- Email: [email protected]
Quantitative imaging of clathrin-mediated endocytosis
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ScienceDirect Quantitative imaging of clathrin-mediated endocytosis Andrea Picco and Marko Kaksonen Clathrin-mediated endocytosis is a process by which eukaryotic cells bend a small region of their plasma membrane to form a transport vesicle that carries specific cargo molecules into the cell. Endocytosis controls the composition of the plasma membrane, imports nutrients and regulates many signalling pathways. The roles of most of the proteins involved in endocytosis have been thoroughly characterised. However, how these proteins cooperate in the cell to drive the endocytic process is not well understood. Microscopy methods have been instrumental in describing the dynamics and the molecular mechanism of endocytosis. Here, we will review the challenges and the recent advances in visualising the endocytic machinery and we will reflect on how the integration of current imaging technologies can lead us toward a quantitative understanding of the molecular mechanisms of endocytosis.
shapes vary slightly between the different organisms. In yeast, the endocytic membrane invagination is a narrow tubule that extends up to 140 nm in length [4] (Figure 1). The resulting vesicle is an ellipsoid of about 60 nm in length. In mammalian cells, the endocytic membrane invaginations are more spherical resulting in a larger vesicle, which is about 100 nm in diameter [5] (Figure 1). In yeast cells, the whole endocytic event, from initiation to the vesicle formation, takes about 1–2 min. Remarkably, all the membrane shape changes take place only within the last 10 s. In mammalian cells, the timing of membrane shaping is longer and more variable but the whole endocytic process spans a similar time range. The size of the protein machinery that envelops the forming vesicle is approximately 200 nm in diameter (Figure 1).
Address Department of Biochemistry and NCCR Chemical Biology, University of Geneva, Geneva, Switzerland
To understand how the endocytic machinery works, we need to know which proteins are involved, and we need to characterise their biochemistry, interactions, and structural properties. Tremendous advances have been made in this direction [1–3]. However, the output of endocytosis is a spatial and temporal deformation of the plasma membrane into a vesicle (Figure 1). Therefore, we need to complement protein structure and biochemistry with data about the supramolecular organisation of the endocytic proteins, and we must visualize when and how these protein structures morph to drive the vesicle formation. This is where imaging becomes essential. However, different imaging methods have distinctive strengths and weaknesses: Transmission electron microscopy (EM) has a resolution down to nanometer level (Figure 1) and can resolve protein and membrane ultrastructures, but has no temporal resolution, because biological samples have to be fixed for EM. Fluorescence microscopy (FM), on the other hand, is excellent for observing dynamic processes as it can be used on living cells. In addition, fluorescent labelling, usually done with fluorescent protein fusions, gives high molecular specificity. However, the spatial resolution of FM is limited to about 200 nm, which is about the size of the whole endocytic machinery (Figure 1). That means that the architecture in which the different proteins assemble and how they operate cannot be directly resolved by normal light microscopy. Superresolution microscopy techniques can boost the resolution down to about 30 nm but this higher spatial resolution typically comes at the expense of temporal resolution. As a result, the supramolecular organisation of endocytic proteins still remains an open question.
Corresponding author: Kaksonen, Marko ([email protected])
Current Opinion in Cell Biology 2018, 53:105–110 This review comes from a themed issue on Membrane trafficking Edited by Anne Spang and Jitu Mayor
https://doi.org/10.1016/j.ceb.2018.06.005 0955-0674/ã 2018 Published by Elsevier Ltd.
Introduction Clathrin-mediated endocytosis is a multifaceted process, where more than 50 different proteins dynamically assemble at the plasma membrane to form a complex, modular machinery [1–3]. In a few minutes, this machinery concentrates transmembrane cargo proteins, bends the plasma membrane into an invagination, and pinches off a vesicle containing the cargoes, which are then trafficked into the cell. There are different endocytic pathways that differ in the kind of proteins they employ, in the speed in which the vesicle is made and in the cargoes which are internalised. The clathrin-mediated endocytosis is so far the best-understood pathway. Most of the existing data about clathrinmediated endocytosis come from studies of cultured mammalian cells and yeasts. Clathrin-mediated endocytosis is well conserved across those species but the membrane www.sciencedirect.com
Electron microscopy Historically, electron microscopy and endocytosis are intimately linked. EM studies pioneered the field by Current Opinion in Cell Biology 2018, 53:105–110
106 Membrane trafficking
Figure 1
Microscopy resolutions SRM > 30 nm
FM > 200 nm
EM > 0.1 nm x100
200 nm
~200 nm
~200 nm
80 nm
40 nm
Budding Yeast ~0.5-2 min.
~10 s ~200 nm
~100 nm
Mammalian cells ~20 s to minutes Clathrin coat
BAR proteins / Dynamin
Actin patch Current Opinion in Cell Biology
The resolutions of fluorescence microscopy (FM), superresolution microscopy (SRM), and electron microscopy (EM) are compared to the different stages of the endocytic process in budding yeast and in mammalian cells.
showing the first evidence of protein uptake via protein coated membrane vesicles forming from the plasma membrane (Figure 2) [6,7]. EM can reveal the cellular membranes and their shapes in detail, therefore being an excellent tool for studying membrane trafficking processes. However, protein structures are difficult to visualize by EM in situ due to their limited contrast, with notable exceptions such as the clathrin cage and actin filaments, which are remarkably well imaged in platinum replica electron microscopy [5,8]. Immuno-EM can provide molecular specificity in situ. For example, a systematic use of immuno-EM resolved the distribution of several endocytic proteins along the invagination axis during endocytosis in yeast [9,10]. Ex situ, cryo-electron microscopy have made it possible to visualise supramolecular structures of purified endocytic proteins at very high resolution [11–13]. Recently, cryo-focussed ion beam milling, cryo-electron tomography and subtomogram averaging together allowed to resolve the supramolecular structure of COPI coated vesicles for the first time in situ [14]. These approaches are likely to revolutionise the imaging of subcellular structures. However, a big challenge in applying EM to dynamic processes such as endocytosis is that EM provides only snapshots of the different stages of the process and the temporal relationship between these snapshots is often not obvious. Current Opinion in Cell Biology 2018, 53:105–110
Fluorescence microscopy The strengths of fluorescence microscopy are temporal resolution and molecular specificity. Proteins can be tagged endogenously with fluorophores and can, therefore, be easily imaged in living cells. When fluorescently labelled endocytic proteins are imaged by fluorescence microscopy, the endocytic sites are visible as diffraction limited spots. The size of these spots does not allow one to resolve the position of individual proteins. However, the abundance of a specific labelled protein can be detected with high sensitivity, down to single molecules and with sub-second temporal resolution. Light microscopy studies in living cells have identified new proteins taking part in the endocytic process, resolved the temporal order in which proteins are recruited at the endocytic site, and measured the protein lifetimes and movement during endocytosis (Figure 2) [15–24]. Total internal reflection fluorescence (TIRF) microscopy is especially suited for studying endocytosis in mammalian cells, because it excites fluorophores only within a 100–200 nm thick section at the bottom of the cells and thereby excludes most of the cellular background fluorescence. The high sensitivity of TIRF imaging allows even single molecules to be detected. For example, the coordinated arrival of the first clathrin triskelia and the AP2 adaptor proteins at early stages of endocytosis could be recorded by TIRF imaging [25]. These studies have given a comprehensive view about when the different proteins assemble at the endocytic sites. www.sciencedirect.com
Imaging endocytosis Picco and Kaksonen 107
Figure 2
Cellular EM 1964
Fluorescence microscopy (FM) 1 μm
–72 s
2s
6s
10 s
18 s
38 s
Evidence of ferritin uptake via endocytosis (Rosenbluth,1964)
Structural cryoEM
58 s
FM of clathrin coated pits (Merrifield,2002)
Deep-etch EM of clathrin coated pits (Heuser,1980) Clathrin lattice model from cryoEM (Fotin, 2004)
Multicolor FM of endocytosis (Kaksonen,2003)
Correlative FM and EM of endocytosis (Kukulski,2010) Reconstruction of a F-BAR domain induced membrane tubule (Frost, 2008)
Today High-speed live superresolution FM of endocytosis (Arasada,2017)
Correlative superresolution FM and EM of endocytosis (Sochacki, 2014)
Tomorrow
Current Opinion in Cell Biology
Electron microscopy (EM) studies in cells gave the first evidence of endocytosis in 1964. Since then, fluorescence microscopy (FM) complemented and later integrated with EM studies to detail the presence, dynamics and position of the different endocytic proteins, while CryoEM studies detailed the structures in which those proteins assembled in vitro. Further integration of these methods in the future may allow resolving the mechanistic details of the endocytic process in cells.
Because of its limited optical resolution, fluorescence microscopy cannot provide direct information about the molecular architecture of the endocytic structures. However, detailed quantitative information can still be gained from live-cell fluorescence microscopy images: the fluorescence intensities of the diffraction limited patches in which endocytic events are imaged can be calibrated to give estimates of absolute numbers of labelled molecules at the endocytic sites [26–30]. Also, the centroid position of www.sciencedirect.com
the fluorescent patches, which estimates the average position of the labelled molecules, can be measured and tracked with a localization precision that is typically in the range of few tens of nm [31]. These quantitative approaches have, so far, been especially powerful for studying endocytosis in yeasts, where fluorescent fusion proteins can be easily expressed from endogenous genomic loci so that every molecule of the protein of interest is labelled. Furthermore, the regular cell shape of yeasts allows one to precisely Current Opinion in Cell Biology 2018, 53:105–110
108 Membrane trafficking
estimate the direction of invagination and vesicle internalisation, which are perpendicular to the cell wall, greatly facilitating the tracking of the endocytic patches [32–34]. In mammalian cells, new genome editing tools are facilitating quantitative live-cell fluorescence imaging approaches [30,35]. However, mammalian cells are more irregularly shaped and typically lie flat on a coverslip and, as a consequence, their endocytic invaginations are mostly oriented perpendicularly to the focal plane, which complicates the direct tracking of the invagination dynamics of the endocytic patches. Differential evanescence nanometry, which uses the ratio between TIRF and wide field epifluorescence excitations, can resolve the position of molecules along the z-axis with a precision of 10 nm [36]. Superresolution microscopy (SRM) techniques can resolve the position of individual fluorophores beyond the diffraction limit [37]. SRM has been used to resolve the distributions in which different proteins assemble at the endocytic sites and how these distributions change as the invagination grows [38,39,40]. However, the temporal resolution and the ability to measure the abundances of the proteins are still limited in SRM. Technical advances in speeding up SRM have recently allowed resolving the distribution of the actin nucleator promoting factors along the axis of endocytic invagination with a time resolution of 1 s in living fission yeast cells [41]. This is exciting as it brings the molecular specificity and time resolution that have been distinctive of FM, toward a resolution that approaches EM.
Integrative microscopy The strengths of each of the different imaging approaches have allowed resolving important details of the endocytic process. However, to understand how the endocytic machinery works as an integrated unit, we need to visualize how the supramolecular protein structures and the membrane change over time. To be able to do so, we believe that it is necessary to integrate different imaging methods to combine their individual strengths (Figure 2). For example, correlative light and electron microscopy (CLEM) has been used to correlate the appearance of different endocytic proteins with the different stages of membrane shaping during invagination and vesicle scission [4,42,43]. The membrane ultrastructures imaged and timed with CLEM can be integrated with the centroid positions of endocytic proteins tracked with high precision in living cells to reconstruct how proteins are distributed along the plasma membrane invagination during time [33]. The integration of SRM and metal-replica EM correlated the distributions of endocytic proteins with the shapes of the clathrin-coated endocytic invaginations in mammalian cells [38,39]. Methods that can reveal membrane shape changes in living cells, such as fast ion conductance microscopy, atomic force microscopy, or fluorescence polarization microscopy, can be integrated with simultaneous fluorescence microscopy of endocytic proteins to relate the invagination formation with the Current Opinion in Cell Biology 2018, 53:105–110
appearance of endocytic proteins [44,45,46]. In addition, methods that can resolve different functional stages of the endocytic machinery will be critical for understanding the mechanisms of endocytosis: Cycles of activation and deactivation of pH sensitive fluorescent dyes can be used to time vesicle scission events in relation to the assembly of different endocytic proteins [47]. Optogenetic initiation of endocytosis in mouse neurons followed by timed rapid freezing and EM can resolve the different stages of ultrafast endocytosis [48]. At the molecular level, conformation specific probes can time protein activities, such as dynamin GTP hydrolysis during vesicle scission [49].
Perspective Where is imaging of endocytosis going next? Different imaging methods are currently being actively developed in many laboratories. The technical developments in cryoEM now allow the characterization of supramolecular structures directly in their cellular context [14]. These advances in cryoEM can help resolve the supramolecular protein structures together with membrane shapes and will therefore be critical in reconstructing how the molecular architecture of the endocytic machinery shapes the membrane. Superresolution light microscopy methods are also constantly being improved allowing us to image cells with increasing resolution: a recent method, MINFLUX, which probes the presence of fluorophores with a local minimum of excitation light, reaches a resolution of 1 nm in vitro [50]. If this resolution can be equaled in vivo, it would bring molecular specificity of fluorescence microscopy to the scale of EM. The SRM methods are thus bridging the spatial resolution gap between light and electron microscopy. Furthermore, developments in fast SRM methods are overcoming the trade-off between spatial and temporal resolution [41,51]. At the cellular and tissue scale, light sheet microscopy methods and adaptive optics are making it possible to image large volumes with good temporal and spatial resolution. One can now image the endocytotic activity throughout a whole cell, or in a cell within a living tissue, with unprecedented speed and resolution [51–53]. In addition to the technical improvements in the individual imaging methods, we believe that the integration of these different methods will push the boundaries of our understanding of the endocytic machinery. The complementary strengths of these different methods will help us to understand the process of endocytosis from its molecular details up to the organismal scale physiology.
Conflict of interest statement Nothing declared.
Acknowledgements This work was supported by the Swiss National Science Foundation (31003A_163267), and the NCCR Chemical Biology. www.sciencedirect.com
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ScienceDirect Quantitative imaging of clathrin-mediated endocytosis Andrea Picco and Marko Kaksonen Clathrin-mediated endocytosis is a process by which eukaryotic cells bend a small region of their plasma membrane to form a transport vesicle that carries specific cargo molecules into the cell. Endocytosis controls the composition of the plasma membrane, imports nutrients and regulates many signalling pathways. The roles of most of the proteins involved in endocytosis have been thoroughly characterised. However, how these proteins cooperate in the cell to drive the endocytic process is not well understood. Microscopy methods have been instrumental in describing the dynamics and the molecular mechanism of endocytosis. Here, we will review the challenges and the recent advances in visualising the endocytic machinery and we will reflect on how the integration of current imaging technologies can lead us toward a quantitative understanding of the molecular mechanisms of endocytosis.
shapes vary slightly between the different organisms. In yeast, the endocytic membrane invagination is a narrow tubule that extends up to 140 nm in length [4] (Figure 1). The resulting vesicle is an ellipsoid of about 60 nm in length. In mammalian cells, the endocytic membrane invaginations are more spherical resulting in a larger vesicle, which is about 100 nm in diameter [5] (Figure 1). In yeast cells, the whole endocytic event, from initiation to the vesicle formation, takes about 1–2 min. Remarkably, all the membrane shape changes take place only within the last 10 s. In mammalian cells, the timing of membrane shaping is longer and more variable but the whole endocytic process spans a similar time range. The size of the protein machinery that envelops the forming vesicle is approximately 200 nm in diameter (Figure 1).
Address Department of Biochemistry and NCCR Chemical Biology, University of Geneva, Geneva, Switzerland
To understand how the endocytic machinery works, we need to know which proteins are involved, and we need to characterise their biochemistry, interactions, and structural properties. Tremendous advances have been made in this direction [1–3]. However, the output of endocytosis is a spatial and temporal deformation of the plasma membrane into a vesicle (Figure 1). Therefore, we need to complement protein structure and biochemistry with data about the supramolecular organisation of the endocytic proteins, and we must visualize when and how these protein structures morph to drive the vesicle formation. This is where imaging becomes essential. However, different imaging methods have distinctive strengths and weaknesses: Transmission electron microscopy (EM) has a resolution down to nanometer level (Figure 1) and can resolve protein and membrane ultrastructures, but has no temporal resolution, because biological samples have to be fixed for EM. Fluorescence microscopy (FM), on the other hand, is excellent for observing dynamic processes as it can be used on living cells. In addition, fluorescent labelling, usually done with fluorescent protein fusions, gives high molecular specificity. However, the spatial resolution of FM is limited to about 200 nm, which is about the size of the whole endocytic machinery (Figure 1). That means that the architecture in which the different proteins assemble and how they operate cannot be directly resolved by normal light microscopy. Superresolution microscopy techniques can boost the resolution down to about 30 nm but this higher spatial resolution typically comes at the expense of temporal resolution. As a result, the supramolecular organisation of endocytic proteins still remains an open question.
Corresponding author: Kaksonen, Marko ([email protected])
Current Opinion in Cell Biology 2018, 53:105–110 This review comes from a themed issue on Membrane trafficking Edited by Anne Spang and Jitu Mayor
https://doi.org/10.1016/j.ceb.2018.06.005 0955-0674/ã 2018 Published by Elsevier Ltd.
Introduction Clathrin-mediated endocytosis is a multifaceted process, where more than 50 different proteins dynamically assemble at the plasma membrane to form a complex, modular machinery [1–3]. In a few minutes, this machinery concentrates transmembrane cargo proteins, bends the plasma membrane into an invagination, and pinches off a vesicle containing the cargoes, which are then trafficked into the cell. There are different endocytic pathways that differ in the kind of proteins they employ, in the speed in which the vesicle is made and in the cargoes which are internalised. The clathrin-mediated endocytosis is so far the best-understood pathway. Most of the existing data about clathrinmediated endocytosis come from studies of cultured mammalian cells and yeasts. Clathrin-mediated endocytosis is well conserved across those species but the membrane www.sciencedirect.com
Electron microscopy Historically, electron microscopy and endocytosis are intimately linked. EM studies pioneered the field by Current Opinion in Cell Biology 2018, 53:105–110
106 Membrane trafficking
Figure 1
Microscopy resolutions SRM > 30 nm
FM > 200 nm
EM > 0.1 nm x100
200 nm
~200 nm
~200 nm
80 nm
40 nm
Budding Yeast ~0.5-2 min.
~10 s ~200 nm
~100 nm
Mammalian cells ~20 s to minutes Clathrin coat
BAR proteins / Dynamin
Actin patch Current Opinion in Cell Biology
The resolutions of fluorescence microscopy (FM), superresolution microscopy (SRM), and electron microscopy (EM) are compared to the different stages of the endocytic process in budding yeast and in mammalian cells.
showing the first evidence of protein uptake via protein coated membrane vesicles forming from the plasma membrane (Figure 2) [6,7]. EM can reveal the cellular membranes and their shapes in detail, therefore being an excellent tool for studying membrane trafficking processes. However, protein structures are difficult to visualize by EM in situ due to their limited contrast, with notable exceptions such as the clathrin cage and actin filaments, which are remarkably well imaged in platinum replica electron microscopy [5,8]. Immuno-EM can provide molecular specificity in situ. For example, a systematic use of immuno-EM resolved the distribution of several endocytic proteins along the invagination axis during endocytosis in yeast [9,10]. Ex situ, cryo-electron microscopy have made it possible to visualise supramolecular structures of purified endocytic proteins at very high resolution [11–13]. Recently, cryo-focussed ion beam milling, cryo-electron tomography and subtomogram averaging together allowed to resolve the supramolecular structure of COPI coated vesicles for the first time in situ [14]. These approaches are likely to revolutionise the imaging of subcellular structures. However, a big challenge in applying EM to dynamic processes such as endocytosis is that EM provides only snapshots of the different stages of the process and the temporal relationship between these snapshots is often not obvious. Current Opinion in Cell Biology 2018, 53:105–110
Fluorescence microscopy The strengths of fluorescence microscopy are temporal resolution and molecular specificity. Proteins can be tagged endogenously with fluorophores and can, therefore, be easily imaged in living cells. When fluorescently labelled endocytic proteins are imaged by fluorescence microscopy, the endocytic sites are visible as diffraction limited spots. The size of these spots does not allow one to resolve the position of individual proteins. However, the abundance of a specific labelled protein can be detected with high sensitivity, down to single molecules and with sub-second temporal resolution. Light microscopy studies in living cells have identified new proteins taking part in the endocytic process, resolved the temporal order in which proteins are recruited at the endocytic site, and measured the protein lifetimes and movement during endocytosis (Figure 2) [15–24]. Total internal reflection fluorescence (TIRF) microscopy is especially suited for studying endocytosis in mammalian cells, because it excites fluorophores only within a 100–200 nm thick section at the bottom of the cells and thereby excludes most of the cellular background fluorescence. The high sensitivity of TIRF imaging allows even single molecules to be detected. For example, the coordinated arrival of the first clathrin triskelia and the AP2 adaptor proteins at early stages of endocytosis could be recorded by TIRF imaging [25]. These studies have given a comprehensive view about when the different proteins assemble at the endocytic sites. www.sciencedirect.com
Imaging endocytosis Picco and Kaksonen 107
Figure 2
Cellular EM 1964
Fluorescence microscopy (FM) 1 μm
–72 s
2s
6s
10 s
18 s
38 s
Evidence of ferritin uptake via endocytosis (Rosenbluth,1964)
Structural cryoEM
58 s
FM of clathrin coated pits (Merrifield,2002)
Deep-etch EM of clathrin coated pits (Heuser,1980) Clathrin lattice model from cryoEM (Fotin, 2004)
Multicolor FM of endocytosis (Kaksonen,2003)
Correlative FM and EM of endocytosis (Kukulski,2010) Reconstruction of a F-BAR domain induced membrane tubule (Frost, 2008)
Today High-speed live superresolution FM of endocytosis (Arasada,2017)
Correlative superresolution FM and EM of endocytosis (Sochacki, 2014)
Tomorrow
Current Opinion in Cell Biology
Electron microscopy (EM) studies in cells gave the first evidence of endocytosis in 1964. Since then, fluorescence microscopy (FM) complemented and later integrated with EM studies to detail the presence, dynamics and position of the different endocytic proteins, while CryoEM studies detailed the structures in which those proteins assembled in vitro. Further integration of these methods in the future may allow resolving the mechanistic details of the endocytic process in cells.
Because of its limited optical resolution, fluorescence microscopy cannot provide direct information about the molecular architecture of the endocytic structures. However, detailed quantitative information can still be gained from live-cell fluorescence microscopy images: the fluorescence intensities of the diffraction limited patches in which endocytic events are imaged can be calibrated to give estimates of absolute numbers of labelled molecules at the endocytic sites [26–30]. Also, the centroid position of www.sciencedirect.com
the fluorescent patches, which estimates the average position of the labelled molecules, can be measured and tracked with a localization precision that is typically in the range of few tens of nm [31]. These quantitative approaches have, so far, been especially powerful for studying endocytosis in yeasts, where fluorescent fusion proteins can be easily expressed from endogenous genomic loci so that every molecule of the protein of interest is labelled. Furthermore, the regular cell shape of yeasts allows one to precisely Current Opinion in Cell Biology 2018, 53:105–110
108 Membrane trafficking
estimate the direction of invagination and vesicle internalisation, which are perpendicular to the cell wall, greatly facilitating the tracking of the endocytic patches [32–34]. In mammalian cells, new genome editing tools are facilitating quantitative live-cell fluorescence imaging approaches [30,35]. However, mammalian cells are more irregularly shaped and typically lie flat on a coverslip and, as a consequence, their endocytic invaginations are mostly oriented perpendicularly to the focal plane, which complicates the direct tracking of the invagination dynamics of the endocytic patches. Differential evanescence nanometry, which uses the ratio between TIRF and wide field epifluorescence excitations, can resolve the position of molecules along the z-axis with a precision of 10 nm [36]. Superresolution microscopy (SRM) techniques can resolve the position of individual fluorophores beyond the diffraction limit [37]. SRM has been used to resolve the distributions in which different proteins assemble at the endocytic sites and how these distributions change as the invagination grows [38,39,40]. However, the temporal resolution and the ability to measure the abundances of the proteins are still limited in SRM. Technical advances in speeding up SRM have recently allowed resolving the distribution of the actin nucleator promoting factors along the axis of endocytic invagination with a time resolution of 1 s in living fission yeast cells [41]. This is exciting as it brings the molecular specificity and time resolution that have been distinctive of FM, toward a resolution that approaches EM.
Integrative microscopy The strengths of each of the different imaging approaches have allowed resolving important details of the endocytic process. However, to understand how the endocytic machinery works as an integrated unit, we need to visualize how the supramolecular protein structures and the membrane change over time. To be able to do so, we believe that it is necessary to integrate different imaging methods to combine their individual strengths (Figure 2). For example, correlative light and electron microscopy (CLEM) has been used to correlate the appearance of different endocytic proteins with the different stages of membrane shaping during invagination and vesicle scission [4,42,43]. The membrane ultrastructures imaged and timed with CLEM can be integrated with the centroid positions of endocytic proteins tracked with high precision in living cells to reconstruct how proteins are distributed along the plasma membrane invagination during time [33]. The integration of SRM and metal-replica EM correlated the distributions of endocytic proteins with the shapes of the clathrin-coated endocytic invaginations in mammalian cells [38,39]. Methods that can reveal membrane shape changes in living cells, such as fast ion conductance microscopy, atomic force microscopy, or fluorescence polarization microscopy, can be integrated with simultaneous fluorescence microscopy of endocytic proteins to relate the invagination formation with the Current Opinion in Cell Biology 2018, 53:105–110
appearance of endocytic proteins [44,45,46]. In addition, methods that can resolve different functional stages of the endocytic machinery will be critical for understanding the mechanisms of endocytosis: Cycles of activation and deactivation of pH sensitive fluorescent dyes can be used to time vesicle scission events in relation to the assembly of different endocytic proteins [47]. Optogenetic initiation of endocytosis in mouse neurons followed by timed rapid freezing and EM can resolve the different stages of ultrafast endocytosis [48]. At the molecular level, conformation specific probes can time protein activities, such as dynamin GTP hydrolysis during vesicle scission [49].
Perspective Where is imaging of endocytosis going next? Different imaging methods are currently being actively developed in many laboratories. The technical developments in cryoEM now allow the characterization of supramolecular structures directly in their cellular context [14]. These advances in cryoEM can help resolve the supramolecular protein structures together with membrane shapes and will therefore be critical in reconstructing how the molecular architecture of the endocytic machinery shapes the membrane. Superresolution light microscopy methods are also constantly being improved allowing us to image cells with increasing resolution: a recent method, MINFLUX, which probes the presence of fluorophores with a local minimum of excitation light, reaches a resolution of 1 nm in vitro [50]. If this resolution can be equaled in vivo, it would bring molecular specificity of fluorescence microscopy to the scale of EM. The SRM methods are thus bridging the spatial resolution gap between light and electron microscopy. Furthermore, developments in fast SRM methods are overcoming the trade-off between spatial and temporal resolution [41,51]. At the cellular and tissue scale, light sheet microscopy methods and adaptive optics are making it possible to image large volumes with good temporal and spatial resolution. One can now image the endocytotic activity throughout a whole cell, or in a cell within a living tissue, with unprecedented speed and resolution [51–53]. In addition to the technical improvements in the individual imaging methods, we believe that the integration of these different methods will push the boundaries of our understanding of the endocytic machinery. The complementary strengths of these different methods will help us to understand the process of endocytosis from its molecular details up to the organismal scale physiology.
Conflict of interest statement Nothing declared.
Acknowledgements This work was supported by the Swiss National Science Foundation (31003A_163267), and the NCCR Chemical Biology. www.sciencedirect.com
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