Increase In Number Of Microtubules Research Articles

Actin filaments regulate microtubule growth at thecentrosome.

The centrosome is the main microtubule-organizing centre. It also organizes a local network of actin filaments. However, the precise function of the actin network at the centrosome is not well understood. Here, we show that increasing densities of actin filaments at the centrosome of lymphocytes are correlated with reduced amounts of microtubules. Furthermore, lymphocyte activation resulted in disassembly of centrosomal actin and an increase in microtubule number. To further investigate the direct crosstalk between actin and microtubules at the centrosome, we performed invitro reconstitution assays based on (i) purified centrosomes and (ii) on the co-micropatterning of microtubule seeds and actin filaments. These two assays demonstrated that actin filaments constitute a physical barrier blocking elongation of nascent microtubules. Finally, we showed that cell adhesion and cell spreading lead to lower densities of centrosomal actin, thus resulting in higher microtubule growth. We therefore propose a novel mechanism, by which the number of centrosomal microtubules is regulated by cell adhesion and actin-network architecture.

Glutamate modulation of dendrite outgrowth: alterations in the distribution of dendritic microtubules.

Glutamate can both facilitate and inhibit dendrite outgrowth in vitro. The major effects of low levels of glutamate occur only on the dendrites (not the axon) of pyramidal neurons and may be important for modulating dendrite outgrowth during neuronal development in vivo. Cytoskeletal changes resulting from glutamate exposure must underlie these changes in dendrite outgrowth. In the present study, hippocampal neuron cultures were used to measure the outgrowth of both axons and immature dendrites in the presence or absence of 50 microM glutamate. Subsequently, neurons were extracted and fixed for immunofluorescent labeling of microtubules and rhodamine phalloidin labeling of microfilaments. Additionally, neurons were prepared for electron microscopy to examine dendritic microtubules at the ultrastructural level. Glutamate led to increased dendrite outgrowth in the short term (4 hr) and dendrite retraction in the long term (8 hr). After short-term glutamate exposures, no obvious morphological changes occur in either the microtubules or microfilaments. However, longer glutamate exposure causes a decrease in the number of microtubules in the distal region of retracting dendrites, and causes an increase in microtubule number in the dendritic shaft of both retracting and growing dendrites. Thus, the microtubule cytoskeleton may be involved in producing the changes in dendrite outgrowth caused by glutamate exposure.

Changes in microtubule number and length during axon differentiation

Hippocampal neurons in culture initially extend several minor processes that are approximately 20 microns in length. The first minor process to grow approximately 10 microns longer than the others will continue to grow rapidly and become the axon (Goslin and Banker, 1989). We sought to define changes in the microtubule (MT) array that occur during axon differentiation. In theory, axon differentiation could involve an increase in MT number, MT length, or some combination of both. To address this issue, we first serially reconstructed the entire MT array of a minor process from a cell whose axon had not yet differentiated. This minor process contained 182 MTs that ranged in length from 0.14 to 20.09 microns. The average MT length was 3.87 +/- 3.83 microns, and the total MT length was 704 microns. We then reconstructed the MT arrays of a minor process and the 56 microns axon from a cell that had undergone axon differentiation. The minor process contained 157 MTs that ranged in length from 0.24 to 17.95 microns. The average MT length was 3.91 +/- 4.84 microns, and the total MT length was 600 microns. The axon contained 1430 MTs that ranged in length from 0.05 to 40.14 microns. The average MT length was 4.02 +/- 5.28 microns, and the total MT length was 5750 microns. These data indicate that a shift occurs toward shorter as well as longer MTs, but that virtually no change in average MT length occurs during axon differentiation. Thus, elongation of existing MTs cannot account for the major expansion of the MT array that occurs as a minor process becomes an axon. In contrast, the number of MTs increases by approximately 10-fold as a minor process differentiates and grows into an axon of the length we analyzed. Based on these data, we conclude that the MT array of a minor process is substantially expanded as it differentiates into an axon, and that the principal mechanism by which this expansion occurs is the copious addition of new MTs.

A quantitative electron microscopic study of changes in microtubule arrays and wall microfibril orientation during in vitro cotton fiber development

ABSTRACT A quantitative electron microscopic (E/M) study of the changes in microtubule arrays and wall microfibril orientation has been done on in vitro grown cotton fibers. Microtubules change orientation during cotton fiber development. During fiber initiation and early elongation, microtubules have a generally random orientation. Microtubules re-orient into shallow pitched helices as elongation and primary wall deposition continue, and into steeply pitched helices during secondary wall deposition. Accompanying the changes in orientation are increases in microtubule length, number, proximity to the plasmalemma and a decreased variability in orientation of the microtubules. Based on these observations, three pivotal stages in microtubule patterns were identified during fiber development: (1) the transition between fiber initiation and elongation, where microtubules develop a shallow pitched helical orientation; (2) the transition between primary and secondary wall synthesis, where microtubules abruptly shift orientation to a steeply pitched helical pattern; and (3) early in secondary wall synthesis, where there is a four fold increase in microtubule number. Microfibrils exhibit changes in orientation similar to the microtubules; however significant differences were found when the precise orientations of microtubules and microfibrils were compared. During secondary wall synthesis, wall microfibrils exhibit some variability in orientation due to inter-fibril bundling, thus indicating that components of the wall may also influence final microfibril orientation.

Increases in microtubule assembly and in tubulin content in mitogenically stimulated mouse splenic T lymphocytes

We have examined the changes in the microtubule and tubulin contents in populations of mouse splenic T lymphocytes stimulated by the mitogen concanavalin A. Indirect immunofluorescence staining with antiserum to tubulin indicated that a more extensive microtubule network was assembled from the centrosome in those cells which had increased in size in response to the mitogen. Direct counts of microtubules from electron micrographs of the centrosome regions of cells showed approximately a 2-fold increase in microtubule number in 48 h stimulated populations and up to a 5-fold increase in the large, fully stimulated, blast cells. Determinations of tubulin and actin contents were made by the measurement of peptides specific to those proteins. As a percentage of total cell protein both of these cytoskeletal proteins increased during the first 24 h of stimulation. Tubulin increased 50% by 24 h and remained high in populations stimulated for 48 h. The tubulin content per cell increased 2.5-fold, from 0.20 to 0.51 μg/10 6 cells, in the 48 h stimulated population. An increase in tubulin content was also seen following the stimulation of nude mouse B lymphocyte populations and of total splenic lymphocyte populations. Our results show that during lymphocyte stimulation there is a large increase in the numbers of microtubules assembled which is correlated with, and appears dependent on, a similar large increase in the cellular tubulin content.

Microtubules, colchicine, and lymphocyte blastogenesis

We have studied the time course of disassembly of microtubules of resting and stimulated mouse lymphocytes caused by the drug colchicine, as well as the effect of this compound on DNA and RNA synthesis of human and mouse lymphocytes. Fine-structure studies with the electron microscope showed a great increase in number of microtubules resulting from stimulation of mouse lymphocytes by the mitogenic lectin Con A. The presence of a network of microtubules was demonstrated in resting lymphocytes by use of the technique of immunofluorescence; this technique was not effective for the study of the microtubules of stimulated lymphocytes in the blast stage. The disappearance of microtubular networks in some cells (approximately 25%) was caused by the protocol of colchicine treatment used in many laboratories (30 min at 10(6) M); a 6- to 8-h treatment was required to cause all cells to lose their microtubules. It is indicated in these findings that there is need for extreme caution in implicating microtubule disruption as the cause of certain colchicine effects, such as that on the Con A-induced inhibition of receptor-ligand migration. The addition of colchicine to stimulated cells at varying times of culture caused marked inhibition of DNA synthesis provided that sufficient time (approximately 20 h for maximum inhibition) elapsed between addition of the drug to the stimulated culture and assay of DNA synthesis. Our data on the time course of inhibition of DNA synthesis by alpha-methyl mannoside (alpha MM) and by colchicine do not exclude the possibility that the latter compound may act partially by affecting the commitment of stimulated lymphocytes to DNA synthesis but they show that it can inhibit well after commitment is complete. The later the time of assay of thymidine incorporation, the more disparate were the curves relating the effects of alpha MM and colchicine to DNA synthesis of human cells. In the case of mouse splenic lymphocytes, there was no resemblance between the time course of the alpha MM and of the colchicine effects. Synthesis of RNA after 12 h of culture of stimulated human lymphocytes was also sensitive to colchicine.

The Terminal Segment of the Seminiferous Tubules and the Blood‐Testis Barrier Before and After Efferent Ductule Ligation in the Rat

The ultrastructure of modified Sertoli cells in the terminal segment of the seminiferous tubules in the rat was studied in material fixed by perfusion through the thoracic or the abdominal aorta. The central lumen of the terminal segment was not distinct under normal conditions, but became quite evident with an increased intratesticular pressure caused by efferent ductule ligation. The modified Sertoli cells were characterized by a marked increase in number of microtubules and microfilaments, extensive inter‐Sertoli cell junctions, vacuolated cytoplasm, and considerably dilated intercellular spaces. Three types of specialized cell contacts were found between modified Sertoli cells: typical inter‐Sertoli cell junctions, desmosome‐like devices, and septate‐like tight junctions. These specialized cell contacts were efficient in preventing lanthanum to penetrate into the lumen of the terminal segment both before and after efferent ductule ligation. Moreover, it was found that typical inter‐Sertoli cell junctions prevented lanthanum penetration into the adluminal compartment of the seminiferous tubules even after efferent ductule ligation.

An electron microscope study of the effects of dibutyryl cyclic AMP on Chinese Hamster Ovary cells

The effect of reverse transformation agents (dibutyryl 3′:5′ cyclic adenosine monophosphate plus synergizing compounds like testosterone) on Chinese Hamster Ovary cells has been studied by scanning and transmission electron microscopy, under conditions of low and high cell density. The disappearance of the cell surface knobs or blebs and the cell elongation previously described by light microscopy are even more striking under the higher resolving power of electron microscopy. The cell elongation is accompanied by and appears to be due to an increase in number of microtubules per unit volume of cytoplasm, and a change in their distribution from a random arrangement to an orderly alignment in parallel to each other and to the long axis of the cell. Increasing cell density, and therefore number of cell contacts in untreated cultures, causes the morphology to change in the direction to be expected if the cellular concentration of cyclic AMP is increased. Ultrastructural details of the cells in the various conditions studied are presented.