Sidney L. Shaw

Nod Factor Elicits Two Separable Calcium Responses in Medicago truncatula Root Hair Cells

Modulation of intracellular calcium levels plays a key role in the transduction of many biological signals. Here, we characterize early calcium responses of wild-type and mutant Medicago truncatula plants to nodulation factors produced by the bacterial symbiont Sinorhizobium meliloti using a dual-dye ratiometric imaging technique. When presented with 1 nm Nod factor, root hair cells exhibited only the previously described calcium spiking response initiating 10 min after application. Nod factor (10 nm) elicited an immediate increase in calcium levels that was temporally earlier and spatially distinct from calcium spikes occurring later in the same cell. Nod factor analogs that were structurally related, applied at 10 nm, failed to initiate this calcium flux response. Cells induced to spike with low Nod factor concentrations show a calcium flux response when Nod factor is raised from 1 to 10 nm. Plant mutants previously shown to be deficient for the calcium spiking response (dmi1 and dmi2) exhibited an immediate, truncated calcium flux with 10 nm Nod factor, demonstrating a competence to respond to Nod factor but an impaired ability to generate a full biphasic response. These results demonstrate that the legume root hair cell exhibits two independent calcium responses to Nod factor triggered at different agonist concentrations and suggests an early branch point in the Nod factor signal transduction pathway.

Nod Factor Inhibition of Reactive Oxygen Efflux in a Host Legume

Hydrogen peroxide (H2O2) efflux was measured from Medicago truncatula root segments exposed to purified Nod factor and to poly-GalUA (PGA) heptamers. Nod factor, at concentrations > 100 pm, reduced H2O2 efflux rates to 60% of baseline levels beginning 20 to 30 min after exposure, whereas the PGA elicitor, at > 75 nm, caused a rapid increase in H2O2 efflux to >200% of baseline rates. Pretreatment of plants with Nod factor alters the effect of PGA by limiting the maximum H2O2 efflux rate to 125% of that observed for untreated plants. Two Nod factor-related compounds showed no ability to modulate peroxide efflux, and tomato (Lycopersicon esculentum), a nonlegume, showed no response to 1 nm Nod factor. Seven M. truncatula mutants, lacking the ability to make nodules, were tested for Nod factor effects on H2O2 efflux. The nfp mutant was blocked for suppression of peroxide efflux, whereas the dmi1 and dmi2 mutants, previously shown to be blocked for early Nod factor responses, showed a wild-type peroxide efflux modulation. These data demonstrate that exposure to Nod factor suppresses the activity of the reactive oxygen-generating system used for plant defense responses.

Localization of Actin mRNA during the Establishment of Cell Polarity and Early Cell Divisions in Fucus Embryos.

Localization of mRNA is a well-described mechanism to account for the asymmetric distribution of proteins in polarized somatic cells and embryos of animals. In zygotes of the brown alga Fucus, F-actin is localized at the site of polar growth and accumulates at the cell plates of the first two divisions of the embryo. We used a nonradioactive, whole-mount in situ hybridization protocol to show the pattern of actin mRNA localization. Until the first cell division, the pattern of actin mRNA localization is identical to that of total poly(A)+ RNA, that is, a symmetrical distribution in the zygote followed by an actin-dependent accumulation at the thallus pole at the time of polar axis fixation. At the end of the first division, actin mRNA specifically is redistributed from the thallus pole to the cell plates of the first two divisions in the rhizoid. This specific pattern of localization in the zygote and embryo involves the redistribution of previously synthesized actin mRNA. The initial asymmetry of actin mRNA at the thallus pole of the zygote requires polar axis fixation and microfilaments but not microtubules, cell division, or polar growth. However, redistribution of actin mRNA from the thallus pole to the first cell plate is insensitive to cytoskeletal inhibitors but is dependent on cell plate formation. The F-actin that accumulates at the rhizoid tip is not accompanied by the localization of actin mRNA. However, maintenance of an accumulation of actin protein at the cell plates of the rhizoid could be explained, at least partially, by a mechanism involving localization of actin mRNA at these sites. The pattern and requirements for actin mRNA localization in the Fucus embryo may be relevant to polarization of the embryo and asymmetric cell divisions in higher plants as well as in other tip-growing plant cells.

Role of the cell wall in the determination of cell polarity and the plane of cell division in Fucus embryos

Recent studies using Fucus embryos indicate that the cell wall relays positional information to the underlying plasma membrane and cytosol to establish a polar axis, initiate polar growth and orient the first cell division plane of the zygote. It is the formation and stabilization of the polar axis in the zygote — not the position of the first division plane — which is critical for the formation of the initial embryonic pattern in Fucus . The pattern of asymmetry in the cell wall set at the time of polar axis fixation may also be the basis for the determination of rhizoid and thallus characteristics in the embryo. The cell division in early Arabidopsis embryos is very similar to that in Fucus embryos, and thus these findings may be pertinent to higher plants.

The mechanics of surface expansion anisotropy in Medicago truncatula root hairs

Wall expansion in tip-growing cells shows variations according to position and direction. In Medicago truncatula root hairs, wall expansion exhibits a strong meridional gradient with a maximum near the pole of the cell. Root hair cells also show a striking expansion anisotropy, i.e. over most of the dome surface the rate of circumferential wall expansion exceeds the rate of meridional expansion. Concomitant measurements of expansion rates and wall stresses reveal that the extensibility of the cell wall must vary abruptly along the meridian of the cell to maintain the gradient of wall expansion. To determine the mechanical basis of expansion anisotropy, we compared measurements of wall expansion with expansion patterns predicted from wall structural models that were either fully isotropic, transversely isotropic, or fully anisotropic. Our results indicate that a model based on a transversely isotropic wall structure can provide a good fit of the data although a fully anisotropic model offers the best fit overall. We discuss how such mechanical properties could be controlled at the microstructural level.

Imaging green fluorescent protein fusion proteins in Saccharomyces cerevisiae

Tagging expressed proteins with the green fluorescent protein (GFP) from Aequorea victoria [1] is a highly specific and sensitive technique for studying the intracellular dynamics of proteins and organelles. We have developed, as a probe, a fusion protein of the carboxyl terminus of dynein and GFP (dynein-GFP), which fluorescently labels the astral microtubules of the budding yeast Saccharomyces cerevisiae. This paper describes the modifications to our multimode microscope imaging system [2,3], the acquisition of three-dimensional (3-D) data sets and the computer processing methods we have developed to obtain time-lapse recordings of fluorescent astral microtubule dynamics and nuclear movements over the complete duration of the 90-120 minute yeast cell cycle. This required low excitation light intensity to prevent GFP photobleaching and phototoxicity, efficient light collection by the microscope optics, a cooled charge-coupled device (CCD) camera with high quantum efficiency, and image reconstruction from serial optical sections through the 6 micron-wide yeast cell to see most or all of the astral molecules. Methods are also described for combining fluorescent images of the microtubules labeled with dynein-GFP with high resolution differential interference contrast (DIC) images of nuclear and cellular morphology [4], and fluorescent images of the chromosomes stained with 4,6-diamidino-2-phenylindole (DAPI) [5].

Microtubule-Associated Proteins MAP65-1 and MAP65-2 Positively Regulate Axial Cell Growth in Etiolated Arabidopsis Hypocotyls

The localization of fluorescently tagged Arabidopsis MAP65-1 and MAP65-2 is monitored in live epidermal hypocotyl cells. Cortical microtubules are shown to bundle via a microtubule-microtubule templating mechanism, and MAP65-1 and MAP65-2 are shown to contribute to the mechanism of axial cell growth in expanding hypocotyls. The Arabidopsis thaliana MAP65-1 and MAP65-2 genes are members of the larger eukaryotic MAP65/ASE1/PRC gene family of microtubule-associated proteins. We created fluorescent protein fusions driven by native promoters that colocalized MAP65-1 and MAP65-2 to a subset of interphase microtubule bundles in all epidermal hypocotyl cells. MAP65-1 and MAP65-2 labeling was highly dynamic within microtubule bundles, showing episodes of linear extension and retraction coincident with microtubule growth and shortening. Dynamic colocalization of MAP65-1/2 with polymerizing microtubules provides in vivo evidence that plant cortical microtubules bundle through a microtubule-microtubule templating mechanism. Analysis of etiolated hypocotyl length in map65-1 and map65-2 mutants revealed a critical role for MAP65-2 in modulating axial cell growth. Double map65-1 map65-2 mutants showed significant growth retardation with no obvious cell swelling, twisting, or morphological defects. Surprisingly, interphase microtubules formed coaligned arrays transverse to the plant growth axis in dark-grown and GA4-treated light-grown map65-1 map65-2 mutant plants. We conclude that MAP65-1 and MAP65-2 play a critical role in the microtubule-dependent mechanism for specifying axial cell growth in the expanding hypocotyl, independent of any mechanical role in microtubule array organization.

A High-Resolution Multimode Digital Microscope System

In this chapter we describe the development of a high-resolution, multimode digital imaging system based on a wide-field epifluorescent and transmitted light microscope and a cooled charge-coupled device (CCD) camera. Taylor and colleagues (Farkas et al., 1993; Taylor et al., 1992) have reviewed the advantages of using multiple optical modes to obtain quantitative information about cellular processes and described instrumentation they have developed for multimode digital imaging. The instrument described here is somewhat specialized for our microtubule and mitosis studies, but it is also applicable to a variety of problems in cellular imaging including tracking proteins fused to the green fluorescent protein (GFP) in live cells (Cubitt et al., 1995; Heim and Tsien, 1996; Olson et al., 1995). For example, the instrument has been valuable for correlating the assembly dynamics of individual cytoplasmic microtubules (labeled by conjugating X-rhodamine to tubulin) with the dynamics of membranes of the endoplasmic reticulum (ER, labeled with DiOC6) and the dynamics of the cell cortex [by differential interference contrast (DIC)] in migrating vertebrate epithelial cells (Waterman-Storer and Salmon, 1997). The instrument has also been important in the analysis of mitotic mutants in the powerful yeast genetic system Saccharo-myces cerevisiae. Yeast cells are a major challenge for high-resolution imaging of nuclear or microtubule dynamics because the preanaphase nucleus is only about 2 μm wide in a cell about 6 μm wide. We have developed methods for visualizing nuclear and spindle dynamics during the cell cycle using high-resolution digitally enhanced DIC (DE-DIC) imaging (Yang et al., 1997; Yeh et al., 1995). Using genetic and molecular techniques. Bloom and coworkers (Shaw et al., 1997a,b) have been able to label the cytoplasmic astral microtubules in dividing yeast cells by expression of cytoplasmic dynein fused to GFP. Overlays of GFP and DIC images of dividing cells have provided the opportunity to see for the first time the dynamics of cytoplasmic microtubules in live yeast cells and how these dynamics and microtubule interactions with the cell cortex change with mitotic cell cycle events in wild-type and in mutant strains (Shaw et al., 1997a,b). Our high-resolution multimode digital imaging system is shown in Fig. 1 and diagrammed in Fig. 2. The legend to Fig. 2 provides model numbers and sources of the key components of the instrument. There are three main parts to the system: a Nikon FXA microscope, a Hamamatsu C4880 cooled CCD camera, and a MetaMorph digital imaging system. First we will consider our design criteria for the instrument, then consider separately the major features of the microscope components, the cooled CCD camera, and the MetaMorph digital imaging system. The reader is referred to other sources for general aspects of microscope optics (Inoue and Oldenbourg, 1993; Keller, 1995; Spencer, 1982); DIC microscopy (Salmon, 1998); fluorescence microscopy (Taylor and Salmon, 1989); video cameras, slow-scan cameras, and video microscopy (Aikens et al., 1989; CLMIB staff, 1995; Inoue, 1986; Inoue and Spring, 1997; Shotten, 1995) and 3D imaging methods (Carrington et al., 1995; Hiroka et al., 1991; Pawley, 1995). Fig. 1 Photograph of the multimode digital imaging system including the Nikon FXA upright microscope sitting on an air table (Newport Corp., Irvine, CA, VW-3660 with 4-inch-high tabletop). Images are captured with a Hamamatsu C4880 cooled CCD camera. Image acquisition, ... Fig. 2 Component parts are L1, 100-watt quartz halogen lamp; S1, Uniblitz shutter (No. 225L2A1Z523398, Vincent Associates, Rochester, NY); G, ground glass diffuser: FBI, manual filter changers including KG4 heat cut and green interference filters; I1, field ... II. Design Criteria A. Fluorescence Considerations When we began building our instrument 4 years ago, our primary objective was to obtain quantitative time-lapse records of spindle microtubule dynamics and chromosome movements for mitosis in live tissue culture cells and in in vitro assembled spindles in Xenopus egg extracts. Fluorescence optical components were chosen, in part, based on the fluorophores that were available for labeling microtubules, chromosomes, and other microtubule- or spindle-associated components. Microtubules could be fluorescently labeled along their lengths by incorporating X-rhodamine-labeled tubulin into the cytoplasmic tubulin pool (Fig. 3; Murray et al., 1996; Salmon et al., 1994). In addition, we needed to use fluorescence photoactivation methods to produce local marks on spindle microtubules to study the dynamics of their assembly (Mitchison and Salmon, 1993; Waters et al., 1996). This is accomplished by the addition of a cagedfluorescein-labeled tubulin to the cytoplasmic pool and fluorescence photoactivation with a 360-nm microbeam as described by Mitchison and coworkers (Mitchison, 1989; Sawin and Mitchison, 1991; Sawin et al., 1992). In extracts and some living cells, chromosomes can be vitally stained with the DNA intercalating dyes DAPI or Hoescht 33342 (Murray et al., 1996; Sawin and Mitchison, 1991). Thus, in fluorescence modes, we needed to be able to obtain images in a “red channel” for X-rhodamine microtubules, a “green channel” for photoactivated fluorescein marks, and a “blue channel” for chromosomes. Fig. 3 Views of a living, dividing yeast, Saccharomyces cerevisiae, by (A) fluorescence of GFP protein bound to nuclear histones and (B) DIC.* (C) Images in DIC of the 0.24-μm spacing between rows of surface pores of the diatom Amphipleura illuminated ... B. Live Cell Considerations Photobleaching and photodamage are a major concern during time-lapse imaging of fluorescently tagged molecules in live cells or in in vitro extracts. Minimizing these problems requires that the specimen be illuminated by light shuttered between camera exposures, that the imaging optics have high transmission efficiencies, that the camera detectors have high quantum efficiency at the imaging wavelengths, and that light can be integrated on the detector to reduce illumination intensity and allow longer imaging sessions without photo-damage. This later point is true not only for epifluorescence but also for transmitted light (phase-contrast or DIC) imaging. In our studies, the detector also needed a high dynamic range (12 bits = 4096 gray levels), as we wanted to be able to quantitate the fluorescence of a single microtubule or the whole mitotic spindle, which could have 1000 or more microtubules. In addition, the red, green, and blue images needed to be in focus at the same position on the detector so that the fluorophores within the specimen could be accurately correlated.

The cell biology of peritrichous flagella in Bacillus subtilis

Bacterial flagella are highly conserved molecular machines that have been extensively studied for assembly, function and gene regulation. Less studied is how and why bacteria differ based on the number and arrangement of the flagella they synthesize. Here we explore the cell biology of peritrichous flagella in the model bacterium Bacillus subtilis by fluorescently labelling flagellar basal bodies, hooks and filaments. We find that the average B. subtilis cell assembles approximately 26 flagellar basal bodies and we show that basal body number is controlled by SwrA. Basal bodies are assembled rapidly (< 5 min) but the assembly of flagella capable of supporting motility is rate limited by filament polymerization (> 40 min). We find that basal bodies are not positioned randomly on the cell surface. Rather, basal bodies occupy a grid‐like pattern organized symmetrically around the midcell and that flagella are discouraged at the poles. Basal body position is genetically determined by FlhF and FlhG homologues to control spatial patterning differently from what is seen in bacteria with polar flagella. Finally, spatial control of flagella in B. subtilis seems more relevant to the inheritance of flagella and motility of individual cells than the motile behaviour of populations.

Requirement of essential Pbp2x and GpsB for septal ring closure in Streptococcus pneumoniae D39

Bacterial cell shapes are manifestations of programs carried out by multi‐protein machines that synthesize and remodel the resilient peptidoglycan (PG) mesh and other polymers surrounding cells. GpsB protein is conserved in low‐GC Gram‐positive bacteria and is not essential in rod‐shaped Bacillus subtilis, where it plays a role in shuttling penicillin‐binding proteins (PBPs) between septal and side‐wall sites of PG synthesis. In contrast, we report here that GpsB is essential in ellipsoid‐shaped, ovococcal Streptococcus pneumoniae (pneumococcus), and depletion of GpsB leads to formation of elongated, enlarged cells containing unsegregated nucleoids and multiple, unconstricted rings of fluorescent‐vancomycin staining, and eventual lysis. These phenotypes are similar to those caused by selective inhibition of Pbp2x by methicillin that prevents septal PG synthesis. Dual‐protein 2D and 3D‐SIM (structured illumination) immunofluorescence microscopy (IFM) showed that GpsB and FtsZ have overlapping, but not identical, patterns of localization during cell division and that multiple, unconstricted rings of division proteins FtsZ, Pbp2x, Pbp1a and MreC are in elongated cells depleted of GpsB. These patterns suggest that GpsB, like Pbp2x, mediates septal ring closure. This first dual‐protein 3D‐SIM IFM analysis also revealed separate positioning of Pbp2x and Pbp1a in constricting septa, consistent with two separable PG synthesis machines.