INDUCTION OF MICROTUBULE ASSEMBLY IN SITU FROM UNPOLYMERIZED TUBULIN POOLS IN SV40 TRANSFORMED 3T3 CELLS

1982 ◽  
pp. 327-341 ◽  
Author(s):  
William J. Deery ◽  
B.R. Brinkley
1987 ◽  
Vol 7 (6) ◽  
pp. 2080-2086
Author(s):  
D I Linzer ◽  
E L Wilder

The serum-inducible expression of proliferin genes in BALB/c 3T3 cells was found to be dependent on both protein synthesis and an extended presence of serum in the medium. Even though no mature proliferin mRNA was detected in serum-starved cells, transcription of the proliferin genes occurred in these resting-cell cultures, indicating that posttranscriptional events may be important for regulating proliferin mRNA levels. These results suggest that protein synthesis after serum stimulation of quiescent mouse fibroblasts is required for posttranscriptional processing or stabilization of proliferin RNA. Proliferin RNA levels were found to be heterogeneous among serum-stimulated cells analyzed by in situ hybridization. This heterogeneity is probably due to asynchrony in the population and may point to a correlation between the time of proliferin expression and the time of entry of a cell into S phase.


1987 ◽  
Vol 7 (6) ◽  
pp. 2080-2086 ◽  
Author(s):  
D I Linzer ◽  
E L Wilder

The serum-inducible expression of proliferin genes in BALB/c 3T3 cells was found to be dependent on both protein synthesis and an extended presence of serum in the medium. Even though no mature proliferin mRNA was detected in serum-starved cells, transcription of the proliferin genes occurred in these resting-cell cultures, indicating that posttranscriptional events may be important for regulating proliferin mRNA levels. These results suggest that protein synthesis after serum stimulation of quiescent mouse fibroblasts is required for posttranscriptional processing or stabilization of proliferin RNA. Proliferin RNA levels were found to be heterogeneous among serum-stimulated cells analyzed by in situ hybridization. This heterogeneity is probably due to asynchrony in the population and may point to a correlation between the time of proliferin expression and the time of entry of a cell into S phase.


1971 ◽  
Vol 50 (3) ◽  
pp. 691-708 ◽  
Author(s):  
N. Scott McNutt ◽  
Lloyd A. Culp ◽  
Paul H. Black

The ultrastructural appearances of normal 3T3, SV40-transformed 3T3 (SV-3T3), and F1A revertant cell lines are compared. Both confluent and subconfluent cultures are described after in situ embedding of the cells for electron microscopy. There is striking nuclear pleomorphism in F1A revertant cells, with many cells having large nuclei compared to the less variable nuclear morphology of both normal 3T3 and SV-3T3 cells. Under the culture conditions used, deep infoldings of the nuclear envelope are prominent in growing cells, e.g., subconfluent normal 3T3 and confluent SV-3T3 cells. Such infoldings are infrequently seen in cultures which display contact inhibition of growth, e.g., normal 3T3 or F1A revertant cells grown just to confluence. In confluent cultures, the cytoplasmic organelles in revertant cells closely resemble those of normal 3T3 cells. In both normal and revertant cells in confluent culture, the peripheral cytoplasm (ectoplasm) has many 70 A filaments (alpha filaments), which are frequently aggregated into bundles. Alpha filaments are also abundant in the ectoplasm near regions of cell-to-cell apposition and in the motile cell processes (filopodia). The abundance and state of aggregation of alpha filaments correlates with contact inhibition of movement and growth in these cell lines since fewer bundles of alpha filaments are seen in growing cells than in contact-inhibited cells. This observation suggests that these filaments may be an important secondary component in the regulation of contact inhibition of movement and, possibly, of growth in normal and revertant cells.


1975 ◽  
Vol 67 (3) ◽  
pp. 904-911 ◽  
Author(s):  
T R Collins ◽  
J C Bartholomew ◽  
M Calvin

A simple method is described for the freeze-fracture in situ of monolayer cultures grown on gold carriers coated with a thin layer of silicon monoxide. Preliminary observations on 3T3 mouse embryo fibroblasts indicate that this technique exposes large areas of cell membrane, making it possible to determine how areas of membrane specialization are related to the cell as a whole and to regions of cellular interaction. 3T3 cells cultured on silicon monoxide show no modification of growth properties compared to cells growing on Falcon plastic, and other cell lines also appear to grow well on this substrate.


1998 ◽  
Vol 51 (8) ◽  
pp. 801-804 ◽  
Author(s):  
MASUO KONDOH ◽  
TAKEO USUI ◽  
TADANORI MAYUMI ◽  
HIROYUKI OSADA
Keyword(s):  

2012 ◽  
Vol 466-467 ◽  
pp. 146-151
Author(s):  
Yuan Cui ◽  
Yan Hui Li ◽  
Qian Duan

Collagen is a kind of protein which can self-assemble into collagen gel in vitro at 37°C in neutral pH. In this work, collagen is extracted from bovine tendon and characterized by amino acid analysis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). When pH of collagen solution is adjusted to 6.6~6.8 and incubated at 37°C for an hour, collagen molecular can be assembling into gel. As a result, collagen fibers in gel observed form electron microscopic (SEM and TEM) look like natural collagen fiber with cross-grain, which is caused by collage molecular assembly behavior. Then mouse embryonic fibroblasts (3T3 cells) are seeded in the gel. Within a period, state of 3T3 cell living in the 3D gel scaffold is observed. As a result, when collagen solution is mixed with 3T3 cells and incubated in vitro, the gel is formed and provides a three-dimensional space for 3T3 cells proliferation at the same time. This study provides a method to make tissue engineering scaffold with cells in situ and predicts its application in soft tissue defect healing.


1977 ◽  
Vol 23 (1) ◽  
pp. 173-192
Author(s):  
R.E. Scott ◽  
P.B. Maercklein ◽  
L.T. Furcht

The topography of plasma membrane intramembranous particles in contact-inhibited and transformed cells is a topic of current controversy. We have reported that particles are aggregated in 3T3 cells and that they are randomly distributed in SV3T3 cells. Other investigators have, however, proposed that these differences result from the effect of cryoprotectants and are blocked by aldehyde fixation. The experiments described in this paper re-examine PMP topography in large samples of 3T3 and SV3T3 cells prepared by a variety of preparative methods. The results confirm that 3T3 cells contain aggregated intramembranous particles and that native SV3T3 cells do not, regardless of whether cells are prepared in glycerol, sucrose, tissue culture medium or following prefixation in paraformaldehyde. Data presented in this paper also show that cytochalasin B induces dose- and time-dependent disaggregations of particles in contact-inhibited 3T3 cells. Particle disaggregation results from exposure of cells in situ for 1–3 h to 1–500 ng/ml cytochalasin B. Removal of the drug by washing with reincubation of cells in medium containing serum results in particle reaggregation within 1 to 3 h. These observations suggest that intramembranous particles in 3T3 cells are aggregated in the native state and that membrane-active drugs modulate their distribution by a mechanism possibly involving disruption of linkages between membrane components and the cytoskeleton.


1973 ◽  
Vol 56 (2) ◽  
pp. 412-428 ◽  
Author(s):  
N. Scott McNutt ◽  
Lloyd A. Culp ◽  
Paul H. Black

A comparison is made of the ultrastructure of the cell periphery in three cloned cell lines: untransformed Balb/c 3T3 cells, SV40-transformed Balb/c 3T3 cells, and revertant cells obtained from the transformed cell line by a selection technique utilizing concanavalin A. Both thin-section and surface replication techniques are used for in situ examination of the cell lines. Microfilaments, 70 Å in diameter (called alpha filaments), are abundant in untransformed and revertant cell lines, particularly in the anterior expansions of the cells, which tend to have many microvilli and small pseudopodia. Alpha filaments are diminished in the anterior expansions of transformed cells, which contain large blunt pseudopodia and relatively few microvilli. Surface replicas confirm the impression gained from thin sections that transformed cells have a greater proportion of their cell surface involved in bulging pseudopodia than either untransformed or revertant cells. Since alpha filaments are shown to bind heavy meromyosin and are similar to F-actin, these filaments are thought to be important in cell motility. These observations suggest that a close relationship exists between decreased alpha filaments, bulging pseudopodia, and loss of contact inhibition of movement in transformed cells.


1983 ◽  
Vol 96 (6) ◽  
pp. 1631-1641 ◽  
Author(s):  
W J Deery ◽  
B R Brinkley

We studied the characteristics of cytoplasmic microtubule reassembly from endogenous tubulin pools in situ using a Brij 58-lysed 3T3 cell system. Cells that were pretreated in vivo with colcemid retain endogenous tubulin in the depolymerized state after lysis. When lysed cells were removed from colcemid block and incubated in GTP-PIPES reassembly buffer at pH 6.9, microtubules repolymerized randomly throughout the cytoplasm, appeared to be free-ended and were generally not associated with the centrosomes. However, tubulin could be induced to polymerize in an organized manner from the centrosomes by increasing the pH to 7.6 in the presence of ATP and cAMP. Microtubules polymerized in ATP had significantly longer lengths than those assembled in GTP or UTP. When cells not treated with colcemid were lysed, the integrity of the cytoplasmic microtubule complex (CMTC) was maintained during subsequent incubation in reassembly buffer. However, in contrast to unlysed, living cells, microtubules of lysed cells were stable to colchicine. A significant fraction of the CMTC was stable to cold-induced disassembly whereas microtubules reassembled after lysis were extremely cold-sensitive. When cells not treated with colcemid were lysed and incubated in millimolar Ca++, microtubules depolymerized from their distal ends and a much reduced CMTC was observed. Ca++ reversal with EGTA rapidly resulted in a reformation of the CMTC apparently by elongation of Ca++ resistant microtubules.


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