Tubulin Polyglycylation: Differential Posttranslational Modification of Dynamic Cytoplasmic and Stable Axonemal Microtubules in Paramecium

Polyglycylation, a posttranslational modification of tubulin, was discovered in the highly stable axonemal microtubules of Paramecium cilia where it involves the lateral linkage of up to 34 glycine units per tubulin subunit. The observation of this type of posttranslational modification mainly in axonemes raises the question as to its relationship with axonemal organization and with microtubule stability. This led us to investigate the glycylation status of cytoplasmic microtubules that correspond to the dynamic microtubules in Paramecium. Two anti-glycylated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49, are shown here to exhibit different affinities toward mono- and polyglycylated synthetic tubulin peptides. Using immunoblotting and mass spectrometry, we show that cytoplasmic tubulin is glycylated. In contrast to the highly glycylated axonemal tubulin, which is recognized by the two mAbs, cytoplasmic tubulin reacts exclusively with TAP 952, and the α- and β- tubulin subunits are modified by only 1–5 and 2–9 glycine units, respectively. Our analyses suggest that most of the cytoplasmic tubulin contains side chain lengths of 1 or 2 glycine units distributed on several glycylation sites. The subcellular partition of distinct polyglycylated tubulin isoforms between cytoplasmic and axonemal compartments implies the existence of regulatory mechanisms for glycylation. By following axonemal tubulin immunoreactivity with anti-glycylated tubulin mAbs upon incubation with a Paramecium cellular extract, the presence of a deglycylation enzyme is revealed in the cytoplasm of this organism. These observations establish that polyglycylation is reversible and indicate that, in vivo, an equilibrium between glycylating and deglycylating enzymes might be responsible for the length of the oligoglycine side chains of tubulin.

Overexpression of a Novel Rho Family GTPase, RacC, Induces Unusual Actin-based Structures and Positively Affects Phagocytosis in Dictyostelium discoideum

Rho family proteins have been implicated in regulating various cellular processes, including actin cytoskeleton organization, endocytosis, cell cycle, and gene expression. In this study, we analyzed the function of a novel Dictyostelium discoideum Rho family protein (RacC). A cell line was generated that conditionally overexpressed wild-type RacC three- to fourfold relative to endogenous RacC. Light and scanning electron microscopy indicated that the morphology of the RacC-overexpressing cells [RacC WT(+) cells] was significantly altered compared with control cells. In contrast to the cortical F-actin distribution normally observed, RacC WT(+) cells displayed unusual dorsal and peripheral F-actin–rich surface blebs (petalopodia, for flower-like). Furthermore, phagocytosis in the RacC WT(+) cells was induced threefold relative to control Ax2 cells, whereas fluid-phase pinocytosis was reduced threefold, primarily as the result of an inhibition of macropinocytosis. Efflux of fluid-phase markers was also reduced in the RacC WT(+) cells, suggesting that RacC may regulate postinternalization steps along the endolysosomal pathway. Treatment of cells with Wortmannin and LY294002 (phosphatidylinositol 3-kinase inhibitors) prevented the RacC-induced morphological changes but did not affect phagocytosis, suggesting that petalopodia are probably not required for RacC-induced phagocytosis. In contrast, inactivating diacylglycerol-binding motif–containing proteins by treating cells with the drug calphostin C completely inhibited phagocytosis in control and RacC WT(+) cells. These results suggest that RacC plays a role in actin cytoskeleton organization and phagocytosis in Dictyostelium.

Caldesmon Inhibits Nonmuscle Cell Contractility and Interferes with the Formation of Focal Adhesions

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca2+–calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca2+–calmodulin or Ca2+–S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.

Chemotactic Peptide-induced Changes of Intermediate Filament Organization in Neutrophils during Granule Secretion: Role of Cyclic Guanosine Monophosphate

In neutrophils activated to secrete with formyl-methionyl-leucyl-phenylalanine, intermediate filaments are phosphorylated transiently by cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G-kinase). cGMP regulation of vimentin organization was investigated. During granule secretion, cGMP levels were elevated and intermediate filaments were transiently assembled at the pericortex to areas devoid of granules and microfilaments. Microtubule and microfilament inhibitors affected intermediate filament organization, granule secretion, and cGMP levels. Cytochalasin D and nocodazole caused intermediate filaments to assemble at the nucleus, rather than at the pericortex. cGMP levels were elevated in neutrophils by both inhibitors; however, with cytochalasin D, cGMP was elevated earlier and granule secretion was excessive. Nocodazole did not affect normal cGMP elevations, but specific granule secretion was delayed. LY83583, a guanylyl cyclase antagonist, inhibited granule secretion and intermediate filament organization, but not microtubule or microfilament organization. Intermediate filament assembly at the pericortex and secretion were partially restored by 8-bromo-cGMP in LY83583-treated neutrophils, suggesting that cGMP regulates these functions. G-kinase directly induced intermediate filament assembly in situ, and protein phosphatase 1 disassembled filaments. However, in intact cells stimulated with formyl-methionyl-leucyl-phenylalanine, intermediate filament assembly is focal and transient, suggesting that vimentin phosphorylation is compartmentalized. We propose that, in addition to changes in microfilament and microtubule organization, granule secretion is also accompanied by changes in intermediate filament organization, and that cGMP regulates vimentin filament organization via activation of G-kinase.

Microtubule self-organization is gravity-dependent

Although weightlessness is known to affect living cells, the manner by which this occurs is unknown. Some reaction-diffusion processes have been theoretically predicted as being gravity-dependent. Microtubules, a major constituent of the cellular cytoskeleton, self-organize in vitro by way of reaction-diffusion processes. To investigate how self-organization depends on gravity, microtubules were assembled under low gravity conditions produced during space flight. Contrary to the samples formed on an in-flight 1 × g centrifuge, the samples prepared in microgravity showed almost no self-organization and were locally disordered.

Identification of two novel transmembrane γ-carboxyglutamic acid proteins expressed broadly in fetal and adult tissues

The proline-rich γ-carboxyglutamic acid (Gla) proteins (PRGPs) 1 and 2 are the founding members of a family of vitamin K-dependent single-pass integral membrane proteins characterized by an extracellular amino terminal domain of approximately 45 amino acids that is rich in Gla. The intracellular carboxyl terminal region of these two proteins contains one or two copies of the sequence PPXY, a motif present in a variety of proteins involved in such diverse cellular functions as signal transduction, cell cycle progression, and protein turnover. In this report, we describe the cloning of the cDNAs for two additional human transmembrane Gla proteins (TMG) of 20–24 kDa named TMG3 and TMG4. These two proteins possess extracellular Gla domains with 13 or 9 potential Gla residues, respectively, followed by membrane-spanning hydrophobic regions and cytoplasmic carboxyl terminal regions that contain PPXY motifs. This emerging family of integral membrane Gla proteins includes proline-rich Gla protein (PRGP) 1, PRGP2, TMG3, and TMG4, all of which are characterized by broad and variable distribution in both fetal and adult tissues. Members of this family can be grouped into two subclasses on the basis of their gene organization and amino acid sequence. These observations suggest novel physiological functions for vitamin K beyond its known role in the biosynthesis of proteins involved in blood coagulation and bone development. The identification and characterization of these proteins may allow a more complete understanding of the teratogenic consequences of exposure in utero to vitamin K antagonists, such as warfarin-based anticoagulants.

The reversible condensation and expansion of the rotavirus genome

Understanding the structural organization of the genome is particularly relevant in segmented double-stranded RNA viruses, which exhibit endogenous transcription activity. These viruses are molecular machines capable of repeated cycles of transcription within the intact capsid. Rotavirus, a major cause of infantile gastroenteritis, is a prototypical segmented double-stranded RNA virus. From our three-dimensional structural analyses of rotavirus examined under various chemical conditions using electron cryomicroscopy, we show here that the viral genome exhibits a remarkable conformational flexibility by reversibly changing its packaging density. In the presence of ammonium ions at high pH, the genome condenses to a radius of ≈180 Å from ≈220 Å. Upon returning to physiological conditions, the genome re-expands and fully maintains its transcriptional properties. These studies provide further insights into the genome organization and suggest that the observed isometric and concentric nature of the condensation is due to strong interactions between the genome core and the transcription enzymes anchored to the capsid inner surface. The ability of the genome to condense beyond what is normally observed in the native virus indicates that the negative charges on the RNA in the native state may be only partially neutralized. Partial neutralization may be required to maintain appropriate interstrand spacing for templates to move around the enzyme complexes during transcription. Genome condensation was not observed either with increased cation concentrations at normal pH or at high pH without ammonium ions. This finding indicates that the observed genome condensation is a synergistic effect of hydroxyl and ammonium ions involving disruption of protein–RNA interactions that perhaps facilitate further charge neutralization and consequent reduction in the interstrand spacing.

Na,K-ATPase β-Subunit Is Required for Epithelial Polarization, Suppression of Invasion, and Cell Motility

The cell adhesion molecule E-cadherin has been implicated in maintaining the polarized phenotype of epithelial cells and suppression of invasiveness and motility of carcinoma cells. Na,K-ATPase, consisting of an α- and β-subunit, maintains the sodium gradient across the plasma membrane. A functional relationship between E-cadherin and Na,K-ATPase has not previously been described. We present evidence that the Na,K-ATPase plays a crucial role in E-cadherin–mediated development of epithelial polarity, and suppression of invasiveness and motility of carcinoma cells. Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK) have highly reduced levels of E-cadherin and β1-subunit of Na,K-ATPase. Forced expression of E-cadherin in MSV-MDCK cells did not reestablish epithelial polarity or inhibit the invasiveness and motility of these cells. In contrast, expression of E-cadherin and Na,K-ATPase β1-subunit induced epithelial polarization, including the formation of tight junctions and desmosomes, abolished invasiveness, and reduced cell motility in MSV-MDCK cells. Our results suggest that E-cadherin–mediated cell-cell adhesion requires the Na,K-ATPase β-subunit's function to induce epithelial polarization and suppress invasiveness and motility of carcinoma cells. Involvement of the β1-subunit of Na,K-ATPase in the polarized phenotype of epithelial cells reveals a novel link between the structural organization and vectorial ion transport function of epithelial cells.

Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization

In humans, SOX9 heterozygous mutations cause the severe skeletal dysmorphology syndrome campomelic dysplasia. Except for clinical descriptions, little is known about the pathogenesis of this disease. We have generated heterozygous Sox9 mutant mice that phenocopy most of the skeletal abnormalities of this syndrome. The Sox9+/− mice died perinatally with cleft palate, as well as hypoplasia and bending of many skeletal structures derived from cartilage precursors. In embryonic day (E)14.5 heterozygous embryos, bending of radius, ulna, and tibia cartilages was already prominent. In E12.5 heterozygotes, all skeletal elements visualized by using Alcian blue were smaller. In addition, the overall levels of Col2a1 RNA at E10.5 and E12.5 were lower than in wild-type embryos. We propose that the skeletal abnormalities observed at later embryonic stages were caused by delayed or defective precartilaginous condensations. Furthermore, in E18.5 embryos and in newborn heterozygotes, premature mineralization occurred in many bones, including vertebrae and some craniofacial bones. Because Sox9 is not expressed in the mineralized portion of the growth plate, this premature mineralization is very likely the consequence of allele insufficiency existing in cells of the growth plate that express Sox9. Because the hypertrophic zone of the heterozygous Sox9 mutants was larger than that of wild-type mice, we propose that Sox9 also has a role in regulating the transition to hypertrophic chondrocytes in the growth plate. Despite the severe hypoplasia of cartilages, the overall organization and cellular composition of the growth plate were otherwise normal. Our results suggest the hypothesis that two critical steps of the chondrocyte differentiation pathway are sensitive to Sox9 dosage. First, an early step presumably at the stage of mesenchymal condensation of cartilage primordia, and second, a later step preceding the transition of chondrocytes into hypertrophic chondrocytes.

Molecular kinesis in cellular function and plasticity

Intracellular transport and localization of cellular components are essential for the functional organization and plasticity of eukaryotic cells. Although the elucidation of protein transport mechanisms has made impressive progress in recent years, intracellular transport of RNA remains less well understood. The National Academy of Sciences Colloquium on Molecular Kinesis in Cellular Function and Plasticity therefore was devised as an interdisciplinary platform for participants to discuss intracellular molecular transport from a variety of different perspectives. Topics covered at the meeting included RNA metabolism and transport, mechanisms of protein synthesis and localization, the formation of complex interactive protein ensembles, and the relevance of such mechanisms for activity-dependent regulation and synaptic plasticity in neurons. It was the overall objective of the colloquium to generate momentum and cohesion for the emerging research field of molecular kinesis.

Ribonucleoprotein infrastructure regulating the flow of genetic information between the genome and the proteome

Following transcription and splicing, each mRNA of a mammalian cell passes into the cytoplasm where its fate is in the hands of a complex network of ribonucleoproteins (mRNPs). The success or failure of a gene to be expressed depends on the performance of this mRNP infrastructure. The entry, gating, processing, and transit of each mRNA through an mRNP network helps determine the composition of a cell's proteome. The machinery that regulates storage, turnover, and translational activation of mRNAs is not well understood, in part, because of the heterogeneous nature of mRNPs. Recently, subsets of cellular mRNAs clustered as members of mRNP complexes have been identified by using antibodies reactive with RNA-binding proteins, including ELAV/Hu, eIF-4E, and poly(A)-binding proteins. Cytoplasmic ELAV/Hu proteins are involved in the stability and translation of early response gene (ERG) transcripts and are expressed predominately in neurons. mRNAs recovered from ELAV/Hu mRNP complexes were found to have similar sequence elements, suggesting a common structural linkage among them. This approach opens the possibility of identifying transcripts physically clustered in vivo that may have similar fates or functions. Moreover, the proteins encoded by physically organized mRNAs may participate in the same biological process or structural outcome, not unlike operons and their polycistronic mRNAs do in prokaryotic organisms. Our goal is to understand the organization and flow of genetic information on an integrative systems level by analyzing the collective properties of proteins and mRNAs associated with mRNPs in vivo.

Alterations in the Cytoskeleton Accompany Aluminum-Induced Growth Inhibition and Morphological Changes in Primary Roots of Maize1

Although Al is one of the major factors limiting crop production, the mechanisms of toxicity remain unknown. The growth inhibition and swelling of roots associated with Al exposure suggest that the cytoskeleton may be a target of Al toxicity. Using indirect immunofluorescence microscopy, microtubules and microfilaments in maize (Zea mays L.) roots were visualized and changes in their organization and stability correlated with the symptoms of Al toxicity. Growth studies showed that the site of Al toxicity was associated with the elongation zone. Within this region, Al resulted in a reorganization of microtubules in the inner cortex. However, the orientation of microtubules in the outer cortex and epidermis remained unchanged even after chronic symptoms of toxicity were manifest. Auxin-induced reorientation and cold-induced depolymerization of microtubules in the outer cortex were blocked by Al pretreatment. These results suggest that Al increased the stability of microtubules in these cells. The stabilizing effect of Al in the outer cortex coincided with growth inhibition. Reoriented microfilaments were also observed in Al-treated roots, and Al pretreatment minimized cytochalasin B-induced microfilament fragmentation. These data show that reorganization and stabilization of the cytoskeleton are closely associated with Al toxicity in maize roots.

Regulation of Actin Tension in Plant Cells by Kinases and Phosphatases1

Changes in the organization and mechanical properties of the actin network within plant and animal cells are primary responses to cell signaling. These changes are suggested to be mediated through the regulation of G/F-actin equilibria, alterations in the amount and/or type of actin-binding proteins, the binding of myosin to F-actin, and the formation of myosin filaments associated with F-actin. In the present communication, the cell optical displacement assay was used to investigate the role of phosphatases and kinases in modifying the tension and organization within the actin network of soybean cells. The results from these biophysical measurements suggest that: (a) calcium-regulated kinases and phosphatases are involved in the regulation of tension, (b) calcium transients induce changes in the tension and organization of the actin network through the stimulation of proteins containing calmodulin-like domains or calcium/calmodulin-dependent regulatory proteins, (c) myosin and/or actin cross-linking proteins may be the principal regulator(s) of tension within the actin network, and (d) these actin cross-linking proteins may be the principal targets of calcium-regulated kinases and phosphatases.

Spindle Dynamics and the Role of γ-Tubulin in Early Caenorhabditis elegans EmbryosV⃞

γ-Tubulin is a ubiquitous and highly conserved component of centrosomes in eukaryotic cells. Genetic and biochemical studies have demonstrated that γ-tubulin functions as part of a complex to nucleate microtubule polymerization from centrosomes. We show that, as in other organisms, Caenorhabditis elegans γ-tubulin is concentrated in centrosomes. To study centrosome dynamics in embryos, we generated transgenic worms that express GFP::γ-tubulin or GFP::β-tubulin in the maternal germ line and early embryos. Multiphoton microscopy of embryos produced by these worms revealed the time course of daughter centrosome appearance and growth and the differential behavior of centrosomes destined for germ line and somatic blastomeres. To study the role of γ-tubulin in nucleation and organization of spindle microtubules, we used RNA interference (RNAi) to deplete C. elegans embryos of γ-tubulin. γ-Tubulin (RNAi) embryos failed in chromosome segregation, but surprisingly, they contained extensive microtubule arrays. Moderately affected embryos contained bipolar spindles with dense and long astral microtubule arrays but with poorly organized kinetochore and interpolar microtubules. Severely affected embryos contained collapsed spindles with numerous long astral microtubules. Our results suggest that γ-tubulin is not absolutely required for microtubule nucleation in C. elegans but is required for the normal organization and function of kinetochore and interpolar microtubules.

Contact interactions between epitheliocytes and fibroblasts: Formation of heterotypic cadherin-containing adhesion sites is accompanied by local cytoskeletal reorganization

Contact interactions between different cell types play a number of important roles in development, for example in cell sorting, tissue organization, and ordered migration of cells. The nature of such heterocellular interactions, in contrast to interactions between cells of the same type, remains largely unknown. In this report, we present experimental data examining the dynamics of heterocellular interactions between epitheliocytes and fibroblasts, which express different cadherin cell adhesion molecules and possess different actin cytoskeletal organizations. Our analysis revealed two striking features of heterocellular contact. First, the active free edge of an epitheliocyte reorganizes its actin cytoskeleton after making contact with a fibroblast. Upon contact with the leading edge of a fibroblast, epitheliocytes disassemble their marginal bundle of actin filaments and reassemble actin filaments into a geometric organization more typical of a fibroblast lamella. Second, epitheliocytes and fibroblasts form cell–cell adhesion structures that have an irregular organization and are associated with components of cell adhesion complexes. The structural organization of these adhesions is more closely related to the type of contacts formed between fibroblasts rather than to those between epitheliocytes. Heterotypic epithelio-fibroblastic contacts, like homotypic contacts between fibroblasts, are transient and do not lead to formation of stable contact interactions. We suggest that heterocellular contact interactions in culture may be regarded as models of how tissue systems consisting of epithelia and mesenchyme interact and become organized in vivo.