Joseph C. Shope

The role of the mesophyll in stomatal responses to light and CO2.

Stomatal responses to light and CO(2) were investigated using isolated epidermes of Tradescantia pallida, Vicia faba and Pisum sativum. Stomata in leaves of T. pallida and P. sativum responded to light and CO(2), but those from V. faba did not. Stomata in isolated epidermes of all three species could be opened on KCl solutions, but they showed no response to light or CO(2). However, when isolated epidermes of T. pallida and P. sativum were placed on an exposed mesophyll from a leaf of the same species or a different species, they regained responsiveness to light and CO(2). Stomatal responses in these epidermes were similar to those in leaves in that they responded rapidly and reversibly to changes in light and CO(2). Epidermes from V. faba did not respond to light or CO(2) when placed on mesophyll from any of the three species. Experiments with single optic fibres suggest that stomata were being regulated via signals from the mesophyll produced in response to light and CO(2) rather than being sensitized to light and CO(2) by the mesophyll. The data suggest that most of the stomatal response to CO(2) and light occurs in response to a signal generated by the mesophyll.

Changes in Surface Area of Intact Guard Cells Are Correlated with Membrane Internalization

Guard cells must maintain the integrity of the plasma membrane as they undergo large, rapid changes in volume. It has been assumed that changes in volume are accompanied by changes in surface area, but mechanisms for regulating plasma membrane surface area have not been identified in intact guard cells, and the extent to which surface area of the guard cells changes with volume has never been determined. The alternative hypothesis—that surface area remains approximately constant because of changes in shape—has not been investigated. To address these questions, we determined surface area for intact guard cells of Vicia faba as they underwent changes in volume in response to changes in the external osmotic potential. We also estimated membrane internalization for these cells. Epidermal peels were subjected to external solutions of varying osmotic potential to shrink and swell the guard cells. A membrane-specific fluorescent dye was used to identify the plasma membrane, and confocal microscopy was used to acquire a series of optical paradermal sections of the guard cell pair at each osmotic potential. Solid digital objects representing the guard cells were created from the membrane outlines identified in these paradermal sections, and surface area, volume, and various linear dimensions were determined for these solid objects. Surface area decreased by as much as 40% when external osmotic potential was increased from 0 to 1.5 MPa, and surface area varied linearly with volume. Membrane internalization was approximated by determining the amount of the fluorescence in the cells interior. This value was shown to increase approximately linearly with decreases in the cells surface area. The changes in surface area, volume, and membrane internalization were reversible when the guard cells were returned to a buffer solution with an osmotic potential of approximately zero. The data show that intact guard cells undergo changes in surface area that are too large to be accommodated by plasma membrane stretching and shrinkage and suggest that membrane is reversibly internalized to maintain cell integrity.

A Monoclonal Antibody to Visualize PtdIns(3,4,5)P3 in Cells

Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] is a second messenger produced in response to agonist stimulation. Traditionally, visualization of phosphoinositide polyphosphates (PtdInsPn) in living cells is accomplished using chimeric green fluorescent protein (GFP)-pleckstrin homology (PH) domain proteins, while PtdInsPn quantitation is accomplished by extraction and separation of radiolabeled cellular PtdInsPns. Here we describe preparation of a covalent protein-PtdIns(3,4,5)P3 immunogen, characterization of binding selectivity of an anti-PtdIns(3,4,5)P3 IgM, and immunodetection of PtdIns(3,4,5)P3 in stimulated mammalian cells. This antibody has greater than three orders of magnitude selectivity for binding PtdIns(3,4,5)P3 relative to its precursor, phosphatidylinositol 4,5-bis-phosphate (PtdIns(4,5)P2), and is therefore optimal for studies of cell function. The immunodetection in platelet-derived growth factor (PDGF)-stimulated NIH 3T3 cells was bench-marked against HPLC analysis of [3H]-myo-inositol-labeled cellular PtdInsPns. In addition, the changes in subcellular amounts and localizations of both PtdIns(3,4,5)P3 and PtdIns(4,5)P2 in stimulated NIH 3T3 fibroblasts and human neutrophils were observed by immunofluorescence. In insulin- or PDGF-stimulated fibroblasts, PtdIns(3,4,5)P3 levels increased in the cytoplasm, peaking at 10 min. In contrast, increases in the PtdIns(4,5)P2 levels were detected in nuclei, corresponding to the production of new substrate following depletion by phosphoinositide (PI) 3-kinase.

Stomatal Responses to Humidity in Isolated Epidermes

The ability of guard cells to hydrate and dehydrate from the surrounding air was investigated using isolated epidermes of Tradescantia pallida and Vicia faba. Stomata were found to respond to the water vapour pressure on the outside and inside of the epidermis, but the response was more sensitive to the inside vapour pressure, and occurred in the presence or absence of living, turgid epidermal cells. Experiments using helium-oxygen air showed that guard cells hydrated and dehydrated entirely from water vapour, suggesting that there was no significant transfer of water from the epidermal tissue to the guard cells. The stomatal aperture achieved at any given vapour pressure was shown to be consistent with water potential equilibrium between the guard cells and the air near the bottom of the stomatal pore, and water vapour exchange through the external cuticle appeared to be unimportant for the responses. Although stomatal responses to humidity in isolated epidermes are the result of water potential equilibrium between the guard cells and the air near the bottom of the stomatal pore, stomatal responses to humidity in leaves are unlikely to be the result of a similar equilibrium.

A Real-Time Fluorogenic Phospholipase A2 Assay for Biochemical and Cellular Activity Measurements

A fluorogenic analog of the PLA(2) substrate PC, named Dabcyl-BODIPY-PC, or simply DBPC, was synthesized with a fluorescence quencher (Dabcyl, 4-[(4-[N,N-dimethylamino]phenyl)azo]benzoic acid) in the sn-1 acyl chain and a BODIPY fluor in the sn-2 acyl chain. DBPC was recognized by sPLA(2) from each of the four sources examined (bee venom, human synovial fluid, cobra venom, and bovine pancreas). A dramatic and quantifiable fluorescence enhancement of DBPC occurred upon phospholipase digestion both in the presence and absence of excess PC. Both real-time and endpoint assays for PLA(2) were sensitive, consistent, and rapid. Thus, DBPC can be used as a sensitive fluorogenic probe for in vitro high-throughput screening assays for PLA(2) activation and inhibition and would expedite studies of PLA(2) in cellular signaling, in vitro screening for drug discovery, and subcellular localization of enzyme activity.

Maximizing Kolmogorov Complexity for accurate and robust bright field cell segmentation

BackgroundAnalysis of cellular processes with microscopic bright field defocused imaging has the advantage of low phototoxicity and minimal sample preparation. However bright field images lack the contrast and nuclei reporting available with florescent approaches and therefore present a challenge to methods that segment and track the live cells. Moreover, such methods must be robust to systemic and random noise, variability in experimental configuration, and the multiple unknowns in the biological system under study.ResultsA new method called maximal-information is introduced that applies a non-parametric information theoretic approach to segment bright field defocused images. The method utilizes a combinatorial optimization strategy to select specific defocused images from each image stack such that set complexity, a Kolmogorov complexity measure, is maximized. Differences among these selected images are then applied to initialize and guide a level set based segmentation algorithm. The performance of the method is compared with a recent approach that uses a fixed defocused image selection strategy over an image data set of embryonic kidney cells (HEK 293T) from multiple experiments. Results demonstrate that the adaptive maximal-information approach significantly improves precision and recall of segmentation over the diversity of data sets.ConclusionsIntegrating combinatorial optimization with non-parametric Kolmogorov complexity has been shown to be effective in extracting information from microscopic bright field defocused images. The approach is application independent and has the potential to be effective in processing a diversity of noisy and redundant high throughput biological data.