Clathrin light chains are required for the gyrating-clathrin recycling pathway and thereby promote cell migration

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Abstract

The clathrin light chain (CLC) subunits participate in several membrane traffic pathways involving both clathrin and actin, through binding the actin-organizing huntingtin-interacting proteins (Hip). However, CLCs are dispensable for clathrin-mediated endocytosis of many cargoes. Here we observe that CLC depletion affects cell migration through Hip binding and reduces surface expression of β1-integrin by interference with recycling following normal endocytosis of inactive β1-integrin. CLC depletion and expression of a modified CLC also inhibit the appearance of gyrating (G)-clathrin structures, known mediators of rapid recycling of transferrin receptor from endosomes. Expression of the modified CLC reduces β1-integrin and transferrin receptor recycling, as well as cell migration, implicating G-clathrin in these processes. Supporting a physiological role for CLC in migration, the CLCb isoform of CLC is upregulated in migratory human trophoblast cells during uterine invasion. Together, these studies establish CLCs as mediating clathrin–actin interactions needed for recycling by G-clathrin during migration.

Abstract

Clathrin light chain (CLC) subunits are dispensable for clathrin-mediated endocytosis of a number of cargoes. Majeed et al. report that CLCs are however required for gyrating-clathrin-dependent recycling of inactive β1-integrins, the absence of which impairs cell migration.

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Natural killer cells and pregnancy.

Ashley Moffett-King (2002)

The fetus is considered to be an allograft that, paradoxically, survives pregnancy despite the laws of classical transplantation immunology. There is no direct contact of the mother with the embryo, only with the extraembryonic placenta as it implants in the uterus. No convincing evidence of uterine maternal T-cell recognition of placental trophoblast cells has been found, but instead, there might be maternal allorecognition mediated by uterine natural killer cells that recognize unusual fetal trophoblast MHC ligands.

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Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral.

R Tsien, G. Baird, David Zacharias (2000)

DsRed is a recently cloned 28-kDa fluorescent protein responsible for the red coloration around the oral disk of a coral of the Discosoma genus. DsRed has attracted tremendous interest as a potential expression tracer and fusion partner that would be complementary to the homologous green fluorescent protein from Aequorea, but very little is known of the biochemistry of DsRed. We now show that DsRed has a much higher extinction coefficient and quantum yield than previously reported, plus excellent resistance to pH extremes and photobleaching. In addition, its 583-nm emission maximum can be further shifted to 602 nm by mutation of Lys-83 to Met. However, DsRed has major drawbacks, such as strong oligomerization and slow maturation. Analytical ultracentrifugation proves DsRed to be an obligate tetramer in vitro, and fluorescence resonance energy transfer measurements and yeast two-hybrid assays verify oligomerization in live cells. Also, DsRed takes days to ripen fully from green to red in vitro or in vivo, and mutations such as Lys-83 to Arg prevent the color change. Many potential cell biological applications of DsRed will require suppression of the tetramerization and acceleration of the maturation.

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Distinct Dynamics of Endocytic Clathrin-Coated Pits and Coated Plaques

Saveez Saffarian, Emanuele Cocucci, Tomas Kirchhausen (2009)

Introduction Clathrin-mediated endocytosis is a mechanism for selective retrieval and internalization of membrane lipids and membrane-bound proteins. Clathrin-coated pits capture their molecular cargo as they invaginate from the cell surface and bud inward to form coated vesicles, in a process that involves a complex sequence of interactions among structural and regulatory proteins and lipids [1]–[3]. The assembly of clathrin-coated pits can be followed in vivo by contemporary live-cell imaging methods, including laser scanning confocal fluorescence microscopy, spinning disk confocal microscopy, and a combination of wide-field (WF) and total internal reflection (TIR) fluorescence microscopy [4]–[16]. These studies have yielded an array of data and models, in part depending on the cell type or imaging method uses, and a range of different views of how coats form, of potential roles for actin and the cytoskeleton, and of possible mechanistic similarities between clathrin-based structures in yeast and mammalian cells (reviewed in [1],[17],[18]). A combination of high- and moderate-resolution molecular structures from X-ray crystallography and electron cryomicroscopy (cryoEM) and dynamic data from live-cell fluorescence imaging of BSC1 cells [5],[14],[19]–[22] has led to the following picture for coated pit formation. At widely distributed locations on the plasma membrane, sequential recruitment of clathrin and its adaptors progressively bends the underlying bilayer as a clathrin lattice assembles; the adaptors selectively capture membrane-bound proteins destined for endocytosis; membrane pinching separates the fully formed coated vesicle from its parent membrane; and an ATP-dependent chaperone uncoats the released vesicle. Important features of this process in BSC1 cells are that most of the endocytic structures are smaller than the diffraction limit of the optical microscope (∼250 nm) and that the coated pits form progressively, over a time between nucleation and pinching of 35–65 s [5]. In other types of cells—HeLa and COS cells in particular—there are, in addition to the rapidly forming coated pits with characteristics similar to those in BSC1 cells, larger and more stable clathrin structures. The latter are present only on the “bottom” (adherent, coverslip proximal) surface of the cell, and we initially thought that they might represent clathrin reservoirs [5]. Relatively flat, extended clathrin arrays, referred to as “clathrin plaques” [23], have been observed by conventional electron microscopy on the bottom surface of HeLa cells and osteoclasts. These plaques probably correspond to the extended hexagonal lattices of clathrin seen in these and other cell types (e.g., Swiss 3T3 cells) when imaged by rapid-freeze, deep-etch electron microscopy [24]–[27]. We believe that the plaques also correspond to the relatively long-lived clathrin structures (lifetimes >3 min) at the substrate-facing surfaces of primary adipose cells imaged in real time by TIR [28], of Swiss 3T3 cells imaged in real time by TIR and WF fluorescence illumination [9],[10], and of HeLa and COS cells as described above [5]. Many of these long-lived clathrin structures display an abrupt inward movement at the end of their lifetime—a process associated with membrane internalization and transferrin uptake [9],[10]. Because no firm distinction has previously been made between the dynamics of clathrin-coated pits and plaques, the observations just summarized have spawned a model in which preformed clathrin lattices on the plasma membrane undergo an extensive structural reorganization, leading to coat curvature, membrane invagination, and vesicle budding [18],[29]. Different results and interpretations on the potential role of actin in clathrin-based endocytosis have accompanied these fundamentally different models for the mechanics of coat assembly. A connection between actin and clathrin-driven membrane uptake was first described in yeast [30],[31]. Acute depolymerization of actin by latrunculin A inhibits clathrin-based endocytosis in some metazoan cells, but not in others [32]. Latrunculin treatment has no detectable effect on the dynamics of endocytic coated pits in BSC1 cells [33], but it strongly inhibits endocytosis in Swiss 3T3 cells, with a marked reduction in the number of clathrin-based endocytic events [15]. Similarly, perturbation of the actin cytoskeleton by genetic means, such as depletion of Hip1R [34], perturbations in Hip1R association with cortactin [8], and depletion of N-WASP [35], has variable effects that depend on cell type. Some clathrin endocytic structures recruit actin or cortactin at the time they bud and disappear, but others, in the same cells, do not [9],[10],[15],[36]–[38]. In an effort to differentiate among distinct modes of clathrin-mediated endocytosis, we have measured the properties of clathrin assemblies on both free (top) and adherent (bottom) lower surfaces of several mammalian cell types. Recently introduced methods in live-cell imaging enable us to make distinctions more readily than has previously been possible. We find that a coated pit grows continuously as an invaginating shell, which pinches off immediately upon completion, as in the usual pictures. The entire process typically takes 30–60 s. A coated plaque grows initially at about the same rate as a coated pit, but without displacement from the cell surface. Its growth reaches a fluctuating plateau, generally two to three times that of a typical pit, remaining in place for up to several minutes before moving uniformly inward a few seconds before membrane pinching. The actin cytoskeleton is not required for normal formation and budding of the rapidly growing pits, but it is essential for the formation, inward movement, and dissolution of plaques. We have thus distinguished two modes of clathrin-coat formation at the plasma membrane, with quite different mechanisms for coat internalization. That is, clathrin is a scaffold for at least two distinct, membrane-associated processes. The distinction allows us to resolve apparent contradictions in the previous literature of clathrin-mediated endocytosis. Results Comparison of Clathrin Dynamics on the Upper and Lower Surfaces of Swiss 3T3 Cells We compared the dynamics of clathrin-containing assemblies on the free surface of Swiss 3T3 cells with their dynamics at the surface in contact with the coverslip (Figure 1 and Videos S1 and S2). For these experiments, we used a previously described cell line in which clathrin is labeled by stable expression of LCa-dsRed [9], and we recorded images using spinning disk confocal imaging. We restricted our analysis to objects that were relatively stationary in x and y, as most of the more motile ones correspond to endosomes [5],[7]. By using exposure times of 100 ms per frame and imaging every 2 to 10 s, we could record for periods of up to 60 min without obvious signs of phototoxicity. All experiments reported here were carried out under conditions in which internalization of transferrin, used as a probe of clathrin-dependent uptake, was not affected by expression of the fluorescent chimeric proteins. 10.1371/journal.pbio.1000191.g001 Figure 1 Formation of clathrin-coated structures on the plasma membrane of cells. (A–E) Time-series images of clathrin assemblies at the top or bottom of Swiss-3T3 cells acquired with a spinning disk confocal microscope (Videos S1 and S2). The fluorescent assemblies were tagged with clathrin LCa-DsRed stably expressed in Swiss-3T3 [9] in the absence (A–D) or presence (E) of transiently expressed auxilin1-EGFP. (A) Representative kymograph obtained from a time series recorded every 5 s (100-ms exposure) for 15 min from the free (top) surface of the cell, showing that the majority of the structures are relatively short-lived. (B) Representative kymograph obtained from a time series recorded every 5 s (100-ms exposure) for 15 min from the adherent (bottom) surface of the cell. All structures are dynamic, but many have longer average lifetimes than those in (A). (C) Selected snapshots from the time series shown in (B) to illustrate examples of clathrin structures displaying the dynamic behavior characteristic of canonical coated pits, of a group of canonical coated pits forming consecutively at a single location (hot spot), and of a plaque. The peak fluorescence intensity of pits assembling in isolation is similar to those forming as part of a hot spot; the overall intensity of the plaque is significantly higher. The acquisition time of each snapshot (in seconds) is indicated. (D) Fluorescence intensity profiles of the clathrin assemblies shown in (C). (E) Fluorescence intensity profiles of clathrin coats tagged with LCa-DsRed and Auxilin1-EGFP to illustrate the dynamics of canonical pits forming in a hot spot and of plaques. A burst of auxilin recruitment coincides with the uncoating step of a canonical pit [22],[43], or with each of the uncoating steps observed within a hot spot; in contrast, variable amounts of auxilin are recruited during the entire life of the plaque, ending with a larger burst. (F) Electron microscopy of clathrin-coated structures on the adherent surface of unroofed BSC1 and HeLa cells. Representative electron micrographs illustrate in (a) the exclusive presence of clathrin-coated pits with various degree of invagination in the plasma membrane of unroofed BSC1 cells; 79 pits were captured in 49 pictures (156 µm2). Representative electron micrographs illustrate in (b and c) the coexistence of clathrin-coated pits and clathrin flat arrays in the plasma membrane of unroofed HeLa cells. 69 pits and 48 sheets were captured in 59 pictures (188 µm2); Bar indicates 100 nm. Nearly all the fluorescent spots on the free surface of the Swiss 3T3 cells belonged to a single class of diffraction-limited objects, as expected for coated pits or vesicles 500 s) clathrin structures were present on the adherent surface, however (Video S5). These spots contained a very small amount of AP-2, with a fluorescent signal barely above background—less than 2% of the signal from plaques in HeLa cells not depleted of AP-2. As there were no conventional coated pits on the top or bottom surface of the cells, we interpret the immobile structures on the bottom surface of AP-2–depleted cells as nascent plaques that were unable to grow, because they had exhausted the extremely limited amount of residual AP-2 present in those cells. We also detected motile clathrin-coated structures in the cytosol and perinuclear regions, with the usual characteristics of endosome- or TGN-associated coats containing AP-1, AP-3, GGAs, or Hrs. We conclude that AP-2 is essential for the formation of conventional coated pits and probably also for the initiation and growth of coated plaques. Recruitment of Auxilin Auxilins 1 and 2 are J-domain cofactors for Hsc70, the uncoating ATPase [42]. Auxilins appear in coated vesicles in a characteristic burst that lasts several seconds, just at the onset of disassembly of the clathrin/AP-2 coat [22],[43]. We find similar bursts of transiently expressed auxilin-1-EGFP in coated vesicles of the LCa-DsRed–expressing Swiss 3T3 cells (Figure 1E), and we can record auxilin bursts at the onset of each of the serial dissolutions of the clathrin signals observed during the lifetime of a hotspot (Figure 1E). Small, variable levels of auxilin are also present in association with clathrin during the lifetime of conventional coated pits and hotspots. These common features of isolated pits and hotspots support the interpretation that the latter correspond to sequential assembly and independent budding of conventional coated pits and vesicles, at positions that all lie within the resolution limit of the microscope. In contrast, coated plaques recruit substantially greater quantities of auxilin during their lifetime and at the onset of their dissolution than do the conventional structures (Figure 1E). We could not detect any direct correlation between variations in clathrin and auxilin fluorescence during the lifetime of the plaques (Figure 1E and unpublished data). We conclude that sequential formation of conventional pits and vesicles and their detachment from a plaque is not an adequate explanation for the fluctuations in clathrin and AP-2 seen during the lifetime of the plaques. Role of Actin Coated pits and plaques differ particularly strikingly in the relationship between their properties and the dynamic state of actin. Treatment of Swiss 3T3 cells with latrunculin A, which depolymerizes the actin cytoskeleton, prevents both formation of new plaques and dissolution of old ones but has no effect on the dynamics of assembly and dissolution of conventional clathrin-coated pits (Figure 2F, Figure S1, and Video S6). These observations confirm our earlier work on clathrin dynamics in BSC1 cells (which lack plaques) [33]. We showed that formation of conventional clathrin-coated pits is not sensitive to loss of actin dynamics after treatment with either latrunculin A or cytochalasin D. Other groups have reported that treatment with latrunculin A of Swiss 3T3 cells (which contain both pits and plaques) prevents coat dissolution at the adhered surface [15]—presumably because the observations were weighted heavily toward the coated plaques. There are two known connections between actin and clathrin assemblies in cells. One is huntingtin-interacting protein 1-related (Hip1R), which has binding sites for F-actin, cortactin, and clathrin light chains [44]–[47]. Interference with the interaction between Hip1R and clathrin light chains by overexpression of a light-chain mutant unable to associate with Hip1R retains the cation-independent mannose-6-phosphate receptor (CI-MPR) in the TGN but has no detectable effect on transferrin or EGF uptake [47]. The other connection between clathrin coats and actin dynamics involves cortactin, which activates the actin nucleation factor Arp2/3 and leads to stimulation of branched actin filament assembly [48]–[50]. It is believed that dynamin recruits cortactin through interaction of the proline-rich C-terminus of dynamin with the SH3-domain of cortactin [51]. To disrupt the interaction between clathrin and Hip1R, we used the dominant-negative clathrin LCb-EED/QQN fused to EGFP [47], a mutant protein that binds normally to clathrin heavy chain but is deficient in its interaction with Hip1R [44],[46] We found that transient overexpression of EGFP-LCb-EED/QQN in Swiss 3T3 cells stably expressing LCb-DsRed or in U373 astrocytes resulted in complete elimination of plaques but that it had no discernable effects on the formation and properties of normal pits (Figures 3B and 4A). In agreement with earlier observations obtained with HeLa cells [47], overexpression of EGFP-LCb-EED/QQN had no discernable effects on the clathrin-dependent receptor-mediated uptake of fluorescent transferrin in astrocytes (Figure S5), a result that is consistent with the small relative contribution of plaques to endocytic traffic (see below). As expected, the mutant light chain colocalized with and replaced wild-type LCa-DsRed (Figure 3A), with an efficiency of approximately 85% as determined by the extent in the decrease of the fluorescence signal of LCa-DsRed at any clathrin spot. Control experiments performed by expression of wild-type EGFP-LCb showed no effects on the formation of clathrin plaques or pits (Figures 3B and 4A) even though the replacement level was similar to that achieved with EGFP-LCb EED/QQN (Figure 3A). 10.1371/journal.pbio.1000191.g003 Figure 3 Disruption of plaque formation by interference with the function of clathrin light chains. Loss of plaques upon expression of EFGP-LCb-EED/QQN (A and B) or due to depletion of clathrin light chains or Hip1R by RNAi (C, D, and E). (A) Effective replacement of LCa-DsRed with EGFP-LCb or EFGP-LCb-EED/QQN. Swiss 3T3 cells stably expressing LCa-DsRed were transfected with a modified form of clathrin EGFP-LCb, in which the conserved three critical residues EED required for the interaction of the light chains with Hip1R were mutated to QQN. As control, cells were transfected with no plasmid or with wild-type EGFP-LCb. The fluorescence images show the characteristic punctate pattern of clathrin light chains elicited by the LCa constructs and, due to replacement, the corresponding loss of LCb fluorescence. The estimated replacement level is 85% (n = 170). (B) Expression of EGFP-LCb-EED/QQN prevents formation of plaques, but not of pits. The panels are representative semi-logarithmic plots of maximum fluorescence versus time for pits and plaques from two Swiss 3T3 cells expressing similar amounts of EGFP-LCb or EGFP-LCb-EED/QQN. The average lifetime of canonical pits on the bottom of the Swiss 3T3 cells expressing EGFP-LCb is 106±31 s (n = 45) and 107±31 s (n = 71) in cells expressing EFGP-LCb-EED/QQN. The maximum fluorescence intensities of these pits are 2,131±968 and 2,526±1,392, respectively. The average lifetime and maximum fluorescence intensity of plaques on the bottom of the Swiss 3T3 cells expressing EGFP-LCb are 291±76 s and 3,701±1,451 (n = 27). The differences in average lifetime or in maximum fluorescence intensity between pits and plaques in cells expressing EGFP-LCb is statistically significant (p 200 s). These and other published tracings [13],[15],[38],[43] show the final interval in the lifetime of these objects but do not illustrate the growth phase. A characteristic of all the structures they examine is an abrupt inward movement coincident with membrane budding shortly before dissolution of the coat. These steps represent bona fide endocytic events, by the criterion that entrapped transferrin-receptor, tagged with a pH-sensitive fluorophore, does not respond to pH changes in the medium once the structure has begun to uncoat [10]. We have now been able to use a similar TIR-WF protocol, but with substantially longer acquisition periods, to capture unambiguously the complete lifecycle of all clathrin structures, whether long or short. We specifically included in our analysis the Swiss 3T3 cell line studied by Merrifield et al. We confirmed that all the long-lived structures at the adherent surface indeed move inward abruptly just before losing their coats, in contrast to the continuous inward movement of clathrin and AP-2 during the growing phase of a canonical coated pit. We conclude that the long-lived endocytic clathrin structures analyzed by Merrifield et al., are the same as the clathrin-coated plaques described here. They are probably also equivalent to the persistent clathrin-containing structures observed by TIR in primary adipocytes [28]. These assemblies, which represent more than half the total clathrin signal at any time point, have variable size, are on average 2.5 times larger than pits, and have lifetimes longer than 3 min. Taking into consideration that plaques are relatively larger and that they invaginate at a considerably smaller frequency than pits, and including the contribution of pits forming on the free surface, we estimate that plaques carry approximately 11% of the transferrin uptake mediated by clathrin. This estimate is in agreement with the insignificant change of transferrin uptake in cells lacking plaques due to perturbations in the interaction between clathrin light chains and Hip1R (this study; see also [47]) or due to Hip1R ablation by RNAi [34]. Do the long-lived coated structures correspond to the gently domed, extended arrays of clathrin seen on the adherent surface of unroofed cells by freeze-etch electron microscopy? Several lines of evidence argue that they do. First, the extended arrays are seen only on the cytoplasmic surfaces of adherent plasma membranes containing plaques as detected by live-cell imaging—e.g., the HeLa cells imaged in Figure 1—and not on the corresponding surfaces of cells—e.g., the BSC1 cells in Figure 1—that do not contain plaques. Second, the sizes of the arrays seen by electron microscopy and the plaques described here are similar. The largest of the hexagonal arrays in published micrographs are about 500–1,000 nm in diameter, and most are smaller (this study and [24]–[27]). The plaques have dimensions that range from diffraction limited to several hundred nanometers. Third, the DiNa measurements described here show that the coated plaques we have characterized are essentially flat, as are those seen by electron microscopy. Fourth, extended fluorescent patches induced during InlB-dependent uptake of Listeria monocytogenes [58] probably correspond to hexagonal flat arrays seen by microscopy during engulfment of latex beads by macrophages [24]. Why do the available electron micrographs of thinly sectioned samples not show deeply invaginated plaques? A simple explanation is provided in the graphical representation presented in Figure S6; the data were obtained from a time-lapse series, and show, for every frame of the video, the number of structures scored as pit or plaque, and the number of pits scored as deeply invaginated (the last 10–15 s of their lifetime) or plaques scored as displaced from the substrate (by 100–150 nm during the last 5 s of their lifetime). This analysis reveals detection of a total of 799 structures scored as pits and 544 as plaques, with 197 images of deeply invaginated pits and only 11 substantially displaced plaques. Thus, the likelihood of visualizing deeply invaginating plaques by electron microscopy of thin sections is extremely low, and presently it is unrealistic to expect acquisition of a sufficient number of images to draw confident conclusions. Coated Plaque Assembly Spontaneous clathrin assembly under suitable conditions in vitro yields closed structures that range in diameter from 60 to 200 nm. Reconstructions from cryoEM images of D6-barrel and tetrahedral coats show that curvature is built into the pucker at the apex of a triskelion and that side-by-side packing of the legs can vary so as to create much flatter lattices [21]. The hexagonal arrays seen by electron microscopy on the inward-facing surface of membranes apposed to plastic substrates or bacterial cell walls are quite imperfect, with frequent defects, as if their enforced planar assembly had introduced strain, just as expected if we were to extrapolate to zero curvature the variation in lateral contacts seen in more sharply curved coat structures. Thus, the extended flat arrays could arise because adhesion to the external substrate resists introduction of curvature, and essentially planar arrays grow until the accumulated strain is best compensated by a defect in the lattice. But the requirement of an intact light-chain-Hip1R-actin linkage for plaques to appear and the correlation of plaques with cell motility both suggest that cytoskeletal interactions may help direct coated plaque formation. AP-2 is also essential, for both coated plaques and canonical coated pits at the plasma membrane (this paper). It is therefore possible that pits and plaques initiate similarly, through an AP-2 dependent mechanism, but that the organization of the actin cytoskeleton dictated by adhesion to a substrate redirects clathrin assembly into planar arrays. To outline a specific mechanism for this redirection would require a more detailed picture than now available of how Hip1R associates with its partners. We note that initiation of clathrin coats that is AP-2 independent can also occur, as the Listeria-directed arrays neither incorporate AP-2 nor require it [59]. Epsin Epsin accumulates along with clathrin during assembly of a canonical coated pit. Our measurements of the relative z-displacement of epsin and clathrin show that the former remains in the plane of the surrounding membrane as the coated pit invaginates. That is, most of epsin does not move into the domed region, nor is it present in the mature coated vesicle. It therefore must accumulate largely around the rim of the pit, as does its partner, Eps15 [55],[56]. A limited number of published [54],[57] and unpublished electron micrographs (L. Traub, personal communication) suggest an accumulation of epsin on the rim of the pits, but the number of available images are not sufficient to draw a firm conclusion. At very high concentrations, epsin can tubulate liposomes, apparently by insertion of an N-terminal amphipathic helix into the outer leaflet of the bilayer [60]. This property has led to the suggestion that a function of epsin in coated-pit formation is to stabilize membrane curvature [60]. The location of most of the epsin, at the base of the invaginating pit, a position at which there is little net curvature, is not consistent with this proposal. Moreover, epsin is present in the essentially uncurved coated plaques, where its fluorescence intensity fluctuates in parallel with clathrin, and it moves inwards together with clathrin when the plaque internalizes. Because there is no curvature, and hence no differential z-displacement, we cannot tell from the DiNa measurements whether epsin is present throughout the plaque, or mostly at its rim as in a canonical coated pit. Published electron micrographs suggest that epsin may be distributed throughout the lattice [54],[57], but the studies have not been extensive enough to draw firm conclusions. Epsin contains multiple ubiquitin-interacting motifs (UIMs), and it is believed to facilitate incorporation of polyubiquitinated cargo into clathrin structures [57], whereas association of epsin with monoubiquitinated substrates is reported to exclude interaction with clathrin coats [61]. How epsin's putative function as a receptor for ubiquitinated cargo relates to its localization remains to be determined. Membrane Budding and Uncoating The clearly defined, inward shift of a coated plaque, which invariably occurs shortly before dissolution, depends on actin and dynamin. As this shift is uniform across the plaque, there is no need to invoke any reorganization of the clathrin lattice into a curved structure [18],[29]; membrane uptake is clearly determined by other processes. Local reorganization of the actin cytoskeleton is the best candidate for the driving force of this invagination. There is considerable evidence that the cortactin-activated, Arp2/3 branching mechanism may have a role [10],[15],[34]. For example, Merrifield et al. [10] report cortactin recruitment at late stages of coat formation, and they link its arrival to internalization. Engqvist-Goldstein et al. [34] show that recruitment of Hip1R into long-lived clathrin coats (which we interpret as coated plaques) follows clathrin accumulation (as might be expected from its direct interaction with light chains), whereas cortactin peaks just before internalization. Our data on coated plaques are in full agreement with these observations. As Hip1R is essential for plaque initiation, we cannot determine whether its linkage to actin is also critical for internalization. Our data suggest that uncoating of a plaque proceeds by an auxilin- and Hsc70-dependent mechanism, as for canonical coated vesicles. Auxilin is recruited during the last seconds of a coated plaque lifetime, but it is also incorporated in variable amounts during the course of assembly. It is possible that auxilin and Hsc70 catalyze exchange of clathrin into and out of the plaque during its lifetime [43]. Plaques, Actin, and Yeast Cells Ample genetic evidence points to a pathway that requires both clathrin and actin for endocytosis in yeast cells (reviewed in [30]). Moreover, live-cell fluorescence microscopy shows that clathrin, actin, and some of their interacting proteins are recruited to distinct sites at the plasma membrane, beginning with clathrin and Las17 (a regulator of Arp2/3) followed by actin and ending with a number of kinases and phosphatases, which facilitate efficient disassembly of different endocytic coat proteins [62]. This sequence of molecular associations parallels inward movement of the clathrin fluorescent spot [4],[11],[62]. Tubular invaginations of up to 50 nm in diameter and 180 nm in length, decorated with clathrin at their tips, can be seen emanating from the plasma membrane [63]. Thus, actin-based activities appear to drive a partially coated clathrin structure inward. Clathrin may provide the membrane anchor, and some additional activity may then drive membrane scission. We suggest that the endocytic pathway and its mechanisms characterized in yeast are closely related to the uptake of coated plaques in more complex eukaryotic cells. Functions of Clathrin-Coated Pits and Coated Plaques The endocytic functions of canonical coated pits have been well documented. The canonical structures are the principal carriers for uptake of transferrin receptor and many other membrane-inserted proteins and for rapid reuptake of membrane components at synapses [2],[3]. Coated plaques can also take up transferrin (and presumably other receptor-bound ligands) [9],[10],[28], but their principal physiological role remains to be determined. When they do form, coated plaques appear to require contact with an external substrate. Conversely, the increased cell motility we see when coated plaques are ablated suggests that loss of plaques is linked to a loss of firm attachment. The close interplay between the actin and clathrin systems responsible for plaque internalization may also have been hijacked by invaders such as certain bacteria [59] and by large viruses [36]. Clathrin, dynamin, actin, and Arp2/3 are all required for invasion by L. monocytogenes and for pinocytosis of beads coated with the invasin InlB [58],[59],[64]. Extended arrays of clathrin have been seen on the adherent surface of osteoclasts [25],[26], on the cytoplasmic face of the invaginated cell membrane during bead uptake [24], and extended clathrin arrays are also present on endosomes [65]. The latter arrays are not associated with the usual heterotetrameric adaptor proteins. In summary, we have established criteria for identifying coated plaques, and we have described many of their properties as detected by live-cell fluorescence microscopy. The properties of coated plaques, as worked out by quantitative analysis of our live-cell data, are distinct from the properties of canonical coated pits and are fully consistent with all published observations of clathrin-coated structures by electron microscopy, including the new electron microscopy data presented here. The participation of actin in coated-plaque internalization is the most obvious difference between these modes of membrane remodeling, but we have documented others, including the dynamics of coat assembly and the relationship to processes such as adhesion and motility. Clathrin is clearly a scaffold for a number of mechanistically distinct, membrane-associated process. Materials and Methods Preparation of Plasmids, Expressor Cells, Immunofluorescence, and RNAi Swiss 3T3 cells expressing LCa-DsRed were kindly provided by Dr. W. Almers, BSC1, and U373 cells expressing σ2-EGFP were previously characterized in [5],[22]. LCa-DsRed was made by fusing rat LCa DNA to the 5′ end of the coding sequence for DsRed (Clonetech). Epsin-EGFP was created by fusing the rat epsin-1 DNA to the 5′ end of the coding sequence of EGFP (Clonetech). Tomato-LCa was created from rat LCa as described in [22]. BSC1 cells stably expressing epsin-EGFP were obtained by transfection using Fugene 6 (Roche Applied Science). A single clone of low-expressing Epsin-EGFP was maintained by selection with G418. All cells were maintained at 37°C and 5% CO2 in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum and 0.4 mg/ml G418. As a control, BSC1 cells were transfected with LCa-DsRed and imaged 48 h after transfection. The dynamics of the clathrin spots under these conditions (Figure S3) show no difference to those in EGFP- or Tomato-LCa–expressing cells. U373 astrocyte cells stably expressing σ2-EGFP were used in conjunction with transient expression of Tomato-LCa and imaged after 48 h of transfection. Swiss 3T3 cells stably expressing LCa-DsRed were used in conjunction with transient expression of epsin-EGFP and imaged after 48 h of transfection. Cells transiently expressing cortactin-EGFP (gift of Dr. D. Drubin), EGFP-LCb, or EGFP-LCb-EED/QQN (gift of Dr. P. McPherson) were also imaged after 48 h of transfection. All transfections were carried out using Fugene 6 according to manufacturer instructions. For immunofluorescence, a monoclonal antibody (mAb), CON.1, specific for clathrin LCa and LCb was generated from hybridoma cells obtained from the American Type Collection. Prior to staining, the cells were fixed with 3.75% paraformaldehyde, and then sequentially labeled with CON.1 and a Cy3-labeled antimouse polyclonal antibody. Depletion of the clathrin light chains was achieved by sRNAi-mediated gene silencing experiments by transfecting U373 astrocytes stably expressing σ2-EGFP with a mixture containing 200 nM of LCa oligo (D-004002-01; Dharmacon) and 200 nM of LCb oligo (D-004003-03; Dharmacon) with Oligofectamine (Invitrogen); the treatment was repeated after 48 h, and the cells were replated after 62 h onto glass coverslips for imaging 10 h after. A scrambled oligo sequence (Dharmacon; gift from Dr. J. Liberman) was used as control. The efficiency of LCa and LCb depletion was determined by measuring the loss of immunofluorescence using CON.1 specific for both light chains. Depletion of Hip1R was achieved by sRNAi-mediated gene silencing experiments by transfecting U373 astrocytes stably expressing σ2-EGFP with 200 nM of a mixture containing Hip1R oligos (L-027079-00; Dharmacon) and with Oligofectamine (Invitrogen) or by first transfecting U3T3 astrocytes with the mixture of Hip1R oligos, which where then transfected 2 d later with EGFP-LCb. Handling of Cells Prior to Imaging Roughly 20,000–40,000 cells were plated on glass #1.5 coverslips (25 mm in diameter) 10 h before the actual imaging experiment. Up to this point, the cells were maintained at 37°C and 5% CO2 in DMEM with 10% fetal bovine serum. Cells were imaged in Gibco's CO2 independent medium and in the absence of phenol red (Invitrogen). The temperature of the sample holder (20/20 Technologies) was kept at 37°C using a Peltier-controlled holding device. The holding device and the stage were surrounded by a custom-designed air-controlled environmental chamber kept at 33–35°C. Imaging All spinning disk confocal imaging experiments were conducted by using the microscope setup previously described [5]. We further modified the microscope by insertion of a computer-controlled Spherical Aberration Correction unit (SAC; Intelligent Imaging Innovations), which drastically reduced the spherical aberration and thus increased the sensitivity. In addition, this setup was modified with lasers equipped at 473 nm and 561 nm (Cobolt) and 660 nm (Crystal Lasers). The lasers input to the fiber optic is controlled through a PCAOM AOTF module (Neos). The emission is collected after the spinning disk unit and passes through a dual view unit (Roper Scientific), equipped with a 565DCXR dichroic mirror and the HQ525/40 and HQ620/50 (Chroma) to separate the fluorescence of EGFP and Tomato/DsRed on two sides of the same CCD chip. The camera has been updated to a Cascade 512B (Roper Scientific), which is operated with multiplication gain and no binning. The Dual view unit and the AOTF allow for simultaneous or for fast switching between excitation with 473 and 561 nm. The sample is only exposed to the laser light while the CCD is acquiring, and is immediately turned off during the frame transfer time of the CCD, which can take 50–200 ms, depending on mode of readout. This shutoff is achieved by synchronizing the AOTF with the camera frame readout TTL signal (Intelligent Imaging Innovations). Using this setup, we have been able to image an exposure as low as 50–100 ms, reducing phototoxicity and allowing longer measurement times. Images were captured with SlideBook 4 software (Intelligent Imaging Innovations). All TIR/WF measurements were carried out using the conditions previously described [14]. Image Processing Clathrin clusters were identified using a sequence of deconvolution, 2D Gaussian and Laplacian filtering followed by thresholding, which created a mask as previously explained in [5],[22]. The masks were tracked using SlideBook 4 (Intelligent Imaging Innovations). The coordinates of the center of these masks versus time were exported along with the images. A MATLAB routine [14] was used to read the intensity profile for each object from the corresponding images. DiNa measurements were carried as previously described [14]. Clathrin-coated structures were selected according to the following criteria [5],[14],[22]: (1) the fluorescent objects appeared and disappeared within the time series; (2) the objects displayed the limited movement expected for membrane-bound clathrin structures in the horizontal plane during their growth phase (500 nm/lifetime); and (3) the objects did not collide with each other. Cell motility as a function of time was determined in cells imaged in WF bright illumination, by tracking the temporal change in the position of the center of the nucleus. Electron Microscopy BSC1 and HeLa cells were plated on glass coverslips and grown for 4 h (BSC1) or overnight (HeLa) at 37°C and 5% CO2 in DMEM with 10% fetal bovine serum. To visualize the adherent surface of the cells, the samples were first broken open (“unroofed”) by standard methods, followed by chemical fixation, rapid freezing, deep etching, and rotary shadowing. [40]. The samples were visualized using a JEOL JEM 1200EX microscope operating at a nominal magnification of 30,000 and 80 kV, and imaged digitally with an AMTKK CCD camera. Supporting Information Figure S1 Effect of latrunculin A on the lifetime and dynamics of pits and plaques. The kymograph is a schematic representation as a function of time of the effect of actin depolymerization on the lifetime of pits (gray) and plaques (black) in the adhered surface of a Swiss 3T3 cell stably expressing LCa-DsRed. The data derived from the time series obtained from a cell imaged using spinning disk confocal microscopy at 10-s intervals and 100-ms exposure time (Video S3). Acquisition of the time series was started at t = 0, and 5 µM latrunculin A was added at t = 540 sec. As shown before with similarly treated BSC1 cells [33], actin depolymerization results in the accumulation of clathrin structures on the plasma membrane but does not affect the dynamics of coated pits. Formation of new plaques ceases, and existing plaques completely freeze upon latrunculin A treatment. (2.12 MB TIF) Click here for additional data file. Figure S2 Intensity profiles of coated pits and plaques. Concatenated tracings of different coated pits (gray) and plaques (black). The characteristic times for intensity fluctuations within the lifetime of a plaque are much shorter than the lifetime of a coated pit. A formal autocorrelation analysis (unpublished data) confirms this conclusion. Thus, the properties of a plaque cannot be attributed to those of a series of coated pits forming one after another at the same location. (2.43 MB TIF) Click here for additional data file. Figure S3 Formation of clathrin structures in BSC1 cells expressing clathrin LCa-DsRed. Images from a time series acquired by spinning disk confocal microscopy from the adherent surface of a BSC1 cell transiently expressing clathrin LCa-DsRed. The cell was imaged 48 h after transfection, using 3-s intervals for a duration of 300-s and 150-ms exposures. The average lifetime of the clathrin structures (53±17 s) is similar to the average lifetime of canonical pits previously characterized in BSC1 cells stably or transiently expressing EGFP-LCa [5]. (1.68 MB TIF) Click here for additional data file. Figure S4 Sizes and lifetimes of clathrin-coated structures forming at the cell surfaces. In these double logarithmic plots, each dot corresponds to the duration (lifetime) and the corresponding size (maximum fluorescence intensity prior to dissolution) of a clathrin or AP-2 fluorescent spot recorded at the top or bottom plasma membrane of adherent cells. Each panel contains data obtained from three cells and includes the observations obtained from the single cells depicted in the experiments associated with Figures 2, 3, and 4. (A) Average lifetime of the canonical coated pits on the top of Swiss 3T3 cells stably expressing Clathrin LCa-DsRed is 54.6±29 s (n = 193). The maximum fluorescence intensity is 1,988±1,387. (B) Average lifetime of the canonical coated pits on the top of U373 astrocytes stably expressing AP-2 σ2-EGFP is 61±31 s (n = 130). The maximum fluorescence intensity is 1,220±607. (C) Average lifetime of the canonical coated pits on the bottom of BSC1 cells stably expressing AP-2 σ2-EGFP is 48±20 s (n = 620). The maximum fluorescence intensity is 1,232±483. (D) Average lifetime of the canonical coated pits and coated plaques on the bottom of Swiss 3T3 cells stably expressing Clathrin LCa-DsRed are 57±26 s (n = 200) and 269±130 s (n = 104), respectively. The corresponding maximum fluorescence intensities are 995±711 and 3,269±2,725. The differences in average lifetime or in maximum fluorescence intensity between pits and plaques in Swiss 3T3 cells are statistically significant (p<0.0001). (E) Average lifetime of the canonical coated pits and coated plaques on the bottom of U373 astrocytes stably expressing AP-2 σ2-EGFP are 62±22 s (n = 525) and 228±104 s (n = 110), respectively. The corresponding maximum fluorescence intensity are 1,447±769 and 2,503±1,270. The differences in average lifetime or in maximum fluorescence intensity between pits and plaques in astrocytes are statistically significant (p<0.0001). (F) Effect of latrunculin A on the lifetime of canonical coated pits and coated plaques on the bottom of Swiss 3T3 cells stably expressing Clathrin LCa-DsRed. The average lifetime and maximum fluorescence intensity of the canonical coated pits after treatment with 5 µM of latrunculin A are 92±40 s (n = 80) and 1,394±1,017, respectively. In contrast, plaques become stable, and their lifetimes are longer than 2,000 s (the extent of the time series). (G) Average lifetime of the canonical coated pits on the bottom of Swiss 3T3 cells stably expressing Clathrin LCa-DsRed and transiently over expressing EGFP-LCb-EED/QQN is 60±22 s (n = 204). The maximum fluorescence intensity is 2,242±1,344. Note the complete absence of coated plaques. (H) Average lifetime of the canonical coated pits on the bottom of U373 astrocytes transiently over expressing EGFP-LCb-EED/QQN is 92.3±33.1 s (n = 402). The maximum fluorescence intensity is 1,219±619. Note the absence of coated plaques. (I) Average lifetime of the canonical coated pits on the bottom of U373 astrocytes depleted of both clathrin light chains by sRNAi treatment is 60±25 s (n = 555). The maximum fluorescence intensity is 674±385. Note the complete absence of coated plaques. (0.21 MB TIF) Click here for additional data file. Figure S5 Relative contribution of plaques to the internalization of transferrin in U373 cells. Cells transiently expressing EGFP-LCb or EGFP-LCb-EED/QQN for 3 d were incubated for 2 min at 37°C with 50 µg/ml Alexa 568 transferrin (Invitrogen). The cells were then washed with fresh DMEM containing 10% FBS for 5 min at 37°C and then fixed with 3% PFA. The whole volume of the cell was imaged using the spinning disk confocal microscope with 300-nm steps between consecutive imaging planes. The amount of internalized transferrin was estimated from the total fluorescence intensity; the small contribution of transferrin remaining at the cell surface was corrected by masking the outer border of the cell. Data shown for each condition are the average plus or minus the standard deviation, from five cells; the differences are not statistically significant. (4.05 MB TIF) Click here for additional data file. Figure S6 Scoring of events corresponding to pits and plaques at early and late stages of invagination or displacement. The data illustrate the distribution of structures scored as pits or plaques in each of the imaging frames. Note that the actual number of pits and plaques traced during the duration of the time series is smaller. They also show the events scored as deeply invaginated pits, imaged during the last 10–15 s of their lifetimes, and of “lifted” plaques (displaced from the substrate), imaged during the last 5 s of their lifetimes. These data were obtained from the time series used to generate Figure 2. The video spans an interval of 250 s (50 frames) and covers 308 µm2 of adherent surface. A total of 799 structures were scored as pits and 544 as plaques; and they include 167 deeply invaginated pits and 11 lifted plaques. (4.60 MB TIF) Click here for additional data file. Video S1 Formation of clathrin-coated structures on the free surface of Swiss 3T3 cells. Time-series images of clathrin assemblies at the plasma membrane on the top of a Swiss 3T3 fibroblast stably expressing clathrin LCa-DsRed imaged using spinning disk confocal microscopy at 5-s intervals and 100-ms exposure time. The video shows 68 frames (340 s). Notice that the mobility of the clathrin assemblies is in part due to overall movement of the cell; some structures are clathrin coats on mobile endosomes. (0.62 MB MOV) Click here for additional data file. Video S2 Formation of clathrin-coated structures on the adherent surface of Swiss 3T3 cells. Time-series images of clathrin assemblies at the plasma membrane on the bottom surface in contact with the coverslip of a Swiss 3T3 fibroblast stably expressing clathrin LCa-DsRed imaged using spinning disk confocal microscopy at 5-s intervals and 100-ms exposure time. Some structures are clathrin coats on mobile endosomes. (2.38 MB MOV) Click here for additional data file. Video S3 Formation of clathrin-coated structures on the adherent surface of Swiss 3T3 cells. Zoom region of Video S2 showing 68 frames (340 s). Examples of one hot spot containing three pits (#s 1, 2, and 3), one plaque (#4) and two coated pits (#5 and 6) are highlighted. Note that coated pit #6 is an object that is independent from coated plaque #4: although their points spread functions partially overlap for a brief moment, the corresponding centers always remain separated. (2.64 MB MOV) Click here for additional data file. Video S4 Comparison of coat dynamics between clathrin pits and plaques. Time-series images of clathrin assemblies at the plasma membrane on the adherent bottom of a U373 astrocyte (left) and a BSC1 cell (right) stably expressing σ2-EGFP imaged by spinning disk confocal microscopy at 10-s intervals and 70-ms exposure time. The video shows 80 frames (800 s). U3T3 cells form pits and plaques whereas plaques are not observed in BSC1 cells. (6.33 MB MOV) Click here for additional data file. Video S5 Clathrin-coat dynamics at the adherent surface of HeLa cells depleted of AP-2. Time-series images of clathrin in HeLa cells whose AP-2s were depleted by using sRNAi specific for μ2. The images were acquired by alternating two channels in order to register the fluorescent signals for the stably expressed σ2-EGFP (left panel) and the transiently expressed Tomato-LCa (right panel). The video was acquired by spinning disk confocal microscopy at 10-s intervals and 200- and 100-ms exposure times, respectively. The video shows 50 frames (500 s). The mobile clathrin-coated structures in the cytosol and perinuclear regions have the usual characteristics of endosome- or TGN-associated coats containing AP-1, AP-3, GGAs, or Hrs. (4.09 MB MOV) Click here for additional data file. Video S6 Effect of latrunculin A on the lifetime and dynamics of pits and plaques. Time-series obtained from a Swiss 3T3 fibroblast stably expressing clathrin LCa-DsRed imaged using spinning disk confocal microscopy at 10-s intervals and 100-ms exposure time. Acquisition of the time series was started at t = 0, and 5 µM latrunculin A was added at t = 540 s. (7.55 MB MOV) Click here for additional data file.

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Journal ID (nlm-ta): Nat Commun

Journal ID (iso-abbrev): Nat Commun

Title: Nature Communications

Publisher: Nature Pub. Group

ISSN (Electronic): 2041-1723

Publication date (Electronic): 23 May 2014

Volume: 5

Electronic Location Identifier: 3891

Affiliations

[1 ]Department of Bioengineering and Therapeutic Sciences, University of California San Francisco , San Francisco, California 94143, USA

[2 ]Department of Pharmaceutical Chemistry, University of California San Francisco , San Francisco, California 94143, USA

[3 ]Department of Microbiology and Immunology, University of California San Francisco , San Francisco, California 94143, USA

[4 ]The G.W. Hooper Foundation, University of California San Francisco , San Francisco, California 94143, USA

[5 ]Department of Biochemistry and Molecular Biology, Thomas Jefferson University , Philadelphia, Pennsylvania 19107, USA

[6 ]Department of Pathology and Centre for Trophoblast Research, University of Cambridge , Cambridge CB2 1QP, UK

[7 ]Department of Orthopedic Surgery, Thomas Jefferson University , Philadelphia, Pennsylvania 19107, USA

[8 ]These authors contributed equally to this work

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[a ] Frances.Brodsky@ 123456ucsf.edu

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Publisher Item ID: ncomms4891

DOI: 10.1038/ncomms4891

PMC ID: 4050264

PubMed ID: 24852344

SO-VID: 97896ea6-b4e1-4bf5-af6b-f98d3f4667e0

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This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

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Date received : 23 March 2014

Date accepted : 14 April 2014

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