A new model of reticulopodial motility and shape: evidence for a microtubule-based motor and an actin skeleton

Cytoskeletal inhibitors were used as probes to test the involvement of microtubules and actin microfilaments in the development, motility, and shape maintenance of the pseudopodial networks (i e, reticulopodia) of the foraminifers Allogromia sp strain NF and Allogromia laticollaris. Agents that disassemble cytoplasmic microtubules (cold, colchicine, and nocodazole) arrest all movement but have variable effects on reticulopodial shape. Electron microscopy reveals a granulofibrillar matrix but few, if any, microtubules in these motility-arrested reticulopods. Allogromiids treated with cytochalasin B or D lose substrate adhesion and undergo dramatic changes in shape and motile behavior, highlighted by the coalescence of reticulopodial cytoplasm into irregularly shaped bodies with chaotic motility. Serial semithick sections of such preparations, viewed by high-voltage electron microscopy, document a striking rearrangement of microtubules within these cytochalasin-induced bodies. All aspects of cytochalasin-altered motility are completely inhibited by colchicine. Actin is present in reticulopodia, as determined by staining with rhodamine-phalloidin; this staining is not observed in cytochalasin-treated organisms. These data provide compelling evidence that microtubules are required for reticulopodial motility. An actin-based cytoskeleton is thought to play a role in maintaining shape, mediating pseudopod/substrate adhesion, and coordinating the various microtubule-dependent processes.     

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.     

Cadmium effects on the organization of microtubular cytoskeleton in interphase and mitotic cells of Allium sativum

We have investigated the effects of cadmium on the microtubular (MT) cytoskeleton in the root tip cells of Allium sativum L. using indirect immunofluorescence microscopy. Cd affected the mechanisms controlling the organization of MT cytoskeleton, as well as tubulin assembly/disassembly processes. Cd induced the formation of abnormal MT arrays, consisting of discontinuous wavy MTs or short MT fragments at the cell periphery. Cadmium caused irregular nuclear disorder in cells where the MT organization and function was disturbed. Furthermore, with increased Cd concentration and duration of treatment the MTs depolymerized more severely, the frequency of abnormal cell increased and the mitotic index decreased progressively. The above findings showed that MT cytoskeleton is one of target sites of Cd toxicity in root tip cells.     

Pb/Cu effects on the organization of microtubule cytoskeleton in interphase and mitotic cells of Allium sativum L.

The effects of lead and copper on the arrangement of microtubule (MT) cytoskeleton in root tip cells of Allium sativum L. were investigated. Batch cultures of garlic were carried out under defined conditions in the presence 10⁻⁴ M Pb/Cu of various duration treatments. With tubulin immunolabelling and transmission electron microscopy (TEM), we found four different types of MT structures depending on the cell cycle stage: the interphase array, preprophase band, mitotic spindle and phragmoplast were typical for the control cells. Pb/Cu affected the mechanisms controlling the organization of MT cytoskeleton, and induces the following aberrations in interphase and mitotic cells. (1) Pb/Cu induced the formation of atypical MT arrays in the cortical cytoplasm of the interphase cells, consisting of skewed, wavy MT bundles, MT fragments and ring-like tubulin aggregations. (2) Pb/Cu disordered the chromosome movements carried out by the mitotic spindle. The outcome was chromosome aberrations, for example, chromosome bridges and chromosome stickiness, as well as inhibition of cells from entering mitosis. (3) Depending on the time of exposure, MTs disintegrated into shorter fragments or they completely disappeared, indicating MT depolymerization. (4) Different metals had different effects on MT organization. MTs were more sensitive to the pressure of Cu ions than Pb. Moreover, TEM observations showed that the MTs were relatively short and in some places wavy when exposed to 10⁻⁴ M Pb/Cu solutions for 1–2 h. In many sections MTs were no longer visible with increasing duration of treatment (>4 h). Based on these results, we suggested that MT cytoskeleton is primarily responsible for Pb/Cu-associated toxicity and tolerance in plants.     

Surface transport properties of reticulopodia: do intracellular and extracellular motility share a common mechanism?

The reticulopodial networks of the foraminiferan protozoans Allogromia sp., strain NF, and A. laticollaris display rapid (up to 11 microns/second) and bidirectional saltatory transport of membrane surface markers (polystyrene microspheres). Electron microscopy shows that microspheres adhere directly to the reticulopodial surface glycocalyx. A videomicroscopic analysis of this phenomenon reveals that microsphere movement is typically independent of pseudopod extension/withdrawal and that particles of different sizes and surface properties display similar motile characteristics. The motile properties of surface-associated microspheres appear identical to those of saltating intracellular organelles. Indeed, in some instances the surface-attached microspheres appear transiently linked in motion to these underlying organelles. Our observations suggest that, in reticulopodia, surface transport of microspheres and intracellular transport of organelles are driven by a common mechanism.     

Root contraction in hyacinth. II. Changes in tubulin levels,microtubule number and orientation associated with differential cell expansion

Root contraction in hyacinth (Hyacinthus orientalis L.) is marked by reoriented cell growth in the cortex of the contractile region. Cellular volume of the inner cortex enlarges fourfold during root contraction. This is associated with large increases in the radial and tangential dimensions and decreases in the longitudinal dimension of the cells. In order to determine the possible role of microtubules (MTs) in these changes we compared tubulin levels and MT numbers and orientation in contracted and non-contracted regions of hyacinth roots. Tubulin content was analysed by a radioimmunoassay; MT numbers and orientation were analyzed by counting profiles in sectioned material using transmission electron microscopy. Contracted tissue was found to have significantly higher levels of tubulin on a per-cell basis than non-contracted tissue, and also increased tubulin levels relative to total protein. The spatial MT frequencies were the same in contracted and non-contracted tissues, indicating a proportional increase in MT numbers in the expanded cells. Although the absolute spatial frequency of MTs was constant, the orientation, as determined by morphometric analysis of MT profiles, was not. While in the longitudinal section plane 42% of the MTs in the non-contracted cells were oblique, in the contracted cells the percentage of MTs presenting oblique profiles increased to 87%. Additionally, a qualitative difference in MTs was observed in contracted cells; electron-opaque material was seen peripherally associated with the MTs of the inner cortex. The changes in tubulin levels and in MT numbers as well as the qualitative differences in the MTs of contracted and non-contracted root regions indicate that, in hyacinth, reoriented cellular enlargement associated with root contraction cannot be explained simply by shifts in the arrangement of preexisting cortical MT arrays, but involves more complex changes in the cytoskeleton.Abbreviations MT(s) microtubule(s) - TEM transmission electron microscopy - RIA radioimmunoassay - M_r apparent molecular mass I=Jernstedt (1984b)     

Digestion of prey in foraminifera is not anomalous: A correlation of light microscopic, cytochemical, and hvem technics to study phagotrophy in two allogromiids

Correlative light, high-voltage electron and conventional electron microscopic methods were used to investigate digestion in two allogromiid foraminiferans, Allogromia sp., strain NF, and A. laticollaris Arnold. Microscopic observations showed that bacterial prey are phagocytosed by reticulopodia and are transported to the allogromiid cell body within blister-like phagosomes. Larger prey (algae, diatoms) are transported along the reticulopodial surface and are either stored extrathalamously or phagocytosed at the oral opening (peduncle). Studies of allogromiids optimally fixed and labeled with an extracellular-space label (colloidal thorium) showed that phagocytosed prey are completely enclosed by a plasma membrane envelope; this finding was corroborated by a serial-section three-dimensional reconstruction of the oral zone of one allogromiid. Cytochemical staining for acid phosphatase showed that lysosomes are absent from reticulopods but abundant in the cell body, particularly in the oral zone cytoplasm. We conclude that digestion in allogromiid foraminiferans is accomplished by a vacuole-based digestive apparatus and not by extracellular digestion within a lacunary system, as has been suggested in earlier studies.     

Mechanical Regulation of Neurite Polarization and Growth: A Computational Study

The densely packed microtubule (MT) array found in neuronal cell projections (neurites) serves two fundamental functions simultaneously: it provides a mechanically stable track for molecular motor-based transport and produces forces that drive neurite growth. The local pattern of MT polarity along the neurite shaft has been found to differ between axons and dendrites. In axons, the neurons’ dominating long projections, roughly 90% of the MTs orient with their rapidly growing plus end away from the cell body, whereas in vertebrate dendrites, their orientations are locally mixed. Molecular motors are known to be responsible for cytoskeletal ordering and force generation, but their collective function in the dense MT cytoskeleton of neurites remains elusive. We here hypothesized that both the polarity pattern of MTs along the neurite shaft and the shaft’s global extension are simultaneously driven by molecular motor forces and should thus be regulated by the mechanical load acting on the MT array as a whole. To investigate this, we simulated cylindrical bundles of MTs that are cross-linked and powered by molecular motors by iteratively solving a set of force-balance equations. The bundles were subjected to a fixed load arising from actively generated tension in the actomyosin cortex enveloping the MTs. The magnitude of the load and the level of motor-induced connectivity between the MTs have been varied systematically. With an increasing load and decreasing motor-induced connectivity between MTs, the bundles became wider in cross section and extended more slowly, and the local MT orientational order was reduced. These results reveal two, to our knowledge, novel mechanical factors that may underlie the distinctive development of the MT cytoskeleton in axons and dendrites: the cross-linking level of MTs by motors and the load acting on this cytoskeleton during growth.     

Actomyosin is Involved in the Organization of the Microtubule Preprophase Band in Arabidopsis Suspension Cultured Cells

The microtubule preprophase bands （PPBs） participate in the sequence of events to position cell plates in most plants. However, the mechanism of PPB formation remains to be clarified. In the present study, the organization of PPBs in Arabidopsis suspension cultured cells was investigated by confocal laser scanning microscopy combined with pharmacological treatments of reagents specific for the cytoskeleton elements. Double staining of F-actin and microtubules （MTs） showed that actin filaments were arranged randomly and no colocalization with cortical MTs was observed in the interphase cells. However, cortical actin filaments showed colocalization with MTs during the formation of PPBs. A broad actin band formed with the broad MT band in the initiation of PPB and narrowed down together with the MT band to form the PPB. Nevertheless, broad MT bands were formed but failed to narrow down in cells treated with the F-actin disruptor latrunculin A. In contrast, in the presence of the F-actin stabilizer phalloidin, PPB formation did not exhibit any abnormality. Therefore, the integrity, but not the dynamics, of the actin cytoskeleton is necessary for the formation of normal PPBs. Treatment with 2, 3-butanedine monoxime, a myosin inhibitor, also resulted in the formation of broad MT bands, indicating that actomyosin may be involved in the rearrangement of MTs to form the PPBs. Double staining of MTs and myosin revealed that myosin concentrated on the PPB region during PPB formation. It is suggested that the actin cytoskeleton at the PPB site may serve as a rack to transport cortical MTs by using myosin when the broad MT band narrows down to form the PPB.     

Dynein-dependent motility of microtubules and nucleation sites supports polarization of the tubulin array in the fungus Ustilago maydis

Microtubules (MTs) are often organized by a nucleus-associated MT organizing center (MTOC). In addition, in neurons and epithelial cells, motor-based transport of assembled MTs determines the polarity of the MT array. Here, we show that MT motility participates in MT organization in the fungus Ustilago maydis. In budding cells, most MTs are nucleated by three to six small and motile gamma-tubulin-containing MTOCs at the boundary of mother and daughter cell, which results in a polarized MT array. In addition, free MTs and MTOCs move rapidly throughout the cytoplasm. Disruption of MTs with benomyl and subsequent washout led to an equal distribution of the MTOC and random formation of highly motile and randomly oriented MTs throughout the cytoplasm. Within 3 min after washout, MTOCs returned to the neck region and the polarized MT array was reestablished. MT motility and polarity of the MT array was lost in dynein mutants, indicating that dynein-based transport of MTs and MTOCs polarizes the MT cytoskeleton. Observation of green fluorescent protein-tagged dynein indicated that this is achieved by off-loading dynein from the plus-ends of motile MTs. We propose that MT organization in U. maydis involves dynein-mediated motility of MTs and nucleation sites.     

The microtubule cytoskeleton does not integrate auxin transport and gravitropism in maize roots

The Cholodny-Went hypothesis of gravitropism suggests that the graviresponse is controlled by the distribution of auxin. However, the mechanism of auxin transport during the graviresponse of roots is still unresolved. To determine whether the microtubule (MT) cytoskeleton is participating in auxin transport, the cytoskeleton was examined and the movement of 3 H-IAA measured in intact and excised taxol, oryzalin, and naphthylphthalamic acid (NPA)-treated roots of Zea mays cv. Merit. Taxol and oryzalin did not inhibit the graviresponse of roots but the auxin transport inhibitor NPA greatly inhibited both auxin transport and graviresponse. NPA had no effect on MT organization in vertical roots, but caused MT reorientation in horizontally placed roots. Regardless of treatment, the organization of MTs in intact roots differed from that in root segments. The MT inhibitors, taxol and oryzalin had opposite effects on the MTs, namely, depolymerization (oryzalin) and stabilization and thickening (taxol), but both treatments caused swelling of the roots. The data indicate that the MT cytoskeleton does not directly interfere with auxin transport or auxin-mediated growth responses in maize roots.     

Quantitative analysis of microtubule transport in growing nerve processes

In neurons, tubulin is synthesized primarily in the cell body, whereas the molecular machinery for neurite extension and elaboration of microtubule (MT) array is localized to the growth cone region. This unique functional and biochemical compartmentalization of neuronal cells requires transport mechanisms for the delivery of newly synthesized tubulin and other cytoplasmic components from the cell body to the growing axon. According to the polymer transport model, tubulin is transported along the axon as a polymer. Because the majority of axonal MTs are stationary at any given moment, it has been assumed that only a small fraction of MTs translocates along the axon by saltatory movement reminiscent of the fast axonal transport. Such intermittent "stop and go" MT transport has been difficult to detect or to exclude by using direct video microscopy methods. In this study, we measured the translocation of MT plus ends in the axonal shaft by expressing GFP-EB1 in Xenopus embryo neurons in culture. Formal quantitative analysis of MT assembly/disassembly indicated that none of the MTs in the axonal shaft were rapidly transported. Our results suggest that transport of axonal MTs is not required for delivery of newly synthesized tubulin to the growing nerve processes.     

A live-cell marker to visualize the dynamics of stable microtubules throughout the cell cycle

The microtubule (MT) cytoskeleton underlies processes such as intracellular transport and cell division. Immunolabeling for posttranslational modifications of tubulin has revealed the presence of different MT subsets, which are believed to differ in stability and function. Whereas dynamic MTs can readily be studied using live-cell plus-end markers, the dynamics of stable MTs have remained obscure due to a lack of tools to directly visualize these MTs in living cells. Here, we present StableMARK (Stable Microtubule-Associated Rigor-Kinesin), a live-cell marker to visualize stable MTs with high spatiotemporal resolution. We demonstrate that a rigor mutant of Kinesin-1 selectively binds to stable MTs without affecting MT organization and organelle transport. These MTs are long-lived, undergo continuous remodeling, and often do not depolymerize upon laser-based severing. Using this marker, we could visualize the spatiotemporal regulation of MT stability before, during, and after cell division. Thus, this live-cell marker enables the exploration of different MT subsets and how they contribute to cellular organization and transport.     

Role of cortical microtubules in the orientation of cellulose microfibril deposition in higher-plant cells

Cortical microtubules (MTs) have been implicated in the morphogenesis of plant cells by regulating the orientation of newly deposited cellulose microfibrils (CMFs). However, the role of MTs in oriented CMF deposition is still unclear. We have investigated the mechanism of CMF deposition with cultured tobacco protoplasts derived from taxol-treated BY-2 cells (taxol protoplasts). The BY-2 protoplasts regenerated patches of beta-l,3-glucan (callose) and fibrils of beta-l,4-glucan (cellulose). Taxol protoplasts possessed the same ordered MT arrays as material cells and regenerated CMFs with patterns almost coincidental with MTs. Electron microscopy revealed that, on the surface of cultured taxol protoplasts, each CMF bundle appeared to be deposited on each cortical MT. These results suggest that MTs may attach directly to the cellulose-synthesizing complexes, by some form of linkage, and regulate the movement of these complexes in higher-plant cells.     

MACF1与成骨细胞骨架之间的相互关系研究

采用激光共聚焦显微术研究微管微丝交联因子（MACF1）与成骨样细胞（MD63及MC3T3）微丝／微管骨架、黏着斑之间的相互关系．结果表明,MACF1不连续地分布于微管纤维上,与微丝骨架部分共定位于胞质中,在很多的成骨细胞中可见MACF1分布于骨架相关的粘着斑处：细胞松弛素B影响了MACF1在成骨细胞中的分布,并有使其向细胞核周围及核内转位的趋势．秋水仙素对MACF1的分布无明显的影响．转染了siRNA—MACFl的MG．63细胞微丝骨架纤维分布不连续、微管骨架纤维分布紊乱．这些结果提示MACF1不仅起交联微丝及微管细胞骨架的作用．而且还可稳定细胞骨架：成骨细胞MACF1的分布更依赖于微丝骨架的完整性．     

Morphologically distinct microtubule ends in the mitotic centrosome of Caenorhabditis elegans

During mitosis, the connections of microtubules (MTs) to centrosomes and kinetochores are dynamic. From in vitro studies, it is known that the dynamic behavior of MTs is related to the structure of their ends, but we know little about the structure of MT ends in spindles. Here, we use high-voltage electron tomography to study the centrosome- and kinetochore-associated ends of spindle MTs in embryonic cells of the nematode, Caenorhabditis elegans. Centrosome-associated MT ends are either closed or open. Closed MT ends are more numerous and are uniformly distributed around the centrosome, but open ends are found preferentially on kinetochore-attached MTs. These results have structural implications for models of MT interactions with centrosomes.     

Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones

We have used multimode fluorescent speckle microscopy (FSM) and correlative differential interference contrast imaging to investigate the actin-microtubule (MT) interactions and polymer dynamics known to play a fundamental role in growth cone guidance. We report that MTs explore the peripheral domain (P-domain), exhibiting classical properties of dynamic instability. MT extension occurs preferentially along filopodia, which function as MT polymerization guides. Filopodial bundles undergo retrograde flow and also transport MTs. Thus, distal MT position is determined by the rate of plus-end MT assembly minus the rate of retrograde F-actin flow. Short MT displacements independent of flow are sometimes observed. MTs loop, buckle, and break as they are transported into the T-zone by retrograde flow. MT breakage results in exposure of new plus ends which can regrow, and minus ends which rapidly undergo catastrophes, resulting in efficient MT turnover. We also report a previously undetected presence of F-actin arc structures, which exhibit persistent retrograde movement across the T-zone into the central domain (C-domain) at approximately 1/4 the rate of P-domain flow. Actin arcs interact with MTs and transport them into the C-domain. Interestingly, although the MTs associated with arcs are less dynamic than P-domain MTs, they elongate efficiently as a result of markedly lower catastrophe frequencies.     

Structural studies of N-terminal mutants of Connexin 32 using (1)H NMR spectroscopy

Microtubules (MTs) control cell replication, material transport and motion in eukaryotic cells, but MT role in several pathologies is still unknown. These functions are related to the MT physico-chemical properties and MT formation mode starting from tubulin molecules. This study describes a new method, based on the computer aided analysis of the electron paramagnetic resonance (EPR) spectra of selected spin probes to obtain structural and dynamical information on tubulins and MTs and the kinetics of MTs formation promoted by guanosine-5'-triphosphate (GTP). It was found that tubulin and MTs avoid radical quenching caused by ethylene glycol tetraacetic acid (EGTA). MT formation showed different kinetics as a function of tubulin concentration. At 5 mg/mL of tubulin, MTs were formed in 8 min. These results are also useful for getting information on MT-drug interactions.     

Role of cortical microtubules in the orientation of cellulose microfibril deposition in higher-plant cells

Summary Cortical microtubules (MTs) have been implicated in the morphogenesis of plant cells by regulating the orientation of newly deposited cellulose microfibrils (CMFs). However, the role of MTs in oriented CMF deposition is still unclear. We have investigated the mechanism of CMF deposition with cultured tobacco protoplasts derived from taxol-treated BY-2 cells (taxol protoplasts). The BY-2 protoplasts regenerated patches of β-l,3-glucan (callose) and fibrils of β-l,4-glucan (cellulose). Taxol protoplasts possessed the same ordered MT arrays as material cells and regenerated CMFs with patterns almost coincidental with MTs. Electron microscopy revealed that, on the surface of cultured taxol protoplasts, each CMF bundle appeared to be deposited on each cortical MT. These results suggest that MTs may attach directly to the cellulose-synthesizing complexes, by some form of linkage, and regulate the movement of these complexes in higher-plant cells.     

川百合花粉管的生殖细胞分裂过程中微管骨架的分布变化

应用透射电镜辅以免疫荧光定位技术研究了川百合 (Lilium davidii Duch.)花粉管中生殖细胞分裂过程中染色体动态和微管分布的关系。在生殖细胞分裂前和有丝分裂前期 ,电镜观察一直未见微管结构 ,但免疫荧光图象显示生殖细胞中有微管蛋白存在。直到分裂的前中期—中期 ,染色体出现 ,它们沿花粉管的长轴前后排列 ,横向的着丝点对相应地一对对地纵向排列。这时 ,生殖细胞中才出现大量微管 ,它们分布于细胞周质区和染色体之间 ,并跨越染色体的整个长度。前中期—中期开始时 ,只有 1～ 2对着丝点从横向转为纵向 ,微管垂直插入着丝点形成着丝点微管 ,而非前人用免疫荧光方法观察到的微管与着丝点侧向联接的图象。随着横向的着丝点对逐渐转变成纵向的过程 ,着丝点微管数量逐渐增多 ,但不形成典型的纺锤体。分裂后期 ,染色体交错分离 ,微管的分布与前中期—中期的基本相同。晚后期 ,染色体呈明显的两群 ,除极区和细胞中央区有微管残余外 ,大部分微管消失。通过染色体长度的测量 ,间接证明了分裂后期 B的存在。分裂末期的晚期 ,核膜形成后 ,在两精核之间的区域 ,微管数量开始增多。此区可能代表用免疫荧光所观察到的微管重叠区。细胞板出现后 ,微管消失