We would like to thank Andrea Kremsreiter for excellent technical assistance. Work in our laboratory is supported by NIH GM096088 and the Vilas Trust of the University of Wisconsin - Madison.

1. Yeast Luminometry Methods
Use strain BYYT of Saccharomyces cerevisiae. It is a yvc1- tok1- derivative of BY4741 (MATa, ura3D0, his3D1, leu2D0, lys2D0, yvc1::HIS3, tok1::kanMX4). Transform cells with the leu-selectable aequorin-expressing plasmid pEVP1/Aeq and the ura-selectable rat-TRPV4-expressing plasmid p416GPDV4, as described in Batiza et al.26 and Loukin et al.24 Use standard methods of plasmid construction and transformation27. Yeast strain and plasmids are available upon request.
<ol>
	<li>Culture 2 ml of yeast cells overnight to post-logarithmic phase in a 30 &#176;C shaker in leu- ura- "DCD" dropout medium28 (a sulfate-depleted variant of the CMD-leu-ura medium29), supplemented with 1M sorbitol. Inoculate 200 &#181;l of these cultures into 1.8 ml of fresh medium supplemented with 2 &#181;M of the luciferin coelenterazine. Grow in the dark at room temperature for another 24 hr without shaking.</li>
	<li>Measure the osmolarity of the culture (typically at 1,400 mOsM) and other solutions (below) using a vapor pressure osmometer.</li>
	<li>Aliquot 20 &#181;l of fresh culture into a 12 mm luminometer tube for a single tube luminometer.</li>
	<li>For the hypotonic shock, add 200 &#181;l of a solution, containing 25 mM NaEGTA (pH 7.2) or NaMES (pH 7.2) and 500-100 mM NaCl, (total osmolarity of 1,200-400 mOsM) depending on the degree of shock desired. Continuously monitor the luminescence before and at least 120 sec after the osmotic downshock, interrupted only by the brief dilution operation. Register luminometer output on a desktop computer as relative luminescence units (RUL).</li>
</ol>
Though not for the study of transgenic TRPV4, we have used a variant of the above protocol in an automated system to examine the responses of a large number of yeast strains. Study of the responses to hypo-30 or hyperosmotic31 stimuli of the yeast deletome (the collection of 4,906 yeast single-gene deletants) using a microplate luminometer and analyzed with corresponding software has been successful.
2. and 3. Oocyte Electrophysiology Methods
Electrophysiology uses basic methods32. PCR amplify the open-reading frame of wild-type or mutant TRPV4 using high-fidelity PfuUtra polymerase and integrated into pGH19 33. This entails a precise insertion of the ORF between the 5' and 3' UTRs of the Xenopus &#946;-globin gene and downstream of a T7 RNA-polymerase promoter. Linearize the construct downstream of the 3' UTR and used as templates in an in vitro T7 RNA polymerase reaction. Use standard molecular-biological techniques34.
Use stage V-VI oocytes from X. laevis. Animal husbandry, partial ovariectomy, defolliculation, and vitelline envelope removal follow standard procedures32,33,35. Inject 2-40 ng of cRNA solution per oocyte using a Drummond Nanoinject II automatic microinjector. Inject ~30 oocytes for each set of experiments. After TRPV4-cRNA injection, incubate the oocytes in the ND96 medium with gentamicin (100 &#181;g/ml) and 1 &#181;M ruthenium red14.
2. Two Electrode Voltage Clamp
<ol>
	<li>Pull borosilicate glass recording pipettes from precision disposable 100 &#181;l micropipettes individually with a pipette puller.</li>
	<li>Pull both voltage-measuring and current-injecting pipette electrodes are pulled to have a tip aperture of ~1 &#181;m diameter.</li>
	<li>Backfill electrodes with 2 M KCl resulting in 0.1-0.2 m&#937; resistance. Mount them on HS2A headstages, which together with a VG-2Ax100 virtual-grounded bath clamp, are connected to a GeneClamp 500 amplifier interface through a Digidata 1440 digitizer. Acquire data using pClamp10 software. </li>
	<li>Place the oocyte to be tested in a 1 ml bath in a fabricated plastic chamber mounted on the stage of a dissecting microscope with 20X magnification. The bath solution is virtual-grounded by a 3 M KCl agar bridge and a chlorinated silver wire placed close to the oocyte and connected to a VG-2A virtual ground stage.</li>
	<li>Construct the chamber for continuous perfusion with plastic tubings as solution inlet and outlet. Connect the inlet tubing is connected to a fabricated perfusion system, which is a manifold of six 50 ml syringe shells, each filled with a different solution. Gravity feed rates of any particular solution is controlled to ~2-3 ml/min using "Keck" ramp clamps on the tubings.</li>
	<li>Mount both electrodes with their headstages on micromanipulators. Perform electrode penetrations of the oocyte by micromanipulation.</li>
</ol>
3. Patch Clamp Examination of Direct Mechanosensitivity
The basic methods of patch clamp including pipette preparation, G&#937;-seal formation, and formation of on-cell or excised modes are those in Hamill et al.36 and Conn32.
<ol>
	<li>Fill borosilicate glass pipettes with an ~1 &#181;m diameter opening at the tip (bubble number 5-6) with 98 mM KCl, 1 mM MgCl2, and 10 mM K+-HEPES, pH 7.2.</li>
	<li>Attach the patch clamp pipette through a plastic tubing to a 5 ml syringe and through a T-joint, also to a manometer. Use a computer to handle both the electrode and the manometer outputs. Acquire data at 10 kHz, and then play back through an eight-pole Bessel filter at 1 kHz for analysis.</li>
	<li>Different patches can be excised from the same oocyte repeatedly. This practice makes the examination of TRPV4's molecular activity more efficient and reliable.</li>
</ol>

<ol>
	<li>Ramsey, I. S., Delling, M., Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+TRP+channels.">An introduction to TRP channels.</a> Annu. Rev. Physiol. 68, 619-647 (2006).</li><li>Nilius, B., Owsianik, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transient+receptor+potential+family+of+ion+channels.">The transient receptor potential family of ion channels.</a> Genome Biol. 12, 218 (2011).</li><li>Everaerts, W., Nilius, B., Owsianik, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vallinoid+transient+receptor+potential+channel+Trpv4:+From+structure+to+disease.">The vallinoid transient receptor potential channel Trpv4: From structure to disease.</a> Prog. Biophys. Mol. Biol. , (2009).</li><li>Nilius, B., Voets, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+puzzle+of+TRPV4+channelopathies.">The puzzle of TRPV4 channelopathies.</a> EMBO Rep.. , (2013).</li><li>Liedtke, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vanilloid+receptor-related+osmotically+activated+channel+(VR-OAC),+a+candidate+vertebrate+osmoreceptor.">Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor.</a> Cell. 103, 525-535 (2000).</li><li>Strotmann, R., Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+novel+human+vanilloid+receptor-like+protein,+VRL-2.">Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2.</a> Physiol. Genomics. 4, 165-174 (2001).</li><li>Rock, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain-of-function+mutations+in+TRPV4+cause+autosomal+dominant+brachyolmia.">Gain-of-function mutations in TRPV4 cause autosomal dominant brachyolmia.</a> Nat. Genet. 40, 999-1003 (2008).</li><li>Krakow, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+gene+encoding+the+calcium-permeable+ion+channel+TRPV4+produce+spondylometaphyseal+dysplasia,+Kozlowski+type+and+metatropic+dysplasia.">Mutations in the gene encoding the calcium-permeable ion channel TRPV4 produce spondylometaphyseal dysplasia, Kozlowski type and metatropic dysplasia.</a> Am. J. Hum. Genet. 84, 307-315 (2009).</li><li>Camacho, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+TRPV4+mutations+in+nonlethal+and+lethal+metatropic+dysplasia.">Dominant TRPV4 mutations in nonlethal and lethal metatropic dysplasia.</a> Am. J. Med. Genet. A. 152, 1169-1177 (2010).</li><li>Voets, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+determinants+of+permeation+through+the+cation+channel+TRPV4.">Molecular determinants of permeation through the cation channel TRPV4.</a> J. Biol. Chem. 277, 33704-33710 (2002).</li><li>Watanabe, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+TRPV4+gating+by+intra-+and+extracellular+Ca2.">Modulation of TRPV4 gating by intra- and extracellular Ca2.</a> Cell Calcium. 33, 489-495 (2003).</li><li>Loukin, S., Zhou, X., Su, Z., Saimi, Y., Kung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wild-type+and+brachyolmia-causing+mutant+TRPV4+channels+respond+directly+to+stretch+force.">Wild-type and brachyolmia-causing mutant TRPV4 channels respond directly to stretch force.</a> J. Biol. Chem. 285, 27176-27181 (2010).</li><li>Gao, X., Wu, L., O'Neil, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-modulated+diversity+of+TRPV4+channel+gating:+activation+by+physical+stresses+and+phorbol+ester+derivatives+through+protein+kinase+C-dependent+and+-independent+pathways.">Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathways.</a> J. Biol. Chem. 278, 27129-27137 (2003).</li><li>Watanabe, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anandamide+and+arachidonic+acid+use+epoxyeicosatrienoic+acids+to+activate+TRPV4+channels.">Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels.</a> Nature. 424, 434-438 (2003).</li><li>Vriens, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+swelling,+heat,+and+chemical+agonists+use+distinct+pathways+for+the+activation+of+the+cation+channel+TRPV4.">Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 396-401 (2004).</li><li>Vincent, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+novel+TRPV4+modulators.">Identification and characterization of novel TRPV4 modulators.</a> Biochem. Biophys. Res. Commun. 389, 490-494 (2009).</li><li>Willette, R. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+activation+of+the+transient+receptor+potential+vanilloid+subtype+4+channel+causes+endothelial+failure+and+circulatory+collapse:+Part+2.">Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: Part 2.</a> J. Pharmacol. Exp. Ther. 326, 443-452 (2008).</li><li>Thorneloe, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orally+active+TRPV4+channel+blocker+prevents+and+resolves+pulmonary+edema+induced+by+heart+failure.">An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure.</a> Sci. Transl. Med. 4, (2012).</li><li>Kang, S. S., Shin, S. H., Auh, C. K., Chun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+skeletal+dysplasia+caused+by+a+constitutive+activated+transient+receptor+potential+vanilloid+4+(TRPV4)+cation+channel+mutation.">Human skeletal dysplasia caused by a constitutive activated transient receptor potential vanilloid 4 (TRPV4) cation channel mutation.</a> Exp. Mol. Med. 44, 707-722 (2012).</li><li>Lamande, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+TRPV4+cause+an+inherited+arthropathy+of+hands+and+feet.">Mutations in TRPV4 cause an inherited arthropathy of hands and feet.</a> Nat. Genet. 43, 1142-1146 (2011).</li><li>Loukin, S., Su, Z., Kung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Basal+Activity+Is+a+Key+Determinant+in+the+Severity+of+Human+Skeletal+Dysplasia+Caused+by+TRPV4+Mutations.">Increased Basal Activity Is a Key Determinant in the Severity of Human Skeletal Dysplasia Caused by TRPV4 Mutations.</a> PLoS One. 6, (2011).</li><li>Loukin, S. H., Su, Z., Kung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypotonic+shocks+activate+rat+TRPV4+in+yeast+in+the+absence+of+polyunsaturated+fatty+acids.">Hypotonic shocks activate rat TRPV4 in yeast in the absence of polyunsaturated fatty acids.</a> FEBS Lett. 583, 754-758 (2009).</li><li>Loukin, S., Su, Z., Zhou, X., Kung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+genetic+analysis+reveals+multiple+gating+mechanisms+of+TRPV4.">Forward genetic analysis reveals multiple gating mechanisms of TRPV4.</a> J. Biol. Chem. 285, 19884-19890 (2010).</li><li>Batiza, A. F., Schulz, T., Masson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+respond+to+hypotonic+shock+with+a+calcium+pulse.">Yeast respond to hypotonic shock with a calcium pulse.</a> J. Biol. Chem. 271, 23357-23362 (1996).</li><li>Amberg, D. C., Burke, D., Strathern, J. 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The authors declare that they have no competing financial interests.

As stated in the Introduction, the methods commonly used to study TRPV4 functions sometimes resulted in inconsistencies and controversies. The two sets of methods described here offer some advantages and can complement the existing methods. While we describe only the studies on TRPV4, the methods may be extended to study other ion channels as well.
1. Yeast Luminometry Methods
Budding yeast has a native Ca2+-influx response to hypo-osmotic shock that is sen...

TRPs (transient receptor potentials) comprise seven subfamilies of cation channels that serve sensory functions1,2. In mammals, TRPV, the vanilloid subfamily of TRPs, has six varieties. TRPV4 (type 4)3,4 responds to heat, certain chemicals, osmotic swelling, or shear stress. The TRPV4 gene was repeatedly isolated by candidate-gene and/or expression cloning5-8. The latter method followed the gene product&#39;s response to hypo-osmolarity. TRPV4 is expressed in nearly all organs and functions in the development, physiology, or pathology of many disparate cell types3,4.
Striking are the &#62;50 human autosomal dominant TRPV4 mutations, causing peripheral neuropathies and/or skeletal dysplasias (abnormalities in skeletal development)9-11. The skeletal dysplasias range from mild brachyomia type 3 (type-3 dwarfism), spondylometaphyseal dysplasia Kozlowski type, to severe dysplasias, some causing neonatal or embryonic death. Though all manners of mechanisms seem possible, none explains the diversity, complexity, variability, and occasional overlaps of these diseases4.
Like other TRP channels1, TRPV4 is a tetramer. In rat or human TRPV4, each subunit is consisted of 871 residues. Its central element is the six transmembrane a helices (S1-S6), which are likely arranged in a manner similar to voltage-gated K+ channels. There, S1 to S4 form a peripheral domain and the S5 and S6 form the permeation pore domain. Between S5 and S6 of TRPV4 is a short pore helix followed by the sequence LDLFKLTIGMGDL, four of which presumably converge to form the cation filter. The 470-residue N-terminal cytoplasmic segment contains 6 ankyrin repeats, known to bind proteins or small ligands. The C-terminal 152-residue cytoplasmic segment includes a calmodulin-binding sequence among other possible sites that bind other elements3.
TRPV4 is a cation channel that essentially excludes anions1. While its physiological function is to transduce stimuli to Ca2+ influx, it is also permeable to other cations with an Eisenman IV permeability sequence, favoring divalent at a PCa : PNa ~7 12. Single-channel conductance rectifies at ~90 pS outward and ~40 pS inward6,13,14. Heterologously expressed current (below) can be activated by hypo-osmotic swelling, shear stress, or warmth15. It is also activated by polyunsaturated fatty acids16,17 and the synthetic phorbal ester 4&#945;PDD 18. At present, the most potent agonist is GSK1016790A 19 and antagonist is GSK2193874, effective in 10-9 to 10-8 M 20, both discovered by high-throughput, small-molecular screen.
Two key areas of TRPV4 research remain confusing: (1) Even as TRPV4 is largely studied for its mechanosensitivity, its molecular basis is controversial. One model describes hypo-osmolarity somehow activates phospholipase A2 (PLA2) to produce the polyunsaturated fatty acid (PUFA) arachidonic acid (AA), which is converted to epoxyeicosatrienoic acid (EET) by an epoxygenase, and the binding of EET activates TRPV4 16,17. Yet, TRPV4 itself has been shown to directly respond to membrane stretch14 (below), providing a simpler explanation. (2) The TRPV4 mutant pathologies are bewildering. At the foundation, one needs to know whether the diseases are due to the loss, the reduction, or the increase of channel activities. Even here, the literature is far from clear. While multiple skeletal-dysplasia alleles were reported to have higher activities, (gain-of-function, GOF)4,21, several were reported to have reduced activities (loss-of-function, LOF)10,22. A systematic study of 14 alleles found them to all be GOF mutations (below)23. The claim that some are LOFs seems to contradict the phenotype of trpV4-/- knockout mouse, which are viable or fertile, with only minor defects, despite a complete loss of TRPV4 function.
A part of these controversies has methodological origin. Laboratories use different methods, or variants of one method, and employ different judging standards. Most commonly, TRPV4 is transiently expressed in cultured mammalian cells (HEK, CHO, HeLa) and the rise of internal [Ca2+] upon agonist or hypotonic stimulation is registered with the Ca2+-sensitive fluorescent dye, Fura-2. The over-reliance on this fluorometric assay has been criticized1. The expression level in different populations, the distribution therein, as well as the effective dye concentration, need to be controlled and documented. More reliable is the direct electrophysiological examinations. Even this, as commonly practiced, is also not without problems. Because the expression levels in individual cultured cells are difficult to control, whole-cell currents have large variations. Further, because the currents are small, reliable statistics will have to rely on large sample sizes, often not practical. Patch-clamp examinations have rarely been performed. Some such recordings show clusters of activity bursts that make statistical evaluation challenging16,17.
To better understand the molecular mechanosensitivity, we have developed two additional systems to examine TRPV4. (1) To isolate TRPV4 away from its usual mammalian partners, we have expressed rat TRPV4 in budding yeast24. Functional expression of TRPV4 in this evolutionarily distant context showed that it could still respond to osmotic force without its usual partners. Because yeast makes no PUFAs such as AA or EET, and its genome has no PLA2 or epoxygenase genes, this expression also shows that they are not required for TRPV4 to sense force. Having TRPV4 in the molecular biologically most amenable eukaryotes also allows efficient forward- or reverse-genetic manipulations25. (2) For in-depth biophysical analyses of TRPV4, we expressed TRPV4 in Xenopus oocytes. Unlike cultured cells, which yield sub-nA to nA (10-10 to 10-9 A) currents, an oocyte expresses currents in &#181;A&#39;s (10-6 to 10-4 A). Much larger signal over noise allows better quantification and more confident comparison. TRPV4, so expressed, can also be examined as individual molecules using a patch clamp. A single oocyte allows repeated patch sampling, again making quantification more reliable. Such studies showed that TRPV4 channel itself can directly be activated by membrane stretch force14. Analyses also showed that 14 representative skeletal-dysplasia alleles are all gain-of-function mutations. Further, the degree of this constitutive Ca2+ leakage parallels the severity of the skeletal diseases23.
Because of their novelty and usefulness, the detailed procedures of these two methods are assembled here to allow replications in future research on TRPV4 or similar channels.

<table border="0" cellpadding="0" cellspacing="0" width="595">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vapor Pressure Osmometer</td>
			<td style="width:93px;">Wescor</td>
			<td style="width:119px;">Wescor 5500</td>
			<td style="width:167px;">Logan, UT, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sirius Single Tube Luminometer </td>
			<td style="width:93px;">Titertek-Berhold</td>
			<td style="width:119px;">Sirius </td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microplate luminometer</td>
			<td style="width:93px;">Titertek-Berthold </td>
			<td style="width:119px;">Mithras LB940</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Luminometry software</td>
			<td style="width:93px;">Titertek-Berthold</td>
			<td style="width:119px;">MikroWin2000</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:216px;">Patch clamp and software</td>
			<td style="width:93px;">Axon Instrument</td>
			<td style="width:119px;">HS2A headstage, VG-2Ax100 virtual grounded bath clamp, GeneClamp 500 amplifier, digidata 1440 digitizer, pClamp10 software </td>
			<td>Union City, CA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">manometer</td>
			<td style="width:93px;">Bio-Tec</td>
			<td style="width:119px;"> </td>
			<td>Winooski, VT, USA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pipet puller</td>
			<td style="width:93px;">Sutter Instrument Co</td>
			<td style="width:119px;">Model P-97</td>
			<td>Novata, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microinjector</td>
			<td style="width:93px;">Drummond Scientific</td>
			<td style="width:119px;">Nanoinject II</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Material:</td>
			<td style="width:93px;"> </td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">luciferin coelenterazine</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;"> </td>
			<td>Carlsbad, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">T7 RNA polymerase reaction</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;">mMessage mMachine</td>
			<td>Austin, TX, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gentamicin</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ruthenium red </td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micropipets </td>
			<td style="width:93px;">Drummond</td>
			<td style="width:119px;">100 microliter micropipets</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">high-fidelity PfuUtra polymerase </td>
			<td style="width:93px;">Stratagene</td>
			<td style="width:119px;"> </td>
			<td>La Jolla, CA, USA</td>
		</tr>
	</tbody>
</table>

yeast luminometric and xenopus oocyte electrophysiological examinations of the molecular mechanosensitivity of trpv4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies in human. Wild-type or mutant TRPV4 transgenes are commonly expressed in cultured mammalian cells and examined by Fura-2 fluorometry and by electrodes. In terms of the mechanism of mechanosensitivity and the molecular bases of the diseases, the current literature is confusing and controversial. To complement existing methods, we describe two additional methods to examine the molecular properties of TRPV4. (1) Rat TRPV4 and an aequorin transgene are transformed into budding yeast. A hypo-osmtic shock of the transformant population yields a luminometric signal due to the combination of aequorin with Ca2+, released through the TRPV4 channel. Here TRPV4 is isolated from its usual mammalian partner proteins and reveals its own mechanosensitivity. (2) cRNA of TRPV4 is injected into Xenopus oocytes. After a suitable period of incubation, the macroscopic TRPV4 current is examined with a two-electrode voltage clamp. The current rise upon removal of inert osmoticum from the oocyte bath is indicative of mechanosensitivity. The microAmpere (10-6 to 10-4 A) currents from oocytes are much larger than the subnano- to nanoAmpere (10-10 to 10-9 A) currents from cultured cells, yielding clearer quantifications and more confident assessments. Microscopic currents reflecting the activities of individual channel proteins can also be directly registered under a patch clamp, in on-cell or excised mode. The same oocyte provides multiple patch samples, allowing better data replication. Suctions applied to the patches can activate TRPV4 to directly assess mechanosensitivity. These methods should also be useful in the study of other types of TRP channels.

In a typical luminometry experiment, a range of 400-1,200 mOsM hypotonic down shocks is achieved by adding 200 &#956;l of shock solutions of different low osmolarity to 20 &#956;l of culture at 1,400 mOsM. The TRPV4 activity is evident when the RLU of the experimental is compared to that of two negative controls: yeast cells transformed with empty p416GPDV4 plasmid or one that contains the TRPV4 gene with a mutation at the ion filter (Figure 1)24.
In a typi...

Watch this Scientific Journal Video about Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4 at JoVE.com

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

To complement commonly used methods to study TRPV4&#8217;s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode.

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

yeast-luminometric-xenopus-oocyte-electrophysiological-examinations

Research

JoVE Journal

Biology

10.0K Views.  University of Wisconsin – Madison.  Per completare i metodi comunemente usati per studiare TRPV4 di, sono descritti due metodi: la sua mechanosensitivity può essere studiato da Ca 2 + luminometria-aequorina in lievito transgenico upon sfida ipo-osmotico. Può pure esaminato TRPV4-RNA oociti di Xenopus iniettati da cellula intera clamp a due elettrodi tensione o Patch Clamp on-cella o modalità asportato. 

Video: Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.
The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16.
The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...