Live-imaging stem-cell homeostasis in the Arabidopsis shoot apex

A precise spatio-temporal regulation of growth and differentiation is crucial to maintain a stable population of stem cells in the shoot apical meristems (SAMs) of higher plants. The real-time and simultaneous observations of dynamics of cell identity transitions, growth patterns, and signaling machinery involved in cell-cell communication is crucial to gain a mechanistic view of stem-cell homeostasis. In this article, I review recent advances in understanding the regulatory dynamics of stem-cell maintenance in Arabidopsis thaliana and discuss future challenges involved in transforming the static maps of genetic interactions into a dynamic framework representing functional molecular and cellular interactions in living SAMs.     

Inhibition of Escherichia coli biofilm formation by self-assembled monolayers of functional alkanethiols on gold

Bacterial biofilms cause serious problems, such as antibiotic resistance and medical device-related infections. To further understand bacterium-surface interactions and to develop efficient control strategies, self-assembled monolayers (SAMs) of alkanethiols presenting different functional groups on gold films were analyzed to determine their resistance to biofilm formation. Escherichia coli was labeled with green florescence protein, and its biofilm formation on SAM-modified surfaces was monitored by confocal laser scanning microscopy. The three-dimensional structures of biofilms were analyzed with the COMSTAT software to obtain information about biofilm thickness and surface coverage. SAMs presenting methyl, L-gulonamide (a sugar alcohol tethered with an amide bond), and tri(ethylene glycol) (TEG) groups were tested. Among these, the TEG-terminated SAM was the most resistant to E. coli biofilm formation; e.g., it repressed biofilm formation by E. coli DH5alpha by 99.5% +/- 0.1% for 1 day compared to the biofilm formation on a bare gold surface. When surfaces were patterned with regions consisting of methyl-terminated SAMs surrounded by TEG-terminated SAMs, E. coli formed biofilms only on methyl-terminated patterns. Addition of TEG as a free molecule to growth medium at concentrations of 0.1 and 1.0% also inhibited biofilm formation, while TEG at concentrations up to 1.5% did not have any noticeable effects on cell growth. The results of this study suggest that the reduction in biofilm formation on surfaces modified with TEG-terminated SAMs is a result of multiple factors, including the solvent structure at the interface, the chemorepellent nature of TEG, and the inhibitory effect of TEG on cell motility.     

Secreted factors from brain endothelial cells maintain glioblastoma stem-like cell expansion through the mTOR pathway

Glioma stem-cells are associated with the brain vasculature. However, the way in which this vascular niche regulates stem-cell renewal and fate remains unclear. Here, we show that factors emanating from brain endothelial cells positively control the expansion of long-term glioblastoma stem-like cells. We find that both pharmacological inhibition of and RNA interference with the mammalian target of rapamycin (mTOR) pathway reduce their spheroid growth. Conversely, the endothelial secretome is sufficient to promote this mTOR-dependent survival. Thus, interfering with endothelial signals might present opportunities to identify treatments that selectively target malignant stem-cell niches.     

Extensive Hair Shaft Growth after Mouse Whisker Follicle Isolation,Cryopreservation and Transplantation in Nude Mice

We previously demonstrated that whole hair follicles could be cryopreserved to maintain their stem-cells differentation potential. In the present study, we demonstrated that cryopreserved mouse whisker hair follicles maintain their hair growth potential. DMSO better cryopreserved mouse whisker follicles compared to glycerol. Cryopreserved hair follicles also maintained the hair follicle-associated-pluripotent (HAP) stem cells, evidenced by P75^NTR expression. Subcutaneous transplantation of DMSO-cryopreserved hair follicles in nude mice resulted in extensive hair fiber growth over 8 weeks, indicating the functional recovery of hair shaft growth of cryopreserved hair follicles.     

Conservation and diversification of HAIRY MERISTEM gene family in land plants

The shoot apical meristems (SAMs) of land plants are crucial for plant growth and organ formation. In several angiosperms, the HAIRY MERISTEM (HAM) genes function as key regulators that control meristem development and stem cell homeostasis. To date, the origin and evolutionary history of the HAM family in land plants remains unclear. Potentially shared and divergent functions of HAM family members from angiosperms and non-angiosperms are also not known. In constructing a comprehensive phylogeny of the HAM family, we show that HAM proteins are widely present in land plants and that HAM proteins originated prior to the divergence of bryophytes. The HAM family was duplicated in a common ancestor of angiosperms, leading to two distinct groups: type I and type II. Type-II HAM members are widely present in angiosperms, whereas type-I HAM members were independently lost in different orders of monocots. Furthermore, HAM members from angiosperms and non-angiosperms (including bryophytes, lycophytes, ferns and gymnosperms) are able to replace the role of the type-II HAM genes in Arabidopsis, maintaining established SAMs and promoting the initiation of new stem cell niches. Our results uncover the conserved functions of HAM family members and reveal the conserved regulatory mechanisms underlying HAM expression patterning in meristems, providing insight into the evolution of key stem cell regulators in land plants.     

Substrate-attached materials are enriched with tetraspanins and are analogous to the structures associated with rear-end retraction in migrating cells

Substrate-attached materials (SAMs) are cellular feet that remain on substrates after the treatment of adherent cells with EGTA. SAMs are thought to contain cell adhesion machineries, but their biochemical properties have not been addressed in detail. To gain insight into the molecular mechanisms operating in cell adhesions, we comprehensively identified the protein components of SAMs by liquid chromatography coupled with tandem mass spectrometry, followed by immunoblot analysis. We found that the tetraspanins CD9, CD81, and CD151 were enriched in SAMs along with other transmembrane proteins that are known to associate with tetraspanins. Notably, integrins were detected in SAMs, but the components of focal adhesions were scarcely detected. These observations are reminiscent of the “footprints” that remain on substrates when the retraction fibers at the rear of migrating cells are released, because such footprints have been reported to contain tetraspanins and integrins but not focal adhesion proteins. In support of this hypothesis, the formation of SAMs was attenuated by inhibitors of ROCK, myosin II and dynamin, all of which are known to participate in rear-end retraction in migrating cells. Furthermore, SAMs left on collagen-coated substrates were found by electron microscopy to be fewer and thinner than those on laminin-coated substrates, reflecting the thin and fragile retraction fibers of cells migrating on collagen. Collectively, these results indicate that SAMs closely resemble the footprints and retraction fibers of migrating cells in their protein components, and that they are yielded by similar mechanisms.     

Electroactive oligoaniline-containing self-assembled monolayers for tissue engineering applications

A novel electroactive silsesquioxane precursor, N-(4-aminophenyl)-N'-(4'-(3-triethoxysilyl-propyl-ureido) phenyl-1,4-quinonenediimine) (ATQD), was successfully synthesized from the emeraldine form of amino-capped aniline trimers via a one-step coupling reaction and subsequent purification by column chromatography. The physicochemical properties of ATQD were characterized using mass spectrometry as well as by nuclear magnetic resonance and UV-vis spectroscopy. Analysis by cyclic voltammetry confirmed that the intrinsic electroactivity of ATQD was maintained upon protonic acid doping, exhibiting two distinct reversible oxidative states, similar to polyaniline. The aromatic amine terminals of self-assembled monolayers (SAMs) of ATQD on glass substrates were covalently modified with an adhesive oligopeptide, cyclic Arg-Gly-Asp (RGD) (ATQD-RGD). The mean height of the monolayer coating on the surfaces was approximately 3 nm, as measured by atomic force microscopy. The biocompatibility of the novel electroactive substrates was evaluated using PC12 pheochromocytoma cells, an established cell line of neural origin. The bioactive, derivatized electroactive scaffold material, ATQD-RGD, supported PC12 cell adhesion and proliferation, similar to control tissue-culture-treated polystyrene surfaces. Importantly, electroactive surfaces stimulated spontaneous neuritogenesis in PC12 cells, in the absence of neurotrophic growth factors, such as nerve growth factor (NGF). As expected, NGF significantly enhanced neurite extension on both control and electroactive surfaces. Taken together, our results suggest that the newly electroactive SAMs grafted with bioactive peptides, such as RGD, could be promising biomaterials for tissue engineering.     

Evolution of shoot apical meristem structures in vascular plants with respect to plasmodesmatal network

Vascular plants have evolved shoot apical meristems (SAMs), whose structures differ among plant groups. To clarify the evolutionary course of the different structural types of SAMs, we compared plasmodesmatal networks in the SAMs for 17 families and 24 species of angiosperms, gymnosperms, and pteridophytes, using transmission electron microscopy (TEM). The plasmodesmata (PD) in almost all cell walls in median longitudinal sections of SAMs were counted, and the PD density per unit area was calculated for each cell wall. Angiosperm and gymnosperm SAMs have low densities, with no difference between stratified (tunica-corpus) and unstratified structures. SAMs of ferns, including Psilotum and Equisetum, have average densities that are more than three times higher than those of seed plants. Interestingly, microphyllous lycopods have both the fern and seed-plant types of PD networks; Selaginellaceae SAMs with single apical cells have high PD densities, while SAMs of Lycopodiaceae and Isoetaceae with plural initial cells have low PD densities, equivalent to those of seed plants. In summary, PD networks are strongly correlated to SAM organizations-SAMs with single and plural initial cells have the fern and seed-plant types of PD, respectively. The two SAM organizations may have evolved separately in lycophytes and euphyllophytes and may be associated with gain or loss of the ability to form secondary PD.     

Specific capture of mammalian cells by cell surface receptor binding to ligand immobilized on gold thin films

Aldehyde-terminated self-assembled monolayers (SAMs) on gold surfaces were modified with proteins and employed to capture intact living cells through specific ligand-cell surface receptor interactions. In our model system, the basic fibroblast growth factor (bFGF) binding receptor was targeted on baby hamster kidney (BHK-21) cells. Negative control and target proteins were immobilized on a gold surface by coupling protein primary amines to surface aldehyde groups. Cell-binding was monitored by phase contrast microscopy or surface plasmon resonance (SPR) imaging. The specificity of the receptor-ligand interaction was confirmed by the lack of cell binding to the negative control proteins, cytochrome c and insulin, and by the disruption of cell binding by treatment with heparitinase to destroy heparan sulfate which plays an essential role in the binding of bFGF to FGF receptors. This approach can simultaneously probe a large number of receptor-ligand interactions in cell populations and has potential for targeting and isolating cells from mixtures according to the receptors expressed on their surface.     

Using Microcontact Printing to Pattern the Attachment of Mammalian Cells to Self-Assembled Monolayers of Alkanethiolates on Transparent Films of Gold and Silver

This paper describes a convenient methodology for patterning substrates for cell culture that allows the positions and dimensions of attached cells to be controlled. The method uses self-assembled monolayers (SAMs) of terminally substituted alkanethiolates (R(CH₂)_11–15S−) adsorbed on optically transparent films of gold or silver to control the properties of the substrates. SAMs terminated in methyl groups adsorb protein and SAMs terminated in oligo(ethylene glycol) groups resist entirely the adsorption of protein. This methodology uses microcontact printing (μCP)—an experimentally simple, nonphotolithographic process—to pattern the formation of SAMs at the micrometer scale; μCP uses an elastomeric stamp having at its surface a pattern in relief to transfer an alkanethiol to a surface of gold or silver in the same pattern. Patterned SAMs having hydrophobic, methyl-terminated lines 10, 30, 60, and 90 μm in width and separated by protein-resistant regions 120 μm in width were prepared and coated with fibronectin; the protein adsorbed only to the methyl-terminated regions. Bovine capillary endothelial cells attached only to the fibronectin-coated, methyl-terminated regions of the patterned SAMs. The cells remained attached to the SAMs and confined to the pattern of underlying SAMs for at least 5–7 days. Because the substrates are optically transparent, cells could be visualized by inverted microscopy and by fluorescence microscopy after fixing and staining with fluorescein-labeled phalloidin.     

The shoot apical meristem: the dynamics of a stable structure

The shoot apical meristem (SAM) is a group of proliferating, embryonic-type cells that generates the aerial parts of the plant. SAMs are highly organized and stable structures that can function for years or even centuries. This is in apparent contradiction to the behaviour of their constituent cells, which continuously proliferate and differentiate. To reconcile the dynamic nature of the cells with the stability of the overall system the existence of elaborate signalling networks has been proposed. This is supported by recent work suggesting that the exchange of signals between cells, rather than a rigidly predetermined genetic program, is required for the establishment and functioning of an organized meristem. Together these interactions form a stable network, set up during embryogenesis, that assures the coordination of cell behaviour throughout development. Besides meristem-specific signalling cascades such as the CLAVATA receptor kinase pathway, which controls meristem size, these interactions involve plant hormones. In particular, cytokinins and auxins are implicated in the maintenance of meristem identity and phyllotaxis, respectively.     

Fluorescence microscopy study of polymorphonuclear leukocyte substrate attached materials

We describe a technique to visualize substrate-attached materials (SAM) of polymorphonuclear leukocytes (PMN) using the fluorescent lipid analog 1,1'-dioctadecyl-3,3,3',3',-tetramethylindocarbocyanine-perchlorate (DiC18Icc). DiC18Icc was incorporated into the membranes of living cells or SAMs. Since cell preparation does not require fixation, SAMs can be rapidly visualized by fluorescence microscopy. SAMs are generated by subjecting attached cells to a shearing force by rinsing with phosphate-buffered saline (PBS). The SAM-labeling protocol identified a membrane compartment as shown by detergent extraction. The SAMs of PMN leukocytes observed with this technique display complex patterns of interconnecting filaments, foci with radiating filaments, and smooth membranous areas with interconnecting filaments. The sensitivity and nondestructive nature of the DiC18Icc-labeling procedure have allowed us to observe filopodia of motile cells. The results are consistent with the hypothesis that locomotion involves a series of attachment and detachment steps. After 60 minutes of locomotion, these trailing filopodia have been measured at lengths up to 100 micron. The amount of membrane associated with these filopodia accounts for roughly 10% of the total membrane area of resting cells. These data set limits for models of membrane flow during chemotaxis.     

The universal properties of stem cells as pinpointed by a simple discrete model

The ability of a few stem-cells to repopulate a severely damaged bone marrow (BM) guarantees the stability of our physical existence, and facilitates successful BM transplantations. What are the basic properties of stem cells that enable the maintenance of the system's homeostasis? In the present work we attempt to answer this question by investigating a discrete (in time and phase-space) dynamical system. The model we present is shown to retrieve the essential properties of homeostasis, as reflected in BM functioning, namely, (a) to produce a constant amount of mature cells, and (b) to be capable of returning to this production after very large perturbations. The mechanism guaranteeing the fulfillment of these properties is extrinsic--negative feedback control in the micro-environment--and does not need additional stochastic assumptions. Nevertheless, the existence of a simple intrinsic control mechanism, a clock which determines the switch to differentiation, ascertains that the system does not admit non-trivial extinction states. This result may help clarifying some of the controversy about extrinsic versus intrinsic control over stem cell fate. It should be stressed that all conclusions are valid for any system containing progenitor and maturing cells.     

Automated tracking of stem cell lineages of Arabidopsis shoot apex using local graph matching

Shoot apical meristems (SAMs) of higher plants harbor stem‐cell niches. The cells of the stem‐cell niche are organized into spatial domains of distinct function and cell behaviors. A coordinated interplay between cell growth dynamics and changes in gene expression is critical to ensure stem‐cell homeostasis and organ differentiation. Exploring the causal relationships between cell growth patterns and gene expression dynamics requires quantitative methods to analyze cell behaviors from time‐lapse imagery. Although technical breakthroughs in live‐imaging methods have revealed spatio‐temporal dynamics of SAM‐cell growth patterns, robust computational methods for cell segmentation and automated tracking of cells have not been developed. Here we present a local graph matching‐based method for automated‐tracking of cells and cell divisions of SAMs of Arabidopsis thaliana. The cells of the SAM are tightly clustered in space which poses a unique challenge in computing spatio‐temporal correspondences of cells. The local graph‐matching principle efficiently exploits the geometric structure and topology of the relative positions of cells in obtaining spatio‐temporal correspondences. The tracker integrates information across multiple slices in which a cell may be properly imaged, thus providing robustness to cell tracking in noisy live‐imaging datasets. By relying on the local geometry and topology, the method is able to track cells in areas of high curvature such as regions of primordial outgrowth. The cell tracker not only computes the correspondences of cells across spatio‐temporal scale, but it also detects cell division events, and identifies daughter cells upon divisions, thus allowing automated estimation of cell lineages from images captured over a period of 72 h. The method presented here should enable quantitative analysis of cell growth patterns and thus facilitating the development of in silico models for SAM growth.     

The formation and patterning of leaves: recent advances

Leaves, the plants major photosynthetic organs, form through the activity of groups of pluripotent cells, termed shoot apical meristems (SAMs), located at the growing tips of plants. Leaves develop with a dorso–ventral asymmetry, with the adaxial surface adjacent to the meristem and the abaxial surface developing at a distance from it. Molecular genetic studies have shown that the correct specification of adaxial/abaxial polarity requires communication between the incipient leaf and the meristem, and that the juxtaposition of adaxial/abaxial fates is necessary for lamina outgrowth (Waites and Hudson 1995; McConnell et al. 2001). Over the last few years, a number of factors that control cell fate specification in the apex have been identified. This review will focus on recent advances on distinct but overlapping aspects of leaf development, namely, the transition from meristem to leaf fate and the specification of abaxial/adaxial polarity and its possible role in leaf growth.     

Roles of the Arabidopsis G protein γ subunit AGG3 and its rice homologs GS3 and DEP1 in seed and organ size control

The size of seeds and organs is coordinately determined by cell proliferation and cell expansion, but the mechanisms that set final seed and organ size are largely unknown in plants. In a recent study, we have demonstrated that the plant specific G protein γ subunit (AGG3) promotes seed and organ growth by increasing the period of proliferative growth in Arabidopsis. AGG3 is localized in plasma membrane and interacts with the G protein β subunit (AGB1). Homologs of AGG3 in rice (GS3 and DEP1/qPE9–1) have been identified as important quantitative trait loci for seed size and yield. However, rice GS3 and DEP1 influence seed and organ growth by restricting cell proliferation. Here, we discuss the possible molecular mechanisms by which Arabidopsis AGG3 and its rice homologs GS3 and DEP1 control seed and organ size.