The work was supported by the Collaborative Research Fund from Shiga Prefecture entitled &#34;Study on water quality and lake-bottom environment for the protection of the soundness of water environment&#34; under the Japanese Grant for Regional Revitalization and the Environment Research and Technology Development Fund (No. 5-1607) of the Ministry of the Environment, Japan.&#160;https://www.kantei.go.jp/jp/singi/tiiki/tiikisaisei/souseikoufukin.html. The authors would like to thank Enago (www.enago.jp) for the English language review.

1. Sampling
<ol>
	<li>Collect lake water&#160;from the surface by a bucket. To target Colacium sp. attached to S. mucronata (Figure 1A-C) filter 0.5-10 L of lake water using a 100 &#181;m nylon mesh net52. 
	NOTE: S. mucronata often densely aggregate in shallow, eutrophic, muddy water, such as among reed (phragmites) areas.</li>
	<li>Store the concentrated samples in 500 mL plastic bottles with 350 mL...

This protocol demonstrated for the first time that photophysiology of Colacium sp. during the attached stage in a natural environment is comparable to its planktonic stage in AF-6 medium. Additionally, gut contents of starved S. mucronata did not affect baseline and Chl-a fluorescence when density was &#8804;5 inds&#183;mL&#8722;1 (Figure 5 and Figure 6). These results suggest this protocol can measure photophysiology of <em...

Chlorophyll variable fluorescence is a useful tool for measuring algal photosystem II (PSII) photophysiology. Algae respond to various environmental stresses, such as excess light and nutrient deficiency, by altering their PSII photophysiology. Fast repetition rate fluorometer (FRRf) is a common method for measuring PSII photophysiology1,2 and estimating primary productivity1,3,4, which enables monitoring phytoplankton PSII photophysiology, as well as primary productivity across wide spatial and temporal scales5,6,7. FRRf can simultaneously measure PSII (&#963;PSII) absorption cross section, reaction center ([RCII]) concentration, maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq&#8242;/Fm&#8242;), and non-photochemical quenching (NPQNSV) (Table 1). In general, Fv/Fm and Fq&#8242;/Fm&#8242; are defined as PSII activity8, while NPQNSV&#160;is defined as relative heat-dissipated energy9.
Importantly, single turnover (ST) flashes of FRRf fully reduce the primary quinone electron acceptor, QA, but not the plastoquinone pool. Conversely, multiple turnover (MT) flashes from a pulse amplitude modulation (PAM) fluorometer can reduce both. The ST method has a clear advantage over the MT method when identifying the possible origins of NPQNSV&#160;by simultaneously measuring recovery kinetics of Fv/Fm, Fq&#8242;/Fm&#8242;, NPQNSV, and &#963;PSII10. To date, several types of FRRf instruments, such as submersible-type, cuvette-type, and flow-through-type, are commercially available. The submersible-type FRRf enables in situ measurements in oceans and lakes, while the cuvette-type FRRf is suitable for measuring small sample volumes. The flow-through type is commonly used to continuously measure the photophysiology of phytoplankton in surface waters.
Given the development of PAM fluorometers, including the cuvette-type, for a broad range of subjects11, PAM fluorometers are still more common than FRRfs in algal photophysiology research12. For example, although the sample chamber structure and cuvette capacity between these tools only differs slightly, the cuvette-type PAM has been applied to phytoplanktons13,14,15, benthic microalgae16,17,18, ice algae19, and epizoic algae20, while the cuvette-type FRRf has been applied primarily to phytoplanktons21,22,23 and a limited number of ice algal communities24,25. Given its effectiveness, cuvette-type FRRf is equally applicable to benthic and epizoic algae. Therefore, expanding its application will provide considerable insight into PSII photophysiology, particularly for lesser-known epizoic algal photophysiology.
Epizoic algae have received little attention, with few studies examining their PSII photophysiology20,26, most likely due to their minor roles in aquatic food webs27,28. However, epibionts, including epizoic algae, can positively influence zooplankton community dynamics, such as increasing reproduction and survival rates29,30, as well as negatively impact processes, such as increasing sinking rate29,31 and vulnerability to visual predators32,33,34,35,36. Therefore, exploring the environmental and biological factors controlling epibiont dynamics in zooplankton communities is crucial.
Among epizoic algae, Colacium Ehrenberg 1834 (Euglenophyta) is a common, freshwater, algal group32,37,38,39 with various life stages, including attached (Figure 1A-D), non-motile planktonic (Figure 1E,F), and motile planktonic stages40,41. During the non-motile planktonic stage, cells live as single-cell planktons, aggregated colonies, or one-layer sheet colonies, covered by mucilage42. In the attached stage, Colacium sp. uses mucilage excreted from the anterior end of the cell37,39,41 to attach to substrate organisms (basibionts), particularly microcrustaceans41,43. Their life cycle also involves detaching from the molted exoskeleton or dead basibiont and swimming with their flagella to find another substrate organism39. Both planktonic and attached stages can increase their population size by mitosis40. Although their attached stage is hypothesized to be an evolutionary trait for gathering resources, such as light44 and trace elements41,45,46, or as a dispersion strategy27, little experimental evidence is available about these aspects37,41,44 and the key attachment mechanisms are largely unknown. For example, Rosowski and Kugrens expected that Colacium obtains manganese (Mn) from substrate copepods41, concentrated in the exoskeleton47.
Here, we describe how to measure PSII photophysiology of planktonic algae and the related application method for targeting attached algae (attaching to zooplankton) with Colacium sp. cells using the cuvette-type FRRf. We use the Act2 system equipped with three light-emitting diodes (LEDs) that provide flash excitation energy centered at 444 nm, 512 nm, and 633 nm48. Here, 444 nm (blue) corresponds to the absorption peak of chrophyll a (Chl-a), while 512 nm (green) and 633 nm (orange) correspond to the absorption peaks of phycoerythrin and phycocyanin, respectively. The fluorescent signal detection peak is 682 nm with 30 nm half bandwidth. Since it is difficult to find the planktonic stage of Colacium sp. in natural environments, their attached stage was collected for the experiments. Among the numerous substrate organisms,Scapholeberis mucronata O.F. M&#252;ller 1776 (Branchiopoda, Daphniidae; Figure 1A,B,G) is one of the simplest to handle due to their slow swimming speed, large body size (400-650 &#181;m), and unique behavior (hanging upside down on the water surface). Therefore, this protocol uses Colacium sp. attached on S. mucronata as a case study of the Colacium-basibiont system. To avoid fluorescence derived from the gut contents, S. mucronata was starved. As a previous study reported that the fluorescence signal from gut contents (ingested algae) displays a five-fold decrease after 40 min49, we expected that 90 min starvation would be enough to minimize the possibility of gut content fluorescence affecting the FRRf measurement with minimum effects of experimental stress to Colacium sp., such as nutrient deficiency. Furthermore, this protocol was applied to clarify the attaching mechanism of Colacium sp. and determine how two metals, calcium (Ca) and manganese (Mn) affect the photophysiology of both planktonic and attached stages. Calcium plays key roles in the photosynthetic pathways50 in multiple ways, and both metals are required to construct the oxygen-evolving complexes of the PSII51. As calcium and manganese are highly concentrated in the carapace of crustacean zooplankton47, we hypothesize that Colacium sp. photophysiology might respond more prominently to Ca and Mn enrichment during the planktonic stage if this life stage obtains these elements from S. mucronata during the attached stage.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acrodisc syringe filter</td>
			<td>Pall Corporation, Ann Arbor, MI, USA</td>
			<td></td>
			<td>0.2 &#956;m pore size</td>
		</tr>
		<tr>
			<td>Act2Run</td>
			<td>CTG Ltd., West Molesey, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Wako</td>
			<td>023-08711</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Wako</td>
			<td>031-25031</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>CaCO3</td>
			<td>Wako</td>
			<td>036-00382</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Wako</td>
			<td>036-05522</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>CoCl2&#183;6H2O</td>
			<td>Wako</td>
			<td>036-03682</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>Concentrated Chlorella</td>
			<td>Recenttec, Tokyo, Japan</td>
			<td></td>
			<td>20 mg C&#183;mL&#8722;1 ; store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>FastOcean Act2</td>
			<td>CTG Ltd., West Molesey, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fe-citrate</td>
			<td>Wako</td>
			<td>093-00952</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>FeCl3&#183;6H2O</td>
			<td>Wako</td>
			<td>091-00872</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>HCLP-880PF</td>
			<td>Nippon Medical and Chemical Instruments 
			&#160;Co., Ltd., Osaka, Japan</td>
			<td></td>
			<td>With LED light bulbs</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Wako</td>
			<td>160-04292</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Wako</td>
			<td>167-04241</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H2O</td>
			<td>Wako</td>
			<td>137-00402</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>MnCl3&#183;4H2O</td>
			<td>Wako</td>
			<td>139-00722</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>Na2EDTA</td>
			<td>Wako</td>
			<td>343-01861</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>Na2MoO4</td>
			<td>Wako</td>
			<td>196-02472</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>NaNO3</td>
			<td>Wako</td>
			<td>191-02542</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>NH4NO3</td>
			<td>Wako</td>
			<td>015-03231</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>Plankton Counter</td>
			<td>Matsunami Glass, Osaka, Japan</td>
			<td>S6300</td>
			<td></td>
		</tr>
		<tr>
			<td>Pylex test tube</td>
			<td>CTG Ltd., West Molesey, UK</td>
			<td></td>
			<td>With rim, 16 x 100 mm</td>
		</tr>
		<tr>
			<td>Vit. B1</td>
			<td>Wako</td>
			<td>203-00851</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>Vit. B12</td>
			<td>Wako</td>
			<td>226-00343</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>Vit. B6</td>
			<td>Wako</td>
			<td>165-05401</td>
			<td>AF-6 medium</td>
		</tr>
		<tr>
			<td>ZnSO4&#183;7H2O</td>
			<td>Wako</td>
			<td>264-00402</td>
			<td>AF-6 medium</td>
		</tr>
	</tbody>
</table>

measuring photophysiology of attached stage of colacium sp. by a cuvette-type fast repetition rate fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf can measure PSII absorption cross-section (&#963;PSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq&#8242;/Fm&#8242;), and non-photochemical quenching (NPQNSV) for various eukaryotic algae and cyanobacteria, almost all FRRf studies to date have focused on phytoplankton. Here, the protocol describes how to measure PSII photophysiology of an epizoic alga Colacium sp.&#160;Ehrenberg 1834 (Euglenophyta), in its attached stage (attached to zooplankton), using cuvette-type FRRf. First, we estimated the effects of substrate zooplankton (Scapholeberis mucronata O.F. M&#252;ller 1776, Cladocera, Daphniidae) on baseline fluorescence and &#963;PSII, Fv/Fm, Fq&#8242;/Fm&#8242;, and NPQNSV of planktonic Colacium sp. To validate this methodology, we recorded photophysiology measurements of attached Colacium sp. on S. mucronata and compared these results with its planktonic stage. Representative results showed how the protocol could determine the effects of calcium (Ca) and manganese (Mn) on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.

There was no significant effect of baseline fluorescence (Figure 5) or Chl-a fluorescence (Figure 6) by S. mucronata up to 5 individuals (inds.) mL&#8722;1. However, Fv/Fm and NPQNSV were significantly affected when S. mucronata was 7.5 inds&#183;mL&#8722;1. Therefore, for measuring the photophysiology of Colacium sp. during the attached stage, w...

Watch this Scientific Journal Video about Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer at JoVE.com

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II photophysiology and primary productivity. Here we describe a protocol to measure PSII photophysiology of epizoic alga, Colacium sp. on substrate zooplankton using cuvette-type FRRf.

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf ...

This protocol measures the photosynthetic activity of attached algae, while they are still attaching to the plankton, just as in nature. The main advantage of this technique is that the measurement of photophysiology can be done in real-time without sample distraction. To examine the effects of zooplankton individuals on baseline fluorescence, prepare adult scapholeberis mucronata from the culture without any attached organisms.Now starve the individuals in FLW at 20 degrees Celsius for at least 90 minutes to avoid fluorescence from the gut content. Pour 1.5-milliliters of FWL into a cuvette. Pick up the desired number of s.mucronata individuals using a pipette under an optical microscope at 100-times magnification and transfer them into the cuvette. Add FLW to bring the sample volume up to 2-milliliters and allow it to acclimate under low light at 20 degrees Celsius is for 15 minutes before the fast repetition rate fluorometer measurement. Then click on act to run"to start the measurement and repeat the measurements more than three times per sample.Read the F0 value from the resulting plot. Pick up colacium species with a molted carapace using a pipette under an optical microscope at 100-times magnification and wash them with FLW. Aseptically inoculate colacium species in the AF-6 medium in a 10-milliliter glass tube on a clean bench.Maintain the culture at a NC2 temperature in a growth chamber, shaking the glass tube gently by hand at least once per day to prevent cell settlement. To examine the effects of zooplankton individuals on chlorophyll a fluorescence from colacium species, use adult s. mucronata without any attached organisms.To avoid fluorescence from the gut contents, starve the individuals in FLW for at least 90 minutes. Set up a cuvette-type fast repetition rate fluorometer. Now pour a 1.5-milliliters of sub sample of pre-cultured colacium species into a cuvette.Then transfer the desired number of s. mucronata individuals into these cuvettes and bring the sample volume up to 2-milliliters with the filtered medium. Acclimate the individuals under low light at 20 degrees Celsius for 15 minutes before taking the fast repetition rate fluorometer measurement.Click on act to run"to start the measurement, repeating the measurements more than three times per sample. Read the F0 and FM values from the resulting plot. To correct the baseline fluorescence, filter the culture medium using a 0.2-micrometer pour-sized filter and measure the fluorescence.Subtract F0 of the baseline sample from F0 and FM of colacium species or modify the blank correction value in the settings in the options tab. Isolate s. mucronata individuals with colacium species using a pipette under an optical microscope.Then wash s. mucronata using FLW. Transfer s.mucronata into 100-milliliters of FLW and keep them under dark conditions at NC2 temperature for 90 minutes for starvation. Pour 1.5-milliliters of FLW into a cuvette. Transfer around ten s.mucronata individuals with colacium species into a cuvette and add FLW to bring the sample up to 2-milliliters. Acclimate the individuals under low light at N2C temperature for 15 minutes and measure chlorophyll a fluorescence, as mentioned in the text manuscript. To enumerate the number of attached cells, fix the sample with 2%glutaraldehyde after taking the fast repetition rate fluorometer measurement.Then take pictures at several focal depths and positions of s. mucronata under a light microscope. There was no significant effect on baseline fluorescence or chlorophyll a fluorescence by s.mucronata up to 5 individuals per milliliter. However, maximum photochemical efficiency and non-photochemical quenching were significantly affected when s. mucronata density was 7.5 individuals per milliliter.Seasonal variation in the phytophysiology of colacium species during the attachment stage and the planktonic stage for the stationary phase in the AF-6 medium showed similar mean maximum photochemical efficiency and non-photochemical quenching. For the attached stage of colacium species under dark conditions, there was no significant difference in photophysiological parameters, except non-photochemical quenching being higher for manganese than calcium after three hours, and at a low light intensity at 21 hours. For the planktonic stage, PS2 absorption cross-section was significantly lower in the manganese than calcium treatment at three hours.Effective photochemical efficiency was significantly higher, but non-photochemical quenching was lower in the manganese treatment than control at 21 hours. Under increasing light, manganese tended to decrease PS2 absorption cross-section at three hours, in non-photochemical quenching at 21 hours, while increasing the effective photochemical efficiency at three hours. Calcium slightly improved non-photochemical quenching under increasing light.However, it decreased the effective photochemical efficiency at three hours. The most important thing is the effect of substrate organisms. If algae attached to different species or materials, the effect can be different.In addition to this protocol, measurement of carbon fixation or oxygen production rates are useful to clarify the response of the calcium productivity to the experimental manipulations. This protocol enable us to clarify how the photosynthetic activity of attached algae responds to environmental changes.

measuring-photophysiology-attached-stage-colacium-sp-cuvette-type

Research

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Biology

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.