A high-speed microscopy approach to single-molecule studies of eukaryote signal transduction

Metabolic processes underlie all forms of life. An organism’s ability to utilise chemical energy to stay alive and eventually reproduce is a central feature of life, regardless of organism length scale. To achieve this, an organism must be adaptable. That is, it must be able to adjust to varying surrounding environmental conditions. Cells must be able to sense this environment, ‘transduce’ the signal and bring about some cell level response. Cells respond to external stimuli by releasing chemical cascades along often intricate signalling pathways which regulate cellular function. In this thesis, I have developed a novel optical microscopy system coupled to microfluidics and image analysis tools to help address challenging biological questions relating to metabolic sensing in eukaryotic life, using Saccharomyces cerevisiæ, as a model system. A set of biophysical tools was developed to monitor signal transduction events in live yeast cells. A bespoke optical microscope was developed that can monitor single living cells and determine their response to controlled variations in environmental nutrient concentration at high sampling speeds comparable to the molecular diffusion time scale in a cells internal environment. High-speed imaging at up to 200 frames per second and exposure times of 4.7 ms can be achieved. An electronic gain of 300x makes the camera system sensitive enough to track diffusion of single or small clusters of fluorescent protein molecules under physiological conditions. A high intensity laser excitation system was developed to deliver the light required to follow single fluorescent proteins in the living cells. A bespoke microfluidics system was built wherein cells can be exposed to rapidly changing extracellular environments and make it possible to follow individual cell responses to changing glucose conditions. Image analysis tools were adapted and developed to facilitate the automated measurement of protein mobility, stoichiometry and copy number, one cell at a time

A high-speed microscopy approach to single-molecule studies of eukaryote signal transduction

Metabolic processes underlie all forms of life. An organism’s ability to utilise chemical energy to stay alive and eventually reproduce is a central feature of life, regardless of organism length scale. To achieve this, an organism must be adaptable. That is, it must be able to adjust to varying surrounding environmental conditions. Cells must be able to sense this environment, ‘transduce’ the signal and bring about some cell level response. Cells respond to external stimuli by releasing chemical cascades along often intricate signalling pathways which regulate cellular function. In this thesis, I have developed a novel optical microscopy system coupled to microfluidics and image analysis tools to help address challenging biological questions relating to metabolic sensing in eukaryotic life, using Saccharomyces cerevisiæ, as a model system. A set of biophysical tools was developed to monitor signal transduction events in live yeast cells. A bespoke optical microscope was developed that can monitor single living cells and determine their response to controlled variations in environmental nutrient concentration at high sampling speeds comparable to the molecular diffusion time scale in a cells internal environment. High-speed imaging at up to 200 frames per second and exposure times of 4.7 ms can be achieved. An electronic gain of 300x makes the camera system sensitive enough to track diffusion of single or small clusters of fluorescent protein molecules under physiological conditions. A high intensity laser excitation system was developed to deliver the light required to follow single fluorescent proteins in the living cells. A bespoke microfluidics system was built wherein cells can be exposed to rapidly changing extracellular environments and make it possible to follow individual cell responses to changing glucose conditions. Image analysis tools were adapted and developed to facilitate the automated measurement of protein mobility, stoichiometry and copy number, one cell at a time

From Animaculum to single-molecules : 300 years of the light microscope

Although not laying claim to being the inventor of the light microscope, Antonj van Leeuwenhoek (1632-1723) was arguably the first person to bring this new technological wonder of the age properly to the attention of natural scientists interested in the study of living things (people we might now term 'biologists'). He was a Dutch draper with no formal scientific training. From using magnifying glasses to observe threads in cloth, he went on to develop over 500 simple single lens microscopes (Baker & Leeuwenhoek 1739 Phil. Trans. 41, 503-519. (doi:10.1098/rstl.1739.0085)) which he used to observe many different biological samples. He communicated his finding to the Royal Society in a series of letters (Leeuwenhoek 1800 The select works of Antony Van Leeuwenhoek, containing his microscopical discoveries in many of the works of nature, vol. 1) including the one republished in this edition of Open Biology. Our review here begins with the work of van Leeuwenhoek before summarizing the key developments over the last ca 300 years, which has seen the light microscope evolve from a simple single lens device of van Leeuwenhoek's day into an instrument capable of observing the dynamics of single biological molecules inside living cells, and to tracking every cell nucleus in the development of whole embryos and plants

Coupling of alpha(1)-Adrenoceptors to ERK1/2 in the Human Prostate

Introduction: alpha(1)-Adrenoceptors are considered critical for the regulation of prostatic smooth muscle tone. However, previous studies suggested further alpha(1)-adrenoceptor functions besides contraction. Here, we investigated whether alpha(1)-adrenoceptors in the human prostate may activate extracellular signal-regulated kinases (ERK1/2). Methods: Prostate tissues from patients undergoing radical prostatectomy were stimulated in vitro. Activation of ERK1/2 was assessed by Western blot analysis. Expression of ERK1/2 was studied by immunohistochemistry. The effect of ERK1/2 inhibition by U0126 on phenylephrine-induced contraction was studied in organ-bath experiments. Results: Stimulation of human prostate tissue with noradrenaline (30 mu M) or phenylephrine (10 mu M) resulted in ERK activation. This was reflected by increased levels of phosphorylated ERK1/2. Expression of ERK1/2 in the prostate was observed in smooth muscle cells. Incubation of prostate tissue with U0126 (30 mu M) resulted in ERK1/2 inhibition. Dose-dependent phenylephrine-induced contraction of prostate tissue was not modulated by U0126. Conclusions: alpha(1)-Adrenoceptors in the human prostate are coupled to ERK1/2. This may partially explain previous observations suggesting a role of alpha(1)-adrenoceptors in the regulation of prostate growth. Copyright (C) 2011 S. Karger AG, Base

Localization and Function of Cannabinoid Receptors in the Corpus Cavernosum: Basis for Modulation of Nitric Oxide Synthase Nerve Activity.

BACKGROUND: Anandamide, a proposed endogenous cannabinoid (CB) agonist, has been shown to enhance neurogenic responses in vitro of the rat corpus cavernosal tissue (CC). However, no information is available on the distribution of CB-receptors or effects by anandamide in CC from primates or humans. OBJECTIVE: To characterize the distribution of CB-receptor isoforms in the human and primate CC and to investigate the effects of anandamide on isolated CC preparations. DESIGN, SETTING, AND PARTICIPANTS: CC tissue was excised from the crura penis of six rhesus monkeys and five patients. Expression and distribution CB1 and CB2 receptors were characterized with Western blot analyses and immunohistochemical investigations. The effects of anandamide on isolated CC preparations were analyzed during pharmacologic and nerve-mediated activation of primate tissue in aerated organ baths. MEASUREMENTS: The expression and localization of CB1 and CB2 receptors in the primate CC and effects of anandamide on nerve-mediated relaxations and pharmacologically evoked contractions. RESULTS AND LIMITATIONS: Western blot experiments revealed CB1 and CB2 receptors at expected band weights. Within and between strands of CC smooth muscle, CB1 and CB2 immunoreactivity (IR) was found in nerve fibers that also expressed IR for nitric oxide synthase (NOS) or transient receptor potential V1 (TRPV1). Neither CB1-IR nor CB2-IR nerves were colocalized with calcitonin-gene-related peptide (CGRP)-containing or tyrosine hydroxylase-containing nerves. No differences were observed between primate and human CC sections. Anandamide (10(-9) to 10(-4) M) had no contractile effects on CC smooth muscle, no relaxant effects on precontracted preparations, and no effect on phenylephrine-induced contractions. However, anandamide (10muM) inhibited electrically evoked smooth-muscle relaxations (34-48%; p</=0.05). CONCLUSIONS: CB1 and CB2 receptors are located on NOS-containing nerves in primate and human CC tissue. In contrast to findings in rats, anandamide antagonized nerve-mediated relaxations of the primate CC, suggesting important species differences for CB-mediated functions. The results also suggest a peripheral mechanism for cannabis-related sexual dysfunction

Update on phosphodiesterase (PDE) isoenzymes as pharmacologic targets in urology: Present and future

Objectives & Methods: Diseases of the human urinary tract represent common morbidities characterized by a high prevalence in the population of most westernized countries. The existeince of a significant number of affected patients and the recent increase in scientific attention has resulted in various experimental and clinical efforts in order to evaluate the mechanisms controlling the function of urinary tract organs. This review attempts to describe the physiology and pharmacology of phosphodiesterase (PDE) isoenzymes with special regard to their (potential) use in disorders of the human urogenital tract. Results: The promising clinical data for the orally active phosphodiesterase (PDE) inhibitors sildenafil, vardenafil and tadalafil, used in the treatment of male erectile dysfunction (MED), has boosted research activities on the significance of the cyclic GMP- and cyclic AMP pathway in other genitourinary tract tissues, such as the bladder, prostate, ureter, urethra, as well as female genital tissues. Based on the more extensive understanding of the pathways controlling the function of the male and female urogenital tract, orally administered phosphodiesterase inhibitors are considered a logical and straightforward approach for treating urological diseases. Due to the unending charge to conceive advanced first-line treatments, new therapeutic options taking into consideration the cyclic nucleotide signaling have been introduced or might be launched in the near future. Upcoming strategies will not only focus on the nitric oxide (No)/cGMP cascade but also on compounds modulating signal transduction mediated by cyclic adenosine monophosphate, as well as combined agents in order to affect multiple peripheral intracellular targets. Conclusions: The article highlights cGMP- and cAMP-pathways, PDE subtypes and their present or putative future clinical significance in urological practice. (c) 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved

Distribution and Function of Cannabinoid Receptors 1 and 2 in the Rat, Monkey and Human Bladder

Purpose: We investigated the distribution of cannabinoid receptor subtypes 1 and 2 in the detrusor of different species and studied the effects of cannabinoid receptor 1 and 2 agonists on bladder function. Materials and Methods: Cannabinoid receptor 1 and 2 expression was studied with Western blot and immunohistochemistry in rat, monkey and human detrusors. Co-staining was done for markers of sensory nerves using calcitonin gene-related peptide (Euro-Diagnostica, Malmo, Sweden) and transient receptor potential vanilloid 1, and for cholinergic nerves using VAChT (Santa Cruz Biotechnology, Santa Cruz, California). Actions of the endogenous cannabinoid receptor-1 and 2 agonist anandamide (Sigma (R)), and the cannabinoid receptor 1 and 2 agonist CP55,940 (Sigma) on isolated detrusor and during cystometry in conscious rats were recorded. Results: Higher expression of cannabinoid receptor 2 but not cannabinoid receptor 1 was noted in the mucosa than in the detrusor. Compared to the detrusor larger amounts of cannabinoid receptor 2 containing nerves that also expressed transient receptor potential vanilloid 1 or calcitonin gene-related peptide were observed in the suburothelium. Nerve fibers containing cannabinoid receptor 2 and VAChT were located in the detrusor. Neither anandamide nor CP55,940 affected isolated detrusor carbachol (Sigma) contractions. Nerve contractions were enhanced by 10 mu M anandamide and decreased by 10 AM CP55,940 (P<0.05). In vivo CP55,940 increased the micturition interval by 46% and threshold pressure by 124% (p <0.05). Anandamide increased threshold pressure by 26% and decreased the micturition interval by 19% (p <0.05 and <0.01, respectively). Conclusions: The distribution of cannabinoid receptor 2 on sensory nerves and in the urothelium, and effects by CP55940 on the micturition interval and threshold pressure suggest a role for cannabinoid receptor 2 in bladder afferent signals. Co-expression of VAChT and cannabinoid receptor 2, and effects by CP55940 on nerve contractions suggest a cannabinoid receptor 2 mediated modulatory effect on cholinergic nerve activity. Anandamide may not be a good tool for cannabinoid receptor studies due to its activity at other receptors

Dysregulation of cGMP-dependent protein kinase 1 (PKG-1) impairs erectile function in diabetic rats: influence of in vivo gene therapy of PKG1 alpha

To investigate the expression of cGMP-dependent protein kinase 1 (PKG1)alpha and PKG1 beta in the corpus cavernosum, and to evaluate the effect of adenoviral gene transfer of PKG1 alpha to the erectile compartment on erectile function in a rat model of diabetes. Diabetic (DM; induced by streptozotocin) male Sprague Dawley rats were transfected with adenoviruses (AdCMV beta gal or AdCMVPKG1 alpha, in 10 rats each) 2 months after the induction of DM. Intracavernosal pressure (ICP) during stimulation of the cavernosal nerve (CN) was assessed, and compared with mean arterial pressure (MAP). Erectile tissue was harvested for Western blot analysis, immunohistochemistry and total PKG activity. Ten age-matched rats without DM served as the control. Compared to controls, AdCMV beta gal-transfected DM rats had significantly lower peak ICP responses, ICP/MAP ratios, and filling rates during CN stimulation. In DM rats transfected with AdCMVPKG1 alpha, peak ICP, ICP/MAP ratios and filling rates were significantly better than in DM rats transfected with the reporter gene. As assessed by Western blot and immunohistochemistry, expression of PKG1 alpha and PKG1 beta was lower in corporal tissue from DM AdCMV beta gal-transfected rats than in controls. PKG1 alpha expression was improved after AdCMVPKG1 alpha gene therapy. Total PKG activity was lower in DM rat corporal tissue than in controls, and PKG1 alpha gene transfer significantly improved DM corporal PKG activity to a value greater than in the control. PKG1 alpha and PKG1 beta activities are reduced in the erectile tissue of the diabetic rat, and gene transfer of PKG1 alpha to the penis restored PKG activity and erectile function in vivo in diabetic rats. Gene therapy procedures targeting PKG1 alpha might be an interesting future therapeutic approach to overcome diabetic erectile dysfunction resistant to oral pharmacotherapy

Transient Receptor Potential A1 and Cannabinoid Receptor Activity in Human Normal and Hyperplastic Prostate: Relation to Nerves and Interstitial Cells.

BACKGROUND: Ion channel transient receptor potential A1 (TRPA1) and cannabinoid (CB) receptors are involved in mechanoafferent signaling from the bladder and the urethra. OBJECTIVE: To characterize TRPA1-, CB1-, and CB2-receptor activities in the human prostate. DESIGN, SETTING, AND PARTICIPANTS: Prostate specimens were obtained from 12 patients undergoing radical prostatectomy. We studied expressions (n=6) of TRPA1, CB1, and CB2 receptors and effects of the TRPA1 agonists allyl isothiocyanate (AI), cinnamaldehyde (CA), sodium hydrogen sulfide (NaHS), and CP 55940 (a CB1/CB2 agonist) on prostatic preparations. MEASUREMENTS: Western blot, immunohistochemistry, and functional experiments were performed. RESULTS AND LIMITATIONS: Western blot detected expected bands for CB1, CB2, and TRPA1. TRPA1 immunoreactivity was located on nerves that were positive for CB1, CB2, calcitonin gene-related peptide (CGRP), nitric oxide synthase (NOS), or vesicular acetylcholine transporter (VAChT). CB1 and CB2 immunoreactivity was found on nerves that were positive for NOS, VAChT, or CGRP. Adrenergic nerves were not immunoreactive for TRPA1, CB1, or CB2. In nodular hyperplasia, nerves containing the above markers were scarce or absent. TRPA1 immunoreactivity was detected in cyclic guanosine monophosphate-positive basal cells of the glandular epithelium. Basal or subepithelial TRPA1-immunoreactive cells contained vimentin and c-kit immunoreactivity. CA and NaHS relaxed precontracted preparations by 55+/-7% and 35+/-3% (n=6 for each). CP 55940, NaHS, AI, capsaicin, and CA decreased nerve contractions up to 27%, 80%, 47%, and 87%, respectively (n=6 for each). CONCLUSIONS: The distribution and function of TRPA1 and CB receptors in prostatic tissue suggest a role for these receptors in mechanoafferent signals, epithelial homeostasis, emission, or inflammation of the human prostate