Spatial relationships of microtubule-organizing centers and the contact area of cytotoxic T lymphocytes and target cells

Specific binding (conjugation) of cytotoxic T lymphocytes (CTL) to target cells (TC) is the first step in a multistage process ultimately resulting in dissolution of the TC and recycling of the CTL. We examined the position of the microtubule organizing center (MTOC) of immune CTL bound to specific TC. Immunofluorescence labeling of freshly prepared CTL-TC conjugates with tubulin antibodies indicated that the MTOC in essentially all conjugated CTL but not in the conjugated TC were oriented towards the intercellular contact site. This finding was corroborated by electron microscopy examination of CTL-TC conjugates fixed either immediately after conjugation or during the lytic process. Antibody-induced caps of membrane antigens of CTL such as H-2 and Thy 1, did not show a similar relationship to the MTOC. Incubation of CTL- TC conjugates, 10-15 min at room temperature, resulted in an apparent deterioration of the microtubular system of conjugated CTL. It is proposed that the CTL plasma membrane proximal to the MTOC is particularly active in forming stable intercellular contacts, resulting in CTL-TC conjugation, and that subsequent modulation of the microtubular system of the CTL may be related to the cytolytic response and to detachment of the effector cell.     

Acentriolar microtubule organization centers and Ran‐mediated microtubule formation pathways are both required in porcine oocytes

Mammalian oocytes lack centrioles but can generate bipolar spindles using several different mechanisms. For example, mouse oocytes have acentriolar microtubule organization centers (MTOCs) that contain many components of the centrosome, and which initiate microtubule polymerization. On the contrary, human oocytes lack MTOCs and the Ran‐mediated mechanisms may be responsible for spindle assembly. Complete knowledge of the different mechanisms of spindle assembly is lacking in various mammalian oocytes. In this study, we demonstrate that both MTOC‐ and Ran‐mediated microtubule nucleation are required for functional meiotic metaphase I spindle generation in porcine oocytes. Acentriolar MTOC components, including Cep192 and pericentrin, were absent in the germinal vesicle and germinal vesicle breakdown stages. However, they start to colocalize to the spindle microtubules, but are absent in the meiotic spindle poles. Knockdown of Cep192 or inhibition of Polo‐like kinase 1 activity impaired the recruitment of Cep192 and pericentrin to the spindles, impaired microtubule assembly, and decreased the polar body extrusion rate. When the Ran^GTP gradient was perturbed by the expression of dominant negative or constitutively active Ran mutants, severe defects in microtubule nucleation and cytokinesis were observed, and the localization of MTOC materials in the spindles was abolished. These results demonstrate that the stepwise involvement of MTOC‐ and Ran‐mediated microtubule assembly is crucial for the formation of meiotic spindles in porcine oocytes, indicating the diversity of spindle formation mechanisms among mammalian oocytes.     

Microtubule distribution in dv, a maize meiotic mutant defective in the prophase to metaphase transition

Microsporogenesis in Zea mays, the meiotic reduction of diploid sporocytes to haploid microspores, proceeds through a well-defined developmental sequence. The ability to generate mutants that affect the process makes this an ideal system for elucidating the role of the cytoskeleton during plant development. We have used immunofluorescence microscopy to compare microtubule distribution in wild-type and mutant microsporocytes. During normal meiosis the distribution of microtubules follows a specific temporal and spatial pattern that reflects the polar nature of microspore formation. Perinuclear microtubule staining increases and the nucleus elongates in the future spindle axis during late prophase I. Metaphase I spindles with highly focused poles align along the long axis of the anther locule. Cytokinesis occurs perpendicular to the spindle axis. The second division axis shifts 90 degrees with respect to the first division plane, thereby yielding an isobilateral tetrad of microspores. Microtubule distribution patterns during meiosis suggest that a nuclear envelope-associated microtubule organizing center (MTOC) controls the organization of cytoplasmic microtubules and contributes to spindle formation. The meiotic mutant dv is defective in the transition from a prophase microtubule array to a metaphase spindle. Instead of converging to form focused poles, the metaphase spindle poles remain diffuse as in prometaphase. This defect correlates with several abnormalities in subsequent developmental events including the formation of multinucleate daughter cells, multiple microspindles during meiosis II, multiple phragmoplasts, polyads of microspores, and cytoplasmic microtubule foci. These results suggest that dv is a mutation that affects MTOC organization.     

Organization, nucleation, and acetylation of microtubules in Xenopus laevis oocytes: a study by confocal immunofluorescence microscopy

Anti-tubulin immunofluorescence and laser-scanning confocal microscopy were used to examine microtubule organization during Xenopus oogenesis (Dumont stages I-VI). Stage I oocytes contained a poorly ordered microtubule array, characterized by concentrations of microtubule in the cortex, surrounding the germinal vesicle, and associated with the mitochondrial mass. No focus of microtubule organization was detectable by optical sectioning or in microtubule regrowth experiments, suggesting that stage I oocytes lack a functional MTOC. The microtubule array becomes progressively more complex and polarized during oogenesis; an extensive array of microtubules and microtubule bundles was apparent in the animal hemisphere of stage VI oocytes, and a less ordered array was observed in the vegetal hemisphere. A dense network of microtubules surrounded the germinal vesicle, apparently extending from a tubulin- and microtubule-rich region of cytoplasm adjacent to the vegetal surface of the GV. The organization of microtubules in normal oocytes, in oocytes recovering from cold-induced microtubule depolymerization, and in enucleated oocytes, suggested that the germinal vesicle serves as an MTOC in stage VI oocytes. Antibodies to acetylated alpha-tubulin revealed numerous acetylated, presumably stable, microtubules in stage I and stage VI oocytes. The array of oocyte microtubules thus might function as a stable framework for the localization of developmentally important molecules and organelles during oogenesis.     

Self-organization of microtubule bundles in anucleate fission yeast cells

Self-organization of cellular structures is an emerging principle underlying cellular architecture. Properties of dynamic microtubules and microtubule-binding proteins contribute to the self-assembly of structures such as microtubule asters. In the fission yeast Schizosaccharomyces pombe, longitudinal arrays of cytoplasmic microtubule bundles regulate cell polarity and nuclear positioning. These bundles are thought to be organized from the nucleus at multiple interphase microtubule organizing centres (iMTOCs). Here, we find that microtubule bundles assemble even in cells that lack a nucleus. These bundles have normal organization, dynamics and orientation, and exhibit anti-parallel overlaps in the middle of the cell. The mechanisms that are responsible for formation of these microtubule bundles include cytoplasmic microtubule nucleation, microtubule release from the equatorial MTOC (eMTOC), and the dynamic fusion and splitting of microtubule bundles. Bundle formation and organization are dependent on mto1p (gamma-TUC associated protein), ase1p (PRC1), klp2p (kinesin-14) and tip1p (CLIP-170). Positioning of nuclear fragments and polarity factors by these microtubules illustrates how self-organization of these bundles contributes to establishing global spatial order.     

Mitosis-specific monoclonal antibodies block cleavage in amphibian embryos

By microinjecting monoclonal antibodies that bind specifically to mitotic and meiotic cells of a variety of species, we studied the biological activity of antigens recognized by these antibodies. The antibodies recognize a family of phosphoprotein antigens that are found throughout the cytoplasm of mitotic cells and particularly at microtubule organizing centers, including centrosomes and kinetochores. Their binding is dependent on phosphorylation of the polypeptides. Immunoglobulins were introduced into Xenopus laevis and Rana pipiens oocytes or cleaving embryos using glass micropipettes. The ability of the antibody-injected oocytes to undergo mitosis or meiosis was compared with those injected with control mouse immunoglobulins. The antibodies failed to block chromosome condensation and germinal vesicle breakdown in progesterone-treated oocytes. However, functional mitotic spindles were not assembled in cleavage stage frog embryos injected with antibodies. In vitro, the binding of the antibodies to the antigens inhibited the dephosphorylation of the antigens by alkaline phosphatase. The antibody binding to the activated microtubule organizing centers (MTOC) seems to block not only the nucleation of microtubules and the organization of the mitotic spindle, but also the dephosphorylation of proteins associated with the MTOC that normally occurs at the mitosis-G1 transition.     

γ-Tubulin: The hub of cellular microtubule assemblies

In eukaryotic cells a specialized organelle called the microtubule organizing center (MTOC) is responsible for disposition of microtubules in a radial, polarized array in interphase cells and in the spindle in mitotic cells. Eukaryotic cells across different species, and different cell types within single species, have morphologically diverse MTOCs, but these share a common function of organizing microtubule arrays. MTOCs effect microtubule organization by initiating microtubule assembly and anchoring microtubules by their slowly growing minus ends, thus ensuring that the rapidly growing plus ends extend distally in each microtubule array. The goal is to define molecular components of the MTOC responsible for regulating microtubule assembly. One approach to defining the molecules responsible for MTOC function is to look for molecules common to all MTOCs. A newly discovered centrosomal protein, γ-tubulin, is found in MTOCs in cells from many different organisms, and has several properties which make it a candidate for both initiation of microtubule assembly and anchorage. The hypothesis that γ-tubulin plays a role in MTOCs in microtubule initiation and anchorage is currently being tested by a variety of experimental approaches.     

Reorientation of the microtubule-organizing center and the Golgi apparatus in cloned cytotoxic lymphocytes triggered by binding to lysable target cells

By immunofluorescence observations with cell couples of cloned murine cytotoxic T lymphocytes (CTL) and target cells, evidence is presented for a rapid reorientation of the microtubule-organizing center (MTOC) and the Golgi apparatus (GA) in the effector cell (but not in the target cell) toward the contact area with the target. The reorientation of the MTOC/GA and the cytotoxic activity of the CTL were inhibited reversibly by nocodazole, a microtubule-disrupting agent. In lectin-formed cell couples of CTL and neuraminidase-treated target cells, the MTOC in essentially all of the CTL was oriented toward the effector-target contact area of a lysable target cell, but was left randomly oriented with a nonlysable target cell. A similar random orientation of the effector-MTOC was also observed in cell couples of cloned natural killer cells and nonlysable targets. These findings indicate that the repositioning of the MTOC and the GA, which is shared by CTL and natural killer cells, is an essential and early event in the onset of the cytolytic mechanism. It is suggested that this reorientation serves the purpose of directing to the bound target cell secretory vesicles derived from the GA that contain cytotoxic substances.     

Self-organization of treadmilling microtubules into a polar array

The centrosome is normally thought to determine the cell center and to dictate the formation of a radial array of microtubules that defines the spatial organization of cytoplasm. However, experiments indicate the existence of a mechanism for organization of a centered microtubule array that is independent of the centrosome. Here, we formulate a model of treadmilling dynamics of non-centrosomal microtubules that predicts a spontaneously established, polarized distribution of microtubule orientation. Based on this model, we propose that the autonomous ability of non-centrosomal microtubules to form a polarized array arises from their treadmilling within the space constrained by the cell boundary.     

A monoclonal antibody to animal centrosomes recognizes components of the basal-body root complex inChlamydomonas

Summary The mammalian centrosome monoclonal antibody MPM-13 recognized component(s) of the well defined MTOC basal-body root complex in the green plantChlamydomonas. The antibody reaction coincided in location with the basal-body root complex and the cruciate nature of the staining pattern corresponded to the configuration of the root microtubules. During mitosis the behaviour of MPM-13 stained material mirrored the duplication, separation and migration to the spindle poles of the basal body-root complex. It is suggested that conserved MTOC components were recognized and that these may have retained a similar, perhaps universal, function in microtubule organization.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidine-2-phenylindole dihydrochloride - mt mating type - MT microtubule - MTOC microtubule organizing centre - PFA paraformaldehyde - PBS phosphate buffered saline     

Interaction of an Overexpressed gamma-Tubulin with Microtubules In Vivo and In Vitro

gamma-Tubulin is an ubiquitous MTOC (microtubule-organizing center) component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for gamma-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus gamma-tubulin. When CHO cells were transiently transfected with the gamma-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such gamma-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that gamma-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas gamma-tubulin purified from insect Sf9 cells (), interaction between gamma-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with gamma-tubulin monomers in vitro were associated with more gold particles conjugated with gamma-tubulin than in controls where no exogenous gamma-tubulin was added. However, binding of gamma-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although gamma-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports (; ), which might be ascribed to the difference in the level of protein expression in transfected cells.     

The many faces of the bouquet centrosome MTOC in meiosis and germ cell development

Meiotic chromosomal pairing is facilitated by a conserved cytoskeletal organization. Telomeres associate with perinuclear microtubules via Sun/KASH complexes on the nuclear envelope (NE) and dynein. Telomere sliding on perinuclear microtubules contributes to chromosome homology searches and is essential for meiosis. Telomeres ultimately cluster on the NE, facing the centrosome, in a configuration called the chromosomal bouquet. Here, we discuss novel components and functions of the bouquet microtubule organizing center (MTOC) in meiosis, but also broadly in gamete development. The cellular mechanics of chromosome movements and the bouquet MTOC dynamics are striking. The newly identified zygotene cilium mechanically anchors the bouquet centrosome and completes the bouquet MTOC machinery in zebrafish and mice. We hypothesize that various centrosome anchoring strategies evolved in different species. Evidence suggests that the bouquet MTOC machinery is a cellular organizer, linking meiotic mechanisms with gamete development and morphogenesis. We highlight this cytoskeletal organization as a new platform for creating a holistic understanding of early gametogenesis, with direct implications to fertility and reproduction.     

Microtubule nucleating capacity of centrosomes in tissue sections.

We used a novel adaptation of methods for microtubule polymerization in vitro to assess the MTOC activity of centrosomes in frozen-sectioned tissues. Remarkably, centrosomes of tissue sections retain the ability to nucleate microtubules even after several years of storage as frozen tissue blocks. Adaptations of these methods allow accurate counts of microtubules from individual cells and the quantitative estimation the MTOC activity of the intact tissue. These methods can be utilized to characterize MTOC activity in normal and diseased tissues and in particular tissues at different stages of development. (J Histochem Cytochem 47:1265-1273, 1999)     

微管骨架在苔藓植物适应干旱胁迫应答中的功能研究

植物体通过一系列生理生化反应的改变来适应干旱胁迫。对干旱/复水及秋水仙素处理后再干旱/复水的仙鹤藓(Atrichum undulatum)原丝体细胞中微管骨架的动态变化进行了研究,发现干旱处理后细胞内微管骨架从有规律排列的较细的丝状形式转换为无规律排列的较粗的微管束;复水后微管骨架的结构和分布与对照细胞中无明显区别;秋水仙素处理后再干旱/复水的细胞中,微管骨架呈分散的棒状或点状分布,而且原丝体丧失了干旱胁迫后正常复水的能力,进而导致细胞不能恢复正常的生理活动。因此认为,微管骨架在仙鹤藓原丝体适应干旱逆境的过程中起着重要作用。     

The dynamics of microtubule repolymerization in a cell: rapid growth from the centrosome and slow recovery of free microtubules

According to the current view, the microtubule system in animal cells consists of two components: microtubules attached to the centrosome (these microtubules stretch radially towards the cell margin), and free microtubules randomly distributed in the cytoplasm without visible association with any microtubule-organizing centers. The ratio of the two sets of microtubules in the whole microtubule array is under discussion. Addressing this question, we have analysed the recovery of microtubules in cultured Vero nucleated cells and cytoplasts, with and without centrosomes in these. Cells were fixed at different time points, and individual microtubules were traced on serial optical sections. During a slow recovery after cold treatment (4 degrees C, for 4 h; recovery at 30 degrees C) polymerization of microtubules started mainly from the centrosome. At early stages of recovery the share of free microtubules made about 10% of all microtubules, and their total length increased slower than the lenght of centrosome-attached microtubules. During a rapid recovery after nocodazole treatment (10 microg/ml, 2 h; recovery in drug-free medium at 37 degrees C), the share of free microtubules was about 35%, but their total length increased slower than the length of centrosome-attached microtubules. In 6-8 min (rapid recovery) or 12-16 min (slow recovery), tips of centrosomal microtubules reached the cell margin, and their increased density made it impossible to recognize individual microtubules. However, under the same conditions in cytoplasts without centrosomes the normal number of microtubules recovered only in 60 min, which enabled us to suppose that the complete recovery of microtubule system in the whole cells may be also rather long. When the first centrosomal microtubules reached the cell margin, the optical density of microtubules started to decrease from the centrosome region towards the cell margin, according to the exponential curve. Later on, the optical density in the centrosome region and near the cell margin remained at the same level, but microtubule density increased in the middle part of the cell, and in 45-60 min the plot of the optical density vs the distance from the centrosome became linear, as in control cells. Since no significant curling of microtubules occurs near the cell margin, the density of microtubules in the endoplasm may increase due only to polymerization of free microtubules. We suppose that in cultured cells the microtubule network recovery proceeds in two stages. At the initial stage, a rapid growth of centrosomal microtubules takes place in addition to the turnover of free microtubules with unstable minus ends. At the second stage, when microtubule growth from the centrosome becomes limited by the cell margin, a gradual extension of free microtubules occurs in the internal cytoplasm.     

Protein complexes at the microtubule organizing center regulate bipolar spindle assembly

Bipolar spindle assembly is essential to genomic stability in dividing cells. Centrosomes or spindle pole bodies duplicated earlier at G1/S remain adjacent until triggered at mitotic onset to become bipolar. Pole reorientation is stabilized by microtubule interdigitation but mechanistic details for bipolarity remain incomplete. To investigate the contribution of spindle pole microtubule organizing center (MTOC) proteins in bipolarity, we applied genetic, structural and molecular biochemical analysis along with timelapse microscopy. Spindle formation was followed by an in vivo growth assay with the conditional allele cut7-22ts, encoding fission yeast mitotic Kinesin-5, essential for bipolarity. By analysis of double and triple mutant strains of MTOC alleles and cut7-22ts we found that stabilized microtubules or increased bundling can rescue cut7-22ts associated bipolarity defects. These changes to microtubule dynamics and organization occurred through two surface domains on γ-tubulin, a helix 11 domain and an adjacent site for binding MTOC protein Alp4. We demonstrate that Kinesin-14 Pkl1, known to oppose bipolarity, can bind to γ-tubulin at helix 11 and that mutation of either of two conserved residues in helix 11 can impair Kinesin-14 binding. Altering the Alp4/γ-tubulin interaction, conserved residues in helix 11 or deletion of pkl1 each are sufficient to rescue bipolarity in our cut7-22ts strain. Our findings provide novel insights into regulation of the bipolar mechanism through the MTOC complex.     

A mutation in gamma-tubulin alters microtubule dynamics and organization and is synthetically lethal with the kinesin-like protein pkl1p

Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. gamma-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in gamma-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30 degrees C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant gamma-tubulin is like the wild-type protein. Prediction of gamma-tubulin structure indicates that non-alpha/beta-tubulin protein-protein interactions could be affected. The kinesin-like protein (klp) Pkl1p localizes to the spindle poles and spindle and is essential for viability of the gamma-tubulin mutant and in multicopy for normal cell morphology at 30 degrees C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for gamma-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of gamma-tubulin that involves non-tubulin protein-protein interactions, presumably with a second motor, MAP, or MTOC component.     

Identification of microtubule-organizing centers in interphase melanophores of Xenopus laevis larvae in vivo.

The morphological characteristics of microtubule-organizing centers (MTOCs) in dermal interphase melanophores of Xenopus laevis larvae in vivo at 51-53 stages of development has been studied using immunostained semi-thick sections by fluorescent microscopy combined with computer image analysis. Computer image analysis of melanophores with aggregated and dispersed pigment granules, stained with the antibodies against the centrosome-specific component (CTR210) and tubulin, has revealed the presence of one main focus of microtubule convergence in the cell body, which coincides with the localization of the centrosome-specific antigen. An electron microscopy of those melanophores has shown that aggregation or dispersion of melanosomes is accompanied by changes in the morphological arrangement of the MTOC/centrosome. The centrosome in melanophores with dispersed pigment exhibits a conventional organization, and their melanosomes are situated in an immediate vicinity of the centrioles. In melanophores with aggregated pigment, MTOC is characterized by a three-zonal organization: the centrosome with centrioles, the centrosphere, and an outlying radial arrangement of microtubules and their associated inclusions. The centrosome in interphase melanophores is presumed to contain a pair of centrioles or numerous centrioles. Because of an inability of detecting additional MTOCs, it has been considered that an active MTOC in interphase melanophores of X. laevis is the centrosome. We assume that remaining intact microtubules in the cytoplasmic processes of mitotic melanophores (Rubina et al., 1999) derive either from the aster or the centrosome active at the interphase.     

Dictyostelium MTOC: Structure and linkage to the nucleus

Summary The microtubule organizing center (MTOC) was isolated fromDictyostelium discoideum to investigate the fine structure of the components as the first step in clarifying its molecular organization and function. The isolation protocol was designed to preserve microtubules bound to the MTOC by using indirect immunofluorescence employing anti--tubulin. After cell lysis with Triton X-100, the MTOCs were isolated in association with the nucleus by centrifugation in a microtubule-stabilizing buffer. The MTOC was found to be bound to the nucleus via an electron-dense fibrous structure, and this linkage could not be destroyed by KI, KCl, or sonication. We named this complex composed of microtubules, MTOC, and the anchor the MTOC-complex. Negative staining of the isolated MTOC-complex revealed that distinct vesicles decorated with 11-nm tacks were associated with microtubules radiating from the MTOC. Fine filaments, 4–5 nm wide, were also present close to the MTOC, aligned parallel to the microtubules. The three-dimensional profile of the central core of the MTOC, examined by transmission electron microscopy of serial thin sections of the isolated MTOC fraction supplemented by a microcomputer analysis, was concluded to be a matchbox-like cuboid (180 × 210 × 370 nm) of 15 layers.We propose that theDictyostelium MTOC is the structural domain of a more complicated unit composed of 1. MTOC, 2. microtubules, and 3. a firm fibrous linkage connecting the MTOC to the nucleus, with the MTOC core being a multilayered cuboid, associated with nodules and surrounded by amorphous electron-dense material including peculiar vesicles with 11 nm-tacks. The possible functions of these domains are discussed.     

The response of the Golgi complex to microtubule alterations: the roles of metabolic energy and membrane traffic in Golgi complex organization

A striking example of the interrelation between the Golgi complex (GC) and microtubules is the reversible fragmentation and dispersal of the GC which occurs upon microtubule depolymerization. We have characterized dispersal of the GC after nocodazole treatment as well as its recovery from the dispersed state by immunofluorescent localization of beta 1, 4-galactosyltransferase in Madin-Darby bovine kidney cells. Immunofluorescent anti-tubulin staining allowed simultaneous examination of the microtubule array. Based on our results, dispersal can be divided into a three-step process: microtubule depolymerization, GC fragmentation, and fragment dispersal. In cells treated with metabolic inhibitors after microtubule depolymerization, neither fragmentation nor dispersal occur, despite the absence of assembled microtubules. Thus, fragmentation is energy dependent and not tightly linked to microtubule depolymerization. The slowing of fragmentation and dispersal by monensin or ammonium chloride, as well as progressive inhibition at less than 34 degrees C, suggest that ongoing membrane traffic is required for these processes. Similarly, recovery may be separated into four steps: microtubule depolymerization, GC fragment centralization, fragment coalescence, and polarization of the reticular GC network. Fragment centralization and coalescence were arrested by metabolic inhibitors, despite the presence of microtubules. Neither monensin nor ammonium choride inhibited GC recovery. Partial inhibition of recovery at reduced temperatures paralleled the extent of microtubule assembly. These data demonstrate that dispersal and recovery are multi-step operations, and that the individual steps differ in temperature dependence, energy dependence, and sensitivity to ionic perturbation. GC distribution and microtubule status have also been clearly dissociate, thereby proving that organization of the GC is an active process that is not simply determined by microtubule binding. Furthermore, the results indicate that ongoing intra-GC membrane traffic may participate in fragmentation and dispersal.