Susan M. Wick

Organized F-Actin Is Essential for Normal Trichome Morphogenesis in Arabidopsis

Actin microfilaments form a three-dimensional cytoskeletal network throughout the cell and constitute an essential throughway for organelle and vesicle transport. Development of Arabidopsis trichomes, unicellular structures derived from the epidermis, is being used as a genetic system in which to study actin-dependent growth in plant cells. The present study indicates that filamentous actin (F-actin) plays an important role during Arabidopsis trichome morphogenesis. For example, immunolocalization of actin filaments during trichome morphogenesis identified rearrangements of the cytoskeletal structure during the development of the mature cell. Moreover, pharmacological experiments indicate that there are distinct requirements for actin- and microtubule-dependent function during trichome morphogenesis. The F-actin–disrupting drug cytochalasin D does not affect the establishment of polarity during trichome development; however, maintenance and coordination of the normal pattern of cell growth are very sensitive to this drug. In contrast, oryzalin, an agent that depolymerizes microtubules, severely inhibits cell polarization. Furthermore, cytochalasin D treatment phenocopies a known class of mutations that cause distorted trichome morphology. Results of an analysis of cell shape and microfilament structure in wild-type, mutant, and drug-treated trichomes are consistent with a role for actin in the maintenance and coordination of an established growth pattern.

Preprophase bands, phragmoplasts, and spatial control of cytokinesis.

SUMMARY Features of preprophase bands (PPBs) of microtubules (MTs), and the spatial relationship between phragmosomes, PPB sites, and developing phragmoplasts during cytokinesis, are reviewed, setting new observations in the context of current knowledge. PPBs in onion root tip cells are present by the beginning of the G2 period of the cell cycle. They are at first wide, but later become more compact, narrower bands. MTs traverse the cytoplasm between the band at the cell cortex and the nuclear envelope. This whole assemblage of nucleus, PPB and intervening MTs remains together when the cell is ruptured during preparation for examination by immunofluorescence microscopy. Double bands are occasionally seen in early stages of PPB development, perhaps as a consequence of double induction from neighbouring cells. Calmodulin is not present in PPBs at a higher concentration than in the general cytoplasm, but it is more abundant in parts of the spindle and in the phragmoplast. The PPB MTs disappear at prophase, but nevertheless the new cell plate fuses with the parental cell walls at the PPB site. This spatial relationship can be disrupted by treatment with CIPC. Another experimental disruption of the relationship, accomplished by making minute wounds in the PPB site of mitotic cells in Tradescantia stamen hairs, is described. In other experiments on these cells the phragmoplast is shown to become tethered to the PPB site when the cell plate is half to three-quarters developed, although the telophase nuclei are free to move. Rhodamine-labelled phalloidin reveals putative F-actin in the phragmoplast of Tradescantia, but not in the gap between the extending phragmoplast and the PPB site. Rhodamine-labelled phalloidin also stains cytoplasmic strands that exist when cytoplasmic streaming occurs before and after (but not during) mitosis. Cytochalasin B treatment blocks incorporation of actin into the phragmoplast, which, however, can still develop, though usually abnormally. The F-actin of the phragmoplast may function in consolidation of the cell plate, rather than in spatial guidance of its growth toward the PPB site at the cell surface.

Selective localization of intracellular Ca2+ with potassium antimonate.

Introduction As a result of concerted efforts by scientists from a wide range of disciplines, we are coming to realize the crucial role played by Ca2 in biological functions. In the realm of biochemistry, especially, there has been recently an explosive rise in our knowledge about Ca2 effects on numerous cellular reactions, processes, and structural components. One emerging concept is that Ca2 ‘ serves as regulator of many of these. In such wellexamined phenomena as muscle contraction, a high degree of compartmentalization of Ca2 , coupled with the cell’s ability to mobilize Ca2 among various compartments (and thus to locally alter levels of reactive Ca2 ), is the means by which this is achieved (2). Often, direct measurement of the dynamics of free Ca2 is technologically difficult. Many have chosen instead to cxamine intracellular Ca2 -binding or Ca2 -sequestration sites in the search for clues on regulatory processes. There exist several histochemical techniques for localization of these sites, among them being in situ precipitation ofCa2 with potassium antimonate. The ideal probe should retain the cell’s in vivo Ca2 distribution, maximize its detection, and minimize interferenee from other reacting species. Realistically speaking, few, if any, techniques in science match up to their ideal: limitations and dangers of artifact abound, and histoehemistry holds no exception. Use of antimonate has received its share of criticism, and, indeed, the variety of eations reported to precipitate with antimonate could easily lead one to conclude that specificity for Ca2 is not possible with the reaction. However, a closer look at the literature reveals that “antimonate precipitation” does not specify a unique procedure, but rather encompasses a bewildering array of variations. A survey of results obtained by others using different buffers, pH’s, antimonate concentrations, fixatives, and tissue pretreatments, as well as our own experience in handling the reagent, indicates that reaction parameters strongly influence retention of and precipitation of physiological cations relative to each other. Thus, while originally proposed and used as a means of loealizing Na (40), antimonate’s use recently has been almost exclusively in studies involving Ca2 localization. As elaborated in this review, careful choice of reaction conditions can make the antimonate technique highly selective for Ca2 in comparison to the other cations that are capable of precipitation. Also, other variations on the antimonate reaction, while not so specific for Ca2 , can be used in conjunction with analytical techniques such as X-ray analysis or chelator treatments to ascertain which of the deposits formed contain Ca2 . By means of several different antimonate procedures, coupled thus with deposit analysis, previous studies have localized Ca2 in a wide variety of tissue and cell types, and cumulatively have revealed Ca2 in nearly every type of membranous organelle, as well as in association with some nonmembranous cellular components (Table 1 ). We believe that a discussion of some parameters of antimonate precipitation is instructive for those considering its use, as well as for those trying to understand results obtained with it in the past. When comparing results obtained in various laboratories, it is often difficult to pinpoint the influence exerted by any single parameter of the technique, since even the most similar protocols usually differ from each other in several details. While we have tried to sort these out as much as possible, there are substantial areas ofunavoidable overlap with material discussed in other sections. In these eases, the reader is requested to cross-refer to appropriate sections for a more detailed analysis of other influencing factors. We hope this cxercise provides evidence that it is possible to employ antimonate as a selective electron microscopic histoehemical stain for localization of exchangeable cellular Ca2 and that, in spite of inevitable limitations, it is a useful tool for exploring Ca2 regulation.

Spatial aspects of cytokinesis in plant cells

Plant cytokinesis in a particular orientation and location can be viewed as having several component stages, often beginning with the establishment of division polarity before karyokinesis occurs. Improved methods for preserving the in situ distribution of actin microfilaments and observations of individual live cells during treatment with cytoskeleton-disrupting drugs are making it possible to elucidate the roles of microtubules and microfilaments in cytokinesis. Current evidence points to involvement of the cytoskeleton throughout the stages of preparation for, and execution of, cytokinesis in many types of plant cell division.

Immunofluorescence microscopy of tubulin and microtubule arrays in plant cells. II. Transition between the pre-prophase band and the mitotic spindle

SummaryChanging patterns of tubulin immunofluorescence as onion root meristematic cells progress from a mature pre-prophase band (PPB) stage into mitosis are reported here. The PPB reaches its narrowest profile at maturity and then remains the same width throughout the rest of the transition. Concomitant with continuation of chromatin condensation and nucleolar breakdown, both initiated earlier in pre-prophase, alignment of fluorescent fibers along the nuclear envelope (NE) changes. Perinuclear microtubules (MTs), which were parallel to the PPB or randomly arranged when first seen at earlier stages of pre-prophase, assume the orientation of spindle MTS at late preprophase. They lie close to the NE and follow the nuclear contour, ultimately converging upon two focal points directly at the NE surface. MTs also can be seen traversing the cytoplasm between nucleus and cell periphery.As spindle initiation proceeds, PPB fluorescence intensity decreases and eventually is exceeded by the NE-associated fluorescence. PPB and spindle arrays co-exist briefly in the transition period, with spindle MTs typically aligned perpendicular to both the axis of the PPB and its constituent MTs. Total disappearance of the PPB occurs only after chromosome condensation is complete and the nucleus is contained within a spherical or ellipsoid cage of fluorescent fibers comprised of two non-overlapping half-spindles. Like the fully formed prophase spindle which follows, the incipient spindle is neither barrel-shaped nor fusiform, but rather displays MTs radiating from the poles in a smooth arc.

Immunofluorescence microscopy of tubulin and microtubule arrays in plant cells. III. Transition between mitotic/ cytokinetic and interphase microtubule arrays

Immunofluorescence microscopy of flowering plant root cells indicates that the earliest interphase microtubules appear during cytokinesis, radiating from the former spindle poles and subsequently from the nuclear envelope. They form networks that have microtubule focal points in the cortex underlying cell faces and in the cytoplasm between the nucleus and cortex. Cortical networks are rapidly replaced by the highly aligned array normally associated with interphase. An antibody that in animal cells identifies the location of pericentriolar material, the site of microtubule initiation, is also localized around the plant cell nuclear envelope at the time that putative early interphase microtubule networks are seen.

Distribution and function of actin in the developing stomatal complex of winter rye (Secale cereale cv. Puma).

SummaryThe dynamics of actin distribution during stomatal complex formation in leaves of winter rye was examined by means of immunofluorescence microscopy of epidermal sheets. This method results in actin localization patterns that are the same as those seen with rhodamine-phalloidin staining, but are more stable. During stomatal development MFs are extensively rearranged, and most of the time the orientation or placement of MFs is distinctly different from that of MTs, the exception being co-localization of MTs and MFs in phragmoplasts. Although MFs show an orientation similar to that of MTs in interphase guard mother cells, no banding of MFs into anything resembling the interphase MT band is observed. From prophase to telophase, a distinct, dense concentration of MFs is found in subsidiary cell mother cells (SMCs) between the nucleus and the region of the cell cortex facing the guard mother cell. Cytochalasin B treatment causes incorrect positioning of the SMC nucleus/daughter nuclei and abarrent placement and orientation of the new cell wall that forms the boundary of the subsidiary cell at cytokinesis. These results suggest that MFs are involved in maintaining the SMC nucleus in its correct position and the SMC spindle in the correct orientation relative to the division site previously delineated by the preprophase band. Because these MFs thus appear to assure that the SMC phragmoplast begins to form in the correct orientation near the division site to which it needs to grow, we suggest that MFs are involved in control of correct placement and orientation of the new cell wall of the subsidiary cell.

Preprophase bands of microtubules and the cell cycle: Kinetics and experimental uncoupling of their formation from the nuclear cycle in onion root-tip cells

We have studied the timing of preprophase band (PPB) development in the division cycle of onion (Allium cepa L.) root-tip cells by combinations of immunofluorescence microscopy of microtubules, microspectrophotometry of nuclear DNA, and autoradiography of [3H]thymidine incorporation during pulse-chase experiments. In normally grown onion root tips, every cell with a PPB had the G2 level of nuclear DNA. Some were in interphase, prior to chromatin condensation, and some had varying degrees of chromatin condensation, up to the stage of prophase at which the PPB-prophase spindle transition occurs. In addition, autoradiography showed that PPBs can be formed in cells which have just finished their S phase, and microspectrophotometry enabled us to detect a population of cells in G2 which had no PPBs, these presumably including cells which had left the division cycle. The effects of inhibitors of DNA synthesis showed that the formation of PPBs is not fully coupled to events of the nuclear cycle. Although the mitotic index decreased 6-10-fold to less than 0.5% when roots were kept in 20 μg·ml-1 aphidicolin for more than 8 h, the percentage of cells containing PPBs did not decrease in proportion: the number of cells in interphase with PPBs increased while the number in prophase decreased. Almost the same phenomena were observed in the presence of 100 μg·ml-1 5-aminouracil and 40 μg·ml-1 hydroxyurea. In controls, all cells with PPBs were in G2 or prophase, but in the presence of aphidicolin, 5-aminouracil or hydroxyurea, some of the interphase cells with PPBs were in the S phase or even in the G1 phase. We conclude that PPB formation normally occurs in G2 (in at least some cases very early in G2) and that this timing can be experimentally uncoupled from the timing of DNA duplication in the cell-division cycle. The result accords with other evidence indicating that the cytoplasmic events of cytokinesis are controlled in parallel to the nuclear cycle, rather than in an obligatorily coupled sequence.

Double immunofluorescence labeling of calmodulin and tubulin in dividing plant cells

SummaryUsing rabbit antibodies to purified spinach calmodulin and monoclonal anti-tubulin, we have investigated via immunofluorescence microscopy the distribution of calmodulin relative to that of tubulin at all stages of the cell cycle in onion and pea root meristematic cells. Calmodulin is associated with the mitotic spindle and with the phragmoplast at cytokinesis, but patterns of calmodulin that coincide with those of interphase microtubule arrays have not been demonstrated with any of the several fixation and processing regimes tested. Calmodulin is not likely to be involved in regulating the formation of the preprophase band of microtubules or its disappearance during spindle formation because a localized cortical band of calmodulin is only very rarely seen in cells at these stages.Prolonged incubation with the calmodulin antagonist calmidazolium has variable effects on calmodulin localization patterns, sometimes reducing immunofluorescence associated with spindles, but not altering phragmoplast patterns. Compound 48/80, another calmodulin inhibitor, appears to inhibit division, and when mitotic spindles and phragmoplasts are found again after a 1–2 day continuous exposure to the drug, some are labeled with anti-calmodulin only very weakly or not at, all. Material treated briefly with 48/80 both before and during fixation likewise shows some reduction in the numbers of dividing cells displaying localization of calmodulin and in the intensity of localized staining.

Effects of various fixatives on the reactivity of plant cell tubulin and calmodulin in immunofluorescence microscopy

SummaryWe have examined the suitability of a variety of fixation regimes for immunofluorescence localization of tubulin and calmodulin in meristematic plant cells. Acrolein and most fixatives that contain glutaraldehyde (GA), while they have been employed by others in immunoenzyme, immunogold or immunofluorescence studies of plant endosperm, animal or plant tissue culture cells, cause unacceptably high background fluorescence of the dense cytoplasm of root meristem cells. Bifunctional imidoester and carbodiimide reagents do not give satisfactory results, either. Fixatives that have produced good results include paraformaldehyde (PFA) with the addition of picric acid or zinc salts or at high pH, a combination of PFA/GA/picric acid, and prefixation in PFA plus a monofunctional imidoester followed by PFA/GA. All of these cross-link the cytoplasm well enough so that cells can withstand isolation procedures, preserve antigenicity, allow antibody penetration and provide good contrast between specific and background fluorescence. The last two fixatives are of particular interest because of the potential they offer for immunoelectron microscopy of densely cytoplasmic, walled cells from tissues.