bluuebunny/crossref_metadata_2025 huggingface dataset:
The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via alpha-actinin: receptor positioning in microvilli does not require interaction with alpha-actinin.
Authors: Pavalko F M , Walker D M , Graham L , Goheen M , Doerschuk C M , Kansas G S
Published: May 2004

The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L-selectin interacts directly with the cytoplasmic actin-binding protein alpha-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified alpha-actinin to L-selectin (Kd = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of alpha-actinin to the L-selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of alpha-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including alpha-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that alpha-actinin binds directly to L-selectin and that vinculin associates by binding to alpha-actinin in vivo to link actin filaments to the L-selectin cytoplasmic domain. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with alpha-actinin or vinculin. Surprisingly, this mutant L-selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of alpha-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L-selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L-selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton.
Developmental expression of fibrillin genes suggests heterogeneity of extracellular microfibrils.
Authors: Zhang H , Hu W , Ramirez F
Published: May 2004

Extracellular microfibrils, alone or in association with elastin, confer critical biomechanical properties on a variety of connective tissues. Little is known about the composition of the microfibrils or the factors responsible for their spatial organization into tissue-specific macroaggregates. Recent work has revealed the existence of two structurally related microfibrillar components, termed fibrillin-1 and fibrillin-2. The functional relationships between these glycoproteins and between them and other components of the microfibrils and elastic fibers are obscure. As a first step toward elucidating these important points, we compared the expression pattern of the fibrillin genes during mammalian embryogenesis. The results revealed that the two genes are differentially expressed, in terms of both developmental stages and tissue distribution. In the majority of cases, fibrillin-2 transcripts appear earlier and accumulate for a shorter period of time than fibrillin-1 transcripts. Synthesis of fibrillin-1 correlates with late morphogenesis and the appearance of well-defined organ structures; fibrillin-2 synthesis, on the other hand, coincides with early morphogenesis and, in particular, with the beginning of elastogenesis. The findings lend indirect support to our original hypothesis stating that fibrillins contribute to the compositional and functional heterogeneity of the microfibrils. The available evidence is also consistent with the notion that the fibrillins might have distinct, but related roles in microfibril physiology. Accordingly, we propose that fibrillin-1 provides mostly force-bearing structural support, whereas fibrillin-2 predominantly regulates the early process of elastic fiber assembly.
A conserved epitope on a subset of SR proteins defines a larger family of Pre-mRNA splicing factors.
Authors: Neugebauer K M , Stolk J A , Roth M B
Published: May 2004

The removal of introns from eukaryotic pre-mRNA occurs in a large ribonucleoprotein complex called the spliceosome. We have generated a monoclonal antibody (mAb 16H3) against four of the family of six SR proteins, known regulators of splice site selection and spliceosome assembly. In addition to the reactive SR proteins, SRp20, SRp40, SRp55, and SRp75, mAb 16H3 also binds approximately 20 distinct nuclear proteins in human, frog, and Drosophila extracts, whereas yeast do not detectably express the epitope. The antigens are shown to be nuclear, nonnucleolar, and concentrated at active sites of RNA polymerase II transcription which suggests their involvement in pre-mRNA processing. Indeed, most of the reactive proteins observed in nuclear extract are detected in spliceosomes (E and/or B complex) assembled in vitro, including the U1 70K component of the U1 small nuclear ribonucleoprotein particle and both subunits of U2AF. Interestingly, the 16H3 epitope was mapped to a 40-amino acid polypeptide composed almost exclusively of arginine alternating with glutamate and aspartate. All of the identified antigens, including the human homolog of yeast Prp22 (HRH1), contain a similar structural element characterized by arginine alternating with serine, glutamate, and/or aspartate. These results indicate that many more spliceosomal components contain such arginine-rich domains. Because it is conserved among metazoans, we propose that the "alternating arginine" domain recognized by mAb 16H3 may represent a common functional element of pre-mRNA splicing factors.
The carboxy termini of Sir4 and Rap1 affect Sir3 localization: evidence for a multicomponent complex required for yeast telomeric silencing.
Authors: Cockell M , Palladino F , Laroche T , Kyrion G , Liu C , Lustig A J , Gasser S M
Published: May 2004

The Silent Information Regulatory proteins, Sir3 and Sir4, and the telomeric repeat-binding protein RAP1 are required for the chromatin-mediated gene repression observed at yeast telomeric regions. All three proteins are localized by immunofluorescence staining to foci near the nuclear periphery suggesting a relationship between subnuclear localization and silencing. We present several lines of immunological and biochemical evidence that Sir3, Sir4, and RAP1 interact in intact yeast cells. First, immunolocalization of Sir3 to foci at the yeast nuclear periphery is lost in rap1 mutants carrying deletions for either the terminal 28 or 165 amino acids of RAP1. Second, the perinuclear localization of both Sir3 and RAP1 is disrupted by overproduction of the COOH terminus of Sir4. Third, overproduction of the Sir4 COOH terminus alters the solubility properties of both Sir3 and full-length Sir4. Finally, we demonstrate that RAP1 and Sir4 coprecipitate in immune complexes using either anti-RAP1 or anti-Sir4 antibodies. We propose that the integrity of a tertiary complex between Sir4, Sir3, and RAP1 is involved in both the maintenance of telomeric repression and the clustering of telomeres in foci near the nuclear periphery.
Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62.
Authors: Paschal B M , Gerace L
Published: May 2004

Protein import into the nucleus is a multistep process that requires the activities of several cytosolic factors. In this study we have purified a cytosolic factor that interacts with the nuclear pore complex glycoprotein p62. Isolation involved biochemical complementation of cytosol depleted of this activity by preadsorption with recombinant p62 and the use of a novel flow cytometry-based assay for quantitation of nuclear import. The purified activity (NTF2) is an apparent dimer of approximately 14-kD subunits and is present at approximately 10(6) copies per cell. We obtained a cDNA encoding NTF2 and showed that the recombinant protein restores transport activity to p62-pretreated cytosol. Our data suggest that NTF2 acts at a relatively late stage of nuclear protein import, subsequent to the initial docking of nuclear import ligand at the nuclear envelope. NTF2 interacts with at least one additional cytosolic transport activity, indicating that it could be part of a multicomponent system of cytosolic factors that assemble at the pore complex during nuclear import.
A conditional allele of the novel repeat-containing yeast nucleoporin RAT7/NUP159 causes both rapid cessation of mRNA export and reversible clustering of nuclear pore complexes.
Authors: Gorsch L C , Dockendorff T C , Cole C N
Published: May 2004

In a screen for Saccharomyces cerevisiae genes required for nucleocytoplasmic transport of messenger RNA, we identified the RAT7 gene (ribonucleic acid trafficking), which encodes an essential protein of 1,460 amino acids. Rat7p is located at the nuclear rim in a punctate pattern characteristic of nucleoporins. Furthermore, the central third of Rat7p contains 22 XXFG and three XFXFG degenerate repeats that are similar to signature GLFG and XFXFG repeats present in a majority of yeast and some mammalian nucleoporins sequenced to date. Shift of a strain bearing the temperature-sensitive rat7-1 allele from 23 degrees C to 37 degrees C resulted in rapid (within 15 minutes) cessation of mRNA export, but did not cause concomitant cytoplasmic accumulation of a reporter protein bearing a nuclear localization signal. This suggests that Rat7p may play a direct role in nucleocytoplasmic export of RNA. Immunofluorescence and thin section electron microscopy revealed that in rat7-1 cells grown at 23 degrees C, the majority of nuclear pore complexes (NPCs) were clustered on one side of the nucleus. No ultrastructural abnormalities of the nuclear envelope were seen. Interestingly, shifting rat7-1 cells to 37 degrees C for 1 h caused the NPCs to disperse, restoring near wild-type NPC distribution. After this temperature shift, the mutant Rat7p was no longer detectable by immunofluorescence.
Stage- and ribosome-specific alterations in nascent chain-Sec61p interactions accompany translocation across the ER membrane.
Authors: Nicchitta C V , Murphy E C , Haynes R , Shelness G S
Published: May 2004

Near-neighbor interactions between translocating nascent chains and Sec61p were investigated by chemical cross-linking. At stages of translocation before signal sequence cleavage, nascent chains could be cross-linked to Sec61p at high (60-80%) efficiencies. Cross-linking occurred through the signal sequence and the mature portion of wild-type and signal cleavage mutant nascent chains. At later stages of translocation, as represented through truncated translocation intermediates, cross-linking to Sec61p was markedly reduced. Dissociation of the ribosome into its large and small subunits after assembly of the precursor into the translocon, but before cross-linking, resulted in a dramatic reduction in subsequent cross-linking yield, indicating that at early stages of translocation, nascent chain-Sec61p interactions are in part mediated through interactions of the ribosome with components of the ER membrane, such as Sec61p. Dissociation of the ribosome was, however, without effect on subsequent translocation. These results are discussed with respect to a model in which Sec61p performs a function essential for the initiation of protein translocation.
Steric masking of a dilysine endoplasmic reticulum retention motif during assembly of the human high affinity receptor for immunoglobulin E.
Authors: Letourneur F , Hennecke S , Démollière C , Cosson P
Published: May 2004

Signals that can cause retention in the ER have been found in the cytoplasmic domain of individual subunits of multimeric receptors destined to the cell surface. To study how ER retention motifs are masked during assembly of oligomeric receptors, we analyzed the assembly and intracellular transport of the human high-affinity receptor for immunoglobulin E expressed in COS cells. The cytoplasmic domain of the alpha chain contains a dilysine ER retention signal, which becomes nonfunctional after assembly with the gamma chain, allowing transport out of the ER of the fully assembled receptor. Juxtaposition of the cytoplasmic domains of the alpha and gamma subunits during assembly is responsible for this loss of ER retention. Substitution of the gamma chain cytoplasmic domain with cytoplasmic domains of irrelevant proteins resulted in efficient transport out of the ER of the alpha chain, demonstrating that nonspecific steric hindrance by the cytoplasmic domain of the gamma chain accounts for the masking of the ER retention signal present in the cytoplasmic domain of the alpha chain. Such a mechanism allows the ER retention machinery to discriminate between assembled and nonassembled receptors, and thus participates in quality control at the level of the ER.
Expression and compartmentalization of caveolin in adipose cells: coordinate regulation with and structural segregation from GLUT4.
Authors: Kandror K V , Stephens J M , Pilch P F
Published: May 2004

Native rat adipocytes and the mouse adipocyte cell line, 3T3-L1, possess transport vesicles of apparently uniform composition and size which translocate the tissue-specific glucose transporter isoform, GLUT4, from an intracellular pool to the cell surface in an insulin-sensitive fashion. Caveolin, the presumed structural protein of caveolae, has also been proposed to function in vesicular transport. Thus, we studied the expression and subcellular distribution of caveolin in adipocytes. We found that rat fat cells express the highest level of caveolin protein of any tissue studied, and caveolin is also expressed at high levels in cardiac muscle, another tissue possessing insulin responsive GLUT4 translocation. Both proteins are absent from 3T3-L1 fibroblasts and undergo a dramatic coordinate increase in expression upon differentiation of these cells into adipocytes. However, unlike GLUT4 in rat adipocytes not exposed to insulin, the majority of caveolin is present in the plasma membrane. In native rat adipocytes, intracellular GLUT4 and caveolin reside in vesicles practically indistinguishable by their size and buoyant density in sucrose gradients, and both proteins show insulin-dependent translocation to the cell surface. However, by immunoadsorption of GLUT4-containing vesicles with anti-GLUT4 antibody, we show that these vesicles have no detectable caveolin, and therefore, this protein is present in a distinct vesicle population. Thus, caveolin has no direct structural relation to the organization of the intracellular glucose transporting machinery in fat cells.
"Synchronized" endocytosis and intracellular sorting in alveolar macrophages: the early sorting endosome is a transient organelle.
Authors: Ward D M , Perou C M , Lloyd M , Kaplan J
Published: May 2004

Incubation of alveolar macrophages in hypoosmotic K(+)-containing buffers results in persistent cell swelling and an inability to undergo regulatory volume decrease. We demonstrate that cells incubated in hypo-K+ show an inhibition of endocytosis without any observed alteration in recycling. The inhibition of endocytosis affected all forms of membrane internalization, receptor and fluid phase. Both increased cell volume and the inhibition of endocytosis could be released upon return of cells to iso-Na+ buffers. The ability to synchronize the endocytic apparatus allowed us to examine hypotheses regarding the origin and maturation of endocytic vesicles. Incubation in hypo-K+ buffers had no effect on the delivery of ligands to degradative compartments or on the return of previously internalized receptors to the cell surface. Thus, membrane recycling and movement of internalized components to lysosomes occurred in the absence of continued membrane influx. We also demonstrate that fluorescent lipids, that had been incorporated into early endosomes, returned to the cell surface upon exposure of cells to hypo-K+ buffers. These results indicate that the early sorting endosome is a transient structure, whose existence depends upon continued membrane internalization. Our data supports the hypothesis that the transfer of material to lysosomes can best be explained by the continuous maturation of endosomes.
Apical and basolateral coated pits of MDCK cells differ in their rates of maturation into coated vesicles, but not in the ability to distinguish between mutant hemagglutinin proteins with different internalization signals.
Authors: Naim H Y , Dodds D T , Brewer C B , Roth M G
Published: May 2004

In polarized epithelial MDCK cells, all known endogenous endocytic receptors are found on the basolateral domain. The influenza virus hemagglutinin (HA) which is normally sorted to the apical plasma membrane, can be converted to a basolateral protein by specific mutations in its short cytoplasmic domain that also create internalization signals. For some of these mutations, sorting to the basolateral surface is incomplete, allowing internalization of two proteins that differ by a single amino acid of the internalization signal to be compared at both the apical and basolateral surfaces of MDCK cells. The rates of internalization of HA-Y543 and HA-Y543,R546 from the basolateral surface of polarized MDCK cells resembled those in nonpolarized cells, whereas their rates of internalization from the apical cell surface were fivefold slower. However, HA-Y543,R546 was internalized approximately threefold faster than HA-Y543 at both membrane domains, indicating that apical endocytic pits in polarized MDCK cells retained the ability to discriminate between different internalization signals. Slower internalization from the apical surface could not be explained by a limiting number of coated pits: apical membrane contained 0.7 as many coated pits per cell cross-section as did basolateral membranes. 10-14% of HA-Y543 at the apical surface of polarized MDCK cells was found in coated pits, a percentage not significantly different from that observed in apical coated pits of nonpolarized MDCK cells, where internalization was fivefold faster. Thus, there was no lack of binding sites for HA-Y543 in apical coated pits of polarized cells. However, at the apical surface many more shallow pits, and fewer deep, mature pits, were observed than were seen at the basolateral. These results suggest that the slower internalization at the apical surface is due to slower maturation of coated pits, and not to a difference in recognition of internalization signals.
A density-sensing factor regulates signal transduction in Dictyostelium.
Authors: Yuen I S , Jain R , Bishop J D , Lindsey D F , Deery W J , Van Haastert P J , Gomer R H
Published: May 2004

Dictyostelium discoideum initiates development when cells overgrow their bacterial food source and starve. To coordinate development, the cells monitor the extracellular level of a protein, conditioned medium factor (CMF), secreted by starved cells. When a majority of the cells in a given area have starved, as signaled by CMF secretion, the extracellular level of CMF rises above a threshold value and permits aggregation of the starved cells. The cells aggregate using relayed pulses of cAMP as the chemoattractant. Cells in which CMF accumulation has been blocked by antisense do not aggregate except in the presence of exogenous CMF. We find that these cells are viable but do not chemotax towards cAMP. Videomicroscopy indicates that the inability of CMF antisense cells to chemotax is not due to a gross defect in motility, although both video and scanning electron microscopy indicate that CMF increases the frequency of pseudopod formation. The activations of Ca2+ influx, adenylyl cyclase, and guanylyl cyclase in response to a pulse of cAMP are strongly inhibited in cells lacking CMF, but are rescued by as little as 10 s exposure of cells to CMF. The activation of phospholipase C by cAMP is not affected by CMF. Northern blots indicate normal levels of the cAMP receptor mRNA in CMF antisense cells during development, while cAMP binding assays and Scatchard plots indicate that CMF antisense cells contain normal levels of the cAMP receptor. In Dictyostelium, both adenylyl and guanylyl cyclases are activated via G proteins. We find that the interaction of the cAMP receptor with G proteins in vitro is not measurably affected by CMF, whereas the activation of adenylyl cyclase by G proteins requires cells to have been exposed to CMF. CMF thus appears to regulate aggregation by regulating an early step of cAMP signal transduction.
Actin filament organization in the fish keratocyte lamellipodium.
Authors: Small J V , Herzog M , Anderson K
Published: May 2004

From recent studies of locomoting fish keratocytes it was proposed that the dynamic turnover of actin filaments takes place by a nucleation-release mechanism, which predicts the existence of short (less than 0.5 microns) filaments throughout the lamellipodium (Theriot, J. A., and T. J. Mitchison. 1991. Nature (Lond.). 352:126-131). We have tested this model by investigating the structure of whole mount keratocyte cytoskeletons in the electron microscope and phalloidin-labeled cells, after various fixations, in the light microscope. Micrographs of negatively stained keratocyte cytoskeletons produced by Triton extraction showed that the actin filaments of the lamellipodium are organized to a first approximation in a two-dimensional orthogonal network with the filaments subtending an angle of around 45 degrees to the cell front. Actin filament fringes grown onto the front edge of keratocyte cytoskeletons by the addition of exogenous actin showed a uniform polarity when decorated with myosin subfragment-1, consistent with the fast growing ends of the actin filaments abutting the anterior edge. A steady drop in filament density was observed from the mid-region of the lamellipodium to the perinuclear zone and in images of the more posterior regions of lower filament density many of the actin filaments could be seen to be at least several microns in length. Quantitative analysis of the intensity distribution of fluorescent phalloidin staining across the lamellipodium revealed that the gradient of filament density as well as the absolute content of F-actin was dependent on the fixation method. In cells first fixed and then extracted with Triton, a steep gradient of phalloidin staining was observed from the front to the rear of the lamellipodium. With the protocol required to obtain the electron microscope images, namely Triton extraction followed by fixation, phalloidin staining was, significantly and preferentially reduced in the anterior part of the lamellipodium. This resulted in a lower gradient of filament density, consistent with that seen in the electron microscope, and indicated a loss of around 45% of the filamentous actin during Triton extraction. We conclude, first that the filament organization and length distribution does not support a nucleation release model, but is more consistent with a treadmilling-type mechanism of locomotion featuring actin filaments of graded length. Second, we suggest that two layers of filaments make up the lamellipodium; a lower, stabilized layer associated with the ventral membrane and an upper layer associated with the dorsal membrane that is composed of filaments of a shorter range of lengths than the lower layer and which is mainly lost in Triton.
Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila.
Authors: Gaertig J , Cruz M A , Bowen J , Gu L , Pennock D G , Gorovsky M A
Published: May 2004

In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of alpha- and beta-tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes. Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of alpha-tubulin at lysine 40, a modification found in most eukaryotes. In Tetrahymena, axonemal alpha-tubulin and numerous other microtubules are acetylated. We completely replaced the single type of alpha-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells. Thus, acetylation of alpha-tubulin at lysine 40 is non-essential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate alpha-tubulin leads us to conclude that: (a) most or all ciliary alpha-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated alpha-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.
Structure of growing microtubule ends: two-dimensional sheets close into tubes at variable rates.
Authors: Chrétien D , Fuller S D , Karsenti E
Published: May 2004

Observation of microtubule growth at different rates by cryo-electron microscopy reveals that the ends range from blunt to long, gently curved sheets. The mean sheet length increases with the growth rate while the width of the distributions increases with the extent of assembly. The combination of a concentration dependent growth rate of the tubulin sheet with a variable closure rate of the microtubule cylinder, results in a model in which stochastic fluctuations in sheet length and tubulin conformation confine GTP-tubulins to microtubule ends. We propose that the variability of microtubule growth rate observed by video microscopy (Gildersleeve, R. F., A. R. Cross, K. E. Cullen, A. P. Fagen, and R. C. Williams. 1992. J. Biol. Chem. 267: 7995-8006, and this study) is due to the variation in the rate of cylinder closure. The curvature of the sheets at the end of growing microtubules and the small oligomeric structures observed at the end of disassembling microtubules, indicate that tubulin molecules undergo conformational changes both during assembly and disassembly.
The basal keratin network of stratified squamous epithelia: defining K15 function in the absence of K14.
Authors: Lloyd C , Yu Q C , Cheng J , Turksen K , Degenstein L , Hutton E , Fuchs E
Published: May 2004

Keratin 5 and keratin 14 have been touted as the hallmarks of the basal keratin networks of all stratified squamous epithelia. Absence of K14 gives rise to epidermolysis bullosa simplex, a human blistering skin disorder involving cytolysis in the basal layer of epidermis. To address the puzzling question of why this disease is primarily manifested in skin rather than other stratified squamous epithelia, we ablated the K14 gene in mice and examined various tissues expressing this gene. We show that a key factor is the presence of another keratin, K15, which was hitherto unappreciated as a basal cell component. We show that the levels of K15 relative to K14 vary dramatically among stratified squamous epithelial tissues, and with neonatal development. In the absence of K14, K15 makes a bona fide, but ultrastructurally distinct, keratin filament network with K5. In the epidermis of neonatal mutant mice, K15 levels are low and do not compensate for the loss of K14. In contrast, the esophagus is unaffected in the neonatal mutant mice, but does appear to be fragile in the adult. Parallel to this phenomenon is that esophageal K14 is expressed at extremely low levels in the neonate, but rises in postnatal development. Finally, despite previous conclusions that the formation of suprabasal keratin filaments might depend upon K5/K14, we find that a wide variety of suprabasal networks composed of different keratins can form in the absence of K14 in the basal layer.
Regulation of a muscle-specific transgene by retinoic acid.
Authors: Xiao Y , Grieshammer U , Rosenthal N
Published: May 2004

Retinoic acid (RA) has been shown to have variable effects on myogenic differentiation in cell culture. The application of RA on primary cultures of embryonic somites, limb buds, and neonatal limbs inhibited myogenic differentiation in a dose-dependent way as indicated by the repression of: (a) myotube formation, (b) myosin heavy chain protein accumulation, (c) myosin light chain (MLC) 1/3, alpha sk-actin and myogenic factor transcript expression. Expression of retinoic acid receptors (RAR) was also affected by RA treatment, specifically RAR gamma transcripts were induced. To further understand the pleiotropic action of RA on myogenesis, we took advantage of two muscle-specific transgene markers which consisted of CAT reporter genes driven by regulatory elements either from the myosin light chain 1/3 locus (MLC-CAT) or the alpha-skeletal actin gene (alpha sk actin-CAT). RA inhibited MLC-CAT transgene but not alpha sk actin-CAT transgene expression in primary cultures from these mice. Analysis of MLC-CAT expression in transgenic mouse primary cultures and in stably transfected C2C12 cells demonstrated that repression of MLC-CAT activity by RA was dependent upon diffusible factors in chick embryo extract. We hypothesize that during development, the pleiotropic effects of RA on myogenesis do not depend solely on the distribution and concentration of RA itself, but are also influenced by extracellular signals in the embryonic environment.
Identification of the low density lipoprotein receptor-related protein (LRP) as an endocytic receptor for thrombospondin-1.
Authors: Godyna S , Liau G , Popa I , Stefansson S , Argraves W S
Published: May 2004

Thrombospondin-1 (TSP1) has potent biological effects on vasculature smooth muscle cells (SMCs) and endothelial cells. The regulation of extracellular accumulation of TSP1 is mediated by a previously obscure process of endocytosis which leads to its lysosomal degradation. Since members of the low density lipoprotein receptor (LDLR) family have been found to mediate endocytosis which leads to degradation of a diverse array of ligands, we evaluated their possible role in the uptake and degradation of TSP1 by vascular SMCs, endothelial-cells and fibroblasts. 125I-TSP1 was found to be internalized and degraded lysosomally by all these cell types. Both the internalization and degradation of 125I-TSP1 could be inhibited by a specific antagonist of the LDLR family, the 39-kD receptor-associated protein (RAP). Antibodies to the LDLR-related protein (LRP) completely blocked the uptake and degradation of 125I-TSP1 in SMCs and fibroblasts but not endothelial cells. Solid-phase binding assays confirmed that LRP bound to TSP1 and that the interaction was of high affinity (Kd = 5 nM). Neither RAP nor LRP antibodies inhibited the binding of 125I-TSP1 to surfaces of SMCs. However, cell surface binding, as well as, endocytosis and degradation could be blocked by heparin or by pre-treatment of the cells with either heparitinase, chondroitinase or beta-D-xyloside. The data indicates that cell surface proteoglycans are involved in the LRP-mediated clearance of TSP1. A model for the clearance of TSP1 by these cells is that TSP1 bound to proteoglycans is presented to LRP for endocytosis. In endothelial cells, however, the internalization of TSP1 was not mediated by LRP but since RAP inhibited TSP1 uptake and degradation, we postulate that another member of the LDLR family is likely to be involved.
BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication.
Authors: Todorov I T , Attaran A , Kearsey S E
Published: May 2004

We have recently cloned and characterized a human member (BM28) of the MCM2-3-5 family of putative relication factors (Todorov, I.T., R. Pepperkok, R.N. Philipova, S. Kearsey, W. Ansorge, and D. Werner. 1994. J. Cell Sci. 107:253-265). While this protein is located in the nucleus throughout interphase, we report here a dramatic alteration in its nuclear binding during the cell cycle. BM28 is retained in the nucleus after Triton X-100 extraction in G1 and early S phase cells, but is progressively lost as S phase proceeds, and little BM28 is retained in detergent-extracted G2 nuclei. BM28 that is resistant to extraction in G1 nuclei is removed by DNase I digestion, suggesting that the protein is chromatin associated. In addition, we present evidence for variations in the electrophoretic mobility of BM28 that may reflect posttranslational modifications of BM28 during the cell cycle. During mitosis, BM28 is present as a fast-migrating form, but on entry into G1, the protein is converted into a slow-migrating form. With the onset of S phase, the slow-migrating form is progressively converted into the fast form. BM28 is phosphorylated at all stages of the cell cycle, but during interphase the fast form is hyperphosphorylated compared with the slow form. These apparent changes in modification may reflect or effect changes in the nuclear binding of BM28. The behavior of BM28 is not dissimilar to related proteins in Saccharomyces cerevisiae, such as Mcm2p, which are excluded from the nucleus after DNA replication. We speculate that BM28 may be involved in the control that limits eukaryotic DNA replication to one round per cell cycle.
Pendulin, a Drosophila protein with cell cycle-dependent nuclear localization, is required for normal cell proliferation.
Authors: Küssel P , Frasch M
Published: May 2004

We describe the dynamic intracellular localization of Drosophila Pendulin and its role in the control of cell proliferation. Pendulin is a new member of a superfamily of proteins which contains Armadillo (Arm) repeats and displays extensive sequence similarities with the Srp1 protein from yeast, with RAG-1 interacting proteins from humans, and with the importin protein from Xenopus. Almost the entire polypeptide chain of Pendulin is composed of degenerate tandem repeats of approximately 42 amino acids each. A short NH2-terminal domain contains adjacent consensus sequences for nuclear localization and cdc2 kinase phosphorylation. The subcellular distribution of Pendulin is dependent on the phase of cell cycle. During interphase, Pendulin protein is exclusively found in the cytoplasm of embryonic cells. At the transition between G2 and M-phase, Pendulin rapidly translocates into the nuclei where it is distributed throughout the nucleoplasm and the areas around the chromosomes. In the larval CNS, Pendulin is predominantly expressed in the dividing neuroblasts, where it undergoes the same cell cycle-dependent redistribution as in embryos. Pendulin is encoded by the oho31 locus and is expressed both maternally and zygotically. We describe the phenotypes of recessive lethal mutations in the oho31 gene that result in a massive decrease or loss of zygotic Pendulin expression. Hematopoietic cells of mutant larvae overproliferate and form melanotic tumors, suggesting that Pendulin normally acts as a blood cell tumor suppressor. In contrast, growth and proliferation in imaginal tissues are reduced and irregular, resulting in abnormal development of imaginal discs and the CNS of the larvae. This phenotype shows that Pendulin is required for normal growth regulation. Based on the structure of the protein, we propose that Pendulin may serve as an adaptor molecule to form complexes with other proteins. The sequence similarity with importin indicates that Pendulin may play a role in the nuclear import of karyophilic proteins and some of these may be required for the normal transmission and function of proliferative signals in the cells.
Oligomerized transferrin receptors are selectively retained by a lumenal sorting signal in a long-lived endocytic recycling compartment.
Authors: Marsh E W , Leopold P L , Jones N L , Maxfield F R
Published: May 2004

Cross-linking of surface receptors results in altered receptor trafficking in the endocytic system. To better understand the cellular and molecular mechanisms by which receptor cross-linking affects the intracellular trafficking of both ligand and receptor, we studied the intracellular trafficking of the transferrin receptor (TfR) bound to multivalent-transferrin (Tf10) which was prepared by chemical cross-linking of transferrin (Tf). Tf10 was internalized about two times slower than Tf and was retained four times longer than Tf, without being degraded in CHO cells. The intracellular localization of Tf10 was investigated using fluorescence and electron microscopy. Tf10 was not delivered to the lysosomal pathway followed by low density lipoprotein but remained accessible to Tf in the pericentriolar endocytic recycling compartment for at least 60 min. The retained Tf10 was TfR-associated as demonstrated by a reduction in surface TfR number when cells were incubated with Tf10. The presence of Tf10 within the recycling compartment did not affect trafficking of subsequently endocytosed Tf. Retention of Tf10 within the recycling compartment did not require the cytoplasmic domain of the TfR since Tf10 exited cells with the same rate when bound to the wild-type TfR or a mutated receptor with only four amino acids in the cytoplasmic tail. Thus, cross-linking of surface receptors by a multivalent ligand acts as a lumenal retention signal within the recycling compartment. The data presented here show that the recycling compartment labeled by Tf10 is a long-lived organelle along the early endosome recycling pathway that remains fusion accessible to subsequently endocytosed Tf.
Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation.
Authors: Gadella T W , Jovin T M
Published: May 2004

The aggregation states of the epidermal growth factor receptor (EGFR) on single A431 human epidermoid carcinoma cells were assessed with two new techniques for determining fluorescence resonance energy transfer: donor photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence lifetime imaging microscopy (FLIM). Fluorescein-(donor) and rhodamine-(acceptor) labeled EGF were bound to the cells and the extent of oligomerization was monitored by the spatially resolved FRET efficiency as a function of the donor/acceptor ratio and treatment conditions. An average FRET efficiency of 5% was determined after a low temperature (4 degrees C) incubation with the fluorescent EGF analogs for 40 min. A subsequent elevation of the temperature for 5 min caused a substantial increase of the average FRET efficiency to 14% at 20 degrees C and 31% at 37 degrees C. In the context of a two-state (monomer/dimer) model for the EGFR, these FRET efficiencies were consistent with minimal average receptor dimerizations of 13, 36, and 69% at 4, 20, and 37 degrees C, respectively. A431 cells were pretreated with the monoclonal antibody mAb 2E9 that specifically blocks EGF binding to the predominant population of low affinity EGFR (15). The average FRET efficiency increased dramatically to 28% at 4 degrees C, indicative of a minimal receptor dimerization of 65% for the subpopulation of high affinity receptors. These results are in accordance with prior studies indicating that binding of EGF leads to a fast and temperature-dependent microclustering of EGFR, but suggest in addition that the high affinity functional subclass of receptors on quiescent A431 cells are present in a predimerized or oligomerized state. We propose that the transmission of the external ligand-binding signal to the cytoplasmic domain is effected by a concerted relative rotational rearrangement of the monomeric units comprising the dimeric receptor, thereby potentiating a mutual activation of the tyrosine kinase domains.
Overexpression of the human NFM subunit in transgenic mice modifies the level of endogenous NFL and the phosphorylation state of NFH subunits.
Authors: Tu P H , Elder G , Lazzarini R A , Nelson D , Trojanowski J Q , Lee V M
Published: May 2004

Neurofilaments (NFs), the major intermediate filaments of central nervous system (CNS) and peripheral nervous system (PNS) neurons, are heteropolymers formed from the high (NFH), middle (NFM), and low (NFL) molecular weight NF subunits. To gain insights into how the expression of NF subunit proteins is regulated in vivo, two transgenes harboring coding sequences for human NFM (hNFM) with or without the hNFM multiphosphorylation repeat domain were introduced into mice. Expression of both hNFM constructs was driven by the hNFM promoter and resulted in increased levels of hNFM subunits concomitant with an elevation in the levels of mouse NFL (mNFL) proteins in the CNS of both lines of transgenic mice. The increased levels of mNFL appear specific to NFM because previous studies of transgenic mice overexpressing either NFL or NFH did not result in increased expression of either of the other two NF subunits. Further, levels of the most heavily phosphorylated isoforms of mouse NFH (mNFH) were reduced in the brains of these transgenic mice, and electron microscopic studies showed a higher packing density of NFs in large-diameter CNS axons of transgenic versus wild-type mice. Thus, reduced phosphorylation of the mNFH carboxy terminal domain may be a compensatory response of CNS neurons to the increase in NFs, and reduced negative charges on mNFH sidearms may allow axons to accommodate more NFs by increasing their packing density. Taken together, these studies imply that NFM may play a dominant role in the in vivo regulation of the levels of NFL protein, the stoichiometry of NF subunits, and the phosphorylation state of NFH. NFM and NFH proteins may assume similar functions in regulation of NF packing density in vivo.
Increased intracellular Ca2+ selectively suppresses IL-1-induced NO production by reducing iNOS mRNA stability.
Authors: Geng Y , Lotz M
Published: May 2004

This study addresses the role of intracellular calcium (Ca2+) in the expression of iNOS, an IL-1 inducible gene in human articular chondrocytes. The calcium ionophore A23187 and ionomycin did not induce NO release or iNOS expression but inhibited dose dependently IL-1-induced NO release with IC50 of 200 nM and 100 nM, respectively. Increased intracellular Ca2+ induced by thapsigargin or cyclopiazonic acid, inhibitors of the endoplasmic reticulum Ca2+ ATPase, had similar inhibitory effects with IC50 of 1 nM and 3 microM, respectively. LPS and TNF alpha induced NO production were also suppressed by these Ca2+ elevating drugs. Levels of IL-1-induced iNOS protein were reduced by A23187, thapsigargin, and cyclopiazonic acid. These drugs as well as Bay K 8644 and KCl inhibited IL-1-induced iNOS mRNA expression. To analyze the role of Ca2+ in the expression of other IL-1 responsive genes in chondrocytes, these Ca2+ modulating drugs were tested for effects on COXII. In contrast to the inhibitory effects on iNOS mRNA, these drugs induced COXII mRNA expression and in combination with IL-1, enhanced COXII mRNA levels. Ca2+ mediated increases in COXII mRNA expression were associated with an increase in COXII protein. The kinetics of Ca2+ effects on IL-1-induced iNOS mRNA levels suggested a posttranscriptional mechanism. Analysis of iNOS mRNA half life showed that it was 6-7 h in IL-1-stimulated cells and decreased by A23187 to 2-3 h. In conclusion, these results show that Ca2+ inhibits IL-1-induced NO release, iNOS protein, and mRNA expression in human articular chondrocytes by reducing iNOS mRNA stability. Under identical conditions increased Ca2+ enhances IL-1-induced COXII gene and protein expression.
Chemoattractant and GTP gamma S-mediated stimulation of adenylyl cyclase in Dictyostelium requires translocation of CRAC to membranes.
Authors: Lilly P J , Devreotes P N
Published: May 2004

We have previously reported that activation of adenylyl cyclase by chemoattractant receptors in Dictyostelium requires, in addition to a heterotrimeric G-protein, a cytosolic protein, designated CRAC (Lilly, P., and P. N. Devreotes. 1994. J. Biol. Chem. 269:14123-14129; Insall, R. H., A. Kuspa, P. J. Lilly, G. Schaulsky, L. R. Levin, W. F. Loomis, and P. N. Devreotes. 1994. J. Cell Biol. 126:1537-1545). In this report, we show that in intact cells, chemoattractants promote translocation of CRAC from the cytosolic to the membrane fraction. However, CRAC is not required at the time of receptor stimulation; it can be added to lysates of activated cells. Treatment of membranes with guanine nucleotides creates binding sites for CRAC. These binding sites can be generated in mutants lacking each of the components of the pathway except the beta-subunit, suggesting that free or "activated" beta gamma-subunits may be a part of the binding site. This hypothesis is consistent with previous observations that CRAC contains a pleckstrin homology domain and that the beta gamma-subunits likely mediate activation of adenylyl cyclase in this system. Thus, CRAC may serve as an adapter, linking the G-protein beta gamma-subunits to activation of the enzyme. GTP gamma S cannot generate CRAC-binding sites when the adenylyl cyclase pathway has been adapted by prior chemoattractant stimulation, suggesting that this is a point of downstream adaptation.
The G protein beta subunit is essential for multiple responses to chemoattractants in Dictyostelium.
Authors: Wu L , Valkema R , Van Haastert P J , Devreotes P N
Published: May 2004

Increasing evidence suggests that the beta gamma-subunit dimers of heterotrimeric G proteins play a pivotal role in transducing extracellular signals. The recent construction of G beta null mutants (g beta-) in Dictyostelium provides a unique opportunity to study the role of beta gamma dimers in signaling processes mediated by chemoattractant receptors. We have shown previously that g beta- cells fail to aggregate; in this study, we report the detailed characterization of these cells. The g beta- cells display normal motility but do not move towards chemattractants. The typical GTP-regulated high affinity chemoattractant-binding sites are lost in g beta- cells and membranes. The g beta- cells do not display chemoattractant-stimulated adenylyl cyclase or guanylyl cyclase activity. These results show that in vivo G beta links chemoattractant receptors to effectors and is therefore essential in many chemoattractant-mediated processes. In addition, we find that G beta is required for GTP gamma S stimulation of adenylyl cyclase activity, suggesting that the beta gamma-dimer activates the enzyme directly. Interestingly, the g beta- cells grow at the same rate as wild-type cells in axenic medium but grow more slowly on bacterial lawns and, therefore, may be defective in phagocytosis.