Influence of proteolytic cleavage of ENaC's γ subunit upon Na+ and K+ handling.

The epithelial Na+ channel (ENaC) γ subunit is essential for homeostasis of Na+, K+, and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146, in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (γQ4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short-circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of γQ4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4 mice was not altered. γQ4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of γ subunit cleavage should not be used in isolation to evaluate ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na+ or high K+ diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC's γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na+ or high K+ diets.

Dietary Na+ depletion up-regulates NKCC1 expression and enhances electrogenic Cl- secretion in rat proximal colon.

The corticosteroid hormone, aldosterone, markedly enhances K+ secretion throughout the colon, a mechanism critical to its role in maintaining overall K+ balance. Previous studies demonstrated that basolateral NKCC1 was up-regulated by aldosterone in the distal colon specifically to support K+ secretion-which is distinct from the more well-established role of NKCC1 in supporting luminal Cl- secretion. However, considerable segmental variability exists between proximal and distal colonic ion transport processes, especially concerning their regulation by aldosterone. Furthermore, delineating such region-specific effects has important implications for the management of various gastrointestinal pathologies. Experiments were therefore designed to determine whether aldosterone similarly up-regulates NKCC1 in the proximal colon to support K+ secretion. Using dietary Na+ depletion as a model of secondary hyperaldosteronism in rats, we found that proximal colon NKCC1 expression was indeed enhanced in Na+-depleted (i.e., hyperaldosteronemic) rats. Surprisingly, electrogenic K+ secretion was not detectable by short-circuit current (ISC) measurements in response to either basolateral bumetanide (NKCC1 inhibitor) or luminal Ba2+ (non-selective K+ channel blocker), despite enhanced K+ secretion in Na+-depleted rats, as measured by 86Rb+ fluxes. Expression of BK and IK channels was also found to be unaltered by dietary Na+ depletion. However, bumetanide-sensitive basal and agonist-stimulated Cl- secretion (ISC) were significantly enhanced by Na+ depletion, as was CFTR Cl- channel expression. These data suggest that NKCC1-dependent secretory pathways are differentially regulated by aldosterone in proximal and distal colon. Development of therapeutic strategies in treating pathologies related to aberrant colonic K+/Cl- transport-such as pseudo-obstruction or ulcerative colitis-may benefit from these findings.

Paraoxonase 2 is an ER chaperone that regulates the epithelial Na+ channel.

The mammalian paraoxonases (PONs) have been linked to protection against oxidative stress. However, the physiological roles of members in this family (PON1, PON2, and PON3) are still being characterized. PON2 and PON3 are expressed in the aldosterone-sensitive distal nephron of the kidney and have been shown to negatively regulate expression of the epithelial sodium channel (ENaC), a trimeric ion channel that orchestrates salt and water homeostasis. To date, the nature of this phenomenon has not been explored. Therefore, to investigate the mechanism by which PON2 regulates ENaC, we expressed PON2 along with the ENaC subunits in fisher rat thyroid (FRT) cells, a system that is amenable to biochemical analyses of ENaC assembly and trafficking. We found that PON2 primarily resides in the endoplasmic reticulum (ER) in FRT cells, and its expression reduces the abundance of each ENaC subunit, reflecting enhanced subunit turnover. In contrast, no effect on the levels of mRNAs encoding the ENaC subunits was evident. Inhibition of lysosome function with chloroquine or NH4Cl did not alter the inhibitory effect of PON2 on ENaC expression. In contrast, PON2 accelerates ENaC degradation in a proteasome-dependent manner and acts before ENaC subunit ubiquitination. As a result of enhanced ENaC subunit ubiquitination and degradation, both channel surface expression and ENaC-mediated Na+ transport in FRT cells were reduced by PON2. Together, our data suggest that PON2 functions as an ER chaperone to monitor ENaC biogenesis and redirects the channel for ER-associated degradation.

Bile acids regulate the epithelial Na+ channel in native tissues through direct binding at multiple sites.

Bile acids, originally known to emulsify dietary lipids, are now established signalling molecules that regulate physiological processes. Signalling targets several proteins that include the ion channels involved in regulating intestinal motility and bile viscosity. Studies show that bile acids regulate the epithelial sodium channel (ENaC) in cultured cell models and heterologous expression systems. ENaC plays both local and systemic roles in regulating extracellular fluids. Here we investigated whether bile acids regulate ENaC expressed in native tissues. We found that taurocholic acid and taurohyodeoxycholic acid regulated ENaC in both the distal nephron and distal colon. We also tested the hypothesis that regulation occurs through direct binding. Using photoaffinity labelling, we found evidence for specific binding to both the ß and γ subunits of the channel. In functional experiments, we found that the α subunit was sufficient for regulation. We also found that regulation by at least one bile acid was voltage-sensitive, suggesting that one binding site may be closely associated with the pore-forming helices of the channel. Our data provide evidence that bile acids regulate ENaC by binding to multiple sites to influence the open probability of the channel. KEY POINTS: Recent studies have shown that bile acids regulate the epithelial sodium channel (ENaC) in vitro. Here we investigated whether bile acids regulate ENaC in native tissues and whether bile acids directly bind the channel. We found that bile acids regulate ENaC expressed in the mouse cortical collecting duct and mouse colon by modulating open probability. Photoaffinity labelling experiments showed specific binding to the ß and γ subunits of the channel, while channels comprising only α subunits were sensitive to taurocholic acid in functional experiments using Xenopus oocytes. Taurocholic acid regulation of ENaC was voltage-dependent, providing evidence for binding to pore-forming helices. Our data indicate that bile acids are ENaC regulatory effectors that may have a role in the physiology and pathophysiology of several systems.

Aldosterone up-regulates basolateral Na+ -K+ -2Cl- cotransporter-1 to support enhanced large-conductance K+ channel-mediated K+ secretion in rat distal colon.

Na+ -K+ -2Cl- cotransporter-1 (NKCC1) facilitates basolateral K+ and Cl- uptake, supporting their efflux across mucosal membranes of colonic epithelial cells. NKCC1 activity has also been shown to be critical for electrogenic K+ secretion induced by aldosterone, which is known to stimulate large-conductance K+ (BK) channel expression in mucosal membranes. This study was aimed to (1) identify whether aldosterone enhances NKCC1 expression specifically to support BK-mediated K+ secretion and (2) to determine whether increased NKCC1 supports electrogenic Cl- secretion in parallel to K+ secretion. Dietary Na+ depletion was used to induce secondary hyperaldosteronism in rats, or aldosterone was administered ex vivo to rat distal colonic mucosae. NKCC1-dependent electrogenic K+ or Cl- secretion was measured as a function of short circuit current (ISC ). qRT-PCR, western blot, and immunofluorescence analyses were performed using standard techniques. Aldosterone enhanced NKCC1 and BKα expression and electrogenic K+ secretion in the distal colon, which was inhibited by either serosal bumetanide (NKCC1 inhibitor) or mucosal iberiotoxin (IbTX; BK channel blocker), but not TRAM-34 (IK channel blocker). Expression of NKCC1 and BKα proteins was enhanced in crypt cells of hyper-aldosterone rats. However, neither NKCC1-dependent Cl- secretion nor CFTR (apical Cl- channel) expression was enhanced by aldosterone. We conclude that aldosterone enhances NKCC1 to support BK-mediated K+ secretion independently of Cl- secretion in the distal colon. The regulation of NKCC1 expression/K+ secretion by aldosterone may be a therapeutic target in treating gastrointestinal disorders associated with alterations in colonic K+ transport, such as colonic pseudo-obstruction, and hyperkalemia associated with renal disease.

Activation of KCNQ (KV7) K+ channels in enteric neurons inhibits epithelial Cl- secretion in mouse distal colon.

Voltage-gated Kv7 (KCNQ family) K+ channels are expressed in many neuronal populations and play an important role in regulating membrane potential by generating a hyperpolarizing K+ current and decreasing cell excitability. However, the role of KV7 channels in the neural regulation of intestinal epithelial Cl- secretion is not known. Cl- secretion in mouse distal colon was measured as a function of short-circuit current (ISC), and pharmacological approaches were used to test the hypothesis that activation of KV7 channels in enteric neurons would inhibit epithelial Cl- secretion. Flupirtine, a nonselective KV7 activator, inhibited basal Cl- secretion in mouse distal colon and abolished or attenuated the effects of drugs that target various components of enteric neurotransmission, including tetrodotoxin (NaV channel blocker), veratridine (NaV channel activator), nicotine (nicotinic acetylcholine receptor agonist), and hexamethonium (nicotinic antagonist). In contrast, flupritine did not block the response to epithelium-targeted agents VIP (endogenous VPAC receptor ligand) or carbachol (nonselective cholinergic agonist). Flupirtine inhibited Cl- secretion in both full-thickness and seromuscular-stripped distal colon (containing the submucosal, but not myenteric plexus) but generated no response in epithelial T84 cell monolayers. KV7.2 and KV7.3 channel proteins were detected by immunofluorescence in whole mount preparations of the submucosa from mouse distal colon. ICA 110381 (KV7.2/7.3 specific activator) inhibited Cl- secretion comparably to flupirtine. We conclude that KV7 channel activators inhibit neurally driven Cl- secretion in the colonic epithelium and may therefore have therapeutic benefit in treating pathologies associated with hyperexcitable enteric nervous system, such as irritable bowel syndrome with diarrhea (IBS-D).

Flupirtine enhances NHE-3-mediated Na+ absorption in rat colon via an ENS-dependent mechanism.

Recent studies in our lab have shown that the KV7 channel activator, flupirtine, inhibits colonic epithelial Cl- secretion through effects on submucosal neurons of the enteric nervous system (ENS). We hypothesized that flupirtine would also stimulate Na+ absorption as a result of reduced secretory ENS input to the epithelium. To test this hypothesis, unidirectional 22Na+ fluxes were measured under voltage-clamped conditions. Pharmacological approaches using an Ussing-style recording chamber combined with immunofluorescence microscopy techniques were used to determine the effect of flupirtine on active Na+ transport in the rat colon. Flupirtine stimulated electroneutral Na+ absorption in partially seromuscular-stripped colonic tissues, while simultaneously inhibiting short-circuit current (ISC; i.e., Cl- secretion). Both of these effects were attenuated by pretreatment with the ENS inhibitor, tetrodotoxin. The Na+/H+ exchanger isoform 3 (NHE-3)-selective inhibitor, S3226, significantly inhibited flupirtine-stimulated Na+ absorption, whereas the NHE-2-selective inhibitor HOE-694 did not. NHE-3 localization near the apical membranes of surface epithelial cells was also more apparent in flupirtine-treated colon versus control. Flupirtine did not alter epithelial Na+ channel (ENaC)-mediated Na+ absorption in distal colonic tissues obtained from hyperaldosteronaemic rats and had no effect in the normal ileum but did stimulate Na+ absorption in the proximal colon. Finally, the parallel effects of flupirtine on ISC (Cl- secretion) and Na+ absorption were significantly correlated with each other. Together, these data indicate that flupirtine stimulates NHE-3-dependent Na+ absorption, likely as a result of reduced stimulatory input to the colonic epithelium by submucosal ENS neurons.NEW & NOTEWORTHY We present a novel mechanism regarding regulation of epithelial ion transport by enteric neurons. Activation of neuronal KV7 K+ channels markedly stimulates Na+ absorption and inhibits Cl- secretion across the colonic epithelium. This may be useful in developing new treatments for diarrheal disorders, such as irritable bowel syndrome with diarrhea (IBS-D).

Increased DMT1 and FPN1 expression with enhanced iron absorption in ulcerative colitis human colon.

Iron deficiency anemia is a common complication of ulcerative colitis (UC) that can profoundly impact quality of life. Most iron absorption occurs in the duodenum via divalent metal transporter 1 (DMT1)-mediated uptake and ferroportin-1 (FPN1)-mediated export across the apical and basolateral membranes, respectively. However, the colon also contains iron transporters and can participate in iron absorption. Studies have shown increased duodenal DMT1 and FPN1 in patients with UC, but there is conflicting evidence about whether expression is altered in UC colon. We hypothesized that expression of colonic DMT1 and FPN1 will also increase to compensate for iron deficiency. Quantitative RT-PCR and Western blot analyses were performed on duodenal and colonic segmental (right colon, transverse colon, left colon, and rectum) biopsies obtained during colonoscopy. DMT1 mRNA and protein abundances in colonic segments were approximately equal to those in the duodenum, whereas colonic FPN1 mRNA and protein abundances of colonic segments were about one-quarter of those of the duodenum. DMT1 specific mRNA and protein abundances were increased twofold, whereas FPN1 mRNA and protein expressions were increased fivefold in UC distal colon. Immunofluorescence studies revealed enhanced expression of apical membrane- and basolateral membrane-localized DMT1 and FPN1 in UC human colon, respectively. Increased DMT1 expression was associated with enhanced 2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea (CISMBI, DMT1 specific inhibitor)-sensitive 59Fe uptake in UC human colon. We conclude from these results that patients with active UC have increased expression of colonic iron transporters and increased iron absorption, which may be targeted in the treatment of UC-related anemia.

Parallel intermediate conductance K+ and Cl- channel activity mediates electroneutral K+ exit across basolateral membranes in rat distal colon.

Transepithelial K+ absorption requires apical K+ uptake and basolateral K+ exit. In the colon, apical H+-K+-ATPase mediates cellular K+ uptake, and it has been suggested that electroneutral basolateral K+ exit reflects K+-Cl- cotransporter-1 (KCC1) operating in parallel with K+ and Cl- channels. The present study was designed to identify basolateral transporter(s) responsible for K+ exit in rat distal colon. Active K+ absorption was determined by measuring 86Rb+ (K+ surrogate) fluxes across colonic epithelia under voltage-clamp conditions. With zero Cl- in the mucosal solution, net K+ absorption was reduced by 38%, indicating that K+ absorption was partially Cl--dependent. Serosal addition of DIOA (KCC1 inhibitor) or Ba2+ (nonspecific K+ channel blocker) inhibited net K+ absorption by 21% or 61%, respectively, suggesting that both KCC1 and K+ channels contribute to basolateral K+ exit. Clotrimazole and TRAM34 (IK channel blockers) added serosally inhibited net K+ absorption, pointing to the involvement of IK channels in basolateral K+ exit. GaTx2 (CLC2 blocker) added serosally also inhibited net K+ absorption, suggesting that CLC2-mediated Cl- exit accompanies IK channel-mediated K+ exit across the basolateral membrane. Net K+ absorption was not inhibited by serosal addition of either IbTX (BK channel blocker), apamin (SK channel blocker), chromanol 293B (KV7 channel blocker), or CFTRinh172 (CFTR blocker). Immunofluorescence studies confirmed basolateral membrane colocalization of CLC2-like proteins and Na+-K+-ATPase α-subunits. We conclude that active K+ absorption in rat distal colon involves electroneutral basolateral K+ exit, which may reflect IK and CLC2 channels operating in parallel.NEW & NOTEWORTHY This study demonstrates that during active electroneutral K+ absorption in rat distal colon, K+ exit across the basolateral membrane mainly reflects intermediate conductance K+ channels operating in conjunction with chloride channel 2, with a smaller, but significant, contribution from K+-Cl- cotransporter-1 (KCC1) activity.

Dextran sulfate sodium-induced chronic colitis attenuates Ca2+-activated Cl- secretion in murine colon by downregulating TMEM16A.

Attenuated Ca2+-activated Cl- secretion has previously been observed in the model of dextran sulfate sodium (DSS)-induced colitis. Prior studies have implicated dysfunctional muscarinic signaling from basolateral membranes as the potential perpetrator leading to decreased Ca2+-activated Cl- secretion. However, in our chronic model of DSS-colitis, cholinergic receptor muscarinic 3 ( Chrm3) transcript (1.028 ± 0.12 vs. 1.029 ± 0.27, P > 0.05) and CHRM3 protein expression (1.021 ± 0.24 vs. 0.928 ± 0.09, P > 0.05) were unchanged. Therefore, we hypothesized that decreased carbachol (CCH)-stimulated Cl- secretion in DSS-induced colitis could be attributed to a loss of Ca2+-activated Cl- channels (CaCC) in apical membranes of colonic epithelium. To establish this chemically-induced colitis, Balb/C mice were exposed to 4% DSS for five alternating weeks to stimulate a more moderate, chronic colitis. Upon completion of the protocol, whole thickness sections of colon were mounted in an Ussing chamber under voltage-clamp conditions. DSS-induced colitis demonstrated a complete inhibition of basolateral administration of CCH-stimulated Cl- secretion that actually displayed a reversal in polarity (15.40 ± 2.22 µA/cm2 vs. -2.47 ± 0.25 µA/cm2). Western blotting of potential CaCCs, quantified by densitometric analysis, demonstrated no change in bestrophin-2 and cystic fibrosis transmembrane regulator, whereas anoctamin-1 [ANO1, transmembrane protein 16A (TMEM16A)] was significantly downregulated (1.001 ± 0.13 vs. 0.510 ± 0.12, P < 0.05). Our findings indicate that decreased expression of TMEM16A in DSS-induced colitis contributes to the decreased Ca2+-activated Cl- secretion in murine colon.

Calcium-Activated Cl- Channel: Insights on the Molecular Identity in Epithelial Tissues.

Calcium-activated chloride secretion in epithelial tissues has been described for many years. However, the molecular identity of the channel responsible for the Ca2+-activated Cl− secretion in epithelial tissues has remained a mystery. More recently, TMEM16A has been identified as a new putative Ca2+-activated Cl− channel (CaCC). The primary goal of this article will be to review the characterization of TMEM16A, as it relates to the physical structure of the channel, as well as important residues that confer voltage and Ca2+-sensitivity of the channel. This review will also discuss the role of TMEM16A in epithelial physiology and potential associated-pathophysiology. This will include discussion of developed knockout models that have provided much needed insight on the functional localization of TMEM16A in several epithelial tissues. Finally, this review will examine the implications of the identification of TMEM16A as it pertains to potential novel therapies in several pathologies.

Proteolytic Cleavage of the ENaC γ Subunit - Impact Upon Na + and K + Handling.

The ENaC gamma subunit is essential for homeostasis of Na + , K + , and body fluid. Dual subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (P O ), in vitro . Cleavage proximal to the tract occurs at a furin recognition sequence ( 143 RKRR 146 in mouse). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143 RKRR 146 mutation to 143 QQQQ 146 ( Q4 ) in 129/Sv mice would reduce ENaC P O , impair flow-stimulated flux of Na + (J Na ) and K + (J K ) in perfused collecting ducts, reduce colonic amiloride-sensitive short circuit current (I SC ), and impair Na + , K + , and body fluid homeostasis. Immunoblot of Q4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, Q4/Q4 male mice on a low Na + diet did not exhibit altered ENaC P O or flow-induced J Na , though flow-induced J K modestly decreased. Colonic amiloride-sensitive I SC in Q4/Q4 mice was not altered. Q4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na + diet. Blood Na + and K + were unchanged on a regular, low Na + , or high K + diet. These findings suggest that biochemical evidence of gamma subunit cleavage should not be used in isolation to evaluate ENaC activity. Further, factors independent of gamma subunit cleavage modulate channel P O and the influence of ENaC on Na + , K + , and fluid volume homeostasis in 129/Sv mice, in vivo .

Mice lacking γENaC palmitoylation sites maintain benzamil-sensitive Na+ transport despite reduced channel activity.

Epithelial Na+ channels (ENaCs) control extracellular fluid volume by facilitating Na+ absorption across transporting epithelia. In vitro studies showed that Cys-palmitoylation of the γENaC subunit is a major regulator of channel activity. We tested whether γ subunit palmitoylation sites are necessary for channel function in vivo by generating mice lacking the palmitoylated cysteines (γC33A,C41A) using CRISPR/Cas9 technology. ENaCs in dissected kidney tubules from γC33A,C41A mice had reduced open probability compared with wild-type (WT) littermates maintained on either standard or Na+-deficient diets. Male mutant mice also had higher aldosterone levels than WT littermates following Na+ restriction. However, γC33A,C41A mice did not have reduced amiloride-sensitive Na+ currents in the distal colon or benzamil-induced natriuresis compared to WT mice. We identified a second, larger conductance cation channel in the distal nephron with biophysical properties distinct from ENaC. The activity of this channel was higher in Na+-restricted γC33A,C41A versus WT mice and was blocked by benzamil, providing a possible compensatory mechanism for reduced prototypic ENaC function. We conclude that γ subunit palmitoylation sites are required for prototypic ENaC activity in vivo but are not necessary for amiloride/benzamil-sensitive Na+ transport in the distal nephron or colon.

Quantifying blood flow and wall shear stresses in the outflow tract of chick embryonic hearts.

Wall shear stresses (WSS) exerted by blood flow on cardiac tissues modulate growth and development of the heart. To study the role of hemodynamic conditions on cardiac morphogenesis, here, we present a methodology that combines imaging and finite element modeling to quantify the in vivo blood flow dynamics and WSS in the cardiac outflow tract (OFT) of early chicken embryos (day 3 out of 21-day incubation period). We found a distinct blood flow field and heterogeneous distribution of WSS in the chicken embryonic heart OFT, which have physiological implications for OFT morphogenesis.

Quantitating morphological changes in biological samples during scanning electron microscopy sample preparation with correlative super-resolution microscopy.

Sample preparation is critical to biological electron microscopy (EM), and there have been continuous efforts on optimizing the procedures to best preserve structures of interest in the sample. However, a quantitative characterization of the morphological changes associated with each step in EM sample preparation is currently lacking. Using correlative EM and superresolution microscopy (SRM), we have examined the effects of different drying methods as well as osmium tetroxide (OsO4) post-fixation on cell morphology during scanning electron microscopy (SEM) sample preparation. Here, SRM images of the sample acquired under hydrated conditions were used as a baseline for evaluating morphological changes as the sample went through SEM sample processing. We found that both chemical drying and critical point drying lead to a mild cellular boundary retraction of ~60 nm. Post-fixation by OsO4 causes at least 40 nm additional boundary retraction. We also found that coating coverslips with adhesion molecules such as fibronectin prior to cell plating helps reduce cell distortion from OsO4 post-fixation. These quantitative measurements offer useful information for identifying causes of cell distortions in SEM sample preparation and improving current procedures.

Methodology for Quantitative Characterization of Fluorophore Photoswitching to Predict Superresolution Microscopy Image Quality.

Single-molecule localization microscopy (SMLM) image quality and resolution strongly depend on the photoswitching properties of fluorophores used for sample labeling. Development of fluorophores with optimized photoswitching will considerably improve SMLM spatial and spectral resolution. Currently, evaluating fluorophore photoswitching requires protein-conjugation before assessment mandating specific fluorophore functionality, which is a major hurdle for systematic characterization. Herein, we validated polyvinyl alcohol (PVA) as a single-molecule environment to efficiently quantify the photoswitching properties of fluorophores and identified photoswitching properties predictive of quality SMLM images. We demonstrated that the same fluorophore photoswitching properties measured in PVA films and using antibody adsorption, a protein-conjugation environment analogous to labeled cells, were significantly correlated to microtubule width and continuity, surrogate measures of SMLM image quality. Defining PVA as a fluorophore photoswitching screening platform will facilitate SMLM fluorophore development and optimal image buffer assessment through facile and accurate photoswitching property characterization, which translates to SMLM fluorophore imaging performance.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM).

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 °C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Effect of Labeling Density and Time Post Labeling on Quality of Antibody-Based Super Resolution Microscopy Images.

Super resolution microscopy (SRM) has overcome the historic spatial resolution limit of light microscopy, enabling fluorescence visualization of intracellular structures and multi-protein complexes at the nanometer scale. Using single-molecule localization microscopy, the precise location of a stochastically activated population of photoswitchable fluorophores is determined during the collection of many images to form a single image with resolution of ~10-20 nm, an order of magnitude improvement over conventional microscopy. One of the key factors in achieving such resolution with single-molecule SRM is the ability to accurately locate each fluorophore while it emits photons. Image quality is also related to appropriate labeling density of the entity of interest within the sample. While ease of detection improves as entities are labeled with more fluorophores and have increased fluorescence signal, there is potential to reduce localization precision, and hence resolution, with an increased number of fluorophores that are on at the same time in the same relative vicinity. In the current work, fixed microtubules were antibody labeled using secondary antibodies prepared with a range of Alexa Fluor 647 conjugation ratios to compare image quality of microtubules to the fluorophore labeling density. It was found that image quality changed with both the fluorophore labeling density and time between completion of labeling and performance of imaging study, with certain fluorophore to protein ratios giving optimal imaging results.

Photoactivated localization microscopy with bimolecular fluorescence complementation (BiFC-PALM) for nanoscale imaging of protein-protein interactions in cells.

Bimolecular fluorescence complementation (BiFC) has been widely used to visualize protein-protein interactions (PPIs) in cells. Until now, however, the resolution of BiFC has been limited by the diffraction of light to ∼250 nm, much larger than the nanometer scale at which PPIs occur or are regulated. Cellular imaging at the nanometer scale has recently been realized with single molecule superresolution imaging techniques such as photoactivated localization microscopy (PALM). Here we have combined BiFC with PALM to visualize PPIs inside cells with nanometer spatial resolution and single molecule sensitivity. We demonstrated that PAmCherry1, a photoactivatable fluorescent protein commonly used for PALM, can be used as a BiFC probe when split between residues 159 and 160 into two fragments. PAmCherry1 BiFC exhibits high specificity and high efficiency even at 37°C in detecting PPIs with virtually no background from spontaneous reconstitution. Moreover, the reconstituted protein maintains the fast photoconversion, high contrast ratio, and single molecule brightness of the parent PAmCherry1, which enables selective PALM localization of PPIs with ∼18 nm spatial precision. With BiFC-PALM, we studied the interactions between the small GTPase Ras and its downstream effector Raf, and clearly observed nanoscale clustering and diffusion of individual KRas G12D/CRaf RBD (Ras-binding domain) complexes on the cell membrane. These observations provided novel insights into the regulation of Ras/Raf interaction at the molecular scale, which would be difficult with other techniques such as conventional BiFC, fluorescence co-localization or FRET.