bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane proximal cleavage of L-selectin: identification of the cleavage site and a 6-kD transmembrane peptide fragment of L-selectin.
Authors: Kahn J , Ingraham R H , Shirley F , Migaki G I , Kishimoto T K
Published: May 2004

Rapid downregulation of L-selectin expression occurs in response to leukocyte activation, and it has been speculated to be an integral process in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. It has previously been proposed that L-selectin is proteolytically cleaved from the cell surface; however, the nature of the cleavage site has been unknown. We have produced polyclonal antisera against the extracellular domain and against the cytoplasmic domain of L-selectin. Both antisera immunoprecipitate the intact form of L-selectin from metabolically labeled phytohemagglutinin-stimulated lymphoblasts and peripheral blood neutrophils. In addition, the anti-cytoplasmic domain serum, but not the antiectodomain serum, immunoprecipitate a 6-kD species from PMA activated lymphoblasts and formylmethionylleucylphenylalanine-activated neutrophils. Conversely, the antiectodomain serum but not the anti-cytoplasmic domain serum immunoprecipitate a 68-kD soluble form of L-selectin from the supernatant of PMA-activated lymphoblasts. The appearance of the 6-kD species on activated cells correlated with the disappearance of the intact form of L-selectin and the appearance of the soluble form of L-selectin. A third polyclonal serum generated against the membrane proximal region of the ectodomain also reacted with the 6-kD species, indicating that this is a transmembrane peptide of L-selectin. That the 6-kD species is derived from L-selectin was confirmed by immunoprecipitation of the 6-kD species from L-selectin transfectants but not from mock transfectants. Radiochemical sequence analysis defined a cleavage site between Lys321 and Ser322, which would predict a transmembrane fragment consistent in size with the observed 6-kD fragment. A Ser-Phe-Ser motif adjacent to the cleavage site is conserved between human, mouse, and rat L-selectin, and a related motif is found proximal to transmembrane domains of other downregulated proteins, such as ACE, CD16-II, and TNF-RII, suggesting the possibility of a common recognition motif.
Monospecific and common glycoprotein ligands for E- and P-selectin on myeloid cells.
Authors: Lenter M , Levinovitz A , Isenmann S , Vestweber D
Published: May 2004

E- and P-selectin are inducible cell adhesion molecules on endothelial cells, which function as Ca(2+)-dependent lectins and mediate the binding of neutrophils and monocytes. We have recently identified a 150-kD glycoprotein ligand for E-selectin on mouse myeloid cells, using a recombinant antibody-like form of mouse E-selectin. Here, we report that this ligand does not bind to an analogous P-selectin fusion protein. Instead, the chimeric P-selectin-IgG protein recognizes a 160-kD glycoprotein on the mouse neutrophil progenitor 32D cl 3, on mature mouse neutrophils and on human HL60 cells. The binding is Ca(2+)-dependent and requires the presence of sialic acid on the ligand. This P-selectin-ligand is not recognized by E-selectin. Removal of N-linked carbohydrate side chains from the 150-kD and the 160-kD monospecific selectin ligands abolishes the binding of both ligands to the respective selectin. Treatment of HL60 cells with Peptide: N-glycosidase F inhibited cell binding to P- and E-selectin. In addition, glycoproteins of 230 and 130 kD were found on mature mouse neutrophils, which bound both to E- and P-selectin in a Ca(2+)-dependent fashion. The signals detected for these ligands were 15-20-fold weaker than those for the monospecific ligands. Both proteins were heavily sialylated and selectin-binding was blocked by removal of sialic acid, but not by removal of N-linked carbohydrates. Our data reveal that E- and P-selectin recognize two categories of glycoprotein ligands: one type requires N-linked carbohydrates for binding and is monospecific for each of the two selectins and the other type binds independent of N-linked carbohydrates and is common for both endothelial selectins.
BEHAB, a new member of the proteoglycan tandem repeat family of hyaluronan-binding proteins that is restricted to the brain.
Authors: Jaworski D M , Kelly G M , Hockfield S
Published: May 2004

Hyaluronan (HA) is a ubiquitous component of the extracellular matrix of all tissues. In the mammalian central nervous system (CNS) HA is present throughout development and into adulthood. While the functions of HA are likely to be mediated by HA-binding proteins, no cell or tissue specific HA-binding proteins have been reported. In an effort to characterize the composition of the extracellular matrix of the CNS, we sought to identify neural HA-binding proteins. We report here the isolation and characterization of a cDNA with a high degree of sequence homology to members of the proteoglycan tandem repeat (PTR) family of HA-binding proteins. Unlike other HA-binding proteins, the expression of this cDNA is restricted to the CNS. We propose the name BEHAB, Brain Enriched HyAluronan Binding protein, for this gene. The expression of BEHAB mRNA is developmentally regulated; expression is first detected in the late embryonic period and peaks during the first two postnatal weeks. In the embryo, BEHAB is expressed at highest levels in mitotically active cells. The sequence of BEHAB has long stretches of identity between rat and cat, suggesting that the encoded protein is functionally important. The size and sequence of BEHAB are consistent with the possibility that it could serve a function like link protein, stabilizing interactions between HA and brain proteoglycans. These observations suggest that existence of other tissue specific HA-binding proteins.
CENP-C is required for maintaining proper kinetochore size and for a timely transition to anaphase.
Authors: Tomkiel J , Cooke C A , Saitoh H , Bernat R L , Earnshaw W C
Published: May 2004

The human autoantigen CENP-C has been demonstrated by immunoelectron microscopy to be a component of the inner kinetochore plate. Here we have used antibodies raised against various portions of CENP-C to probe its function in mitosis. We show that nuclear microinjection of anti-CENP-C antibodies during interphase causes a transient arrest at the following metaphase. Injection of the same antibodies after the initiation of prophase, however, does not disrupt mitosis. Correspondingly, indirect immunofluorescence using affinity-purified human anti-CENP-C antibodies reveals that levels of CENP-C staining are reduced at centromeres in cells that were injected during interphase, but appear unaffected in cells which were injected during mitosis. Thus, we suggest that the injected antibodies cause metaphase arrest by reducing the amount of CENP-C at centromeres. Examination of kinetochores in metaphase-arrested cells by electron microscopy reveals that the number of trilaminar structures is reduced. More surprisingly, the few remaining kinetochores in these cells retain a normal trilaminar morphology but are significantly reduced in diameter. In cells arrested for extended periods, these small kinetochores become disrupted and apparently no longer bind microtubules. These observations are consistent with an involvement of CENP-C in kinetochore assembly, and suggest that CENP-C plays a critical role in both establishing and/or maintaining proper kinetochore size and stabilizing microtubule attachments. These findings also support the idea that proper assembly of kinetochores may be monitored by the cell cycle checkpoint preceding the transition to anaphase.
A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus.
Authors: Wilson B S , Nuoffer C , Meinkoth J L , McCaffery M , Feramisco J R , Balch W E , Farquhar M G
Published: May 2004

The Golgi apparatus is a dynamic organelle whose structure is sensitive to vesicular traffic and to cell cycle control. We have examined the potential role for rab1a, a GTPase previously associated with ER to Golgi and intra-Golgi transport, in the formation and maintenance of Golgi structure. Bacterially expressed, recombinant rab1a protein was microinjected into rat embryonic fibroblasts, followed by analysis of Golgi morphology by fluorescence and electron microscopy. Three recombinant proteins were tested: wild-type rab, mutant rab1a(S25N), a constitutively GDP-bound form (Nuoffer, C., H. W. Davidson, J. Matteson, J. Meinkoth, and W. E. Balch, 1994. J. Cell Biol. 125: 225-237), and mutant rab1a(N124I) defective in guanine nucleotide binding. Microinjection of wild-type rab1a protein or a variety of negative controls (injection buffer alone or activated ras protein) did not affect the appearance of the Golgi, as visualized by immunofluorescence of alpha-mannosidase II (Man II), used as a Golgi marker. In contrast, microinjection of the mutant forms promoted the disassembly of the Golgi stacks into dispersed vesicular structures visualized by immunofluorescence. When S25N-injected cells were analyzed by EM after immunoperoxidase labeling, Man II was found in isolated ministacks and large vesicular elements that were often surrounded by numerous smaller unlabeled vesicles resembling carrier vesicles. Golgi disassembly caused by rab1a mutants differs from BFA-induced disruption, since beta-COP remains membrane associated, and Man II does not redistribute to the ER. BFA can still cause these residual Golgi elements to fuse and disperse, albeit at a slower rate. Moreover, BFA recovery is incomplete in the presence of rab1 mutants or GTP gamma S. We conclude that GTP exchange and hydrolysis by GTPases, specifically rab1a, are required to form and maintain normal Golgi stacks. The similarity of Golgi disassembly seen with rab1a mutants to that occurring during mitosis, may point to a molecular basis involving rab1a for fragmentation of the Golgi apparatus during cell division.
Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network.
Authors: Riederer M A , Soldati T , Shapiro A D , Lin J , Pfeffer S R
Published: May 2004

Newly synthesized lysosomal enzymes bind to mannose 6-phosphate receptors (MPRs) in the TGN, and are carried to prelysosomes, where they are released. MPRs then return to the TGN for another round of transport. Rab9 is a ras-like GTPase which facilitates MPR recycling to the TGN in vitro. We show here that a dominant negative form of rab9, rab9 S21N, strongly inhibited MPR recycling in living cells. The block was specific in that the rates of biosynthetic protein transport, fluid phase endocytosis and receptor-mediated endocytosis were unchanged. Expression of rab9 S21N was accompanied by a decrease in the efficiency of lysosomal enzyme sorting. Cells compensated for the presence of the mutant protein by inducing the synthesis of both soluble and membrane-associated lysosomal enzymes, and by internalizing lysosomal enzymes that were secreted by default. These data show that MPRs are limiting in the secretory pathway of cells expressing rab9 S21N and document the importance of MPR recycling and the rab9 GTPase for efficient lysosomal enzyme delivery.
Expression and polarized targeting of a rab3 isoform in epithelial cells.
Authors: Weber E , Berta G , Tousson A , St John P , Green M W , Gopalokrishnan U , Jilling T , Sorscher E J , Elton T S , Abrahamson D R
Published: May 2004

Pathways of polarized membrane traffic in epithelial tissues serve a variety of functions, including the generation of epithelial polarity and the regulation of vectorial transport. We have identified a candidate regulator of polarized membrane traffic in epithelial cells (i.e., rab3B), which is a member of the rab family of membrane traffic regulators. Rab3B is highly homologous to a brain-specific rab3 isoform (rab3A) that targets in a polarized fashion to the presynaptic nerve terminal, where it probably regulates exocytosis. The coding region for human rab3B was cloned from epithelial mRNA using a reverse-transcription polymerase chain reaction strategy. This cDNA clone hybridized to a single mRNA species in Northern blots of poly(A)+ RNA isolated from epithelial cell lines. A rab3B-specific antibody that was raised against recombinant fusion protein recognized a 25-kD band in immunoblots of cell lysates prepared from cultured epithelial cells (e.g., T84 and HT29-CL19A), but not from a variety of nonepithelial cells (e.g., PC12 neuroendocrine cells). Immunofluorescence analysis confirmed that rab3B protein is preferentially expressed in cultured epithelial cells as well as in a number of native epithelial tissues, including liver, small intestine, colon, and distal nephron. Rab3B localized to the apical pole very near the tight junctions between adjacent epithelial cells within all of these cell lines and native epithelial tissues, as determined by immunofluorescence and immunoelectron microscopic analysis. Moreover, this pattern of intracellular targeting was regulated by cell contact; namely, rab3B was reversibly retrieved from the cell periphery as epithelial cell contact was inhibited by reducing the extracellular Ca2+ concentration. Our results indicate that neurons and epithelial cells express homologous rab3 isoforms that target in a polarized fashion within their respective tissues. The pattern and regulation of rab3B targeting in epithelial cells implicates this monomeric GTPase as a candidate regulator of apical and/or junctional protein traffic in epithelial tissues.
Separation of subcellular compartments containing distinct functional forms of MHC class II.
Authors: Qiu Y , Xu X , Wandinger-Ness A , Dalke D P , Pierce S K
Published: May 2004

Antigen processing in B lymphocytes entails initial binding of antigen to the surface Ig and internalization of the antigen into acidic compartments where the antigen is degraded, releasing peptides for binding to major histocompatibility complex class II molecules. Using subcellular fractionation techniques we show that functional, processed antigen-class II complexes capable of activating antigen-specific T cells in vitro are first formed in dense vesicles cosedimenting with lysosomes which are distinct from early endosomes and the bulk of late endosomes. With time, processed antigen-class II complexes appear in vesicles sedimenting with early endosomes and finally cofractionate with plasma membrane. A separate compartment is identified which contains major histocompatibility complex class II receptive to peptide binding but which does not have access to processed antigen in the B cell. These class II molecules are in the so-called "floppy" form in contrast to the class II molecules in the very dense vesicles which are in the "compact" form. These results demonstrate a correlation between the floppy and compact forms of class II molecules and their association with processed antigen and show that floppy and compact forms of class II reside in distinct and physically separable subcellular compartments.
DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1.
Authors: Lukas J , Müller H , Bartkova J , Spitkovsky D , Kjerulff A A , Jansen-Dürr P , Strauss M , Bartek J
Published: May 2004

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.
Cytokine-mediated protein kinase C activation is a signal for lineage determination in bipotential granulocyte macrophage colony-forming cells.
Authors: Whetton A D , Heyworth C M , Nicholls S E , Evans C A , Lord J M , Dexter T M , Owen-Lynch P J
Published: May 2004

Granulocyte macrophage colony-forming cells (GM-CFC) have the potential to develop into either macrophages and/or neutrophils. With a highly enriched population of these cells we have found that although GM-CFC are equally responsive to macrophage colony stimulating factor (M-CSF) and stem cell factor (SCF) in terms of DNA synthesis, M-CSF stimulated the development of colonies containing macrophages in soft gel assays, while SCF promoted neutrophilic colony formation. When SCF and M-CSF were combined, mainly macrophage development was stimulated both in soft agar colony-forming assays and liquid cultures. An analysis of some potential signaling mechanisms associated with cytokine-mediated developmental decisions in GM-CFC revealed that M-CSF, but not SCF, was able to chronically stimulate phosphatidylcholine breakdown and diacylglycerol production, indicating that protein kinase C (PKC) may be involved in the action of M-CSF. Furthermore, M-CSF, but not SCF, can increase the levels of PKC alpha (PKC alpha) expression and stimulate the translocation of PKC alpha to the nucleus. When the PKC inhibitor, calphostin C, was added to GM-CFC cultured in M-CSF then predominantly neutrophils were produced, conversely PKC activators added with SCF stimulated macrophage development. The data indicate a role for PKC in M-CSF-stimulated macrophage development from GM-CFC.
ADP ribosylation factor and a 14-kD polypeptide are associated with heparan sulfate-carrying post-trans-Golgi network secretory vesicles in rat hepatocytes.
Authors: Nickel W , Huber L A , Kahn R A , Kipper N , Barthel A , Fasshauer D , Söling H D
Published: May 2004

Constitutive secretory vesicles carrying heparan sulfate proteoglycan (HSPG) were identified in isolated rat hepatocytes by pulse-chase experiments with [35S]sulfate and purified by velocity-controlled sucrose gradient centrifugation followed by equilibrium density centrifugation in Nycodenz. Using this procedure, the vesicles were separated from plasma membranes, Golgi, trans-Golgi network (TGN), ER, endosomes, lysosomes, transcytotic vesicles, and mitochondria. The diameter of these vesicles was approximately 100-200 nm as determined by electron microscopy. A typical coat structure as described for intra-Golgi transport vesicles or clathrin-coated vesicles could not be seen, and the vesicles were not associated with the coat protein beta-COP. Furthermore, the vesicles appear to represent a low density compartment (1.05-1.06 g/ml). Other constitutively secreted proteins (rat serum albumin, apolipoprotein E, and fibrinogen) could not be detected in purified HSPG-carrying vesicles, but banded in the denser fractions of the Nycodenz gradient. Moreover, during pulse-chase labeling with [35S]methionine, labeled albumin did not appear in the post-TGN vesicle fraction carrying HSPGs. These findings indicate sorting of HSPGs and albumin into different types of constitutive secretory vesicles in hepatocytes. Two proteins were found to be tightly associated with the membranes of the HSPG carrying vesicles: a member of the ADP ribosylation factor family of small guanine nucleotide-binding proteins and an unknown 14-kD peripheral membrane protein (VAPP14). Concerning the secretory pathway, we conclude from these results that ADP ribosylation factor proteins are not only involved in vesicular transport from the ER via the Golgi to the TGN, but also in vesicular transport from the TGN to the plasma membrane.
Retinoic acid disrupts the Golgi apparatus and increases the cytosolic routing of specific protein toxins.
Authors: Wu Y N , Gadina M , Tao-Cheng J H , Youle R J
Published: May 2004

All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.
Internalization and sorting of a fluorescent analogue of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms.
Authors: Martin O C , Pagano R E
Published: May 2004

We examined the uptake and intracellular transport of the fluorescent glucosylceramide analogue N-[5-(5,7-dimethyl BODIPYTM)-1-pentanoyl]-glucosyl sphingosine (C5-DMB-GlcCer) in human skin fibroblasts, and we compared its behavior to that of the corresponding fluorescent analogues of sphingomyelin, galactosylceramide, and lactosylceramide. All four fluorescent analogues were readily transferred from defatted BSA to the plasma membrane during incubation at 4 degrees C. When cells treated with C5-DMB-GlcCer were washed, warmed to 37 degrees C, and subsequently incubated with defatted BSA to remove fluorescent lipid at the cell surface, strong fluorescence was observed at the Golgi apparatus, as well as weaker labeling at the nuclear envelope and other intracellular membranes. Similar results were obtained with C5-DMB-galactosylceramide, except that labeling of the Golgi apparatus was weaker than with C5-DMB-GlcCer. Internalization of C5-DMB-GlcCer was not inhibited by various treatments, including ATP depletion or warming to 19 degrees C, and biochemical analysis demonstrated that the lipid was not metabolized during its internalization. However, accumulation of C5-DMB-GlcCer at the Golgi apparatus was reduced when cells were treated with a nonfluorescent analogue of glucosylceramide, suggesting that accumulation of C5-DMB-GlcCer at the Golgi apparatus was a saturable process. In contrast, cells treated with C5-DMB-analogues of sphingomyelin or lactosylceramide internalized the fluorescent lipid into a punctate pattern of fluorescence during warming at 37 degrees C, and this process was temperature and energy dependent. These results with C5-DMB-sphingomyelin and C5-DMB-lactosylceramide were analogous to those obtained with another fluorescent analogue of sphingomyelin in which labeling of endocytic vesicles and plasma membrane lipid recycling were documented (Koval, M., and R. E. Pagano. 1990. J. Cell Biol. 111:429-442). Incubation of perforated cells with C5-DMB-sphingomyelin resulted in prominent labeling of the nuclear envelope and other intracellular membranes, similar to the pattern observed with C5-DMB-GlcCer in intact cells. These observations are consistent with the transbilayer movement of fluorescent analogues of glucosylceramide and galactosylceramide at the plasma membrane and early endosomes of human skin fibroblasts, and suggest that both endocytic and nonendocytic pathways are used in the internalization of these lipids from the plasma membrane.
The kinetic aspects of intracellular fluorescence labeling with TMA-DPH support the maturation model for endocytosis in L929 cells.
Authors: Illinger D , Kuhry J G
Published: May 2004

TMA-DPH (1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene), a hydrophobic fluorescent membrane probe, interacts with living cells by instantaneous incorporation into the plasma membrane, where it becomes fluorescent. It then follows the intracellular constitutive membrane traffic and acts as a bulk membrane marker of the endocytic pathway (Illinger, D., P. Poindron, P. Fonteneau, M. Modolell, and J. G. Kuhry. 1990. Biochim. Biophys. Acta. 1030:73-81; Illinger, D., P. Poindron, and J. G. Kuhry. 1991. Biol. Cell. 73:131-138). As such, TMA-DPH displays particular properties mainly due to partition between membranes and aqueous media. From these properties, original arguments can be inferred in favor of the maturation model for the endocytic pathway, against that of pre-existing compartments, in L929 cultured mouse fibroblasts. (a) TMA-DPH labeling is seen to progress from the cell periphery to perinuclear regions during endocytosis without any noticeable loss in fluorescence intensity; with a vesicle shuttle model this evolution would be accompanied by probe dilution with a decrease in the overall intracellular fluorescence intensity, and the labeling of the inner (late) compartments could in no way become more intense than that of the peripheral (early) ones. (b) From TMA-DPH fluorescence anisotropy assays, it is concluded that membrane fluidity is the same in the successive endocytic compartments as in the plasma membrane, which probably denotes a similar phospholipidic membrane composition, as might be expected in the maturation model. (c) TMA-DPH internalization and release kinetics are more easily described with the maturation model.
Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins.
Authors: Thomas J L , Holowka D , Baird B , Webb W W
Published: May 2004

Large scale aggregation of fluorescein-labeled immunoglobulin E (IgE) receptor complexes on the surface of RBL cells results in the co-aggregation of a large fraction of the lipophilic fluorescent probe 3,3'-dihexadecylindocarbocyanine (diI) that labels the plasma membranes much more uniformly in the absence of receptor aggregation. Most of the diI molecules that are localized in patches of aggregated receptors have lost their lateral mobility as determined by fluorescence photobleaching recovery. The diI outside of patches is mobile, and its mobility is similar to that in control cells without receptor aggregates. It is unlikely that the co-aggregation of diI with IgE receptors is due to specific interactions between these components, as two other lipophilic probes of different structures are also observed to redistribute with aggregated IgE receptors, and aggregation of two other cell surface antigens also results in the coredistribution of diI at the RBL cell surface. Quantitative analysis of CCD images of labeled cells reveals some differences in the spatial distributions of co-aggregated diI and IgE receptors. The results indicate that cross-linking of specific cell surface antigens causes a substantial change in the organization of the plasma membrane by redistributing pre-existing membrane domains or causing their formation.
The biologically active form of the sea urchin egg receptor for sperm is a disulfide-bonded homo-multimer.
Authors: Ohlendieck K , Partin J S , Lennarz W J
Published: May 2004

Since many cell surface receptors exist in their active form as oligomeric complexes, we have investigated the subunit composition of the biologically active sperm receptor in egg plasma membranes from Strongylocentrotus purpuratus. Electrophoretic analysis of the receptor without prior reduction of disulfide bonds revealed that the surface receptor exists in the form of a disulfide-bonded multimer, estimated to be a tetramer. These findings are in excellent agreement with the fact that the NH2-terminus of the extracellular domain of the sperm receptor is rich in cysteine residues. Studies with cross-linking agents of various length and hydrophobicity suggest that no other major protein is tightly associated with the receptor. Given the multimeric structure of the receptor, we investigated the effect of disulfide bond reduction on its biological activity. Because in quantitative bioassays fertilization was found to be inhibited by treatment of eggs with 5 mM dithiothreitol, we undertook more direct studies of the effect of reduction on properties of the receptor. First, we studied the effect of addition of isolated, pure receptor on fertilization. Whereas the non-reduced, native receptor complex inhibited fertilization in a dose-dependent manner, the reduced and alkylated receptor was inactive. Second, we tested the ability of the isolated receptor to mediate binding of acrosome-reacted sperm to polystyrene beads. Whereas beads coated with native receptor bound sperm, those containing reduced and alkylated receptor did not. Thus, these results demonstrate that the biologically active form of the sea urchin sperm receptor consists only of 350 kD subunits and that these must be linked as a multimer via disulfide bonds to produce a complex that is functional in sperm recognition and binding.
Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae.
Authors: Lillie S H , Brown S S
Published: May 2004

Myo2 protein (Myo2p), an unconventional myosin in the budding yeast Saccharomyces cerevisiae, has been implicated in polarized growth and secretion by studies of the temperature-sensitive myo2-66 mutant. Overexpression of Smy1p, which by sequence is a kinesin-related protein, can partially compensate for defects in the myo2 mutant (Lillie, S. H. and S. S. Brown, 1992. Nature (Lond.). 356:358-361). We have now immunolocalized Smy1p and Myo2p. Both are concentrated in regions of active growth, as caps at incipient bud sites and on small buds, at the mother-bud neck just before cell separation, and in mating cells as caps on shmoo tips and at the fusion bridge of zygotes. Double labeling of cells with either Myo2p or Smy1p antibody plus phalloidin was used to compare the localization of Smy1p and Myo2p to actin, and by extrapolation, to each other. These studies confirmed that Myo2p and Smy1p colocalize, and are concentrated in the same general regions of the cell as actin spots. However, neither colocalizes with actin. We noted a correlation in the behavior of Myo2p, Smy1p, and actin, but not microtubules, under a number of circumstances. In cdc4 and cdc11 mutants, which produce multiple buds, Myo2p and Smy1p caps were found only in the subset of buds that had accumulations of actin. Mutations in actin or secretory genes perturb actin, Smy1p and Myo2p localization. The rearrangements of Myo2p and Smy1p correlate temporally with those of actin spots during the cell cycle, and upon temperature and osmotic shift. In contrast, microtubules are not grossly affected by these perturbations. Although wild-type Myo2p localization does not require Smy1p, Myo2p staining is brighter when SMY1 is overexpressed. The myo2 mutant, when shifted to restrictive temperature, shows a permanent loss in Myo2p localization and actin polarization, both of which can be restored by SMY1 overexpression. However, the lethality of MYO2 deletion is not overcome by SMY1 overexpression. We noted that the myo2 mutant can recover from osmotic shift (unlike actin mutants; Novick, P., and D. Botstein. 1985. Cell. 40:405-416). We have also determined that the myo2-66 allele encodes a Lys instead of a Glu at position 511, which lies at an actin-binding face in the motor domain.
Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins.
Authors: White T W , Bruzzone R , Wolfram S , Paul D L , Goodenough D A
Published: May 2004

Gap junctions are collections of intercellular channels composed of structural proteins called connexins (Cx). We have examined the functional interactions of the three rodent connexins present in the lens, Cx43, Cx46, and Cx50, by expressing them in paired Xenopus oocytes. Homotypic channels containing Cx43, Cx46, or Cx50 all developed high conductance. heterotypic channels composed of Cx46 paired with either Cx43 or Cx50 were also well coupled, whereas Cx50 did not form functional channels with Cx43. We also examined the functional response of homotypic and heterotypic channels to transjunctional voltage and cytoplasmic acidification. We show that all lens connexins exhibited sensitivity to cytoplasmic acidification as well as to voltage, and that voltage-dependent closure of heterotypic channels for a given connexin was dramatically influenced by its partner connexins in the adjacent cell. Based on the observation that Cx43 can discriminate between Cx46 and Cx50, we investigated the molecular determinants that specify compatibility by constructing chimeric connexins from portions of Cx46 and Cx50 and testing them for their ability to form channels with Cx43. When the second extracellular (E2) domain in Cx46 was replaced with the E2 of Cx50, the resulting chimera could no longer form heterotypic channels with Cx43. A reciprocal chimera, where the E2 of Cx46 was inserted into Cx50, acquired the ability to functionally interact with Cx43. Together, these results demonstrate that formation of intercellular channels is a selective process dependent on the identity of the connexins expressed in adjacent cells, and that the second extracellular domain is a determinant of heterotypic compatibility between connexins.
Role of muscle insulin-like growth factors in nerve sprouting: suppression of terminal sprouting in paralyzed muscle by IGF-binding protein 4.
Authors: Caroni P , Schneider C , Kiefer M C , Zapf J
Published: May 2004

The protracted absence of muscle activation initiates complex cellular and molecular reactions aimed at restoring functional neuromuscular transmission and preventing degenerative processes. A central aspect of these reactions is the sprouting of intramuscular nerves in the vicinity of inactivated muscle fibers. Sprouts emerging from terminal nerve branches and nodes of Ranvier can reestablish functional contacts with inactive muscle fibers, and this is an essential restorative process in pathological conditions of the neuromuscular system. Due to their rapid upregulation in inactive skeletal muscle fibers and their ability to induce nerve sprouting in adult muscle, insulin-like growth factors (IGFs) are candidate signaling molecules to promote restorative reactions in the neuromuscular system. In this study we have exploited the high affinity and specificity of IGF-binding protein 4 (IGF-BP4) and IGF-BP5 for IGF1 and IGF2 to determine whether these growth factors are involved in the nerve sprouting reaction in paralyzed skeletal muscle. In tissue culture experiments with sensory- and motoneurons we demonstrate that the neurite promoting activity of IGF1 is blocked by IGF-BP4, and that a similar IGF-BP-sensitive activity is detected in muscle extracts from paralyzed, but not from control muscle. In in vivo experiments, we show that local delivery of IGF-BP4 to Botulinum toxin A-paralyzed skeletal muscle effectively prevents nerve sprouting in that muscle. Our findings indicate that muscle IGFs play an essential role in intramuscular nerve sprouting. In addition, these findings suggest that IGFs are major signaling factors from inactivated muscle to promote local restorative reactions, including interstitial cell proliferation and nerve sprouting.
Association of the transmembrane TGF-alpha precursor with a protein kinase complex.
Authors: Shum L , Reeves S A , Kuo A C , Fromer E S , Derynck R
Published: May 2004

A variety of growth factors including transforming growth factor-alpha (TGF-alpha) are synthesized as transmembrane precursors. The short cytoplasmic domain of the transmembrane TGF-alpha precursor lacks any apparent motif associated with signal transduction. However, the sequence conservation of this cytoplasmic domain and its abundance of cysteine residues, reminiscent of the cytoplasmic domains of CD4 and CD8, suggest a biological function. In this study, we showed that transmembrane TGF-alpha was rapidly internalized after interaction with a specific antibody and that this internalization was greatly decreased when the COOH-terminal 31 amino acids were removed. Chemical cross-linking experiments revealed two associated proteins of 86 and 106 kD which coimmunoprecipitated with the TGF-alpha precursor. The association of p86 was dependent on the presence of the COOH-terminal cytoplasmic 31 amino acids of the TGF-alpha precursor, whereas p106 still remained associated when this segment was deleted. In addition, p106 was tyrosine-phosphorylated and exposed on the cell surface. The protein complex associated with transmembrane TGF-alpha displayed kinase activities towards tyrosine, serine, and threonine residues. These activities were not associated with transmembrane TGF-alpha when the COOH-terminal segment was truncated. The association of a protein kinase complex with transmembrane TGF-alpha may provide the basic elements for a "reverse" mode of signaling through the cytoplasmic domain of this growth factor, which may lead to two-directional communication during ligand-receptor interaction.
Interactions of tensin with actin and identification of its three distinct actin-binding domains.
Authors: Lo S H , Janmey P A , Hartwig J H , Chen L B
Published: May 2004

Tensin, a 200-kD phosphoprotein of focal contacts, contains sequence homologies to Src (SH2 domain), and several actin-binding proteins. These features suggest that tensin may link the cell membrane to the cytoskeleton and respond directly to tyrosine kinase signalling pathways. Here we identify three distinct actin-binding domains within tensin. Recombinant tensin purified after overexpression by a baculovirus system binds to actin filaments with Kd = 0.1 microM, cross-links actin filaments at a molar ratio of 1:10 (tensin/actin), and retards actin assembly by barbed end capping with Kd = 20 nM. Tensin fragments were constructed and expressed as fusion proteins to map domains having these activities. Three regions from tensin interact with actin: two regions composed of amino acids 1 to 263 and 263 to 463, cosediment with F-actin but do not alter the kinetics of actin assembly; a region composed of amino acids 888-989, with sequence homology to insertin, retards actin polymerization. A claw-shaped tensin dimer would have six potential actin-binding sites and could embrace the ends of two actin filaments at focal contacts.
Growth site localization of Rho1 small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae.
Authors: Yamochi W , Tanaka K , Nonaka H , Maeda A , Musha T , Takai Y
Published: May 2004

The Rho small GTP-binding protein family regulates various actomyosin-dependent cell functions, such as cell morphology, locomotion, cytokinesis, membrane ruffling, and smooth muscle contraction. In the yeast Saccharomyces cerevisiae, there is a homologue of mammalian RhoA, RHO1, which is essential for vegetative growth of yeast cells. To explore the function of the RHO1 gene, we isolated a recessive temperature-sensitive mutation of RHO1, rho1-104. The rho1-104 mutation caused amino acid substitutions of Asp 72 to Asn and Cys 164 to Tyr of Rho1p. Strains bearing the rho1-104 mutation accumulated tiny- or small-budded cells in which cortical actin patches were clustered to buds at the restrictive temperature. Cell lysis and cell death were also seen with the rho1-104 mutant. Indirect immunofluorescence microscopic study demonstrated that Rho1p was concentrated to the periphery of the cells where cortical actin patches were clustered, including the site of bud emergence, the tip of the growing buds, and the mother-bud neck region of cells prior to cytokinesis. Indirect immunofluorescence study with cells overexpressing RHO1 suggested that the Rho1p-binding site was saturable. A mutant Rho1p with an amino acid substitution at the lipid modification site remained in the cytoplasm. These results suggest that Rho1 small GTP-binding protein binds to a specific site at the growth region of cells, where Rho1p exerts its function in controlling cell growth.
KIF3A is a new microtubule-based anterograde motor in the nerve axon.
Authors: Kondo S , Sato-Yoshitake R , Noda Y , Aizawa H , Nakata T , Matsuura Y , Hirokawa N
Published: May 2004

Neurons are highly polarized cells composed of dendrites, cell bodies, and long axons. Because of the lack of protein synthesis machinery in axons, materials required in axons and synapses have to be transported down the axons after synthesis in the cell body. Fast anterograde transport conveys different kinds of membranous organelles such as mitochondria and precursors of synaptic vesicles and axonal membranes, while organelles such as endosomes and autophagic prelysosomal organelles are conveyed retrogradely. Although kinesin and dynein have been identified as good candidates for microtubule-based anterograde and retrograde transporters, respectively, the existence of other motors for performing these complex axonal transports seems quite likely. Here we characterized a new member of the kinesin super-family, KIF3A (50-nm rod with globular head and tail), and found that it is localized in neurons, associated with membrane organelle fractions, and accumulates with anterogradely moving membrane organelles after ligation of peripheral nerves. Furthermore, native KIF3A (a complex of 80/85 KIF3A heavy chain and a 95-kD polypeptide) revealed microtubule gliding activity and baculovirus-expressed KIF3A heavy chain demonstrated microtubule plus end-directed (anterograde) motility in vitro. These findings strongly suggest that KIF3A is a new motor protein for the anterograde fast axonal transport.
On the localization of voltage-sensitive calcium channels in the flagella of Chlamydomonas reinhardtii.
Authors: Beck C , Uhl R
Published: May 2004

This study was undertaken to prove that voltage-sensitive calcium channels controlling the photophobic stop response of the unicellular green alga Chlamydomonas reinhardtii are exclusively found in the flagellar region of the cell and to answer the question as to their exact localization within the flagellar membrane. The strategy used was to amputate flagella to a variable degree without perturbing the electrical properties of the cell and measure flagellar currents shortly after amputation and during the subsequent regeneration process. Under all conditions, a close correlation was found between current size and flagellar length, strongly suggesting that the channels that mediate increases in intraflagellar calcium concentration are confined to and distributed over the total flagellar length. Bald mutants yielded tiny flagellar currents, in agreement with the existence of residual flagellar stubs. In the presence of the protein synthesis inhibitor cycloheximide, flagellar length and flagellar currents also recovered in parallel. Recovery came to an earlier end, however, leveling off at a time when in the absence of cycloheximide only half maximal values were achieved. This suggests the existence of a pool of precursors, which permits the maintenance of a constant ratio between voltage-sensitive calcium channels and other intraflagellar proteins.
Evidence for an interplay between cell cycle progression and the initiation of differentiation between life cycle forms of African trypanosomes.
Authors: Matthews K R , Gull K
Published: May 2004

Successful transmission of the African trypanosome between the mammalian host blood-stream and the tsetse fly vector involves dramatic alterations in the parasite's morphology and biochemistry. This differentiation through to the tsetse midgut procyclic form is accompanied by re-entry into a proliferative cell cycle. Using a synchronous differentiation model and a variety of markers diagnostic for progress through both differentiation and the cell cycle, we have investigated the interplay between these two processes. Our results implicate a relationship between the trypanosome cell cycle position and the perception of the differentiation signal and demonstrate that irreversible commitment to the differentiation occurs rapidly after induction. Furthermore, we show that re-entry into the cell cycle in the differentiating population is synchronous, and that once initiated, progress through the differentiation pathway can be uncoupled from progress through the cell cycle.
A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg.
Authors: Lin Y , Mahan K , Lathrop W F , Myles D G , Primakoff P
Published: May 2004

A typical mammalian egg is surrounded by an outer layer of about 3,000 cumulus cells embedded in an extracellular matrix rich in hyaluronic acid. A current, widely proposed model is that the fertilizing sperm, while it is acrosome intact, passes through the cumulus cell layer and binds to the egg zona pellucida. This current model lacks a well-supported explanation for how sperm penetrate the cumulus layer. We report that the sperm protein PH-20 has a hyaluronidase activity and is present on the plasma membrane of mouse and human sperm. Brief treatment with purified, recombinant PH-20 can release all the cumulus cells surrounding mouse eggs. Acrosome intact mouse sperm incubated with anti-PH-20 antibodies can not pass through the cumulus layer and thus can not reach the zona pellucida. These results, indicating that PH-20 enables acrosome intact sperm to penetrate the cumulus barrier, reveal a mechanism for cumulus penetration, and thus provide the missing element in the current model.