Tubulin paracrystals in intermitotic nuclei of the foraminifer Allogromia laticollaris Arnold after treatment with vinblastine

Electron microscopic investigations on the foraminifer Allogromia laticollaris showed that after treatment with 10(-3) M vinblastine tubulin paracrystals can be demonstrated in intermitotic nuclei. As these paracrystals are either membrane coated or lie free in the karyoplasm, and as in the perinuclear cytoplasm, membrane coated paracrystals can be demonstrated as well, it is assumed that the cytoplasmic tubulin which is composing the intranuclear division spindle can transverse the intact nuclear envelope via vesicle transport.     

Metabolism in the protozoon Allogromia laticollaris Arnold

The foraminiferan Allogramia laticollaris, deprived of its natural food, Chlorella, incorporated and metabolized D-glucose added to culture fluid; L-glucose and inulin were not taken up. Fructose 6-phosphate and ATP were also incorporated, probably in a non-degraded form, and metabolized. Equal distribution of radioactivity of [gamma-32P]-ATP added to the medium was observed within 10 min. Intracellular glucose concentrations, and rates of respiration, glucose phosphorylation, and glycolysis were determined. Glucose utilization was stimulated with increasing concentrations of glucose, whereas respiration remained constant. The respiratory quotient increased in accordance with the higher contribution of glucose to energy supply. A. laticollaris can, then, be used for experimental studies of substrate utilization of a synthetic medium.     

Carotene Wax Pigments in the Foraminiferan Allogromia laticollaris Arnold

SYNOPSIS. It is evident from the results obtained by UV spectroscopy and chemical analyses that the pigments of the foraminiferan Allogromia laticollaris are carotene waxes of the xanthophyll type. Since none of the pigments was found in the food organisms, one can exclude the possibility that these pigments are derived from food. The origin of the pigments is discussed.     

Studies on the motility of the foraminifera. I. Ultrastructure of the reticulopodial network of Allogromia laticollaris (Arnold)

Allogromia laticollaris, a benthic marine foraminifer, extends numerous trunk filopodia that repeatedly branch, anastomose, and fuse again to form the reticulopodial network (RPN), within which an incessant streaming of cytoplasmic particles occurs. The motion of the particles is saltatory and bidirectional, even in the thinnest filopodia detected by optical microscopy. Fibrils are visible by differential interference microscopy, and the PRN displays positive birefringence in polarized light. These fibrils remain intact after lysis and extraction of the RPN in solutions that stabilize microtubules (MTs). Electron micrographs of thin sections through these lysed and stabilized cytoskeletal models reveal bundles of MTs. The RPNs of living Allogromia may be preserved by standard EM fixatives only after acclimatization to calcium-free seawater, in which the streaming is normal. The MTs in the RPN are typically arranged in bundles that generally lie parallel to the long axis of the trunk and branch filopodia. Stereo electron micrographs of whole-mount, fixed, and critical-point-dried organisms show that the complex pattern of MT deployment reflects the pattern of particle motion in both flattened and highly branched portions of the RPN. Cytoplasmic particles, some of which have a fuzzy coat, are closely associated with, and preferentially oriented along, either single MTs or MT bundles. Thin filaments (approximately 5 nm) are also observed within the network, lying parallel to and interdigitating with the MTs, and in flattened terminal areas of the filopodia. These filaments do not bind skeletal muscle myosin S1 under conditions that heavily decorate actin filaments in controls (human blood platelets), and are approximately 20% too thin to be identified ultrastructurally as F-actin.     

Studies on the motility of the foraminifera. II. The dynamic microtubular cytoskeleton of the reticulopodial network of Allogromia laticollaris

Lamellipodia have been induced to form within the reticulopodial networks of Allogromia laticollaris by being plated on positively charged substrata. Video-enhanced, polarized light, and differential interference contrast microscopy have demonstrated the presence of positively birefringent fibrils within these lamellipodia. The fibrils correspond to the microtubules and bundles of microtubules observed in whole-mount transmission electron micrographs of lamellipodia. Microtubular fibrils exhibit two types of movements within the lamellipodia: lateral and axial translocations. Lateral movements are often accompanied by reversible lateral associations between adjacent fibrils within a lamellipodium. This lateral association-dissociation of adjacent fibrils has been termed 'zipping' and 'unzipping'. Axial translocations are bidirectional. The axial movements of the microtubular fibrils can result in the extension of filopodia by pushing against the plasma membrane of the lamellipodia. Shortening, or complete withdrawal, of such filopodia is accomplished by the reversal of the direction of the axial movement. The bidirectional streaming characteristic of the reticulopodial networks also occurs within the lamellipodia. In these flattened regions the streaming is clearly seen to occur exclusively in association with the intracellular fibrils. Transport of both organelles and bulk hyaline cytoplasm occurs bidirectionally along the fibrils.     

Enzyme Activity Pattern and Mitochondrio-cytoplasmic Relations in Protozoa. Comparative Study of Tetrahymena pyriformis, Allogromia laticollaris, and Labyrinthula coenocystis

SYNOPSIS. Enzyme activity patterns were determined in Tetrahymena pyriformis, Allogromia laticollaris , and Labyrinthula coenocystis. The activities of enzymes belonging to certain metabolic pathways (glycolysis, fatty acid oxidation, tricarboxylic acid cycle) had constant proportions in each species, whereas their absolute levels differed. The activities of intramitochondrial enzymes were compared with the mitochondrial to total cytoplasmic volume ratio. Despite the very different fine structure of the mitochondria in the 3 protozoa, there was good correlation between the activities of citric acid cycle enzymes and the mitochondrial volume.     

Plasmodium gallinaceum: critical role for microtubules in the transformation of zygotes into Ookinetes

The role of microtubules and microfilaments in the transformation of spherical zygotes of Plasmodium gallinaceum (avian malaria parasite) into vermiform ookinetes has been studied by using specific drugs (taxol, colchicine, and cytochalasin-B). Both taxol and colchicine completely abolished the transformation of zygotes into ookinetes. The inhibitory effect was seen only if the drugs were added during the initial 6 hr of total time (20-24 hr) required for complete transformation; the addition of drugs after 6-8 hr of initiation of transformation had no effect. Electron microscopy revealed that microtubules were depolymerized by colchicine treatment, whereas in taxol-treated cells there was an extensive array of cytoplasmic and nuclear microtubules which appeared to be clumped in bundles. In contrast to the effects of taxol and colchicine, cytochalasin-B, which affects the microfilament system, had no effect on the transformation. Protein synthesis and expression of two ookinete-specific surface proteins were not affected in the drug-inhibited parasites. Zygotes treated with taxol for 4 hr at room temperature failed to develop into oocysts when they were subsequently fed to mosquitoes. These studies demonstrate a critical role for microtubules in the initial stages of transformation of zygotes into ookinetes.     

Actin filaments connected with the microtubules of lipotubuloids, cytoplasmic domains rich in lipid bodies and microtubules

Summary. Lipotubuloids, i.e., cytoplasmic domains containing an agglomeration of lipid bodies surrounded by half-unit membrane, entwined and held together by a system of microtubules, have been found in the ovary epidermis of Ornithogalum umbellatum. Ultrastructural studies demonstrated thin filaments in lipotubuloids that are probably actin filaments arranged parallel to microtubules. It is suggested that interaction of actin filaments with the microtubules determines the driving force for the rotary motion characteristic of lipotubuloids, as this movement is sensitive to cytochalasin B. Correspondence: Department of Cytophysiology, University of Łódź, Pilarskiego 14, 90-231 Łódź, Poland.     

New arrays of cytoplasmic microtubules in the fission yeastSchizosaccharomyces pombe

Summary New arrays of microtubules in the fission yeastSchizosaccharomyces pombe, which distribute in the cell in a cell cycle-dependent manner, were characterized using conventional and confocal laser scanning immunofluorescence microscopy. During the interphase and prophase, we observed abundant cytoplasmic microtubules between cell poles, a peripheral network of randomly and helically distributed cortical microtubules, and perinuclear microtubules surrounding the nucleus. At the anaphase and telophase, an equatorial ring containing tubulin was visualized. This ring colocalized with an actin contractile ring, suggesting that they may control the plane of cell division cooperatively.Abbreviations MT(s) microtubule(s) - cMT(s) cytoplasmic microtubule(s) - CLSM confocal laser scanning microscopy - DAPI 4,6-diamidino-2-phenylindole     

Role of cytoplasmic dynein in the axonal transport of microtubules and neurofilaments

Recent studies have shown that the transport of microtubules (MTs) and neurofilaments (NFs) within the axon is rapid, infrequent, asynchronous, and bidirectional. Here, we used RNA interference to investigate the role of cytoplasmic dynein in powering these transport events. To reveal transport of MTs and NFs, we expressed EGFP-tagged tubulin or NF proteins in cultured rat sympathetic neurons and performed live-cell imaging of the fluorescent cytoskeletal elements in photobleached regions of the axon. The occurrence of anterograde MT and retrograde NF movements was significantly diminished in neurons that had been depleted of dynein heavy chain, whereas the occurrence of retrograde MT and anterograde NF movements was unaffected. These results support a cargo model for NF transport and a sliding filament model for MT transport.     

Analysis of cytoplasmic microtubules and flagellar roots in the zoospores ofAllomyces macrogynus

Summary Cytoskeletal and flagellar microtubules in the zoospores of the aquatic fungusAllomyces macrogynus are resistant to microtubule depolymerizing drugs. Consequently, we have analyzed the partial composition and organization of microtubules (Mts) in the cytoplasm and flagellar apparatus in the zoospores ofA. macrogynus. Evidence from two-dimensional gel electrophoresis demonstrated the presence of two -tubulin isoforms in axonemal and cytoplasmic Mts. In addition, a monoclonal antibody specific for acetylated -tubulin was used on one-dimensional protein blots to show that acetylated -tubulins are present in isolated zoospore cell bodies and axonemes. Immunofluorescence microscopy observations using this monoclonal antibody demonstrated that flagellar, kinetosomal, and cytoplasmic Mts were labeled. The nature of Mts in the flagellar apparatus was studied ultrastructurally. InA. macrogynus, the flagellar apparatus consists of the kinetosome, rhizopolast (striated flagellar rootlet), axoneme, and 9 sets of triplet Mts which radiate anteriorly from the proximal end of the kinetosome (microtubular rootlet), Analysis of the rhizoplast indicated that this structure does not contain Mts. The rhizoplast, which connects the functional kinetosome with a single, large basal mitochrondrion, consists of four electron-opaque bands. Serial-sectioning indicated that the rhizoplast is always adjacent to kinetosome triplets 1, 2, and 9, and thus lies perpendicular to the plane of flagellar beat. These results suggest that the primary function of the rhizoplast is to organize the kinetosome and mitochondrion with respect to one another and to bias flagellar beat in the appropriate orientation for cell motility.Abbreviations BSA bovine serum albumin - BCA bicinchoninic acid - DS dilute salts - EGTA ethylene glycol-bis-(-aminoethyl ether)-N,N-tetracetic acid - EM electron microscopy - Mes 2-(N-morpholinomethane sulfonic acid - Mt microtubule - NP-40 Nonidet P-40 - 1-D PAGE one-dimensional polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - 2-D PAGE two-dimensional polyacrylamide gel electrophoresis - Tween-20 polyoxyethylenesorbitan monolaurate     

Specific visualization of tubulin-containing structures in tissue culture cells by immunofluorescence. Cytoplasmic microtubules, vinblastine-induced paracrystals, and mitotic figures.

Antibody against tubulin from the outer doublets of sea urchin sperm flagella reacts with tubulin-containing structures in mammalian cells. Thus cytoplasmic microtubules, vinblastine-induced paracrystals and the full spectrum of mitotic figures can be visualized by immunofluorescence. These results show that the tubulin structure has been highly conserved during evolution.     

Formation of cytoplasmic turns between two closely spaced transmembrane helices during membrane protein integration into the ER membrane

The helical hairpin, two closely spaced transmembrane helices separated by a short turn, is a recurring structural element in integral membrane proteins, and may serve as a compact unit that inserts into the membrane en bloc. Previously, we have determined the propensities of the 20 natural amino acids, when present in the middle of a long hydrophobic stretch, to induce the formation of a helical hairpin with a lumenally exposed turn during membrane protein assembly into the endoplasmic reticulum membrane. Here, we present results from a similar set of measurements, but with the turn placed on the cytoplasmic side of the membrane. We find that a significantly higher number of turn-promoting residues need to be present to induce a cytoplasmic turn compared to a lumenal turn, and that, in contrast to the lumenal turn, the positively charged residues Arg and Lys are the strongest turn-promoters in cytoplasmic turns. These results suggest that the process of turn formation between transmembrane helices is different for lumenal and cytoplasmic turns.     

微管解聚对生长因子在DNA合成中的作用

PPP (platelet-poor plasma) alone can not stimulate DNA synthesis in Go C3H/10T1/2 cells.50 ng/ml of EGF promoted partial Go cells to enter S phase. However, there was an apparent synergic effect of simultaneous treatment with 50 ng/ml EGF and 5%PPP, their synergic effect to stimulate DNA synthesis in Go cells was the same as 10% calf serum. Taxol can resist the depolymerization of microtubules. After treatment with taxol (10 mumol/L), the progression from Go to S phase in C 3 H 10 T 1/2 cells was inhibited. This inhibition was especially exhibited at early stage of transition from Go to S phase. The result indicated that Go cells can not enter S phase without the depolymerization of microtubules. It showed that DNA synthesis was stimulated by the simultaneous treatment with colcemid (0.1 microgram/ml) and growth factors (50 ng/ml EGF + 5% PPP or 10% Calf serum). But without the stimulation of growth factors, the unique effect of depolymerization of microtubules can not stimulate DNA synthesis. The results present evidence indicating that the depolymerization of microtubules has the potency to elevate DNA synthesis in Go cells stimulated by growth factors. This potency was also appeared at early stage of progression from Go to S phase. We suggest that the depolymerization of cytoplasmic microtubules and synergic effect of growth factors are involved in account for the transition from Go to S phase in C 3 H 10 T 1/2 cells.     

Tubulin assembly sites and the organization of cytoplasmic microtubules in cultured mammalian cells

The number, distribution, and nucleating capacity of microtubule- organizing centers (MTOCs) has been investigated in a variety of cultured mammalian cells. Most interphase cells contain a single MTOC that is localized at the centrosome region and corresponds to the centriole and pericentriolar material. MTOCs, like centrioles, become duplicated during the S phase of the cell cycle and are equationally distributed to daughter cells in mitosis. Multiple MTOCs were rarely observed in cultured cells except in one cell line (neuroblastoma), which also displayed an equally large number of centrioles in the cytoplasm. The kinetics of microtubule assembly and the tubulin nucleating capacity of MTOCs was assayed by incubating tubulin- depleted, permeabilized 3T3 and simian virus 40-transformed 3T3 cells with phosphocellulose-purified 65 brain tubulin and microtubule assembly buffer. Initiation and assembly of 65 tubulin occurred in association with the cells' endogenous MTOCs, and the length, number, and distribution of microtubules generated about the organizing centers were regulated and cell specific. Our results are consistent with the notion that the specification of microtubule length, number, and spatial arrangement resides largely in the MTOCs and surrounding cytoplasm and not in the tubulin subunits.     

Organization of the flagellar apparatus and associate cytoplasmic microtubules in the quadriflagellate alga Polytomella agilis

The organization of microtubular systems in the quadriflagellate unicell Polytomella agilis has been reconstructed by electron microscopy of serial sections, and the overall arrangement confirmed by immunofluorescent staining using antiserum directed against chick brain tubulin. The basal bodies of the four flagella are shown to be linked in two pairs of short fibers. Light microscopy of swimming cells indicates that the flagella beat in two synchronous pairs, with each pair exhibiting a breast-stroke-like motion. Two structurally distinct flagellar rootlets, one consisting of four microtubules in a 3 over 1 pattern and the other of a striated fiber over two microtubules, terminate between adjacent basal bodies. These rootlets diverge from the basal body region and extend toward the cell posterior, passing just beneath the plasma membrane. Near the anterior part of the cell, all eight rootlets serve as attachment sites for large numbers of cytoplasmic microtubules which occur in a single row around the circumference of the cell and closely parallel the cell shape. It is suggested that the flagellar rootless may function in controlling the patterning and the direction of cytoplasmic microtubule assembly. The occurrence of similar rootlet structures in other flagellates is briefly reviewed.