ArticlePDF Available

Investigating G Protein-Coupled Receptor Endocytosis and Trafficking by TIR-FM

Authors:

Abstract and Figures

G protein-coupled receptors (GPCRs) represent the largest and most versatile family of signaling receptors. Their actions are highly regulated, both under physiological conditions and in response to clinically relevant drugs. A key element in this regulation is control of the number of functional receptors at the cell surface. Major processes that mediate this regulation are vesicular endocytosis and exocytosis of receptors. These trafficking events involve a concerted series of steps, some of which occur on a rapid timescale similar to that of functional signaling itself. Here, we describe and discuss an optical imaging approach, based on evanescent field or total internal reflection-fluorescence microscopy (TIR-FM), to investigate receptor endocytosis and recycling at the level of discrete membrane fission and fusion events. TIR-FM facilitates the study of receptor trafficking events near the plasma membrane with much greater spatial and temporal resolution than afforded by traditional methods. TIR-FM has already provided new insight to GPCR regulation, and we believe that this method has great potential for addressing a variety of questions in GPCR biology.
Content may be subject to copyright.
Investigating GPCR endocytosis and trafficking by TIR-FM
Guillermo A. Yudowski and Mark von Zastrow
Abstract
G protein-coupled receptors (GPCRs) represent the largest and most versatile family of signaling
receptors. Their actions are highly regulated, both under physiological conditions and in response
to clinically relevant drugs. A key element in this regulation is control of the number of functional
receptors at the cell surface. Major processes that mediate this regulation are endocytosis and
recycling of receptors. These trafficking events involve a concerted series of steps, and it is
increasingly clear that some of these steps can occur on a rapid timescale comparable to functional
signaling itself. Here, we discuss and describe an optical imaging approach, based on evanescent
field or total internal reflection fluorescence microscopy (TIR-FM), to investigate receptor
endocytosis and recycling at the level of discrete membrane fission and fusion events. TIR-FM
facilitates the study of receptor trafficking events near the plasma membrane with much greater
spatial and temporal resolution than afforded by traditional methods. TIR-FM has already
provided new insight to GPCR regulation, and we believe that this method has great potential for
addressing a variety of questions in GPCR biology.
Keywords
Fluorescence microscopy; live imaging; total internal reflection; trafficking; endocytosis;
recycling; receptors
1. Introduction
Membrane trafficking of signaling receptors is critical to many aspects of animal
physiology. Rapid internalization of surface receptors is often stimulated by agonist-induced
activation of receptors, and is thought to control signaling both from the plasma membrane
and intracellular compartments(1). The functional importance of endocytic trafficking has
been well established for members of the GPCR superfamily. Many physiological responses
mediated by GPCRs, particularly in the nervous system, occur on a relatively rapid time
scale (seconds to minutes). This time scale is significantly shorter than the kinetics of most
receptor trafficking events estimated using traditional methods. It is increasingly clear that
certain GPCR trafficking events, particular those occurring in the endocytic pathway, can
occur with kinetics that are in a similar range as acute signaling. Further, there is increasing
evidence that endocytic trafficking contributes to the regulation of receptor number in
particular domains of complex cells, such as in controlling receptor number near spatially
separated chemical synapses(2). These realizations have motivated increased interest in
methods for examining GPCR trafficking with higher temporal and spatial resolution than
afforded by traditional methods. Developments in methods and reagents for live
fluorescence imaging have greatly facilitated progress in this direction.
We will focus on the application of total internal reflection-fluorescence microscopy (TIR-
FM) to study GPCR trafficking in the endocytic pathway. Reflection of light at a refractive
interface generates an evanescent field that diminishes exponentially with distance from the
interface. The evanescent field creates a shallow field of illumination, extending in practice
100 nm from the reflective surface. If this surface is a coverslip supporting dissociated
NIH Public Access
Author Manuscript
Methods Mol Biol. Author manuscript; available in PMC 2012 March 20.
Published in final edited form as:
Methods Mol Biol
. 2011 ; 756: 325–332. doi:10.1007/978-1-61779-160-4_19.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
cells in a culture preparation, the evanescent field of illumination is useful for selectively
exciting fluorescent probes located in the basal plasma membrane and extending a short
distance into the cytoplasm (Figure 1). TIR-FM facilitates observation of events occurring in
the plasma membrane, and in a shallow region of cytoplasm immediately adjacent to the
plasma membrane, with high signal-to-background ratio because fluorescent molecules
located deeper within cells or in the culture medium are not excited (3–5). Combined with
recent improvements in electronic sensor technology, such as the widespread availability of
high sensitivity CCD cameras, TIR-FM is capable of investigating membrane events
involving small numbers of receptors and with practical time resolution on the order of tens
of milliseconds. Newly developed automated systems help to maintain a steady focal plane
during acquisition, which is essential for quantitative imaging over prolonged intervals and
at physiological temperatures. This combination of technological advances, once in the
domain of only highly specialized laboratories, is increasingly available and provides a
highly useful platform with which to study surface receptor trafficking at the single cell
level.
The availability of a wide range of biologically compatible fluorescent probes, including
genetically encoded fluorescent proteins, enable molecular specificity combined with spatio-
temporal resolution that is useful for analyzing surface receptor dynamics. These tags have
been widely applied in cell biology and are extensively reviewed elsewhere (6–8). Here we
focus on imaging a pH sensitive variant of the green fluorescent protein called superecliptic
phluorin (SpH or SEP) (9, 10) fused to the amino-terminal extracellular domain of the
human beta-2 adrenergic receptor (SEP- β2AR). SEP- β2AR is highly fluorescent at the
neutral pH of the extracellular media, but its fluorescence is rapidly and reversibly quenched
in the mildly acidic environment of the endocytic and recycling pathways. This property of
SEP- β2AR facilitates the detection of discrete endocytic and exocytic events mediating
surface receptor removal and insertion.
2. Materials
2.1 Cell culture
1. HEK-293 cells passage 20–50 (ATCC:CRL-1573)
2. 35 mm disposable MatTek glass bottom dishes
(http://www.glass-bottom-dishes.com/)
3. Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM) supplemented with
10% fetal bovine serum (Sigma)
4. Lipofectamine 2000 (Invitrogen)
5. Opti-MEM imaging buffer supplemented with 20 mM HEPES (Invitrogen)
6. Poly-D-Lysine (Sigma P0899)
7. Isoproterenol (Sigma I6504 ())
2.2. Imaging equipment and settings
1. Inverted fluorescence microscope (Nikon TE2000E) with Perfect Focus and TIRF
objectives: 60x/1.45 Oil - Plan Apo TIRF; 100x/1.49 Oil - Plan Apo TIRF. Nikon
TIRF system with 440 nm, 488 nm, 514 nm, 561 nm lasers.
2. EM-CCD cameras Photometrics Quant EMCCD (www.photomet.com ) or
iXonEM+ EMCCD 897 Camera from Andor (www.andor.com)
Yudowski and von Zastrow Page 2
Methods Mol Biol. Author manuscript; available in PMC 2012 March 20.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
3. Objective and Petri dish heaters with temperature controller to maintain media
temperature at 37°C (Bioscience Tools)
4. Excitation and emission settings for TIRF: GFP = 488 nm laser excitation (2
mWatt) mCherry = 561 nm laser excitation (2–4 mWatt) 525/50 band pass, 527/21
nm and 645/24 nm dual bandpass emission filter.
5. Exposure time: continuous 100 msec. exposure for receptor recycling, camera EM
gain is set constant to obtain comparable results: X299, binning: 1×1, image:
512×512, pre-amp-gain=4.90, horizontal readout=10 vertical readout time=3.3,
temperature= 75. BitDepth=14 bits for Andor iXonEM+.
3. Methods
3.1 Cell Prepration
1. Dissolve Poly-D-Lysine in sterile water (50 μg/ml) and place 2ml overnight at
room temperature. Wash PDL with sterile water (3 washes) and dry the culture
dishes.
2. Seed HEK-293 cells onto the coated dishes.
3. Transfect with SEP- β2AR (11) construct (1 μg per dish) using lipofectamine 2000
following manufacturer protocol 72 hours prior to imaging.
4. The day of the imaging, replace incubation media 15 to 30 minutes before
experiments with Opti-MEM or a low fluorescence media and return cells to the
incubator. Remove phenol red, serum, folic acid and riboflavin and other possible
interference from the imaging media. EGFP Photostability should also taken into
account during media selection (12).
3.2 Live-cell imaging
5Start by initializing microscope, lasers, camera and temperature control devices
30 to 45 minutes before any data acquisition.
6Select the proper TIRF objective and add a small amount of immersion oil
(TYPE DF from Cargille) on the objective and fit the glass bottom dish on the
stage of the microscope and to the heating ring element. NOTE: temperature of
the imaging media must be monitored and kept constant when dishes are
imaged, changes in temperature will affect trafficking kinetics.
7First, find cells using transmission light to get them into focus minimizing
photobleaching. Second, illuminate cells in epifluorescent mode to find cells
expressing tagged receptors and then switch to TIRF illumination. Move the
laser away from the center of the optical path and continue to achieve total
internal reflection. Find the plasma membrane by adjusting the focal plane.
NOTE: Finding the exact angle for TIRF is the most critical step in this
protocol. Cells illuminated in TIRF will present sharp edges and a increased
signal to noise ratio when compared with out of TIRF or oblique illumination
(Compared fig 2A versus 2B)
8Find cells in the ideal fluorescence range for your experiments and begin data
acquisition. NOTE: TIR-FM is intrinsically very sensitive, due to the low level
of background fluorescence. We generally strive for the lowest expression level
and illumination intensity that is sufficient for later analysis. In our experiments,
using an Andor iXonEM+, we have found that an intensity signal of ~2000 (out
of a maximum of 16384 in the 14-bit readout mode) to be more than adequate.
Yudowski and von Zastrow Page 3
Methods Mol Biol. Author manuscript; available in PMC 2012 March 20.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
We recommend using a polyclonal cell line that stably expresses your receptor
of interest, to allow rapid identification of cells in a suitably range of
fluorescence intensity.
9Acquisition settings for imaging agonist-induced clustering and endocytosis of
receptors: Intermittent illumination and acquisition of100msec exposures every
3 seconds. Total time: 10 minutes.
10 Initiate data acquisition and acquire 10 to 30 frames before agonist addition.
11 Add agonist (10μM isoproterenol) with minimum disturbance to the cell either
by an automated perfusion system or by careful addition of the agonist diluted in
pre-warmed imaging media. Manual agonist addition should not be performed
directly on top of the imaging area/cells but outside of the imaging area.
12 For resolving discrete fusion events mediating SEP- β2AR recycling, cells are
exposed to the presence of 10μM isoproterenol for 10 minutes in the incubator.
This step induces receptor internalization and loads the endocytic pathway
inducing extensive recycling events that can be subsequently observed.
13 Acquisition settings for observing discrete recycling events: Continuous
illumination and acquisition of serial 100msec exposures, using the CCD in
frame-transfer readout mode. Total imaging time: 60 seconds.
14 Save acquired data. Note: careful consideration must be given to file
management and storage. Tags, metadata and thorough indexing will help future
data retrieval and analysis. See http://www.openmicroscopy.org for open source
tools to support data management.
3.3 Analysis
Data management and analysis are critical steps in live cell microscopy. Detailed discussion
of image analysis methods is beyond the present scope and is addressed elsewhere (5, 13,
14). Examples include orthogonal views of image series as kymographs, useful for visually
representing the time dependence of trafficking events (figure 2D), and intensity-versus-time
measurements to follow the dynamics of individual events (Figure 2E). Additional examples
can be found in the recent literature; e.g.,(11, 15–17). Practical image analysis has been
greatly aided by the development of computer software specifically intended for this
application. We typically use ImageJ, an excellent open source program developed by the
NIH, which is supported by additional code written by an extensive user base and is
available to the scientific community free of charge (rsbweb.nih.gov/ij/). We also
recommend Micromanager (www.micro-manager.org) for controlling the microscope and
peripheral devices during image acquisition. Micromanager is an open source program that
is remarkably powerful and flexible, so is readily adapted to a variety of microscope
systems, and it runs as an integrated plug-in linked to ImageJ.
Acknowledgments
The authors thank members of the von Zastrow laboratory and Dr. Kurt Thorn, Director of the UCSF/Nikon
Imaging Center, for valuable discussion. The work discussed was supported by research grants from the NIH
(DA023444 to G.A.Y., DA010711 to M.v.Z.).
References
1. Sorkin A, von Zastrow M. Endocytosis and signalling: intertwining molecular networks. Nat Rev
Mol Cell Biol. 2009; 10(9):609–22. [PubMed: 19696798]
Yudowski and von Zastrow Page 4
Methods Mol Biol. Author manuscript; available in PMC 2012 March 20.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
2. Shcherbakova OG, Hurt CM, Xiang Y, et al. Organization of beta-adrenoceptor signaling
compartments by sympathetic innervation of cardiac myocytes. J Cell Biol. 2007; 176(4):521–33.
[PubMed: 17296797]
3. Steyer JA, Almers W. A real-time view of life within 100 nm of the plasma membrane. Nat Rev
Mol Cell Biol. 2001; 2(4):268–75. [PubMed: 11283724]
4. Schmoranzer J, Goulian M, Axelrod D, Simon SM. Imaging Constitutive Exocytosis with Total
Internal Reflection Fluorescence Microscopy 10.1083/jcb.149.1.23. J Cell Biol. 2000; 149(1):23–
32. [PubMed: 10747084]
5. Goldman, RD.; Spector, DL. Live cell imaging: a laboratory manual. Cold Spring Harbor, N.Y:
Cold Spring Harbor Laboratory Press; 2005.
6. Lippincott-Schwartz J, Altan-Bonnet N, Patterson GH. Photobleaching and photoactivation:
following protein dynamics in living cells. Nat Cell Biol. 2003; (Suppl):S7–14. [PubMed:
14562845]
7. Miyawaki A. Innovations in the imaging of brain functions using fluorescent proteins. Neuron.
2005; 48(2):189–99. [PubMed: 16242400]
8. Giepmans BN, Adams SR, Ellisman MH, Tsien RY. The fluorescent toolbox for assessing protein
location and function. Science. 2006; 312(5771):217–24. [PubMed: 16614209]
9. Miesenbock G, De Angelis DA, Rothman JE. Visualizing secretion and synaptic transmission with
pH-sensitive green fluorescent proteins. Nature. 1998; 394(6689):192–5. [PubMed: 9671304]
10. Sankaranarayanan S, De Angelis D, Rothman JE, Ryan TA. The use of pHluorins for optical
measurements of presynaptic activity. Biophys J. 2000; 79(4):2199–208. [PubMed: 11023924]
11. Yudowski GA, Puthenveedu MA, von Zastrow M. Distinct modes of regulated receptor insertion
to the somatodendritic plasma membrane. Nat Neurosci. 2006; 9(5):622–7. [PubMed: 16604070]
12. Bogdanov AM, Bogdanova EA, Chudakov DM, Gorodnicheva TV, Lukyanov S, Lukyanov KA.
Cell culture medium affects GFP photostability: a solution. 2009; 6(12):859–60.
13. Waters JC. Accuracy and precision in quantitative fluorescence microscopy 10.1083/jcb.
200903097. J Cell Biol. 2009; 185(7):1135–48. [PubMed: 19564400]
14. BOLTE S, CORDELIÈRES FP. A guided tour into subcellular colocalization analysis in light
microscopy. Journal of Microscopy. 2006; 224(3):213–32. [PubMed: 17210054]
15. Yudowski GA, Puthenveedu MA, Leonoudakis D, et al. Real-time imaging of discrete exocytic
events mediating surface delivery of AMPA receptors. J Neurosci. 2007; 27(41):11112–21.
[PubMed: 17928453]
16. Puthenveedu MA, von Zastrow M. Cargo regulates clathrin-coated pit dynamics. Cell. 2006;
127(1):113–24. [PubMed: 17018281]
17. Pucadyil TJ, Schmid SL. Real-time visualization of dynamin-catalyzed membrane fission and
vesicle release. Cell. 2008; 135(7):1263–75. [PubMed: 19084268]
Yudowski and von Zastrow Page 5
Methods Mol Biol. Author manuscript; available in PMC 2012 March 20.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 1. Schematic view showing the main features of a TIR-FM imaging system
A standard wide field microscope is used. The evanescent illumination field is generated by
total internal reflection at the cover slip/sample interface. This requires illuminating the
cover slip with a collimated light source at the critical angle, and is achieved in a typical
“through-the-objective” system by focusing a laser beam near the edge of the back focal
plane of a high numerical aperture objective. The evanescent field generated at the reflective
interface falls off rapidly with distance, selectively exciting fluorophores located at or near
the plasma membrane. This results in a signal-to-background ratio that is substantially
higher than can be achieved in wide field imaging using standard epifluorescence
illumination, and generally higher than that obtainable using confocal fluorescence
microscopy.
Yudowski and von Zastrow Page 6
Methods Mol Biol. Author manuscript; available in PMC 2012 March 20.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 2. Examples of GPCR localization observed by TIR-FM
A. Example of SEP- β2AR –expressing HEK293 cells imaged using epifluorescence
illumination. Two adjacent cells are shown. B. TIR-FM view of the same field, showing the
distinct footprints of each cell on the cover slip. C. TIR-FM view of the same field acquired
1 min after adding agonist (1 μM isoproterenol) to the imaging bath. The region outlined by
the white square is show at higher magnification in the inset. The fluorescent spot
surrounded by the yellow circle represents a clathrin-coated pit containing SEP- β2ARs. D.
Kymograph showing SEP- β2AR dynamics in these representative cells, with increasing
time going from left to right in the image. The vertical arrow indicates the addition of
isoproterenol to the culture medium. The SEP- β2AR fluorescence intensity pattern shifts
from a diffuse appearance to defined horizontal lines, representing receptor clustering into
clathrin-coated pits. An example is indicated by the arrowhead at left. The lines disappear
shortly after endocytic scission of coated pits, as the SEP- β2AR –containing endocytic
vesicles produced by this scission event move rapidly out of the evanescent illumination
field. An example is indicated by the arrowhead at right. E. Plot of the time course of
maximum fluorescence intensity measured in the circled region indicated in panel C, called
ΔF because the value measured in an adjacent (non-clustering) region of the plasma
membrane is subtracted. Left arrow indicates the time at which isoproterenol was added,
Yudowski and von Zastrow Page 7
Methods Mol Biol. Author manuscript; available in PMC 2012 March 20.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
showing the time course of SEP- β2AR concentration in the coated pit. Right arrow
indicates the time at which the spot of SEP- β2AR fluorescence disappears from the
evanescent illumination field following endocytic scission.
Yudowski and von Zastrow Page 8
Methods Mol Biol. Author manuscript; available in PMC 2012 March 20.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
... To test this hypothesis, we utilized TIRF microscopy of live cells to investigate the endocytosis of CB1Rs at the single pit level 33 . CB1Rs tagged at the extracellular amino terminus with the pH-sensitive super-ecliptic phluorin 34,35 (SEP-CB1R) stably expressed in HEK293 cells were exposed to saturating concentrations of either the synthetic agonist WIN 55,212-2 or the endogenous eicosanoid 2-arachidonoylglycerol (2-AG), both considered to be high-efficacy agonists of G protein activation 36,37 . ...
... Image processing and analysis. Analysis of SEP-CB1, Ds-Red-CCP, mCherry-Caveolae or RFP-b-Arrestin2 endocytic events was performed as described before 33,66,67 . Briefly, raw images were first background-subtracted and flat fieldcorrected 33 . ...
... Analysis of SEP-CB1, Ds-Red-CCP, mCherry-Caveolae or RFP-b-Arrestin2 endocytic events was performed as described before 33,66,67 . Briefly, raw images were first background-subtracted and flat fieldcorrected 33 . Individual endocytic events were quantified by an observer blinded to experimental details, multiple times manually and using the particle tracking algorithm two-dimensional spot tracker 68 . ...
Article
Full-text available
G protein-coupled receptors (GPCRs) are the major transducers of external stimuli and key therapeutic targets in many pathological conditions. When activated by different ligands, one receptor can elicit multiple signalling cascades that are mediated by G proteins or β-arrestin, a process defined as functional selectivity or ligand bias. However, the dynamic mechanisms underlying β-arrestin signalling remain unknown. Here by studying the cannabinoid receptor 1 (CB1R), we identify ligand-specific endocytic dwell times, that is, the time during which receptors are clustered into clathrin pits together with β-arrestins before endocytosis, as the mechanism controlling β-arrestin signalling. Agonists inducing short endocytic dwell times produce little or no β-arrestin signalling, whereas those eliciting prolonged dwell times induce robust signalling. Remarkably, extending CB1R dwell times by preventing endocytosis substantially increased β-arrestin signalling. These studies reveal how receptor activation translates into β-arrestin signalling and identify a mechanism to control this pathway.
... We previously used total internal reflection fluorescence (TIRF) microscopy to directly visualize the exocytosis of the B2 adrenergic receptor (B2AR), the mu opioid receptor (MOR), and the GluR1-containing AMPA-type receptor in dissociated neuronal cultures with high temporal resolution (Yudowski and von Zastrow, 2011;Roman-Vendrell and Yudowski, 2015). During these studies we identified transient and persistent modes of exocytosis. ...
... Hippocampal cultures transfected with TfR-SEP were imaged by TIRF microscopy at 10 Hz in a controlled environment. To avoid imaging nonneuronal cells, transfected neurons were first morphologically identified under epifluorescence light before switching to TIRF illumination (Yudowski and von Zastrow, 2011). With TIRF microscopy, individual exocytotic events were easily observed as rapid discrete increases in fluorescence intensity (Figures 1A,B) (Supplemental Movie 1). ...
Article
Full-text available
Transmembrane proteins are continuously shuttled from the endosomal compartment to the neuronal plasma membrane by highly regulated and complex trafficking steps. These events are involved in many homeostatic and physiological processes such as neuronal growth, signaling, learning and memory among others. We have previously shown that endosomal exocytosis of the B2 adrenergic receptor (B2AR) and the GluR1-containing AMPA receptor to the neuronal plasma membrane is mediated by two different types of vesicular fusion. A rapid type of exocytosis in which receptors are delivered to the plasma membrane in a single kinetic step, and a persistent mode in which receptors remain clustered at the insertion site for a variable period of time before delivery to the cell surface. Here, by comparing the exocytosis of multiple receptors in dissociated hippocampal and striatal cultures, we show that persistent events are a general mechanism of vesicular delivery. Persistent events were only observed after 10 days in vitro, and their frequency increased with use of the calcium ionophore A23187 and with depolarization induced by KCl. Finally, we determined that vesicles producing persistent events remain at the plasma membrane, closing and reopening their fusion pore for a consecutive release of cargo in a mechanism reminiscent of synaptic kiss-and-run. These results indicate that the delivery of transmembrane receptors to the cell surface can be dynamically regulated by kiss-and-run exocytosis.
... GRIA1, TfR, and β2 adrenergic receptor) have been observed to exhibit similar kinetic profiles in neurons, while long display events were rarely seen in astrocytes and fibroblasts (Jullie et al., 2014). That said, there is also a relationship between the type of cargo and event kinetics in neurons, as for example, different GPCRs exhibited different signal shapes (Yudowski and von Zastrow, 2011). The decay kinetics of fast fusion events may represent a rapid dispersion of AMPARs on the neuronal surface after the transport organelle has collapsed into the plasma membrane. ...
... These limitations have inspired researchers to introduce alternative methods to control the temperature of samples. These include objective heaters, in which a flexible heating element is wrapped around the microscope objective to heat the air surrounding the Petri dish [10,11]. Using a similar principle, objective coolers have been introduced, in which water tubes are wrapped around the microscope objective [12][13][14]. ...
Article
Full-text available
Water jacket systems are routinely used to control the temperature of Petri dish cell culture chambers. Despite their widespread use, the thermal characteristics of such systems have not been fully investigated. In this study, we conducted a comprehensive set of theoretical, numerical and experimental analyses to investigate the thermal characteristics of Petri dish chambers under stable and transient conditions. In particular, we investigated the temperature gradient along the radial axis of the Petri dish under stable conditions, and the transition period under transient conditions. Our studies indicate a radial temperature gradient of 3.3 °C along with a transition period of 27.5 min when increasing the sample temperature from 37 to 45 °C for a standard 35 mm diameter Petri dish. We characterized the temperature gradient and transition period under various operational, geometric, and environmental conditions. Under stable conditions, reducing the diameter of the Petri dish and incorporating a heater underneath the Petri dish can effectively reduce the temperature gradient across the sample. In comparison, under transient conditions, reducing the diameter of the Petri dish, reducing sample volume, and using glass Petri dish chambers can reduce the transition period.
... The NIS Elements Advanced Research Microscope Imaging Software (Nikon, Melville, NY) was used for image acquisition. Fluorescent intensity analysis from the equatorial plane was carried out using the Fiji imaging processing software (Image J, University of Wisconsin-Madison, Madison, WI).Kinetics of CB 2 R endocytosis and recycling by TIRF.To assess the kinetic properties of CB 2 R internalization, SEP-hCB2R HEK293 cells were subjected to total internal reflection fluorescent microscopy (TIRF) as described elsewhere(Yudowski and von Zastrow, 2011;Delgado-Peraza et al., 2016b). Briefly, cells seeded in PDL coated 35 mm glass-bottom dishes (MatTek, Ashland, MA) were pre-incubated with Opti-MEM supplemented with 20 mM HEPES. ...
Article
Full-text available
G protein-coupled receptors (GPCRs) mediate their complex functions through activation of signaling cascades from receptors localized at the cell surface and endosomal compartments. These signaling pathways are modulated by heterotrimeric G proteins and the scaffold proteins beta-arrestin 1 and 2. However, in contrast to the events occurring at the cell surface, our knowledge of the mechanisms controlling signaling from receptors localized at intracellular compartments is still very limited. Here, we sought to investigate the intracellular signaling from cannabinoid 2 receptor (CB2R). First, we show that receptor internalization is required for agonist induced phosphorylation of ERK1/2. Then, we demonstrate that ERK1/2 activation is mediated by beta-arrestin 1 from receptors localized exclusively at Rab4/5 compartments. Finally, we identify the retromer complex as a gatekeeper, terminating beta-arrestin 1 mediated ERK phosphorylation. These findings extend our understanding of the events controlling signaling from endocytosed receptors and identify the retromer as a modulator of beta-arrestin mediated signaling from CB2R.
... In particular, TIRF microscopy offers the possibility of studying membrane proteins with higher spatial and temporal resolution than conventional epifluorescence or confocal fluorescence microscopy. Moreover, events occurring within the plasma membrane like receptor-dimerization 33,43,44 or in close proximity to the membrane such as internalization can be studied in living cells under nearly physiological conditions 32,45 . ...
Article
Full-text available
G protein-coupled receptors (GPCRs), including the dopamine receptors, represent a group of important pharmacological targets. Upon agonist binding, GPCRs frequently undergo internalization, a process that is known to attenuate functional responses upon prolonged exposure to agonists. In this study, internalization was visualized by means of total internal reflection fluorescence (TIRF) microscopy at a level of discrete single events near the plasma membrane with high spatial resolution. A novel method has been developed to determine the relative extent of internalized fluorescent receptor-ligand complexes by comparative fluorescence quantification in living CHO cells. The procedure entails treatment with the reducing agent sodium borohydride, which converts cyanine-based fluorescent ligands on the membrane surface to a long-lived reduced form. Because the highly polar reducing agent is not able to pass the cell membrane, the fluorescent receptor-ligand complexes located in internalized compartments remain fluorescent under TIRF illumination. We applied the method to investigate differences of the short (D2S) and the long (D2L) isoforms of dopamine D2 receptors in their ability to undergo agonist-induced internalization.
... Analysis was performed using the public domain NIH Image program ImageJ/FIJI software, which is freely available at http://fiji.sc/Fiji as described before Roman-Vendrell et al., 2014;Yudowski and von Zastrow, 2011) . Briefly, raw images were first background-subtracted and flat field-corrected. ...
Article
Full-text available
Activation of G protein-coupled receptors (GPCR) result in multiple waves of signaling which are mediated by heterotrimeric G proteins and the scaffolding proteins β-arrestin 1/ 2. Ligands can elicit full or subsets of cellular responses, a concept defined sometimes as ligand bias or functional selectivity. However, our current understanding of beta-arrestin mediated signaling is still very limited. Here we provide a comprehensive view of β-arrestin mediated signaling from the cannabinoid receptor 1 (CB1R). Utilizing a signaling biased receptor, we define the cascades, specific receptor kinases and molecular mechanism underlying β-arrestin mediated signaling: We identify the interaction kinetics of CB1R and β-arrestin 1 during their endocytic trafficking as directly proportional to its efficacy. Finally, we demonstrate that signaling results in the control of genes clustered around prosurvival and proapoptotic functions among others. Together, these studies constitute a comprehensive description of β-arrestin mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control β-arrestin mediated signaling.
... BK perimembrane localization was determined by TIRF microscopy. Imaging was performed as described before (Guillermo A Yudowski & von Zastrow, 2011) with a Nikon Ti-E inverted microscope (Melville, NY) using a CFI-APO 100× oil immersion TIRF objective (NA=1.49) with a 0.5× optical magnification. ...
Article
Full-text available
The large conductance Ca(2+) - and voltage-activated K(+) channel (BK) is an important player in molecular and behavioral alcohol tolerance. Trafficking and surface expression of ion channels contribute to the development of addictive behaviors. We have previously reported that internalization of the BK channel is a component of molecular tolerance to ethanol (EtOH). Using primary cultures of hippocampal neurons, we combine total internal reflection fluorescence microscopy, electrophysiology, and biochemical techniques to explore how exposure to EtOH affects the expression and subcellular localization of endogenous BK channels over time. Exposure to EtOH changed the expression of endogenous BK channels in a time-dependent manner at the perimembrane area (plasma membrane and/or the area adjacent to it), while total protein levels of BK remain unchanged. These results suggest a redistribution of the channel within the neurons rather than changes in synthesis or degradation rates. Our results showed a temporally nonlinear effect of EtOH on perimembrane expression of BK. First, there was an increase in BK perimembrane expression after 10 minutes of EtOH exposure that remained evident after 3 hours, although not correlated to increases in functional channel expression. In contrast, after 6 hours of EtOH exposure, we observed a significant decrease in both BK perimembrane expression and functional channel expression. Furthermore, after 24 hours of EtOH exposure, perimembrane levels of BK had returned to baseline. We report a complex time-dependent pattern in the effect of EtOH on BK channel trafficking, including successive increases and decreases in perimembrane expression and a reduction in active BK channels after 3 and 6 hours of EtOH exposure. Possible mechanisms underlying this multiphasic trafficking are discussed. As molecular tolerance necessarily underlies behavioral tolerance, the time-dependent alterations we see at the level of the channel may be relevant to the influence of drinking patterns on the development of behavioral tolerance. Copyright © 2015 by the Research Society on Alcoholism.
... This concept is applied by DiscoverRx (Fremont, CA) in their commercial Pathhunter GPCR internalization assay. Additionally, there also exist internalization assays that take advantage of the acidic environment of endosomes by tagging receptors with pH-sensitive fluorescent proteins (26,33,34). In these assays, fluorescence is quenched when GPCRs enter endosomes, which can be measured using a microscope. ...
Article
Full-text available
Abstract The quantity of G protein-coupled receptors (GPCRs) expressed on the cell surface is an important factor regulating receptor signaling. Maturation, internalization, recycling and degradation together determine the net amount of receptor surface expression. Understanding every aspect of the receptor lifecycle will facilitate the development of therapeutic applications. A number of assays for measuring the surface expression of GPCRs are currently available. This minireview summarizes the currently available assays and their suitability and usage for measuring GPCR surface expression.
Article
Full-text available
Selective activation of dopamine D1 receptors (D1Rs) has been pursued for 40 years as a therapeutic strategy for neurologic and psychiatric diseases due to the fundamental role of D1Rs in motor function, reward processing, and cognition. All known D1R-selective agonists are catechols, which are rapidly metabolized and desensitize the D1R after prolonged exposure, reducing agonist response. As such, drug-like selective D1R agonists have remained elusive. Here we report a novel series of selective, potent non-catechol D1R agonists with promising in vivo pharmacokinetic properties. These ligands stimulate adenylyl cyclase signaling and are efficacious in a rodent model of Parkinson's disease after oral administration. They exhibit distinct binding to the D1R orthosteric site and a novel functional profile including minimal receptor desensitization, reduced recruitment of β-arrestin, and sustained in vivo efficacy. These results reveal a novel class of D1 agonists with favorable drug-like properties, and define the molecular basis for catechol-specific recruitment of β-arrestin to D1Rs.
Article
Full-text available
It is generally accepted that the functional compartmentalization of eukaryotic cells is reflected by the differential occurrence of proteins in their compartments. The location and physiological function of a protein are closely related; local information of a protein is thus crucial to understanding its role in biological processes. The visualization of proteins residing on intracellular structures by fluorescence microscopy has become a routine approach in cell biology and is increasingly used to assess their colocalization with well-characterized markers. However, image-analysis methods for colocalization studies are a field of contention and enigma. We have therefore undertaken to review the most currently used colocalization analysis methods, introducing the basic optical concepts important for image acquisition and subsequent analysis. We provide a summary of practical tips for image acquisition and treatment that should precede proper colocalization analysis. Furthermore, we discuss the application and feasibility of colocalization tools for various biological colocalization situations and discuss their respective strengths and weaknesses. We have created a novel toolbox for subcellular colocalization analysis under ImageJ, named JACoP, that integrates current global statistic methods and a novel object-based approach.
Article
Full-text available
Total internal reflection fluorescence microscopy has been applied to image the final stage of constitutive exocytosis, which is the fusion of single post-Golgi carriers with the plasma membrane. The use of a membrane protein tagged with green fluorescent protein allowed the kinetics of fusion to be followed with a time resolution of 30 frames/s. Quantitative analysis allowed carriers undergoing fusion to be easily distinguished from carriers moving perpendicularly to the plasma membrane. The flattening of the carriers into the plasma membrane is seen as a simultaneous rise in the total, peak, and width of the fluorescence intensity. The duration of this flattening process depends on the size of the carriers, distinguishing small spherical from large tubular carriers. The spread of the membrane protein into the plasma membrane upon fusion is diffusive. Mapping many fusion sites of a single cell reveals that there are no preferred sites for constitutive exocytosis in this system.
Article
Advances in molecular biology, organic chemistry, and materials science have recently created several new classes of fluorescent probes for imaging in cell biology. Here we review the characteristic benefits and limitations of fluorescent probes to study proteins. The focus is on protein detection in live versus fixed cells: determination of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy. Small organic fluorescent dyes, nanocrystals ("quantum dots"), autofluorescent proteins, small genetic encoded tags that can be complexed with fluorochromes, and combinations of these probes are highlighted.
Article
Cell signalling and endocytic membrane trafficking have traditionally been viewed as distinct processes. Although our present understanding is incomplete and there are still great controversies, it is now recognized that these processes are intimately and bidirectionally linked in animal cells. Indeed, many recent examples illustrate how endocytosis regulates receptor signalling (including signalling from receptor tyrosine kinases and G protein-coupled receptors) and, conversely, how signalling regulates the endocytic pathway. The mechanistic and functional principles that underlie the relationship between signalling and endocytosis in cell biology are becoming increasingly evident across many systems.
Article
The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Förster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.
Article
The GTPase dynamin assembles at the necks of budded vesicles in vivo and functions in membrane fission. We have developed fluid supported bilayers with excess membrane reservoir, (SUPER) templates, to assay vesicle formation and membrane fission. Consistent with previous studies, in the absence of GTP, dynamin assembles in spirals, forming long membrane tubules. GTP addition triggers disassembly, but not membrane fission, arguing against models in which fission is mediated by concerted and global GTP-driven conformational changes. In contrast, under physiological conditions in the constant presence of GTP, dynamin mediates membrane fission. Under these conditions, fluorescently labeled dynamin cooperatively organizes into self-limited assemblies that continuously cycle at the membrane and drive vesicle release. When visualized at the necks of emergent vesicles, self-limited dynamin assemblies display intensity fluctuations and persist for variable time periods before fission. Thus, self-limited assemblies of dynamin generated in the constant presence of GTP catalyze membrane fission.
Article
In neural systems, information is often carried by ensembles of cells rather than by individual units. Optical indicators provide a powerful means to reveal such distributed activity, particularly when protein-based and encodable in DNA: encodable probes can be introduced into cells, tissues, or transgenic organisms by genetic manipulation, selectively expressed in anatomically or functionally defined groups of cells, and, ideally, recorded in situ, without a requirement for exogenous cofactors. Here we describe sensors for secretion and neurotransmission that fulfil these criteria. We have developed pH-sensitive mutants of green fluorescent protein ('pHluorins') by structure-directed combinatorial mutagenesis, with the aim of exploiting the acidic pH inside secretory vesicles to monitor vesicle exocytosis and recycling. When linked to a vesicle membrane protein, pHluorins were sorted to secretory and synaptic vesicles and reported transmission at individual synaptic boutons, as well as secretion and fusion pore 'flicker' of single secretory granules.