Abundance Of Microtubules Research Articles

Restrain-stress-induced Biochemical and Transmission Electron Microscopic Changes in Albino Rat’s Brain

Restrained-stress in adult male albino rats for 24 hours in continuity resulted in increased lipid peroxidation (maximum 37.03% in cerebellum) and 8-fold increase (statistically significant p<0.001in brainstem) of fluorescent pigment intensity. Greater affinity of darkly stained luxol-fast positive Purkinje neurons suggests decreased metabolic activity as a result of stress. TEM revealed an increase in the incidence of pleomorphic electrodense bodies in neurons of different brain parts. In cerebellum, dense bodies even exhibited fingerprint pattern due to engulfment of damaged cytomembranes subsequent to increased lipid peroxidation. The electronluscent vacuoles located either at or near the periphery or inside the electrondense bodies, were likely to represent the digested material consequent upon the action of action of hydrolytic enzymes. The increase in the number of coated and smooth vesicles and abundance of microtubules in the cytoplasm of neurons of stressed rats exhibited ultrastructural features in support of their being involved in the transfer of membranes and membrane-associated molecules in the intracellular compartments. Increase in GERL-like profiles could also be detected in cingulate cortex, hypothalamus, and cerebellum after 24 hours restrain of rats. Interestingly, GERL displayed continuity with different cellular organelles in cerebellum of stressed rats. These findings supported the concept advocated by Paavola that GERL’s lytic enzymes engulf membranes and trapped lysosomes thus producing aggregate type inclusions of electrodense bodies.

Ultrastructural features of aberrant glial cells isolated from the spinal cord of paralytic rats expressing the amyotrophic lateral sclerosis-linked SOD1G93A mutation.

In the rat model of amyotrophic lateral sclerosis expressing the G93A superoxide dismutase-1 mutation, motor neuron death and rapid paralysis progression are associated with the emergence of a population of aberrant glial cells (AbAs) that proliferate in the degenerating spinal cord. Targeting of AbAs with anti-neoplasic drugs reduced paralysis progression, suggesting a pathogenic potential contribution of these cells accelerating paralysis progression. In the present study, analyze the cellular and ultrastructural features of AbAs following their isolation and establishment in culture during several passages. We found that AbAs exhibit permanent loss of contact inhibition, absence of intermediate filaments and abundance of microtubules, together with an important production of extracellular matrix components. Remarkably, AbAs also exhibited exacerbated ER stress together with a significant abundance of lipid droplets, as well as autophagic and secretory vesicles, all characteristic features of cellular stress and inflammatory activation. Taken together, the present data show AbA cells as a unique aberrant phenotype for a glial cell that might explain their pathogenic and neurotoxic effects.

Intrinsic microtubule GTP-cap dynamics in semi-confined systems: kinetochore–microtubule interface

In order to quantify the intrinsic dynamics associated with the tip of a GTP-cap under semi-confined conditions, such as those within a neuronal cone and at a kinetochore-microtubule interface, we propose a novel quantitative concept of critical nano local GTP-tubulin concentration (CNLC). A simulation of a rate constant of GTP-tubulin hydrolysis, under varying conditions based on this concept, generates results in the range of 0-420s(-1). These results are in agreement with published experimental data, validating our model. The major outcome of this model is the prediction of 11 random and distinct outbursts of GTP hydrolysis per single layer of a GTP-cap. GTP hydrolysis is accompanied by an energy release and the formation of discrete expanding zones, built by less-stable, skewed GDP-tubulin subunits. We suggest that the front of these expanding zones within the walls of the microtubule represent soliton-like movements of local deformation triggered by energy released from an outburst of hydrolysis. We propose that these solitons might be helpful in addressing a long-standing question relating to the mechanism underlying how GTP-tubulin hydrolysis controls dynamic instability. This result strongly supports the prediction that large conformational movements in tubulin subunits, termed dynamic transitions, occur as a result of the conversion of chemical energy that is triggered by GTP hydrolysis (Satarić et al., Electromagn Biol Med 24:255-264, 2005). Although simple, the concept of CNLC enables the formulation of a rationale to explain the intrinsic nature of the "push-and-pull" mechanism associated with a kinetochore-microtubule complex. In addition, the capacity of the microtubule wall to produce and mediate localized spatio-temporal excitations, i.e., soliton-like bursts of energy coupled with an abundance of microtubules in dendritic spines supports the hypothesis that microtubule dynamics may underlie neural information processing including neurocomputation (Hameroff, J Biol Phys 36:71-93, 2010; Hameroff, Cognit Sci 31:1035-1045, 2007; Hameroff and Watt, J Theor Biol 98:549-561, 1982).

Microtubule Dependent Mechano-Transduction Drives Oxidative Stress and Calcium Dysregulation in Dystrophic Skeletal Muscle

Duchenne muscular dystrophy (DMD) is a common X-linked progressive muscle wasting disease characterized by an increased susceptibility to stretch-induced damage. While an abnormal regulation of reactive oxygen species (ROS) and calcium (Ca2+) signaling cascades are established contributors, little mechanistic insight has been revealed for how mechanical stress induces the dysregulation of these signaling pathways. With novel tools to mechanically manipulate enzymatically isolated skeletal muscle fibers, we show for the first time that a small physiologic stretch of a single mdx myofiber produces an almost instantaneous increase in ROS. The inhibition of NADPH oxidase 2 (NOX2) abrogated the stretch-induced ROS consistent with the recently reported X-ROS signaling in the heart (Prosser et al Science, 2011); however X-ROS was not detectable in wild-type fibers. Our group and others have demonstrated that the microtubule cytoskeleton is an important mechano-transduction element in muscle. Our investigation shows increased microtubule density in mdx muscle that displays X-ROS signaling. In this regard, microtubule depolymerization with colchicine was sufficient to inhibit X-ROS signaling in mdx myocytes implicating microtubules as critical proximate mechano-transducers in this pathway. Accordingly, proteins necessary for X-ROS (tubulin subunits and the NOX2 subunits gp91phox and rac1) are increased in dystrophic muscle. An important consequence of X-ROS signaling in mdx is increased sarcolemmal Ca2+ influx which is abrogated by the stretch-activated channel blocker GsMTx4 or microtubule destabilization. Lastly, we find increased surface membrane stiffness in mdx (measured by atomic force microscopy) suggesting that the abundance of microtubules contributes to important functional changes in skeletal muscle properties including stiffness which impacts X-ROS signaling. Our discoveries suggest that X-ROS may underscore the pathogenic stretch-dependent signaling in DMD and thus may provide novel therapeutic targets. Funding: RJK- T32AR007592, CWW- RC2 NR011968, WJL- R01 HL106059, R01 HL36974

Decrease in expression of beta‐tubulin and microtubule abundance in flight muscles during diapause in adults of Culex pipiens

The cDNA encoding beta-tubulin in the mosquito Culex pipiens has high similarity with the beta-tubulins reported in other insects. In this study, we examine expression of this gene and microtubule abundance in relation to diapause and low temperature. While non-diapausing mosquitoes express beta-tubulin highly in their thoracic muscles, expression is quite low during adult diapause. The abundance of microtubules was also much lower in flight muscles of diapausing adults than in flight muscles from non-diapausing individuals, as confirmed by laser confocal microscopy of tubulins stained using indirect immunofluorescence. Low temperatures decreased microtubule abundance in midguts of non-diapausing mosquitoes, but microtubule abundance in diapausing mosquitoes was already low and remained unchanged by low temperature exposure. Overall, pixel intensity averages were higher in the flight muscles than in the midguts, and again low temperatures decreased microtubule abundance in the flight muscles of non-diapausing females, while levels remained consistently low in diapausing females. These results clearly indicate that a decrease in microtubule abundance is evoked both by the programming of diapause and, in non-diapausing females, by exposure to low temperatures. Quite possibly the reduced microtubule abundance in the flight muscles and reduced expression of beta-tubulin are functionally correlated to the reduction in flight activity that is associated with low temperature and diapause.

ABA Inhibits Embryo Cell Expansion and Early Cell Division Events During Coffee (Coffea arabica ‘Rubi’) Seed Germination

Coffee seed germination represents an interplay between the embryo and the surrounding endosperm. A sequence of events in both parts of the seed determines whether germination will be successful or not. Following previous studies, the aim here was to further characterize the morphology of endosperm degradation and embryo growth with respect to morphology and cell cycle, and the influence of abscisic acid on these processes. Growth of cells in a fixed region of the axis was quantified from light micrographs. Cell cycle events were measured by flow cytometry and by immunocytochemistry, using antibodies against beta-tubulin. Aspects of the endosperm were visualized by light and scanning electron microscopy. The embryonic axis cells grew initially by isodiametric expansion. This event coincided with reorientation and increase in abundance of microtubules and with accumulation of beta-tubulin. Radicle protrusion was characterized by a shift from isodiametric expansion to elongation of radicle cells and further accumulation of beta-tubulin. Early cell division events started prior to radicle protrusion. Abscisic acid decreased the abundance of microtubules and inhibited the growth of the embryo cells, the reorganization of the microtubules, DNA replication in the embryonic axis, the formation of a protuberance and the completion of germination. The endosperm cap cells had smaller and thinner cell walls than the rest of the endosperm. Cells in the endosperm cap displayed compression followed by loss of cell integrity and the appearance of a protuberance prior to radicle protrusion. Coffee seed germination is the result of isodiametric growth of the embryo followed by elongation, at the expense of integrity of endosperm cap cells. The cell cycle, including cell division, is initiated prior to radicle protrusion. ABA inhibits expansion of the embryo, and hence subsequent events, including germination.

Projections of low spontaneous rate, high threshold auditory nerve fibers to the small cell cap of the cochlear nucleus in cats

The marginal shell of the anteroventral cochlear nucleus houses small cells that are distinct from the overlying microneurons of the granule cell domain and the underlying projection neurons of the magnocellular core. This thin shell of small cells and associated neuropil receives auditory nerve input from only the low (<18 spikes/s) spontaneous rate (SR), high threshold auditory nerve fibers; high SR, low threshold fibers do not project there. It should be noted, that most of these auditory nerve terminations reside in the neuropil and intermix with dendrites that originate outside the shell. Consequently, electron microscopy is necessary to determine the synaptic targets. For this report, the terminations of intracellularly labeled low SR auditory nerve fibers in the small cell of cats cap were mapped through serial sections using a light microscope. The terminals were then examined with an electron microscope and found to form synapses with the somata and dendrites of small cells. Moreover, the small cell dendrites were identifiable by an abundance of microtubules and the presence of polyribosomes that were free or associated with membranous cisterns. These data contribute to the concept of a high threshold feedback circuit to the inner ear, and reveal translational machinery for local control of activity-dependent synaptic modification.

Mutations in the APC tumour suppressor gene cause chromosomal instability.

Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the beta-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.

Evidence that collapsin response mediator protein-2 is involved in the dynamics of microtubules.

Collapsin response mediator protein-2 (CRMP-2) is a member of the CRMP/TOAD/Ulip/DRP family of cytosolic phosphoproteins involved in neuronal differentiation and axonal guidance. CRMP-2 mediates the intracellular response to collapsin 1/semaphorin 3A, a repulsive extracellular guidance cue for axonal outgrowth. The mutation of UNC-33, a Caenorhabditis elegans homolog of CRMP-2, results in abnormality of microtubules in neurites, but the mechanism of CRMP-2 action remains to be clarified. Here, we report that overexpression of human CRMP-2 in Neuro2a cells, a mouse neuroblastoma cell line, results in blebbing of the cytoplasm. Furthermore, some cells exhibited intranuclear inclusions, which were labeled with antibodies to CRMP-2 and tubulin. CRMP-2 was found to be associated with microtubule bundles in the spindles at the metaphase and in the midbodies at the late telophase in mitotic cells. Thus, it is most likely that failure of complete disassembly of the spindle microtubules during mitosis is responsible for the formation of these intranuclear inclusions. We suggest that CRMP-2 functions by regulating the dynamics of microtubules.

Ducts of the labral glands of Leptestheria dahalacensis (Crustacea: Branchiopoda: Spinicaudata).

Inside the labrum of Leptestheria dahalacensis are situated three types of large epidermal gland cells, whose ducts open onto the outer dorsal surface of the labrum. SEM revealed that the thin ducts of the A-type gland cells open out behind the epipharynx at the end of small, conically shaped protuberances, the two paired ducts of the B-type gland cells lead into the distal portion of the labrum, and the external opening of the single duct of the C-type gland cells lies on the dorsal lobe of the labrum. The ducts of the three different gland cell types have the same fundamental constitution, but vary in diameter. Each secretory unit consists of a pair of gland cells (A, B, or C) and a secretory duct. The duct is formed by ring-shaped folding of one anteroposteriorly elongated epidermal cell (duct cell), whose ends adhere closely to one another. A further ring-folded epidermal cell (accessory cell), but flattened in shape, is interposed, like a sleeve-connection, between the gland cells and the duct cell. The reservoirs of gland cells open into the lumen of the duct. Discontinuous deposits of highly electron-dense matter are present on the plasma membrane of the accessory cell delimiting the initial part of the duct lumen, while the plasma membrane of the duct cell facing the lumen is cuticularized. The cytoplasm of the accessory cell, on examination by TEM, appears quite similar to that of the duct cell, except for the different distribution and greater abundance of microtubules. Similarly organized tricellular tegumental glands also commonly occur in other Crustacea, both Malacostraca and non-Malacostraca. Possible functions of secretions from the three different types of gland cells present in the labrum of L. dahalacensis are discussed.

Microtubule-organizing centers in the mitotic melanophores of Xenopus laevis larvae in vivo: ultrastructural study.

Mitotic melanophores of Xenopus laevis larvae at 51-53 stages of development were morphologically studied using light and electron microscopy, with special reference to their microtubule-organizing centers. These melanophores represented a highly branched cell shape in mitosis, each cell process is distributed with melanosomes without exhibiting any responsiveness to hormonal (melatonin) stimulation, and upon completion of mitosis, recovered the ability to translocate these granules in response to such a stimulus. At the metaphase, these cells contained bipolar or multipolar spindles, whose poles were composed of three zones: the centrosome with centrioles; the centrosphere; and an outlying radial arrangement of microtubules and their associated inclusions. In these mitotic melanophores, a number of microtubules are distributed within the radially stretching cell processes, whereas an abundance of microtubules reside in the spindles. Possible origins of the microtubules observed in these cytoplasmic processes are discussed in relation to the loss of the ability of pigment translocation during mitosis.

Embryology ofCymbidium sinense:the Microtubule Organization of Early Embryos

InCymbidium sinense, the pattern of embryo development is unusual in that oblique cell divisions result in the formation of several suspensor cells prior to the development of the embryo proper. Characteristic changes in microtubular distribution can be found within the zygote and the proembryo during their development. After fertilization, the ellipsoid-shaped zygote has randomly distributed microtubules within its cytoplasm. As the zygote takes on a more rounded appearance, microtubules organize into a dense meshwork. Furthermore, microtubule bundles appear at the chalazal region of the cell prior to the first mitotic division of the zygote. At the preprophase stage of mitosis, a preprophase band of microtubules appears in the cytoplasm of the zygote. The zygote divides obliquely and unequally and gives rise to an apical cell and a slightly larger basal cell. Many randomly-aligned microtubules can be found in the cortex of the basal cell. The increase in the abundance of microtubules coincides with the isotropic expansion of the basal cell. The early division of the basal cell and subsequent division of the apical cell results in the formation of a four-celled embryo, of which three cells near the micropylar pole develop as suspensor cells. In the suspensor cells, the microtubules tend to orient in the same direction as the long axis of the cell. In addition, prominent microtubules can also be found near the adjoining cell walls of the four-celled embryo. The terminal cell is highly cytoplasmic with abundant microtubules within the cell. Subsequent divisions of the terminal cell give rise to additional suspensor cells and the embryo proper. In the mature embryo, five suspensor cells are usually present; one eventually grows through the micropyle of the inner integument and four grow towards the chalazal pole. The cortical microtubules of suspensor cells redistribute from a longitudinal to a transverse direction as they grow towards their respective poles.

Organization of microtubules and microfilaments in protoplasts from suspension cells ofNicotiana plumbaginifolia: a quantitative analysis

SUMMARY The cortical cytoskeleton of suspension cells and protoplasts of Nicotiana plumbaginifolia was studied by means of fluorescent probes by a light microscope and by means of dry-cleaved preparations by an electron microscope. No differences in the organization of the cortical cytoskeleton of the suspension cells and protoplasts were observed. Generally, the cytoskeleton showed highly organized domains within superficially random-like patterns. Microtubules formed extensive bundles with up to 10 elements per cross-linked bundle. About 65 cross-bridges occurred per micrometre. The cross-links were 25–50 nm long and 5–20 nm in diameter. On average, 56% of the entire microtubular length was present in the cross-linked bundles. The length and abundance of microtubules ranged between 0·8 and 14·28 μm and 0·93–4·29 μm μm–2, respectively. Microtubules were regularly aligned with microfilaments, either as parallel or as ‘stitched’ filaments. These alignments did not occur over distances longer than 1·46 μm. The distribution of the coated pits appeared to depend on microtubular distribution. Coated pits were often connected with the microtubules by means of putative actin filaments.

Posttranslational modification of distinct microtubule subpopulations during cell polarization and differentiation in the mouse preimplantation embryo.

During the course of preimplantation development, the cells of the mouse embryo undergo both a major subcellular reorganization (at the time of compaction) and, subsequently, a process of differentiation as the phenotypes of trophectoderm and inner cell mass cell types diverge. We have used antibodies specific for tyrosinated (Kilmartin, J. V., B. Wright, and C. Milstein. 1982. J. Cell Biol. 93:576-582) and acetylated (Piperno, G., and M. T. Fuller. 1985. J. Cell Biol. 101:2085-2094) alpha-tubulin in immunofluorescence studies and found that subsets of microtubules can be distinguished within and between cells during the course of these events. Whereas all microtubules contained tyrosinated alpha-tubulin, acetylated alpha-tubulin was detected only in a subpopulation, located predominantly in the cell cortices. Striking differences developed between the distribution of the two populations during the course of development. Firstly, whereas the microtubule population as a whole tends to redistribute towards the apical domain of cells as they polarize during compaction (Houliston, E., S. J. Pickering, and B. Maro. 1987. J. Cell Biol. 104:1299-1308), the microtubules recognized by the antiacetylated alpha-tubulin antibody became enriched in the basal part of the cell cortex. After asymmetric division of polarized cells to generate two distinct cell types (termed inside and outside cells) we found that, despite the relative abundance of microtubules in outside cells, acetylated microtubules accumulated preferentially in inside cells. Treatment with nocodazole demonstrated that within each cell type acetylated microtubules were the more stable ones; however, the difference in composition of the microtubule network between cell types was not accompanied by a greater stability of the microtubule network in inside cells.

Transitional cells at the junction of seminiferous tubules with the rete testis of the rat: their fine structure, endocytic activity, and basement membrane.

Transitional cells line the intermediate region of rat seminiferous tubules situated between the rete testis and the seminiferous epithelium proper. These tall elongated cells orient themselves in a downstream direction and converge on one another distally in the lumen of the rete testis where they form a distinct papillalike structure through which a narrow patent lumen is apparent. In addition to widely dispersed Golgi apparatus and mitochondria, these cells contain an abundance of microtubules, cisternae of endoplasmic reticulum, and a distinct lobulated nucleus showing clumps of chromatin and a prominent nucleolus. The endocytic activity of these cells was examined by employing adsorptive (cationic ferritin, concanavalin A ferritin) and fluid-phase tracers (native ferritin, horseradish peroxidase-colloidal gold complex, and concanavalin A ferritin in presence of alpha methyl-D-mannoside). Such tracers were injected separately into the lumen of the rete testis, and the animals were killed at 2, 5, 15, and 30 min and 1, 2, and 6 hr after injection. At 2 min, both adsorptive and fluid-phase tracers were found within coated and uncoated pits of the apical plasma membrane of these cells as well as in large, subsurface, uncoated spherical, C-shaped, and tubular membranous elements. At 5 min the tracers were seen in endosomes of different sizes; while at 15 min and 30 min, pale and dense multivesicular bodies of small and large sizes, respectively, were labeled. At 1-hr and longer time intervals secondary lysosomes became labeled. While both fluid-phase and adsorptive tracers followed the same pathway and fate, binding to the apical and lateral plasma membranes of the transitional cells and to the membrane delimiting coated and uncoated pits was observed only with the adsorptive tracers. These results demonstrate that the transitional cells are actively involved in both fluid-phase and adsorptive endocytosis, which may play an important role in modifying the composition of the luminal fluid. The transitional cells of the distal zone of the intermediate region rest on an elaborate basement membrane (BM) complex which includes a thin BM immediately underlying these cells, a thick distal layer of BM, and strands of BM spanning the distance between the two in the form of a loose anastomotic network. Use of antisera against heparan sulfate proteoglycan, laminin, and type IV collagen revealed the presence of all three components within all areas of the BM complex. In the meshes of the anastomotic BM network, extracellular vesicles were observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Abundance of microtubules in preprophase bands of some Triticum species

Root tip procambial cells of Triticum speltoides, T. tauschii, T. turgidum and T. aestivum have been investigated ultrastructurally for the detection of preprophase microtubule bands (PMBs) and to estimate the number of microtubules comprising the bands. The species selected are phylogenetically related but differ in the ploidy level. It was found that all species develop well-defined PMBs prior to mitosis. Estimations of microtubule abundance in the PMBs was carried out in midpreprophase cells, a stage judged by a feature of the nucleus in which electron-transparent canals are formed around the initial condensations of the chromatin material and the nucleoli. Triticum speltoides bears the smaller average number of microtubules per PMB and T. aestivum the greater. The results indicate that the increase follows the upgrade of the number of chromosome sets. It is suggested that the average number of microtubules of PMBs is related to the ploidy level.

Taxol-induced neuropathy: further ultrastructural studies of nerve fibre changes in situ.

Nerve fibre changes have been further monitored morphologically in rat sciatic nerves which had been locally injected with taxol, an antimitotic drug known to promote microtubule assembly. Taxol caused a slowly progressive accumulation of microtubules over a three week period of experimentation, the effect being more pronounced in Schwann cells. Schwann cells of myelinated fibres became detached from nodes of Ranvier and were applied to naked internodes as bulbous cells replete with microtubules and smooth endoplasmic reticulum (ER). In some cases, the smooth ER was believed to arise from rough ER after the displacement of ribosomes. These cells also contained myelin fragments from the original internodes. Microtubules displayed unique relationships with cytoplasmic membranes and were seen to exist as regular 22 nm microtubules, as obliquely sectioned profiles or as incomplete, trough-shaped structures. Internodes devoid of myelin were common, and naked axons covered only by basal lamina appeared swollen and invariably contained an abundance of microtubules. Schwann cells lacking axons had multilobate nuclei and possessed complex arrays comprising smooth ER membranes and microtubules. In areas where microtubules were absent, these membranes compacted to form intracellular myelin. Intermediate filaments existed in lower numbers than normal within both axons and Schwann cells. Arrested mitoses were occasionally seen and it is speculated that together with the immobilization of the cell by the over-abundant polymerization of tubulin, incomplete mitosis was an underlying cause for the observed lack of remyelination over the 21-day period of study. These results suggest that perturbations in microtubule synthesis might dramatically affect Schwann cell behaviour and myelin proliferation.

Increased organization of cytoskeleton accompanying the aging of human fibroblasts in vitro

Fibroblastic cells of human origin have a limited lifespan in culture. One of the senescence-associated phenotypic changes is an increase in the abundance of cytoplasmic filaments. Human skin fibroblasts (strain 0011), derived from an 8-week-old male fetus, were passaged according to a predetermined schedule and examined at successive population doubling levels. In young rapidly growing cultures, fluorescence microscopy with NBD-Phallacidin shows a normal organization of the actin-containing fibers, microtubules and intermediate filaments, as has been described previously. At stages close to the end of the in vitro lifespan of the cell strain, large flat fibroblasts are the predominant cell type in culture. These large senescent fibroblasts contain numerous prominent actin fibers traversing the entire long axis of the cytoplasm. The fibers are often located adjacent to each other and appear to form a sheet on the ventral side of the cytoplasm. Staining of senescent cells with anti-tubulin antibody reveals an increase in the abundance of microtubules per cell and the distribution pattern is altered through the increase in the number of organization centers. Intermediate filaments are also more abundant and display tightly packed fibrillar sheets or bundles. Electron microscopic studies have confirmed the increased organization of microfilaments into bundles in senescent cells. These results suggest that during in vitro senescence, the increase in cell size is correlated with increased organization of the cytoskeleton. The presence of a rigid cytoskeletal structure may contribute in part to the inability of the senescent cell to replicate.

The long term culture of bulk-isolated bovine oligodendroglia from adult brain

Oligodendroglia isolated from adult bovine brain by the method of Farooq et al. could be plated on polylysine-coated plastic dishes with an efficiency of 55–80%, and maintained in culture for as long as 4 months. The addition of cytosine arabinoside to the nutrient medium resulted in cultures that were approximately 90% oligodendroglia and 10% large fibroblasts. From 50 g of white matter 100 − 160 × 10 6 oligodendroglia, containing approximately 6–10 mg protein, could be obtained in culture. These small round cells started to send out processes at 5 days in vitro and by 2 weeks they formed an extensive network of processes. By immunofluorescence, all cells of this morphology were positive for galactocerebroside (GC) and myelin basic protein (MBP), and negative for glial filament protein and fibronectin. Most of the large flat cells were positive for fibronectin and negative for GC, MBP and glial filament protein. As the cultures aged the oligodendroglia tented to clump and blebs formed on the surface of both perikarya and processes. By 4 months they showed evidence of degeneration and detached from the substrate. Electron microscopic examination showed that the cells had the appearance typical of oligodendroglia in situ. The somata were round to elliptical, with eccentrically placed nuclei, and were larger than freshly isolated cells. They grew directly on the substrate or on the surface of the fibroblasts. In older cultures the cells formed tight nests. The somata were enveloped by sheets of oligodendrocyte cytoplasm, sometimes having a myelin-like appearance. Gap junctions and small desmosomes were seen between oligodendroglial processes and between oligodendroglia and fibroblasts. The cytoplasm was characterized by a prominent Golgi apparatus, many mitochondria and lysosomes, scattered rough endoplasmic reticulum, free ribosomes, frequent centrioles and an abundance of microtubules. In cells from older cultures large vacuoles were common, and rarely they had multilamellar walls with alternating major and minor dense lines resembling myelin.

Microtubules in differentiating sieve elements of Gossypium hirsutum

Changes in distribution, orientation, and abundance of microtubules (MT) in relation to sieve element differentiation in root protophloem and stem and midvein metaphloem of Gossypium hirsutum were studied at the ultrastructural level. Depending on their stage of differentiation the sieve elements were divided into four previously characterized groups: Stage 1—the least differentiated cells; Stage 2—the differentiating cells; Stage 3—the nearly mature cells containing nuclear remnants; and Stage 4—the mature cells. In dividing cells of Stage 1, MT are observed only as part of the phragmoplast. After the division is completed MT, oriented at right angles to the long axis of the cell, begin to appear along the lateral walls. The number of MT increases, reaching a plateau and remaining relatively constant in successively older Stage 1 sieve elements. The increase in cell wall thickness is most obvious at the plateau phase, when the maximum numbers of MT are observed. The protoplast of a sieve element in Stage 1 contains a normal nucleus, dispersed plastids and mitochondria, and scattered ER cisternae. At Stage 1, MT in adjacent parenchyma cells are not abundant. During Stage 2 of sieve element differentiation, when nuclear chromatin aggregates, plastids and mitochondria assume a peripheral position in the cell, and ER cisternae accumulate into convoluted masses, the number of MT along the lateral walls decreases. In adjacent parenchyma cells the MT are abundant at this stage. MT have also been observed adjacent to sieve plates of cells in Stages 1 and 2. These MT appeared in transverse and longitudinal views in the same sections.