Social Patterning of Screening Uptake and the Impact of Facilitating Informed Choices : Psychological and Ethical Analyses Screening for unsuspected disease has both possible benefits and harms for those who participate . Historically the benefits of participation have been emphasized to maximize uptake reflecting a public health approach to policy ; currently policy is moving towards an informed choice approach involving giving information about both benefits and harms of participation . However , no research has been conducted to evaluate the impact on health of an informed choice policy . Using psychological models , the first aim of this study was to describe an explanatory framework for variation in screening uptake and to apply this framework to assess the impact of informed choices in screening . The second aim was to evaluate ethically that impact . Data from a general population survey -LRB- n = 300 -RRB- of beliefs and attitudes towards participation in diabetes screening indicated that greater orientation to the present is associated with greater social deprivation and lower expectation of participation in screening . The results inform an explanatory framework of social patterning of screening in which greater orientation to the present focuses attention on the disadvantages of screening , which tend to be immediate , thereby reducing participation . This framework suggests that an informed choice policy , by increasing the salience of possible harms of screening , might reduce uptake of screening more in those who are more deprived and orientated to the present . This possibility gives rise to an apparent dilemma where an ethical decision must be made between greater choice and avoiding health inequality . Philosophical perspectives on choice and inequality are used to point to some of the complexities in assessing whether there really is such a dilemma and if so how it should be resolved . The paper concludes with a discussion of the ethics of paternalism . Introduction Approaches to Screening A major component of current public health strategy is the provision of screening programmes to allow prevention and early treatment of serious disease . A characteristic of screening is that it involves the possibility of immediate harm in return for the possibility of future benefit . For example breast screening with mammography has a number of substantial possible immediate harms -LSB- 26 , 38 , 50 -RSB- . In 2001 five percent of mammography screens in the US gave a false positive result leading to further testing -LSB- 53 -RSB- . Even when further tests result in a diagnosis of breast cancer , about 20 percent of the cases identified will be of ductal carcinoma in situ which has an uncertain course without treatment -LSB- 50 -RSB- . Many such cancers are not life threatening but , once detected , are usually treated as such -LSB- 53 -RSB- . Such treatments are unpleasant and damaging to health and women diagnosed with ductal carcinoma in situ have similar anxiety levels to those with early invasive breast cancer -LSB- 39 -RSB- . In contrast , only a small number of people , relative to the total screened , have their lives saved , even over a considerable period of time . Estimates suggest that 2451 women aged between 50 and 59 need to be screened as recommended over a five-year period to save one life -LSB- 41 -RSB- . Because only a few cases of disease will be detected within a healthy population , large numbers need to be screened in order to have an impact on overall population health . Thus high uptake of screening has been encouraged by emphasizing the benefits of participation , reflecting a public health approach to screening -LSB- 27 -RSB- . Recently , in the UK , there has been a policy change towards promoting informed choices particularly in screening : There is a responsibility to ensure that those who accept an invitation -LSB- to screening -RSB- do so on the basis of an informed choice , and appreciate that in accepting an invitation or participating in a programme to reduce their risk of a disease there is a risk of an adverse outcome -LSB- 32 -RSB- . One of the factors influencing the move to informed choices has been a concern about patient autonomy -LSB- 16 -RSB- . The issue of autonomy has become more salient with the rise of ` patient-centred medicine ' in reaction to concerns with traditional medical practice and its emphasis on the role of the health professional in medical decision-making . -LSB- 37 -RSB- Informed Choice in Screening Informed choices have been defined as those based on relevant knowledge , consistent with the decision-maker 's values and behaviourally implemented -LSB- 28 -RSB- . To make an informed choice participants need information about their personal risks of developing the condition , what having the screening test will be like , accuracy of the test , and what will happen if the screening test is positive -LSB- 15 -RSB- . While the place of this information in facilitating informed choices is obvious , the role of values may be less so . People will , however , attach different values to the possible outcomes of screening . For example those who are more and less socially deprived may place a different value on early diagnosis . The consequences of being diagnosed with a serious condition can have very different implications for those who have material resources and those who do n't , even within a universal healthcare system . While those who have a high level of material resources can use those resources to ameliorate the impact of a diagnosis of serious illness , for those who are more socially deprived such a diagnosis may bring the prospect of increasing poverty and uncertainty and they might thus prefer to delay knowledge of an illness for as long as possible -LSB- 24 -RSB- . There has been no research to describe or evaluate the impact of an informed choice policy in screening . One possible impact is that it will reduce uptake of the screening programme with a greater reduction in some groups , for example those who are more socially deprived and already have lower participation in screening . Such a decline in the uptake of screening could be evaluated negatively as contributing to a decline in the overall health of the population or positively as reflecting an increase in the autonomy of the individual . This paper seeks to describe and evaluate the possible impact of an informed choice policy in screening . Explaining Screening Uptake Uptake of screening is lowest in those who are most socially deprived . This is a consistent finding across different screening programmes and healthcare systems -LSB- 20 , 29 , 48 -RSB- . Lower uptake among the most socially deprived is , at least in part , a consequence of a lack of material resources , such as transport costs to the screening centre . But when the lack of such resources is controlled for in statistical analyses , differences in uptake remain -LSB- 23 -RSB- . Psychological characteristics , among other factors , contribute to explaining those differences -LSB- 48 , 52 -RSB- . One such psychological characteristic might be time orientation . Psychologists suggest that people use information about the timeframe in which an event occurs to process information about the event and to make decisions . However , people 's responses to the specific timeframe in which an event might occur , vary . Individuals have preferences for certain timeframes which influence their information processing and evaluation of actions and the possible outcomes of those actions . These preferences are called time orientation . While a number of different time orientations have been identified -LSB- 55 -RSB- , evidence for their existence is strongest for two of these , future and present orientation . Those who have high future orientation think more about the future and have an awareness of the effects of current actions on future outcomes compared to those with lower future orientation -LSB- 42 -RSB- . Future orientation is associated with the practice of health-related behaviours , such as physical activity and healthy eating , that might involve immediate cost for possible future gain -LSB- 22 , 25 , 34 , 42 -RSB- . Those who have high present orientation think more about immediate outcomes of their behaviour than those who are less present orientated -LSB- 42 -RSB- . High present orientation is also associated with a limited sense of control and fatalism about life events -LSB- 42 -RSB- and unhealthy behaviours , such as substance abuse , which result in immediate rather than future rewards -LSB- 18 , 42 -RSB- . Future and present orientation are largely independent , of one another with each being associated with different patterns of thought and behaviour -LSB- 18 , 56 -RSB- . Because the possible harms of screening are immediate while the possible benefits occur in the future , time orientation may contribute to explaining screening uptake . Increasing information about the possible harms and limited benefits of screening may therefore have a differential impact on those with different time orientations . Given that time orientation and social deprivation are associated -LSB- 33 , 47 -RSB- , this effect may vary by social deprivation . Aims The first aim of this study is to describe the possible impact of an informed choice policy on screening uptake by exploring the relationships between social deprivation , present orientation and expectations of participation in screening . The second aim is to evaluate that impact from different ethical perspectives . Method Design A questionnaire-based descriptive survey . Sample A total of 300 participants was recruited . The sample was structured to reflect the English population in terms of age and sex with one third of the sample being drawn from each of the North , South and Midlands of England . Procedure Home-based interviews were conducted by a research agency . Questionnaires were completed by interviewers on behalf of participants . Materials In the first part of the interview information about diabetes screening , based on that used in previous research -LSB- 34 -RSB- , was presented to participants -LRB- Appendix 1 -RRB- , after which the following measures were completed : Expectations of participation in screening . The mean response to three items measuring intention to participate in screening was assessed on five-point response scales -LSB- 34 , 35 -RSB- giving a measure with good reliability -LRB- Cronbach 's alpha : .85 -RRB- . Time orientation . A brief , nine item version of the Stanford Time Perspective Inventory -LRB- Crockett et al. in submission -RRB- . The Stanford Time Perspective Inventory -LRB- STPI -RRB- has been extensively validated and used in the study of health behaviour -LSB- 9 , 21 , 42 , 56 -RSB- . This brief STPI consists of nine items , measured on five-point rating scales , comprising two subscales with adequate reliability : five items measuring future orientation -LRB- Cronbach 's alpha : .67 -RRB- and four items measuring present orientation -LRB- Cronbach 's alpha : .62 -RRB- . Social deprivation : A brief three item measure asking participants to indicate : Possession of educational qualificationsHome ownership -LRB- including having a mortgage -RRB- . Those who neither owned their homes nor had any educational qualifications were considered to have the greatest social deprivation -LRB- scored as 2 -RRB- ; those who either owned their homes or had educational qualifications were considered to have intermediate levels of social deprivation -LRB- scored as 1 -RRB- ; and , those who both owned their homes and had educational qualifications were considered to have the least social deprivation -LRB- scored as 0 -RRB- . This measure was derived from our previous research which indicated that individual level measures of social deprivation showed greater associations with psychological characteristics than did neighbourhood measures such as the Indices of Multiple Deprivation -LRB- Crockett et al. in preparation -RRB- . Analysis Statistical analyses were conducted using SPSS version 12 . Associations between social deprivation , future and present time orientation and expectations of participation in diabetes screening were examined using Spearman 's rank correlations . Results The associations between social deprivation , time orientation and expectations of participation in diabetes screening are shown in Table 1 . Table 1Associations between expectations of participation in screening , social deprivation and present orientation -LRB- Spearman 's rho correlation -RRB- Present orientationFuture orientationExpectations of diabetes screeningSocial deprivationa − .245 *** .067.218 *** Present orientation − .301 *** − .265 *** Future orientation .054 *** P < 0.001 amost deprived scored as 0 , intermediate group scored as 1 , least deprived group scored as 2 Future orientation was not significantly associated with social deprivation -LRB- r = − .067 , n = 300 , P = .247 -RRB- or with expectations of participation in screening -LRB- r = .054 , n = 300 , P = .349 -RRB- . However present orientation was significantly associated with both social deprivation -LRB- r = .245 , n = 300 , P < 0.001 -RRB- and expectations of participation in screening -LRB- r = − .265 , n = 300 , P < 0.001 -RRB- . Because only present orientation showed associations with social deprivation and expectations of participation in screening , further findings are presented for present orientation only . To illustrate the association between social deprivation , uptake of screening and present orientation , two figures were plotted . Figure 1 -LRB- a -RRB- shows the mean expectation of participation in diabetes screening at each level of deprivation . As social deprivation increases , expectations of participation decrease . A one-point difference between those most and those least deprived indicates a substantial effect of social deprivation on expectations of participation in screening . Figure 1 -LRB- b -RRB- shows the mean expectation of participation between those with high and low levels of present orientation , indicating that those with low present orientation express higher expectations of participating in screening . Figure 1 -LRB- b -RRB- does not indicate such large effects as seen in Fig. 1 -LRB- a -RRB- suggesting that other variables must contribute to the impact of social deprivation and expectations of participation in screening . Fig. 1Expectations of participation in screening and -LRB- a -RRB- social deprivation , -LRB- b -RRB- present orientation , High and low present orientation calculated by means of a mean split Additional analyses using another part of the data set presented here indicate present orientation explains part of the association between social deprivation and uptake -LRB- Crockett et al. in submission -RRB- . Summary The results of this study suggest that present orientation is associated with both social deprivation and with uptake of screening , such that present orientation partially accounts for the relationship between greater social deprivation and lower expectations of participation in screening . The results indicate that psychological factors can contribute to an explanatory framework of screening uptake . This framework suggests that there is an association between social deprivation and present orientation and that decisions about uptake of screening are influenced by the time orientation of those who are invited . This framework further suggests that making the more immediate possible harms of screening more salient , as would happen in an informed choice policy , could reduce uptake of screening in those who are more socially deprived . This framework can be used to identify and evaluate the possible impact of an informed choice policy in screening . Evaluation The Possible Impact of Informed Choices on Inequality The study results suggest that the implementation of an informed choice policy , which makes salient the possible immediate harms of participation , might lead to decreases in uptake of screening among those who are more socially deprived . This decrease is unlikely to be matched by a similar decrease among those who are less deprived , not only because they are less present orientated but also because evidence suggests that those who are more educated , and typically less deprived , are more aware of the limited benefits of screening programmes -LSB- 12 -RSB- . A differential decline in uptake of screening is an issue of particular concern . Those who are more socially deprived already have poorer health -LSB- 7 , 17 -RSB- and are more likely to develop diabetes and its complications -LSB- 3 , 8 -RSB- . A reduction in uptake of screening among those who are most deprived might widen the existing gap in physical health between those who are more and less socially deprived , running counter to UK government policy of reducing such inequalities -LSB- 10 , 11 -RSB- . If an informed choice policy does reduce the rate of screening of the most deprived , then there appears to be an ethical dilemma . On the one hand , there can be greater choice but at the ethical cost of increased inequality ; on the other hand , greater inequality can be avoided , but at the ethical cost of less informed choice . In the rest of this paper , we aim to describe this dilemma more fully and offer some thoughts about how , in the face of it , an informed choice might be evaluated . We should say at the start that our aims are limited and that we do not try , nor think it possible , to evaluate an informed choice policy fully in the space available . Our discussion is aimed at those who feel an initial ethical pull both toward reducing inequality and toward informed choice . Among those who feel this pull are the UK government , whose policies are explicitly designed to try to achieve both . Thus we do not aim to persuade those libertarians or elitists who oppose taxation-funded screening programs altogether . Nor do we intend the ethical claims we make to be applicable in all times and places . In aiming our discussion at those who are attracted both to equality and choice , we do not suggest that the nature of these values is at all obvious . Indeed , one of our major points will be that any evaluation of an informed choice depends on further specification of these values . It might well turn out , as these values are specified , that the dilemma between choice and equality is merely apparent and that a decision between them in the context of screening does not have to be made . After the ethical characterization of the choice and equality dilemma , we critique a public health and paternalistic approach to screening that is sceptical about the value of choice . Is There a Dilemma ? As mentioned , it appears that there is a dilemma for those who value both choice and equality if an informed choice policy reduces the rate of screening of the most deprived without significantly reducing the rates for everybody else . It appears that the health of the most deprived would decline relative to others and thus inequality of health would increase . Whether there is actually a dilemma depends partly on what happens to screening rates in practice , but it also partly depends on the characterization of the ethical values , as we shall now show . In the first place , it is not obvious , even if there are differential impacts on screening rates , that choice would produce greater inequality of health . While maximizing informed choices might result in decreased physical health , by promoting personal autonomy they might increase psychological well-being . Personal autonomy has been related to two specific psychological constructs -LSB- 51 -RSB- : perceived control , which has been linked to positive outcomes including coping , personal adjustment and success or failure in a variety of areas of life -LSB- 46 -RSB- ; and self-efficacy , which is the sense of having mastery over the action needed to achieve a particular end -LSB- 4 -RSB- . Self-efficacy is associated with a number of health-related behaviours including uptake of screening -LSB- 13 , 14 , 19 , 25 -RSB- . Those who are more socially deprived are not only disadvantaged in terms of physical health but commonly also have lower levels of positive psychological characteristics , such as self-efficacy , which contribute to an individual 's overall well-being and are related to the ability to make autonomous choices in a variety of health and life domains -LSB- 5 -RSB- . The point here can be made in two different ways . One is to say that health , as a philosophical concept , is about more than narrowly defined physical health and that the psychological benefits of greater choice for the most deprived are gains in health to them . This leaves it moot whether a reduction in their rates of screening is worse for them in terms of their health , and thus moot whether choice increases inequalities in health . A second way , which avoids the complicated conceptual debates about what health is , puts the point as one about the determinants of health . Even on a relatively narrow construal of health as physical health the psychological gains of choice , such as increased sense of control -LSB- 51 -RSB- , might improve the physical health of the most deprived -LSB- 1 -RSB- , again leaving it moot whether choice increases inequalities in health . Even if there is a conflict between choice and equality of health , it does not follow that there is a conflict between choice and the value of equality . Again , whether there is a conflict depends on further specification of the value . There is a large debate in political philosophy called the ` equality of what ? ' debate -LSB- 44 -RSB- . Fundamentally , however health is characterized and however equality is characterized , health would only be one item in the metric of inequality . Suppose that an informed choice policy causes the health of the most deprived to be lower than it would otherwise have been . The policy also causes their choice to go up . Whether the result is to make them worse off than they would otherwise have been is a complicated question that depends on the relative value of health and choice . Possibly the result should be counted as a gain in equality , since it increases the choice of the most deprived , although possibly not . The point here is that , from the viewpoint of equality , concentrating on inequalities in health is concentrating on only part of the picture . Not only is the evaluation of choice complicated by debates about health and the metric of equality , it is also complicated by debates over how to understand the value of equality . It is possible to give only an incomplete and sketchy account of this here . Consider three views : utilitarianism , prioritarianism , and egalitarianism . Utilitarianism recommends policies that maximize aggregate welfare -LSB- 45 -RSB- . It would recommend reducing inequalities of health if this would indeed maximize welfare . Many utilitarians believe that greater equality would maximize welfare -LSB- 6 -RSB- . As for the policy of informed choice , utilitarians would recommend it if it led to greater welfare than the alternative and oppose it if it reduced welfare . In the next section , on paternalism , we make some points about the effects of choice on welfare . Prioritarianism recommends policies that improve the position of the worst off . Prioritarians disagree among themselves about the extent of the priority that should be given to the worst off , for instance whether small gains to the worst off outweigh large gains to everyone else -LSB- 30 , 31 -RSB- . However prioritarians decide to evaluate gains to the worst off , there is no necessary connection between their view and favouring equality . Improving the position of the worst off might reduce inequalities in health , but it might also increase them -LSB- 36 , 40 -RSB- . Egalitarians , by contrast , believe that inequality is in itself bad . For them , there is some value in reducing inequality even if it is bad for some and good for no one -LSB- 49 -RSB- . This is not to say that egalitarians would , all-things-considered , want greater equality if it produced no gain in anyone 's welfare . Their attitude to inequalities would depend on the other values they hold -LSB- 36 -RSB- . In the face of this sketch of various positions , it is clear that evaluating any inequality produced by an informed choice policy requires more detail about those effects . That inequality increases does not on its own justify opposing the policy . Suppose inequality increases because the most deprived stay the same , on whatever is the right metric for measuring people 's positions , but the position of others improves . Utilitarians and prioritarians should endorse the policy . Egalitarians might oppose it but also might not , depending on the other values they hold . Suppose the position of the most deprived goes down and others ' positions improve . Egalitarians and prioritarians would probably oppose it , although that might depend on how much the most deprived lose and how much others gain . Utilitarians will weigh up the gains and losses to see what maximizes welfare . In this section we have pointed out some of the complexities of evaluating the policy of informed choice even if we assume some initial ethical commitment to both choice and equality . In particular , we have shown that the initial statement of the dilemma between choice and equality is too crude , and that there might be no dilemma in the end . In describing the complexities we do not want to suggest that a rationally defensible overall evaluation is impossible , only that it is perhaps more difficult than one might think . In the next section , we continue the theme of complexity by pointing to the for a brisk paternalistic public health approach that says that if choice reduces health it must be bad . Informed Choice , Public Health , and Paternalism If an informed choice policy really does reduce the screening rates of the most deprived -- or , indeed , of the general population -- then the argument might be made that the policy is ethically wrong because it would increase morbidity and mortality . The argument might be put in terms of public health , that the disease burden of society would increase , or more directly in paternalistic terms , that individuals would be net worse off for having and making the choice not to be screened , and that either way , an informed choice should not be implemented . While we can not show decisively that this argument is mistaken , this section points to some of the serious difficulties that face it . We should state at the outset that we understand paternalism in the sense commonly used in political philosophy , where an action is paternalistic only if it is both motivated by concern for the target 's interests and in some way bypasses the target 's own decision-making , for instance by coercing the target or failing to disclose relevant information -LSB- 54 -RSB- . Firstly , at the population level any loss of life resulting from an informed choice policy has to be set alongside the negative impacts of a public health approach to screening including death as a consequence of that approach . Emphasizing the benefits of screening results in limited understanding of the harms of screening , including the possibility of false negatives and in turn this leads to an overestimation of the reliability of screening results by both the public and health professionals -LSB- 38 -RSB- . This may lead to symptoms of disease being ignored following a negative screening result , delaying diagnosis and treatment -LSB- 2 , 38 -RSB- . Essentially the same points can be made if the argument is put instead in paternalistic terms , that individuals choose against their best interests when they choose against screening . From the point of view of any given individual facing the choice of whether to undergo screening , not being screened carries a certain risk of morbidity and mortality and so does being screened . Given other costs and benefits of screening , it might be quite rationally self-interested for some individuals to decide to undergo screening while others choose against it . It is likely that in many cases those who are more deprived would indeed value increased life expectancy and it would therefore be appropriate to give those from lower socioeconomic groups information about their higher risk of dying from the disease to inform their personal evaluation of the benefits and harms of participation . However , it is also possible that those who are more socially deprived may value increased life expectancy differently to those who are less socially deprived resulting in a preference to delay diagnosis for as long as possible , even if , in the long term this results in a shortened life expectancy -LSB- 24 -RSB- . Thus it is much harder than might be thought to show that those who choose against screening must be acting against their better interests . Moreover , as we pointed out in the previous subsection , the value of autonomous choice can be intertwined with people 's interests in that having choices can have psychological , and health-promoting , benefits given perceived control and self-efficacy have been linked to good health -LSB- 1 , 5 -RSB- . This is a further reason to doubt that restricting individuals ' informed choice would promote their overall welfare . The argument from the benefits of screening against informed choice , in either public health or paternalistic forms , arguably also undervalues autonomy . For many political philosophers , and people more generally , being able to make choices for oneself is good in itself . The value of informed choice is not simply in the extent to which is allows us to choose what is really in our interests . In other dimensions of life , such a choice of career or partner , many people want to make their own choices even if they are less good at selecting than some disinterested observer -LSB- 43 -RSB- . Perhaps these reasons to value informed choice carry over to the choice of screening even if people do sometimes get it wrong . A persuasive paternalistic argument must show that , given the possibility of an informed choice , the people would tend to choose against their interests and that this would justify denying them the choice . We have not shown that no such argument could be made , but we have tried to show the severity of the difficulties that face it . Conclusions This paper has shown how psychological research can contribute to assessing the possible impact of an informed choice policy on screening uptake . It suggests that an informed choice policy could lead to a decrease in uptake of screening amongst those who are most socially deprived , resulting in decreases in the physical health of this group . From a public health perspective any decrease in physical health is a matter of concern , particularly if that decrease is greater in those who are more socially deprived . From an informed choice perspective such a decrease in uptake could be interpreted as indicating that people are making autonomous choices based not only on good knowledge , but also in line with their own values . Those who are more socially deprived are more present orientated . They therefore value actions that have positive outcomes immediately and thus , once they understand that the benefits of screening are not immediate , may be less likely to participate in the screening programme . These results have been evaluated from different philosophical perspectives on health inequality and on choice . This evaluation has not attempted to provide a definitive assessment of whether the introduction of an informed choice policy in screening can be justified in the light of the likely impact on physical health inequalities outcomes across the population . Rather , the evaluation has sought to describe the way in which philosophical approaches to choice and to health inequality can be used to inform further discussions about choosing an informed choice approach to screening over a public health approach .

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O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O 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Morphogenesis of post-Golgi transport carriers The trans-Golgi network -LRB- TGN -RRB- is one of the main , if not the main , sorting stations in the process of intracellular protein trafficking . It is therefore of central importance to understand how the key players in the TGN-based sorting and delivery process , the post-Golgi carriers -LRB- PGCs -RRB- , form and function . Over the last few years , modern morphological approaches have generated new insights into the questions of PGC biogenesis , structure and dynamics . Here , we present a view by which the `` lifecycle '' of a PGC consists of several distinct stages : the formation of TGN tubular export domains -LRB- where different cargoes are segregated from each other and from the Golgi enzymes -RRB- ; the docking of these tubular domains onto molecular motors and their extrusion towards the cell periphery along microtubules ; the fission of the forming PGC from the donor membrane ; and the delivery of the newly formed PGC to its specific acceptor organelle . It is now important to add the many molecular machineries that have been described as operating at the TGN to this `` morphofunctional map '' of the TGN export process . Introduction The Golgi complex serves as the central station in the biosynthetic pathway , where proteins are sorted for their different destinations , such as various domains of the cell surface and the endosomal -- lysosomal system . This delivery of cargo proteins from the Golgi complex to their target compartments is carried out by dynamic , membrane-bound organelles that are frequently called either `` transport carriers '' or `` post-Golgi carriers '' -LRB- PGCs -RRB- -LRB- Luini et al. 2005 -RRB- . As these PGCs have an important role in the process of intracellular transport , their morphology , living dynamics and molecular compositions have become the focus of significant interest , particularly over the last few years . These PGC transport organelles were originally defined through the development of green fluorescent protein -LRB- GFP -RRB- technology and living-cell imaging -LRB- Lippincott-Schwartz et al. 2000 -RRB- , whereby the first fluorescent cargo protein that was followed in living cells revealed a new world of images of these relatively large and highly dynamic structures that travel from the Golgi complex to the plasma membrane -LRB- Wacker et al. 1997 ; Hirschberg et al. 1998 ; Nakata et al. 1998 -RRB- . Then , with time , the list of cargo molecules that could be visualized in vivo expanded , further exposing the unexpected complexity of the post-Golgi transport pathways . Finally , further technical advances resulted in the combination of video and electron microscopy -LRB- correlative light-electron microscopy , CLEM -RRB- , which provided the means to define the morphogenesis of these PGCs at the ultrastructural level . As a result of these studies we now know that PGCs arise from specific membrane domains of the Golgi complex that lack resident Golgi enzymes , forming what are known as the `` PGC precursors '' -LRB- Hirschberg et al. 1998 ; Keller et al. 2001 ; Polishchuk et al. 2003 ; Puertollano et al. 2003 -RRB- . The shapes and sizes of PGCs that even carry the same cargo can vary across a wide range , and under the light microscope , most of them are seen to be clearly larger that plasma-membrane associated clathrin vesicles and 100-nm fluorescent beads -LRB- Hirschberg et al. 1998 -RRB- . The smaller PGCs usually have a size of 300 -- 400 nm , although some of the larger ones can reach several microns in length . Video microscopy has also revealed that many of these carriers appear globular , although they frequently stretch out into tubular shapes during their translocation through the cytosol ; thus PGCs have frequently been termed as `` pleiomorphic '' structures . In transporting their cargoes to the correct acceptor compartments , the movement of PGCs is mediated via microtubules . However , carriers can also form and support post-Golgi transport without microtubules , although the correct targeting of their cargo proteins is usually compromised under these conditions -LRB- Rindler et al. 1987 ; Kreitzer et al. 2003 -RRB- . Finally , the life cycle of a PGC can be schematically imagined to consist of three stages : -LRB- 1 -RRB- its formation -LRB- which can in turn be further divided into several substages ; see below -RRB- ; -LRB- 2 -RRB- its transition through the cytosol ; and -LRB- 3 -RRB- its docking and fusion with the target membrane -LRB- Polishchuk et al. 2000 -RRB- . The first of these steps , the formation of the PGC , appears to be the most complex , and it also probably remains the least understood . Since the question of the morphogenesis of a PGC is closely intertwined with that of the structure of the organelle from which it originates , the trans-Golgi network -LRB- TGN -RRB- , both of these issues will now be discussed in an integrated fashion . The trans-Golgi network and the origin of PGCs The process of PGC morphogenesis has been characterized in detail using video and electron microscopy , as well as with the combination of these techniques -LRB- CLEM -RRB- . As indicated above , this process comprises the formation , the extrusion and the fission of the export domain from the TGN , thus generating the free carrier -LRB- Polishchuk et al. 2003 ; see also Fig. 1 -RRB- . The first step -LRB- the formation of the tubular export domains -RRB- also includes the segregation of the cargo proteins from the Golgi resident enzymes . These events appear to be common to the proteins directed towards different post-Golgi compartments , such as basolateral plasma membrane -LRB- Hirschberg et al. 1998 ; White et al. 2001 ; Polishchuk et al. 2003 -RRB- , the apical plasma membrane -LRB- Keller et al. 2001 -RRB- and the endosomal -- lysosomal system -LRB- Puertollano et al. 2003 -RRB- . These export domains usually contain TGN markers ; i.e. they are part of the TGN -LRB- Polishchuk et al. 2003 ; Puertollano et al. 2003 -RRB- . Of note , these three post-Golgi compartments are the ones that have been best characterized for the PGCs leaving the TGN , with the total number and types of pathways for cargo exit from the TGN at present not known , although there are at least six , and probably more -LRB- Rodriguez-Boulan et al. 2005 -RRB- . Fig. 1Formation of post-Golgi transport carriers . Subsequent frames extracted from a time-lapse sequence illustrating the different stages of PGC biogenesis -LRB- arrows -RRB- : a formation of the tubular domain containing the cargo VSVG-YFP and devoid of the Golgi-resident protein galatosyltransferase-CFP . b Extrusion of this tubular domain from the Golgi complex . c , d Fission of the domain from the parental Golgi membranes . e Thin section of a cell expressing the TGN38-HRP construct . Arrows indicate tubular PGC precursor that has been pulled out of the TGN area of the Golgi complex Although the existence of the TGN has been known for many years -LRB- Griffiths and Simons 1986 -RRB- , its precise structure and identity still remain to be ascertained . In one view , which predominated in the field until recently , the TGN was considered to consist of essentially an anastomosing tubular network that emanates from -LRB- or that results from the breakdown of -RRB- the trans-most Golgi cisterna , and that projects mainly in the trans direction -LRB- Griffiths et al. 1989 , 1985 ; Clermont et al. 1995 -RRB- . Rambourg and colleagues have thoroughly described the TGN in several different cell types . In these descriptions , this trans-most cisterna also tends to `` peel off '' from the rest of the Golgi stack , and together with the other morphological characteristics of the Golgi stack , this is a feature that is suggestive -LRB- Clermont et al. 1995 -RRB- of the cisternal progression-maturation trafficking model -LRB- Bannykh and Balch 1997 ; Mironov et al. 1997 ; Bonfanti et al. 1998 ; Losev et al. 2006 ; Matsuura-Tokita et al. 2006 -RRB- . Thus , in a simple version of this scheme , the TGN would result from the final stages of maturation of the Golgi cisternae . This would include the partial transformation of this trans-most cisternae into a tubular network , and the various carriers that leave the TGN for their different destinations would all originate form different domains of this tubular network . A more complex and more recent view of the organization at the trans face of the Golgi complex comes from studies using electron microscopy tomography of cryofixed , freeze-substituted cells -LRB- Ladinsky et al. 1999 , 2002 -RRB- . These studies have indicated that the TGN derives not only from the last trans cisterna , but instead from the three trans-most cisternae -LRB- Roth et al. 1985 -RRB- , from where tubules emanate into the trans space of the Golgi stacks . Remarkably , only the trans-most cisterna exhibits clathrin-coated buds , with the other buds appearing to have different , yet-to-be identified , types of coats -LRB- Ladinsky et al. 1999 -RRB- . Thus , only the trans-most cisterna would be responsible for the clathrin - and AP-1-dependent trafficking towards the endo-lysosomes , and although all of these three trans-most cisternae of the Golgi stacks would serve as the classical TGN , each of them could be specialized in the packaging and export of specific cargo proteins . This might have important functional and mechanistic implications for the proteins sorting and export process . Another remarkable TGN feature that has been revealed by these tomographic studies is that these three trans-most cisternae can intercalate with the cisternae of the endoplasmic reticulum -LRB- ER -RRB- -LRB- Ladinsky et al. 1999 , 2002 -RRB- . These have been proposed to have important roles in the transfer of lipids -LRB- through specialized contact sites -RRB- between the ER itself and the Golgi complex . It will be important to establish whether the morphological discrepancies described above reflect fundamental differences in the organization of the export of cargo in different cell systems . Indeed , it is possible that the first model -LRB- the TGN as a network resulting from the tubular disassembly of the last trans-most cisterna -RRB- might actually only represent a special case of the more complete three-trans-cisterna model , and that both of these two different models of TGN may apply , depending on the cell type and the functional conditions . In support of this possibility , it is clear that the TGN can vary significantly in both size and composition across different cell types . For example , cells with a well-developed endo-lysosomal system and without secretory granules exhibit an extensive tubular TGN , while the tubular component of the TGN is reduced in cells that are specialized in regulated secretion -LRB- Clermont et al. 1995 -RRB- . This appears to occur in the latter because most of the TGN membranes are used for the packaging of secretory granules , and the tubules only form the thin bridges between the granule precursors . Such an organization simplifies the release of the granules , which occurs through the rupture of these tubular elements -LRB- Clermont et al. 1995 -RRB- . Similarly , human fibroblasts have large collagen-containing distensions that are connected to tubular elements in the trans-Golgi area -LRB- Polishchuk et al. 2003 -RRB- . Thus , the morphology and size of the TGN apparently depend on the predominant type and amount of cargo protein departing from the Golgi complex . The morphology of the TGN depends also on its secretory status . When exit from the TGN is blocked by a lowering of the temperature to 20 °C in the presence of abundant cargo -LRB- Matlin and Simons 1983 -RRB- , the volume and surface areas of the TGN increase greatly -LRB- Griffiths et al. 1989 -RRB- ; while in the absence of cargo , the 20 °C block results in all three of the trans-most cisternae producing bulging exit domains -LRB- Ladinsky et al. 2002 -RRB- . At physiological temperatures , three-dimensional -LRB- 3D -RRB- analyses of the Golgi stacks have revealed well developed , tubular -- reticular , TGN-like membranes at the trans side of the Golgi complex in actively secreting cells -LRB- Trucco et al. 2004 -RRB- , while the TGN is nearly absent in quiescent cells -LRB- Trucco et al. 2004 -RRB- . Thus , the TGN is a very dynamic structure , the shape and size of which is potently regulated by the extent of traffic flowing through it . To discover the regulatory mechanisms is an important challenge for the future . Another challenge will be to define whether the three-cisterna organization really underlies a functional specialization of these cisternae in the sorting and packaging of different cargo classes , as suggested by the presence of clathrin buds only on the last cisterna of the Golgi stack . So far , the lack of immunolabeling studies does not allow determining whether different trans cisternae really do contain different transport proteins . In addition to being an organelle of the biosynthetic pathways , the TGN is involved in endocytic transport routes -LRB- Griffiths and Simons 1986 ; Mellman and Simons 1992 ; Pavelka et al. 1998 -RRB- . Several studies with different cell types showed that the TGN and Golgi stacks can contain plasma-membrane constituents and internalized materials -LRB- Stoorvogel et al. 1988 ; van Weert et al. 1997 -RRB- . These exchanges with the endocytic systems could well introduce a further level of structural complications , since the recycling endosomes , in particular , reside in the Golgi area and are tubular in nature . In conclusion , both the dynamics and the structure of the TGN are variable and incompletely defined , thereby presenting an additional layer of difficulty to our understanding of the biogenesis of the carriers that depart from this organelle . Formation of PGC precursors at the TGN There are two key aspects to the formation of the PGC precursors : their morphogenesis at the TGN ; and the segregation between different types of cargo proteins that are targeted for different destinations , as well as between the Golgi-resident proteins , such as the Golgi enzymes . Regarding PGCs morphogenesis , in principle , the tubular carrier precursors might form through the mechanical pulling force exerted by microtubule-based motors on a flatter , parent , membrane domain -LRB- e.g. , a Golgi cisterna -RRB- . This has been shown to be mechanistically possible for both artificial and natural membranes -LRB- Roux et al. 2002 -RRB- . If this is the case , the PGC precursors would be expected to be essentially simple linear tubular structures -LRB- Roux et al. 2002 -RRB- . The second possibility is that these precursors are actually formed from the tubular subdomains of the TGN , which are generated prior to docking and extrusion by microtubules . In this case , the GPCs should reflect the morphologically complex structure of their parent membranes . Recent studies have provided compelling support in favor of the latter model -LRB- Polishchuk et al. 2003 -RRB- . First , both free PGCs and their precursors at the TGN comprise complex tubulo-reticular structures , which have often been described as the main component of the TGN in many cell types -LRB- Clermont et al. 1995 -RRB- . For example , PGCs carrying VSVG show a mostly tubular morphology , and can have a complex structure that even contains clearly visible fenestrae -LRB- Polishchuk et al. 2003 -RRB- ; this would be expected of membranes that derive from protrusions of the TGN . Indeed , PGC precursors that have been visualized using CLEM , appear to be comprised of tubular segments that are interconnected with complex branching and fenestrated membranes ; they are also seen to be continuous with the parent membranes of the Golgi stack -LRB- Polishchuk et al. 2003 -RRB- . Similarly , carriers containing the apical cargo protein hemagglutinin -LRB- HA -RRB- frequently have a tubular morphology as well as HA-positive domains at the TGN -LRB- Puertollano et al. 2001 -RRB- . Thus structural similarities between PGC precursors and the TGN appear to be a common feature of different types of PGCs . This strongly suggests that PGCs form via the fission of an entire precursor domain -LRB- or a large part thereof -RRB- from the rest of the TGN membranes . Thus the question regarding the mechanism by which the originally flat Golgi membranes are converted into highly bent , tubular -- reticular TGN structures arises . Essentially , this can be achieved by one of two mechanisms : either by the action of proteins that can bend these membranes into tubules , in a manner that is possibly similar to that of amphiphysin , endophilin , sorting nexins and others -LRB- Antonny 2006 -RRB- , or via the alteration of the lipid composition of these trans-Golgi membranes , which can be mediated , in turn , by several processes . For instance , changes in lipid content , and hence membrane curvature , can be modulated via the lipid-metabolizing enzymes that reside at the Golgi complex -LRB- for review , see De Matteis and Godi 2004 ; Luini et al. 2005 -RRB- . The transmembrane or inter-organelle transfer of lipids can contribute to the generation of particular lipid environments in the membranes of the TGN . In this respect , it is important to note that numerous contact sites between the ER and the trans-cisternae of the Golgi complex have been detected by electron microscopy tomography -LRB- Ladinsky et al. 1999 , 2002 -RRB- . Such contact sites can be explored for lipid transfer between the ER and trans-Golgi that is mediated by specific lipid-transfer proteins . Also , since the cisterna-like morphology of Golgi compartments can be stabilized by large polymers of Golgi enzymes -LRB- Nilsson et al. 1996 -RRB- , the loss of oligomerized Golgi enzymes in the trans-Golgi should , in principle , favor cisterna-to-tubule transformation of cargo-containing membranes . Moreover , this process can be accompanied by the loss of the stacking mechanisms at the trans side of the Golgi complex . Here , GRASP65 and GRASP55 are two proteins that have been suggested to be involved in the maintenance of cisterna juxtapositioning , and they are located mainly at the cis and medial Golgi , rather than the trans-Golgi -LRB- Barr et al. 1997 ; Shorter et al. 1999 -RRB- . So this intercisternal `` glue '' may be gradually lost as a cisterna progresses towards the trans pole of the Golgi complex . This has been confirmed both in mammals and yeast by the observation that the trans-most cisterna frequently peels off from the main Golgi stack -LRB- Clermont et al. 1995 ; Mogelsvang et al. 2003 -RRB- . It is possible that more than two or more mechanisms act in synergy to provide this transition from flat Golgi cisternae to the tubulo-reticular TGN morphology . In addition to the formation of tubular domains at the exit face of the Golgi complex , this is also the level at which cargo proteins that are directed to different post-Golgi destinations should be sorted . The classical view in the membrane transport field implies that sorting at the TGN -LRB- as well as throughout the whole secretory pathway -RRB- is driven mainly by the coat-adaptor-protein machinery , which interacts specifically with amino-acid signals of certain transmembrane cargo proteins ; this then provides the mechanical force for budding and fission of transport vesicle -LRB- Mellman and Warren 2000 -RRB- . This holds true for the endo-lysosome-directed carriers that have been thoroughly characterized . These carriers consist of clusters of clathrin-coated buds that are connected by tubular regions , and thus exhibit a grape-like structure . In contrast , PGCs carrying a cargo like the G-protein of vesicular stomatitis virus -LRB- VSVG -RRB- form in a coat and an AP-independent manner . Both PGCs and their precursors do not show β-COP or γ - , δ - and ε-adaptins at their membranes -LRB- Polishchuk et al. 2003 -RRB- . Other adaptors , such as the GGAs , are excluded from VSVG carriers as well -LRB- Puertollano et al. 2003 ; Polishchuk et al. 2003 -RRB- . Similarly , coats and adaptors have also never been detected on PGCs that are carrying proteins to the apical surface in polarized cells -LRB- Kreitzer et al. 2003 -RRB- . Thus , these carriers should form either by virtue of some still-unknown adaptors that can not yet be visualized by electron microscopy , or by their association with specific lipid microdomains that are involved in sorting -LRB- Schuck and Simons 2004 -RRB- . This might be the case for proteins directed to the apical surface in polarized epithelial cells , the concentration of which at the TGN appear to be through their partition into cholesterol - and sphingolipid-rich membrane domains that are known as `` rafts '' -LRB- Schuck and Simons 2004 -RRB- . The scission of PGCs from the TGN The morpho-dynamics of the fission process are also fairly complex . Observations in living cells have revealed that the fission of PGCs frequently coincides with mechanical pulling of carrier precursor from the TGN along microtubules . Apparently , the pulling force which the molecular motors such as kinesin -LRB- see below -RRB- can apply to the TGN membranes is important to facilitate the extension of PGC precursors from the Golgi body and for the later fission of the PGC -LRB- Kreitzer et al. 2000 ; Polishchuk et al. 2003 -RRB- . In cell-free systems , the addition of kinesin to Golgi membranes -LRB- and even to liposomes -RRB- together with microtubules induces the formation of tubule-like membranes that are similar to PGC precursors -LRB- Roux et al. 2002 -RRB- , while a block of kinesin function by microinjection of an inhibitory antibody -LRB- Kreitzer et al. 2000 -RRB- or expression of the headless kinesin mutant -LRB- Nakata and Hirokawa 2003 -RRB- prevent PGC formation from the Golgi complex . Kinesin has been seen to be associated with the tip of PGC precursors , although it can also attach to other points along the PGC precursor membrane -LRB- Polishchuk et al. 2003 -RRB- . The movement of kinesins along microtubules can then create tension within the PGC precursor that will facilitate the fission process . Indeed , based on in vitro data , membranes under tension have recently been proposed to have an important role in fission -LRB- Roux et al. 2006 -RRB- . However , PGCs can also form when microtubules have been destroyed by nocodozole treatment ; in this case , the pulling force to create membrane tension in fission-prone regions might be provided by actin motors -LRB- Warner et al. 2003 ; Sahlender et al. 2005 -RRB- . Live cell imaging and CLEM have also shown that fission does not take place randomly along the membranes of PGC precursors . In this case , which regions of a PGC precursor can be defined as prone to fission ? First , this depends on the geometry of the precursor membranes . Our data suggest that fission usually takes place at the thinnest parts of the PGC precursor -LRB- Fig. 2a -RRB- , which at the electron microscopy level corresponding to thin tubular segments of membranes -LRB- Polishchuk et al. 2003 -RRB- . In contrast , fission does not take place at the TGN regions with a complex morphology -LRB- i.e. , in those containing tubular networks and branching tubules , or in thick vacuolar regions -RRB- . Obviously , the precise points of fission will define not only the compositions of the PGC carriers , but also their morphology . If fission occurs close to the tip of the TGN tubule , a carrier will be smaller in size . In contrast , larger PGCs can form by cleavage at the base of the PGC precursors -LRB- Fig. 2a -RRB- . Similarly , endosome-directed PGCs can apparently detach from the TGN as simple cargo-containing vesicles if the fission occurs at the neck of the clathrin-coated buds -LRB- Fig. 2b -RRB- . However , many clathrin-positive PGCs have a grape-like morphology -LRB- tubule with several buds -RRB- , suggesting that entire chunks of TGN membranes containing 2 -- 3 clathrin-coated buds can be cleaved from the Golgi complex -LRB- Polishchuk et al. 2006 -RRB- . Fig. 2Fission of post-Golgi transport carriers . a TGN precursors of post-Golgi carriers are pulled along microtubules by kinesin . The fission -LRB- red line -RRB- of the carriers occurs at the thinnest parts of the PGC precursor , which correspond to thin tubular segments of the TGN membrane at the electron microscopy level . In contrast , fission does not take place at the TGN regions with a complex morphology -LRB- i.e. , containing tubular networks and fenestrae , or in thick vacuolar regions -RRB- . If fission occurs close to the tip of a PGC precursor , the carrier will be smaller in size -LRB- 1 -RRB- . In contrast , larger PGCs can be formed by cleavage at the bottom of a PGC precursor -LRB- 2 -RRB- . b PGCs directed to endosomes detach from the TGN as simple clathrin-coated vesicles if fission -LRB- red line -RRB- occur at the neck of the clathrin-coated bud -LRB- 1 -RRB- . Alternatively , entire chunks of the TGN membrane containing 2-3 clathrin-coated buds can be cleaved from the Golgi complex Another factor that might be important for PGC shaping is the type of cargo that is embedded in the PGC . For example , procollagen-I usually forms quite large aggregates that are visible within 300 -- 400-nm-diameter membrane distensions of the Golgi membranes . As a consequence , similar distensions have been detected in most collagen-containing PGCs -LRB- Polishchuk et al. 2003 ; Canty et al. 2004 -RRB- . Movement of PGCs from the Golgi complex to the target membrane After fission from the TGN , PGCs move to the acceptor membrane . Different members of the kinesin superfamily -LRB- Kamal et al. 2000 ; Nakata and Hirokawa 2003 ; Teng et al. 2005 -RRB- , and also the other microtubule motor dynein -LRB- Tai et al. 1999 -RRB- , have been shown to drive post-Golgi transport of specific cargo to various destinations . This high fidelity of cargo selection by molecular motors at the TGN and their further delivery to the correct surface or intracellular domain may be regulated by interactions of motor protein directly with the cargo -LRB- Kamal et al. 2000 ; Teng et al. 2005 -RRB- or with components of the sorting machinery at the TGN -LRB- Nakagawa et al. 2000 -RRB- . As an example , transport of HA and annexin 13b to the apical surface in epithelial cells relies on raft-associated motor KIFC3 -LRB- Noda et al. 2001 -RRB- . KIF13A operates in the other post-Golgi route that is used for the transport of the mannose-6-phosphate receptor -LRB- Nakagawa et al. 2000 -RRB- . A number of neuronal proteins , including bAPP , GAP43 and vamp-2 , require KIF5 for their correct targeting -LRB- Nakata and Hirokawa 2003 -RRB- , while the microtubule minus-end-directed motor dynein has been shown to support rhodopsin transport in rod photoreceptors -LRB- Tai et al. 1999 ; Yeh et al. 2006 -RRB- . Selection of specific cargoes by motors could be driven by various mechanisms . The simplest would use a direct interaction between the motor and a specific domain of a cargo protein , as is seen for the dynein light chain and the cytoplasmic tail of rhodopsin -LRB- Tai et al. 1999 -RRB- . Alternatively , adaptor proteins could serve as a bridge between a motor and its cargo . For example , KIF13A transports the mannose-6-phosphate receptor through its interaction with the AP-1 complex -LRB- Nakagawa et al. 2000 -RRB- . Finally , both motor and cargo could associate with the same specific lipid microdomain , as for instance , for KIFC3 and the apically targeted annexin XIIIb . Another issue that needs to be addressed is whether any of these sorting processes take place in the PGCs while they are moving toward their acceptor compartment . This happens , for example , with the maturation of secretory granules when the mannose-6-phosphate receptor is concentrated and sorted from the secretory granules by clathrin-coated vesicles -LRB- Klumperman et al. 1998 -RRB- . So several strategies have been used to determine whether similar sorting events happen with PGCs . Mature exocytic carriers can be arrested before their fusion with the plasma membrane either by microinjection of an anti-NSF antibody or by treatment with tannic acid -LRB- which fixes the plasma membrane but does not penetrate inside the cell -RRB- . In contrast to secretory granules , the comparison of mature and newly formed PGCs did not reveal significant changes in either their ultrastructure or their composition -LRB- Polishchuk et al. 2003 , 2004 -RRB- . Similarly , mature Golgi-to-endosome carriers were accumulated in cells upon endosome inactivation . However , they did not show any significant transformation , except for a very moderate reduction in the area covered by clathrin -LRB- Polishchuk et al. 2006 -RRB- . Live-cell imaging of subconfluent MDCK cells has shown that PCGs that initially contained both a basolateral marker -LRB- VSVG-CFP -RRB- and an apical marker -LRB- GPI-YFP -RRB- did not sort out either of these cargoes into any separate structures , and instead delivered both of the proteins to the plasma membrane -LRB- Polishchuk et al. 2004 -RRB- . GPI-GFP was then sorted from the basolateral surface to the apical membrane through transcytosis -LRB- Polishchuk et al. 2004 -RRB- . On the other hand , the partitioning of two proteins from a common PGC into separate carriers has also been reported -LRB- Jacob and Naim 2001 -RRB- . This suggests that sorting from the PGC may exist , but that it should depend on the nature of the cargo proteins being transported . Intermediate stations in post-Golgi transport The complexity of sorting events in the post-Golgi space appears to be even worse since the discovery that certain cargoes may pass through the endosomal compartments before their arrival at the plasma membrane . Sporadic reports in the past have suggested that some secretory proteins do not move from the Golgi complex directly to the plasma membrane , but instead pass through an endocytic intermediate on their way to the cell surface -LRB- Leitinger et al. 1995 -RRB- . This indirect `` through-endosome '' delivery of cargo to the plasma membrane might be also facilitated by the intimate association of TGN membranes with number of endocytic compartments in the perinuclear area of the cell -LRB- Marsh et al. 2001 -RRB- . The list of the proteins using this pathway has been recently updated , and it has now been shown that in MDCK epithelial cells , VSVG , the LDL receptor and E-cadherin can be detected in the endosomes before their exit to the plasma membrane -LRB- Ang et al. 2004 ; Lock and Stow 2005 -RRB- . These findings , however , promote a number of further questions . The first is whether this transport route is ubiquitous -LRB- i.e. , does it exist in different cells ? -RRB- . The second question is whether different cargoes move through the same endosomal compartment on their way to the cell surface in epithelial cells ? A number of proteins -LRB- such as VSVG and the LDL receptor -RRB- have been reported to use a Rab8-positive sub-population of endosomes as an intermediate station on their way to the basolateral membrane in epithelial cells -LRB- Ang et al. 2003 -RRB- . These proteins require the AP-1B adaptor complex to be properly sorted from these Rab8 endosomes towards the basolateral surface domain -LRB- Ang et al. 2003 -RRB- . Other cargoes -LRB- such as E-cadherin , for example -RRB- move to the plasma membrane through a Rab11 endocytic compartment -LRB- Lock and Stow 2005 -RRB- . It remains to be determined , however , whether there is any cross-talk between these Rab8 - and Rab11-dependent routes . This possibility apparently exists , since Rab8 and Rab11 endosomes are both accessible to transferrin -LRB- Ang et al. 2004 ; Lock and Stow 2005 -RRB- . Finally it is important to clarify as to what extent this through-endosome transport route is used by different cargo proteins in epithelial cells . This issue has been partially addressed by the silencing of the μ1B subunit of the AP-1B adaptor complex , which resides at the endosomes and is required for the correct delivery of many basolateral proteins . The use of RNA interference has revealed , however , that a number of cargoes -LRB- such as transferrin and Fc receptors , for example -RRB- can be targeted correctly even in the absence of AP-1B , which suggests that these proteins can move directly from the Golgi complex to the basolateral surface without crossing the endocytic routes -LRB- Gravotta et al. 2007 -RRB- . The existence of more than one basolateral pathway has also been supported by the observation that transport of different basolateral proteins can be selectively regulated by different PKD isoforms -LRB- Yeaman et al. 2004 -RRB- . Thus , further efforts need to be made to understand to what extent an endocytic post-Golgi compartment is involved in the sorting and transport events of cell-surface proteins . Conclusions The extensive characterization of PGC morphology by video and electron microscopy has provided a framework for the positioning of the molecular machineries in the morpho-functional maps of various trafficking segments . At the same time , many molecular players in TGN-to-plasma membrane transport have been identified . The attribution of each of the molecular components to each of the pathways is probably now the main challenge . We believe that the development of specific assays will significantly assist in the achieving of this objective . Video microscopy of GFP-tagged cargo proteins has allowed us to evaluate the process of PGC formation , as well as the speed and directionality of PGC movement through the cytosol . Other assays use tannic acid treatment -LRB- Polishchuk et al. 2004 ; Jakob et al. 2006 -RRB- or horse-radish-peroxidase-based endosome immobilization -LRB- Ang et al. 2004 ; Polishchuk et al. 2006 -RRB- to prevent the fusion of PGCs with their target membranes . These conditions allow us to trap mature PGCs and to compare their compositions to newly formed carriers and the TGN membranes . Moreover , preferential sites of PGC docking and fusion can also be easily identified in this way . The combination of these experimental approaches with specific molecular inhibitors now allows us to attribute a protein of interest to one of the steps in post-Golgi transport .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

trans-golgi network
post-golgi carriers
post-golgi transport