Microtubules regulate the organization of actin filaments at the cortical region in root hair cells ofHydrocharis

Summary We studied the mechanism controlling the organization of actin filaments (AFs) inHydrocharis root hair cells, in which reverse fountain streaming occurs. The distribution of AFs and microtubules (MTs) in root hair cells were analyzed by fluorescence microscopy and electron microscopy. AFs and MTs were found running in the longitudinal direction of the cell at the cortical region. AFs were observed in the transvacuolar strand, but not MTs. Ultrastructural studies revealed that AFs and MTs were colocalized and that MTs were closer to the plasma membrane than AFs. To examine if MTs regulate the organization of AFs, we carried out a double inhibitor experiment using cytochalasin B (CB) and propyzamide, which are inhibitors of AFs and MTs, respectively. CB reversibly inhibited cytoplasmic streaming while propyzamide alone had no effect on it. However, after treatment with both CB and propyzamide, removal of CB alone did not lead to recovery of cytoplasmic streaming. In these cells, AFs showed a meshwork structure. When propyzamide was also removed, cytoplasmic streaming and the original organization of AFs were recovered. These results strongly suggest that MTs are responsible for the organization of AFs inHydrocharis root hair cells.     

去核过程中CHO细胞波形纤维的分布

本文用豚鼠抗小牛晶状体波形纤维蛋白的血清抗体,对经细胞松驰素B(CB)和秋水仙素等药物处理后再离心去核的CHO细胞及其核体、胞质体进行了间接免疫荧光染色,并对核体做了电镜观察。CB处理后离心的细胞的免疫荧光染色显示,去核过程中核的后方始终伴有强烈的荧光,核体上也有强荧光斑。在核体的电镜材料中同样观察到了中等纤维。经CB和秋水仙素合并处理后离心的细胞,去核效果比仅用CB处理有明显的增强,免疫荧光染色表明,核后的荧光并不因秋水仙素处理而消失。实验结果表明:1.微丝对维持细胞表面的完整性有重要作用,CB能破坏微丝故有利于离心去核。2.中等纤维与核之间存在密切联系,这种联系在核膜的某些区域比较集中、牢固,不易为离心力所破坏。3.微管对核固着作用有重要意义,细胞核可能通过中等纤维与微管相连而抛锚在胞质中,故秋水仙素可增强去核作用。微管对维持细胞表面强度可能也有一定作用。     

Blue light-dependent nuclear positioning in Arabidopsis thaliana leaf cells

The plant nucleus changes its intracellular position not only upon cell division and cell growth but also in response to environmental stimuli such as light. We found that the nucleus takes different intracellular positions depending on blue light in Arabidopsis thaliana leaf cells. Under dark conditions, nuclei in mesophyll cells were positioned at the center of the bottom of cells (dark position). Under blue light at 100 mumol m(-2) s(-1), in contrast, nuclei were located along the anticlinal walls (light position). The nuclear positioning from the dark position to the light position was fully induced within a few hours of blue light illumination, and it was a reversible response. The response was also observed in epidermal cells, which have no chloroplasts, suggesting that the nucleus has the potential actively to change its position without chloroplasts. Light-dependent nuclear positioning was induced specifically by blue light at >50 mumol m(-2) s(-1). Furthermore, the response to blue light was induced in phot1 but not in phot2 and phot1phot2 mutants. Unexpectedly, we also found that nuclei as well as chloroplasts in phot2 and phot1phot2 mutants took unusual intracellular positions under both dark and light conditions. The lack of the response and the unusual positioning of nuclei and chloroplasts in the phot2 mutant were recovered by externally introducing the PHOT2 gene into the mutant. These results indicate that phot2 mediates the blue light-dependent nuclear positioning and the proper positioning of nuclei and chloroplasts. This is the first characterization of light-dependent nuclear positioning in spermatophytes.     

Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia

Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501- 520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.     

Fertilization in Torenia fournieri: Actin organization and nuclear behavior in the central cell and primary endosperm

Studies of the living embryo sacs of Torenia fournieri reveal that the actin cytoskeleton undergoes dramatic changes that correlate with nuclear migration within the central cell and the primary endosperm. Before pollination, actin filaments appear as short bundles randomly distributed in the cortex of the central cell. Two days after anthesis, they become organized into a distinct actin network. At this stage the secondary nucleus, which is located in the central region of the central cell, possesses an associated array of short actin filaments. Soon after pollination, the actin filaments become fragmented in the micropylar end and the secondary nucleus is located next to the egg apparatus. After fertilization, the primary endosperm nucleus moves away from the egg cell and actin filaments reorganize into a prominent network in the cytoplasm of the primary endosperm. Disruption of the actin cytoskeleton with latrunculin A and cytochalasin B indicates that actin is involved in the migration of the nucleus     

Fertilization in Torenia fournieri: Actin organization and nuclear behavior in the central cell and primary endosperm

Studies of the living embryo sacs of Torenia fournieri reveal that the actin cytoskeleton undergoes dramatic changes that correlate with nuclear migration within the central cell and the primary endosperm. Before pollination, actin filaments appear as short bundles randomly distributed in the cortex of the central cell. Two days after anthesis, they become organized into a distinct actin network. At this stage the secondary nucleus, which is located in the central region of the central cell, possesses an associated array of short actin filaments. Soon after pollination, the actin filaments become fragmented in the micropylar end and the secondary nucleus is located next to the egg apparatus. After fertilization, the primary endosperm nucleus moves away from the egg cell and actin filaments reorganize into a prominent network in the cytoplasm of the primary endosperm. Disruption of the actin cytoskeleton with latrunculin A and cytochalasin B indicates that actin is involved in the migration of the nucleus in the central cell. Our data also suggest that the dynamics of actin cytoskeleton may be responsible for the reorganization of the central cell and primary endosperm cytoplasm during fertilization.     

A Possible role of the nucleus in cytochalasin B-Induced capping

The possible influence of the nucleus on Cytochalasin B (CB)-induced capping of antibodies to surface antigens on L cells SV40-3T3 and NRK La 334 cells was studied. The cap formation induced by CB, was generally localized opposite the nucleus which was displaced against the cell periphery. To be able to observe the nuclear membrane in relation to the capping process we have taken advantage of an antiserum specific for antigens in the nuclear membrane but lacking reactivity to the plasma membrane and intranuclear antigens. This approach indicated that the CB-induced capping caused an accumulation of nuclear membrane antigens in the area of the nucleus facing the cap. The CB-induced local accumulation of nuclear membrane antigens required intact cells and could not be induced by binding of antibodies to the nuclear membrane followed by exposure to CB. Whatever the basis for the CB-induced altered reactivity of the anti-nuclear membrane antibodies (folding of the nuclear periphery, for example) this result indicated that the nuclear membrane was affected by CB capping. The possible role of the nucleus in the CB-induced capping process was further investigated in enucleated cells. The results obtained indicate that such cells both when enucleated in suspension and adherent to a surface did not exhibit CB capping. This disappearance of CB capping did probably not reflect decreased cell viability, previous exposure of the cells to CB during the enucleation procedure or a decreased capacity of the enucleated cells to bind CB.     

An Ultrastructural Study on Triggering of Cell Division in Young Pollen Protoplast Culture of Hemerocallis fulva L.

The ultrastructural changes of young pollen protoplasts under culture condition in Hemerocallis fulva were studied. In comparison with the original pollen grains, the pollen protoplasts had been completely deprived of pollen wall, but kept the internal structure intact, including a large vacuole, a thin layer of cytoplasm and a peripherally located nucleus. After 8 days of culture a few pollen protoplasts were triggered to cell division: some of them were just undergoing mitosis with clearly visible chromosomes and spindle fibers; the others already divided into 2-celled units. The two daughter cells were equal or unequal in size but with similar distribution of organelles inside. Besides cell division, there were also free nuclear division, amitosis and formation of micronuclei indicating a diversity of division modes in pollen protoplast culture, A series of changes occurred during the process of induction of cell division, such as locomotion of the nucleus toward the central position, disappearence of the large vacuole, increase of electron density of cytoplasm, increase and activation of organelles, diminishing of starch granules in plastids, etc. However, the regeneration of surface wall was not sufficient it contained mostly vesicles with only a few microfibrits. The wall separating the two daughter cells were either complete or incomplete. The weak capability of wall formation is supposed to be one of the major obstacles which has so far restricted sustained cell divisions of young pollen protoplasts under current culture condition.     

Organization of Microtubules during the Transition from Cytokinesis to Interphase in Protonemal Cells of Adiantum capillus-veneris L.

The reorganization of microtubules (MTs) from cytokinesis tointerphase was examined in protonemal cells of the fern Adiantumcapillus-veneris. During the reorganization, many MTs fannedout from the nuclear envelope towards the cell periphery. Newlyformed cortical MTs were located only near the nucleus and werearranged randomly. The randomly arranged cortical MTs were thenreplaced by an interphase array of cortical MTs that were orientedpredominantly parallel to the cell axis. At the boundary betweenthe new and the old cell wall, clusters of MTs were observedafter the formation of cortical MTs. Re-formation of MTs after depolymerization of MTs was also examined.Clusters of short MTs appeared only at the nuclear envelopewhen MTs had been depolymerized by exposure of cells to 100µM propyzamide at 0°C. Few MTs were formed at theboundary between the new and old cell walls. These results suggestthat, even in fern cells, the nuclear envelope might act asMT-organizing center during the establishment of the interphasearray of MTs. (Received June 21, 1995; Accepted January 23, 1996)     

Response of yeast protoplasts to their mating partners

The mating process between two protoplasts or between a protoplast and a cell in the yeastSaccharomyces cerevisiœ was manifested by a specific morphological response of only the cell partner. The cells produced projections, up to 5 μm long, to meet their protoplast partners. The protoplasts responded, after a period of nonspecific hernia-like growth, by ceasing to grow and assuming oval or spherical shapes. They never formed mating projections, apparently due to the absence of complete cell walls. Similarly to the cells, nuclear division in protoplasts was arrested and the nucleus migrated towards the plasma membrane at the site of protoplast-cell contact. Cytoplasmic microtubules were directed to this site, indicating the position of the spindle pole body (SPB) on the nucleus adjacent to the plasma membrane. Actin patches accumulated also in this region. These cytological features of the protoplasts were reminiscent of the reorganization of the cytoskeleton and nucleus characteristic of mating cells. This implies that the ability of protoplasts to produce and receive mating signals was unaffected by protoplasting. Fusion, however, was not initiated due to the absence of the complete cell wall in one of the partners. Thus, the cell wall appeared to be necessary for the expression of polarized growth during mating and for cell fusion. Dedicated to Professor O. Nečas on the occasion of his 70th birthday     

Actin-filled nuclear invaginations indicate degree of cell de-differentiation

For years the existence of nuclear actin has been heavily debated, but recent data have clearly demonstrated that actin, as well as actin-binding proteins (ABPs), are located in the nucleus. We examined live EGFP-actin-expressing cells using confocal microscopy and saw the presence of structures strongly resembling actin filaments in the nuclei of MDA-MB-231 human mammary epithelial tumor cells. Many nuclei had more than one of these filamentous structures, some of which appeared to cross the entire nucleus. Extensive analysis, including fluorescence recovery after photobleaching (FRAP), showed that all EGFP-actin in the nucleus is monomeric (G-actin) rather than filamentous (F-actin) and that the apparent filaments seen in the nucleus are invaginations of cytoplasmic monomeric actin. Immunolocalization of nuclear pore complex proteins shows that similar invaginations are seen in cells that are not overexpressing EGFP-actin. To determine whether there is a correlation between increased levels of invagination in the cell nuclei and the state of de-differentiation of the cell, we examined a variety of cell types, including live Xenopus embryonic cells. Cells that were highly de-differentiated, or cancerous, had an increased incidence of invagination, while cells that were differentiated had few nuclear invaginations. The nuclei of embryonic cells that were not yet differentiated underwent multiple shape changes throughout interphase, and demonstrated numerous transient invaginations of varying sizes and shapes. Although the function of these actin-filled invaginations remains speculative, their presence correlates with cells that have increased levels of nuclear activity.     

Break of symmetry in regenerating tobacco protoplasts is independent of nuclear positioning

Nuclear migration and positioning are crucial for the morphogenesis of plant cells. We addressed the potential role of nuclear positioning for polarity induction using an experimental system based on regenerating protoplasts, where the induction of a cell axis de novo can be followed by quantification of specific regeneration stages. Using overexpression of fluorescently tagged extranuclear (perinuclear actin basket, kinesins with a calponin homology domain (KCH)) as well as intranuclear (histone H2B) factors of nuclear positioning and time‐lapse series of the early stages of regeneration, we found that nuclear position is no prerequisite for polarity formation. However, polarity formation and nuclear migration were both modulated in the transgenic lines, indicating that both phenomena depend on factors affecting cytoskeletal tensegrity and chromatin structure. We integrated these findings into a model where retrograde signals are required for polarity induction. These signals travel via the cytoskeleton from the nucleus toward targets at the plasma membrane.     

Nesprin-3: a versatile connector between the nucleus and the cytoskeleton

The cytoskeleton is connected to the nuclear interior by LINC (linker of nucleoskeleton and cytoskeleton) complexes located in the nuclear envelope. These complexes consist of SUN proteins and nesprins present in the inner and outer nuclear membrane respectively. Whereas SUN proteins can bind the nuclear lamina, members of the nesprin protein family connect the nucleus to different components of the cytoskeleton. Nesprin-1 and -2 can establish a direct link with actin filaments, whereas nesprin-4 associates indirectly with microtubules through its interaction with kinesin-1. Nesprin-3 is the only family member known that can link the nuclear envelope to intermediate filaments. This indirect interaction is mediated by the binding of nesprin-3 to the cytoskeletal linker protein plectin. Furthermore, nesprin-3 can connect the nucleus to microtubules by its interactions with BPAG1 (bullous pemphigoid antigen 1) and MACF (microtubule-actin cross-linking factor). In contrast with the active roles that nesprin-1, -2 and -4 have in actin- and microtubule-dependent nuclear positioning, the role of nesprin-3 is likely to be more passive. We suggest that it helps to stabilize the anchorage of the nucleus within the cytoplasm and maintain the structural integrity and shape of the nucleus.     

萱草幼嫩花粉原生质体培养启动细胞分裂的超微结构研究

萱草(Hemerocallis fulva L.)幼嫩花粉,即后期小孢子原生质体在培养8天时进入有丝分裂或已形成二个细胞。此外,还观察到游离核分裂、无丝分裂、微核形成等现象。这显示了花粉原生质体分裂方式的多样性。在启动分裂时发生一系列变化:如细胞核移位、大液泡消失、细胞质电子密度增加、细胞器增多、质体不含淀粉等。再生的细胞壁含许多小泡,很少纤丝,表现出现有培养条件下壁的形成能力薄弱。这是今后改进培养技术需要特别注意的问题。     

Actin distribution in somatic embryos and embryogenic protoplasts of white spruce (Picea glauca)

Summary Examination of unfixed immature somatic embryos of white spruce (Picea glauca) with fluorescent rhodamine-labeled phalloidin revealed an extensive network of fine actin microfilaments (MFs) in the embryonal region which were not detected in specimens fixed with formaldehyde. Transition cells linking the embryonal region and suspensor cells contained fine MFs as well as bundles of MFs. The large, highly vacuolated suspensor cells were characterized by actin MF cables only. Treatment of embryos with cytochalasin B (CB) removed the fine MFs from the embryonal region and transition cells, but many MF cables in suspensor cells were resistant. Full recovery from CB treatment was observed in most somatic embryos. Embryogenic protoplasts capable of regenerating to somatic embryos in culture were released from only the embryonal region of somatic embryos. Both uninucleate and multinucleate embryogenic protoplasts retained the extensive network of fine actin MFs. In contrast, protoplasts derived from vacuolated suspensor cells and vacuolated free-floating cells contained thick MF bundles and were not embryogenic. Distinct MF cages enclosed nuclei in multinucleate protoplasts and may be responsible for preventing nuclear fusion. Microspectrophotometric analyses showed that the DNA contents of embryonal cells in the embryo and embryogenic protoplasts were similar and characteristic of rapidly dividing cell populations. However, transition and suspensor cells which released nonembryogenic protoplasts appeared to be arrested in G₁, and suspensor cells showed signs of DNA degradation.     

Communication between the cytoskeleton and the nuclear envelope to position the nucleus

In most eukaryotic cells, the nucleus is localized to a specific location. This highlight article focuses on recent advances describing the mechanisms of nuclear migration and anchorage. Central to nuclear positioning mechanisms is the communication between the nuclear envelope and the cytoskeleton. All three components of the cytoskeleton-microtubules, actin filaments and intermediate filaments-are involved in nuclear positioning to varying degrees in different cell types. KASH proteins on the outer nuclear membrane connect to SUN proteins on the inner nuclear membrane. Together they transfer forces between the cytoskeleton and the nuclear lamina. Once at the outer nuclear membrane, KASH proteins can interact with the cytoskeleton. Nuclear migrations are a component of many cellular migration events and defects in nuclear positioning lead to human diseases, most notably lissencephaly.     

Spatial determinants in morphogenesis: recovery from plasmolysis in the diatom Ditylum

Ditylum cells are enclosed in a rigid wall consisting of two "valves" (end walls) connected by "girdle bands." A hollow spine, the Labiate Process (LP), extends from each valve and a stable cytoplasmic strand connects its base with the nucleus. We investigated whether cells might possess "spatial determinants" for controlling their internal organization and wall morphogenesis. Upon plasmolysis, cells contracted into a spherical protoplast detached from the wall. Recovery was initiated by growing filopodia that "searched" the inside of the wall. Some attached to the inside corners, generating tension that could temporarily displace the protoplast. Others consolidated into the strand connecting nucleus with the LP. The protoplasts soon expanded and cells recovered: some divided immediately, the rest within 24 h. When recently divided cells were plasmolysed, their nascent valves were exocytosed. These were ignored by the filopodia during recovery. Later, protoplasts secreted a new valve, while the nascent valves were discarded. The interphase microtubule (MT) cytoskeleton radiates from a central Microtubule Center. A thicker bundle connects the nucleus to each LP. Plasmolysis destroyed the MT cytoskeleton; its re-establishment matched growth of the filopodia. The anti-MT drug oryzalin prevented filopodial extension while existing filopodia retracted, except those stabilized by attachment to the corners of the cell and the LP. Several anti-actin agents had relatively little effect. However, one, mycalolide B, caused the nucleus to be extruded from the protoplast by a bundle of MTs. We conclude that the geometry of the wall could provide spatial information to which the MT-cytoskeleton/filopodia respond.