This protocol describes a methodology to assess the function of mechanically activated ion channels in native urothelial cells using the fluorescent Ca2+ sensor GCaMP5G.
Mechanically activated ion channels are biological transducers that convert mechanical stimuli such as stretch or shear forces into electrical and biochemical signals. In mammals, mechanically activated channels are essential for the detection of external and internal stimuli in processes as diverse as touch sensation, hearing, red blood cell volume regulation, basal blood pressure regulation, and the sensation of urinary bladder fullness. While the function of mechanically activated ion channels has been extensively studied in the in vitro setting using the patch-clamp technique, assessing their function in their native environment remains a difficult task, often because of limited access to the sites of expression of these channels (e.g., afferent terminals, Merkel cells, baroreceptors, and kidney tubules) or difficulties applying the patch-clamp technique (e.g., the apical surfaces of urothelial umbrella cells). This protocol describes a procedure to assess mechanically evoked Ca2+ transients using the fluorescent sensor GCaMP5G in an ex vivo urothelial preparation, a technique that could be readily adapted for the study of mechanically evoked Ca2+ events in other native tissue preparations.
Epithelial cells in the urinary tract are subjected to mechanical forces as the urinary filtrate travels through the nephrons, and urine is pumped out of the renal pelvis and travels through the ureters to be stored in the urinary bladder. It has been long recognized that mechanical forces (e.g., shear stress and stretch) exerted by fluids on the epithelial cells that line the urinary tract regulate the reabsorption of protein in the proximal tubule and of solutes in the distal nephron1,2,3,4,5,6,7,8,9,10,11,12,13, as well as the storage of urine in the urinary bladder and micturition14,15,16,17.
The conversion of mechanical stimuli into electrical and biochemical signals, a process referred to as mechanotransduction, is mediated by proteins that respond to the deformation of cellular structures or the associated extracellular matrix18,19,20,21. Mechanically activated ion channels are unique in the sense that they transition from a closed state to an open permeable state in response to changes in membrane tension, pressure, or shear stress18,19,20,21,22. In addition, Ca2+ transients can be initiated by integrin-mediated mechanotransduction or by activation of mechanoresponsive adhesion systems at cell-cell junctions23,24,25,26. Ion channel function is usually assessed with the patch-clamp technique, which involves the formation of a gigaohm seal between the cell membrane and the patch pipette27. However, cells located in deep tissue layers with a dense extracellular matrix (e.g., kidney tubules) or surrounded by a physical barrier (e.g., glycocalyx) are difficult to access with a glass micropipette. Likewise, cells embedded or that are integral parts of tissues with poor mechanical stability (e.g., the urothelium) can not be readily studied with the patch-clamp technique. Because many mechanically activated ion channels are permeable to Ca2+, an alternative approach is to assess their activity by fluorescent microscopy using a Ca2+-sensitive dye or genetically encoded calcium indicators (GECIs) such as GCaMP. Recent efforts in protein engineering have significantly increased the dynamic range, sensitivity, and response of GECIs28,29,30, and advances in genetics have allowed their expression in specific cell populations, making them ideally suited to study mechanotransduction.
The urothelium, the stratified epithelium that covers the interior of the urinary bladder, functions as a barrier, preventing the diffusion of urinary solutes into the bladder interstitium, but also functions as a transducer, sensing bladder fullness and communicating these events to the underlying nerves and musculature16. Previous studies have shown that the communication between the urothelium and underlying tissues requires the mechanically activated ion channels Piezo1 and Piezo231. To assess mechanically induced Ca2+ transients in urothelial cells, a new technique described that uses adenoviral gene transfer to express the Ca2+ sensor GCaMP5G in urothelial cells was developed. This technique employs a mucosal sheet preparation that provides easy access to the outermost umbrella cell layer and a computer-assisted system for the simultaneous mechanical stimulation of individual cells with a closed glass micropipette and recording of changes in fluorescence over time.
Care and handling of the animals were carried out in accordance with the University of Pittsburgh Institutional Animal Care and Use Committee. Female, 2-4-month-old C57Bl/6J mice were used for the present study. The mice were obtained commercially (see Table of Materials).
1. Equipment assembly and setup
2. In situ transduction and isolation of the bladder mucosa
3. Mechanical stimulation of individual urothelial cells and Ca2+ imaging
4. Data analysis
The present protocol describes a technique to assess mechanically evoked Ca2+ transients in umbrella cells using the fluorescent Ca2+ sensor GCaMP5G. Adenoviral transduction was employed to express GCaMP5G in urothelial cells due to its high efficiency and because it produces an elevated level of expression. Fluorescent images of stained cryosections from a transduced bladder are shown in Figure 2D. For these experiments, GCaMP5G expression is highest in the umbrella cell layer. A sequence of representative images captured during an experiment where an umbrella cell was poked are shown in Figure 3A. The first image in Figure 3A shows a fluorescent view of the stimulating pipette positioned on the top of an umbrella cell of a bladder mucosa expressing GCaMP5G. Mechanical stimulation of the umbrella cell expressing GCaMP5G causes a deformation (see image Figure 3A at 12.5 s), followed by a rapid increase in the fluorescence emission (see image Figure 3A at 13.5 s). The change in fluorescence was plotted as a function of time (Figure 3B). As reported previously31, poking evokes a Ca2+ response in most umbrella cells tested in bladders from control and wild-type mice transduced with GCaMP5G. Deleting Piezo1 and Piezo2 from umbrella cells reduced the peak amplitude of the Ca2+ response31. When comparing the effect of different treatments or genetic backgrounds on mechanically evoked Ca2+ responses, it is essential to standardize the experimental conditions, including transduction of the bladder, mounting of the tissue, size of the tip of the pipette used for indentation, mechanical stimulus duration and amplitude, and imaging conditions.
Figure 1: Experimental setup to record mechano-activated Ca2+ transients in urothelial cells. The rig consists of an upright microscope, fluorescent light source, excitation and emission filters, and a CMOS camera. The imaging software controls the system. A piezoelectric actuator controlled by a single channel open-loop piezo controller is used to move the poking micropipette. Glass micropipettes are mounted in a pipette holder affixed to the piezoelectric actuator. The piezoelectric actuator and associated micropipette are mounted on a micromanipulator (not shown). The piezo open-loop controller is remotely operated by an analog/digital converter and electrophysiology software. A recording protocol in the imaging software delivers a TTL signal via a digital I/O device, and this initiates the stimulation protocol in the electrophysiology software that controls the piezoelectric and, subsequently, image capture. Please click here to view a larger version of this figure.
Figure 2: Isolation and mounting of the bladder mucosa for Ca2+ imaging. (A) Mucosal sheet preparation with an attached catheter. The bladder mucosa was stripped from the underlying muscular layer with fine forceps. (B) Magnified image of the stripped bladder mucosa shown in panel (A). (C) The mucosa was pinned down with the urothelium facing up with 0.15 mm insect pins to a silicone elastomer insert. (D) Confocal immunofluorescence cross-section images of the mucosal sheet preparation stained with an antibody against GFP and a secondary donkey anti-rabbit conjugated antibody (green), rhodamine-phalloidin (red), and DAPI (blue). The bladder mucosal preparation includes part of the lamina propria (LP). Arrows indicate the location of the umbrella cell layer (Ub). Scale bars = 50 µm. (E) Photograph of the experimental setup. a, in-line heater; b, piezoelectric actuator; c, heater elements and controller; d, pipette holder and micropipette; e, mucosal sheet attached to the silicone insert in a tissue cultured dish and dish incubator. Please click here to view a larger version of this figure.
Figure 3. Recording and analysis of mechano-activated Ca2+ transients in urothelial cells. (A) Sequence of fluorescent images captured at different time points during the course of an experiment. Note that mechanical stimulation of the umbrella cell causes a deformation followed by a rapid increase in the emission of GCaMP5G (arrow). The borders of the stimulated cell (ROI) are marked in red. (B) Change in fluorescence intensity (ΔF/F) over time for eight independent mechanically stimulated cells in a bladder mucosal preparation from a control mouse. The time of indentation (12.5 s) is marked with a blue arrow. The data in (B) have been modified from Dalghi et al.31. Please click here to view a larger version of this figure.
All organisms, and seemingly most cell types, express ion channels that respond to mechanical stimuli20,33,34,35,36,37. The function of these mechanically activated channels has been predominantly assessed with the patch-clamp technique. However, due to accessibility issues, patch-clamp studies of mechanically activated ion channels have been largely restricted to dissociated cells and cell lines. Since many mechanically activated ion channels are permeable to Ca2+, we took advantage of recent advances in microscopy, biosensing, and micromanipulation to develop an imaging method to assess the function of mechanically activated channels in an ex vivo urothelial preparation. In this protocol, urothelial cells are transduced with an adenovirus encoding for GCaMP5G. Adenoviral vectors are ideally suited for studying Ca2+ signaling in the urothelium as they provide a high rate of transduction and high levels of expression of GCaMP5G in urothelial cells (Figure 2D). Under these conditions, background fluorescence is relatively low. If adenoviral transduction is used to express a genetically encoded Ca2+ sensor, special emphasis should be given to optimizing the conditions, so that only the cells of interest express the sensor. As an alternative, expression of genetically encoded Ca2+ indicators (GECIs) could be achieved by crossing mice carrying a floxed transgene encoding for the protein of interest (e.g., GCaMP5G) and mice expressing Cre recombinase under the control of a suitable promotor. This approach does not require surgical procedures and has the advantage of providing targeted expression and even levels of expression of proteins in cells expressing Cre recombinase.
The protocol described here uses an upright wide-field microscope to assess mechanically activated Ca2+ transients in umbrella cells stimulated with a closed fire-polished micropipette. The method could be adapted to study mechanotransduction in deeper layers of the urothelium or even cells in the lamina propria with an upright confocal or two-photon microscope. Using a confocal microscope may provide the necessary resolution to define subcellular changes in intracellular Ca2+ that occur in response to mechanical stimulation. This protocol employs a closed fire-polished micropipette to poke individual umbrella cells. The main advantage of using a micropipette for stimulation is that the mechanical perturbation exerted on the tissue is transitory and limited to the cell of interest and the surrounding area. Whereas cell stretching systems can be used to mechanically stimulate cells grown on PDMS membranes, inherent limitations impede the use of such devices for imaging experiments with ex vivo preparations. These limitations include challenges with mounting and imaging a mouse bladder and, given the folded nature of the bladder mucosa, difficulties in maintaining focus on the cells of interest while stretching the tissue.
The protocol to measure mechanically activated Ca2+ transients in urothelial preparations has some limitations. First, the rig includes relatively expensive components, and its assembly and setup require expertise with microscopes, micromanipulators, analog-digital converters, and relatively specialized software. Like patch-clamping, this technique requires the investigator to learn how to use a micromanipulator and make glass micropipettes for mechanical stimulation. However, unlike the patch-clamp technique, which involves the formation of a high resistance seal between the pipette and the membrane, the procedure described here to measure mechanically activated Ca2+ transients is relatively simple and only requires the investigator to position the stimulatory pipette close to the surface of the cell to be stimulated. Therefore, a trained investigator can potentially assess the response to the poking of 8-10 cells in 1 hour, which is inconceivable with patch-clamp technique.
Given the importance that mechanically activated ion channels have in normal body function and disease states, new methods are needed to study these channels in their native milieu. As described in this protocol, imaging methods can provide unique insight into how these channels sense changes in their environment and generate physiological responses. Given the accessibility of the mucosal surface of the tube- or sac-shaped organs, the method described here could be adapted to study mechanotransduction in other settings, including the gut, urogenital tract, blood vessels, etc. Thus, studying mechanical responses in the body may be broadly useful.
The authors have nothing to disclose.
This work was supported by NIH grants R01DK119183 (to G.A. and M.D.C.) and S10OD028596 (to G.A.) and by the Cell Physiology and Model Organisms Kidney Imaging Cores of the Pittsburgh Center for Kidney Research (P30DK079307).
20x Objective | Olympus | UMPlanFL N | |
24 G ¾” catheter | Medline | Suresite IV slide | |
4x Objective | Olympus | UPlanFL N | |
Analog/digital converter | Molecular Devices | Digidata 1440A | |
Anti-GFP antibody | Abcam | Ab6556 | |
Beam splitter | Chroma | T495lpxr | |
Bipolar temperature controller | Warner Instruments | TC-344B | |
CaCl2 | Fluka | 21114-1L | 1 M solution |
cellSens software | Olympus | Imaging software | |
CMOS camera | Hamamatsu | ORCA fusion | |
Donkey anti-rabbit conjugated to Alexa Fluor 488 | Jackson ImmunoResearch | 711-545-152 | |
Excel | Microsoft Corporation | ||
Filter | Chroma | ET470/40X | |
Glass capillaries Corning 8250 glass | Warner Instruments | G85150T-4 | |
Glucose | Sigma | G8270 | |
HEPES | Sigma | H4034 | |
Inline heater | Warner Instruments | SH-27B | |
KCl | Sigma | 793590 | |
Light source | Sutter Instruments | Lambda XL | |
Manifold pump tubing | Fisherbrand | 14-190-510 | ID 1.52 mm |
Manifold pump tubing | Fisherbrand | 14-190-533 | ID 2.79 mm |
MgCl2 | Sigma | M9272 | |
Mice | Jackson Lab | 664 | 2-4 months old female C57BL/6J |
Microforge | Narishige | MF-830 | |
Micromanipulator | Sutter Instruments | MP-285 | |
Microscope | Olympus | BX51W | |
Mounting media with DAPI | Invitrogen | S36964 | Slowfade Diamond Antifade with DAPI |
NaCl | Sigma | S7653 | |
pClamp software | Molecular Devices | Version 10.4 | Patch-clamp electrophysiology data acquisition and analysis software |
Peristaltic pump | Gilson | Minipuls 3 | |
Piezoelectric actuator | Thorlabs | PAS005 | |
Pipette holder | World Precision Instruments | ||
Pipette puller | Narishige | PP-830 | |
Quick exchange heated base with perfusion and adapter ring kit | Warner Instruments | QE-1 | Quick exchange platform fits 35 mm dish |
Rhodamine-phalloidin | Invitrogen | R415 | |
Sigma-Plot | Systat Software Inc | Version 14.0 | Scientific graphing and data analysis software |
Silicone elastomer | Dow | Sylgard 184 | |
Single channel open-loop piezo controller | Thorlabs | MDT694B | |
Square grid holder pad | Ted Pella | 10520 | |
Suture | AD Surgical | S-S618R13 | 6-0 Sylk |
Teflon mounting rod | Custom made | Use to mount the piezoelectric actuator in the micromanipulator | |
Tubing | Fisher Scientific | 14171129 | Tygon S3 ID 1/16 IN, OD 1/8 IN |
USB Digital I/O device | National Instruments | NI USB-6501 |