Cy3-ATP labeling of unfixed, permeabilized mouse hair cells

ATP-utilizing enzymes play key roles in hair bundles, the mechanically sensitive organelles of sensory hair cells in the inner ear. We used a fluorescent ATP analog, EDA-ATP-Cy3 (Cy3-ATP), to label ATP-binding proteins in two different preparations of unfixed hair-cell stereocilia of the mouse. In the first preparation, we lightly permeabilized dissected cochleas, then labeled them with Cy3-ATP. Hair cells and their stereocilia remained intact, and stereocilia tips in rows 1 and 2 were labeled particularly strongly with Cy3-ATP. In many cases, vanadate (Vi) traps nucleotides at the active site of myosin isoforms and presents nucleotide dissociation. Co-application with Vi enhanced the tip labeling, which is consistent with myosin isoforms being responsible. By contrast, the actin polymerization inhibitors latrunculin A and cytochalasin D had no effect, suggesting that actin turnover at stereocilia tips was not involved. Cy3-ATP labeling was substantially reduced—but did not disappear altogether—in mutant cochleas lacking MYO15A; by contrast, labeling remained robust in cochleas lacking MYO7A. In the second preparation, used to quantify Cy3-ATP labeling, we labeled vestibular stereocilia that had been adsorbed to glass, which demonstrated that tip labeling was higher in longer stereocilia. We found that tip signal was reduced by ~ 50% in Myo15ash2/sh2 stereocilia as compared to Myo15ash2/+stereocilia. These results suggest that MYO15A accounts for a substantial fraction of the Cy3-ATP tip labeling in vestibular hair cells, and so this novel preparation could be utilized to examine the control of MYO15A ATPase activity in situ.

A Novel High-Throughput Screening Tool in Saccharomyces Cerevisiae

<p>The elucidation of drug targets and their biological effects can be aided by the identification of yeast deletion mutants that confer hypersensitivity to the drug. However, the biological activities of some compounds are reduced by the mechanisms of the pleotropic drug resistance (PDR) network. For this reason a PDR-deficient strain with deletions in the master transcriptional regulators of the PDR network, PDR1 and PDR3 was created. This double deletion mutant strain was robotically mass mated against the non-essential deletion mutant array (DMA) to create genome wide nonessential PDR-deficient DMA (PD-DMA). No phenotypic enhancements were observed with Δpdr1Δpdr3 double mutant and the various deletion strains of the DMA. Increased genome-wide sensitivity of the PDR-deficient mutants was demonstrated by screening pools of PD-DMA and the DMA against cycloheximide, a Pdr5p substrate and rapamycin which is not. Similar sensitivities were observed for the non-PDR substrate rapamycin for the two deletion mutant pools while sensitivity to Pdr5p substrate cycloheximide was significantly more sensitive in the PD-DMA. The genome-wide increased sensitivity of the PDR-deficient mutants was further assessed by screening the LOPAC library of pharmacologically active compounds against pools of PD-DMA and the wild-type background DMA. The DMA screen identified 5 of 1280 compounds having bioactivity whilst the PD-DMA screen identified 25 compounds including the 5 identified by the DMA. The 20 additional compounds identified in the PDR-deficient background were inactive at the concentrations used in the wild-type background. The PD-DMA was then used in chemical genetic profiling assays; namely solid-phase chemical genetic profiling and DNA barcode microarray experiments. The PD-DMA was screened against natural products, rapamycin and cycloheximide with well characterized chemical genetic profiles. The PDR-deficient strains hypersensitivity to rapamycin and cycloheximide were indicative of TORC1 pathway inhibition and translational elongation inhibition, respectively. This was consistent with literature as rapamycin is an inhibitor of TORC1 and it mimics nutrient starvation response and cycloheximide inhibits eukaryotic translational elongation. These results validated the utility of PD-DMA as a hypersensitive genome-wide deletion reagent for chemical genetic profiling. Following validation of the PD-DMA, biochemical assays were performed on latrunculin-A, a less well characterised marine natural product and Plakortide-T a marine natural product with novel activity. These inhibitory drugs were shown to be PDR substrates with biological activity at low nanomolar concentrations. Latrunculin-A, was ~28 fold more potent in the PDR-deficient strain. In contrast, plakortide-T was biologically active only in the PDR-deficient background and the PDR mediated efflux did not involve the major efflux transporters. DNA barcode microarray experiments performed with latrunculin-A identified several hypersensitive deletion mutants consistent with cytoskeletal disruption specifically, actin microfilament assembly. Latrunculin-A is known to bind monomeric G-actin and inhibit actin polymerisation. However, in novel findings tubulin cytoskeleton disassembly was also shown to be mediated by latrunculin-A. Plakortide-T belongs to a class of compounds that disrupts calcium homeostasis. DNA barcode microarray experiments performed identified 56 deletion mutants hypersensitive to Plakortide-T, however, none were involved in calcium homeostasis and the deletion mutants were not over represented in any of the GO terms. Plakortide-T caused hypersensitivity in several deletion mutants of genes encoding for mitochondrial proteins. This activity however, did not generate reactive oxygen species as increased oxidation of free thiols or induction of the oxidative stress response was not observed. Plakortide-T was shown to induce an increase in cytosolic calcium detected by the nuclear localisation of Crz1p, a transcription factor activated in response to increased cytosolic calcium. This activation was dependent on functional calcineurin which further validates this response is an increase in cytosolic calcium.</p>

A Novel High-Throughput Screening Tool in Saccharomyces Cerevisiae

<p>The elucidation of drug targets and their biological effects can be aided by the identification of yeast deletion mutants that confer hypersensitivity to the drug. However, the biological activities of some compounds are reduced by the mechanisms of the pleotropic drug resistance (PDR) network. For this reason a PDR-deficient strain with deletions in the master transcriptional regulators of the PDR network, PDR1 and PDR3 was created. This double deletion mutant strain was robotically mass mated against the non-essential deletion mutant array (DMA) to create genome wide nonessential PDR-deficient DMA (PD-DMA). No phenotypic enhancements were observed with Δpdr1Δpdr3 double mutant and the various deletion strains of the DMA. Increased genome-wide sensitivity of the PDR-deficient mutants was demonstrated by screening pools of PD-DMA and the DMA against cycloheximide, a Pdr5p substrate and rapamycin which is not. Similar sensitivities were observed for the non-PDR substrate rapamycin for the two deletion mutant pools while sensitivity to Pdr5p substrate cycloheximide was significantly more sensitive in the PD-DMA. The genome-wide increased sensitivity of the PDR-deficient mutants was further assessed by screening the LOPAC library of pharmacologically active compounds against pools of PD-DMA and the wild-type background DMA. The DMA screen identified 5 of 1280 compounds having bioactivity whilst the PD-DMA screen identified 25 compounds including the 5 identified by the DMA. The 20 additional compounds identified in the PDR-deficient background were inactive at the concentrations used in the wild-type background. The PD-DMA was then used in chemical genetic profiling assays; namely solid-phase chemical genetic profiling and DNA barcode microarray experiments. The PD-DMA was screened against natural products, rapamycin and cycloheximide with well characterized chemical genetic profiles. The PDR-deficient strains hypersensitivity to rapamycin and cycloheximide were indicative of TORC1 pathway inhibition and translational elongation inhibition, respectively. This was consistent with literature as rapamycin is an inhibitor of TORC1 and it mimics nutrient starvation response and cycloheximide inhibits eukaryotic translational elongation. These results validated the utility of PD-DMA as a hypersensitive genome-wide deletion reagent for chemical genetic profiling. Following validation of the PD-DMA, biochemical assays were performed on latrunculin-A, a less well characterised marine natural product and Plakortide-T a marine natural product with novel activity. These inhibitory drugs were shown to be PDR substrates with biological activity at low nanomolar concentrations. Latrunculin-A, was ~28 fold more potent in the PDR-deficient strain. In contrast, plakortide-T was biologically active only in the PDR-deficient background and the PDR mediated efflux did not involve the major efflux transporters. DNA barcode microarray experiments performed with latrunculin-A identified several hypersensitive deletion mutants consistent with cytoskeletal disruption specifically, actin microfilament assembly. Latrunculin-A is known to bind monomeric G-actin and inhibit actin polymerisation. However, in novel findings tubulin cytoskeleton disassembly was also shown to be mediated by latrunculin-A. Plakortide-T belongs to a class of compounds that disrupts calcium homeostasis. DNA barcode microarray experiments performed identified 56 deletion mutants hypersensitive to Plakortide-T, however, none were involved in calcium homeostasis and the deletion mutants were not over represented in any of the GO terms. Plakortide-T caused hypersensitivity in several deletion mutants of genes encoding for mitochondrial proteins. This activity however, did not generate reactive oxygen species as increased oxidation of free thiols or induction of the oxidative stress response was not observed. Plakortide-T was shown to induce an increase in cytosolic calcium detected by the nuclear localisation of Crz1p, a transcription factor activated in response to increased cytosolic calcium. This activation was dependent on functional calcineurin which further validates this response is an increase in cytosolic calcium.</p>

Cy3-ATP Labeling of Unfixed, Permeabilized Hair Cells

ATP-utilizing enzymes play key roles in hair bundles, the mechanically sensitive organelles of sensory hair cells in the inner ear. We used a fluorescent ATP analog, EDA-ATP-Cy3 (Cy3-ATP), to label ATP-binding proteins in two different preparations of unfixed hair-cell stereocilia. In the first preparation, we lightly permeabilized dissected cochleas, then labeled them with Cy3-ATP. Hair cells and their stereocilia remained intact, and stereocilia tips in rows 1 and 2 were labeled particularly strongly with Cy3-ATP. Co-application with vanadate (VO43-) enhanced the tip labeling, which is consistent with myosin isoforms being responsible; by contrast, the actin polymerization inhibitors latrunculin A and cytochalasin D had no effect, suggesting that actin turnover at stereocilia tips was not involved. Cy3-ATP labeling was substantially reduced—but did not disappear altogether—in mutant cochleas lacking MYO15A; by contrast, labeling remained robust in cochleas lacking MYO7A. In the second preparation, used to quantify Cy3-ATP labeling, we labeled vestibular stereocilia that had been adsorbed to glass, which demonstrated that tip labeling was higher in longer stereocilia. We found that tip signal was reduced by ~50% in Myo15ash2/sh2 stereocilia as compared to Myo15ash2/+ stereocilia of the same length range. These results suggest that MYO15A accounts for a substantial fraction of the Cy3-ATP tip labeling in vestibular hair cells, and so this novel preparation could be utilized to examine the control of MYO15A ATPase activity in situ.

Measuring expression heterogeneity of single-cell cytoskeletal protein complexes

Multimeric cytoskeletal protein complexes orchestrate normal cellular function. However, protein-complex distributions in stressed, heterogeneous cell populations remain unknown. Cell staining and proximity-based methods have limited selectivity and/or sensitivity for endogenous multimeric protein-complex quantification from single cells. We introduce micro-arrayed, differential detergent fractionation to simultaneously detect protein complexes in hundreds of individual cells. Fractionation occurs by 60 s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Proteins are measured with a ~5-hour immunoassay. Co-detection of cytoskeletal protein complexes in U2OS cells treated with filamentous actin (F-actin) destabilizing Latrunculin A detects a unique subpopulation (~2%) exhibiting downregulated F-actin, but upregulated microtubules. Thus, some cells may upregulate other cytoskeletal complexes to counteract the stress of Latrunculin A treatment. We also sought to understand the effect of non-chemical stress on cellular heterogeneity of F-actin. We find heat shock may dysregulate filamentous and globular actin correlation. In this work, our assay overcomes selectivity limitations to biochemically quantify single-cell protein complexes perturbed with diverse stimuli.

Rab11FIP1 maintains Rab35 at the intercellular bridge to promote actin removal and abscission

Cytokinesis occurs at the end of mitosis/meiosis wherein the cytoplasms of daughter cells are separated. Prior to abscission, an intercellular bridge containing the remaining furrowing machinery, mitotic spindle and actin cytoskeleton connects the two daughter cells. To remove this actin and allow for separation of daughter cells, Rab35 vesicles, loaded with the actin oxidizer MICAL1 and the inositol polyphosphate 5-phosphatase OCRL are recruited to the midbody in a fine-tuned spatiotemporal manner. Importantly however, the means by which these vesicles are recruited is currently unclear. Here, we demonstrate that Rab11FIP1 is recruited to the midbody after Rab35 to scaffold it at the bridge and maintain Rab35 in this region. In the absence of Rab11FIP1, Rab35 dramatically drops from the midbody, inducing defects such as cytokinetic delays and binucleation due to actin overaccumulation at the intercellular bridge, defects which can be rescued with Latrunculin A treatment. Importantly, we show that Rab11FIP1 is critical for Rab35 function in actin removal prior to cytokinesis.

β-Adrenergic control of sarcolemmal CaV1.2 abundance by small GTPase Rab proteins

The number and activity of Cav1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca2+-induced Ca2+ release and myocardial contraction. β-Adrenergic receptor (βAR) activation stimulates sarcolemmal insertion of CaV1.2. This supplements the preexisting sarcolemmal CaV1.2 population, forming large “superclusters” wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca2+ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early and recycling endosome-localized, presynthesized CaV1.2 channels. βAR-activation decreased CaV1.2/endosome colocalization in ventricular myocytes, as it triggered “emptying” of endosomal CaV1.2 cargo into the t-tubule sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that CaV1.2 are often inserted into the sarcolemma as preformed, multichannel clusters. Similarly, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by coexpression of constitutively active Rab4a, halved by coexpression of dominant-negative Rab11a, and abolished by coexpression of dominant-negative mutant Rab4a. In ventricular myocytes, βAR-stimulated recycling of CaV1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented βAR-activated Ca2+ current augmentation. Moreover, βAR-regulation of CaV1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that βAR-stimulation triggers an on-demand boost in sarcolemmal CaV1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for βAR-regulation of cardiac CaV1.2.

β-adrenergic control of sarcolemmal CaV1.2 abundance by small GTPase Rab proteins

The number and activity of Cav1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca2+-induced Ca2+ release and myocardial contraction. β-adrenergic receptor (βAR) activation stimulates sarcolemmal insertion of CaV1.2 channels. This supplements the pre-existing sarcolemmal CaV1.2 population, forming large ‘super-clusters’ wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca2+ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early- and recycling endosome-localized, pre-synthesized CaV1.2 channels. βAR-activation decreased CaV1.2/endosome colocalization in ventricular myocytes, as it triggered ‘emptying’ of endosomal CaV1.2 cargo into the sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that CaV1.2 are often inserted into the sarcolemma as pre-formed, multi-channel clusters. Likewise, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by co-expression of constitutively-active Rab4a, halved by co-expression of dominant-negative Rab11a, and abolished by co-expression of dominant-negative mutant Rab4a. In ventricular myocytes, βAR-stimulated recycling of CaV1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented βAR-activated Ca2+ current augmentation. Moreover, βAR-regulation of CaV1.2 was abolished when recycling was halted by co-application of nocodazole and latrunculin-A. These findings reveal that βAR-stimulation triggers an on-demand boost in sarcolemmal CaV1.2 abundance via targeted, Rab4a and Rab11a-dependent insertion of CaV1.2 channels is essential for βAR-regulation of cardiac CaV1.2.Significance StatementThe L-type voltage-gated Ca2+ channel CaV1.2 is essential for excitation-contraction coupling in the heart. During the fight-or-flight response, CaV1.2 channel activity is augmented as a result of PKA-mediated phosphorylation, downstream of β-adrenergic receptor (βAR) activation. We discovered that enhanced sarcolemmal abundance of CaV1.2 channels, driven by stimulated insertion/recycling of specific CaV1.2 containing endosomes, is essential for βAR-mediated regulation of these channels in the heart. These data reveal a new conceptual framework of this critical and robust pathway for on-demand tuning of cardiac EC-coupling during fight-or-flight.

Smooth Muscle α-Actin Expression in Mitral Valve Interstitial Cells is Important for Mediating Extracellular Matrix Remodeling

Background: Mitral valve prolapse (MVP) affects 3–6% of the total population including those with connective tissue disorders. Treatment is limited, and patients commonly require surgery which can be impermanent and insuperable. Abnormal prolapse of mitral valve leaflets into the left atria is caused by disturbances to the composition and organization of the extracellular matrix (ECM), that weaken biomechanics. This process, known as myxomatous degeneration is characterized by an abnormal accumulation of proteoglycans, in addition to collagen fiber disruption and elastic fiber fragmentation. The underlying mechanisms that promote myxomatous degeneration to the point of biomechanical failure are unknown, but previous histological studies of end-stage diseased tissue have reported abnormal α-smooth muscle actin (SMA) in a subset of heart valve interstitial cells (VICs); however, the contribution of these abnormal cells to MVP pathogenesis has not been extensively examined. Methods: In vivo and in vitro approaches were used. Mice harboring a Fbn1C1039G mutation mimic human Marfan Syndrome and develop MVP. Using these mice, temporal and spatial changes in SMA expression relative to myxomatous degeneration were examined using histological techniques. In parallel in vitro experiments, SMA expression was downregulated in primary porcine mitral VICs directly using siRNA, and indirectly using the actin depolymerizing agent Latrunculin A. In addition, the regulation of SMA in VICs by mechanical stiffness was explored relative to ECM remodeling. Results: We show, in mitral valves from Fbn1C1039G/+ mice, that abnormal increases in SMA expression in VICs are evident during early postnatal stages of disease, prior to significant myxomatous degeneration as indicated at later stages by increased proteoglycans and collagen type I (Col1a1). Furthermore, abnormal SMA expression continues to increase during the course of pathogenesis and is localized to the mid belly region of the mitral valve leaflets from 10 weeks. Using an in vitro approach, we demonstrate that reduced SMA function by direct siRNA or indirect Latrunculin A treatment attenuates proteoglycan and Col1a1 expression in porcine mitral VICs. While upstream, we provide insights to show that SMA is regulated by mechanical tension in VICs to promote changes in ECM homeostasis. Conclusions: Together, our data show that in VICs, SMA, an actin binding protein, is important for mediating ECM remodeling associated with phenotypes observed in myxomatous degeneration, and its expression is regulated by mechanical tension. These novel insights could inform the development of future non-surgical therapeutics to halt the progression of mitral valve degeneration thereby avoiding end-stage prolapse.