Improved Visualization of Oral Microbial Consortia.

Bacteria on the tongue dorsum (TD) form consortia tens to hundreds of microns in diameter organized around a core of epithelial cells. Whole-mount preparations have been instrumental in revealing their organization and specific microbial associations. However, their thickness and intricate 3-dimensional complexity present challenges for a comprehensive spatial analysis. To overcome these challenges, we employed a complementary approach: embedding in hydrophilic plastic followed by sectioning and postsectioning labeling. Samples were labeled by hybridization with multiplexed fluorescent oligonucleotide probes and visualized by spectral imaging and linear unmixing. Application of this strategy to TD biofilms improved the visualization of bacteria that were difficult to resolve in whole-mount imaging. Actinomyces, previously detected as patches, became resolved at the single-cell level. The filamentous taxa Leptotrichia and Lachnospiraceae, located at the core of the consortium, were regularly visualized whereas previously they were rarely detected when using whole mounts. Streptococcus salivarius, heterogeneously detected in whole mounts, were regularly and homogenously observed. Two-dimensional images provide valuable information about the organization of bacterial biofilms. However, they offer only a single plane of view for objects that can extend to hundreds of microns in thickness, and information obtained from such images may not always reflect the complexity of a 3-dimensional object. We combined serial physical sectioning with optical sectioning to facilitate the 3-dimensional reconstruction of consortia, spanning over 100 µm in thickness. Our work showcases the use of hydrophilic plastic embedding and sectioning for examining the structure of TD biofilms through spectral imaging fluorescence in situ hybridization. The result was improved visualization of important members of the human oral microbiome. This technique serves as a complementary method to the previously employed whole-mount analysis, offering its own set of advantages and limitations. Addressing the spatial complexity of bacterial consortia demands a multifaceted approach for a comprehensive and effective analysis.

[The spatial organization of centrosome-attached and free microtubules in 3T3 fibroblasts].

Microtubules spatial organization is essential for different cellular processes to proceed normally. It is supposed traditionally, that the fibroblasts have radial microtubule array consisting of long microtubules running from the centrosome. However, the detailed analysis of the microtubule array in the internal cytoplasm has never been performed. In the current study we used laser photobleaching for the analysis of the spatial organization of microtubules in the internal cytoplasm of cultured 3T3 fibroblasts. Cells were injected with Cy-3-labeled tubulin, and then in the bleached zone growth of microtubules in the centrosome region and in the peripheral parts of cytoplasm was analyzed. In most cases microtubules growth in the bleached zone occurred rectilinearly, on the distance up to 5 microm they seldom bend more than 10-15 degrees. We considered a growing fragment of the microtubule as a vector with the beginning in the point of occurrence and with the end in a point where growth terminated (or the end point after 30 s if microtubule's persistent growth proceeded longer). We defined the direction of microtubules growth in different parts of the cell using these vectors and measured the angle of their deviation from the vector of comparison. In the area of the centrosome we directed the vector of comparison inside of the bleached zone from the centrosome to the beginning of the growing microtubule segment; in fibroblast lamella and in fibroblast trailing part we used, the vector of comparison was directed along the long axis of the cell from its geometrical center to periphery. The microtubules growing immediately from the centrosome grew along the cell radius. However at a distance of 10 microm from the centrosome radially growing microtubules gave 40% from the overall number, and at a distance of 20 microm--only 25%. The rest of microtubules grew in different directions, with the preferred angle between their growth direction and cell radius around 90 degrees. Fibroblast lamella and trailing part 80% of all microtubules grew along the cell long axis or at the angle no more than 20 degrees, and 10-15% of microtubules grew along cell axis but towards the centrosome. Thus, in 3T3 fibroblasts the radial system of microtubules is perturbed starting from the distance of several microns from the centrosome. In the internal cytoplasm the microtubule system is completely disordered, and in the stretched parts of the polarized cell (lamella, trailing edge) the microtubule system again becomes well organized--microtubules are preferentially oriented along the long cell axis. From the results obtained we conclude that orderliness of microtubules at the periphery of the fibroblast is not a consequence of their growth from the centrosome, but their orientation is preset by local factors.

Self-organization of a propulsive actin network as an evolutionary process.

The leading edge of motile cells is propelled by polymerization of actin filaments according to a dendritic nucleation/array treadmilling mechanism. However, little attention has been given to the origin and maintenance of the dendritic array. Here we develop and test a population-kinetics model that explains the organization of actin filaments in terms of the reproduction of dendritic units. The life cycle of an actin filament consists of dendritic nucleation on another filament (birth), elongation by addition of actin subunits and, finally, termination of filament growth by capping protein (death). The regularity of branch angle between daughter and mother filaments endows filaments with heredity of their orientation. Fluctuations of branch angle that become fixed in the actin network create errors of orientation (mutations) that may be inherited. In our model, birth and death rates depend on filament orientation, which then becomes a selectable trait. Differential reproduction and elimination of filaments, or natural selection, leads to the evolution of a filament pattern with a characteristic distribution of filament orientations. We develop a procedure based on the Radon transform for quantitatively analyzing actin networks in situ and show that the experimental results are in agreement with the distribution of filament orientations predicted by our model. We conclude that the propulsive actin network can be understood as a self-organizing supramolecular ensemble shaped by the evolution of dendritic lineages through natural selection of their orientation.

Dendritic organization of actin comet tails.

Polymerization of actin filaments is necessary for both protrusion of the leading edge of crawling cells and propulsion of certain intracellular pathogens, and it is sufficient for generating force for bacterial motility in vitro. Motile intracellular pathogens are associated with actin-rich comet tails containing many of the same molecular components present in lamellipodia, and this suggests that these two systems use a similar mechanism for motility. However, available structural evidence suggests that the organization of comet tails differs from that of lamellipodia. Actin filaments in lamellipodia form branched arrays, which are thought to arise by dendritic nucleation mediated by the Arp2/3 complex. In contrast, comet tails have been variously described as consisting of short, randomly oriented filaments, with a higher degree of alignment at the periphery, or as containing long, straight axial filaments with a small number of oblique filaments. Because the assembly of pathogen-associated comet tails has been used as a model system for lamellipodial protrusion, it is important to resolve this apparent discrepancy. Here, using a platinum replica approach, we show that actin filament arrays in comet tails in fact have a dendritic organization with the Arp2/3 complex localizing to Y-junctions as in lamellipodia. Thus, comet tails and lamellipodia appear to share a common dendritic nucleation mechanism for protrusive motility. However, comet tails differ from lamellipodia in that their actin filaments are usually twisted and appear to be under significant torsional stress.

Cadherin-mediated regulation of microtubule dynamics.

Epithelial polarization and neuronal outgrowth require the assembly of microtubule arrays that are not associated with centrosomes. As these processes generally involve contact interactions mediated by cadherins, we investigated the potential role of cadherin signalling in the stabilization of non-centrosomal microtubules. Here we show that expression of cadherins in centrosome-free cytoplasts increases levels of microtubule polymer and changes the behaviour of microtubules from treadmilling to dynamic instability. This effect is not a result of cadherin expression per se but depends on the formation of cell-cell contacts. The effect of cell-cell contacts is mimicked by application of beads coated with stimulatory anti-cadherin antibody and is suppressed by overexpression of the cytoplasmic cadherin tail. We therefore propose that cadherins initiate a signalling pathway that alters microtubule organization by stabilizing microtubule ends.

Detyrosinated (Glu) microtubules are stabilized by an ATP-sensitive plus-end cap.

Many cell types contain a subset of long-lived, 'stable' microtubules that differ from dynamic microtubules in that they are enriched in post-translationally detyrosinated tubulin (Glu-tubulin). Elevated Glu tubulin does not stabilize the microtubules and the mechanism for the stability of Glu microtubules is not known. We used detergent-extracted cell models to investigate the nature of Glu microtubule stability. In these cell models, Glu microtubules did not incorporate exogenously added tubulin subunits on their distal ends, while >70% of the bulk microtubules did. Ca(2+)-generated fragments of Glu microtubules incorporated tubulin, showing that Glu microtubule ends are capped. Consistent with this, Glu microtubules in cell models were resistant to dilution-induced breakdown. Known microtubule end-associated proteins (EB1, APC, p150(Glued) and vinculin focal adhesions) were not localized on Glu microtubule ends. ATP, but not nonhydrolyzable analogues, induced depolymerization of Glu microtubules in cell models. Timelapse and photobleaching studies showed that ATP triggered subunit loss from the plus end. ATP breakdown of Glu microtubules was inhibited by AMP-PNP and vanadate, but not by kinase or other inhibitors. Additional experiments showed that conventional kinesin or kif3 were not involved in Glu microtubule capping. We conclude that Glu microtubules are stabilized by a plus-end cap that includes an ATPase with properties similar to kinesins.

Immunostructural evidence for the template mechanism of microtubule nucleation.

Two opposing models have been proposed to explain how the gamma-tubulin ring complex (gammaTuRC) induces microtubule nucleation. In the 'protofilament' model, the gammaTuRC induces nucleation as a partially or completely straightened protofilament that is incorporated longitudinally into the wall of the nascent microtubule, whereas the 'template' model proposes that the gammaTuRC acts as a helical template that constitutes the base of the newly-formed polymer. Here we appraise these two models, using high-resolution structural and immunolocalization methods. We show that components of the gammaTuRC localize to a narrow zone at the extreme minus end of the microtubule and that these ends terminate in a pointed cap. Together, these results strongly favour the template model of microtubule nucleation.

The 300-kDa intermediate filament-associated protein (IFAP300) is a hamster plectin ortholog.

Plectin is a high-molecular-weight cytoskeleton-associated protein that was initially identified in intermediate filament (IF)-enriched fractions of rat C6 glioma cells. At the cellular level, plectin has been found to associate with IF networks and IF-associated structures that are involved in cell-cell and cell-substrate adhesions. IFAP300 is an IF-associated protein that was initially identified in hamster cells by a monoclonal antibody directed against a high molecular weight protein present in IF-enriched cytoskeletal preparations. Plectin and IFAP300 display similar distribution patterns within cells as determined by immunofluorescence. Based upon this and the finding that their biochemical properties are similar, it has been suggested that they may actually be orthologous proteins. In this paper we demonstrate that this is the case. Cloning and sequencing of most of the hamster plectin cDNA demonstrates that plectin is found in hamster cells and that its sequence is highly conserved between species. Using immunological cross-reactivity, epitope mapping, and immunoelectron microscopy, we show that IFAP300 is actually the hamster ortholog of plectin.

Actin machinery: pushing the envelope.

The reconstitution of microbial rocketing motility in vitro with purified proteins has recently established definitively that no myosin motor is required for protrusion. Instead, actin polymerization, in conjunction with a small number of proteins, is sufficient. A dendritic pattern of nucleation controlled by the Arp2/3 complex provides an efficient pushing force for lamellipodial motility.

Speckle microscopy: when less is more.

Fluorescent speckle microscopy is a new and simplified method for generating fiduciary marks on cellular structures. It promises to become the method of choice for studying polymer movement and dynamics in vivo.

[Microtubule dynamics in cultured cells]. / Dinamika mikrotrubochek v kul'tiviruemykh kletkakh.

The behavior of microtubules in cultured cells in a cooled matrix after the microinjection of fluorescent tubulin was studied using a frame recording by a digital camcorder. In the cell lamella, thepositive ends of individual microtubules extend and shorten at random. The histograms of rate distribution have an almost normal distribution with a mode around 0. The maximum rate of lengthening and shortening reaches 30 and 50 microns/min, respectively. The positive ends of microtubules in PtK1 cells were in an equilibrium state, while in murine embryonic fibroblasts and Vero cells, they were displaced, usually, to the cell edge. Free microtubules were present in the cells of all three cultures. In the epithelial cells, they were numerous and relatively stable, while in the fibroblasts, they occurred rarely and were depolymerized at the proximal end. Free microtubules in PtK1 cells appeared, mostly due to spontaneous assembly in the cytoplasm, not in the relationship with the preexisting microtubules, and, more rarely, due to breakage of long microtubules. Separation of microtubules from the centrosome is a very rare event. Unlike positive ends that were characterized by dynamic instability, negative ends were stable and were sometimes depolymerized. When long microtubules were broken, new negative ends were formed that were, as a rule, stable, while in the lamella of fibroblasts (in murine embryonic fibroblasts and Vero cells), new negative ends were immediately depolymerized: free microtubules existed in these cells no more than 1-2 min. A diffusion model has been proposed where the behavior of microtubule ends is considered as unidimensional diffusion. The coefficient of diffusion of positive ends in the epithelial cells is several times less than in the fibroblasts, thus suggesting a higher rate of tubulin metabolism in the fibroblasts, as compared to the epithelium. The results obtained indicate that for the exchange of long microtubules, the dynamic instability is not sufficient. In the fibroblasts, their exchange takes place, mostly, at the expense of depolymerization of the liberating negative ends, which agrees with the previously proposed conveyer hypothesis of microtubule assembly on the centrosome.

The role of Xgrip210 in gamma-tubulin ring complex assembly and centrosome recruitment.

The gamma-tubulin ring complex (gammaTuRC), purified from the cytoplasm of vertebrate and invertebrate cells, is a microtubule nucleator in vitro. Structural studies have shown that gammaTuRC is a structure shaped like a lock-washer and topped with a cap. Microtubules are thought to nucleate from the uncapped side of the gammaTuRC. Consequently, the cap structure of the gammaTuRC is distal to the base of the microtubules, giving the end of the microtubule the shape of a pointed cap. Here, we report the cloning and characterization of a new subunit of Xenopus gammaTuRC, Xgrip210. We show that Xgrip210 is a conserved centrosomal protein that is essential for the formation of gammaTuRC. Using immunogold labeling, we found that Xgrip210 is localized to the ends of microtubules nucleated by the gammaTuRC and that its localization is more distal, toward the tip of the gammaTuRC-cap structure, than that of gamma-tubulin. Immunodepletion of Xgrip210 blocks not only the assembly of the gammaTuRC, but also the recruitment of gamma-tubulin and its interacting protein, Xgrip109, to the centrosome. These results suggest that Xgrip210 is a component of the gammaTuRC cap structure that is required for the assembly of the gammaTuRC.

Two components of actin-based retrograde flow in sea urchin coelomocytes.

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase-specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a "push-pull" mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior.

Speckle microscopic evaluation of microtubule transport in growing nerve processes.

Assembly of microtubules is fundamental to neuronal morphogenesis. Microtubules typically form crosslinked bundles in nerve processes, precluding resolution of single microtubules at the light microscopic level. Therefore, previous studies of microtubule transport in neurites have had to rely on indirect approaches. Here we show that individual microtubules can be visualized directly in the axonal shafts of Xenopus embryo neurons by using digital fluorescence microscopy. We find that, although the array of axonal microtubules is dynamic, microtubules are stationary relative to the substrate. These results argue against a model in which newly synthesized tubulin is transported down the axon in the form of microtubules.

Progress in protrusion: the tell-tale scar.

The crawling movement of a cell involves protrusion of its leading edge, in coordination with the translocation of its cell body, and depends upon a cytoplasmic machinery able to respond to signals from the environment. Protrusion is now understood to be driven by actin polymerization, and signalling from membrane receptors to actin has been shown to be mediated by the Rho family of GTPases. However, a major gap in our understanding of regulated motility has been how to connect the signalling pathway to the motile machinery itself. Recent structural, biochemical and genetic studies have identified some of the missing links and provided a strong working model for the pathways and mechanisms by which the signals are interpreted and implemented.

Centrosomal and non-centrosomal microtubules.

While microtubule (MT) arrays in cells are often focused at the centrosome, a variety of cell types contain a substantial number of non-centrosomal MTs. Epithelial cells, neurons, and muscle cells all contain arrays of non-centrosomal MTs that are critical for these cells' specialized functions. There are several routes by which non-centrosomal MTs can arise, including release from the centrosome, cytoplasmic assembly, breakage or severing, and stabilization from non-centrosomal sites. Once formed, MTs that are not tethered to the centrosome must be organized, which can be accomplished by means of self-organization or by capture and nucleation of MTs where they are needed. The presence of free MTs requires stabilization of minus ends, either by MT-associated proteins or by an end-capping complex. Although some of the basic elements of free MT formation and organization are beginning to be understood, a great deal of work is still necessary before we have a complete picture of how non-centrosomal MT arrays are assembled in specific cell types.

Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia.

The leading edge (approximately 1 microgram) of lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 micrometer++ of the lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181-6186) for lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex.

Contribution of plus and minus end pathways to microtubule turnover.

Turnover is important for the maintenance and remodeling of the cytoskeleton during the processes of cell morphogenesis, mitosis and motility. Microtubule (MT) turnover is thought to occur by dynamic instability, growth and shortening at distal (plus) ends. Recent observation of MT release from the centrosome and depolymerization from proximal (minus) ends indicates the existence of a minus end pathway. To evaluate the relative contributions of plus and minus end pathways to turnover, we analyzed MT dynamics in a model system, the fish melanophore, a large non-motile cell with a regular radial array of long MTs. MT ends were tracked in digital fluorescence time-lapse sequences and life histories of individual MTs were analyzed using random walk theory generalized to the case of diffusion with drift. Analysis of plus end dynamics gave an apparent diffusion coefficient of D=7.5 microm2/minute. The random walk model predicts that the half-time for turnover driven solely by plus end dynamics will depend strongly on position in the cell. Based on the experimentally determined value of D, turnover of MTs near the center of a typical melanophore of radius 70 microm was calculated to require over 5 hours, a paradoxically long time. To examine MT behavior deep in the cytoplasm, we developed a novel, sequential subtraction mode of image analysis. This analysis revealed a subpopulation of MTs which shortened from their minus ends, presumably after constitutive release from the centrosome. Given the relative slowness of plus end dynamics to turn over the root of a long MT, the turnover of MTs near the cell center is determined primarily by the minus-end pathway. MTs released from the centrosome become replaced by newly nucleated ones. The relative contributions of plus and minus end pathways was estimated from the diffusion coefficient, D, for the plus end, the length distribution of MTs, t he frequency of free minus ends, and the rate of minus-end shortening. We conclude that, in large animal cells with a centrosomally focussed array of MTs, turnover occurs by a combination of plus and minus end pathways, the plus end dominating at the cell periphery and the minus end dominating near the cell center.

Network contraction model for cell translocation and retrograde flow.

Kinetic and structural analysis of the actin-myosin II system in mammalian fibroblasts and fish epidermal keratocytes suggests that the cell's motility machinery arises behind the leading edge in the form of myosin filament clusters immersed in an actin filament network. We discuss how the contraction of this actin-myosin II network is related to the formation of actin-myosin filament bundles, cell translocation and retrograde flow.