Anni Vedeler

The characterization of free, cytoskeletal and membrane-bound polysomes in Krebs II ascites and 3T3 cells.

SummaryPolysomes from Krebs II ascites and 3T3 cells were separated into three populations by using a sequential extraction method. Free polysomes were released by using a combination of low salt (25 mM KCl) and NP-40 detergent in the lysis buffer. The cytoskeletal bound polysomes were subsequently released by raising the salt concentration to 130 mM and finally, polysomes bound to the membranes of the endoplasmic reticulum were extracted by the combined treatment with Triton X-100 and deoxycholate. The results presented here illustrate that the three polysome-containing fractions differ in many parameters such as polysome profiles, cytoskeletal components and phospholipid content. When polyA-containing mRNA was isolated from the three polysome fractions and translated in an in vitro system, some differences were observed in the patterns of proteins being synthesized.

Annexin A2 binds to the localization signal in the 3′ untranslated region of c‐myc mRNA

Messenger RNA trafficking, which provides a mechanism for local protein synthesis, is dependent on cis‐acting sequences in the 3′ untranslated regions (3′UTRs) of the mRNAs concerned acting together with trans‐acting proteins. The C‐MYC transcription factor is a proto‐oncogene product involved in cell proliferation, differentiation and apoptosis. Localization of c‐myc mRNA to the perinuclear cytoplasm and its association with the cytoskeleton is determined by a signal in the 3′UTR. Here we show the specific binding of a trans‐acting factor to the perinuclear localization element in the 3′UTR of c‐myc mRNA and identify this protein as annexin A2. Gel retardation and UV cross‐linking experiments showed that proteins in fibroblast extracts formed complexes with the region of c‐myc 3′UTR implicated in localization; a protein of ≈ 36 kDa exhibited specific, Ca2+‐dependent binding. Binding was reduced by introduction of a mutation that abrogates localization. Using RNA‐affinity columns followed by gel electrophoresis and mass spectrometry this protein was identified as annexin A2. The RNA–protein complex formed by cell extracts was further retarded by anti‐(annexin A2). Purified annexin A2 bound to the same region of the c‐myc 3′UTR but binding was reduced by introduction of a mutation, as with cell extracts. It is proposed that binding of annexin A2 to the localization signal in the c‐myc mRNA leads to association with the cytoskeleton and perinuclear localization. The data indicate a novel functional role for the RNA‐binding properties of annexin A2 in perinuclear localization of mRNA and the association with the cytoskeleton.

Insulin and step-up conditions cause a redistribution of polysomes among free, cytoskeletal-bound and membrane-bound fractions in krebs II ascites cells

From 30 min to 1h of step-up conditions there was a redistribution of polysomes between free, cytoskeletal-bound and membrane-bound fractions such that more polysomes were recovered bound to the cytoskeleton and less in the free fraction. After 1h incubation with insulin there was a higher proportion of polysomes in the cytoskeletal fraction with a decrease occurring in the membrane-bound fraction. At 2h little change was observed in the presence of insulin while a large increase occurred in the cytoskeletal-bound fraction and a decrease in membrane-bound polysomes was seen in cells incubated in the absence of insulin. The results indicate that the proportions of polysomes in the three different fractions can be modulated by physiological stimuli, such as media replenishment and insulin.

Ubiquitinated annexin A2 is enriched in the cytoskeleton fraction

Annexin A2 is a multifunctional protein and its cellular functions are regulated by post‐translational modifications and ligand binding. When purified from porcine intestinal mucosa and transformed mouse Krebs II cells, SDS–PAGE revealed high‐molecular‐mass forms in addition to the 36 kDa protomer. These forms were identified as poly‐/multi‐ubiquitin conjugates of annexin A2, and ubiquitination represents a novel post‐translational modification of this protein. Subcellular fractionation of mouse Krebs II cells revealed an enrichment of annexin A2‐ubiquitin conjugates in the Triton X‐100 resistant cytoskeleton fraction, suggesting that ubiquitinated annexin A2 may have a role associated with its function as an actin‐binding protein.

The Sequestration of mRNA in the Cytoskeleton and Other Subcellular Structures

Publisher Summary This chapter reviews the subcellular compartmentalization of mRNAs. Before release from the nucleus, mRNA molecules are subjected to posttranscriptional processing: capping, polyadenylation, and splicing. Transport of mRNA from the nucleus to the cytoplasm is a heterogenous mechanism that involves several proteins and factors that influence these proteins. After their transport into the cytoplasm, mRNA molecules were sequestered into two compartments of polysomes: cytosolic or free polysomes and those associated with the rough endoplasmic reticulum (ER)—that is, membrane-bound polysomes. Several studies from different and specialized biological systems have indicated that mRNA and ribosomes are orientated with cellular components in a specific manner. The presence of specific mRNAs in cytoskeletal-bound polysomes requires a mechanism that directs the RNA species to the cytoskeletal compartment rather than to the ER or the cytoplasmic (“free”) compartment. The translational system in eukaryotic mitochondria has many unique characteristics that clearly distinguish it from the protein synthetic apparatus present in the cytoplasm of both prokaryotic and eukaryotic cells.

Nucleotide binding to elongation factor 2 inactivated by diphtheria toxin

Binding of guanosine nucleotides to purified native and ADP‐ribosylated wheat germ EF‐2 was measured. Both forms of EF‐2 bound [3H]GDP to the same extent. [3H]GDP binding to native but not to ADP‐ribosylated EF‐2 was reduced in the presence of GTP and ribosomes. Binding of [γ‐32P]GTP to EF‐2 was significantly reduced upon ADP‐ribosylation. ADP‐ribosylation almost abolished both the stimulatory effect of ribosomes on GTP binding to EF‐2 and the ability of EF‐2 to form a high‐affinity complex with GuoPP(CH2)P and ribosomes. Low‐affinity complex formation between EF‐2 · GDP and ribosomes was not influenced by ADP‐ribosylation. The results indicate that the inhibition of the elongation process caused by the toxin is probably due to the inability of modified EF‐2 to exchange GDP with GTP.

Insulin induces changes in the subcellular distribution of actin and 5′-nucleotidase

SummaryAn increase in the amount of actin associated with the plasma membrane was visualized by immunocytochemistry 5 min after the addition of insulin to Krebs II ascites tumour cells maintained in serum-free medium. At 1 h of incubation the rim of fluorescence at the plasma membrane as measured by image analysis, was about 30% more intense than in control cells indicating that the initial accumulation of actin at the plasma membrane was not of a transient nature. Since an increase in the total cellular actin content in ascites cells did not occur until after a lag period of about 15 min then the increased amount of actin at the plasma membrane seen at 5 min was attributed to a stimulation of the polymerization of actin. An increase in the association of actin at the plasma membrane was also observed in 3T3 fibroblasts in areas of membrane ruffling, while in some cells there was also increased actin accumulation in the perinuclear area. The putative plasma membrane-microfilament linking protein 5′-nucleotidase was shown to be present in association with actin in the cytoskeletal fraction. Incubation of cells with insulin resulted in a shift of the enzyme toward the bottom of gradients indicating association with actin filaments of a greater length. The results demonstrate that insulin causes a stimulation of actin polymerization and that the hormone can be therefore assigned a role in the regulation of the cytoskeleton.

Changes in distribution of actin mRNA in different polysome fractions following stimulation of MPC-11 cells.

Individual mRNA species have been shown to differ both with respect to localization in the cell, and in their distribution upon stimulation of cells with different signals. In this study we have examined the distribution of actin mRNA in the free, cytoskeletal-bound, and membrane-bound RNA fractions, both in starved cells, and in response to stimulation by feeding. These results were then compared with mRNAs for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and histone H4. The results we obtained showed that actin mRNA was located in the free RNA fraction in starved cells, while upon stimulation it was located both in the free, and in the cytoskeletal fraction; no redistribution of GAPDH mRNA occurred between the three RNA fractions, while H4 mRNA showed a different localization upon stimulation. Incubation with the drugs actinomycin-D and cycloheximide showed that an altered localization of actin mRNA from free in starved cells to free and cytoskeletal mRNA fractions following stimulation, was dependent on RNA synthesis, and not on protein synthesis.

Morphological changes in Krebs II ascites tumour cells induced by insulin are associated with differences in protein composition and altered amounts of free, cytoskeletal-bound and membrane-bound polysomes

A three-step sequential detergent/salt extraction procedure was used in order to isolate three distinct subcellular fractions containing free (FP), cytoskeletal-bound (CBP) and membrane-bound polysomes (MBP), respectively, from Krebs II ascites cells (Vedeler et al., Mol Cell Biochem 100: 183–193,1991). The purpose was to study changes in the distribution of polysomes in these three fractions during long-term incubation with insulin under either stationary conditions or in roller suspension culture- Insulin caused a redistribution of polysomes between FP, CBP and MBP fractions. The hormone appeared to promote an entry of ribosomes into polysomes both in CBP and MBP populations. When cells were grown in stationary culture in the presence of insulin and thus promoted to attach to the substratum and undergo morphological changes, a diversion of ribosomes from CBP into MBP was observed. The level of protein synthesis was apparently very high in this latter fraction since more then 70% of ribosomes were in polysomes. Morphological changes observed following insulin treatment were accompanied by a shift of certain proteins among subcellular fractions (for example actin and p35). The fibronectin content was about 20% higher in attached compared to non-attached cells. The results suggest that morphological changes induced by stimulation with insulin are associated with an increased activity of MBP, presumably reflecting a requirement for an increased synthesis of membrane proteins. (Mol Cell Biochem 118: 131–140, 1992)

Ribosomal proteins sustain morphology, function and phenotype in acute myeloid leukemia blasts

Translation of mRNA is a prerequisite for cell proliferation, differentiation and viability. We have studied the effect of ribosome protein factors (GPRE) on acute myeloid leukemia (AML) blast cells. Ribosomes were isolated from MPC-11 cells using ultra-centrifugation. GPRE were extracted using a high KCl procedure. Blast cells from six AML patients were grown in suspension cultures for 24 and 96 h. GPRE or granulocyte macrophage-colony stimulating factor (GM-CSF) were added at the start of the incubation. GPRE, but not GM-CSF, prevented chromatin condensation and fragmentation of blast cell nuclei in AML-M2, -M4 and -M5 and the loss of nucleoli in AML-M2 and -M5. The fraction of phagocytosing blast cells in AML-M1, -M2, -M4 and -M5 was increased by GPRE. GPRE stimulated opsonin-dependent and -independent attachment and internalisation of N. meningitidis. GPRE increased the fraction of blasts expressing CD11b and CD32 in AML-M2 and -M5. GPRE diminished the fraction of AML-M5 cells bearing CD35 and CD32. GPRE also decreased the fraction of CD11c-bearing AML-M2 and -M5 cells. GM-CSF potentiated effects of GPRE in AML-M1, -M2, -M4 and -M5. GPRE and GM-CSF in combination affected phagocytosis and surface antigen expression in blast cells that were not influenced by either factor alone. Neither GPRE nor GM-CSF induced terminal differentiation or DNA-synthesis. We conclude that GPRE affects AML blast cell morphology, function and surface molecule expression, possibly by inhibiting apoptosis. The effects of GPRE may be mediated by ribosomal proteins that regulate translation and modulate the subcellular distribution of mRNA species.