Immunocytochemical study of endocytotic structures accumulated in HeLa cells transformed with a temperature-sensitive mutant of dynamin.

Dynamin is a 100-kD GTPase, which is required for clathrin-mediated endocytosis. Recent studies have revealed that dynamin is closely involved in clathrin-coated vesicle formation. In this study we investigated the ultrastructure of endocytotic structures accumulated in HeLa cells that were transformed with a temperature-sensitive (ts) mutant of dynamin to clarify which step was blocked in dynts cells. Endocytosis of transferrin receptors was restricted at the level of surface-connected membrane structures. Tubular and vesicular membrane invaginations were accumulated in the cells' peripheral regions, suggesting that the endocytosis was blocked just before the pinching-off steps in coated vesicle formation. The "collared" tubes, which were reported to be localized in nerve terminals in shibirets1 flies and GTPgammaS-treated synaptosomes, were not observed in the dynts cells even at nonpermissive temperature. The distribution pattern of dynamin in deeply invaginated coated pits in dynts cells was similar to that in dynwt cells but not to that in dynK44A cells, which are other endocytosis-defective mutant cells. These morphological data suggest that dynts blocked the pinching-off steps in clathrin-coated vesicle formation, which may be caused by a different mechanism from that of dynK44A cells.     

Elevated endosomal pH in HeLa cells overexpressing mutant dynamin can affect infection by pH-sensitive viruses

Many viruses gain access to the cell via the endosomal route and require low endosomal pH for infectivity. The GTPase dynamin is essential for clathrin-dependent endocytosis, and in HeLa cells overexpressing the nonfunctional dynamin^K44A mutant the formation of clathrin-coated vesicles is halted. HRV2, a human minor group rhinovirus, is internalized by members of the low-density lipoprotein receptor family in a clathrin-independent manner. The low endosomal pH then leads to conversion of the capsid to C-antigen, which is required for release (uncoating) and transfer of the viral RNA into the cytosol and de novo synthesis of infectious virus. We here demonstrate that overexpression of dynamin^K44A reduces this antigenic conversion and results in diminished viral synthesis. In contrast, lysosomal degradation is unaffected. The kinetics of the formation of C-antigen in vitro and in vivo suggest that the pH in endosomes is elevated by about 0.4 units upon overexpression of dynamin^K44A. As a consequence, HRV2 uncoating is diminished early after internalization but attains control levels upon prolonged internalization. Thus, overexpression of dynamin^K44A, in addition to trafficking defects, results in an elevated endosomal pH and thereby affects virus infection and most likely endosomal sorting and processing.     

Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells

We describe here the properties of a mutant of Chinese hamster ovary cells that expresses a conditional-lethal mutation affecting dense lysosomes. This mutant, termed V.24.1, is a member of the End4 complementation group of temperature-sensitive mutants selected for resistance to protein toxins (Colbaugh, P. A., C.-Y. Kao, S.-P. Shia, M. Stookey, and R. K. Draper. 1988. Somatic Cell Mol. Genet. 14:499-507). Vesicles present in postnuclear supernatants prepared from V.24.1 cells harvested at the restrictive temperature had a 50% reduction in acidification activity, assessed by the ATP-stimulated accumulation of the dye acridine orange in acidic vesicles. To investigate whether specific populations of vesicles were impaired in acidification, we measured acidification activity in three subcellular fractions prepared from Percoll gradients: one containing endosomal and Golgi markers, one containing buoyant lysosomes, and the third containing dense lysosomes. Activity in dense lysosomes was reduced by 90%, activity in the buoyant lysosome fraction was unaffected, and activity in the endosome-Golgi fraction was mildly reduced. The activity of three lysosomal enzymes--beta-hexosaminidase, beta-galactosidase, and beta-glucocerebrosidase--was also reduced in dense lysosomes but nearly normal in the buoyant lysosome fraction. However, beta-hexosaminidase and beta-glucocerebrosidase activity was increased two- to threefold in the endosome-Golgi fraction. We conclude that the lesion selectively impairs dense lysosomes but has little effect on properties of buoyant lysosomes.     

Dynamic instability of microtubules requires dynamin 2 and is impaired in a Charcot-Marie-Tooth mutant

Dynamin is a fission protein that participates in endocytic vesicle formation. Although dynamin was originally identified as a microtubule-binding protein, the physiological relevance of this function was unclear. Recently, mutations in the ubiquitously expressed dynamin 2 (dyn2) protein were found in patients with Charcot-Marie-Tooth (CMT) disease, which is an inherited peripheral neuropathy. In this study, we show that one of these mutations, 551Δ3, induces prominent decoration of microtubules with the mutant dyn2. Dyn2 was required for proper dynamic instability of microtubules, and this was impaired in cells expressing the 551Δ3 mutant, which showed a remarkable increase in microtubule acetylation, a marker of stable microtubules. Depletion of endogenous dyn2 with a small interfering RNA also resulted in the accumulation of stable microtubules. Furthermore, the formation of mature Golgi complexes, which depends on microtubule-dependent membrane transport, was impaired in both dyn2 knockdown cells and cells expressing the 551Δ3 mutant. Collectively, our results suggest that dyn2 regulates dynamic instability of microtubules, which is essential for organelle motility, and that this function may be impaired in CMT disease.     

Characterization of a temperature-sensitive mutant of BALB-c 3T3 cells

A temperature-sensitive mutant, designated ts-1, has been isolated from BALB/c 3T3 mouse cells; this is the first such mutant of the cell line to be reported. The mutant is similar to the original cell line in morphology, growth rate, and contact inhibition at the permissive temperature (33 °C). When ts-1 is studied at the non-permissive temperature (38-38.5 °C), however, cell division ceases. Incorporation of radioactive thymidine or uridine under conditions in which the amount of isotope incorporated reflects the amount of the appropriate macromolecule present in the culture shows gradual cessation corresponding to the inhibition of cell division. On the other hand, in pulse experiments, incorporation of radioactive thymidine or uridine continues, although at a diminished rate (50%). These results suggest substantial turnover of DNA and RNA at the non-permissive temperature.The cessation of growth of ts-1 upon shift to 38.5 °C is markedly dependent on the cell number at the time of shift. Experiments in which cell number is kept constant but initial area of inoculation or volume of medium are varied indicate that the cell number per unit area is most important. Though the biochemical basis for this ‘cell-cooperation’ is unknown, these results might explain the apparent low incidence of mutants recovered in our studies thus far.     

Potassium-induced reverse transformation of cells infected with a temperature-sensitive transformation mutant virus

High potassium concentrations altered the morphology and the ability to grow in soft agar in 6m2 cells, a clone of rat kidney cells infected with a temperature-sensitive mutant of Moloney sarcoma virus. Approximately 60% of cells exhibited normal morphology in the presence of 94.8 mM potassium in isotonic medium at the temperature permissive for transformation, whereas 100% were normal at 72 mM potassium in hypertonic media. A significant reduction of growth in soft agar was also induced with these conditions. However, the synthesis ratio of virus-specified transforming protein to marker viral protein was not altered. Na+K+-ATPase might play a role in this reverse-transformation process.     

Analysis of the protein glycosylation defect of a temperature-sensitive cell cycle mutant by the use of mutant cells overexpressing the human epidermal growth factor receptor after transfection of the gene

A temperature-sensitive mutant with a defect in glycoprotein synthesis and a cell cycle (G1)-specific arrest at the nonpermissive temperature (Tenner et al., J. Cell. Physiol., 90:145-160, 1977; Tenner and Scheffler, J. Cell. Physiol., 98:251-266, 1979) was investigated further after a human epidermal growth factor (EGF) receptor gene had been transfected and amplified in these cells. While a temperature shift-up lead to an immediate arrest in the biosynthesis of mature EGF receptor and its appearance on the plasma membrane, the observed turnover of the preexisting receptor was too slow to account for the arrest of DNA synthesis in these mutant cells. Tunicamycin could in fact mimic the effect of a temperature shift on the biosynthesis of EGF receptor, but it did not have the same rapid effect on DNA synthesis and cell cycle progression. These mutants have also been shown to induce a set of stress proteins or glucose-regulated proteins, GRPs (Lee et al., J. Cell. Physiol., 129:277-282, 1986). The question is addressed whether the defect in glycoprotein synthesis is the primary defect and a possible cause of the induction of the GRPs, or whether a more basic defect at the level of the endoplasmic reticulum (ER) is responsible for the complex phenotype of the mutant. Our results argue in favor of a primary defect which indirectly affects N-linked glycosylation of proteins, as well as several other functions associated with the ER. We hypothesize that the defect affects the calcium distribution between ER and cytosol, since the calcium ionophore A23187 has an effect similar to that of a temperature shift.     

A recurring temperature-sensitive mutant class of BHK-21 cells

A particular technique designed to select for temperature-sensitive DNA-synthesis mutants of BHK cells has yielded primarily a class of mutants which do not show complementation in cell hybrids.     

Changes in RNA polymerase II in a cell cycle-specific temperature-sensitive mutant of hamster cells

tsAF8 cells are a temperature-sensitive mutant of BHK cells that arrest at the nonpermissive temperature in the G₁ phase of the cell cycle. The activity of solubilized RNA polymerase II and its ability to bind [³H]-γ-amanitin decrease in tsAF8 cells at 40.6°, with a half-life of ～ 10 hr. No appreciable changes occur in these two parameters in tsAF8 cells at 34° or in BHK cells at either 34° or 40.6°. Protein synthesis is not appreciably affected for at least 24 hr after tsAF8 cells are shifted to 40.6°. These results indicate that in tsAF8 cells at the nonpermissive temperature, there is a defect in either the synthesis, the assembly, or the stability of RNA polymerase II, and that the loss of RNA polymerase II molecules is not due to widespread cellular damage.     

A temperature-sensitive mutant of cultured mouse cells defective in chromosome condensation

A temperature-sensitive mutant, designated ts85, was isolated from a mouse mammary carcinoma cell line, FM3A. The ts85 cells grew at 33 °C (permissive temperature) with a doubling time of 18 h, which was almost the same as with wild-type cells, whereas the cell number scarcely increased at all at 39 °C (non-permissive temperature). When the ts85 cells were shifted from 33 to 39 °C, their DNA synthesis fell to below 1% of the initial value in 14 h. RNA or protein synthesis, however, was maintained at the initial levels for at least 14 h at 39 °C. Cytofluorometric analysis of asynchronous cultures and studies with synchronous cultures suggested that the bulk of the cells cultured at 39 °C for 12–18 h were arrested in late S and G2 phases. Electron microscopic observations revealed that chromatin was abnormally condensed into fragmented and compact forms, particularly around nucleoli, in about 80% of cells of an asynchronous culture incubated at 39 °C for 16 h. Cells in mitosis were not detected in such cultures and nuclear membrane and nucleoli were still intact. Such abnormal chromosome condensation was not observed in the ts85 cells at 33 °C or in wild-type cells at either temperature. Since these findings suggest that a ts gene product of ts85 cells is necessary for chromosome condensation, ts85 cells may represent a useful tool for establishing the mechanisms of chromosome condensation. The interrelationship between abnormal chromosome condensation and reduction in DNA synthesis of the ts85 cells is discussed.     

Transfer of a mutant viral thymidine kinase gene results in temperature-sensitive mouse cells.

Temperature-sensitive cell lines were obtained by DNA-mediated transfer of the thymidine kinase (TK) gene from a mutant, ts1117, of herpes simplex virus type 1. The cells died at 39 degrees C in selective medium which contained low levels (1 microgram/ml) of thymidine. In this lethal condition, no revertants were detected among 10(8) cells. It was shown by in vitro analysis of the TK activity that the temperature-sensitive cell line contains an enzyme whose activity is temperature sensitive and relatively unaffected by dTTP. The viral enzyme has these properties. The effect of the lethal growth conditions in the cell line was characterized by cell cycle analysis and rescue experiments which involved a shift to the permissive conditions. The successful transfer of the mutant viral TK activity to cells provides an additional selective marker for gene transfer.     

Effects of mutant rat dynamin on endocytosis

The process of wound repair in monolayers of the intestinal epithelial cell line, Caco-2BBe, was analyzed by a combination of time-lapse differential interference contrast (DIC) video and immunofluorescence microscopy, and laser scanning confocal immunofluorescence microscopy (LSCIM). DIC video analysis revealed that stab wounds made in Caco-2BBe monolayers healed by two distinct processes: (a) Extension of lamellipodia into the wounds; and (b) Purse string closure of the wound by distinct arcs or rings formed by cells bordering the wound. The arcs and rings which effected purse string closure appeared sharp and sheer in DIC, spanned between two and eight individual cells along the wound border, and contracted in a concerted fashion. Immunofluorescence analysis of the wounds demonstrated that the arcs and rings contained striking accumulations of actin filaments, myosin-II, villin, and tropomyosin. In contrast, arcs and rings contained no apparent enrichment of microtubules, brush border myosin-I immunogens, or myosin- V. LSCIM analysis confirmed the localization of actin filaments, myosin- II, villin, and tropomyosin in arcs and rings at wound borders. ZO-1 (a tight junction protein), also accumulated in arcs and rings around wounds, despite the fact that cell-cell contacts are absent at wound borders. Sucrase-isomaltase, an apically-localized integral membrane protein, maintained an apical localization in cells where arcs or rings were formed, but was found in lamellipodia extending into wounds in cells where arcs failed to form. Time-course, LSCIM quantification of actin, myosin II, and ZO-1 revealed that accumulation of these proteins within arcs and rings at the wound edge began within 5 minutes and peaked within 30-60 minutes of wounding. Actin filaments, myosin-II, and ZO-1 achieved 10-, 3-, and 4-fold enrichments, respectively, relative to cell edges which did not border wounds. The results demonstrate that wounded Caco-2BBe monolayers assemble a novel cytoskeletal structure at the borders of wounds. The results further suggest that this structure plays at least two roles in wound repair; first, mediation of concerted, purse string movement of cells into the area of the wound and second, maintenance of apical/basolateral polarity in cells which border the wound.     

Modification of gap junctions in cells transformed by a temperature-sensitive mutant of Rous sarcoma virus

Summary Prompted by our observation that a reduction in junctional permeance is one of the earlier events in the process of neoplastic transformation of a cell line by Rous sarcoma virus, we analyzed the gap junctions, from these cells to determine if the basis of the reduction is a loss of junctional channels. The cells (normal rat kidney, or NRK) are infected with a temperature-sensitive mutant of Rous sarcoma virus, allowing one easily to manipulate the cells into and out of the transformed state, and hence also to manipulate the junctional permeance. Using freeze-fracture electron microscopy, we found that the number and size of the junctions did not change in parallel with the permeance changes we had previously characterized. There is, however, a significant rearrangement of the junctional particles to a more random configuration when the cells are transformed and a reversal to the more ordered pattern when the cells are shifted back to the normal phenotype. These changes do parallel the changes in junctional permeance. We conclude that the permeance of existing junctional channels is modified and that the change in permeance may involve a change in the interaction of the junctional channels with each other and/or the surrounding lipid domain.     

A temperature-sensitive cell-cycle mutant of mammalian cells, tsJT16, is defective in a function operating soon after growth stimulation

A temperature-sensitive cell-cycle mutant, tsJT16, which has been isolated from Fischer rat fibroblasts, was defective in the function(s) that operated soon after growth stimulation. When G0-arrested tsJT16 was stimulated to proliferate, it entered the S phase after 12-15 h at 34 degrees C but failed to do so at 40 degrees C. The function mutated in tsJT16 was required to be normal for the first 4 h or less for cells to transit from the G0 to S phase. The induction of cell-cycle-dependent genes such as c-fos, c-myc and ornithine decarboxylase was observed at both temperatures after growth stimulation. Although an increase in total protein synthesis occurred at both temperatures after growth stimulation, synthesis of one protein (p70) (pI 7.8 and Mr 70,000) was inhibited at 40 degrees C. Synthesis of p70 was negligible in G0-arrested cells and blocked by actinomycin D in serum-stimulated cells at 34 degrees C. These results suggest that tsJT16 has a ts defect in one of the signal transduction processes to induce gene activation. tsJT16 was also defective in progression of the G1 phase of growing cells, consistent with the previous results in which growth stimuli were required at G1 for continuation of proliferation.     

Lysolecithin as a modifier of induced pinocytosis in Amoeba proteus

Lysolecithin was found to modify cation-induced pinocytosis in Amoeba proteus. It is shown here that lysolecithin (LPC) in the concentration range of 10(-15) to 10(-10) g/ml has the same effect on Na+ -induced pinocytosis as cAMP and a pinocytosis regulating factor (PRF) which is secreted by the amoeba. Thus, LPC activated Na+-induced pinocytosis in starved amoebae and decreased the sensitivity to the inducer in normal cells. Pinocytosis depressed by treatment with EGTA or dibucaine became normal upon addition of LPC to the inducer. These effects were also obtained with lysolecithin isolated from the amoeba. It is suggested that PRF and amoeba LPC may be closely related and that phospholipase activity of the amoeba may regulate its capacity for pinocytosis.     

Environment-dependent long-range structural distortion in a temperature-sensitive point mutant

Extensive environment-dependent rearrangement of the helix-turn-helix DNA recognition region and adjacent L-tryptophan binding pocket is reported in the crystal structure of dimeric E. coli trp aporepressor with point mutation Leu75Phe. In one of two subunits, the eight residues immediately C-terminal to the mutation are shifted forward in helical register by three positions, and the five following residues form an extrahelical loop accommodating the register shift. In contrast, the second subunit has wildtype-like conformation, as do both subunits in an isomorphous wildtype control structure. Treated together as an ensemble pair, the distorted and wildtype-like conformations of the mutant apoprotein agree more fully than either conformation alone with previously reported NOE measurements, and account more completely for its diverse biochemical and biophysical properties. The register-shifted segment Ile79-Ala80-Thr81-Ile82-Thr83 is helical in both conformations despite low helical propensity, suggesting an important structural role for the steric constraints imposed by β-branched residues in helical conformation.     

Disappearance of a basic chromosomal protein from cells of a mouse temperature-sensitive mutant defective in histone phosphorylation

The amount of a basic nuclear protein which migrates a little slower than histone H1 in urea-acetic acid-polyacrylamide gel electrophoresis and a little faster than H1 in sodium dodecylsulfate-polyacrylamide gel electrophoresis, decreases when cells of a temperature-sensitive mutant, ts85, derived from a mouse carcinoma cell line, are incubated at the nonpermissive temperature (39°C). This protein appears again, when cells cultured at 39°C are shifted down to the permissive temperature, indifferent to the added cycloheximide. Neither in wild type nor in a revertant of ts85, the protein disappeared at 39°C. Since the ts85 cells were found to be defective in chromosome condensation and in the phosphorylation of histone H1 at 39°C (1,2), this basic protein may relate to the both events.     

Altered phospholipid metabolism in a temperature-sensitive mutant of a thermophilic bacillus

The phospholipid metabolism of a temperature-sensitive mutant of a thermophilic bacillus was studied after the shift from a permissive (58°C) to a restrictive (65°C) growth temperature. During the short period of growth of the mutant at 65°C, the proportions of cardiolipin and its 3-acyl derivative (lyso-cardiolipin) increased, and the proportions of phosphatidylglycerol and phosphatidylethanolamine decreased on cell dry weight basis. In ³²P incorporation and turnover experiments, phosphatidylglycerol showed the most rapid uptake and loss of the label. Turnover of cardiolipin, limited to a short period, ceased 18 min after the shift, as did the turnover of phosphatidylethanolamine. In the absence of net phospholipid synthesis, there was a quantitative conversion of phosphatidylglycerol to cardiolipin and an increase in the proportion of lyso-cardiolipin. Chloramphenicol, added to the medium at the time of the shift, reduced the rate of phospholipid synthesis, prevented the increase in the proportions of cardiolipin and lyso-cardiolipin, and slowed the decrease in the proportions of the other two phospholipids. The results indicated a defect in the regulatory mechanism(s) of phospholipid metabolism in the mutant at the restrictive temperature.Nonstandard Abbreviations WT parental strain, thermophilic bacillus - TS-13 temperature-sensitive mutant of a thermophilic bacillus - CL cardiolipin - PG phosphatidylglycerol - PE phosphatidylethanolamine - l-CL lyso-cardiolipin