MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
(e-mail: ben{at}mrc-lmb.cam.ac.uk)
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Summary |
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Key words: Caveosome, Endocytosis, Lipid raft
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Introduction |
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Endocytosis comprises multiple mechanisms that allow cells to internalize macromolecules and particles into transport vesicles derived from the plasma membrane (Conner and Schmid, 2003; Fig. 1). Together, these mechanisms have to control entry into the cell in a co-ordinated and specific manner, and they play a crucial role in many cellular processes. New data on endocytosis of raft markers not only provide insights into these mechanisms, but also constitute a good testing ground for the validity of competing models of lipid raft organization and function. The interplay between studies of specific endocytic pathways and more general consideration of the nature of lipid rafts and organization of the plasma membrane provides the basis of this Commentary. Recent data have also shed light on the role of rafts and caveolae in pathogen entry, and on post-endocytic trafficking of raft proteins and lipids. These topics are beyond the scope of this Commentary and are discussed elsewhere (Nichols et al., 2001
; Puri et al., 2001
; Lamaze et al., 2001
; Nichols, 2001; Sabharanjak et al., 2002
; Fivaz et al., 2002
; Duncan and Abraham, 2002; Nabi and Le, 2003
; Sharma et al., 2003
; Chazal and Gerlier, 2003
).
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Clathrin-independent endocytosis |
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A role for rafts? |
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First, nearly all molecules that are known to be internalized independently of clathrin are found in biochemically defined lipid rafts, and archetypical substrates for uptake by clathrin-coated pits, such as the low-density lipoprotein (LDL) and transferrin receptors, are not (Nichols and Lippincott-Schwartz, 2001). Raft components might thus be taken up preferentially by clathrin-independent endocytosis, or might be excluded from clathrin-coated pits.
Second, one widely used criterion for a functional involvement of lipid rafts is pharmacological depletion of cholesterol from cell membranes. Such treatment reduces the propensity of proteins and lipids to accumulate in DRM fractions (Simons and Ikonen, 1997; Edidin, 2003
), and cholesterol depletion blocks uptake of many of the molecules reported to be internalized independently of clathrin-coated pits (Lamaze et al., 2001
; Nichols et al., 2001
; Nichols and Lippincott-Schwartz, 2001
; Puri et al., 2001
; Sabharanjak et al., 2002
; Di Guglielmo et al., 2003
; Venkatesan et al., 2003
). Interpretation of these data in terms of a functional role for lipid rafts is somewhat complicated by the facts that cholesterol depletion is likely to have more effects on cell physiology than specifically perturbing lipid rafts and, when carried out stringently enough, will also inhibit uptake by clathrin-coated pits (Rodal et al., 1999
; Subtil et al., 1999
). A sceptic might additionally argue that, even in those cases where a clear differential effect on clathrin-independent uptake has been observed, this does not directly demonstrate a requirement for clustering of the relevant molecules in the same microdomain. Nevertheless, the greater cholesterol sensitivity of clathrin-independent endocytosis is likely to reflect differences in sensitivity to biophysical properties of the plasma membrane and is consistent with a functional role for rafts.
Third, caveolae, defined as morphological entities (small, uncoated invaginations in the plasma membrane), frequently contain the membrane protein caveolin 1 (Rothberg et al., 1992; Harder and Simons, 1997
), and the findings that caveolin 1 binds to cholesterol and is unusually resistant to detergent extraction (Sargiacomo et al., 1993
; Murata et al., 1995
) led to the suggestion that caveolae constitute a type of lipid raft (Harder and Simons, 1997
). The discoveries that treatment with a phosphatase inhibitor (okadaic acid) apparently causes budding of caveolae and associated proteins into the cell (Parton et al., 1994
; Thomsen et al., 2002
) and that dynamin, a GTPase involved in budding of clathrin-coated pits, is also found at the necks of caveolae (Oh et al., 1998
; Henley et al., 1998
) has generated much interest in the potential role of these structures as endocytic vesicles. Consequently, much research on cholesterol-sensitive, clathrin-independent endocytosis has focused on uptake in caveolae rather than other potential mechanisms (Fig. 1).
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Caveolar endocytosis |
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Two non-exclusive models go some way towards explaining these observations (Fig. 2). Experiments based largely on overexpression of caveolin 1 suggest that caveolin 1 negatively regulates caveolar budding. Uptake of gp60, a receptor for albumin localized to caveolae in endothelial cells, is inhibited by overexpression of caveolin 1 (Minshall et al., 2000). Clathrin-independent uptake of cholera toxin B subunit (CTB), which binds to the sphingolipid GM1, is also decreased by such overexpression (Le and Nabi, 2003
). Conversely, decreased expression of caveolin 1 in transformed NIH-3T3 cells correlates with increased clathrin-independent uptake of autocrine motility factor, and this effect is reversed when caveolin 1 expression is artificially increased (Le et al., 2002
). These data led Nabi and Le to propose that morphologically defined caveolar invaginations in the plasma membrane that do not contain caveolin 1 exist as transient intermediates during budding into the cell, and that these intermediates are stabilized by caveolin 1, thereby slowing the rate of budding (Nabi and Le, 2003
). This model is appealing because it provides a good explanation for the observation that cells lacking caveolin 1 lack abundant caveolae but maintain high rates of endocytosis of markers that, in caveolin-positive cells, are internalized by clathrin-independent, caveolae-related mechanisms (Orlandi and Fishman, 1998
; Lamaze et al., 2001
; Nichols, 2002
). A second way of explaining the data is to view caveolar endocytosis as a highly regulated process. There could, for example, be two functionally distinct populations of caveolin-positive caveolae, one population being largely static and the other actively budding from the plasma membrane. Little published data directly support this model, but live-cell microscopy does reveal the existence of a small pool of very active caveolin-containing vesicles (Mundy et al., 2002
). It is worth reiterating that these models are in no way exclusive and emphasizing that both invoke currently uncharacterized machinery to generate caveolar invaginations, or to regulate budding of caveolin-containing caveolae (Fig. 2). Discovery of further proteins required for caveolar endocytosis is clearly required to facilitate further progress in this area.
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Caveolar budding might be regulated by reversible phosphorylation. Treatment with a phosphatase inhibitor (okadaic acid) causes massive mobilization of previously static caveolae, and a Src-family kinase inhibitor, genistein, blocks presumptively caveolar endocytosis (Parton et al., 1994; Puri et al., 2001
; Thomsen et al., 2002
; Sharma et al., 2003
). How these effects are mediated is not clear. Caveolin 1 is phosphorylated on at least one tyrosine residue (Y14) in response to a variety of stimuli, including insulin, angiotensin II, osmotic shock and oxidative stress (Mastick et al., 1995
; Aoki et al., 1999
; Volonte et al., 2001
), probably by non-receptor tyrosine kinases such as c-Fyn, c-Src and c-Abl (Sanguinetti and Mastick, 2003
). However, caveolin 1 is thought to interact with several different signalling receptors (reviewed by Liu et al., 2002
), and a direct functional role for caveolin 1 phosphorylation in endocytosis, rather than an interaction with signalling molecules (Cao et al., 2002
), has yet to be demonstrated.
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Caveosomes |
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The emergence of caveosomes as a distinct organelle raises the question of the functional significance of uptake by a caveolar mechanism as opposed to clathrin-coated pits. The idea that different types of endocytosis have markedly different functions is supported by work on the uptake of cholera toxin (Orlandi and Fishman, 1998; Nichols et al., 2001
). For cholera toxin to be toxic it must reach the Golgi apparatus (Orlandi and Fishman, 1998
). Several studies have now shown that cholera toxin is taken up into the cell by multiple mechanisms (Torgersen et al., 2001
; Wolf et al., 2002
). One key experiment, by Orlandi and Fishman, used pharmacological perturbation of uptake via clathrin-coated pits or caveolae to show that changes in total uptake of the toxin do not simply correlate with changes in its toxicity. Rather, a block in uptake via clathrin-coated pits has little effect on toxicity, whereas selective inhibition of caveolar (cholesterol-sensitive) endocytosis is sufficient to prevent toxicity (Orlandi and Fishman, 1998
). These data argue against significant intracellular mixing of toxin taken up through caveolar endocytosis and clathrin-coated pits.
Recent experiments following endocytosis and signalling from the transforming growth factor ß (TGFß) receptor also highlight a functional distinction between delivery to caveosomes and classical early endosomes (Di Guglielmo et al., 2003). Wrana and colleagues showed that the TGFß receptor is constitutively endocytosed both by clathrin-coated pits into EEA1-positive early endosomes and by a clathrin-independent mechanism into caveosomes. Signal transduction by the TGFß receptor is affected differently by blocks in these different modes of endocytosis: uptake by clathrin-coated pits promotes TGFß signalling, whereas uptake into caveosomes leads to receptor turnover. Accordingly, accessory proteins involved in TGFß receptor signalling, such as SARA and Smad2, are found in EEA1-positive endosomes, and proteins that target the receptor for degradation, such as Smad7 and Smurf2, are localized to caveosomes. TGFß receptor can bind directly to caveolin 1, and this interaction could play a role in the targeting of TGFß receptor to caveolin-1-positive organelles (Razani et al., 2001b
). How this interaction might be regulated, and how a significant pool of TGFß receptor remains free to enter clathrin-coated pits, is not fully understood. It seems unlikely that caveolin 1 is critical for TGFß signalling per se, because TGFß receptor presumably continues to function more or less normally in mice lacking caveolin 1, which are surprisingly normal developmentally (Drab et al., 2001
; Razani et al., 2001a
). In addition to TGFß receptor, caveolin 1 is thought to interact with a diverse array of signalling receptors, including the insulin receptor (Mastick et al., 1995
; Bickel, 2002
). Endocytosis of many of these molecules into caveosomes has yet to be investigated, but the relatively subtle phenotypes of caveolin-1-null mice, in terms of, for example, insulin tolerance and fatty acid metabolism (Cohen et al., 2003
), again suggest that the function of these various interactions may not be essential for signalling, but rather, as is apparently the case for TGFß receptor, may be more to do with regulating receptor turnover. This suggestion is supported by the observation of significantly reduced levels of insulin receptor in caveolin-1-null mice. Moreover, this observation agrees well with the ideas that caveolin 1 negatively regulates endocytosis and that endocytosis to caveosomes is involved in targeting receptors for degradation (Nabi and Le, 2003
; Di Guglielmo et al., 2003
).
Studies suggesting that ligand-induced uptake of chemokine receptors CCR5 and CXCR4 occurs by different mechanisms provide an additional indication that uptake into caveosomes and by clathrin-coated pits have different consequences. CCR5 accumulates in caveosomes whereas CXCR4 uses clathrin-coated pits to enter the cell (Venkatesan et al., 2003). Venkatesan et al. suggest that this is important for modulating the signalling potential of the receptors. Uptake to caveosomes is a slow process relative to uptake by clathrin-coated pits, which is consistent with the study of TGFß signalling described above, as well as previous work (Nichols et al., 2001
; Nichols, 2002
). Another set of experiments suggesting a specific function for uptake to caveosomes followed the uptake of the GPI-linked proteoglycan glypican 1 (Cheng et al., 2002
). Dissection of the endocytic cycle followed by glypican 1 showed that delivery of glypican 1 to caveosomes is specifically associated with exposure to heparanase activity and progressive S-nitrosylation of the protein core. This suggests that the appropriate enzymes are somehow recruited to caveosomes (Cheng et al., 2002
).
The studies discussed above raise several intriguing questions about the properties of caveosomes. It seems likely that specific proteins can be recruited to caveosomes, but how this is achieved is not at all clear. Classical, transferrin-containing endosomes are characterized by the presence of phosphatidyl-inositol-3-phosphate (PtdIns3P) (Simonsen et al., 2001), but the fact that proteins containing a domain that specifically binds to this lipid, the FYVE domain (Misra et al., 2001
; Di Guglielmo et al., 2003
), are not recruited to caveosomes suggests a different mechanism. A related question is to what extent, and how, proteins and lipids are sorted during endocytosis to caveosomes.
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Rafts and sorting during clathrin-independent endocytosis |
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One can of course imagine combinations of the above models, and there may be more than one type of lipid raft. A consideration of the way in which cells sort biochemically defined raft markers to different endocytic pathways is potentially useful in trying to begin to discriminate between the various possibilities. Some raft proteins and lipids for example, TGFß receptor and GM1, the sphingolipid to which CTB binds enter cells by both clathrin-coated pits and uptake into caveosomes (Torgersen et al., 2001; Nichols, 2002
; Di Guglielmo et al., 2003
). However, others for example exogenously added short-acyl chain fluorescent sphingolipid analogs (Sharma et al., 2003
), chemokine receptor CCR5 (which is palmitoylated) (Venkatesan et al., 2003
), and at least some GPI-linked proteins (Nichols, 2002
) are largely endocytosed into caveosomes. It has also been reported that GPI-linked proteins are taken up by a clathrin-independent mechanism into organelles devoid of caveolin 1 [termed GPI-enriched endosomal compartments, GEECs; Sabharanjak et al. (Sabharanjak et al., 2002
); discussed further below]. The general picture that emerges is one in which molecules that can partition into biochemically defined rafts have a variety of different endocytic itineraries. Whether clathrin-independent endocytic mechanisms such as caveolar endocytosis are truly specific for raft proteins and lipids is not fully understood, because uptake of non-raft molecules has yet to be carefully studied in this respect. The best characterized is the transferrin receptor, which is largely excluded from caveosomes (Pelkmans et al., 2001
). However, this may be because the receptor is largely concentrated within clathrin-coated pits, little free receptor being found in the rest of the plasma membrane. The issue of whether non-raft proteins are actively excluded during uptake to caveosomes thus remains to be addressed. Given the diversity of endocytic trafficking outlined above, raft molecules are unlikely to reside exclusively in the same stable microdomain. In addition, the finding that the same molecule can enter the cell by different mechanisms complicates the simple model where raft membrane is endocytosed to caveosomes and non-raft membrane enters by clathrin-coated pits. It is probably premature to postulate a single raft-specific or raft-mediated endocytic pathway, and better techniques to assay incorporation into rafts and to perturb potential raft functions are required.
Experiments carried out in our laboratory have used measurements of FRET as a tool to follow organization of GPI-linked proteins and CTB relative to clathrin-coated pits and caveolin in the plasma membrane of live cells (Nichols, 2003). A significant fraction of total membrane CTB is likely to be present in cholesterol-sensitive clusters, and these clusters are quite efficiently excluded from clathrin-coated pits. However, some CTB is clearly internalized by clathrin-coated pits, which leads to the hypothesis that CTB is found in two pools within the plasma membrane clustered and unclustered and that only the unclustered CTB can enter clathrin-coated pits. Surprisingly, no change in the distribution of CTB appears to be associated with regions of the plasma membrane containing a high concentration of caveolae (see Parton, 1994
). These data are then consistent with a model where exclusion from clathrin-coated pits is an important factor in the sorting of raft markers indeed, exclusion from clathrin-coated pits might be sufficient to generate apparently selective uptake by other mechanisms. At least some GPI-linked proteins are also likely to be excluded from clathrin-coated pits (Nichols et al., 2001
), but the extent to which GPI-linked proteins are themselves clustered in the plasma membrane is controversial and requires further investigation (Kenworthy and Edidin, 1998
; Varma and Mayor, 1998
; Kenworthy et al., 2000
; Dietrich et al., 2002
). Thus, although one can conclude that clusters of lipid are excluded from clathrin-coated pits, the question of whether these clusters contain more than one protein molecule remains to be addressed, and all of the current data are as consistent with the lipid shell model outlined above as with the more typical view of 50 nm rafts.
It may be that crosslinking of membrane components by other factors is sufficient to generate raft-like clusters. Two separate papers show that specific, protein-mediated, crosslinking of membrane proteins [anthrax toxin receptor (Abrami et al., 2003) and the B cell receptor (Stoddart et al., 2002
)] can cause incorporation into biochemically defined rafts, and is associated with uptake by clathrin-coated pits. However, it is hard to be confident that acquisition of resistance to detergent extraction is the key or primary effect of crosslinking, because crosslinking might well also promote interaction between clathrin-associated machinery and cytosolic domains of the relevant proteins. The fact that cholesterol depletion in both studies blocks crosslinking-dependent recruitment to clathrin-coated pits does suggest a role for rafts, but cholesterol depletion is likely to have effects on the biophysical properties of membranes beyond perturbing rafts. Indeed, as has already been mentioned, separate experiments show that cholesterol can, under some conditions, inhibit all clathrin-mediated endocytosis (Rodal et al., 1999
; Subtil et al., 1999
).
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Further clathrin-independent pathways |
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Overexpression of dominant-negative mutants of the small GTPases RhoA, Rac and Cdc42 has also been used as a tool to distinguish different endocytic pathways. As mentioned above, dominant-negative Cdc42 blocks uptake of GPI-linked proteins to GEECs (Sabharanjak et al., 2002). Internalization of interleukin 2 (IL-2) receptors, which like GPI-linked proteins are found in biochemically defined rafts, is specifically blocked by the equivalent mutant of RhoA, which indicates another potential endocytic mechanism (Lamaze et al., 2001
). IL-2 receptors are internalized normally in cells lacking caveolin 1, but the suggestion that caveolar endocytosis does not necessarily require caveolin 1 protein (Nichols, 2002
; Nabi and Le, 2003
) means that IL-2 receptors might still use this pathway and accumulate in caveosomes; this question remains to be answered. The complexities in the use of Rho GTPases as specific reagents to block endocytosis (Qualmann and Mellor, 2003
) are highlighted by the findings that constitutively activated RhoA and Rac can downregulate clathrin-mediated uptake (Lamaze et al., 1996
) and that dominant-negative Rac (but not Cdc42 or RhoA) can block clathrin-independent uptake of the ATPase ATP7A (Cobbold et al., 2003
). The identification of effectors, exchange factors or activators for these GTPases specific to a particular endocytic pathway would be a significant step forward.
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Conclusions |
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Acknowledgments |
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References |
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