatment where, after removal of BFA at 16 degrees C, resident Golgi enzymes fail to exit the ER, a process presumed to require the formation of vesicles. We propose that at 16 degrees C there may be general inhibition in the process of vesicle formation, whereas the process of vesicle fusion is not affected.
Bidirectional transcytosis determines the steady state distribution of the transferrin receptor at opposite plasma membrane domains of BeWo cells
Authors: Cerneus DP , Strous GJ , van der Ende A
Published: May 2004

Trophoblast-like BeWo cells form well-polarized epithelial monolayers, when cultured on permeable supports. Contrary to other polarized cell systems, in which the transferrin receptor is found predominantly on the basolateral cell surface, BeWo cells express the transferrin receptor at both apical and basolateral cell surfaces (Cerneus, D.P., and A. van der Ende. 1991. J. Cell Biol. 114: 1149-1158). In the present study we have addressed the question whether BeWo cells use a different sorting mechanism to target transferrin receptors to the cell surface, by examining the biosynthetic and transcytotic pathways of the transferrin receptor in BeWo cells. Using trypsin and antibodies to detect transferrin receptors at the cell surface of filter-grown BeWo cells, we show that at least 80% of newly synthesized transferrin receptor follows a direct pathway to the basolateral surface, demonstrating that the transferrin receptor is efficiently intracellularly sorted. After surface arrival, pulse-labeled transferrin receptor equilibrates between apical and basolateral cell surfaces, due to ongoing transcytotic transport in both directions. The subsequent redistribution takes over 120 min and results in a steady state distribution with 1.5-2.0 times more transferrin receptors at the basolateral surface than at the apical surface. By monitoring the fate of surface-bound 125I-transferrin, internalized either from the apical or basolateral surface transcytosis of the transferrin receptor was studied. About 15% of 125I-transferrin is transcytosed in the basolateral to apical direction, whereas 25% is transcytosed in the opposite direction, indicated that the fraction of receptors involved in transcytosis is roughly twofold higher for the apical receptor pool, as compared to the basolateral pool. Upon internalization, both apical and basolateral receptor pools become redistributed on both surfaces, resulting in a twofold higher number of transferrin receptors at the basolateral surface. Our results indicate that in BeWo cells bidirectional transcytosis is the main factor in surface distribution of transferrin receptors on apical and basolateral surfaces, which may represent a cell type-specific, post-endocytic, sorting mechanism.
GPI- and transmembrane-anchored influenza hemagglutinin differ in structure and receptor binding activity
Authors: Kemble GW , Henis YI , White JM
Published: May 2004

We investigated the influence of a glycosylphosphatidylinositol (GPI) anchor on the ectodomain of the influenza hemagglutinin (HA) by replacing the wild type (wt) transmembrane and cytoplasmic domains with a GPI lipid anchor. GPI-anchored HA (GPI-HA) was transported to the cell surface with equal efficiency and at the same rate as wt-HA. Like wt-HA, cell surface GPI-HA, and its ectodomain released with the enzyme PI-phospholipase C (PI-PLC), were 9S trimers. Compared to wt-HA, the GPI-HA ectodomain underwent additional terminal oligosaccharide modifications; some of these occurred near the receptor binding pocket and completely inhibited the ability of GPI-HA to bind erythrocytes. Growth of GPI-HA-expressing cells in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM) abrogated the differences in carbohydrate modification and restored the ability of GPI-HA to bind erythrocytes. The ectodomain of GPI-HA produced from cells grown in the presence or absence of dMM underwent characteristic low pH-induced conformational changes (it released its fusion peptides and became hydrophobic and proteinase sensitive) but at 0.2 and 0.4 pH units higher than wt-HA, respectively. These results demonstrate that although GPI-HA forms a stable trimer with characteristics of the wt, its structure is altered such that its receptor binding activity is abolished. Our results show that transmembrane and GPI-anchored forms of the same ectodomain can exhibit functionally important differences in structure at a great distance from the bilayer.
Three-dimensional solution structure of Acanthamoeba profilin-I
Authors: Vinson VK , Archer SJ , Lattman EE , Pollard TD , Torchia DA
Published: May 2004

We have determined a medium resolution three-dimensional solution structure of Acanthamoeba profilin-I by multidimensional nuclear magnetic resonance spectroscopy. This 13-kD actin binding protein consists of a five stranded antiparallel beta sheet flanked by NH2- and COOH-terminal helices on one face and by a third helix and a two stranded beta sheet on the other face. Data from actin-profilin cross-linking experiments and the localization of conserved residues between profilins in different phyla indicate that actin binding occurs on the molecular face occupied by the terminal helices. The other face of the molecule contains the residues that differ between Acanthamoeba profilins-I and II and may be important in determining the difference in polyphosphoinositide binding between these isoforms. This suggests that lipids and actin bind to different faces of the molecule.
Suppression of vinculin expression by antisense transfection confers changes in cell morphology, motility, and anchorage-dependent growth of 3T3 cells
Authors: Rodríguez Fernández JL , Geiger B , Salomon D , Ben-Ze'ev A
Published: May 2004

The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, is markedly modulated in cells during growth activation, differentiation, motility and cell transformation. The stimulation of quiescent cells by serum factors and the culturing of cells on highly adhesive matrices induce vinculin gene expression, whereas the transformation of fibroblast and epithelial cells often results in decreased vinculin expression (reviewed in Rodríguez Fernández, J. L., B. Geiger, D. Salomon, I. Sabanay, M. Zöller, and A. Ben-Ze'ev. 1992. J. Cell Biol. 119:427). To study the effect of reduced vinculin expression on cell behavior, 3T3 cells were transfected with an antisense vinculin cDNA construct, and clones displaying decreased vinculin levels down to 10-30% of control levels were isolated. These cells showed a round phenotype with smaller and fewer vinculin-positive plaques localized mostly at the cell periphery. In addition, they displayed an increased motility compared to controls, manifested by a faster closure of "wounds" introduced into the monolayer, and by the formation of longer phagokinetic tracks. Moreover, the antisense transfectants acquired a higher cloning efficiency and produced larger colonies in soft agar than the parental counterparts. The results demonstrate that the regulation of vinculin expression in cells can affect, in a major way, cell shape and motility, and that decreased vinculin expression can induce cellular changes reminiscent of those found in transformed cells.
Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments
Authors: Ching GY , Liem RK
Published: May 2004

We report here on the in vivo assembly of alpha-internexin, a type IV neuronal intermediate filament protein, in transfected cultured cells, comparing its assembly properties with those of the neurofilament triplet proteins (NF-L, NF-M, and NF-H). Like the neurofilament triplet proteins, alpha-internexin coassembles with vimentin into filaments. To study the assembly characteristics of these proteins in the absence of a preexisting filament network, transient transfection experiments were performed with a non-neuronal cell line lacking cytoplasmic intermediate filaments. The results showed that only alpha-internexin was able to self-assemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. alpha-internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with alpha-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments.
Neurofilaments are obligate heteropolymers in vivo
Authors: Lee MK , Xu Z , Wong PC , Cleveland DW
Published: May 2004

Neurofilaments (NFs), composed of three distinct subunits NF-L, NF-M, and NF-H, are neuron-specific intermediate filaments present in most mature neurons. Using DNA transfection and mice expressing NF transgenes, we find that despite the ability of NF-L alone to assemble into short filaments in vitro NF-L cannot form filament arrays in vivo after expression either in cultured cells or in transgenic oligodendrocytes that otherwise do not contain a cytoplasmic intermediate filament (IF) array. Instead, NF-L aggregates into punctate or sheet like structures. Similar nonfilamentous structures are also formed when NF-M or NF-H is expressed alone. The competence of NF-L to assemble into filaments is fully restored by coexpression of NF-M or NF-H to a level approximately 10% of that of NF-L. Deletion of the head or tail domain of NF-M or substitution of the NF-H tail onto an NF-L subunit reveals that restoration of in vivo NF-L assembly competence requires an interaction provided by the NF-M or NF-H head domains. We conclude that, contrary to the expectation drawn from earlier in vitro assembly studies, NF-L is not sufficient to assemble an extended filament network in an in vivo context and that neurofilaments are obligate heteropolymers requiring NF-L and NF-M or NF-H.
Subtractive immunization yields monoclonal antibodies that specifically inhibit metastasis
Authors: Brooks PC , Lin JM , French DL , Quigley JP
Published: May 2004

Subtractive immunization allowed the isolation and characterization of monoclonal antibodies that specifically inhibit metastasis but not proliferation of highly metastatic human tumor cells. The tolerizing agent cyclophosphamide was used to suppress the immune system in mice to dominant immunodeterminants present on a non-metastatic variant (M-) of the human epidermoid carcinoma cell line (HEp3). Mice were then inoculated with a highly metastatic variant (M+) of HEp3 to enhance an immune response to antigenic determinants present on metastatic cells. Hybridomas were generated and screened by ELISA for differential reactivity to M+ HEp3 over M- HEp3 cells. This experimental approach, termed subtractive immunization (S.I.), was compared to a control immunization protocol, which eliminated the cyclophosphamide treatment. The S.I. protocol resulted in an eight-fold increase in the proportion of mAbs that react with molecules enriched on the surface of the M+ HEp3 cells. Two of the mAbs derived from the S.I. protocol, designated DM12-4 and 1A5, were purified and examined for their effect in a metastasis model system in which chick embryos are transplanted with primary HEp3 tumors. Purified mAbs DM12-4 and 1A5, inoculated i.v. into the embryos, inhibited spontaneous metastasis of HEp3 cells by 86 and 90%, respectively. The mAbs are specifically anti-metastatic in that they have no effect on the growth of HEp3 cells in vitro nor did they inhibit primary tumor growth in vivo. The mAbs recognize M+ HEp3 cell surface molecules of 55 kD and 29 kD, respectively. These data demonstrate that the S.I. protocol can be used for the development of unique mAbs that are reactive with antigenic determinants whose expression is elevated on metastatic human tumor cells and which function mechanistically in the metastatic cascade.
A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins.
Authors: Gambill B D , Voos W , Kang P J , Miao B , Langer T , Craig E A , Pfanner N
Published: May 2004

The role of mitochondrial 70-kD heat shock protein (mt-hsp70) in protein translocation across both the outer and inner mitochondrial membranes was studied using two temperature-sensitive yeast mutants. The degree of polypeptide translocation into the matrix of mutant mitochondria was analyzed using a matrix-targeted preprotein that was cleaved twice by the processing peptidase. A short amino-terminal segment of the preprotein (40-60 amino acids) was driven into the matrix by the membrane potential, independent of hsp70 function, allowing a single cleavage of the presequence. Artificial unfolding of the preprotein allowed complete translocation into the matrix in the case where mutant mt-hsp70 had detectable binding activity. However, in the mutant mitochondria in which binding to mt-hsp70 could not be detected the mature part of the preprotein was only translocated to the intermembrane space. We propose that mt-hsp70 fulfills a dual role in membrane translocation of preproteins. (a) Mt-hsp70 facilitates unfolding of the polypeptide chain for translocation across the mitochondrial membranes. (b) Binding of mt-hsp70 to the polypeptide chain is essential for driving the completion of transport of a matrix-targeted preprotein across the inner membrane. This second role is independent of the folding state of the preprotein, thus identifying mt-hsp70 as a genuine component of the inner membrane translocation machinery. Furthermore we determined the sites of the mutations and show that both a functional ATPase domain and ATP are needed for mt-hsp70 to bind to the polypeptide chain and drive its translocation into the matrix.
Distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different Na+,K(+)-ATPase distributions in polarized epithelia.
Authors: Marrs J A , Napolitano E W , Murphy-Erdosh C , Mays R W , Reichardt L F , Nelson W J
Published: May 2004

In simple epithelia, the distribution of ion transporting proteins between the apical or basal-lateral domains of the plasma membrane is important for determining directions of vectorial ion transport across the epithelium. In the choroid plexus, Na+,K(+)-ATPase is localized to the apical plasma membrane domain where it regulates sodium secretion and production of cerebrospinal fluid; in contrast, Na+,K(+)-ATPase is localized to the basal-lateral membrane of cells in the kidney nephron where it regulates ion and solute reabsorption. The mechanisms involved in restricting Na+,K(+)-ATPase distribution to different membrane domains in these simple epithelia are poorly understood. Previous studies have indicated a role for E-cadherin mediated cell-cell adhesion and membrane-cytoskeleton (ankyrin and fodrin) assembly in regulating Na+,K(+)-ATPase distribution in absorptive kidney epithelial cells. Confocal immunofluorescence microscopy reveals that in chicken and rat choroid plexus epithelium, fodrin, and ankyrin colocalize with Na+,K(+)-ATPase at the apical plasma membrane, but fodrin, ankyrin, and adducin also localize at the lateral plasma membrane where Na+,K(+)-ATPase is absent. Biochemical analysis shows that fodrin, ankyrin, and Na+,K(+)-ATPase are relatively resistant to extraction from cells in buffers containing Triton X-100. The fractions of Na+,K(+)-ATPase, fodrin, and ankyrin that are extracted from cells cosediment in sucrose gradients at approximately 10.5 S. Further separation of the 10.5 S peak of proteins by electrophoresis in nondenaturing polyacrylamide gels revealed that fodrin, ankyrin, and Na+,K(+)-ATPase comigrate, indicating that these proteins are in a high molecular weight complex similar to that found previously in kidney epithelial cells. In contrast, the anion exchanger (AE2), a marker protein of the basal-lateral plasma membrane in the choroid plexus, did not cosediment in sucrose gradients or comigrate in nondenaturing polyacrylamide gels with the complex of Na+,K(+)-ATPase, ankyrin, and fodrin. Ca(++)-dependent cell adhesion molecules (cadherins) were detected at lateral membranes of the choroid plexus epithelium and colocalized with a distinct fraction of ankyrin, fodrin, and adducin. Cadherins did not colocalize with Na+,K(+)-ATPase and were absent from the apical membrane. The fraction of cadherins that was extracted with buffers containing Triton X-100 cosedimented with ankyrin and fodrin in sucrose gradients and comigrated in nondenaturing gels with ankyrin and fodrin in a high molecular weight complex. Since a previous study showed that E-cadherin is an instructive inducer of Na+,K(+)-ATPase distribution, we examined protein distributions in fibroblasts transfected with B-cadherin, a prominent cadherin expressed in the choroid plexus epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)
Assembly of flagellar radial spoke proteins in Chlamydomonas: identification of the axoneme binding domain of radial spoke protein 3.
Authors: Diener D R , Ang L H , Rosenbaum J L
Published: May 2004

Radial spokes of the eukaryotic flagellum extend from the A tubule of each outer doublet microtubule toward the central pair microtubules. In the paralyzed flagella mutant of Chlamydomonas pf14, a mutation in the gene for one of 17 polypeptides that comprise the radial spokes results in flagella that lack all 17 spoke components. The defective gene product, radial spoke protein 3 (RSP3), is, therefore, pivotal to the assembly of the entire spoke and may attach the spoke to the axoneme. We have synthesized RSP3 in vitro and assayed its binding to axonemes from pf14 cells to determine if RSP3 can attach to spokeless axonemes. In vitro, RSP3 binds to pf14 axonemes, but not to wild-type axonemes or microtubules polymerized from purified chick brain tubulin. The sole axoneme binding domain of RSP3 is located within amino acids 1-85 of the 516 amino acid protein; deletion of these amino acids abolishes binding by RSP3. Fusion of amino acids 1-85 or 42-85 to an unrelated protein confers complete or partial binding activity, respectively, to the fusion protein. Transformation of pf14 cells with mutagenized RSP3 genes indicates that amino acids 18-87 of RSP3 are important to its function, but that the carboxy-terminal 140 amino acids can be deleted with little effect on radial spoke assembly or flagellar motility.
The relationship between cell size and cell fate in Volvox carteri.
Authors: Kirk M M , Ransick A , McRae S E , Kirk D L
Published: May 2004

In Volvox carteri development, visibly asymmetric cleavage divisions set apart large embryonic cells that will become asexual reproductive cells (gonidia) from smaller cells that will produce terminally differentiated somatic cells. Three mechanisms have been proposed to explain how asymmetric division leads to cell specification in Volvox: (a) by a direct effect of cell size (or a property derived from it) on cell specification, (b) by segregation of a cytoplasmic factor resembling germ plasm into large cells, and (c) by a combined effect of differences in cytoplasmic quality and cytoplasmic quantity. In this study a variety of V. carteri embryos with genetically and experimentally altered patterns of development were examined in an attempt to distinguish among these hypotheses. No evidence was found for regionally specialized cytoplasm that is essential for gonidial specification. In all cases studied, cells with a diameter > approximately 8 microns at the end of cleavage--no matter where or how these cells had been produced in the embryo--developed as gonidia. Instructive observations in this regard were obtained by three different experimental interventions. (a) When heat shock was used to interrupt cleavage prematurely, so that presumptive somatic cells were left much larger than they normally would be at the end of cleavage, most cells differentiated as gonidia. This result was obtained both with wild-type embryos that had already divided asymmetrically (and should have segregated any cytoplasmic determinants involved in cell specification) and with embryos of a mutant that normally produces only somatic cells. (b) When individual wild-type blastomeres were isolated at the 16-cell stage, both the anterior blastomeres that normally produce two gonidia each and the posterior blastomeres that normally produce no gonidia underwent modified cleavage patterns and each produced an average of one large cell that developed as a gonidium. (c) When large cells were created microsurgically in a region of the embryo that normally makes only somatic cells, these large cells became gonidia. These data argue strongly for a central role of cell size in germ/soma specification in Volvox carteri, but leave open the question of how differences in cell size are actually transduced into differences in gene expression.
Tissue polarity genes of Drosophila regulate the subcellular location for prehair initiation in pupal wing cells.
Authors: Wong L L , Adler P N
Published: May 2004

The Drosophila wing is decorated with a regular array of distally pointing hairs. In the pupal wing, the hairs are formed from micro-villus like prehairs that contain large bundles of actin filaments. The distal orientation of the actin bundles reveals the proximal-distal polarity within the pupal wing epithelium. We have used F-actin staining to examine early stages of prehair development in both wild-type and mutant pupal wings. We have found a striking correlation between hair polarity and the subcellular location for assembly of the prehair. In a wild-type wing, all of the distally pointing hairs are derived from prehairs that are formed at the distal vertex of the hexagonally shaped pupal wing cells. Mutations in six tissue polarity genes result in abnormal hair polarity on the adult wing, and all also alter the subcellular location for prehair initiation. Based on their cellular phenotypes, we can place these six genes into three phenotypic groups. Double mutant analysis indicates that these phenotypic groups correspond to epistasis groups. This suggests that the tissue polarity genes function in or on a pathway that controls hair polarity by regulating the subcellular location for prehair formation.
Mutation of putative divalent cation sites in the alpha 4 subunit of the integrin VLA-4: distinct effects on adhesion to CS1/fibronectin, VCAM-1, and invasin.
Authors: Masumoto A , Hemler M E
Published: May 2004

To investigate the functional significance of putative integrin divalent cation binding sites, several mutated alpha 4 subunit cDNAs were constructed. Mutants contained the conservative substitution of Glu for Asp or Asn at the third position in each of three putative divalent cation sites. Transfection of wild-type or mutated alpha 4 into K562 cells yielded comparable expression levels and immunoprecipitation profiles. However, for all three alpha 4 mutants, adhesion to CS1/fibronectin was greatly diminished in either the presence or absence of the stimulatory anti-beta 1 mAb TS2/16. Constitutive adhesion to vascular cell adhesion molecule (VCAM) 1 was also diminished but, unlike CS1 adhesion, was restored upon TS2/16 stimulation. In contrast, adhesion to the bacterial protein invasin was minimally affected by any of the three mutations. For each of the mutants, the order of preference for divalent cations was unchanged compared to wild-type alpha 4, on CS1/fibronectin (Mn2+ > Mg2+ > Ca2+), on VCAM-1 (Mn2+ > Mg2+ = Ca2+) and on invasin (Mg2+ = Ca2+). However for the three mutants, the efficiency of divalent cation utilization was decreased. On VCAM-1, 68-108 microM Mn2+ was required to support half-maximal adhesion for the mutants compared with 14-18 microM for wild-type alpha 4. These results indicate (a) that three different ligands for VLA-4 show widely differing sensitivities to mutations within putative divalent cation sites, and (b) each of the three putative divalent cation sites in alpha 4 have comparable functional importance with respect to both divalent cation usage and cell adhesion.
Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis.
Authors: Lazebnik Y A , Cole S , Cooke C A , Nelson W G , Earnshaw W C
Published: May 2004

We have developed a cell-free system that induces the morphological transformations characteristic of apoptosis in isolated nuclei. The system uses extracts prepared from mitotic chicken hepatoma cells following a sequential S phase/M phase synchronization. When nuclei are added to these extracts, the chromatin becomes highly condensed into spherical domains that ultimately extrude through the nuclear envelope, forming apoptotic bodies. The process is highly synchronous, and the structural changes are completed within 60 min. Coincident with these morphological changes, the nuclear DNA is cleaved into a nucleosomal ladder. Both processes are inhibited by Zn2+, an inhibitor of apoptosis in intact cells. Nuclear lamina disassembly accompanies these structural changes in added nuclei, and we show that lamina disassembly is a characteristic feature of apoptosis in intact cells of mouse, human and chicken. This system may provide a powerful means of dissecting the biochemical mechanisms underlying the final stages of apoptosis.
Redistribution of clathrin-coated vesicle adaptor complexes during adipocytic differentiation of 3T3-L1 cells.
Authors: Chakrabarti R , Joly M , Corvera S
Published: May 2004

Mechanisms for intracellular retention of proteins are induced during adipocytic differentiation of 3T3-L1 cells. To investigate the potential role of clathrin lattices in these retention processes, we performed a morphological and biochemical analysis of coated vesicle components in 3T3-L1 cells. Optical sectioning and image restoration revealed a marked increase in the staining of clathrin and beta adaptins in the perinuclear region of cells with differentiation. In addition, predominance of beta (subunit of the AP-2, plasma membrane adaptor) over beta' (subunit of the AP-1, Golgi adaptor) adaptin was observed in immunoblots of clathrin-coated vesicles purified from nondifferentiated fibroblasts, and this ratio was reversed in coated vesicles purified from differentiated adipocytes. These results indicate that the relative abundance of TGN-derived clathrin lattices increases markedly during adipocytic differentiation. Subcellular fractionation indicated that cytosolic AP-1 and AP-2 adaptors comprised approximately 70% of the total cellular adaptor pool. Interestingly, neither the concentration nor the relative ratio of cytosolic AP-1 to AP-2 adaptors increased significantly during differentiation. These data suggest that the increase in TGN-derived lattices results from differentiation-induced mechanisms for enhanced assembly or stabilization of adaptors on Golgi membranes. Interestingly, double-immunofluorescence microscopy also revealed that whereas extensive colocalization between clathrin and beta adaptins occurred both in fibroblasts and adipocytes, structures stained only with anti-adaptin antibody could be detected. Taken together these results suggest that membranes coated with adaptors, but not clathrin, can exist in these cells.
Mannose 6-phosphate-independent targeting of lysosomal enzymes in I-cell disease B lymphoblasts.
Authors: Glickman J N , Kornfeld S
Published: May 2004

B lymphocytes from patients with I-cell disease (ICD) maintain normal cellular levels of lysosomal enzymes despite a deficiency of the enzyme UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. We find that an ICD B lymphoblastoid cell line targets about 45% of the lysosomal protease cathepsin D to dense lysosomes. This targeting occurs in the absence of detectable mannose 6-phosphate residues on the cathepsin D and is not observed in ICD fibroblasts. The secretory protein pepsinogen, which is closely related to cathepsin D in both amino acid sequence and three-dimensional structure, is mostly excluded from dense lysosomes, indicating that the lymphoblast targeting pathway is specific. Carbohydrate residues are not required for lysosomal targeting, since a non-glycosylated mutant cathepsin D is sorted with comparable efficiency to the wild type protein. Analysis of a number of cathepsin D/pepsinogen chimeric proteins indicates that an extensive polypeptide determinant in the cathepsin D carboxyl lobe can confer efficient lysosomal sorting when introduced into the pepsinogen sequence. This determinant overlaps but is not identical to the recognition marker for phosphotransferase. These results indicate that a specific protein recognition event underlies Man-6-P-independent lysosomal sorting in ICD lymphoblasts.
A temperature-sensitive NUP116 null mutant forms a nuclear envelope seal over the yeast nuclear pore complex thereby blocking nucleocytoplasmic traffic.
Authors: Wente S R , Blobel G
Published: May 2004

NUP116 encodes a 116-kD yeast nuclear pore complex (NPC) protein that is not essential but its deletion (nup116 delta) slows cell growth at 23 degrees C and is lethal at 37 degrees C (Wente, S. R., M. P. Rout, and G. Blobel. 1992. J. Cell Biol. 119:705-723). Electron microscopic analysis of nup116 delta cells shifted to growth at 37 degrees C revealed striking perturbations of the nuclear envelope: a double membrane seal that was continuous with the inner and outer nuclear membranes had formed over the cytoplasmic face of the NPCs. Electron-dense material was observed accumulating between the cytoplasmic face of these NPCs and the membrane seal, resulting in "herniations" of the nuclear envelope around individual NPCs. In situ hybridization with poly(dT) probes showed the accumulation of polyadenylated RNA in the nuclei of arrested nup116 delta cells, sometimes in the form of punctate patches at the nuclear periphery. This is consistent with the electron microscopically observed accumulation of electron-dense material within the nuclear envelope herniations. We propose that nup116 delta NPCs remain competent for export, but that the formation of the membrane seals over the NPCs blocks nucleocytoplasmic traffic.
Three-dimensional reconstruction of caldesmon-containing smooth muscle thin filaments.
Authors: Vibert P , Craig R , Lehman W
Published: May 2004

Caldesmon is known to inhibit actomyosin ATPase and filament sliding in vitro, and may play a role in modulating smooth muscle contraction as well as in diverse cellular processes including cytokinesis and exocytosis. However, the structural basis of caldesmon action has not previously been apparent. We have recorded electron microscope images of negatively stained thin filaments containing caldesmon and tropomyosin which were isolated from chicken gizzard smooth muscle in EGTA. Three-dimensional helical reconstructions of these filaments show actin monomers whose bilobed shape and connectivity are very similar to those previously seen in reconstructions of frozen-hydrated skeletal muscle thin filaments. In addition, a continuous thin strand of density follows the long-pitch actin helices, in contact with the inner domain of each actin monomer. Gizzard thin filaments treated with Ca2+/calmodulin, which dissociated caldesmon but not tropomyosin, have also been reconstructed. Under these conditions, reconstructions also reveal a bilobed actin monomer, as well as a continuous surface strand that appears to have moved to a position closer to the outer domain of actin. The strands seen in both EGTA- and Ca2+/calmodulin-treated filaments thus presumably represent tropomyosin. It appears that caldesmon can fix tropomyosin in a particular position on actin in the absence of calcium. An influence of caldesmon on tropomyosin position might, in principle, account for caldesmon's ability to modulate actomyosin interaction in both smooth muscles and non-muscle cells.