Organization of the actin cytoskeleton during pollen development inGasteria verrucosa (Mill.) H. Duval visualized with rhodamine-phalloidin

The three-dimensional organization of the microfilamental cytoskeleton of developingGasteria pollen was investigated by light microscopy using whole cells and fluorescently labelled phalloidin. Cells were not fixed chemically but their walls were permeabilized with dimethylsulphoxide and Nonidet P-40 at premicrospore stages or with dimethylsulphoxide, Nonidet P-40 and 4-methylmorpholinoxide-monohydrate at free-microspore and pollen stages to dissolve the intine.Four strikingly different microfilamentous configurations were distinguished. (i) Actin filaments were observed in the central cytoplasm throughout the successive stages of pollen development. The network was commonly composed of thin bundles ramifying throughout the cytoplasm at interphase stages but as thick bundles encaging the nucleus prior to the first and second meiotic division. (ii) In released microspores and pollen, F-actin filaments formed remarkably parallel arrays in the peripheral cytoplasm. (iii) In the first and second meiotic spindles there was an apparent localization of massive arrays of phalloidin-reactive material. Fluorescently labelled F-actin was present in kinetochore fibers and pole-to-pole fibers during metaphase and anaphase. (iv) At telophase, microfilaments radiated from the nuclear envelopes and after karyokinesis in the second meiotic division, F-actin was observed in phragmoplasts.We did not observe rhodamine-phalloidin-labelled filaments in the cytoplasm after cytochalasin-B treatment whereas F-actin persisted in the spindle. Incubation at 4° C did not influence the existence of cytoplasmic microfilaments whereas spindle filaments disappeared. This points to a close interdependence of spindle microfilaments and spindle tubules.Based on present data and earlier observations on the configuration of microtubules during pollen development in the same species (Van Lammeren et al., 1985, Planta165, 1-11) there appear to be apparent codistributions of F-actin and microtubules during various stages of male meiosis inGasteria verrucosa.Abbreviation DMSO dimethylsulfoxide     

Cytoskeletal pattern changes during branch formation in a centrifugedAdiantum protonema

A protonemal branch was induced on a side wall of a fern filamentous protonema by cell centrifugation and subsequent polarized-red light irradiation as described in a previous paper (Wada 1995, J. Plant Res. 108: 501–509). Changes in microtubule (MT) and microfilament (MF) patters during the branch development were observed under fluorescence microscopy. A ring-like band of cortical MTs (MT-ring) and MFs similar to a preprophase band or a subapical ring structure (Murataet al. 1987) appeared transiently at the future branching site before cell swelling, the first visible step of branch formation. At this stage, the nucleus was located far from the branching site and the MT-ring appeared to be connected to the nucleus by endoplasmic MFs as well as with endoplasmic MTs. The MT-ring disappeared when cell wall swelling occurred. When the cell wall swelling began, a fan-like pattern of cortical MTs emanating from the new growing tip was established and the MTs reached the opposite flank of the protonema. When a new branch started to elongate and the nucleus moved into the branch, a faint subapical ring of MTs appeared at the subapical part of the new branch. Strands of MTs and MFs emanating from the nuclear front end reached a part of the subapical ring.     

Calmodulin and wound healing in the coenocytic green alga Ernodesmis verticillata (Kützing) Børgesen

The involvement of calmodulin (CaM) in wound-induced cytoplasmic contractions in E. verticillata was investigated. Indirect immunofluorescence of CaM in intact cells showed a faint, reticulate pattern of fluorescence in the cortical cytoplasm. Diffuse fluorescence was evident deeper within the cytoplasm. In contracted cells, CaM co-localizes with actin in the cortical cytoplasm in extensive, longitudinal bundles of microfilaments (MFs), and in an actin-containing reticulum. No association of CaM with tubulin was ever observed in the cortical cytoplasm at any stage of wound-healing. When contraction rates in wounded cells are measured, a lag period of 2 min is followed by a rapid, steady rate of movement over the subsequent 10 min. The delay in the initiation of longitudinal contraction corresponds to the time necessary for the assembly of the longitudinal MF bundles. Cytoplasmic motility was inhibited in a dose-dependent manner by CaM antagonists. In these inhibited cells, MF bundles did not assemble, or were poorly formed. In the latter case, CaM was always found associated with MFs. These results indicate a direct spatial and temporal correlation between CaM and actin, and a potential role for CaM in regulating the formation of functional MF bundles during wound-induced cytoplasmic contraction in Ernodesmis.Abbreviations CaM calmodulin - DMSO dimethyl sulfoxide - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - MF(s) microfilament(s) - MT(s) microtubule(s) - TFP trifluoperazine - w-5 N-(6-aminohexyl)-1-naphthalenesulfonamide - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide We are especially grateful to: Dr. J.A. West (University of California, Berkeley) for the original algal isolates; Dr. L. Van Eldik (Vanderbilt University School of Medicine) and Dr. J.L. Lessard (University of Cincinnati College of Medicine) for graciously providing CaM and actin antibodies, respectively; Dr. S.J. Roux (University of Texas, Austin) for the gift of purified oat CaM; Dr.H. Green (Smith, Kline and French Laboratories, Philadelphia, Penn., USA) for providing the trifluoperazine; and M.E.T. Scioli for assistance with the statistical analyses. Portions of this work were supported by National Science Foundation grant DCB 8402345 and U.S. Department of Agriculture grant 87-CRCR-1-2545 to J.W.L.     

去包埋剂制片术揭示小麦珠被组织中微丝的跨胞分布

以DGD(diethylene glycol distearate)包埋、去包埋剂制片法,结合常规电镜制备术,观察研究了小麦珠被组织中胞间连丝的结构与分布特征及其与微丝的关系。试验结果表明,同层珠被细胞间壁上胞间连丝的分布密度比上下层珠被间的要显著地高得多;珠被中胞间连丝保持固有的正常结构,孔径30—40 nm,变动幅度不大,观察不到如作者以前在小麦珠心和胚乳中所见的那种胞间连丝经结构修饰而扩大、转变为开放态的景象。对去包埋剂制片的观察进一步揭示,从胞质骨架网络表层有纤细微丝以分散状向外伸展突入分界壁,时而可见各个微丝横贯分界壁而连接相邻骨架网络的图象。联系珠被中正常胞间连丝结构的稳定性与各个微丝以分散状跨越分界壁,讨论了微丝参与胞间连丝结构的可能性与可能模式。     

Studies on microplasmodia ofPhysarum polycephalum

Summary The new technique of fluorescent analog cytochemistry was used to investigate the cell surface morphology (RITC-WGA staining), the organization of the microfilament system (Rh-phalloidin staining) and the spatial distribution of mitochondria (Rh-123 staining) in the various growth stages of axenically cultured living and fixed microplasmodia ofPhysarum polycephalum. The differentiation degree of the cell surface is generally size- and age-dependent: the invagination system develops by degrees from small spherical stages (50–100 m) without invaginations to large vein-like or dumbbell-shaped specimens (300–1,000 (m long) with extensive invagination systems. The microfilaments are always organized in a cortical system along the entire cell surface and sometimes in a fibrillar system as well, extending throughout the cytoplasmic matrix. Results on living microplasmodia demonstrate that the cortical microfilament system is mainly involved in motive force generation for changes of cell surface morphology and protoplasmic streaming activity, whereas the fibrillar system rather serves a stabilizing and adhering function. Moreover, the functional differences of the two microfilament systems are indicated by the position of a large population of stationary mitochondria in close vicinity to the cell surface, thus pointing to a reasonable arrangement of the energy-supplying and energy-transforming system.     

Cytochalasin effects on structure and movement in the pollen tube of Iris

Summary Cytochalasins B and D in the concentration range 0.5–10 g ml^–1 produced similar effects on growth, movement and cytoplasmic structure in the pollen tubes of Iris spp. cultured in vitro. Continuous video recording showed that at 5 g ml_–1, CB was capable of stopping organelle circulation in as short a period as 20 s. The usually elongated vegetative nuclei were also arrested, and subsequently contracted irregularly. Generative cells were not radically changed in shape, but occasionally moved erratically before being halted. Detailed examination of CD- and CB-treated tubes regarded as being capable of recovering growth upon transfer to normal medium revealed several characteristic effects on cytoplasmic structure. Fibrils presumed to consist of, or contain, microfilament bundles are readily visible in the older parts of the living tube where they form the pathways of organelle movement; these were either condensed into amorphous columns or fragmented by treatment. In the distal parts of the tube, the cytoplasm had contracted into amorphous masses which continued to show very slow shape changes. With the arrest of extension growth, pectin accumulated over the tube tip and in patches along the flanks. In a medium containing 1 mM ATP, recovery from treatment was achieved in some instances within l min. Organelle circulation in the younger tubes was resumed, and fresh adventive tube tips were formed. The fibrillar system of the older tubes was not restored, however; instead, the cytoplasm in these zones formed aggregates which underwent continuous amoeboid movement, the organelles within moving rapidly in irregular trajectories with no indication of the resumption of the original long-range cyclotic flux. Some possible implications of the results are briefly discussed.     

Verticillium dahliae toxin induced alterations of cytoskeletons and nucleoli in Arabidopsis thaliana suspension cells

Summary. In plant cells, cytoskeletons play important roles in response to biotic and abiotic stresses. However, little is known about the dynamics of cytoskeletons when cells are attacked by unphysical stress factors such as elicitors and toxins. We report here that the toxin of Verticillium dahliae (VD toxin) induced changes of microfilaments (MFs) and microtubules (MTs) in Arabidopsis thaliana suspension-cultured cells. When cells were treated with a low concentration of VD toxin, MFs were disrupted ordinally from the cortex to the perinuclear region, and then recovered spontaneously; but the MTs persisted. The MFs in the perinuclear region showed more resistance to VD toxin than the cortical ones. In contrast, when cells were treated with a high concentration of VD toxin, MFs and MTs were disrupted sooner and more severely and did not recover spontaneously. Treatments with high concentrations of VD toxin also induced changes of nucleoli. At the early stages of treatment, a nucleus had a single ring-shaped nucleolus. At the later stages, multiple smaller and more brightly fluorescing nucleoli emerged in a single nucleus. Disrupted MFs could be recovered by removing the VD toxin before the ringshaped nucleoli appeared. All these results showed that MFs and MTs play important roles in the early defense responses against VD toxin in Arabidopsis suspension cells. The cytoskeletons may be used as sensors and effectors monitoring the defense reactions. The changes of nucleoli induced by VD toxin should be important characteristics of cell death. Correspondence and reprints: Department of Plant Sciences, College of Biological Sciences, China Agricultural University, Beijing 100094, People’s Republic of China.     

Trans-scale Granular Modelling of Cytoskeleton: a Mini-Review

Living cells are the functional unit of organs that controls reactions to their exterior. However, the mechanics of living cells can be difficult to characterize due to the crypticity of their microscale structures and associated dynamic cellular processes. Fortunately, multiscale modelling provides a powerful simulation tool that can be used to study the mechanical properties of these soft hierarchical, biological systems. This paper reviews recent developments in hierarchical multiscale modeling technique that aimed at understanding cytoskeleton mechanics. Discussions are expanded with respects to cytoskeletal components including: intermediate filaments, microtubules and microfilament networks. The mechanical performance of difference cytoskeleton components are discussed with respect to their structural and material properties. Explicit granular simulation methods are adopted with different coarse-grained strategies for these cytoskeleton components and the simulation details are introduced in this review.     

Participation of Rho, ROCK-2, and GAP activities during actin microfilament rearrangements in Entamoeba histolytica induced by fibronectin signaling

In Entamoeba histolytica little is known about the microfilament rearrangements formed by actin and ABPs. Fibronectin regulates many aspects of cell behavior involving the actin cytoskeleton and members of the Rho family of small GTPases. Using trophozoites interacted with fibronectin and glass, we present evidence related with the formation and regulation of different microfilament rearrangements and their cellular distribution, the effect of actin affecting drugs on these arrangements, and on trophozoites adhesion; we also demonstrate that actin isoforms are induced after adhesion, and also the selective participation of specific actin binding proteins such as ABP-120 and phospho-paxillin, regarding their location in the different actin structures. In addition, we show results that confirm the participation of EhRho, ROCK-2, and GAP activities. We propose that fibronectin induced signaling in E. histolytica trophozoites have important consequences in the actin cytoskeleton that may affect its behavior during the invasive process in the host.