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PubMed2001

Genetic interaction between yeast Saccharomyces cerevisiae release factors and the decoding region of 18 S rRNA.

Functional and structural similarities between tRNA and eukaryotic class 1 release factors (eRF1) described previously, provide evidence for the molecular mimicry concept. This concept is supported here by the demonstration of a genetic interaction between eRF1 and the decoding region of the ribosomal RNA, the site of tRNA-mRNA interaction. We show that the conditional lethality caused by a mutation in domain 1 of yeast eRF1 (P86A), that mimics the tRNA anticodon stem-loop, is rescued by compensatory mutations A1491G (rdn15) and U1495C (hyg1) in helix 44 of the decoding region and by U912C (rdn4) and G886A (rdn8) mutations in helix 27 of the 18 S rRNA. The rdn15 mutation creates a C1409-G1491 base-pair in yeast rRNA that is analogous to that in prokaryotic rRNA known to be important for high-affinity paromomycin binding to the ribosome. Indeed, rdn15 makes yeast cells extremely sensitive to paromomycin, indicating that the natural high resistance of the yeast ribosome to paromomycin is, in large part, due to the absence of the 1409-1491 base-pair. The rdn15 and hyg1 mutations also partially compensate for inactivation of the eukaryotic release factor 3 (eRF3) resulting from the formation of the [PSI+] prion, a self-reproducible termination-deficient conformation of eRF3. However, rdn15, but not hyg1, rescues the conditional cell lethality caused by a GTPase domain mutation (R419G) in eRF3. Other antisuppressor rRNA mutations, rdn2(G517A), rdn1T(C1054T) and rdn12A(C526A), strongly inhibit [PSI+]-mediated stop codon read-through but do not cure cells of the [PSI+] prion. Interestingly, cells bearing hyg1 seem to enable [PSI+] strains to accumulate larger Sup35p aggregates upon Sup35p overproduction, suggesting a lower toxicity of overproduced Sup35p when the termination defect, caused by [PSI+], is partly relieved.

Anti-Bacterial Agents↗
PubMed1994

Role of topoisomerase II in the structural and functional evolution of mitogen-stimulated lymphocyte nuclei.

To examine the role of DNA topoisomerase II (Topo II) in the mitogenic activation of mouse lymphocytes, we applied the Topo II inhibitor VM26 throughout the stimulation period and monitored morphological and functional parameters of lymphocyte activation. Cell viability and the usual increase in cell size were little affected at doses between 0.05 and 0.5 microM. DNA synthesis, however, was already significantly inhibited at 0.05 microM, with RNA synthesis inhibited to a lesser extent. Light microscope autoradiography showed that a smaller proportion of cells entered S phase, with each S phase cell incorporating less [3H]thymidine. In immunofluorescence studies, the nucleolar antigen fibrillarin was reduced, although only minor effects on the snRNP Sm antigen and the internal component labeled by antibody PI1 were observed. At the electron microscope level, nucleoli were remodeled and chromatin became aggregated. At a high dose of VM26 (5 microM), cells showed the expected high levels of apoptosis and strong inhibition in all activation parameters assayed. The results support the hypothesis that the Topo II beta isoform is involved in the very early phases of lymphocyte activation, with function of the Topo II alpha isoform, which is more sensitive to VM26, being required for progression through S phase.

Animals↗
PubMed2003

Approaches for a sustainable use of the bioactive potential in sponges: analysis of gene clusters, differential display of mRNA and DNA chips.

In recent years, analyses of the genome organization of marine sponges have begun that have led to the elucidation of selected genes and gene arrangements that exist in gene clusters (e.g. the receptor tyrosine kinase cluster and the allograft inflammatory factor cluster). Most of these studies were performed with the demosponge Suberites domuncula; but Geodia cydonium (Demospongiae), Aphrocallistes vastus (Hexactinellida) and Sycon raphanus (Calcarea) were also investigated. Both S. domuncula and G. cydonium possess a surprisingly large genome of approximately 1.7 pg DNA per haploid set. Taking the high gene density in these sponges into account and considering that predominantly single-copy DNA exists, the gene number of S. domuncula and G. cydonium was estimated to be approximately 300,000. Presumably, the large gene number in the sponge genome is due to regional gene duplication; so far evidence for a transposition in sponges has been presented. Data indicate that only 0.25 % of the total sponge genome comprises CA/TG microsatellites, and until now also no SINEs/transposable elements have been identified. Due to the rapid progress in the field of molecular biology of sponges the application of sponge genes for expression studies by DNA-array techniques (microarray) has become possible. These achievements will be further supported by the systematic analysis of the expressed genome of sponges; the results will be (partially) released (http://spongebase.uni-mainz.de/cgi-bin/blast/blastserver.cgi). In our efforts employing the results from the analysis of the genome to molecular biotechnology, we applied the technique of differential display of mRNA. One example, the effect of silicate on gene expression in S. domuncula, is outlined here. Future results will allow the identification of the genes involved in the synthesis of bioactive compounds from sponges [Porifera]. This progress will contribute considerably to a fruitful and fast development in the field of molecular marine biotechnology.

Amino Acid Sequence↗
PubMed1991

Effect of alanine supply on hepatic protein synthesis in animals maintained on a protein free diet.

In contrast to what it is observed during starvation, animals maintained on a protein-free isocaloric diet showed an increase in the rate of hepatic peptide chain elongation as determined by measuring the ribosomal transit time in vivo. The loss of body nitrogen per se is insufficient to generate the signal(s) which arrests hepatic peptide chain elongation. This observation suggests that it is an increase in gluconeogenic demand, and not the negative nitrogen balance, which is implicated in determining reciprocal changes in the rate of protein synthesis. The rate of protein synthesis, as expressed per mg of DNA, does not change in protein deprived animals, while the RNA to DNA ratio decreased. These data also agree with a higher ribosomal efficiency at the elongation step. The animals maintained on a protein-free diet have a decreased hepatic content of protein and an increased concentration of valine, indicating an increased proteolysis. The enhanced rate of polypeptide elongation observed in animals kept on a protein-free diet was accompanied by decreases in the state of aggregation of polyribosomes and in the ability of liver extracts to form eIF-2 catalyzed ternary complexes. These observations suggest that the activity of the hepatic initiation factor in vivo may not be rate limiting. The administration of alanine in vivo to animals maintained on a protein-free diet showed a preferential effect in reaggregating polyribosomes. This action was neither accompanied by detectable effects on the rate of eIF-2 catalyzed ternary complexes formation nor by significant changes in the rate of elongation.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1984

Nonaqueous fractionation of HeLa cells in glycols.

A further modification of Behrens ' method of nonaqueous cell fractionation, using glycols as media for homogenization and centrifugation, was presented. HeLa cells were frozen in melting Freon-12 ( CCl2F2 ), dried under vacuum at -30 degrees C, sonicated in hexylene glycol at -35 degrees C, and centrifuged through either propylene glycol or a mixture of the two glycols at -40 degrees C. The centrifugation yielded a nuclear pellet and a cytoplasmic supernatant. The supernatant was recentrifuged at -10 degrees C, yielding a cytoplasmic pellet. The success of the method depended on the temperature-dependent viscosities of the glycols and on the aggregation properties of cell structures in cold glycols. The allowed ranges of low temperatures were critical but not difficult to use; methods are given for sonication and for centrifugation. The two pellet fractions together contained 90% or more of cellular proteins and nucleic acids. Distribution of [3H]uridine-labeled nucleic acids showed that the first pellet (nuclei) contained over 95% of the nuclear markers, DNA, and ribosomal RNA precursors, plus about 10% of the cytoplasmic marker, 18 S ribosomal RNA. The cytoplasmic pellet contained less than 5% of the nuclear markers. Two enzyme activities were tested; DNA polymerase, a mostly soluble nuclear marker frequently eluted in aqueous fractionation, and lactate dehydrogenase, a cytoplasmic marker. The two enzymes each lost activity in propylene glycol but not in a mixture of 90% hexylene glycol and 10% propylene glycol, so the glycol mixture was used as a centrifugation medium when studying enzymes. The glycol mixture sometimes gave more cytoplasmic material, up to 20% of the 18 S ribosomal RNA, in the nuclear pellet. The fractionation showed, as expected, that DNA polymerase activity was 95% nuclear and lactate dehydrogenase activity was more than 68% cytoplasmic. The concentration of cytoplasmic material afforded by the glycol method allowed the detection of a small amount (approx. 5%) of DNA polymerase activity not associated with nuclei. The chief reason for use of the glycol method instead of other methods of cell fractionation is that easily solubilized cellular material can be recovered in concentrated pellet form in the appropriate nuclear or cytoplasmic fraction.

Cell Fractionation↗
PubMed2006

Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway.

It has been thought that early inner cell mass (ICM) is a homogeneous population and that cell position in the ICM leads to the formation of two lineages, epiblast (EPI) and primitive endoderm (PE), by E4.5. Here, however, we show that the ICM at E3.5 is already heterogeneous. The EPI- and PE-specific transcription factors, Nanog and Gata6, were expressed in the ICM in a random "salt and pepper" pattern, as early as E3.5, in a mutually exclusive manner. Lineage tracing showed predominant lineage restriction of single ICM cells at E3.5 to either lineage. In embryos lacking Grb2 where no PE forms, Gata6 expression was lost and all ICM cells were Nanog positive. We propose a model in which the ICM develops as a mosaic of EPI and PE progenitors at E3.5, dependent on Grb2-Ras-MAP kinase signaling, followed by later segregation of the progenitors into the appropriate cell layers.

Animals↗
PubMed2003

Localization of cartilage linking protein 1 during primary neurulation in the chick embryo.

Primary neurulation is a form-shaping event during the early development of the vertebrate embryo in which the neural plate is rolled up into the neural tube, the rudiment of the central nervous system. In an effort to identify genes specifically expressed in tissues lateral to the chick neural plate--tissues known to generate extrinsic forces for primary neurulation--we designed a subtractive scheme and identified a positive clone as the gene encoding chick cartilage linking protein 1 (CRTL1). CRTL1 (also known as link protein) is a small glycoprotein of the extracellular matrix that was originally identified for its role in stabilizing aggregates of aggrecan and hyaluronan in cartilage. In addition to being expressed in cartilage, CRTL1 is also immunolocalized in several noncartilaginous tissues as assessed with the 4B6 monoclonal antibody. Using the 4B6 antibody and the G9 riboprobe derived from our subtraction, we report the detailed distribution of CRTL1 protein and crtl1 transcripts during primary neurulation in chick embryos. This report emphasizes and briefly discusses important differences between the RNA expression pattern and the domains of accumulation of the protein. CRTL1 prominently accumulates in the basal lamina of the epidermal ectoderm just lateral to the neural plate. Based on the crucial role of the interface between this tissue and the neuroepithelium in the formation of the neural folds, and because of the biophysical role of hyaluronan in tissue morphogenesis, we propose that crtl1 represents is an excellent candidate neurulation gene, worthy of further study.

Animals↗
PubMed2003

Bernard-soulier syndrome with a homozygous 13 base pair deletion in the signal peptide-coding region of the platelet glycoprotein Ib(beta) gene.

We report a family with Bernard-Soulier syndrome with a homozygous mutation within the GPIb(beta) gene. The proband was a 24-year-old Japanese male who has suffered from life-long bleeding tendency. The patient's sister also had severe bleeding episodes. The proband and the affected sister had no apparent complications including organic or skeletal anomaly, or mental disturbance. They had thrombocytopenia [(35-40) x 10(9)/l] with giant platelets. In addition to platelet size, electron microscopic analysis revealed abnormalities in the internal structures of platelets. Ristocetin-induced platelet aggregation was defective. Flow cytometric analysis and western blot analysis showed that glycoprotein IX was nearly absent in platelets, whereas GPIb(alpha) and GPV were detectable. Genetic studies revealed a 13 base pair deletion in the signal peptide-coding sequence of GPIb(beta). The deletion would cause a frame-shift, resulting in the appearance of a stop codon following an indifferent polypeptide sequence. Analysis of platelet RNA showed that the mutant GPIb(beta) gene was transcribed. The propositus and his affected sister were homozygous for the deletion, whereas their unaffected father and mother were heterozygotes. The molecular defects of this family would help understand the relevance of GPIb(beta) for complex formation of the glycoprotein Ib/IX/V receptor.

Adult↗
PubMed2004

Inhibition of MUC4 expression suppresses pancreatic tumor cell growth and metastasis.

The MUC4 mucin is a high molecular weight membrane-bound glycoprotein. It is aberrantly expressed in pancreatic tumors and tumor cell lines with no detectable expression in the normal pancreas. A progressive increase of MUC4 expression has also been observed in pancreatic intraepithelial neoplasia, suggesting its association with disease development. Here, we investigated the consequences of silencing MUC4 expression in an aggressive and highly metastatic pancreatic tumor cell line CD18/HPAF that expresses high levels of MUC4. The expression of MUC4 was down-regulated by the stable integration of a plasmid-construct expressing antisense-MUC4 RNA. A decrease in MUC4 expression, confirmed by Western blot and immunofluorescence analyses, resulted in diminished growth and clonogenic ability of antisense-MUC4-transfected (EIAS19) cells compared with parental, empty vector (ZEO) and sense transfected (ES6) control cells. In addition, EIAS19 cells displayed a significant decrease in tumor growth and metastatic properties when transplanted orthotopically into the immunodeficient mice. In vitro biological assays for motility, adhesion, and aggregation demonstrated a 3-fold decrease in motility of EIAS19 cells compared with control cells, whereas these cells adhered more and showed an increase in cellular aggregation. Interestingly, MUC4 down-regulation also correlated with the reduced expression of its putative interacting partner, HER2/neu, in antisense-MUC4-transfected cells. In conclusion, the present work demonstrates, for the first time, a direct association of the MUC4 mucin with the metastatic pancreatic cancer phenotype and provides experimental evidence for a functional role of MUC4 in altered growth and behavioral properties of the tumor cell.

Animals↗
PubMed2004

The antimetastatic role of thrombomodulin expression in islet cell-derived tumors and its diagnostic value.

Islet cell tumors, endocrine neoplasm arising from pancreatic islets of Langerhans, are histologically difficult to diagnose as benign or malignant. Molecular markers are associated with the clinical characteristics that most of insulinoma are usually benign tumors, whereas other islet cell tumors are malignant but have not been identified. In this context, we newly found that an endothelial anticoagulant thrombomodulin was expressed in the normal islet beta cells and insulinoma, but not of other islet components or noninsulinoma islet cell tumors. Clinically, all of the subjects (n=15) of the insulinoma group showed no metastasis together with thrombomodulin expression in the lesions, whereas the other islet cell tumor groups showed a high incidence of metastasis (82%) and a low expression rate of thrombomodulin (6%). To examine the functional role of thrombomodulin, especially regarding the clinical characteristics of islet cell tumors, we tested the effect of exogenous thrombomodulin overexpression on cell adhesiveness and proliferation using MIN6 insulinoma cell line. In cell-based experiments, thrombomodulin overexpression reduced cell proliferation and enhanced Ca2+-independent cell aggregation, possibly through direct interaction with neural cell adhesion molecule. Taken together, these results are suggesting that thrombomodulin may act as antimetastatic molecule of insulinomas. In addition, thrombomodulin is a clinically useful molecular marker not only for identifying beta-cell-origin islet cell tumors (i.e., insulinomas) but also for predicting disease prognosis of islet cell tumors.

Adolescent↗
PubMed1999

Ecotropic viral integration site-1 is activated during, and is sufficient for, neuroectodermal P19 cell differentiation.

Expression of the ecotropic viral integration site-1 (Evi1) proto-oncogene during murine embryonal development is observed by in situ hybridization in primary head folds and neural crest-derived cells associated with the peripheral nervous system and embryonic mesoderm. To elucidate whether expression of Evi1 is involved in early neuroectodermal or mesodermal differentiation, we used murine embryonal carcinoma P19 cells as a model for the study of early embryonic differentiation. After retinoic acid (RA) treatment with aggregation, expression of Evi1 was detected during neural differentiation in P19 cells. However, Evi1 was not expressed in P19 cells during mesodermal differentiation after DMSO treatment with aggregation. Enforced expression of Evi1 in P19 cells induced neuron-specific microtubule-associated protein-2 microtubule-associated protein-2 and TrkA expression in the absence of RA under monolayer culture. After incubation with RA with aggregation, the Evi1 clones expressed microtubule-associated protein-2 continuously but did not express glial fibrillary acidic protein as an astrocyte marker protein until 12 days of culture. Thus, the overexpression of Evi1 leads to neural differentiation of P19 cells and blocks further differentiation into astrocytes by RA treatment, suggesting that Evi1 might be an important transcription factor for regulation of early neuroectodermal differentiation.

Animals↗
PubMed2006

Destabilization of the VCP-Ufd1-Npl4 complex is associated with decreased levels of ERAD substrates.

p97/VCP associated with Ufd1-Npl4 is considered a key player in ER-associated degradation (ERAD). RNA interference (RNAi) of one component of the Ufd1-Npl4 heterodimer destabilizes the VCP-Ufd1-Npl4 complex inducing proteasome-dependent degradation of the other component and releasing free VCP. In contrast to RNAi of VCP, RNAi of Ufd1 or Npl4 depleting approximately 90% of the VCP-Ufd1-Npl4 complexes does not induce unfolded protein response, indicating that the Ufd1-Npl4 dimer is not involved in the regulation of ER function by VCP. RNAi of Ufd1 or Npl4 is associated with a 2-fold increase in the levels of polyubiquitinated proteins, which form dispersed aggregates often associated with calnexin-positive structures. However, contrary to the effects of proteasome inhibition, RNAi of Ufd1 or Npl4 does not induce an accumulation of alpha-TCR and delta-CD3, two ERAD substrates overexpressed in HeLa cells. Instead, a 60-70% decrease in their levels is observed. The decrease in alpha-TCR levels is associated with a 50% decrease of its half-life. Upregulation of the putative channel forming protein, derlin-1, may contribute to the increased degradation of ERAD substrates. To explain our findings, we propose a model, where association of emerging ERAD substrates with VCP-Ufd1-Npl4 is not required for their degradation but has a regulatory role.

Adaptor Proteins, Vesicular Transport↗
PubMed1976

Urea-extractable protein from human epidermis.

The major protein fraction extracted from human epidermis by urea has the following characteristics: (1) A molecular weight of approximately 600 000. (2) A copper content of 0.03% giving a minimum molecular weight of 212 000. (3) A non-protein moiety indicated by (a) protein/dry weight ratio (b) anomalous ultraviolet absorption spectrum (c) high-voltage electrophoresis after mild alkaline or acid treatment (d) an excess of imidazole groups by Pauly reaction compared with the number of histidine residues by amino acid analysis. The non-protein component is not urocanic acid or RNA as has been reported (Bernstein, I.A. (1970), J. Soc. Cosmet, Chem. 21, 583-594 and Sibrack, L. A., Gray, R. H. and Bernstein, I. A. (1974), J. Invest. Dermatol. 62, 394-405). (4) The presence of interchain disulphide linkages indicated by polyacrylamide gel electrophoresis in sodium dodecyl sulphate with and without beta-mercaptoethanol. (5) Polyacrylamide gel electrophoresis in presence and absence of 6 M urea suggests that aggregates are broken down in urea.

Amino Acids↗
PubMed1992

Membrane inserted APP fragments containing the beta A4 sequence of Alzheimer's disease do not aggregate.

Previously we have shown that the COOH-terminal fragment (A4CT) of the Alzheimer amyloid protein precursor (APP), which at the NH2-terminus carries the sequence of the amyloid beta A4 protein, forms highly insoluble aggregates [EMBO J. (1988) 7, 949-957]. Here we report that aggregation is prevented if A4CT is expressed in vitro with a signal sequence at the NH2-terminus (SPA4CT) under conditions which allow membrane insertion. Aggregates from SPA4CT are obtained after removal of membranes by chloroform/methanol extraction or heating.

Alzheimer Disease↗
PubMed1995

Morphogenesis in memory formation: synaptic and cellular mechanisms.

We review some of the evidence for structural changes in synapses in response to environmental stimulation. These include changes in synapse number, in distribution of presynaptic vesicles, in synaptic bouton size, and complex changes in the shape and size of synaptic contact zones. Increased numbers of postsynaptic polyribosomal aggregates (PRA) are correlated histologically with developmental plasticity. We discuss the role that dendritically targeted mRNAs and polyribosomes might play in providing rapid, localized synthesis of proteins necessary for structural change. Using synaptoneurosomes, we have demonstrated that depolarization leads to a rapid (1-2 min) increase in PRA and in [35S]methionine incorporation into polypeptides. We have shown that this process is initiated by metabotropic glutamate receptors, which trigger phosphatidyl inositol hydrolysis, leading to release of internal Ca2+ stores and activation of protein kinase C. Entry of external Ca2+, however, seems to downregulate polyribosomal aggregation, via a calmodulin-dependent mechanism, suggesting that translation may be controlled by interaction of ionotropic receptors, voltage-dependent calcium channels, and metabotropic receptors.

Animals↗
PubMed2006

The chemokine receptor homologue encoded by US27 of human cytomegalovirus is heavily glycosylated and is present in infected human foreskin fibroblasts and enveloped virus particles.

Human cytomegalovirus (HCMV), a member of the beta-herpesvirus family, encodes four homologues of cellular G protein-coupled receptors (GPCRs). One of these, the protein product of HCMV open reading frame (ORF) UL33, has been identified in HCMV-infected cells and virus particles and shown to be heat-aggregatable and N-glycosylated. Another, the product of ORF US28, has been functionally characterized as a beta-chemokine receptor. Here we report the use of RT-PCR, coupled in vitro transcription-translation, immunoprecipitation, and Western immunoassays to (i) show that RNA from the open reading frame US27 appears predominantly during the late phase of replication; (ii) identify the protein encoded by HCMV US27 in infected cells and enveloped virus particles; (iii) demonstrate that the US27-encoded protein is heterogeneously N-glycosylated and resolves as two species following treatment with peptide N-glycosidase F; and (iv) show that both the recombinant and deglycoylated infected cell US27 protein aggregate when heated in the presence of SDS prior to electrophoresis in polyacrylamide gels, a property which is abrogated with the addition of urea to sample buffer.

Cells, Cultured↗
PubMed2004

Caveolin-1 interacts with 5-HT2A serotonin receptors and profoundly modulates the signaling of selected Galphaq-coupled protein receptors.

5-Hydroxytryptamine 2A (5-HT(2A)) serotonin receptors are important for a variety of functions including vascular smooth muscle contraction, platelet aggregation, and the modulation of perception, cognition, and emotion. In a search for 5-HT(2A) receptor-interacting proteins, we discovered that caveolin-1 (Cav-1), a scaffolding protein enriched in caveolae, complexes with 5-HT(2A) receptors in a number of cell types including C6 glioma cells, transfected HEK-293 cells, and rat brain synaptic membrane preparations. To address the functional significance of this interaction, we performed RNA interference-mediated knockdown of Cav-1 in C6 glioma cells, a cell type that endogenously expresses both 5-HT(2A) receptors and Cav-1. We discovered that the in vitro knockdown of Cav-1 in C6 glioma cells nearly abolished 5-HT(2A) receptor-mediated signal transduction as measured by calcium flux assays. RNA interference-mediated knockdown of Cav-1 also greatly attenuated endogenous Galpha(q)-coupled P2Y purinergic receptor-mediated signaling without altering the signaling of PAR-1 thrombin receptors. Cav-1 appeared to modulate 5-HT(2A) signaling by facilitating the interaction of 5-HT(2A) receptors with Galpha(q). These studies provide compelling evidence for a prominent role of Cav-1 in regulating the functional activity of not only 5-HT(2A) serotonin receptors but also selected Galpha(q)-coupled receptors.

Animals↗
PubMed2006

Alpha-crystallin expression affects microtubule assembly and prevents their aggregation.

The molecular chaperones alphaA- and alphaB-crystallins are important for cell survival and genomic stability and associate with the tubulin cytoskeleton. The mitotic spindle is abnormally assembled in a number of alphaA-/- and alphaB-/- lens epithelial cells. However, no report to date has studied the effect of alpha-crystallin expression on tubulin/microtubule assembly in lens epithelial cells. In the current work we tested the hypothesis that the absence of alphaA- and alphaB-crystallins alters microtubule assembly. Microtubules were reconstituted from freshly dissected explants of wild-type, alphaA-/-, alphaB-/-, and alpha(A/B) -/- (DKO) mouse lens epithelia and examined by electron microscopic and biochemical analyses. The wild-type microtubules were 4 mum long and approximately 25 nm wide and had a characteristic protofilament structure, but alphaB-/- microtubules were 2.5-fold longer. Microtubule-associated proteins (MAPs) extracted from microtubules by washing with salt included transketolase, alpha-enolase, and betaB2-crystallin. In DKO lens epithelial microtubules but not in wild-type, alphaA-/- or alphaB-/- microtubules, extraction of the MAPs gave very long (14-20 microm) "polyfilament" assemblies that were tightly bundled. Addition of exogenous alpha-crystallin (alphaA+ alphaB) was ineffective in preventing polyfilament formation. However, normal microtubule structure could be restored by including MAPs derived from wild-type lens epithelial cells during microtubule reconstitution. Intriguingly, these data suggest that alpha-crystallin may interact with MAPs to inhibit aggregation of microtubules in lens epithelial cells. Sedimentation analysis and 90 degrees light scattering measurements showed that alpha-crystallin suppressed tubulin assembly in vitro. Alpha-crystallin did not have a strong effect on the GTPase activity of purified tubulin. SDS-PAGE analysis showed that alpha-crystallin prevented heat-induced aggregation of tubulin, suggesting that alpha-crystallin may affect microtubule assembly by maintaining the pool of unassembled tubulin.

Animals↗