Intracellular Journey of Cell Surface Receptors
Imaging Intracellular Trafficking using Photoconvertible Fluorescent Proteins
- Fig. 1: A. Examples of images and quantification of the internalization of a cell surface receptor (TCRζ) fused to PA-mCherry after a single round of 405 nm illumination (upper row) or repetitively photoactivated 8 sec for 200 sec (lower row) with a region of interest at the edge of Jurkat T cells (white dashed line). B: Number of TCRζ-positive after standard (blue) or repetitive (cyan) photoactivation. Scale bars = 5 µm.
- Fig. 2: A. Principle of two-photon specific photoconversion of cargo proteins of interest within intracellular compartments. B. Jurkat T cells transfected with TCRζ- mCherry (magenta) and TCRζ-PSCFP2 (cyan) were activated on glass coverslips and fixed 20 minutes post activation. Z-stacks of cells were imaged before (left) and after (right) photoactivation with one-photon 405 nm excitation (first row) or two-photon 800 nm excitation at the indicated z-depth. Dashed white line indicates photoactivated region. Adapted with permissions from G. M. I. Redpath et al et al. Flotillins promote T cell receptor sorting through a fast Rab5–Rab11 endocytic recycling axis, Nat. Commun. 10 4392 (2019).
Imaging intracellular trafficking in live cells is challenging. Mostly because labelling of cargo proteins with conventional fluorescent proteins does not provide temporal or directional information about the vesicles that transport these cargoes. The use of photoswitchable or photoactivatable fluorescent proteins enable to “shine the light” on specific membranes, vesicles or intracellular compartments at a given time and location. The transport of cargoes can thereby be visualized and quantified in order to obtain key information about the mechanism underpinning intracellular trafficking.
Since photoactivatable GFP was engineered in 2002 , the past seventeen years have seen the development of a multitude of photoswitchable (PS) or photoactivatable (PA) fluorescent proteins of various spectra, increasing photostability and quantum yields [2-5]. They were first designed with live cell imaging in mind, because of the obvious edge they provide cell biologists by allowing visualization and measure of protein dynamics.
However, PA/PS proteins perfectly illustrate the gap that often stands between powerful tools developed by research groups that specialize in method development and their application to address biologically relevant questions. The Medline database references almost every publication in life and biomedical sciences and can be publicly accessed with the search engine PubMed. A quick search on PubMed with the keywords “Photoconvertible”, “Photoactivatable” or “Photoswitchable fluorescent proteins” reveals that the number of studies reporting development, characterization or methodologies related to PS/PA proteins outweighs papers using them to gain novel insights into biological questions by at least forty to one. Biologists too often privilege the use of a more widespread and characterized approach, fluorescence recovery after bleaching (FRAP). Partially because nowadays most commercial fluorescence microscopes include step-by-step wizards to generate and analyze FRAP data. PS/PA fluorescent proteins benefited from a renewed interest ten years ago with the advent of photoactivatable localization microscopy [6,7], which further shifted the balance of publications towards method papers .
Hence, these powerful tools have been continuously developed and improved, but too sparsely used to do what they had been initially designed for.
Imaging intracellular trafficking in live cells involves several challenges. The main problem is that when cargo proteins are labelled with conventional fluorescent proteins such as GFP, the fluorescent signal will not provide any information about the time the cargo was packaged into a transport vesicle. It will not reveal either if this vesicle is heading towards intracellular compartments from the plasma membrane after having been internalized, from one endosome to another or being delivered from intracellular stores to the plasma membrane. However, an accurate quantification of these parameters is required to understand the mechanisms regulating how proteins are transported through the cell. PS/PA fluorescent proteins represent the ideal tool to access this information. Cargo proteins fused to PS/PA fluorescent proteins can be revealed at a specific time and location within the cell. It is then possible to visualize the cellular trafficking of these proteins from the region where the photoactivation/conversion has been performed to a target destination of interest.
Accordingly, several studies have made use of PS/PA fluorescent proteins to follow proteins of interest while they are transported within various cell types [9-11]. Two recent publications [12, 13] describe a global microscopy approach based on the light-induced activation of photoactivatable fluorescent proteins to visualize, and most importantly to quantify, the endocytic trafficking of proteins associated with the plasma membrane. This approach has allowed to extract quantitative information about every step of the endocytic recycling process: 1) internalization of receptors from the plasma membrane (endocytosis); 2) incorporation of these receptors into intracellular compartments; 3) return of the receptor from intracellular compartment to the plasma membrane (endocytic recycling).
To investigate endocytosis, the cell surface protein of interest is fused to PA-mCherry. Defined regions of the cells are illuminated for a few seconds with 405 nm laser using a confocal microscope. These regions are selected on the edges of the cell, which consist mostly of plasma membrane and contain very little intracellular material, in order to photoactivate only proteins within the plasma membrane. The PA-mCherry that is revealed at the plasma membrane initially shows a globally uniform distribution before being eventually packaged into vesicles, which appear as bright punctate structures (fig. 1). These structures can easily be counted throughout a time series by any analysis routine that identifies particles. The number of vesicles that form and the speed at which they form provide direct information about the processes that mediate the endocytosis of the protein fused to PA-mCherry. This approach can also be used to identify the cellular machinery supporting endocytosis. Components of this machinery are fused to EGFP and a cross-channel nearest neighbor analysis can be used to determine if the endocytosed particles identified in the red channel (the membrane protein fused to PA-mCherry) and the particles in the green channel (an endocytic protein fused to EGFP) are the same vesicles or not.
Incorporation into Intracellular Compartments
The target compartments – or endosomes – of internalized cell surface proteins determine if these proteins will be sent for degradation or are reused, and thereby returned to the cell surface. PA/PS fluorescent proteins also allow to quantify how much of an internalized protein reaches a given endosome. As when investigating endocytosis, membrane proteins fused to PA-mCherry are photoactivated at the edge of the cell, but this time repetitively (every 5-10 seconds), in order to reveal large amounts of internalized proteins. Markers of specific endosomes labelled with EGFP are used to define a mask. The fluorescence intensity of the PA-mCherry signal within the EGFP-mask indicates if the internalized protein has reached the endosome, how quickly it enters the compartment, as well as how long it remains in this specific compartment before continuing its endocytic journey.
Transport of Membrane Proteins from Intracellular Compartment to the Cell Surface
While the mechanisms that regulate the fusion of vesicles with the plasma membrane have been extensively investigated, much less is known regarding how cargoes are loaded into transport vesicles that will bring them to the cell surface for such fusion. Two-photon illumination of fluorescent proteins can answer this question, because it allows the selective photoconversion of membrane proteins strictly within intracellular compartment and nowhere else in the cell (fig. 2). The membrane proteins of interest are fused to PS-CFP2, which in our hands proved to be more resistant to photobleaching than PA-mCherry and therefore is more suitable for this approach. Similar to the process for visualizing the incorporation of internalized proteins into endosomes, the cells express markers of endosomes of interest labelled with mCherry. The mCherry signal is used as an “aiming light” to define which area inside the cell will be illuminated with 800 nm light to visualize if the transport to the plasma membrane occurs from this compartment of interest. The photoconverted PS-CFP2 signal is measured by confocal imaging at the plasma membrane (using 488 nm excitation). The intensity of this signal is directly related to transport of the PS-CFP2-fused membrane protein from the endosome where the photoconversion has been performed to the cell surface. Additionally, vesicles positive for converted PS-CFP2 can be imaged at a focal plan that is axially located between the endosome of interest and the plasma membrane to uncover the identity of the vesicles transporting the cargo protein. A cross-channel nearest-neighbour analysis between photoconverted vesicles and vesicles positive for mCherry can be used to determine if the membrane protein of interest is transported in the endosomal population where PS-CFP2 was photoconverted. Altogether, this approach allows direct quantification of the magnitude, kinetics and molecular identity regulating the transport of membrane proteins to the cell surface.
While PA/PS proteins tend to be used more for single molecule localization microscopy, they are a very powerful tool to investigate cellular trafficking. They allow to follow and quantify the entire endocytic journey of a given membrane protein in live cells. Hence, PA/PS proteins can be used to obtain crucial information about the cellular mechanisms regulating the shuttling of membrane proteins between the plasma membrane and intracellular compartments to fully understand how endocytic trafficking regulates cell function.
Gregory Redpath1, Manuela Ecker2, Jérémie Rossy3
1Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand
2EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, Australia
3Biotechnology Institute Thurgau at the University of Konstanz, Kreuzlingen, Switzerland
Dr. Jérémie Rossy
Biotechnology Institute Thurgau
University of Konstanz
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