bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional differences between L- and T-plastin isoforms.
Authors: Arpin M , Friederich E , Algrain M , Vernel F , Louvard D
Published: May 2004

Fimbrins/plastins are a family of highly conserved actin-bundling proteins. They are present in all eukaryotic cells including yeast, but each isoform displays a remarkable tissue specificity. T-plastin is normally found in epithelial and mesenchymal cells while L-plastin is present in hematopoietic cells. However, L-plastin has been also found in tumor cells of non-hematopoietic origin (Lin, C.-S., R. H. Aebersold, S. B. Kent, M. Varma, and J. Leavitt. 1988. Mol. Cell. Biol. 8:4659-4668; Lin, C.-S., R. H. Aebersold, and J. Leavitt. 1990. Mol. Cell. Biol. 10: 1818-1821). To learn more about the biological significance of their tissue specificity, we have overproduced the T- and L-plastin isoforms in a fibroblast-like cell line, CV-1, and in a polarized epithelial cell line, LLC-PK1. In CV-1 cells, overproduction of T- and L-plastins induces cell rounding and a concomitant reorganization of actin stress fibers into geodesic structures. L-plastin remains associated with microfilaments while T-plastin is almost completely extracted after treatment of the cells with non-ionic detergent. In LLC-PK1 cells, T-plastin induces shape changes in microvilli and remains associated with microvillar actin filaments after detergent extraction while L-plastin has no effect on these structures and is completely extracted. The effect of T-plastin on the organization of microvilli differs from that of villin, another actin-bundling protein. Our experiments indicate that these two isoforms play differing roles in actin filament organization, and do so in a cell type-specific fashion. Thus it is likely that these plastin isoforms play fundamentally different roles in cell function.
Neogenin, an avian cell surface protein expressed during terminal neuronal differentiation, is closely related to the human tumor suppressor molecule deleted in colorectal cancer.
Authors: Vielmetter J , Kayyem J F , Roman J M , Dreyer W J
Published: May 2004

Using a monoclonal antibody, we have identified and characterized a previously unknown cell surface protein in chicken that we call neogenin and have determined its primary sequence. The deduced amino acid sequence and structure of neogenin characterize it as a member of the immunoglobulin (Ig) superfamily. Based on amino acid sequence similarities, neogenin is closely related to the human tumor suppressor molecule DCC (deleted in colorectal cancer). Neogenin and DCC define a subgroup of Ig superfamily proteins structurally distinct from other Ig molecules such as N-CAM, Ng-CAM, and Bravo/Nr-CAM. As revealed by antibody staining of tissue sections and Western blots, neogenin expression correlates with the onset of neuronal differentiation. Neogenin is also found on cells in the lower gastrointestinal tract of embryonic chickens. DCC has been observed in human neural tissues and has been shown to be essential for terminal differentiation of specific cell types in the adult human colon. These parallels suggest that neogenin, like DCC, is functionally involved in the transition from cell proliferation to terminal differentiation of specific cell types. Since neogenin is expressed on growing neurites and downregulated at termination of neurite growth, it may also play an important role in many of the complex functional aspects of neurite extension and intercellular signaling.
TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors.
Authors: Miettinen P J , Ebner R , Lopez A R , Derynck R
Published: May 2004

The secreted polypeptide transforming growth factor-beta (TGF-beta) exerts its multiple activities through type I and II cell surface receptors. In epithelial cells, activation of the TGF-beta signal transduction pathways leads to inhibition of cell proliferation and an increase in extracellular matrix production. TGF-beta is widely expressed during development and its biological activity has been implicated in epithelial-mesenchymal interactions, e.g., in branching morphogenesis of the lung, kidney, and mammary gland, and in inductive events between mammary epithelium and stroma. In the present study, we investigated the effects of TGF-beta on mouse mammary epithelial cells in vitro. TGF-beta reversibly induced an alteration in the differentiation of normal mammary epithelial NMuMG cells from epithelial to fibroblastic phenotype. The change in cell morphology correlated with (a) decreased expression of the epithelial markers E-cadherin, ZO-1, and desmoplakin I and II; (b) increased expression of mesenchymal markers, such as fibronectin; and (c) a fibroblast-like reorganization of actin fibers. This phenotypic differentiation displays the hallmarks of an epithelial to mesenchymal transdifferentiation event. Since NMuMG cells make high levels of the type I TGF-beta receptor Tsk7L, yet lack expression of the ALK-5/R4 type I receptor which has been reported to mediate TGF-beta responsiveness, we evaluated the role of the Tsk7L receptor in TGF-beta-mediated transdifferentiation. We generated NMuMG cells that stably overexpress a truncated Tsk7L type I receptor that lacks most of the cytoplasmic kinase domain, thus function as a dominant negative mutant. These transfected cells no longer underwent epithelial to mesenchymal morphological change upon exposure to TGF-beta, yet still displayed some TGF-beta-mediated responses. We conclude that TGF-beta has the ability to modulate E-cadherin expression and induce a reversible epithelial to mesenchymal transdifferentiation in epithelial cells. Unlike other transdifferentiating growth factors, such as bFGF and HGF, these changes are accompanied by growth inhibition. Our results also implicate the Tsk7L type I receptor as mediating the TGF-beta-induced epithelial to mesenchymal transition.
Expression of variant fibronectins in wound healing: cellular source and biological activity of the EIIIA segment in rat hepatic fibrogenesis.
Authors: Jarnagin W R , Rockey D C , Koteliansky V E , Wang S S , Bissell D M
Published: May 2004

We have examined the cell-specific expression of two fibronectin isoforms, EIIIA and EIIIB, during experimental hepatic fibrosis induced by ligation of the biliary duct. AT the mRNA level, EIIIA and EIIIB were undetectable in normal liver but expressed early injury, preceding fibrosis. The cellular sources of these changes were determined by fractionating the liver at various time points after bile duct ligation into its constituent cell populations and extracting RNA from the fresh isolates. EIIIA-containing fibronectin mRNA was undetectable in normal sinusoidal endothelial cells but increased rapidly within 12 h of injury. By contrast, the EIIIB form was restricted to hepatic lipocytes (Ito or fat-storing cells) and appeared only after a lag of 12-24 h: it was minimal in sinusoidal endothelial cells. Both forms were minimal in hepatocytes. At the protein level, EIIIA-containing fibronectin was markedly increased within two days of injury and exhibited a sinusoidal distribution. Secretion of this form by endothelial cells was confirmed in primary culture. Matrices deposited in situ by endothelial cells from injured liver accelerated the conversion ("activation") of normal lipocytes to myofibroblast-like cells, and pretreatment of matrices with monoclonal antibody to the EIIIA segment blocked this response. Finally, recombinant fibronectin peptide containing the EIIIA segment was stimulatory to lipocytes in culture. We conclude that expression of EIIIA fibronectin by sinusoidal endothelial cells is a critical early event in the liver's response to injury and that the EIIIA segment is biologically active, mediating the conversion of lipocytes to myofibroblasts.
Measurements of growth cone adhesion to culture surfaces by micromanipulation.
Authors: Zheng J , Buxbaum R E , Heidemann S R
Published: May 2004

Neurons were grown on plastic surfaces that were untreated, or treated with polylysine, laminin, or L1 and their growth cones were detached from their culture surface by applying known forces with calibrated glass needles. This detachment force was taken as a measure of the force of adhesion of the growth cone. We find that on all surfaces, lamellipodial growth cones require significantly greater detachment force than filopodial growth cones, but this differences is, in general, due to the greater area of lamellipodial growth cones compared to filopodial growth cones. That is, the stress (force/unit area) required for detachment was similar for growth cones of lamellipodial and filopodial morphology on all surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces, which had a significantly lower stress of detachment than on other surfaces. Surprisingly, the forces required for detachment (760-3,340 mudynes) were three to 15 times greater than the typical resting axonal tension, the force exerted by advancing growth cones, or the forces of retraction previously measured by essentially the same method. Nor did we observe significant differences in detachment force among growth cones of similar morphology on different culture surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces. These data argue against the differential adhesion mechanism for growth cone guidance preferences in culture. Our micromanipulations revealed that the most mechanically resistant regions of growth cone attachment were confined to quite small regions typically located at the ends of filopodia and lamellipodia. Detached growth cones remained connected to the substratum at these regions by highly elastic retraction fibers. The closeness of contact of growth cones to the substratum as revealed by interference reflection microscopy (IRM) did not correlate with our mechanical measurements of adhesion, suggesting that IRM cannot be used as a reliable estimator of growth cone adhesion.
E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton.
Authors: Hülsken J , Birchmeier W , Behrens J
Published: May 2004

beta-Catenin is involved in the formation of adherens junctions of mammalian epithelia. It interacts with the cell adhesion molecule E-cadherin and also with the tumor suppressor gene product APC, and the Drosophila homologue of beta-catenin, armadillo, mediates morphogenetic signals. We demonstrate here that E-cadherin and APC directly compete for binding to the internal, armadillo-like repeats of beta-catenin; the NH2-terminal domain of beta-catenin mediates the interaction of the alternative E-cadherin and APC complexes to the cytoskeleton by binding to alpha-catenin. Plakoglobin (gamma-catenin), which is structurally related to beta-catenin, mediates identical interactions. We thus show that the APC tumor suppressor gene product forms strikingly similar associations as found in cell junctions and suggest that beta-catenin and plakoglobin are central regulators of cell adhesion, cytoskeletal interaction, and tumor suppression.
Antibodies to the kinesin motor domain and CENP-E inhibit microtubule depolymerization-dependent motion of chromosomes in vitro.
Authors: Lombillo V A , Nislow C , Yen T J , Gelfand V I , McIntosh J R
Published: May 2004

Chromosomes can move with the ends of depolymerizing microtubules (MTs) in vitro, even in the absence of nucleotide triphosphates (Coue, M., V. A. Lombillo, and J. R. McIntosh. 1991. J. Cell Biol. 112:1165-1175.) Here, we describe an immunological investigation of the proteins important for this form of motility. Affinity-purified polyclonal antibodies to kinesin exert a severe inhibitory effect on depolymerization-dependent chromosome motion. These antibodies predominantly recognize a polypeptide of M(r) approximately 250 kD on immunoblots of CHO chromosomes and stain kinetochores as well as some vesicles that are in the chromosome preparation. Antibodies to CENP-E, a kinetochore-associated kinesin-like protein, also recognize a 250-kD electrophoretic component, but they stain only the kinetochroe region of isolated chromosomes. Polyclonal antibodies that recognize specific domains of the CENP-E polypeptide affect MT disassembly-dependent chromosome motion in different ways; antibodies to the head or tail portions slow motility threefold, while those raised against the neck region stop motion completely. Analogous antibodies that block conventional, ATP-dependent motility of cytoplasmic dynein (Vaisberg, G., M. P. Koonce, and J. R. McIntosh. 1993. J. Cell Biol. 123:849-858) have no effect on disassembly-dependent chromosome motion, even though they bind to kinetochores. These observations suggest that CENP-E helps couple chromosomes to depolymerizing MTs. A similar coupling activity may allow spindle MTs to remain kinetochore-bound while their lengths change during both prometaphase and anaphase A.
The role of microtubules in growth cone turning at substrate boundaries.
Authors: Tanaka E , Kirschner M W
Published: May 2004

To understand the role of microtubules in growth cone turning, we observed fluorescently labeled microtubules in neurons as they encountered a substrate boundary. Neurons growing on a laminin-rich substrate avoided growing onto collagen type IV. Turning growth cones assumed heterogeneous morphologies and behaviors that depended primarily in their extent of adhesion to the substrate. We grouped these behaviors into three categories-sidestepping, motility, and growth-mediated reorientation. In sidestepping and motility-mediated reorientation, the growth cone and parts of the axon were not well attached to the substrate so the acquisition of an adherent lamella caused the entire growth cone to move away from the border and consequently reoriented the axon. In these cases, since the motility of the growth cone dominates its reorientation, the microtubules were passive, and reorientation occurred without significant axon growth. In growth-mediated reorientation, the growth cone and axon were attached to the substrate. In this case, microtubules reoriented within the growth cone to stabilize a lamella. Bundling of the reoriented microtubules was followed by growth cone collapse to form new axon, and further, polarized lamellipodial extension. These observations indicate that when the growth cone remains adherent to the substrate during turning, the reorientation and bundling of microtubules is an important, early step in growth cone turning.
Nuclear spreads: I. Visualization of bipartite ribosomal RNA domains.
Authors: Garcia-Blanco M A , Miller D D , Sheetz M P
Published: May 2004

Visualization of nuclear architecture is key to the understanding of the association between RNA synthesis and processing. This architecture is obscured by the high density of components in most nuclei. We have developed a method of spreading nuclei and nucleoli that reduces overlap of weakly associated components. Strong interactions among nuclear components are not disrupted by this method. Spread nucleoli remained structurally distinct and functionally competent in ribosomal RNA synthesis. Nascent ribosomal RNA colocalized with RNA polymerase I and fibrillarin, a protein required for processing of ribosomal RNA. Colocalization of nascent transcripts and fibrillarin was seen in nucleoli spread over several microns, suggesting a strong interaction. These data suggest that nucleoli are superassemblies of bipartite domains, each composed of a ribosomal RNA synthesis center tightly associated with areas likely to be involved in ribosomal RNA processing.
Roles of hepatocyte growth factor/scatter factor and the met receptor in the early development of the metanephros.
Authors: Woolf A S , Kolatsi-Joannou M , Hardman P , Andermarcher E , Moorby C , Fine L G , Jat P S , Noble M D , Gherardi E
Published: May 2004

Several lines of evidence suggest that hepatocyte growth factor/scatter factor (HGF/SF), a soluble protein secreted by embryo fibroblasts and several fibroblast lines, may elicit morphogenesis in adjacent epithelial cells. We investigated the role of HGF/SF and its membrane receptor, the product of the c-met protooncogene, in the early development of the metanephric kidney. At the inception of the mouse metanephros at embryonic day 11, HGF/SF was expressed in the mesenchyme, while met was expressed in both the ureteric bud and the mesenchyme, as assessed by reverse transcription PCR, in situ hybridization, and immunohistochemistry. To further investigate the expression of met in renal mesenchyme, we isolated 13 conditionally immortal clonal cell lines from transgenic mice expressing a temperature-sensitive mutant of the SV-40 large T antigen. Five had the HGF/SF+/met+ phenotype and eight had the HGF/SF-/met+ phenotype. None had the HGF/SF+/met- nor the HGF/SF-/met- phenotypes. Thus the renal mesenchyme contains cells that express HGF/SF and met or met alone. When metanephric rudiments were grown in serum-free organ culture, anti-HGF/SF antibodies (a) inhibited the differentiation of metanephric mesenchymal cells into the epithelial precursors of the nephron; (b) increased cell death within the renal mesenchyme; and (c) perturbed branching morphogenesis of the ureteric bud. These data provide the first demonstration for coexpression of the HGF/SF and met genes in mesenchymal cells during embryonic development and also imply an autocrine and/or paracrine role for HGF/SF and met in the survival of the renal mesenchyme and in the mesenchymal-epithelial transition that occurs during nephrogenesis. They also confirm the postulated paracrine role of HGF/SF in the branching of the ureteric bud.
Viral proteins E1B19K and p35 protect sympathetic neurons from cell death induced by NGF deprivation.
Authors: Martinou I , Fernandez P A , Missotten M , White E , Allet B , Sadoul R , Martinou J C
Published: May 2004

To study molecular mechanisms underlying neuronal cell death, we have used sympathetic neurons from superior cervical ganglia which undergo programmed cell death when deprived of nerve growth factor. These neurons have been microinjected with expression vectors containing cDNAs encoding selected proteins to test their regulatory influence over cell death. Using this procedure, we have shown previously that sympathetic neurons can be protected from NGF deprivation by the protooncogene Bcl-2. We now report that the E1B19K protein from adenovirus and the p35 protein from baculovirus also rescue neurons. Other adenoviral proteins, E1A and E1B55K, have no effect on neuronal survival. E1B55K, known to block apoptosis mediated by p53 in proliferative cells, failed to rescue sympathetic neurons suggesting that p53 is not involved in neuronal death induced by NGF deprivation. E1B19K and p35 were also coinjected with Bcl-Xs which blocks Bcl-2 function in lymphoid cells. Although Bcl-Xs blocked the ability of Bcl-2 to rescue neurons, it had no effect on survival that was dependent upon expression of E1B19K or p35.
NCAM polypeptides in heart development: association with Z discs of forms that contain the muscle-specific domain.
Authors: Byeon M K , Sugi Y , Markwald R R , Hoffman S
Published: May 2004

Previous studies of neural cell adhesion molecule (NCAM) cDNAs have revealed an alternatively spliced set of small exons (12A, 12B, 12C, and 12D) that encode a region in the extracellular portion of the molecule known as the muscle-specific domain (MSD). The entire MSD region can be expressed in skeletal muscle, heart, and skin; only exons 12A and 12D have been found in brain. These studies did not reveal which NCAM polypeptides contain the MSD region or the immunohistochemical distribution of these NCAM molecules. To address these questions, we prepared antibodies against the oligopeptides encoded by exons 12A and 12B and by exons 12C and 12D, and we used these antibodies to study the forms of NCAM containing the MSD region expressed during embryonic chicken heart development. These antibodies recognize certain forms of NCAM found in the heart, but they do not recognize brain NCAM. In the heart, each of the splice variants of NCAM (large cytoplasmic domain, small cytoplasmic domain, and small surface domain) that differ in their mode of attachment to the plasma membrane or in the size of their cytoplasmic domain is expressed in a form that contains and in a form that lacks the MSD region. No microheterogeneity is observed in the size of NCAM molecules containing the MSD region, even at the level of cyanogen bromide fragments, suggesting that exons 12A-D are expressed as a single unit. Depending on the site and the stage of development, the percent of NCAM molecules containing the MSD region can vary from nearly 0 to 100%. In general, this percentage increases during development. In immunohistochemical studies of hearts from stage 18 embryos, forms of NCAM containing the MSD region colocalized with Z discs. No other adhesion molecules were found in this distribution at this early stage of development. Studies on isolated cells in vitro demonstrate that the colocalization with Z discs of NCAM molecules containing the MSD region does not depend on cell-cell contact, and they raise the possibility that this form of NCAM is involved in cell-extracellular matrix interactions. The association of NCAM molecules containing the MSD region with Z discs suggests that this form of NCAM is involved in early myofibrillogenesis.
Type X collagen gene expression in mouse chondrocytes immortalized by a temperature-sensitive simian virus 40 large tumor antigen.
Authors: Lefebvre V , Garofalo S , de Crombrugghe B
Published: May 2004

Mouse endochondral chondrocytes were immortalized with a temperature-sensitive simian virus 40 large tumor antigen. Several clonal isolates as well as pools of immortalized cells were characterized. In monolayer cultures at the temperature permissive for the activity of the large tumor antigen (32 degrees C), the cells grew continuously with a doubling time of approximately 2 d, whereas they stopped growing at nonpermissive temperatures (37 degrees C-39 degrees C). The cells from all pools and from most clones expressed the genes for several markers of hypertrophic chondrocytes, such as type X collagen, matrix Gla protein, and osteopontin, but had lost expression of type II collagen mRNA and failed to be stained by alcian blue which detects cartilage-specific proteoglycans. The cells also contained mRNAs for type I collagen and bone Gla protein, consistent with acquisition of osteoblastic-like properties. Higher levels of mRNAs for type X collagen, bone Gla protein, and osteopontin were found at nonpermissive temperatures, suggesting that the expression of these genes was upregulated upon growth arrest, as is the case in vivo during chondrocyte hypertrophy. Cells also retained their ability to respond to retinoic acid, as indicated by retinoic acid dose-dependent and time-dependent increases in type X collagen mRNA levels. These cell lines, the first to express characteristic features of hypertrophic chondrocytes, should be very useful to study the regulation of the type X collagen gene and other genes activated during the last stages of chondrocyte differentiation.
Calnexin and BiP act as sequential molecular chaperones during thyroglobulin folding in the endoplasmic reticulum.
Authors: Kim P S , Arvan P
Published: May 2004

Before secretion, newly synthesized thyroglobulin (Tg) folds via a series of intermediates: disulfide-linked aggregates and unfolded monomers-->folded monomers-->dimers. Immediately after synthesis, very little Tg associated with calnexin (a membrane-bound molecular chaperone in the ER), while a larger fraction bound BiP (a lumenal ER chaperone); dissociation from these chaperones showed superficially similar kinetics. Calnexin might bind selectively to carbohydrates within glycoproteins, or to hydrophobic surfaces of secretory proteins while they form proper disulfide bonds (Wada, I., W.-J. Ou, M.-C. Liu, and G. Scheele, J. Biol. Chem. 1994. 269:7464-7472). Because Tg has multiple disulfides, as well as glycans, we tested a brief exposure of live thyrocytes to dithiothreitol, which resulted in quantitative aggregation of nascent Tg, as analyzed by SDS-PAGE of cells lysed without further reduction. Cells lysed in the presence of dithiothreitol under non-denaturing conditions caused Tg aggregates to run as reduced monomers. For cells lysed either way, after in vivo reduction, Tg coprecipitated with calnexin. After washout of dithiothreitol, nascent Tg aggregates dissolved intracellularly and were secreted ultimately. 1 h after washout, > or = 92% of labeled Tg was found to dissociate from calnexin, while the fraction of labeled Tg bound to BiP rose from 0 to approximately 40%, demonstrating a "precursor-product" relationship. Whereas intralumenal reduction was essential for efficient Tg coprecipitation with calnexin, Tg glycosylation was not required. These data are among the first to demonstrate sequential chaperone function involved in conformational maturation of nascent secretory proteins within the ER.
Depletion of calcium from the lumen of endoplasmic reticulum reversibly inhibits passive diffusion and signal-mediated transport into the nucleus.
Authors: Greber U F , Gerace L
Published: May 2004

Nuclear pore complexes provide channels for molecular transport across the nuclear envelope. Translocation of most proteins and RNAs through the pore complex is mediated by signal- and ATP-dependent mechanisms, while transport of small molecules is accomplished by passive diffusion. We report here that depletion of calcium from the lumen of the endoplasmic reticulum and nuclear envelope with ionophores or the calcium pump inhibitor thapsigargin rapidly and potently inhibits signal mediated transport of proteins into the nucleus. Lumenal calcium depletion also inhibits passive diffusion through the pore complex. Signal-mediated protein import and passive diffusion are rapidly restored when the drugs depleting lumenal calcium are removed and cells are incubated at 37 degrees C in calcium-containing medium. These results indicate that loss of calcium from the lumen of the endoplasmic reticulum and nuclear envelope reversibly affects properties of pore complex components located on the nuclear/cytoplasmic membrane surfaces, and they provide direct functional evidence for conformational flexibility of the pore complex. These methods will be useful for achieving reversible inhibition of nucleocytoplasmic trafficking for in vivo functional studies, and for studying the structure of the passive diffusion channel(s) of the pore complex.
Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments.
Authors: Schafer D A , Hug C , Cooper J A
Published: May 2004

The actin filaments of myofibrils are highly organized; they are of a uniform length and polarity and are situated in the sarcomere in an aligned array. We hypothesized that the barbed-end actin-binding protein, CapZ, directs the process of actin filament assembly during myofibrillogenesis. We tested this hypothesis by inhibiting the actin-binding activity of CapZ in developing myotubes in culture using two different methods. First, injection of a monoclonal antibody that prevents the interaction of CapZ and actin disrupts the non-striated bundles of actin filaments formed during the early stages of myofibril formation in skeletal myotubes in culture. The antibody, when injected at concentrations lower than that required for disrupting the actin filaments, binds at nascent Z-disks. Since the interaction of CapZ and the monoclonal antibody are mutually exclusive, this result indicates that CapZ binds nascent Z-disks independent of an interaction with actin filaments. In a second approach, expression in myotubes of a mutant form of CapZ that does not bind actin results in a delay in the appearance of actin in a striated pattern in myofibrils. The organization of alpha-actinin at Z-disks also is delayed, but the organization of titin and myosin in sarcomeres is not significantly altered. We conclude that the interaction of CapZ and actin is important for the organization of actin filaments of the sarcomere.
Identification and partial characterization of mitotic centromere-associated kinesin, a kinesin-related protein that associates with centromeres during mitosis.
Authors: Wordeman L , Mitchison T J
Published: May 2004

Using antipeptide antibodies to conserved regions of the kinesin motor domain, we cloned a kinesin-related protein that associates with the centromere region of mitotic chromosomes. We call the protein MCAK, for mitotic centromere-associated kinesin. MCAK appears concentrated on centromeres at prophase and persists until telophase, after which time the localization disperses. It is found throughout the centromere region and between the kinetochore plates of isolated mitotic CHO chromosomes, in contrast to two other kinetochore-associated microtubule motors: cytoplasmic dynein and CENP-E (Yen et al., 1992), which are closer to the outer surface of the kinetochore plates. Sequence analysis shows MCAK to be a kinesin-related protein with the motor domain located in the center of the protein. It is 60-70% similar to kif2, a kinesin-related protein originally cloned from mouse brain with a centrally located motor domain (Aizawa et al., 1992). MCAK protein is present in interphase and mitotic CHO cells and is transcribed as a single 3.4-kb message.
Macromolecular interactions in the nucleoporin p62 complex of rat nuclear pores: binding of nucleoporin p54 to the rod domain of p62.
Authors: Buss F , Stewart M
Published: May 2004

Nuclear pore complexes are constructed from a large number of different proteins, called collectively nucleoporins. One of these nucleoporins, p62, has an alpha-helical coiled-coil COOH-terminal rod domain linked to an NH2-terminal domain that contains a series of degenerate pentapeptide repeats. In nuclear pores p62 forms a tight complex with at least two other proteins, p58 and p54, which can be extracted from isolated rat liver nuclei (Finlay, D. R., E. Meier, P. Bradley, J. Horecka, and D. J. Forbes. 1991. J. Cell Biol. 114:169-183). We have used a range of methods to demonstrate a strong binding between p62 and p54 in this complex and show that the rod domain of p62 appears to constitute the principal binding site for p54. Whole p62 and its rod domain expressed in Escherichia coli both bind strongly to p54 in blot-overlay assays. Most of the epitopes on the p62 rod recognized by polyclonal antisera are masked in the complex, whereas epitopes on the NH2-terminal domain of p62 are still exposed, both in the isolated complex and also in nuclear pores stained in situ by immunofluorescence in isolated rat nuclei. Moreover, it has been possible to exchange recombinant p62 rod for some of the native p62 in complexes partially dissociated by 4 M urea. Overall these results suggest a key role for the p62 rod domain in maintaining the structural integrity of the complex and also suggest a molecular model for the complex. This model is consistent with data that indicate that the analogous coiled-coil region of yeast nucleoporin NSP1 may function in a similar way.
The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane.
Authors: Miller J D , Tajima S , Lauffer L , Walter P
Published: May 2004

The signal recognition particle receptor (SR) is required for the cotranslational targeting of both secretory and membrane proteins to the endoplasmic reticulum (ER) membrane. During targeting, the SR interacts with the signal recognition particle (SRP) which is bound to the signal sequence of the nascent protein chain. This interaction catalyzes the GTP-dependent transfer of the nascent chain from SRP to the protein translocation apparatus in the ER membrane. The SR is a heterodimeric protein comprised of a 69-kD subunit (SR alpha) and a 30-kD subunit (SR beta) which are associated with the ER membrane in an unknown manner. SR alpha and the 54-kD subunits of SRP (SRP54) each contain related GTPase domains which are required for SR and SRP function. Molecular cloning and sequencing of a cDNA encoding SR beta revealed that SR beta is a transmembrane protein and, like SR alpha and SRP54, is a member of the GTPase superfamily. Although SR beta defines its own GTPase subfamily, it is distantly related to ARF and Sar1. Using UV cross-linking, we confirm that SR beta binds GTP specifically. Proteolytic digestion experiments show that SR alpha is required for the interaction of SRP with SR. SR alpha appears to be peripherally associated with the ER membrane, and we suggest that SR beta, as an integral membrane protein, mediates the membrane association of SR alpha. The discovery of its guanine nucleotide-binding domain, however, makes it likely that its role is more complex than that of a passive anchor for SR alpha. These findings suggest that a cascade of three directly interacting GTPases functions during protein targeting to the ER membrane.
The Hansenula polymorpha PER8 gene encodes a novel peroxisomal integral membrane protein involved in proliferation.
Authors: Tan X , Waterham H R , Veenhuis M , Cregg J M
Published: May 2004

We previously described the isolation of mutants of the methylotrophic yeast Hansenula polymorpha that are defective in peroxisome biogenesis. Here, we describe the characterization of one of these mutants, per8, and the cloning of the PER8 gene. In either methanol or methylamine medium, conditions that normally induce the organelles, per8 cells contain no peroxisome-like structures and peroxisomal enzymes are located in the cytosol. The sequence of PER8 predicts that its product (Per8p) is a novel polypeptide of 34 kD, and antibodies against Per8p recognize a protein of 31 kD. Analysis of the primary sequence of Per8p revealed a 39-amino-acid cysteine-rich segment with similarity to the C3HC4 family of zinc-finger motifs. Overexpression of PER8 results in a markedly enhanced increase in peroxisome numbers. We show that Per8p is an integral membrane protein of the peroxisome and that it is concentrated in the membranes of newly formed organelles. We propose that Per8p is a component of the molecular machinery that controls the proliferation of this organelle.
Cytoplasmic determinants involved in direct lysosomal sorting, endocytosis, and basolateral targeting of rat lgp120 (lamp-I) in MDCK cells.
Authors: Höning S , Hunziker W
Published: May 2004

Rat lysosomal glycoprotein 120 (lgp120; lamp-I) is a transmembrane protein that is directly delivered from the trans-Golgi network (TGN) to the endosomal/lysosomal system without prior appearance on the cell surface. Its short cytosolic domain of 11 residues encodes determinants for direct lysosomal sorting, endocytosis and, in polarized cells, basolateral targeting. We now characterize the structural requirements in the cytosolic domain required for sorting of lgp120 into the different pathways. Our results show that the cytoplasmic tail is sufficient to mediate direct transport from the trans-Golgi network (TGN) to lysosomes and that a G7-Y8-X-X-I11 motif is crucial for this sorting event. While G7 is only critical for direct lysosomal sorting in the TGN, Y8 and I11 are equally important for lysosomal sorting, endocytosis, and basolateral targeting. Thus, a small motif of five amino acids in the cytoplasmic tail of lgp120 can be recognized by the sorting machinery at several cellular locations and direct the protein into a variety of intracellular pathways.
Glycosyl phosphatidylinositol-dependent cross-linking of alpha-agglutinin and beta 1,6-glucan in the Saccharomyces cerevisiae cell wall.
Authors: Lu C F , Montijn R C , Brown J L , Klis F , Kurjan J , Bussey H , Lipke P N
Published: May 2004

The cell adhesion protein alpha-agglutinin is bound to the outer surface of the Saccharomyces cerevisiae cell wall and mediates cell-cell contact in mating. alpha-Agglutinin is modified by addition of a glycosyl phosphatidylinositol (GPI) anchor as it traverses the secretory pathway. The presence of a GPI anchor is essential for cross-linking into the wall, but the fatty acid and inositol components of the anchor are lost before cell wall association (Lu, C.-F., J. Kurjan, and P. N. Lipke, 1994. A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin. Mol. Cell. Biol. 14:4825-4833). Cell wall association of alpha-agglutinin was accompanied by an increase in size and a gain in reactivity to antibodies directed against beta 1,6-glucan. Several kre mutants, which have defects in synthesis of cell wall beta 1,6-glucan, had reduced molecular size of cell wall alpha-agglutinin. These findings demonstrate that the cell wall form of alpha-agglutinin is covalently associated with beta 1,6-glucan. The alpha-agglutinin biosynthetic precursors did not react with antibody to beta 1,6-glucan, and the sizes of these forms were unaffected in kre mutants. A COOH-terminal truncated form of alpha-agglutinin, which is not GPI anchored and is secreted into the medium, did not react with the anti-beta 1,6-glucan. We propose that extracellular cross-linkage to beta 1,6-glucan mediates covalent association of alpha-agglutinin with the cell wall in a manner that is dependent on prior addition of a GPI anchor to alpha-agglutinin.
Mechanical function of dystrophin in muscle cells.
Authors: Pasternak C , Wong S , Elson E L
Published: May 2004

We have directly measured the contribution of dystrophin to the cortical stiffness of living muscle cells and have demonstrated that lack of dystrophin causes a substantial reduction in stiffness. The inferred molecular structure of dystrophin, its preferential localization underlying the cell surface, and the apparent fragility of muscle cells which lack this protein suggest that dystrophin stabilizes the sarcolemma and protects the myofiber from disruption during contraction. Lacking dystrophin, the muscle cells of persons with Duchenne muscular dystrophy (DMD) are abnormally vulnerable. These facts suggest that muscle cells with dystrophin should be stiffer than similar cells which lack this protein. We have tested this hypothesis by measuring the local stiffness of the membrane skeleton of myotubes cultured from mdx mice and normal controls. Like humans with DMD mdx mice lack dystrophin due to an x-linked mutation and provide a good model for the human disease. Deformability was measured as the resistance to indentation of a small area of the cell surface (to a depth of 1 micron) by a glass probe 1 micron in radius. The stiffness of the membrane skeleton was evaluated as the increment of force (mdyne) per micron of indentation. Normal myotubes with an average stiffness value of 1.23 +/- 0.04 (SE) mdyne/micron were about fourfold stiffer than myotubes cultured from mdx mice (0.34 +/- 0.014 mdyne/micron). We verified by immunofluorescence that both normal and mdx myotubes, which were at a similar developmental stage, expressed sarcomeric myosin, and that dystrophin was detected, diffusely distributed, only in normal, not in mdx myotubes. These results confirm that dystrophin and its associated proteins can reinforce the myotube membrane skeleton by increasing its stiffness and that dystrophin function and, therefore, the efficiency of therapeutic restoration of dystrophin can be assayed through its mechanical effects on muscle cells.
Syntrophin binds to an alternatively spliced exon of dystrophin.
Authors: Ahn A H , Kunkel L M
Published: May 2004

Dystrophin, the protein product of the Duchenne muscular dystrophy locus, is a protein of the membrane cytoskeleton that associates with a complex of integral and membrane-associated proteins. Of these, the 58-kD intracellular membrane-associated protein, syntrophin, was recently shown to consist of a family of three related but distinct genes. We expressed the cDNA of human beta 1-syntrophin and the COOH terminus of human dystrophin in reticulocyte lysates using an in vitro transcription/translation system. Using antibodies to dystrophin we immunoprecipitated these two interacting proteins in a variety of salt and detergent conditions. We demonstrate that the 53 amino acids encoded on exon 74 of dystrophin, an alternatively spliced exon, are necessary and sufficient for interaction with translated beta 1-syntrophin in our assay. On the basis of its alternative splicing, dystrophin may thus be present in two functionally distinct populations. In this recombinant expression system, the dystrophin relatives, human dystrophin related protein (DRP or utrophin) and the 87K postsynaptic protein from Torpedo electric organ, also bind to translated beta 1-syntrophin. We have found a COOH-terminal 37-kD fragment of beta 1-syntrophin sufficient to interact with translated dystrophin and its homologues, suggesting that the dystrophin binding site on beta 1-syntrophin occurs on a region that is conserved among the three syntrophin homologues.
Mammalian alpha 1- and beta 1-syntrophin bind to the alternative splice-prone region of the dystrophin COOH terminus.
Authors: Suzuki A , Yoshida M , Ozawa E
Published: May 2004

The carboxy-terminal region of dystrophin has been suggested to be crucially important for its function to prevent muscle degeneration. We have previously shown that this region is the locus that interacts with the sarcolemmal glycoprotein complex, which mediates membrane anchoring of dystrophin, as well as with the cytoplasmic peripheral membrane protein, A0 and beta 1-syntrophin (Suzuki, A., M. Yoshida, K. Hayashi, Y. Mizuno, Y. Hagiwara, and E. Ozawa. 1994. Eur. J. Biochem. 220:283-292). In this work, by using the overlay assay technique developed previously, we further analyzed the dystrophin-syntrophin/A0 interaction. Two forms of mammalian syntrophin, alpha 1- and beta 1-syntrophin, were found to bind to very close but discrete regions on the dystrophin molecule. Their binding sites are located at the vicinity of the glycoprotein-binding site, and correspond to the amino acid residues encoded by exons 73-74 which are alternatively spliced out in some isoforms. This suggests that the function of syntrophin is tightly linked to the functional diversity among dystrophin isoforms. Pathologically, it is important that the binding site for alpha 1-syntrophin, which is predominantly expressed in skeletal muscle, coincides with the region whose deletion was suggested to result in a severe phenotype. In addition, A0, a minor component of dystrophin-associated proteins with a molecular mass of 94 kD which is immunochemically related to syntrophin, binds to the same site as beta 1-syntrophin. Finally, based on our accumulated evidence, we propose a revised model of the domain organization of dystrophin from the view point of protein-protein interactions.
Tropomyosin is essential in yeast, yet the TPM1 and TPM2 products perform distinct functions.
Authors: Drees B , Brown C , Barrell B G , Bretscher A
Published: May 2004

Sequence analysis of chromosome IX of Saccharomyces cerevisiae revealed an open reading frame of 166 residues, designated TPM2, having 64.5% sequence identity to TPM1, that encodes the major form of tropomyosin in yeast. Purification and characterization of Tpm2p revealed a protein with the characteristics of a bona fide tropomyosin; it is present in vivo at about one sixth the abundance of Tpm1p. Biochemical and sequence analysis indicates that Tpm2p spans four actin monomers along a filament, whereas Tpmlp spans five. Despite its shorter length, Tpm2p can compete with Tpm1p for binding to F-actin. Over-expression of Tpm2p in vivo alters the axial budding of haploids to a bipolar pattern, and this can be partially suppressed by co-over-expression of Tpm1p. This suggests distinct functions for the two tropomyosins, and indicates that the ratio between them is important for correct morphogenesis. Loss of Tpm2p has no detectable phenotype in otherwise wild type cells, but is lethal in combination with tpm1 delta. Over-expression of Tpm2p does not suppress the growth or cell surface targeting defects associated with tpm1 delta, so the two tropomyosins must perform an essential function, yet are not functionally interchangeable. S. cerevisiae therefore provides a simple system for the study of two tropomyosins having distinct yet overlapping functions.
Both synchronous and asynchronous muscle isoforms of projectin (the Drosophila bent locus product) contain functional kinase domains.
Authors: Ayme-Southgate A , Southgate R , Saide J , Benian G M , Pardue M L
Published: May 2004

In Drosophila, the large muscle protein, projectin, has very different localizations in synchronous and asynchronous muscles, suggesting that projectin has different functions in different muscle types. The multiple projectin isoforms are encoded by a single gene; however they differ significantly in size (as detected by gel mobility) and show differences in some peptide fragments, presumably indicating alternative splicing or termination. We now report additional sequence of the projectin gene, showing a kinase domain and flanking regions highly similar to equivalent regions of twitchin, including a possible autoinhibitory region. In spite of apparent differences in function, all isoforms of projectin have the kinase domain and all are capable of autophosphorylation in vitro. The projectin gene is in polytene region 102C/D where the bentD phenotype maps. The recessive lethality of bentD is associated with a breakpoint that removes sequence of the projectin kinase domain. We find that different alleles of the highly mutable recessive lethal complementation group, l(4)2, also have defects in different parts of the projectin sequence, both NH2-terminal and COOH-terminal to the bentD breakpoint. These alleles are therefore renamed as alleles of the bent locus. Adults heterozygous for projectin mutations show little, if any, effect of one defective gene copy, but homozygosity for any of the defects is lethal. The times of death can vary with allele. Some alleles kill the embryos, others are larval lethal. These molecular studies begin to explain why genetic studies suggested that l(4)2 was a complex (or pseudoallelic) locus.
A MAP kinase necessary for receptor-mediated activation of adenylyl cyclase in Dictyostelium.
Authors: Segall J E , Kuspa A , Shaulsky G , Ecke M , Maeda M , Gaskins C , Firtel R A , Loomis W F
Published: May 2004

Analysis of a developmental mutant in Dictyostelium discoideum which is unable to initiate morphogenesis has shown that a protein kinase of the MAP kinase/ERK family affects relay of the cAMP chemotactic signal and cell differentiation. Strains in which the locus encoding ERK2 is disrupted respond to a pulse of cAMP by synthesizing cGMP normally but show little synthesis of cAMP. Since mutant cells lacking ERK2 contain normal levels of both the cytosolic regulator of adenylyl cyclase (CRAC) and manganese-activatable adenylyl cyclase, it appears that this kinase is important for receptor-mediated activation of adenylyl cyclase.