Macromolecular uptake is a spontaneous event during mitosis in cultured fibroblasts: implications for vector-dependent plasmid transfection

The process through which macromolecules penetrate the plasma membrane of mammalian cells remains poorly defined. We have examined whether natural cellular events modulate the capacity of cells to take up agents applied extraneously. Herein, we report that during mitosis and in a cell type-independent manner, cells exhibit a natural ability to absorb agents present in the extracellular environment up to 150 kDa as assessed using fluorescein isothiocyanate-dextrans. This event is exclusive to the mitotic period and not observed during G0, G1, S, or G2 phase. During mitosis, starting in advanced prophase, oligonucleotides, active enzymes, and polypeptides are efficiently taken into mitotic cells. This uptake of macromolecules during mitosis still takes place in the presence of cytochalasin D or nocodazole, showing no requirement for intact microtubules or actin filaments in this process. However, cell rounding up, which still takes place in the presence of either of these drugs in mitotic cells, appears to be a key event in this process. Indeed, limited trypsinization of adherent cells mimics both the cell retraction and macromolecule uptake observed as cells enter mitosis. A plasmid DNA encoding green fluorescent protein (3.3Mda) coated with an 18 amino acid peptide is efficiently expressed when applied onto synchronized G2/M fibroblasts, whereas little or no expression is observed when the coated plasmid is applied onto asynchronous cell cultures. This shows that such coating peptides are only efficient for their encapsulating and protective effect on the plasmid DNA to be "vectorized" rather than acting as true vectors.     

Binding and degradation of alpha 2-macroglobulin by cultured fibroblasts

We studied the interactions of alpha 2-macroglobulin, a major protease inhibitor of plasma and of serum-containing culture medium, with cultured fibroblasts. Iodinated human alpha 2-macroglobulin bound specifically to washed cell layers of cultured human fibroblasts. At 0--4 degrees C, binding was saturated at a concentration of 10--20 micrograms/ml. At 37 degrees C, radiolabel appeared in the medium in a form soluble in 10% trichloroacetic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that ingested iodinated alpha 2-macroglobulin transiently forms a complex with a trypsin-like protease. Indirect immunofluorescence demonstrated alpha 2-macroglobulin in vacuoles of fibroblasts grown in 10% human serum or incubated with purified alpha 2-macroglobulin. Fibroblasts transformed by SV-40 (VA-13 cells) bound and degraded less 125I-labeled alpha 2-macroglobulin than non-transformed fibroblasts and had fewer vacuoles containing alpha 2-macroglobulin. These observations indicate that cultured fibroblasts bind, take up by endocytosis, and degrade alpha 2-macroglobulin. Binding and endocytosis of alpha 2-macroglobulin by a cell may be a means of modulating proteases in the microenvironment of the cell and during endocytosis.     

Localized accumulation of tubulin during semi-open mitosis in the Caenorhabditis elegans embryo

The assembly of microtubules inside the cell is controlled both spatially and temporally. During mitosis, microtubule assembly must be activated locally at the nascent spindle region for mitotic spindle assembly to occur efficiently. In this paper, we report that mitotic spindle components, such as free tubulin subunits, accumulated in the nascent spindle region, independent of spindle formation in the Caenorhabditis elegans embryo. This accumulation coincided with nuclear envelope permeabilization, suggesting that permeabilization might trigger the accumulation. When permeabilization was induced earlier by knockdown of lamin, tubulin also accumulated earlier. The boundaries of the region of accumulation coincided with the remnant nuclear envelope, which remains after nuclear envelope breakdown in cells that undergo semi-open mitosis, such as those of C. elegans. Ran, a small GTPase protein, was required for tubulin accumulation. Fluorescence recovery after photobleaching analysis revealed that the accumulation was accompanied by an increase in the immobile fraction of free tubulin inside the remnant nuclear envelope. We propose that this newly identified mechanism of accumulation of free tubulin-and probably of other molecules-at the nascent spindle region contributes to efficient assembly of the mitotic spindle in the C. elegans embryo.     

Receptor-mediated endocytosis of alpha 2-macroglobulin in cultured fibroblasts

Alpha 2-macroglobulin is internalized into cultured fibroblasts by receptor-mediated endocytosis. This ligand binds initially to diffusely distributed receptors on the cell surface which cluster rapidly into bristle-coated pits. Within a few minutes at 37 degrees C, these complexes are internalized into uncoated cytoplasmic vesicles, called receptosomes, which move about in the cell by saltatory motion. These vesicles interact with the Golgi-endoplasmic reticulum-lysosome system in the cell to deliver the ligand to newly formed lysosomes within 30--60 min.     

Colchicine-resistant assembly of tubulin in plant mitosis

Summary Tubulin distribution in c-mitoses (induced by 1 mM colchicine) has been studied by indirect immunofluorescence with monoclonal antibodies inAllium cepa L. meristems proliferating under steady state kinetics. Two hours after colchicine treatment was initiated tubulin is detected in approximately 25% of the cells as arrowheads on the kinetochores, as if these structures stabilize microtubules against disassembly. Total disassembly of microtubules occurs in 70% of the c-mitoses six hours after the initiation of the colchicine treatment, when restitution nuclei also start appearing. After 2 to 14 hours of colchicine treatment, tubulin is detected in about 30% of the c-mitoses, both in small kinetochores-like dots and in a strand which apparently connects sister kinetochores. Other larger microtubule-like structures, up to 20 m long, apparently unassociated with kinetochores, are assembled in the presence of cholchicine in c-mitoses after 10 hours. Such structures disappear when chromosomes decondense and the nuclear envelope reforms in the restitution nucleus; they do not seem to be related to interphase cortical microtubules which reappear in control telophase.     

Loss of tubulin during cold treatment of cultured carrot cells

When carrot cells, Daucus carota L. cv. Kintoki, in suspension culture were chilled on ice, more than 90% of the cortical microtubules disappeared within an hour. Colchicine-binding activity in the soluble extract decreased gradually during the prolonged cold treatment and after 4 days reached a minimum level. In SDS-polyacrylamide gel electrophoretograms of the cold-treated cell extracts, the bands corresponding to tubulin subunits disappeared, whereas most of the other protein bands remained. The level of tubulin was restored within a day after the cells were returned to 267deg;C. The relative ratio of the two major β-tubulin isoforms was reversed during the cold treatment, indicating that the cold sensitivity of tubulin differs according to its molecular species. When the cells were returned to 26°C, the ratio was restored to the original state within 24 h.     

Interaction of annexin A6 with alpha actinin in cardiomyocytes

Background  

Annexins are calcium dependent phospholipid binding proteins that are expressed in a wide variety of tissues and implicated in various extra- and intracellular processes. In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal. Conflicting reports from transgenic mice overexpressing annexin A6 or null mice lacking annexin A6 showed imbalances in intracellular calcium turnover and disturbed cardiac contractility. However, few studies have focussed on the signalling module of annexin A6 in the heart either in normal or in pathological state.     

The ATP-dependent helicase RUVBL1/TIP49a associates with tubulin during mitosis

RUVBL1/TIP49a/Pontin52 is a recently identified multi-functional protein with 2 ATP binding (WALKER) sites, which is essential for cell proliferation. We recovered and identified RUVBL1/TIP49a as a tubulin-binding protein from Triton X-100 lysates of U937 promonocytic cells by protein affinity chromatography and tryptic peptide microsequencing. Performing co-immunoprecipitation using newly generated RUVBL1/TIP49a-specific antibodies (mAb and rabbit polyclonal Ab) and RUVBL1/TIP49a-GST fusion protein-pull down assays we demonstrate co-precipitation of alpha- and gamma tubulin with RUVBL1/TIP49a. Confocal immunoflourescence microscopy reveals that RUVBL1/TIP49a was present not only in the nucleus, as expected, but was also concentrated at the centrosome and at the mitotic spindle in colocalization with tubulin. The topology of RUVBL1/TIP49a at the mitotic spindle varied, depending on the mitotic stage. The protein was localized at the centrosome and at the polar and astral microtubules in metaphase, and was detectable at the zone of polar tubule interdigitation in anaphase B and telophase. During cytokinesis the protein reappeared at the area of decondensing chromosomes. Whereas preincubation of U937 cells with colcemid resulted in inhibition of mitotic spindle formation with subsequent loss of RUVBL1/TIP49a mitotic spindle staining, no relevant influence of colcemid on RUVBL1/TIP49a-tubulin binding was observed. An agonistic effect of RUVBL1/TIP49a on in vitro tubulin assembly is demonstrated. Our results reveal a new functional aspect of RUVBL1/TIP49a.     

Assembly of nucleolus-derived foci in various cultured mammalian cells during mitosis

During mitosis, rebuilding of the nucleolus is a step-wise process that, above all, includes an assembly of nucleolus-derived foci (NDF) in the cytoplasm of telophase cells. In this study, we performed a comparative analysis of NDF formation in mitotic cells of various mammalian cell cultures, such as green monkey CV1 cells, human HeLa cells, mouse 3T3 cells, and pig PK cells, both in control and following inhibition of rRNA synthesis by actinomycin D or by an adenosine analogue, DRB. The results obtained show that in all examined cell types NDF are formed shortly after or simultaneously with the onset of chromosome segregation to the poles of the mitotic spindle. However, an efficiency of NDF assembly, i.e. the number of NDF per anaphase or telophase cell, and the portion of anaphase and telophase cells containing NDF vary in different cell cultures, being most prominent in CVI and HeLa cells. In these cells, the vast majority of NDF accumulate several proteins of the mature nucleolus, such as B23/nucleophopmin, C23/nucleotin, fibrillarin, and, to a lesser extent, Nop52. The rRNA harbored by NDF is synthesized several hours prior mitosis, and plays an essential role the maintenance of NDF structural integrity. Starting from early stages of the assembly onwards, the NDF are predominantly located in the area occupied by aster microtubules of the mitotic spindle.     

离体培养小鼠神经细胞在有丝分裂过程中的膜电位变化（简报）

Primarily cultured dorsal root ganglion cells and olfactory bulb cells were dissected from 12 to 14-d-old fetal C57 BL/6 J mice. After the cells were cultured for about two weeks, the growing status of cells were observed and the membrane potentials(MP) were recorded. The results show that mean value of the MP in anaphase was -68 +/- 3.1 mV (SE, n = 3), same as in interphase: Beginning from telophase, the cell membrane in the equator contracted gradually to become a concave ditch, and the MP decreased obviously, the mean value was -23.3 +/- 3.3 mV (SE, n = 6). After this, MP recovered gradually, till it divided into two sister cells. MP which were recorded separately in two sister cells were similar. But usually MP did not recovered to their normal values immediately.     

Autoregulation of tubulin synthesis in hepatocytes and fibroblasts

Microtubule polymer levels in mouse 3T6 fibroblasts and primary cultures of rat hepatocytes can be manipulated by treatment of cells with long term, low doses of colcemid. Such treatment produces a rather uniform population of cells with microtubules of reduced lengths. Using this system, we demonstrate (a) that the rate of tubulin synthesis is sensitive to small changes (10%) in microtubule polymer mass and (b) that the percent of inhibition of synthesis is proportional to the level of soluble tubulin. Experiments with hepatocytes indicate that not only synthesis but the stability of tubulin protein was also regulated to maintain a specific level of tubulin. Treatment of hepatocytes with colcemid or other microtubule-depolymerizing drugs reduced the half-life of tubulin from 50 to 2 h, whereas taxol, which stabilizes microtubules, increased the half-life. To assess the consequences of altering microtubule polymer mass, we have analyzed the effect of controlled depolymerization of microtubules in rat hepatocytes on the processing of endocytosed ligands and found it sensitive to small changes in microtubule polymer levels.     

Posttranslational modifications of alpha tubulin: detyrosination and acetylation differentiate populations of interphase microtubules in cultured cells

Subsets of microtubules enriched in posttranslationally detyrosinated (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski. 1984. Cell. 38:779) or acetylated (Piperno, G., M. Le Dizet, and X. Chang. 1987. J. Cell Biol. 104:298), alpha tubulin have previously been described in interphase cultured cells. In this study an immunofluorescence comparison of these minor populations of microtubules revealed that, in African green monkey kidney epithelial cells (TC-7 line), the population of microtubules enriched in detyrosinated tubulin was virtually coincident with the population enriched in acetylated alpha tubulin. In some cell types, however, such as human HeLa or marsupial PtK-2 cells, only one posttranslationally modified form of tubulin, i.e., acetylated or detyrosinated, respectively, was detectable in microtubules. In TC-7 cells, although both modifications were present, dissimilar patterns and kinetics of reappearance of microtubules enriched in detyrosinated and acetylated tubulin were observed after recovery of cells from microtubule-depolymerizing treatments or from mitosis. Thus, a minor population of microtubules exists in cultured cells that contains an elevated level of tubulin modified in either one or two ways. While these two modifications occur primarily on the same subset of microtubules, they differ in their patterns of formation in vivo.     

Spectrin redistributes to the cytosol and is phosphorylated during mitosis in cultured cells

Dramatic changes in morphology and extensive reorganization of membrane-associated actin filaments take place during mitosis in cultured cells, including rounding up; appearance of numerous actin filament-containing microvilli and filopodia on the cell surface; and disassembly of intercellular and cell-substratum adhesions. We have examined the distribution and solubility of the membrane-associated actin-binding protein, spectrin, during interphase and mitosis in cultured CHO and HeLa cells. Immunofluorescence staining of substrate-attached, well-spread interphase CHO cells reveals that spectrin is predominantly associated with both the dorsal and ventral plasma membranes and is also concentrated at the lateral margins of cells at regions of cell-cell contacts. In mitotic cells, staining for spectrin is predominantly in the cytoplasm with only faint staining at the plasma membrane on the cell body, and no discernible staining on the membranes of the microvilli and filopodia (retraction fibers) which protrude from the cell body. Biochemical analysis of spectrin solubility in Triton X-100 extracts indicates that only 10-15% of the spectrin is soluble in interphase CHO or HeLa cells growing attached to tissue culture plastic. In contrast, 60% of the spectrin is soluble in mitotic CHO and HeLa cells isolated by mechanical "shake-off" from nocodazole-arrested synchronized cultures, which represents a four- to sixfold increase in the proportion of soluble spectrin. This increase in soluble spectrin may be partly due to cell rounding and detachment during mitosis, since the amount of soluble spectrin in CHO or HeLa interphase cells detached from the culture dish by trypsin-EDTA or by growth in spinner culture is 30-38%. Furthermore, mitotic cells isolated from synchronized spinner cultures of HeLa S3 cells have only 2.5 times as much soluble spectrin (60%) as do synchronous interphase cells from these spinner cultures (25%). The beta subunit of spectrin is phosphorylated exclusively on serine residues both in interphase and mitosis. Comparison of steady-state phosphorylation levels of spectrin in mitotic and interphase cells demonstrates that solubilization of spectrin in mitosis is correlated with a modest increase in the level of phosphorylation of the spectrin beta subunit in CHO and HeLa cells (a 40% and 70% increase, respectively). Two-dimensional phosphopeptide mapping of CHO cell spectrin indicates that this is due to mitosis-specific phosphorylation of beta-spectrin at several new sites. This is independent of cell rounding and dissociation from other cells and the substratum, since no changes in spectrin phosphorylation take place when cells are detached from culture dishes with trypsin-EDTA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Actin and tubulin in normal and cystic fibrosis fibroblasts

Actin and tubulin contents of early passage, confluent human fibroblast cultures have been determined. Actin comprised 5.87 ± 0.81% of the total protein of IMR-90 fibroblasts which was not significantly different than the actin contents of two cystic fibrosis fibroblast cultures GM0142 and GM1348 (5.64 ± 0.90% of total protein). However, a significant difference between the amount of tubulin in IMR-90 fibroblasts (7.17 ± 0.25% of total protein) and the amount of tubulin in cystic fibrosis fibroblasts (4.51 ± 0.64% of total protein) was found.