Fibulin binds to itself and to the carboxyl-terminal heparin-binding region of fibronectin.

Fibulin is a recently described extracellular matrix (ECM) and plasma glycoprotein (Argraves, W. S., Tran, H., Burgess, W. H., and Dickerson, K. (1990) J. Cell Biol. 111, 3155-3164). In this report, ligand affinity chromatography and solid-phase binding analyses were performed to determine which ECM protein(s) interact with fibulin. Fibulin-Sepharose bound two polypeptides of 240 and 100 kDa from the culture medium of metabolically radiolabeled fibroblasts. These two proteins were identified as fibronectin (FN) and fibulin, respectively, based on their electrophoretic behavior and reactivity with monoclonal antibodies. Consistent with the findings of affinity chromatography, fibulin bound to surfaces coated with FN (either plasma or cellular form) or fibulin but not with other ECM proteins, such as laminin, merosin, and types I and IV collagen. The binding of fibulin to solid-phase FN was estimated to have a Kd of 139 nM, whereas the Kd for self-interaction was 322 nM. Evaluation of proteolytic fragments from all regions of FN allowed a fibulin-binding site to be localized within a 23-kDa heparin-binding fragment containing type III repeats 13-14. Heparin did not compete for the interaction between fibulin and FN, suggesting that the binding sites for fibulin and heparin are distinct.     

Mobilization and recruitment of HP1β: A bimodal response to DNA breakage

The pathways that signal double-strand DNA breaks (DSBs) in mammalian cells are central to the maintenance of genome integrity. We have reported (Ayoub et al., Nature 2008; 453: 682-6) that the rapid mobilization of the heterochromatin protein, HP1β, within seconds from DSB sites promotes chromatin changes like H2AX phosphorylation that trigger this response. Notably, this paper and a subsequent report (Ayoub et al., Cell Cycle 2009; 8: 1494-500), demonstrate that transient HP1β mobilization is followed by its accumulation over time at DSB sites. Indeed, two recent papers (Luijsterburg et al., J Cell Biol 2009; 185:577-86 and Zarebski et al., Cytometry A May 2009) suggest that HP1 recruitment to damage sites, rather than its rapid mobilization, is the predominant behaviour exhibited by this protein. Here, we present new experimental analyses which corroborate that fluorophore-tagged HP1β exhibits two distinct behaviours at DSB sites in living cells – rapid, transient mobilization, most evident in heterochromatic regions, followed by slower recruitment. Experimental methods allowing visualization of these behaviours are described. Interestingly, chemical inhibition of the DNA-damage responsive enzyme, casein kinase 2 (CK2), suppresses HP1β mobilization while permitting recruitment. Our findings reconcile recent findings in a new model, wherein rapid HP1β mobilization from DSBs mediated by its phosphorylation on Thr51 by CK2, is followed by, and may overlap with, its accumulation at these sites via the chromoshadow domain, independent of Thr51. Our analyses provide fresh insight into the earliest events that trigger the DNA damage response in mammalian cells.     

Xenopus crescent encoding a Frizzled-like domain is expressed in the Spemann organizer and pronephros

The Spemann organizer can be subdivided into head- and trunk-inducing tissues along the anteroposterior axis (Mangold, 1933. Naturwiisenschaften 43, 761-766; Spemann, 1931. Wilhelm Roux Arch. Entwicklungsmech. Org. 123, 389-517). Recent studies have suggested that head formation is brought about by repression of both Wnt and BMP signalling (Glinka et al., 1998. Nature 391, 357-362; Glinka et al., 1997. Nature 389, 517-519). Several Wnt inhibitors secreted from the head organizer region have been identified in Xenopus, such as Cerberus (Bouwmeester et al., 1996. Nature 382, 595-601), Frzb-1 (Leyns et al., 1997. Cell 88, 747-756; Lin et al., 1997. Proc. Natl. Acad. Sci. USA 94, 11196-11200), and Dkk-1 (Glinka et al., 1998. Nature 391, 357-362), supporting this two-inhibitor model. To isolate genes expressed in the head organizer, we screened a prechordal plate cDNA library by sequencing and expression pattern, and isolated the Xenopus ortholog of chick crescent encoding a Frizzled-like domain that is related to Wnt-binding regions of the Frizzled-family proteins. Expression of Xenopus crescent was first detected in the Spemann organizer region at the early gastrula stage and later in prechordal plate cells lining the boundary of mesoderm and ectoderm layers and in the anterior endoderm. At tailbud stages, the expression in the endomesoderm region was diminished, while expression in the pronephros became detectable. In animal cap assays, crescent gene was synergistically upregulated by coexpression of Xlim1, Ldb1, and Siamois, but not by Activin treatment.     

Beta 1 integrins isolated from embryonic chicken fibroblasts bind to monomers and polymers of type I collagen.

The avian integrin beta 1 subfamily consists of multiple alpha-beta subunit heterodimers. We employed two different physical states of type I collagen, monomers and fibrils, in the isolation and characterization of avian collagen integrins. Affinity chromatography showed that three integrins, tentatively designated alpha 155 beta 1 (band 1), alpha 5a beta 1, and alpha 3 beta 1 (band 2), bind fibrillar and monomeric collagen under physiological ionic conditions and require divalent cations for binding activity. Sodium chloride gradients (0-0.5 M) were used to assess the functional ability of the integrins to remain bound to the two forms of type I collagen. The results show that integrins elute from the two forms of collagen with distinct fractionation profiles. One integrin, alpha 155 beta 1, binds fibrillar collagen with relatively higher affinity than the other beta 1 receptors. This same avian integrin, alpha 155 beta 1, is immunoreactive with an antiserum (Hynes et al., 1989) raised against a peptide that corresponds to the entire alpha 5 cytoplasmic domain, and coincidently, part of the alpha 6 cytoplasmic domain (de Curtis et al., 1991). Cell biological studies employing double immunofluorescence show that integrins recognized by this antiserum co-localize with extracellular deposits of type I collagen.     

Location of 64K collagen producer chondrocytes in developing chicken embryo tibiae.

The synthesis of a new low-molecular-weight collagen by cultured chicken embryo chondrocytes has been recently demonstrated (Capasso et al., Exp. Cell Res. 142:197-206, 1982; Gibson et al., J. Cell Biol. 93:767-774, 1982; Schmid and Conrad, J. Biol. Chem. 257:12444-12450, 1982). In this paper we report results on the location of chondrocytes synthesizing this new collagen (64K collagen) in the developing chicken embryo. The 64K collagen is synthesized in very large amounts by cells concentrated at the diaphysis of 9-day-old and at the epiphysis of 17-day-old embryo tibiae. These regions are characterized by a remodeling of the cartilage matrix leading to the replacement of the cartilage with bone tissue; therefore, this collagen appears to be a marker of a specific developmental stage of chondrocytes. The origin of cells competent for the synthesis of the 64K collagen is also discussed.     

Cortex shatters the glass ceiling

Recreating developmental structures in vitro has been a primary challenge for stem cell biologists. Recent studies in Cell Stem Cell (Eiraku et al., 2008) and Nature (Gaspard et al., 2008) demonstrate that embryonic stem cells can recapitulate early cortical development, enabling them to generate specific cortical subtypes.     

Transduction of the chemotactic signal to the actin cytoskeleton of Dictyostelium discoideum

Dictyostelium discoideum amebae chemotax toward folate during vegetative growth and toward extracellular cAMP during the aggregation phase that follows starvation. Stimulation of starving amebae with extracellular cAMP leads to both actin polymerization and pseudopod extension (Hall et al., 1988, J. Cell. Biochem. 37, 285-299). We have identified an actin nucleation activity (NA) from starving amebae that is regulated by cAMP receptors and controls actin polymerization (Hall et al., 1989, J. Cell Biol., in press). We show here that NA from vegetative cells is also regulated by chemotactic receptors for folate. Our studies indicate that NA is an essential effector in control of the actin cytoskeleton by chemotactic receptors. Guided by a recently proposed model for signal transduction from the cAMP receptor (Snaar-Jagalska et al., 1988, Dev. Genet. 9, 215-225), we investigated which of three signaling pathways activates the NA effector. Treatment of whole cells with a commercial pertussis toxin preparation (PT) inhibited cAMP-stimulated NA. However, endotoxin contamination of the PT appears to account for this effect. The synag7 mutation and caffeine treatment do not inhibit activation of NA by cAMP. Thus, neither activation of adenylate cyclase nor a G protein sensitive to PT treatment of whole cells is necessary for the NA response. Actin nucleation activity stimulated with folate is normal in vegetative fgdA cells. However, cAMP suppresses rather than activates NA in starving fgdA cells. This indicates that the components of the actin nucleation effector are present and that a pathway regulating the inhibitor(s) of nucleation remains functional in starving fgdA cells. The locus of the fgdA defect, a G protein implicated in phospholipase C activation, is directly or indirectly responsible for transduction of the stimulatory chemotactic signal from cAMP receptors to the nucleation effector in Dictyostelium.     

Origin of cortical microtubules organized at M/G1 interface: recruitment of tubulin from phragmoplast to nascent microtubules

The origin of cortical microtubules (CMTs) was investigated in transgenic BY-2 cells stably expressing a GFP (green fluorescent protein) -tubulin fusion protein (BY-GT16). In a previous study, we found that CMTs were initially organized in the perinuclear regions but then elongated to reach the cell cortex where they formed bright spots, and that the appearance of parallel MTs from the bright spots was followed by the appearance of transverse MTs (Kumagai et al., Plant Cell Physiol. 42, 723-732, 2001). In this study, we investigated the migration of tubulin to the reorganization sites of CMTs at the M/G1 interface. After synchronization of the BY-GT16 cells by aphidicolin, the localization of GFP-tubulin was monitored and analyzed by deconvolution microscopy. GFP-tubulin was found to accumulate on the nuclear surface near the cell plate at the final stage of phragmoplast collapse. Subsequently, GFP-tubulin accumulated again on the nuclear surface opposite the cell plate, where nascent MTs elongated to the cell cortex. The significance of these observations on the mode of CMT organization is discussed.     

Localization of spindle checkpoint proteins in cells undergoing mitosis with unreplicated genomes

CHO cells can be arrested with hydoxyurea at the beginning of the DNA synthesis phase of the cell cycle. Subsequent treatment with the xanthine, caffeine, induces cells to bypass the S-phase checkpoint and enter unscheduled mitosis [Schlegel and Pardee,1986, Science 232:1264-1266]. These treated cells build a normal spindle and distribute kinetochores, unattached to chromosomes, to their daughter cells [Brinkley et al.,1988, Nature 336:251-254; Zinkowski et al.,1991, J Cell Biol 113:1091-1110; Wise and Brinkley,1997, Cell Motil Cytoskeleton 36:291-302; Balczon et al.,2003, Chromosoma 112:96-102]. To investigate how these cells distribute kinetochores to daughter cells, we analyzed the spindle checkpoint components, Mad2, CENP-E, and the 3F3 phosphoepitope, using immunofluorescence and digital microscopy. Even though the kinetochores were unpaired and DNA was fragmented, the tension, alignment, and motor components of the checkpoint were found to be present and localized as predicted in prometaphase and metaphase. This unusual mitosis proves that a cell can successfully localize checkpoint proteins and divide even when kinetochores are unpaired and fragmented.     

Increased phosphorylation of tyrosine in vinculin does not occur upon transformation by some avian sarcoma viruses.

The level of phosphotyrosine in vinculin was determined in chicken embryo fibroblasts transformed by various strains of avian sarcoma virus. As previously reported (Sefton et al., Cell 24:165-174, 1981), vinculin was phosphorylated at tyrosine residues in most cultures examined, but the level varied greatly and no detectable change was found in cultures infected with Fujinami sarcoma virus or UR2 sarcoma virus. Regardless of the level of vinculin phosphorylation, the number of organized microfilament bundles was found to be decreased in all transformed cells. These results strongly suggest that tyrosine phosphorylation of vinculin is not an obligatory step in cell transformation by this class of oncogenes, nor is it correlated with the associated cytoskeletal disarray.     

Cellular Redistribution of Protein Tyrosine Phosphatases LAR and PTPσ by Inducible Proteolytic Processing

Most receptor-like protein tyrosine phosphatases (PTPases) display a high degree of homology with cell adhesion molecules in their extracellular domains. We studied the functional significance of processing for the receptor-like PTPases LAR and PTPσ. PTPσ biosynthesis and intracellular processing resembled that of the related PTPase LAR and was expressed on the cell surface as a two-subunit complex. Both LAR and PTPσ underwent further proteolytical processing upon treatment of cells with either calcium ionophore A23187 or phorbol ester TPA. Induction of LAR processing by TPA in 293 cells did require overexpression of PKCα. Induced proteolysis resulted in shedding of the extracellular domains of both PTPases. This was in agreement with the identification of a specific PTPσ cleavage site between amino acids Pro₈₂₁ and Ile₈₂₂. Confocal microscopy studies identified adherens junctions and desmosomes as the preferential subcellular localization for both PTPases matching that of plakoglobin. Consistent with this observation, we found direct association of plakoglobin and β-catenin with the intracellular domain of LAR in vitro. Taken together, these data suggested an involvement of LAR and PTPσ in the regulation of cell contacts in concert with cell adhesion molecules of the cadherin/catenin family. After processing and shedding of the extracellular domain, the catalytically active intracellular portions of both PTPases were internalized and redistributed away from the sites of cell–cell contact, suggesting a mechanism that regulates the activity and target specificity of these PTPases. Calcium withdrawal, which led to cell contact disruption, also resulted in internalization but was not associated with prior proteolytic cleavage and shedding of the extracellular domain. We conclude that the subcellular localization of LAR and PTPσ is regulated by at least two independent mechanisms, one of which requires the presence of their extracellular domains and one of which involves the presence of intact cell–cell contacts. A key element in the regulation of cell–cell and cell– matrix contacts is the tyrosine phosphorylation of proteins that are localized in focal adhesions and at intercellular junctions (for reviews see Kemler, 1993; Clark and Brugge, 1995). While much is known about the protein tyrosine kinases involved in the phosphorylation of cell adhesion components, very little information exists about the identity of protein tyrosine phosphatases (PTPases),¹ which are responsible for the dephosphorylation and thereby regulation of these structural complexes. Probable candidates are those receptor-like PTPases that contain cell adhesion molecule-like extracellular domains and could therefore regulate their intrinsic phosphatase activity in response to cell contact. Recent reports suggest that some PTPases do, in fact, possess properties that resemble those of classical cell adhesion molecules (for review see Brady-Kalnay and Tonks, 1995). A direct involvement in cell–cell contact has so far been demonstrated for PTPμ (Brady-Kalnay et al., 1993; Gebbink et al., 1993) and PTPκ (Sap et al., 1994), for which a homophilic interaction between their extracellular domains was found. The localization of PTPμ (Brady-Kalnay et al., 1995; Gebbink et al., 1995), PTPκ (Fuchs et al., 1996), and PCP-2 (Wang et al., 1996) was restricted to sites of cell–cell contact and surface expression of PTPμ (Gebbink et al., 1995), and PTPκ (Fuchs et al., 1996) was increased in a cell density-dependent manner. Moreover, a direct association of PTPκ (Fuchs et al., 1996) and PTPμ (Brady-Kalnay et al., 1995) with members of the cadherin/catenin family suggests that proteins of the cell adhesion complex represent physiological substrates for these PTPases. A possible regulatory function in cell–matrix adhesion has been proposed for LAR, another receptor-like PTPase, which associated with focal cell–substratum adhesions via the newly identified LAR interacting protein 1, LIP-1 (Serra-Pages et al., 1995).PTPμ (Gebbink et al., 1991), PTPκ (Jiang et al., 1993; Fuchs et al., 1996), PTPδ (Krueger et al., 1990; Mizuno et al., 1993, Pulido et al., 1995a), PCP-2 (Wang et al., 1996), and LAR (Streuli et al., 1988, Pot et al., 1991) are members of the so-called type II receptor-like PTPases. The extracellular domains of these PTPases contain a variable number of Ig-like and fibronectin type III-like (FNIII) domains (for review see Charbonneau and Tonks, 1992). With the exception of PCP-2 (Wang et al., 1996), these PTPases also share characteristics in their biosynthesis. They all underwent proteolytic processing by a furin-like endoprotease and were expressed at the cell surface in two subunits which were not covalently linked (Streuli et al., 1992; Yu et al., 1992; Jiang et al., 1993; Brady-Kalnay and Tonks, 1994; Gebbink et al., 1995; Pulido et al., 1995a; Fuchs et al., 1996). It was shown for LAR that the E subunit, which contains the cell adhesion molecule-like extracellular domain, was shed from the cell surface when cells were grown to a high density (Streuli et al., 1992). This shedding of the E subunit of LAR was the result of an additional proteolytic processing step that could also be induced by treatment of the cells with the phorbol ester TPA (Serra-Pages et al., 1995). An accumulation of E subunits in the supernatant of cells was also observed for PTPμ (Gebbink et al., 1995) and PTPδ (Pulido et al., 1995a), and this suggests a common mechanism in the regulation of type II PTPases. However, the effect of proteolytic processing on either the catalytic activity, the substrate specificity, or the cellular localization of these PTPases has not yet been determined. We report here that PTPσ, a recently identified new member of the family of receptor-like type II PTPases (Pan et al., 1993; Walton et al., 1993; Yan et al., 1993; Ogata et al., 1994; Zhang et al., 1994), underwent biosynthesis and proteolytic processing in a manner that resembled that of the most closely related PTPase LAR. Moreover, further proteolytic processing of PTPσ as well as of LAR could be induced by treatment of the cells with TPA or the calcium ionophore A23187. Transient expression studies indicated that TPA-induced processing of LAR, but not PTPσ, was dependent on the coexpression of PKCα. Inducible processing of both PTPases took place in the extracellular segment of the P subunit in a juxtamembrane position and led to the shedding of the E subunit. Both LAR and PTPσ were predominantly localized in regions of cell–cell contact and accumulated in dot-like structures that could be identified as adherens junctions and desmosomes by colocalization with plakoglobin (Cowin et al., 1986). Moreover, plakoglobin and β-catenin, another component of E-cadherin–containing cell adhesion complexes in adherens junctions, associated directly with the intracellular domain of LAR in vitro. The inducible shedding of the E subunit of LAR and PTPσ was followed by a redistribution of the PTPases within the cell membrane and by an internalization of the cleaved P subunits. It therefore represents a mechanism through which the phosphatase activity of these PTPases could be regulated in response to cell–cell contact. The cell adhesion molecule-like character of LAR and PTPσ was further supported by the fact that the internalization of LAR and PTPσ occurred independently of the proteolytic processing if cells were grown in calcium-depleted growth medium. The analogies in specific localization as well as internalization behavior of PTPσ and LAR, with molecules of the cadherin/catenin family, strongly suggest a direct involvement of PTPσ and LAR in the formation or maintenance of intercellular contacts.     

New "hogs" in Hedgehog transport and signal reception

A recent paper in Cell (Yao et al., 2006) and two papers in Developmental Cell (Tenzen et al., 2006; Zhang et al., 2006) identify a new receptor component for Hedgehog, a key morphogen in embryonic development. Many other proteins that bind to Hedgehog in the extracellular matrix or on the cell surface have been identified. In light of these recent discoveries, we discuss how these factors control the stability, transport, reception, and availability of Hedgehog in modulating Hedgehog-mediated responses.     

Small RNAs loom large during reprogramming

In two independent Cell Stem Cell reports, the Morrisey and Mori groups show that human and mouse somatic cells can be reprogrammed to produce induced pluripotent stem cells by expressing microRNAs, completely eliminating the need for ectopic protein expression (Anokye-Danso et al., 2011; Miyoshi et al., 2011).     

Converting human skin cells to neurons: a new tool to study and treat brain disorders?

Recent publications in Cell Stem Cell (Son et?al., 2011; Ambasudhan et?al., 2011), PNAS (Pfisterer et?al., 2011), and Nature (Caiazzo et?al., 2011; Pang et?al., 2011; Yoo et?al., 2011) report that functional neurons can be directly generated from human fibroblast cells without going through the pluripotent state.     

FGF and EGF act synergistically to induce proliferation in BC3H1 myoblasts

BC3H1 muscle cells proliferate when grown in high concentrations of FBS (20%). Lowering the FBS concentration to 0.5% causes the cells to stop proliferating and is permissive for the morphological and biochemical differentiation of BC3H1 cells. Exposure of differentiated BC3H1 myocytes to high concentrations of serum or to the purified growth factors FGF or TGF-b induced a shutdown of this differentiation program but did not induce cell proliferation (Olson et al., J. Cell Biol., 103:1799-1805, 1986; Lathrop et al., J. Cell Biol., 100:1540-1547, 1985, and J. Cell Biol., 101:2194-2198, 1985). We explored the possibility that BC3H1 cells require factors to act synergistically to induce proliferation. We found that EGF and FGF function in a synergistic fashion to stimulate BC3H1 proliferation. Moreover, the temporal requirement for these growth factors suggest that they are functioning as competence and progression factors for BC3H1 cell proliferation.     

The development and subsequent elimination of aberrant peripheral axon projections in Semaphorin3A null mutant mice

Semaphorin3A (previously known as Semaphorin III, Semaphorin D, or collapsin-1) is a member of the semaphorin gene family, many of which have been shown to guide axons during nervous system development. Semaphorin3A has been demonstrated to be a diffusible chemorepulsive molecule for axons of selected neuronal populations in vitro. Analysis of embryogenesis in two independent lines of Semaphorin3A knockout mice support the hypothesis that this molecule is an important guidance signal for neurons of the peripheral nervous system (M. Taniguchi et al., 1997, Neuron 19, 519-530; E. Ulupinar et al., 1999, Mol. Cell. Neurosci. 13, 281-292). Surprisingly, newborn Semaphorin3A null mutant mice exhibit no significant abnormalities (O. Behar et al., 1996, Nature 383, 525-528). In this study we have tested the hypothesis that guidance abnormalities that occurred during early stages of Semaphorin3A null mice development are corrected later in development. We have found that the extensive abnormalities formed during early developmental stages in the peripheral nervous system are largely eliminated by embryonic day 15.5. We demonstrate further that at least in one distinct anatomical location these abnormalities are mainly the result of aberrant projections. In conclusion, these findings suggest the existence of correction mechanisms that eliminate most sensory axon pathfinding errors early in development.