Plasma Membrane-Associated Actin in Bright Yellow 2Tobacco Cells . Evidence for Interaction with Microtubules (original) (raw)
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Plasma Membrane-Associated Actin in Bright Yellow 2 Tobacco Cells
Plant Physiology, 1998
Plasma membrane ghosts form when plant protoplasts attached to a substrate are lysed to leave a small patch of plasma membrane. We have identified several factors, including the use of a mildly acidic actin stabilization buffer and the inclusion of glutaraldehyde in the fixative, that allow immunofluorescent visualization of extensive cortical actin arrays retained on membrane ghosts made from tobacco (Nicotiana tabacum L.) suspension-cultured cells (line Bright Yellow 2). Normal microtubule arrays were also retained using these conditions. Membrane-associated actin is random; it exhibits only limited coalignment with the microtubules, and microtubule depolymerization in whole cells before wall digestion and ghost formation has little effect on actin retention. Actin and microtubules also exhibit different sensitivities to the pH and K ؉ and Ca 2؉ concentrations of the lysis buffer. There is, however, strong evidence for interactions between actin and the microtubules at or near the plasma membrane, because both ghosts and protoplasts prepared from taxol-pretreated cells have microtubules arranged in parallel arrays and an increased amount of actin coaligned with the microtubules. These experiments suggest that the organization of the cortical actin arrays may be dependent on the localization and organization of the microtubules.
Chapter 2 Actin-Microtubule Interaction in Plants
2018
Interactions between actins and microtubules play an important role in many fundamental cellular processes in eukaryotes. Although several studies have shown actins and microtubules to be involved in specific cellular activities, little is known about how actins and microtubules contribute together to a given process. Preprophase band formation, which plays an essential role in plant division site determination, is a cellular process that lends itself to studies of actin-microtubule interactions and how they contribute to important cellular functions. Recently, we have analyzed microtubule-associated microfilaments during preprophase band formation in onion cotyledon epidermal cells using a combination of high-pressure freezing/freeze substitution and electron tomography. Quantitative analysis of our electron tomography data showed that relatively short single microfilaments form bridges between two adjacent microtubules in the process of narrowing of the preprophase microtubule ban...
Plant and Cell Physiology, 2004
The roles of actin microfilaments (MFs) in the organization of microtubules (MTs) at the M/G 1 interface were investigated in transgenic tobacco BY-2 cells stably expressing a GFP-tubulin fusion protein, using the MF-disrupting agent, Bistheonellide A (BA). When MFs were disrupted by BA treatment, cortical MTs (CMTs) did not become reorganized even 3 h after phragmoplast collapse, whereas nontreated cells completed CMT reorganization within 1 h. Furthermore, in the absence of MFs, the tubulin proteins did not show appropriate recruitment but remained at the site where the phragmoplast had existed, or extra-phragmoplasts were organized. These extra-phragmoplasts could functionally form extra-cell plates. This is the first observation of the formation of multiple cell plates during one nuclear division, and of phragmoplast generation irrespective of the position of the mitotic spindle or nuclei. The significance of these observations on the role of MFs at the M/G 1 interface is discussed.
Plant Cell Reports, 2006
Excellent visualisation of microtubules and actin filaments was obtained in fixed tobacco BY-2 suspension cells after optimising a protocol for whole mount immunolabelling. The procedure is based on modification of fixation, cell wall digestion, dimethyl sulfoxide (DMSO) treatment, post fixation, and blocking. The most critical aspects of successful preservation and visualization of cytoskeletal elements appeared to be: a two-step fixation with paraformaldehyde and glutaraldehyde before enzymatic cell wall digestion and a post fixation with aldehydes thereafter. The method allows the improved visualization of the organisation of the microtubular and actin filament arrays during the successive stages of cell division and at interphase. Although we present the application of our protocols for cytoskeleton labelling, the excellent results show the potential of using this method for the analysis of various proteins and molecules in plant cells.
Protoplasma, 1980
The arrangement of cortical microtubules in tobacco protoplasts is described using the following techniques: 1. Transmission electron microscopy (TEM) of thin sections of whole protoplasts, 2. TEM of negatively stained protoplast ghosts, and 3. Indirect immunofluorescence microscopy of protoplast ghosts. Ghosts were prepared by attaching freshly isolated protoplasts to glass coverslips or formvar/carbon-coated grids with poly-L-lysine and then bursting them either osmotically or by detergent treatment in the presence of a microtubule stabilizing buffer. Osmotic bursting of protoplasts yielded large pieces of plasma membrane with attached microtubules. These preparations proved very useful for measuring the density and length of cortical microtubules. Detergent treatment dissolved the plasma membrane and altered the distribution of cortical microtubules.
Protoplasma, 1989
Double labeling with fluorescent probes showed that in the cortical cytoplasm of pollen tubes of Nicotiana tabacum and Lilium longiflorum, the microtubules and actin filaments co-localize for the most part. They displayed complex net-axial or helical distributions. They structural association of microtubules and actin filaments implies a functional relationship with respect to organelle movement and/or the organization of the cortical cytoplasm and cell surface.
The Journal of Cell Biology, 1981
Microtubules participate as morphogenetic tools in two basic processes by which plants develop their characteristic forms : (a) production of new cells in specific sites and with specific initial shapes by partitioning of parental cells, and (b) further shaping of the progeny during their expansion and differentiation . In respect of (a), microtubules are present in the mitotic spindle, where they develop in the absence of centrioles (14). Immediately before the division cycle they are deposited as a transitory "pre-prophase band" (PPB) , which in its positioning predicts the site and plane of the future cytokinesis. At telophase another microtubule system contributes to the organization of the phragmoplast, which contains the new partitioning wall, or cell plate. In respect of(b), there are many instances of congruence between the orientation of microtubules in the cell cortex during interphase ("interphase cortical arrays") and the orientation of currently deposited microfibrils of cell wall material (see 12 and 15 for recent summaries) . The inference is that the cell exerts geometrical control over its expansion by setting up specifically oriented microtubule arrays. These in turn guide wall deposition, thereby regulating the mechanical properties of the wall and determining its spatial reaction to the turgor forces that drive cell expansion.
Journal of Plant Research
In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global-local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global-local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.
Protoplasma, 2003
The mode of cytokinesis, especially in determining the site of cell division, is not well understood in higher-plant cells. The division site appears to be predicted by the preprophase band of microtubules that develop with the phragmosome, an intracellular structure of the cytoplasm suspending the nucleus and the mitotic apparatus in the center. As the preprophase band disappears during mitosis, it is thought to leave some form of "memory" on the plasma membrane to guide the growth of the new cell plate at cytokinesis. However, the intrinsic nature of this "memory" remains to be clarified. In addition to microtubules, microfilaments also dynamically change forms during cell cycle transition from the late G 2 to the early G 1 phase. We have studied the relationships between microtubules and microfilaments in tobacco BY-2 cells and transgenic BY-2 cells expressing a fusion protein of green-fluorescent protein and tubulin. At the late G 2 phase, microfilaments colocalize with the preprophase band of microtubules. However, an actin-depleted zone which appears at late prometaphase is observed around the chromosomes, especially at metaphase, but also throughout anaphase. To study the functions of the actin-depleted zone, we disrupted the microfilament structures with bistheonellide A, a novel macrolide that depolymerizes microfilaments very rapidly even at low concentrations. The division planes became disorganized when the drug was added to synchronized BY-2 cells before the appearance of the actin-depleted zone. In contrast, the division planes appeared smooth, as in control cells, when the drug was added after the appearance of the actin-depleted zone. These results suggest that the actin-depleted zone may participate in the demarcation of the division site at the final stage of cell division in higher plants.