Molecular dissection of plant cytokinesis and phragmoplast structure: a survey of GFP-tagged proteins

To identify molecular players implicated in cytokinesis and division plane determination, the Arabidopsis thaliana genome was explored for potential cytokinesis genes. More than 100 open reading frames were selected based on similarity to yeast and animal cytokinesis genes, cytoskeleton and polarity genes, and Nicotiana tabacum genes showing cell cycle-controlled expression. The subcellular localization of these proteins was determined by means of GFP tagging in tobacco Bright Yellow-2 cells and Arabidopsis plants. Detailed confocal microscopy identified 15 proteins targeted to distinct regions of the phragmoplast and the cell plate. EB1- and MAP65-like proteins were associated with the plus-end, the minus-end, or along the entire length of microtubules. The actin-binding protein myosin, the kinase Aurora, and a novel cell cycle protein designated T22, accumulated preferentially at the midline. EB1 and Aurora, in addition to other regulatory proteins (homologs of Mob1, Sid1, and Sid2), were targeted to the nucleus, suggesting that this organelle operates as a coordinating hub for cytokinesis.     

The relative effect of citral on mitotic microtubules in wheat roots and BY2 cells

The plant volatile monoterpene citral is a highly active compound with suggested allelopathic traits. Seed germination and seedling development are inhibited in the presence of citral, and it disrupts microtubules in both plant and animal cells in interphase. We addressed the following additional questions: can citral interfere with cell division; what is the relative effect of citral on mitotic microtubules compared to interphase cortical microtubules; what is its effect on newly formed cell plates; and how does it affect the association of microtubules with γ‐tubulin? In wheat seedlings, citral led to inhibition of root elongation, curvature of newly formed cell walls and deformation of microtubule arrays. Citral’s effect on microtubules was both dose‐ and time‐dependent, with mitotic microtubules appearing to be more sensitive to citral than cortical microtubules. Association of γ‐tubulin with microtubules was more sensitive to citral than were the microtubules themselves. To reveal the role of disrupted mitotic microtubules in dictating aberrations in cell plates in the presence of citral, we used tobacco BY2 cells expressing GFP‐Tua6. Citral disrupted mitotic microtubules, inhibited the cell cycle and increased the frequency of asymmetric cell plates in these cells. The time scale of citral’s effect in BY2 cells suggested a direct influence on cell plates during their formation. Taken together, we suggest that at lower concentrations, citral interferes with cell division by disrupting mitotic microtubules and cell plates, and at higher concentrations it inhibits cell elongation by disrupting cortical microtubules.     

Building bridges: formin1 of Arabidopsis forms a connection between the cell wall and the actin cytoskeleton

Actin microfilament (MF) organization and remodelling is critical to cell function. The formin family of actin binding proteins are involved in nucleating MFs in Arabidopsis thaliana. They all contain formin homology domains in the intracellular, C‐terminal half of the protein that interacts with MFs. Formins in class I are usually targeted to the plasma membrane and this is true of Formin1 (AtFH1) of A. thaliana. In this study, we have investigated the extracellular domain of AtFH1 and we demonstrate that AtFH1 forms a bridge from the actin cytoskeleton, across the plasma membrane and is anchored within the cell wall. AtFH1 has a large, extracellular domain that is maintained by purifying selection and that contains four conserved regions, one of which is responsible for immobilising the protein. Protein anchoring within the cell wall is reduced in constructs that express truncations of the extracellular domain and in experiments in protoplasts without primary cell walls. The 18 amino acid proline‐rich extracellular domain that is responsible for AtFH1 anchoring has homology with cell‐wall extensins. We also have shown that anchoring of AtFH1 in the cell wall promotes actin bundling within the cell and that overexpression of AtFH1 has an inhibitory effect on organelle actin‐dependant dynamics. Thus, the AtFH1 bridge provides stable anchor points for the actin cytoskeleton and is probably a crucial component of the signalling response and actin‐remodelling mechanisms.     

The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat <Emphasis Type="Italic">Triticum aestivum L.</Emphasis>

We have studied the response of interphase and mitotic microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) to cold stress (1 h at 0°C) and acclimation to cold (3–48 h at 0°C). We show that, in general, interphase microtubules are more resistant to cold then mitotic arrays in both cultivars. During cold stress, no changes are detected in the microtubule system of interphase cells of spring wheat, whereas the density of endoplasmic microtubules increases in interphase cells of winter wheat. During mitosis, the density of the kinetochore fibers of the spindle decreases in the cells of both cultivars, but it is prevailing in the cells of spring cultivar of wheat. During acclimation to cold, the disorganization of the cortical microtubule bundles and the enhanced growth of the endoplasmic microtubule network, which is comprised of microtubule converging centers, are observed in cells of both cultivars. However, the mitotic microtubule systems of winter and spring cultivars respond differently to cold acclimation. During prophase, a diffuse tubulin “halo,”followed by the assembly of microtubule converging centers, accumulate at the perinuclear area in the cells of winter wheat. In cells of spring cultivar, the prophase spindle is only detected during initial stages of cold acclimation. During metaphase, aberrant mitotic spindles, abnormal metaphase plates, and the excessive appearance of microtubule converging centers are observed in cells of both cultivars. Acclimation induces the disorganization of the phragmoplast and the formation of multiple microtubule converging centers during telophase in the cells of both cultivars. Microtubule converging centers are detected at the perinuclear area of daughter cells in winter wheat and in the cortical cytoplasm in spring wheat. The excessive formation of microtubule converging centers suggests the activation of microtubule assembly during prolonged exposure to low temperature. Our data also demonstrates common pathways of microtubule response to cold treatment (0°C).     

Organelle inheritance in plant cell division: the actin cytoskeleton is required for unbiased inheritance of chloroplasts, mitochondria and endoplasmic reticulum in dividing protoplasts

Nuclear inheritance is highly ordered, ensuring stringent, unbiased partitioning of chromosomes before cell division. In plants, however, little is known about the analogous cellular processes that might ensure unbiased inheritance of non-nuclear organelles, either in meristematic cell divisions or those induced during the acquisition of totipotency. We have investigated organelle redistribution and inheritance mechanisms during cell division in cultured tobacco mesophyll protoplasts. Quantitative analysis of organelle repositioning observed by autofluorescence of chloroplasts or green fluorescent protein (GFP), targeted to mitochondria or endoplasmic reticulum (ER), demonstrated that these organelles redistribute in an ordered manner before division. Treating protoplasts with cytoskeleton-disrupting drugs showed that redistribution depended on actin filaments (AFs), but not on microtubules (MTs), and furthermore, that an intact actin cytoskeleton was required to achieve unbiased organelle inheritance. Labelling the actin cytoskeleton with a novel GFP-fusion protein revealed a highly dynamic actin network, with local reorganisation of this network itself, appearing to contribute substantially to repositioning of chloroplasts and mitochondria. Our observations show that each organelle exploits a different strategy of redistribution to ensure unbiased partitioning. We conclude that inheritance of chloroplasts, mitochondria and ER in totipotent plant cells is an ordered process, requiring complex interactions with the actin cytoskeleton.     

Fine structure of gels prepared from an actin-binding protein and actin: comparison to cytoplasmic extracts and cortical cytoplasm in amoeboid cells of cortical cytoplasm in amoeboid cells of Dictyostelium discoideum

We have identified the three-dimensional ultrastructure of actin gels that are formed in well-characterized cell extracts and mixtures of purified actin and the 120K actin-binding protein and compared these to the ultrastructure of the cytoplasmic matrix in regions of nonextracted Dictyostelium amoebae that are rich in actin and 120K. This ultrastructural characterization was achieved by using critical-point-dried whole-mount preparations. All three preparations--gelled extracts, purified proteins, and cortical cytoplasm--are composed of filament networks. The basic morphological feature of these networks is the presence of contacts between convergent filaments resulting in "T" or "X" shaped contacts. The finding that actin-containing gels are composed of filament networks, where the primary interaction occurs between convergent filaments, reconciles the known requirement of F actin for gelation with the amorphous appearance of these gels in thin sections. Increasing the molar ratio of 120K dimer to actin monomer increases the number of contacts between filaments per unit volume and decreases the lengths of filaments between contacts. This indicates that 120K stabilizes interactions between filaments and is consistent with biochemical evidence that 120K crosslinks actin filaments. The cortical network in situ resembles more closely networks formed in 120K-rich extracts than networks assembled in mixtures of purified 120K and actin. The heterogeneity of filament diameters and variation of network density are properties shared by extracts and the cytomatrix in situ while networks found in purified 120K-actin gels have filament diameters and densities that are more uniform. These differences are certainly due to the more complex composition of cell extracts and cortical cytoplasm as compared to that of purified 120K-actin gels.     

The Effect of cytoskeleton inhibitors on coccolith morphology in Coccolithus braarudii and Scyphosphaera apsteinii

The calcite platelets of coccolithophores (Haptophyta), the coccoliths, are among the most elaborate biomineral structures. How these unicellular algae accomplish the complex morphogenesis of coccoliths is still largely unknown. It has long been proposed that the cytoskeleton plays a central role in shaping the growing coccoliths. Previous studies have indicated that disruption of the microtubule network led to defects in coccolith morphogenesis in Emiliania huxleyi and Coccolithus braarudii. Disruption of the actin network also led to defects in coccolith morphology in E. huxleyi, but its impact on coccolith morphology in C. braarudii was unclear, as coccolith secretion was largely inhibited under the conditions used. A more detailed examination of the role of actin and microtubule networks is therefore required to address the wider role of the cytoskeleton in coccolith morphogenesis. In this study, we have examined coccolith morphology in C. braarudii and Scyphosphaera apsteinii following treatment with the microtubule inhibitors vinblastine and colchicine (S. apsteinii only) and the actin inhibitor cytochalasin B. We found that all cytoskeleton inhibitors induced coccolith malformations, strongly suggesting that both microtubules and actin filaments are instrumental in morphogenesis. By demonstrating the requirement for the microtubule and actin networks in coccolith morphogenesis in diverse species, our results suggest that both of these cytoskeletal elements are likely to play conserved roles in defining coccolith morphology.     

Two proteins from Saccharomyces cerevisiae: Pfy1 and Pkc1, play a dual role in activating actin polymerization and in increasing cell viability in the adaptive response to oxidative stress

In this work, we show that the proteins Pkc1 and Pfy1 play a role in the repolarization of the actin cytoskeleton and in cell survival in response to oxidative stress. We have also developed an assay to determine the actin polymerization capacity of total protein extracts using fluorescence recovery after photobleaching techniques and actin purified from rabbit muscle. This assay allowed us to demonstrate that Pfy1 promotes actin polymerization under conditions of oxidative stress, while Pkc1 induces actin polymerization and cell survival under all the conditions tested. Our assay also points to a relationship between Pkc1 and Pfy1 in the actin cytoskeleton polymerization that is required to adapt to oxidative stress.     

Effect of membrane stiffness and cytoskeletal element density on mechanical stimuli within cells: an analysis of the consequences of ageing in cells

A finite element model of a single cell was created and used to compute the biophysical stimuli generated within a cell under mechanical loading. Major cellular components were incorporated in the model: the membrane, cytoplasm, nucleus, microtubules, actin filaments, intermediate filaments, nuclear lamina and chromatin. The model used multiple sets of tensegrity structures. Viscoelastic properties were assigned to the continuum components. To corroborate the model, a simulation of atomic force microscopy indentation was performed and results showed a force/indentation simulation with the range of experimental results. A parametric analysis of both increasing membrane stiffness (thereby modelling membrane peroxidation with age) and decreasing density of cytoskeletal elements (thereby modelling reduced actin density with age) was performed. Comparing normal and aged cells under indentation predicts that aged cells have a lower membrane area subjected to high strain as compared with young cells, but the difference, surprisingly, is very small and may not be measurable experimentally. Ageing is predicted to have a more significant effect on strain deep in the nucleus. These results show that computation of biophysical stimuli within cells are achievable with single-cell computational models; correspondence between computed and measured force/displacement behaviours provides a high-level validation of the model. Regarding the effect of ageing, the models suggest only small, although possibly physiologically significant, differences in internal biophysical stimuli between normal and aged cells.     

Demarcation of the cortical division zone in dividing plant cells

Somatic cytokinesis in higher plants involves, besides the actual construction of a new cell wall, also the determination of a division zone. Several proteins have been shown to play a part in the mechanism that somatic plant cells use to control the positioning of the new cell wall. Plant cells determine the division zone at an early stage of cell division and use a transient microtubular structure, the preprophase band (PPB), during this process. The PPB is formed at the division zone, leaving behind a mark that during cytokinesis is utilized by the phragmoplast to guide the expanding cell plate toward the correct cortical insertion site. This review discusses old and new observations with regard to mechanisms implicated in the orientation of cell division and determination of a cortical division zone.     

Thematic Minireview Series: The State of the Cytoskeleton in 2015

The study of cytoskeletal polymers has been an active area of research for more than 70 years. However, despite decades of pioneering work by some of the brightest scientists in biochemistry, cell biology, and physiology, many central questions regarding the polymers themselves are only now starting to be answered. For example, although it has long been appreciated that the actin cytoskeleton provides contractility and couples biochemical responses with mechanical stresses in cells, only recently have we begun to understand how the actin polymer itself responds to mechanical loads. Likewise, although it has long been appreciated that the microtubule cytoskeleton can be post-translationally modified, only recently have the enzymes responsible for these modifications been characterized, so that we can now begin to understand how these modifications alter the polymerization and regulation of microtubule structures. Even the septins in eukaryotes and the cytoskeletal polymers of prokaryotes have yielded new insights due to recent advances in microscopy techniques. In this thematic series of minireviews, these topics are covered by some of the very same scientists who generated these recent insights, thereby providing us with an overview of the State of the Cytoskeleton in 2015.     

DNA replication and the development of preprophase bands in soybean protoplast cultures

Nuclear DNA replication and the development of preprophase bands (PPBs) are two chronologically close processes during the higher plant cell cycle. However, it is not clear whether occurrence of PPBs is coupled with DNA replication. A soybean protoplast culture with a high frequency of PPBs was used to study the relationship between the two processes when treated with aphidicolin, a potent and specific inhibitor of eukaryotic DNA polymerase-α. When DNA replication was partially inhibited by 10 mg l^-1 aphidicolin, both the percentage of cells with PPBs and the mitotic index (MI) decreased in absolute terms, but there were proportionately more PPBs than mitoses. Since PPBs change in appearance as they develop, they were divided into categories of early (interphase associated) and late (prophase associated). The increased PPB/MI ratio was associated with an increased proportion of early stage PPBs relative to late stage PPBs. When DNA replication was completely blocked by 50 mg l^-1 aphidicolin, both MI and the percentage of cells with PPBs were close to zero. These results suggest that development of PPBs was to a large extent coupled DNA replication. We propose that the increased PPB/MI ratio at 10 mg l^-1 aphidicolin was due to a linkage between the duration of interphase and the time period in which early stage PPBs are visible. The increased duration of early PPBs partially compensates for the reduced number of nuclei reaching the stage of PPB initiation. Furthermore, in cultures containing aphidicolin, the percentage of PPBs with simultaneous perinuclear fluorescence (PNF, accumulation of microtubules on nuclear envelope) was reduced and whenever PNF was prominent and dense on the nuclear envelope the nucleus showed chromatin condensation. These observations indicated that the transition from PPB to PNF and then to the prophase spindle is closely related to the progress of the nuclear cycle.     

冰冻切片法在植物微管骨架研究中的应用

介绍了冰冻切片法研究植物微管骨架的一般程序和技术上的一些改进,结果证明,改进的冰冻切片技术,可以对植物不同类型的细胞进行很好的标记。实验结果表明,甘蔗正在迅速伸长的幼叶分布的微管类型主要是与细胞伸长轴方向垂直的周质微管,幼叶基部尤其是第三幼叶基部分布的主要是与细胞伸长轴方向平行的周质微管。表明冰冻切片法在植物微管骨架的研究中具有广阔的应用前景。     

The quantitation of G- and F-actin in cultured cells

An improved method to quantitate the amounts of filamentous (F-actin) and monomeric (globular) actin (G-actin) in cultured cells was developed. Cells are lysed into a myosin-containing buffer and F-actin is removed by centrifugation. The pelleted F-actin is then depolymerized to G-actin in a 1 mM ATP-containing buffer for 1 h before measuring the levels of G-actin using the DNase I inhibition assay. Partitioning of G-actin in the supernatant (greater than 95%) and recovery of actin in both fractions (greater than 85%) were measured by adding [3H]actin to cultured cells. Actin in the separated fractions is stable for at least 72 h at 0 degree C. Asynchronous monolayer cultures of Chinese hamster ovary (CHO) cells contain 2.5 +/- 0.2% of the total protein as actin with 72.4 +/- 5.7% as F-actin. About 10% of this F-actin is not associated with the readily sedimented Triton-cytoskeleton. CHO cells grown in suspension contain 55.8% of the actin as F-actin; following plating about 90 min is required for these cells to flatten and for the F-actin level to reach the monolayer value of about 70%.     

The morphogenesis of lobed plant cells in the mesophyll and epidermis: organization and distinct roles of cortical microtubules and actin filaments

The morphogenesis of lobed plant cells has been considered to be controlled by microtubule (MT) and/or actin filament (AF) organization. In this article, a comprehensive mechanism is proposed, in which distinct roles are played by these cytoskeletal components. First, cortical MT bundles and, in the case of pavement cells, radial MT arrays combined with MT bundles determine the deposition of local cell wall thickenings, the cellulose microfibrils of which copy the orientation of underlying MTs. Cell growth is thus locally prevented and, consequently, lobes and constrictions are formed. Arch-like tangential expansion is locally imposed at the external periclinal wall of pavement cells by the radial arrangement of cellulose microfibrils at every wall thickening. Whenever further elongation of the original cell lobes occurs, AF patches assemble at the tips of growing lobes. Intercellular space formation is promoted or prevented by the opposite or alternate, respectively, arrangement of cortical MT arrays between neighboring cells. The genes that are possibly involved in the molecular regulation of the above morphogenetic procedure by MT and AF array organization are reviewed.     

The actin cytoskeleton coordinates the signal transduction and antigen processing functions of the B cell antigen receptor

The B cell antigen receptor （BCR） is the sensor on the B cell surface that surveys foreign molecules （antigen） in our bodies and activates B cells to generate antibody responses upon encountering cognate antigen. The binding of antigen to the BCR induces signaling cascades in the cytoplasm, which provides the first signal for B cell activation. Subsequently, BCRs internalize and target bound antigen to endosomes, where antigen is processed into T cell recognizable forms. T helper cells generate the second activation signal upon binding to antigen presented by B cells. The optimal activation of B cells requires both signals, thereby depending on the coordination of BCR signaling and antigen transport functions. Antigen binding to the BCR also induces rapid remodeling of the cortical actin network of B cells. While being initiated and controlled by BCR signaling, recent studies reveal that this actin remodeling is critical for both the signaling and antigen processing functions of the BCR, indicating a role for actin in coordinating these two pathways. Here we will review previous and recent studies on actin reorganization during BCR activation and BCR- mediated antigen processing, and discuss how actin remodeling translates BCR signaling into rapid antigen uptake and processing while providing positive and negative feedback to BCR signaling.     

The adenomatous polyposis coli protein unambiguously localizes to microtubule plus ends and is involved in establishing parallel arrays of microtubule bundles in highly polarized epithelial cells

Loss of full-length adenomatous polyposis coli (APC) protein correlates with the development of colon cancers in familial and sporadic cases. In addition to its role in regulating β-catenin levels in the Wnt signaling pathway, the APC protein is implicated in regulating cytoskeletal organization. APC stabilizes microtubules in vivo and in vitro, and this may play a role in cell migration (Näthke, I.S., C.L. Adams, P. Polakis, J.H. Sellin, and W.J. Nelson. 1996. J. Cell Biol. 134:165–179; Mimori-Kiyosue, Y., N. Shiina, and S. Tsukita. 2000. J. Cell Biol. 148:505–517; Zumbrunn, J., K. Inoshita, A.A. Hyman, and I.S. Näthke. 2001. Curr. Biol. 11:44–49) and in the attachment of microtubules to kinetochores during mitosis (Fodde, R., J. Kuipers, C. Rosenberg, R. Smits, M. Kielman, C. Gaspar, J.H. van Es, C. Breukel, J. Wiegant, R.H. Giles, and H. Clevers. 2001. Nat. Cell Biol. 3:433–438; Kaplan, K.B., A. Burds, J.R. Swedlow, S.S. Bekir, P.K. Sorger, and I.S. Näthke. 2001. Nat. Cell Biol. 3:429–432). The localization of endogenous APC protein is complex: actin- and microtubule-dependent pools of APC have been identified in cultured cells (Näthke et al., 1996; Mimori-Kiyosue et al., 2000; Reinacher-Schick, A., and B.M. Gumbiner. 2001. J. Cell Biol. 152:491–502; Rosin-Arbesfeld, R., G. Ihrke, and M. Bienz. 2001. EMBO J. 20:5929–5939). However, the localization of APC in tissues has not been identified at high resolution. Here, we show that in fully polarized epithelial cells from the inner ear, endogenous APC protein associates with the plus ends of microtubules located at the basal plasma membrane. Consistent with a role for APC in supporting the cytoskeletal organization of epithelial cells in vivo, the number of microtubules is significantly reduced in apico-basal arrays of microtubule bundles isolated from mice heterozygous for APC.