The cytoskeletal framework of sea urchin eggs and embryos: developmental changes in the association of messenger RNA

Extraction of sea urchin eggs and embryos with Triton X-100 generated a cytoskeletal framework (CSK) composed of a cortical filamentous network and an internal system of filaments associated with ribosomes. The CSK contained only 10-20% of the cellular protein, RNA, and lipid. A specific subset of proteins was enriched in the CSK. Several lines of evidence suggest that mRNA is a component of the CSK of both eggs and embryos. First, the CSK contained poly(A) sequences which hybridized with [3H]poly(U). Second, the CSK contained polyribosomes. Finally, RNA extracted from the CSK showed translational activity in an in vitro system. The nonhistone messages present in the CSK were qualitatively similar to those solubilized by detergent, as determined by separation on polyacrylamide gels of the products of in vitro translation. In the unfertilized egg, most mRNA was present as nonpolyribosomal messenger ribonucleoprotein complexes which, along with monoribosomes, were efficiently extracted by Triton X-100. The converse was found in blastulae, as most of the mRNA was present as polyribosomes associated with the CSK, although monoribosomes were still efficiently extracted by detergent. These results indicate a correlation between the activation of protein synthesis in eggs and the association of polyribosomes with the CSK.     

Preparation and properties of a complex from rat liver of polyribosomes with components of the cytoskeleton.

Gel filtration with 1% agarose (Bio-Gel A-150m) separates polyribosomes bound to microsomal membranes from 'free' polyribosomes when these fractions are prepared by standard centrifugal techniques. However, when polyribosomes contained in an unfractionated postmitochondrial supernatant are run on an identical column, over 90% of the total polyribosomes are present as aggregates, designated 'membrane-cytomatrix', which are eluted in the column void volume. Polyribosomes are not released from these aggregates on removal of microsomal phospholipids by treatment of postmitochondrial supernatant with 1% Triton X-100, a neutral detergent. The aggregates are disrupted by the usual ultracentrifugation techniques used in subcellular fractionation. After treatment of membrane-cytomatrix with Triton X-100 to remove phospholipids and membrane proteins, 58% of the polyribosomes still remain associated with protein-containing complexes in the form of a cytomatrix and are not 'free'. Preparations of both membrane-cytomatrix and cytomatrix are capable of sustained protein synthesis. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that the cytoskeletal proteins actin and myosin are present in the cytomatrix. Incubation of cytomatrix preparations with the actin-depolymerizing agent deoxyribonuclease I caused release of the polyribosomes. Polyribosome release by deoxyribonuclease I was prevented by prior incubation with phalloidin, which is known to stabilize F-actin. Thus polyribosomes are associated with cytoskeletal elements in rat liver, and this association is dependent on polymeric forms of actin.     

Desmin filaments are stably associated with the outer nuclear surface in chick myoblasts

Eukaryotic cells have highly organized, interconnected intracellular compartments. The nuclear surface and cytoplasmic cytoskeletal filaments represent compartments involved in such an association. Intermediate filaments are the major cytoskeletal elements in this association. Desmin is a muscle-specific structural protein and one of the earliest known muscle-specific genes to be expressed during cardiac and skeletal muscle development. Desmin filaments have been shown to be associated with the nuclear surface in the myogenic cell line C2C12. Previous studies have revealed that mice lacking desmin develop imperfect muscle, exhibiting the loss of nuclear shape and positioning. In the present work, we have analyzed the association between desmin filaments and the outer nuclear surface in nuclei isolated from pectoral skeletal muscle of chick embryos and in primary chick myogenic cell cultures by using immunofluorescence microscopy, negative staining, immunogold, and transmission electron microscopy. We show that desmin filaments remain firmly attached to the outer nuclear surface after the isolation of nuclei. Furthermore, positive localization of desmin persists after gentle washing of the nuclei with high ionic strength solutions. These data suggest that desmin intermediate filaments are stably and firmly connected to the outer nuclear surface in skeletal muscles cells in vivo and in vitro.     

Association of rapidly-labelled RNAs with actin in nuclear matrix from mouse L5178Y cells

More than 90% of rapidly-labelled nuclear RNA was associated with a nuclear matrix prepared from mouse leukemia L5178Y cells. The binding was not affected with up to 4 M NaCl; however, these RNAs were released from the nuclear matrix by treatment with a low ionic strength buffer (5 mM Tris-HCl buffer, pH 7.5, containing 1 mM ATP, 1 mM dithiothreitol, 0.2 mM ethylenediaminetetraacetic acid (EDTA) and 0.4 mM calcium chloride), without destruction of the sphere of the nuclear matrix. Actin filaments in the nuclear matrix were depolymerized with this buffer accompanied with rapidly-labelled RNAs. When the depolymerization was inhibited by slight modifications of the low ionic strength buffer (replacement of ATP by the same concentration of GTP; replacement of calcium ion by the same concentration of magnesium ion; addition of 20 micrograms/ml of phalloidine, which is a specific inhibitor of actin depolymerization), the release of rapidly-labelled RNAs from the nuclear matrix was also inhibited. The complex containing rapidly-labelled RNAs and matrix proteins was solubilized by a sonication from the nuclear matrix, and subjected to cesium chloride equilibrium centrifugation. Rapidly-labelled RNAs were concentrated on the bottom of the gradient accompanied with a small number of proteins (68K, 60K, 43K and 40K). The 43K protein was identified as actin by immunoblotting. By RNase digestion before equilibrium centrifugation, actin in the bottom fractions disappeared. These results suggest that rapidly-labelled RNAs anchor on the actin filaments in the nuclear matrix.     

Isolation of hamster brain polyribosomes-cytoskeleton complexes

We have developed a method for the isolation of a brain subcellular fraction enriched in both highly aggregated polyribosomes and cytoskeletal proteins. This method is based on gentle homogenization of brain tissue and low speed centrifugation. The mechanism of association of polyribosomes to cytoskeletal structures has been studied by in vitro treatment of this fraction with polyribosome-disaggregating agents. RNase and EDTA, while succeeding in completely disrupting them into monosomes or subunits, did not release them from cytoskeleton. Puromycin showed no noticeable effect.Abbreviations CSK cytoskeleton - DOC sodium deoxycholate - PR polyribosome - PRCSC polyribosomes-cytoskeleton complex     

The effects of human cytomegalovirus infection on cytoskeleton-associated polysomes

Human cytomegalovirus (HCMV) infection induces disruption of the host cell's cytoskeleton (CSK). This disruption is accompanied by three transient phases of actin depolymerization that occur at 20 min, 5 to 10 h and 48 to 72 h post infection (pi). During the 20 min peak of actin depolymerization, the level of cellular polysomes associated with the CSK was reduced, due to release of ribosomes from CSK-associated polysomes. Cellular mRNAs previously existing in these polysomes, however, remained associated with the CSK. Also during this period, nuclear to cytoplasmic transport of host cellular mRNA as well as the association of newly synthesized mRNA with the CSK was temporarily delayed. By 60 min pi, ribosomes, preexisting host cellular mRNA, and newly synthesized mRNAs (host and viral) had reestablished a distribution in the infected cell comparable to that of uninfected cells. Sedimentation profiles of soluble and CSK fractions at various times throughout the viral infection indicated that, although the amount of polysomes associated with the CSK at 20 min pi was reduced, essentially all HCMV and all host cell polysomes present were associated with the CSK. The majority of HCMV DNA hybridizable poly(A)+ RNAs were associated with the CSK throughout the viral infection. These early events appear to correlate with a transient interruption of host cellular mRNA translation early in infection and may represent a process whereby HCMV gene expression becomes competitive with that of the host cell.     

Interactions of cytoskeletal elements with the plasma membrane of Sarcoma 180 ascites tumor cells

The association of cytoskeletal proteins with cell surface envelopes from Sarcoma 180 ascites cells has been studied by several techniques previously used successfully in studying the interaction of spectrin with erythrocyte membranes. By electron microscopy the envelopes exhibit irregular exterior surfaces and the presence of substantial amounts of “fuzz” at the interior surface. Extraction of the envelopes at low ionic strength and alkaline pH fragments the membranes and depletes them of the “fuzz” with concomitant elution of four major polypeptides of mol. wt >300 000 (Band E), 250 000, 100 000 and 43 000 D. The last three of these have been tentatively identified as actin-binding protein (ABP), α-actinin and actin. Membrane-associated myosin is not eluted under these conditions. Neither actin nor myosin is eluted under conditions commonly used to depolymerize them. However, myosin can be eluted at high salt concentrations if the envelopes have been previously extracted and fragmented with alkaline buffer as above. Extraction of the envelopes with Triton X-100 removes 60% of the membrane lipid and 70–80% of lactoperoxidase-iodinated cell surface proteins without removal of significant amounts of the cytoskeletal proteins. The Triton residues maintain the shape of the original envelopes but have lost the trilaminar membrane structure. Proteolysis of intact envelopes with trypsin or papain cleaves the high molecular weight polypeptides in the order E > ABP > myosin. Fragmentation occurs with cleavage of E or ABP, but does not appear to require cleavage of myosin. Actin and α-actinin are not appreciably cleaved when associated with the membrane. The results, combined with previous observations, suggest an extensive complex of cytoskeletal proteins attached to the membrane interior surface.     

Isolation and properties of rat plasma lecithin-cholesterol acyltransferase

Lecithin-cholesterol acyltransferase was purified from rat plasma and the properties of this enzyme during the purification procedures and those of the purified enzyme were investigated in comparison with the human enzyme. The rat enzyme was not adsorbed on hydroxyapatite, which was employed for the purification of the human enzyme. When purified human enzyme was incubated at 37 degrees C in 0.1 mM phosphate buffer (pH 7.4; ionic strength, 0.00025), no alteration of enzyme activity was observed for up to 6 h. In the case of the rat enzyme, however, approximately 40% of the enzyme activity was lost under the same conditions. The human enzyme and rat enzyme were both retained on a Sepharose 4B column to which HDL3 was covalently linked, in 39 mM phosphate buffer, pH 7.4. Although the human enzyme was eluted from the column in 1 mM phosphate buffer, the rat enzyme was dissociated from the column at a lower buffer concentration (0.1 mM phosphate buffer). These findings indicate that the rat enzyme effectively associated with HDL3 in 39 mM phosphate buffer, pH 7.4, but the association was more sensitive to increase of ionic strength compared with that of the human enzyme.     

Cytoskeleton involvement in the distribution of mRNP complexes and small cytoplasmic RNAs

These studies were designed to determine whether small cytoplasmic RNAs and two different mRNAs (actin mRNA and histone H4 mRNA) were uniformly distributed among various subcellular compartments. The cytoplasm of HeLa S3 cells was fractionated into four RNA-containing compartments. The RNAs bound to the cytoskeleton were separated from those in the soluble cytoplasmic phase and each RNA fraction was further separated into those bound and those not bound to polyribosomes. The four cytoplasmic RNA fractions were analysed to determine which RNA species were present in each. The 7 S RNAs were found in all cytoplasmic fractions, as were the 5 S and 5.8 S ribosomal RNAs, while transfer RNA was found largely in the soluble fraction devoid of polysomes. On the other hand a group of prominent small cytoplasmic RNAs (scRNAs of 105-348 nucleotides) was isolated from the fraction devoid of polysomes but bound to the cytoskeleton. Actin mRNA was found only in polyribosomes bound to the cytoskeleton. This mRNA was released into the soluble phase by cytochalasin B treatment, suggesting a dependence upon actin filament integrity for cytoskeletal binding. A significant portion of several scRNAs was also released from the cytoskeleton by cytochalasin B treatment. Analysis of the spatial distribution of histone H4 mRNAs, however, revealed a more widely dispersed message. Although most (60%) of the H4 mRNA was associated with polyribosomes in the soluble phase, a significant amount was also recovered in both of the cytoskeleton bound fractions either associated or free of polyribosome interaction. Treatment with cytochalasin B suggested that only cytoskeleton bound, untranslated H4 mRNA was dependent upon the integrity of actin filaments for cytoskeletal binding.     

Messenger RNA is translated when associated with the cytoskeletal framework in normal and VSV-infected HeLa cells

When the cytoskeletal framework is prepared from suspension-grown HeLa by extraction with nonionic detergent, all the polyribosomes are associated with the framework while 80% of tRNA and the major portion of monoribosomes as well as 75% of the cell proteins are found in the soluble fraction. The mRNA of polyribosomes is bound to the cytoskeleton and these molecules remain attached even after polyribosomes are disassembled in vivo prior to extraction. Although all actively translating message molecules are attached to the framework, about one quarter of the poly(A)+ mRNA is free of the framework. The binding of message to the skeleton may be obligatory for translation. Upon infection with VSV, all the viral polyribosomes but not all the viral messages of the infected cell are associated with the cytoskeletal framework. Pulse-chase labeling shows that VSV messages initially associate with the framework and then later detach and cease translation. The mRNA for the viral glycoprotein (G), known to translate only on ribosomes bound to endoplasmic reticulum, is also retained by the detergent-extracted structure. It appears that the protein substructure of the endoplasmic reticulum which binds polyribosomes is a component of the cytoskeletal framework.     

Translation and the cytoskeleton: a mechanism for targeted protein synthesis

This review describes the critical evidence that in eukaryotic cells polyribosomes, mRNAs and components of the protein synthetic machinery are associated with the cytoskeleton. The role of microtubules, intermediate filaments and microfilaments are discussed; at present most evidence suggests that polyribosomes interact with the actin filaments. The use of non-ionic detergent/deoxycholate treatment in the isolation of cytoskeletal-bound polysomes is described and the conclusion reached that at low salt concentrations this leads to mixed preparations of polysomes derived from both the cytoskeleton and the endoplasmic reticulum. At present the best approach for isolation of cytoskeletal-bound polysomes appears to involve extraction with salt concentrations greater than 130 mM after an initial non-ionic detergent treatment. Such polysomes appear to be enriched in certain mRNAs and thus it is suggested that they are involved in translation of a unique set of proteins. The evidence for mRNA localisation is presented and the role of the cytoskeleton in transport and localisation of RNA discussed. Recent data on the role of the 3 untranslated region in the targeting of mRNAs both to particular regions of the cell and for translation on cytoskeletal-bound polysomes is described. The hypothesis is developed that the association of polysomes with the cytoskeleton is the basis of a mechanism for the targeting of mRNAs and the compartmentalization of protein synthesis.Abbreviations CBP cytoskeletal-bound polysomes - FP free polysomes - MBP membrane-bound polysomes - ER endoplasmic reticulum     

Ionic strength-dependent interaction of rat liver casein kinase I with plasma membrane

Casein kinase I binding to rat liver plasma membrane was rapidly released from membrane by increasing the ionic strength above physiological level. The released activities at 250-300 mM NaCl were 3-4-fold higher than those obtained under lower ionic strength below 100 mM NaCl. This reaction occurred nonenzymatically and was reversible. By lowering the ionic strength from 250 mM to 50 mM NaCl by dilution at least 50% of the released enzyme was rebound to plasma membrane. By gel filtration analysis, most of the released enzyme activity under higher NaCl concentration was recovered around the molecular mass of 35,000 which corresponded to that of casein kinase I. This enzyme showed the tendency to aggregate under lower ionic strength (50 mM NaCl), but existed as monomer under higher ionic strength (250 mM NaCl). These results suggest that the release of casein kinase I from plasma membrane and the rebinding to membrane induced by the alteration of ionic strength seem to be an important regulatory mechanism in determining the subcellular distribution of this enzyme.     

Influence of pH and ionic strength on formation and stability of emulsions containing oil droplets coated by beta-lactoglobulin-alginate interfaces

Emulsions of 0.1 wt % corn oil-in-water containing oil droplets coated by beta-lactoglobulin (0.009 wt % beta-Lg, 5 mM phosphate buffer, pH 7.0) were prepared in the absence and presence of sodium alginate (0 or 0.004 wt %). The pH (3-7) and ionic strength (0-250 mM NaCl) of these emulsions were adjusted, and the particle charge, particle size, and creaming stability were measured. Alginate adsorbed to the beta-Lg-coated droplets from pH 3 to 6, which was attributed to electrostatic attraction between the anionic polymer and cationic patches on the droplet surfaces. Droplets coated by beta-Lg-alginate had better stability to flocculation than those coated by beta-Lg alone, especially around the isoelectric point of the adsorbed proteins and at low ionic strengths (< 100 mM NaCl). At pH 5, alginate molecules desorbed from the droplet surfaces at high salt concentrations due to weakening of the electrostatic attraction.     

Vesicle release from rat red cell ghosts and increased association of cell membrane proteins with cytoskeletons induced by cadmium

When rat red cell ghosts were incubated with 0.1-0.5 mM CdCl2 in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, they became irregular in shape and released small vesicles. The release of vesicles was dependent on the incubation temperature and Cd2+ concentration. The maximum release occurred at 37 degrees C in the presence of 0.2 mM Cd2+. The protein composition of Cd2+-induced vesicles was similar to that of the vesicles released from ATP-depleted red cells. Upon incubation with 0.1-0.2 mM Cd2+, more than 90% of the Cd2+ added to the incubation buffer was recovered in ghosts and 15-20% of the ghost Cd2+ was located on the cytoskeletons prepared by washing ghosts with 0.5% Triton X-100 solution containing 0.1 M KCl and 10 mM Tris-HCl (pH 7.4). Moreover, the cytoskeletons prepared from Cd2+-treated ghosts markedly contained cell membrane proteins, bands 2.1, 3, 4.2 and 4.5, and glycophorins. The association of bands 3 and 4.2 with cytoskeletons increased with increasing concentrations of Cd2+ added to the incubation buffer and saturated at 0.2 mM Cd2+. The solubilization of cytoskeletal proteins, bands 1, 2 and 5, from ghosts at low ionic strength was almost completely suppressed by preincubation of ghosts with 0.1 mM Cd2+. HgCl2, PbCl2 and ZnCl2 at 0.2 mM each also produced an increased association of cell membrane proteins with cytoskeletons, whereas CaCl2 and MgCl2 did not.     

Actin in triton-treated cortical preparations of unfertilized and fertilized sea urchin eggs

Triton-treated cortical fragments of unfertilized and fertilized sea urchin eggs prepared in the presence of greater than or equal to 5 mM EGTA contain 15-30% of the total egg actin. However, actin filaments are not readily apparent by electron microscopy on the cortical fragments of unfertilized eggs but are numerous on those of fertilized eggs. The majority of the actin associated with cortical fragments of unfertilized eggs is solubilized by dialysis against a low ionic strength buffer at pH 7.5. This soluble actin preparation (less than 50% pure actin) does not form proper filaments in 0.1 M KCl and 3 mM MgCl2, whereas actin purified from this preparation does, as judged by electron microscopy. Optical diffraction analysis reveals that these purified actin filaments have helical parameters very similar to those of muscle actin. Furthermore, the properties of the purified actin with regard to activation of myosin ATPase are similar to those of actin from other cell types. The possibility that actin is maintained in a nonfilamentous form on the inner surface of the unfertilized egg plasma membrane and is induced to assemble upon fertilization is discussed.     

Small cytoplasmic RNAs and their location within the cytoplasm

These studies were designed to establish the location of various species of small RNAs within the subcellular cytoplasmic compartments. Four cytoplasmic RNA-containing compartments were examined: (A) cytoskeleton-bound polyribosomal ribonucleoprotein (RNP) complexes, (B) soluble-phase polyribosomal RNP complexes, (C) cytoskeleton-bound free RNP complexes, (D) soluble-phase free RNP complexes. The presence of the small cytoplasmic RNA (scRNA) population and histone H4 and actin mRNAs in each compartment was examined to determine their spatial distribution within the cytoplasm. The 7S signal recognition RNA and the 5S and 5.8S rRNAs were distributed among all four compartments, while 4S tRNAs were localized largely in fraction D. Fraction C contained a group of seven abundant scRNAs, of approximately 105-348 nucleotides in length, which were localized almost entirely within the cytoskeleton-bound free RNP compartment. Actin mRNAs were localized in fraction A, the actively translating cytoskeleton-bound compartment. Actin mRNAs were localized in fraction A, the actively translating cytoskeleton-bound compartment. Following cytochalasin B treatment, actin mRNAs were released into the soluble phase, implicating a dependence on the integrity of actin filaments in its binding. Such treatment also released several of the scRNAs from their cytoskeleton-bound location. In contrast histone H4 mRNAs were much more widely dispersed, being present in all four cytoskeletal compartments. Approximately 60% of the H4 mRNAs, however, were localized within the soluble-phase polyribosomes in fraction B. Cytochalasin B treatment released only the small portion of untranslated histone H4 mRNA associated with the cytoskeleton in fraction C, suggesting that the binding of these H4 mRNAs was dependent in some manner upon the integrity of actin filaments.     

Soluble respiratory syncytial virus fusion protein in the fully cleaved, pretriggered state is triggered by exposure to low-molarity buffer

The paramyxovirus fusion (F) glycoprotein is anchored in the virion membrane in a metastable, pretriggered form. Once triggered, the F protein undergoes a dramatic conformational extension that inserts its hydrophobic fusion peptide into the target cell membrane, then folds back on itself to bring the membranes together and initiate fusion. Unlike most other paramyxoviruses, the respiratory syncytial virus (RSV) F protein alone is sufficient to mediate membrane fusion and virus infection. To study the triggering mechanism of the RSV F protein, we have generated a soluble F (sF) protein by replacing the transmembrane and cytoplasmic tail domains with a 6His tag. The sF protein is secreted efficiently from 293T cells in a fully cleaved form. It is recognized by neutralizing monoclonal antibodies, appears spherical by electron microscopic analysis, and is not aggregated, all consistent with a native, pretriggered trimer. The sF protein was purified on a Ni(+2) column and eluted with 50 mM phosphate buffer containing 500 mM NaCl and 250 mM imidazole. Dialysis against 10 mM buffer caused the sF protein to trigger, forming "hat pin"-shaped molecules that aggregated as rosettes, characteristic of the posttriggered form. Further dialysis experiments indicated that the efficiency of triggering correlated well with the reduction of buffer molarity. Reduction of buffer molarity by dilution also resulted in exposure of the fusion peptide, as detected by liposome association, confirming sF protein triggering. Mutation of the furin cleavage site adjacent to the fusion peptide prevented liposome association, further confirming that association is via the fusion peptide.     

Heparan sulfate proteoglycans in the substratum adhesion sites of human neuroblastoma cells: modulation of affinity binding to fibronectin

Tissue culture substratum adhesion sites from EGTA-detached Platt human neuroblastoma cells were extracted with a buffer containing ocytlglucoside, NaCl, guanidine hydrochloride, and a variety of protease inhibitors, an extraction which resulted in quantitative solubilization of the 35SO4 = -radiolabeled proteoglycans and 3H-leucine-radiolabeled proteins. Of the sulfate-radiolabeled material, the vast majority was heparan sulfate proteoglycan (Kav = 0.15 on Sepharose C14B columns) and the remainder was chondroitin sulfate chains (no single chains of heparan sulfate were observed). This extract was then fractionated on DEAE-Sephadex columns under two different buffer elution conditions. Under DEAE-I conditions in low ionic strength acetate buffer, two major peaks of 35SO4 = -radiolabeled material (A,B) and a minor peak (C) could be resolved in the NaCl gradient; however, three-fourths of the material required 4 M guanidine hydrochloride to elute it from the column (peak D). Under DEAE-II conditions in acetate buffer supplemented with 8 M urea, the vast majority of the proteoglycan material could be eluted in the NaCl gradient as peak AB. Peak D material was shown to contain aggregated proteoglycan, along with nonproteoglycan protein, which high concentrations of urea or guanidine could dissociate, but not nonionic or zwitterionic detergents. Three different affinity chromatography systems were used to further characterize these components. Approximately 60% of peak A heparan sulfate proteoglycan from DEAE-I binds to the hydrophobic matrix, octyl-Sepharose, while 80% of the proteoglycan in DEAE-I peak D binds to this hydrophobic column. A sizable fraction of peak A proteoglycan fails to bind to plasma fibronectin but does bind to platelet factor-4 affinity columns. In contrast, peak AB proteoglycan from DEAE-II columns yields a much higher proportion of molecules which do bind to fibronectin. To examine the basis for these differences in affinity binding, nonproteoglycan protein from these adhesion sites was mixed with peak AB proteoglycan prior to affinity chromatography; proteoglycan binding to fibronectin decreased markedly while binding to platelet factor-4 was unaffected. This modulating activity involves the binding of nonproteoglycan protein in adhesion site extracts to both fibronectin on the column, as well as to heparan sulfate proteoglycan itself, and it could not be mimicked by a number of known proteins in adhesion site extracts or several other proteins. These results demonstrate selectivity and specificity in this modulation and indicate that a previously unidentified protein(s) is responsible.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fluorescent measurement of desmin intermediate filament assembly

Intermediate filaments (IF) are cytoskeletal elements that are believed to play a major role in the specification and maintenance of cell form. Although previously thought to be stable and static because of their relative insolubility in physiological solvents, IF have recently been shown to have dynamic properties not unlike those of other cytoskeletal elements. The methodology for measuring this dynamic behavior, however, has been mostly borrowed from studies of other filament proteins and are poorly suited to IF because of their unusual physicochemical properties. In this report we introduce a fluorescence assay for quantifying in vitro IF assembly. Desmin subunits labeled with iodoacetamidofluorescein (IAF) to approximately 0.4 mol/mol retain the ability to polymerize into filaments indistinguishable from unlabeled IF in the electron microscope. By spectrophotometry, however, up to 90% of the starting fluorescence is quenched upon maximal IF assembly from IAF-desmin subunits. This quench is proportional to the total concentration of desmin subunits and is a sensitive measure of the assembly process. The critical concentration of assembly, measured at 170 mM NaCl, 1 mM MgCl2, 10 mM Tris-HCl, pH 7.0, is 0.2 microM. This indicates that a significant level of unpolymerized desmin exists in steady-state equilibrium with polymerized filaments under these conditions and suggests that IF subunit-filament equilibria may play a role in cytoskeletal dynamics.     

Anion exchange purification of plasmid DNA using expanded bed adsorption

Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH=8.0) buffer at an upward flow of 300 cmh^-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh^-1. Purification factors of 36±1 fold, 26±0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed–values of 35±2 and 5±0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step.