Imaging and imagination : understanding the endo-lysosomal system Lysosomes are specialized compartments for the degradation of endocytosed and intracellular material and essential regulators of cellular homeostasis . The importance of lysosomes is illustrated by the rapidly growing number of human disorders related to a defect in lysosomal functioning . Here , we review current insights in the mechanisms of lysosome biogenesis and protein sorting within the endo-lysosomal system . We present increasing evidence for the existence of parallel pathways for the delivery of newly synthesized lysosomal proteins directly from the trans-Golgi network -LRB- TGN -RRB- to the endo-lysosomal system . These pathways are either dependent or independent of mannose 6-phosphate receptors and likely involve multiple exits for lysosomal proteins from the TGN . In addition , we discuss the different endosomal intermediates and subdomains that are involved in sorting of endocytosed cargo . Throughout our review , we highlight some examples in the literature showing how imaging , especially electron microscopy , has made major contributions to our understanding of the endo-lysosomal system today . Introduction Lysosomes are the terminal degradative compartments of the endocytic pathway . They receive extracellular components via endocytosis and intracellular material via autophagy , as well as via the biosynthetic pathway -LRB- Eskelinen 2005 ; Klionsky 2007 ; Luzio et al. 2007 ; Sachse et al. 2002a -RRB- . By modulating the degradation of a multitude of proteins , lysosomes are key organelles in the regulation of cellular homeostasis . Moreover , these catabolic functions are complemented by a group of cell type-specific lysosome-related organelles , which store and/or secrete key factors involved in a diversity of functions , such as blood clotting , antigen presentation , pigmentation and alveolar surface tension regulation -LRB- dell ' Angelica et al. 2000a ; Raposo et al. 2007 ; Weaver et al. 2002 -RRB- . Because of their many functions , defects in lysosome functioning can have devastating consequences , as is illustrated by the rapidly growing number of human disorders displaying a primary or secondary defect in the lysosomal system . To date , lysosomal aberrations have been found in the ` classical ' lysosomal storage disorders , as well as in muscular and neurological disorders , numerous types of cancer , immune-deficiency diseases and pigmentation-bleeding disorders -LRB- Futerman and van Meer 2004 ; Kroemer and Jäättelä 2005 ; Mohamed and Sloane 2006 ; Nixon 2005 ; Saftig et al. 2001 -RRB- . Unraveling the intrinsic complexity of the endo-lysosomal system is a major challenge for cell biologists . Here , we will review some of the current insights in the mechanisms involved in lysosome biogenesis and functioning . Imaging of the endo-lysosomal system combined with imaginative visions has been instrumental for furthering the conceptual understanding of the endo-lysosomal system . Therefore , on the special occasion of the 50th anniversary of the Journal of Histochemistry and Cell Biology , we will also highlight some examples in the literature showing how imaging , especially electron microscopy -LRB- EM -RRB- , has contributed to our understanding of the endo-lysosomal system today . The discovery of the lysosome The famous story of the discovery of the lysosome reads as a compelling detective novel -LRB- Bainton 1981 ; de Duve 2005 -RRB- . A major question that was faced by the cell biological society of the fifties was how cells could exert proteolytic activity without being digested by autolysis . The first indications on the existence of lysosomes came from studies designed to answer an entirely different question-i.e. to clarify the action of insulin on the liver-in which acid phosphatase activity -LRB- nowadays known as a lysosomal activity -RRB- measurements were used as a control . A combination of experiments from the labs of Christian de Duve and Albert Claude led to the intriguing observation that acid phosphatase in fresh fractions , prepared by a relatively gentle protocol , showed astonishingly low activity levels . By contrast , samples that were obtained by a crude fractionation protocol or stored for some days before assaying showed elevated levels of phosphatase activity . When this same hide-and-seek strategy was found for four additional hydrolytic enzymes , all with an acidic pH optimum and the same sedimentation characteristics as acid phosphatase , de Duve concluded that these enzymes must be contained together in particles surrounded by a membrane ` to prevent the enzymes getting out and the substrate getting in ' . The term lysosome , for lytic particle or digestive body , was introduced -LRB- de Duve et al. 1955 -RRB- . Novikoff , together with de Duve , provided the first EM pictures of these liver fractions that in addition to numerous mitochondria also showed an enrichment of electron dense bodies -LRB- Fig. 1 -RRB- . These were tentatively recognized as the ` pericanalicular dense bodies ' previously found in intact liver cells -LRB- Novikoff et al. 1956 -RRB- . In these same years , Gomori had developed a protocol for acid phosphatase cytochemistry -LRB- Gomori 1952 -RRB- , which was subsequently optimized for EM by Novikoff , Holt , Barka and Anderson -LRB- Barka and Anderson 1962 ; Holt 1959 ; Novikoff 1961 -RRB- . The circle was closed when the acid phosphatase staining was applied to the ultrastructural level , thereby providing the direct evidence that acid phosphatase activity localized to the dense bodies of the liver parenchyma cells -LRB- Essner and Novikoff 1961 -RRB- -LRB- Fig. 2 -RRB- , as well as similar bodies in various other cell types . Fig. 1Reproduction of the first electron micrograph ever that indicated the existence of lysosomes -LRB- arrows -RRB- , as originally published in 1956 by Novikoff and colleagues . The sample represents a liver fraction that also contains mitochondria -LRB- MT -RRB- and endoplasmic reticulum -LRB- ER -RRB- . Reproduced from Novikoff et al. -LRB- 1956 -RRB- Fig. 2Reproduction from Essner and Novikoff -LRB- 1961 -RRB- . This picture shows for the first time at the ultrastructural level that acid phosphatase activity is localized in lysosomes -LRB- LY -RRB- of liver cells . BC bile canaliculus , ER endoplasmic reticulum , EX extraneous precipitate , MI microvilli , V vacuole Another major clue was provided by Werner Straus . He found that ` droplets ' of unknown function within the proximal tubules of the kidney contained reabsorbed -LRB- i.e. endocytosed -RRB- material as well as acid phosphatase activity and other lysosomal hydrolases -LRB- Straus 1954 , 1956 -RRB- . This observation provided the first link between lysosomal digestion and endocytic uptake of extracellular material . Hence , the concept was born of the lysosome as a membrane-bound organelle that contains acid hydrolases involved in the digestion of substances that enter the cell via endocytosis -LRB- de Duve 1959 -RRB- . It explained that cells digest without autolysis by setting apart the degradative enzymes together with their endocytosed substrates in a membrane-bound compartment . The answer to the riddle was found . The endocytic pathway in a nutshell Lysosomes receive extracellular substances for degradation via endocytosis : the invagination and pinching-off of membrane-bound vesicles from the plasma membrane -LRB- Fig. 3a -RRB- . There are at least five highly controlled different entries into the cell , of which the ` classical ' clathrin-mediated pathway is best-documented . However , the importance of non-clathrin-mediated pathways is becoming increasingly evident -LRB- Conner and Schmid 2003 ; Kirkham and Parton 2005 ; Mayor and Pagano 2007 ; Nichols 2003 -RRB- . Clathrin-mediated endocytosis begins with the sorting of receptors into a clathrin-coated pit at the plasma membrane , which then buds off to form a transport vesicle . These vesicles fuse with the first intracellular sorting station , the early endosome -LRB- Fig. 3a -RRB- . Early endosomes have a mildly acidic pH that triggers the dissociation of some ligands from their receptors . Emptied receptors enter membrane tubules that emerge from the endosomal vacuole , resulting in transport via the recycling endosome back to the plasma membrane or , alternatively , the trans-Golgi network -LRB- TGN -RRB- -LRB- Fig. 3a -RRB- . Receptors that remain ligand-bound are sequestered into small 50 -- 80 nm diameter intraluminal vesicles -LRB- ILVs -RRB- that bud from the endosomal-limiting membrane into the endosomal lumen . Through a series of such vesicle fusion and fission events involving protein sorting , early endosomes gradually transform into late endosomes , a process called endosomal maturation . The formation of ILVs can continue up to the late endosome , which is why during endosomal maturation the number of these internal vesicles increases and late endosomes are often referred to as multivesicular bodies -LRB- MVBs , Fig. 3a -RRB- . Degradation of cargo starts in late endosomes/MVBs and continues in the lysosome . Late endosomes/MVBs develop into lysosomes by multiple fusion and fission cycles -LRB- Futter et al. 1996 ; Luzio et al. 2007 -RRB- . After degradation of the lysosomal content , transporter molecules translocate sugars , amino acids and lipids across the limiting lysosomal membranes into the cytosol where they function as building blocks in the synthesis of new products . Fig. 3Sorting and transport within the biosynthetic and endo-lysosomal pathways . a Schematic representation of the interplay between the biosynthetic pathway and endo-lysosomal system depicting the main endosomal compartments that are discussed in the text . b Detailed overview of the various sorting steps at the early endosome . At the endosomal vacuole or sorting endosome , receptor -- ligand complexes destined for degradation accumulate in the bilayered clathrin coat -LRB- 1 -RRB- , upon which they are sorted into intraluminal vesicles -LRB- ILV -RRB- that form by inward budding of the vacuolar limiting membrane . Recycling cargo by default enters the membrane tubules of the tubular sorting endosome -LRB- 2 -RRB- from which it can recycle back to the plasma membrane via the recycling endosome , or is actively sorted towards late endosomes/lysosomes -LRB- AP-3 pathway for lysosomal membrane proteins -RRB- or TGN -LRB- AP-1 pathway -RRB- . A third exit from the vacuolar endosome is provided by the endosome-to-TGN carriers -LRB- 3 -RRB- that mediate SNX1-dependent recycling of MPRs and sortilin to the TGN . c From the TGN , multiple pathways arise that mediate transport to the endo-lysosomal system and plasma membrane . It is still an open question to what extent these pathways use different or overlapping molecular machinery and transport carriers . AP adaptor protein complex , ER endoplasmic reticulum , M6P mannose 6-phosphate , MPR mannose 6-phosphate receptor , SAP sphingolipid activator protein , SNX1 sorting nexin 1 , TGNtrans-Golgi network Defining early and late endosomes When studying the endo-lysosomal system , one is forced to continuously pose the question how to discriminate between the distinct endosomal intermediates . The distinction ` early ' and ` late ' endosomes often has too limited discriminating power to describe the dynamic continuum of the endo-lysosomal pathway . In a recent EM study -LRB- Mari et al. 2007 -RRB- , a novel method to distinguish early and late endosomes was introduced by combining morphological , kinetic and molecular criteria . Early endosomes are by definition accessible to internalized transferrin -LRB- Tf -RRB- that recycles to the plasma membrane . As a result , late endosomes contain only marginal levels of Tf . With this in mind , the number of ILVs within the endosomal vacuoles was correlated with the occurrence of internalized Tf . Tf-positive early endosomal vacuoles on average contained one to eight ILVs , whereas endosomal vacuoles with nine or more ILVs were mostly devoid of Tf and therefore designated as late endosomes . A similar analysis with various other established early endosomal marker proteins corroborated and extended this definition . For example , the early endosomal antigen 1 -LRB- EEA1 -RRB- appeared to be connected with only the very early maturation stages of early endosomes -LRB- one to five ILVs -RRB- , whereas Rab11 appeared only on the later maturation stages of early endosomes -LRB- four ILVs or more -RRB- . The early endosomal markers Rab4 and Hrs were more evenly distributed . Although the interpretation of this study is currently limited to the cell line in which the analysis was performed , i.e. the human hepatoma cell line HepG2 , these observations illustrate that distinct stages of endosomes can be better monitored with increasing means of analysis . TGN-to-endosome transport pathways To fulfill their degradative function , lysosomes must also receive lysosomal proteins . These are synthesized in the endoplasmic reticulum and Golgi complex , after which they enter the TGN -LRB- Fig. 3a -RRB- . From the TGN , multiple pathways to the endosomal system arise , mediating delivery of essential components to various endosomal intermediates . Thus , lysosomes receive their components via both the endocytic pathway and biosynthetic pathway . Two classes of proteins together are essential for lysosomal functioning : the soluble lysosomal hydrolases and lysosomal membrane proteins . Each lysosomal hydrolase targets specific substrates for degradation and presently more than 50 different types are known . The heavily glycosylated lysosomal membrane proteins encompass a group of proteins with various functions . The major lysosomal membrane proteins -LRB- Marsh et al. 1987 -RRB- are the lysosome-associated membrane proteins -LRB- LAMP -RRB- -1 and -2 , the lysosomal integral membrane protein -LRB- LIMP -RRB- -2 and the tetraspanin CD63 -LSB- originally identified as platelet-activating glycoprotein -LRB- Pltgp40 -RRB- and also often referred to as melanoma-associated antigen ME491 , LIMP-1 or LAMP-3 -RSB- . Initially , lysosomal membrane proteins were thought to mainly play a role in lysosomal stability and integrity , but currently new functions emerge involving chaperone-mediated autophagy and macroautophagy -LRB- reviewed by Eskelinen et al. 2003 -RRB- . Thus , the proper targeting of lysosomal proteins from the TGN to the endo-lysosomal system is an essential process in the biogenesis and maintenance of lysosomes . The best-known pathway for TGN-to-endosome transport is the delivery of soluble lysosomal hydrolases by the 300 kD cation-independent -LRB- CI -RRB- and 46 kD cation-dependent -LRB- CD -RRB- mannose 6-phosphate receptors -LRB- MPR -RRB- -LRB- Kornfeld and Mellman 1989 ; von Figura 1991 -RRB- . However , as will be outlined in the next paragraphs , increasing evidence indicates the existence of multiple additional or alternative pathways from the TGN to endosomes . MPR-dependent transport of soluble lysosomal hydrolases Both the CI and CD-MPR are present in almost all mammalian cell types . The lumenal domain of the CI-MPR is composed of 15 repetitive units , with two mannose 6-phosphate -LRB- M6P -RRB- - binding sites , whereas the CD-MPR has a lumenal domain of 159 amino acids , resembling one such unit , and has a single M6P-binding site . The M6P-binding sites recognize M6P-moieties , which are specifically present on the precursor forms of newly synthesized lysosomal hydrolases . Both receptors predominantly function as non-covalently bound homodimers . A specific role for each MPR in the targeting of lysosomal hydrolases has not been established , but distinct lysosomal hydrolases can exhibit different affinities for CI or CD-MPR -LRB- Qian et al. 2008 ; Sleat and Lobel 1997 -RRB- . CI-MPR can endocytose extracellular lysosomal hydrolases , whereas under physiological conditions CD-MPR is not involved in endocytosis . CD-MPR shows enhanced ligand binding in the presence of divalent cations -LRB- Hoflack and Kornfeld 1985a , b -RRB- . Interestingly , the CI-MPR also has several other functions ; it regulates the levels of circulating insulin-like growth factor II by binding it at the plasma membrane for delivery to lysosomes . Furthermore , it facilitates the activation of the precursor of transforming growth factor-β1 , which is a growth inhibitor , and also binds retinoic acid , thereby mediating retinoic acid-induced apoptosis and growth inhibition , further supporting the idea that CI-MPR could be a tumor suppressor -LRB- reviewed by Ghosh et al. 2003a -RRB- . Together , the two MPRs mediate the targeting of the huge majority of newly synthesized lysosomal hydrolases from the TGN to the endo-lysosomal system -LRB- Fig. 3c -RRB- -LRB- Hoflack and Kornfeld 1985a , b ; Sahagian et al. 1981 -RRB- . In steady state , both CD and CI-MPR have been localized by immuno-EM to the TGN , plasma membrane , endosomes and associated vesicles -LRB- Bleekemolen et al. 1988 ; Geuze et al. 1984 , 1985 ; Griffiths et al. 1988 ; Klumperman et al. 1993 , 1998 -RRB- . Sorting of MPRs at the TGN was long thought to predominantly depend on binding to the heterotetrameric adaptor-protein complex -LRB- AP -RRB- -1 , which also mediates the recruitment of clathrin -LRB- Fig. 4 -RRB- . The discovery of the Golgi-localized , γ-ear-containing , Arf-binding family of proteins -LRB- GGA -RRB- as clathrin adaptors changed this view -LRB- Boman et al. 2000 ; dell ' Angelica et al. 2000b ; Hirst et al. 2000 ; Poussu et al. 2000 -RRB- . GGAs are monomeric proteins and three different forms are known in mammals , GGA1 , GGA2 and GGA3 . GGAs and AP-1 might function in parallel to generate distinct MPR-containing vesicles at the TGN , thereby allowing delivery to different endosomal compartments . Alternatively or additionally , GGAs may facilitate entry of MPR into clathrin-coated vesicles by interacting with AP-1 . A major clue in favor of the latter hypothesis was provided by immuno-EM showing that GGAs and AP-1 colocalize in the same clathrin-coated TGN buds -LRB- Doray et al. 2002 ; Puertollano et al. 2003 -RRB- . Furthermore , the three GGAs were found to interact with each other and also to bind directly with their hinge domain to the γ-ear domain of AP-1 . Depletion of each GGA resulted in decreased levels of the other GGAs , their redistribution to the cytosol and impaired sorting of CI-MPR and the lysosomal hydrolase cathepsin D -LRB- Doray et al. 2002 ; Ghosh et al. 2003b -RRB- . However , these findings do not rule out the possibility that different adaptor proteins can also act separately to a certain extent . Interestingly , it is not precisely known where the MPRs deliver their bound ligands . A small percentage of the MPRs travels to the cell surface , from where they are internalized by clathrin-mediated endocytosis and routed to the early endosomes . However , the majority takes a direct intracellular pathway to the endo-lysosomal system , and it is generally believed that both early and late endosomes can be recipients for TGN-derived MPR . The involvement of multiple adaptors could provide a means to establish these distinct pathways . Fig. 4Immuno-EM of a human hepatoma cell -LRB- HepG2 -RRB- showing the occurrence of CIMPR -LRB- represented by 10 nm gold particles -RRB- in AP-1 -LRB- represented by 15 nm gold particles -RRB- coated TGN membranes -LRB- arrowheads -RRB- . CI-MPR and AP-1 positive membranes also show the characteristic cytoplasmic dense coat indicative for the presence of clathrin . The TGN area is enclosed by distinct golgi stacks -LRB- G -RRB- . Bar , 200 nm MPR-independent transport of soluble lysosomal hydrolases In addition to the well-characterized MPR pathway , several additional transport pathways for lysosomal hydrolases exist -LRB- Fig. 3c -RRB- . For example , the lysosomal hydrolases lysosomal acid phosphatase and β-glucocerebrosidase do not acquire M6P residues and their transport is not depending on MPRs -LRB- Aerts et al. 1988 ; Waheed et al. 1988 -RRB- . Lysosomal acid phosphatase is a type I transmembrane protein that follows the constitutive secretory pathway to the plasma membrane and reaches the lysosome via endocytosis . In lysosomes , the transmembrane precursor is processed into a soluble form , but both proteins are enzymatically active -LRB- Waheed et al. 1988 -RRB- . Lysosomal acid phosphatase contains the tyrosine-based motif YRHV in its cytosolic domain , which is required for endocytosis , but does not mediate direct TGN-to-endosome targeting -LRB- Obermüller et al. 2002 ; Peters et al. 1990 ; Pohlmann et al. 1988 -RRB- . β-Glucocerebrosidase has no membrane spanning domain -LRB- Erickson et al. 1985 -RRB- . After synthesis , it is glycosylated upon which it becomes membrane-associated -LRB- Rijnboutt et al. 1991 -RRB- . The machinery required for proper transport of β-glucocerebrosidase was long unknown . Recently , Paul Saftig and colleagues unexpectedly identified the lysosomal membrane protein LIMP-2 as a specific receptor for β-glucocerebrosidase . LIMP-2 binds β-glucocerebrosidase in a pH-dependent manner , enabling association in the ER and guiding it all the way to the lysosome , where the complex likely dissociates because of the acidic pH -LRB- Reczek et al. 2007 -RRB- . Whether LIMP-2 recycles between endosomes and TGN is not known . This study for the first time implies a lysosomal membrane protein in the transport of a lysosomal hydrolase . It will be very interesting to establish whether LIMP-2 acts as a more general binding partner for lysosomal hydrolases . In addition , characterization of the molecular machinery required for transport will provide further insight into this TGN-to-endosome transport pathway . Lysosomal hydrolases that are normally modified with a M6P residue can also traffic in an MPR-independent way to the lysosomes -LRB- Fig. 3c -RRB- . This became apparent by comparing different cell types from patients with I-cell disease , also known as Mucolipidosis type II . In this disease , soluble lysosomal hydrolases do not acquire M6P groups due to a deficient activity of the enzyme GlcNAc-phosphotransferase -LRB- Hasilik et al. 1981 ; Reitman et al. 1981 -RRB- . As a consequence , the lysosomal hydrolases follow the default constitutive pathway to the plasma membrane -LRB- Fig. 3c -RRB- and are thus secreted . However , in cells of non-mesenchymal origin , most lysosomal hydrolases are still targeted correctly to the lysosome -LRB- Fig. 5 -RRB- -LRB- Little et al. 1987 ; Owada and Neufeld 1982 ; Waheed et al. 1982 -RRB- , suggesting the existence of alternative pathways . The same phenomenon was observed in a very recently established mouse model system for I-cell disease , achieved by GlcNAc-phosphotransferase knockout -LRB- Gelfman et al. 2007 -RRB- . Furthermore , in mice that are deficient of both MPRs , the intracellular activities of several lysosomal hydrolases are comparable to the control situation -LRB- Dittmer et al. 1999 -RRB- . These findings indicate that lysosomal hydrolases can follow a pathway to the lysosome that is not mediated by MPRs . Fig. 5 Immuno-EM of a B lymphoblast derived from a patient with I-cell disease showing a lysosome -LRB- L -RRB- positively labeled for the lysosomal hydrolase cathepsin D -LRB- represented by 10 nm gold particles -RRB- . This picture indicates that although the MPR pathway is impaired in these cells , lysosomal enzymes can still reach lysosomes . Bar 200 nm The mechanisms and pathways of MPR-independent transport are still poorly defined . Recent studies , however , have implicated a role for the multiligand receptor sortilin as an alternative receptor for a subset of lysosomal proteins , called sphingolipid activator proteins -LRB- SAPs -RRB- , which are non-enzymatic cofactors required for the degradation of glycosphingolipids . Two SAPs are known ; the precursor protein prosaposin , which renders saposin A , B , C and D after proteolysis , and GM2 activator protein -LRB- AP -RRB- , an essential cofactor for β-hexosaminidase A. Studies from Lefrancois and colleagues have shown that sortilin interacts with both prosaposin and GM2AP , which is independent of M6P tags , whereas depletion of sortilin induced their secretion -LRB- Lefrancois et al. 2003 -RRB- . It remains to be established , however , whether sortilin is the exclusive receptor for SAPs , or whether MPR has an additional role in their transport . Interestingly , sortilin , together with SorLA and SorCS1 -- 3 , forms a protein family sharing homology to the lumenal domain of the yeast vacuolar sorting protein Vps10p , which directs carboxypeptidase Y to the vacuole -LRB- Marcusson et al. 1994 -RRB- . Immuno-localization studies positioned sortilin predominantly in endosomes and the TGN -LRB- Mari et al. 2007 -RRB- , where it colocalized with CI-MPR in AP-1 and GGA3 positive clathrin-coated transport carriers , indicating that sortilin and MPRs travel via the same carriers to the endo-lysosomal system . Currently , the functions of the SorCS-subgroup are unknown , but since SorCS3 is predominantly localized at the plasma membrane it is unlikely to be involved in TGN-to-endosome trafficking -LRB- Westergaard et al. 2005 -RRB- . Whether sortilin or possibly SorLA could act as alternative receptors for lysosomal hydrolases is still unclear . TGN-to-lysosome transport of lysosomal membrane proteins Lysosomal membrane proteins that exit the TGN can be transported to lysosomes following either a direct or indirect pathway , involving passage over the plasma membrane . Virtually nothing is known about the carriers and molecular machinery that mediate the direct route of lysosomal membrane proteins to the lysosomes . Moreover , the contribution of this pathway remains debated -LRB- Carlsson and Fukuda 1992 ; Janvier and Bonifacino 2005 -RRB- . Important for the sorting of lysosomal membrane proteins to lysosomes are the tyrosine and dileucine-based consensus motifs within their cytosolic tails . LAMP-1 , LAMP-2 and CD63 -LRB- LIMP-1 -RRB- all bear a YXXØ motif , with an essential tyrosine residue , hydrophobic XX residues and the Ø residue being a bulky hydrophobic amino acid -LRB- reviewed by Bonifacino and Traub 2003 -RRB- . The YXXØ motif is required for endocytosis at the plasma membrane , but has also been implicated in the direct targeting from TGN to lysosomes . For TGN sorting , however , additional characteristics seem to be required ; a glycine residue preceding the critical tyrosine , the XX residues being acidic and the entire motif positioned six to nine residues from the transmembrane domain . LIMP-2 , the recently identified receptor for β-glucocerebrosidase , has a dileucine-based sorting motif DXXLL , also referred to as acidic cluster-dileucine motif , which has also been implicated in both internalization and TGN-to-lysosome targeting . The YXXØ motifs are recognized by the μ subunits of AP-1 , AP-2 , AP-3 and AP-4 and the DXXLL motif by the VHS domain of the GGAs -LRB- reviewed by Bonifacino and Traub 2003 -RRB- . The role of adaptor proteins in TGN sorting of lysosomal membrane proteins remains to be established . Segregation of LAMP-1 into AP-1 positive vesicles was observed in a biochemical study and also confirmed by immuno-EM , indicating exit from the TGN in clathrin-coated vesicles -LRB- Höning et al. 1996 ; Hunziker and Geuze 1996 -RRB- . However , sorting of LAMP-1 is not strictly dependent on AP-1 . In mice lacking the μ1A subunit , which impairs AP-1 binding and clathrin assembly at the TGN , there was no significant increase in plasma membrane levels of LAMP-1 -LRB- Meyer et al. 2000 -RRB- , whereas the protein was still found in lysosomes . Depletion of clathrin in HeLa cells did result in increased levels of LAMPs at the cell surface , but not a complete redistribution of LAMPs to the plasma membrane -LRB- Janvier and Bonifacino 2005 -RRB- , indicating the existence of a direct TGN-to-lysosome pathway independent of clathrin -LRB- Karlsson and Carlsson 1998 -RRB- . Interestingly , depletion of AP-1 in HeLa cells , like in mice , again had only modest effects on LAMP transport over the surface -LRB- Janvier and Bonifacino 2005 -RRB- . Together , these data indicate that in addition to AP-1 and clathrin , other factors might be involved in direct TGN-to-lysosome transport of LAMPs . The presence of a clathrin-independent TGN-to-endosome pathway was also indicated by studies on cells that present antigens , like B lymphocytes , macrophages and dendritic cells , in the context of major histocompatibility complex -LRB- MHC -RRB- class II molecules -LRB- Fig. 3c -RRB- . The newly synthesized MHC II molecules are transported from the TGN either via the plasma membrane or directly to so-called MHC II loading compartments -LRB- MIICs -RRB- that are lysosome-related organelles representing early and late endosomes in these cells -LRB- Kleijmeer et al. 1997 -RRB- . For proper transport of the integral membrane protein MHC II from TGN to MIICs , association with the type 2 transmembrane protein , invariant chain -LRB- Cresswell 1996 -RRB- , is important . Invariant chain contains -LSB- DE -RSB- XXX -LSB- LI -RSB- - type signals in its C-terminus , but also the cytoplasmic tail of class II was found to contain endosomal sorting information -LRB- Bakke and Nordeng 1999 ; Bonifacino and Traub 2003 -RRB- . The TGN-to-endosome pathway of MHC II/invariant chain was studied in detail in B lymphoblasts by a combined biochemical and immuno-EM approach -LRB- Glickman et al. 1996 -RRB- . This revealed that MHC II , together with cathepsin D , in both normal and I-cell disease B lymphoblasts entered TGN exit domains that did not contain AP-1 or clathrin and were devoid of CD-MPR . Further studies are required to establish the molecular machinery of this pathway . Interestingly , a recent study linked the TGN exit of lysosomal membrane proteins to GGA3 , the same component involved in the exit of MPRs . GGAs are monoubiquitinated at their GAT -LRB- GGA and TOM -RRB- domain , which supports the idea that ubiquitin is a universal sorting signal , acting at various intracellular sites . The lysosomal-associated protein transmembrane-5 -LRB- LAPTM5 -RRB- , which is specifically expressed in hematopoietic cells , associates in the TGN with both the ubiquitin ligase Nedd4 and GGA3 -LRB- Pak et al. 2006 -RRB- . This association is required for efficient targeting of LAPTM5 to lysosomes and indicates that ubiquitination of GGA3 is possibly accomplished by Nedd4 . The finding that GGAs can be involved in TGN-to-lysosome transport of a lysosomal membrane protein might explain the mild effect of AP-1 knockdown . Clearly , further studies are required to clarify this direct TGN-to-endosome transport pathway of lysosomal membrane proteins . The early endosome : from CURL to tubular sorting endosome The combined data on TGN-to-endosome transport of lysosomal hydrolases and lysosomal membrane proteins suggest the existence of multiple TGN exits for direct delivery to the endosomal system -LRB- Fig. 3c -RRB- . There may be several reasons for such different pathways . For example , they allow custom tailored regulation of the delivery of distinct types of cargoes and provide a back-up system in case a given pathway is no longer available -LRB- such as in I-cell disease -RRB- . The existence of alternative TGN-to-endosome pathways would also allow different cargoes to enter the endo-lysosomal system at distinct stages , which may coincide with their function and mode of sorting . In addition to understanding TGN exits , it is therefore equally important to define the recipient endo-lysosomal intermediates in lysosome biogenesis . With the refining of EM techniques , our understanding of the endo-lysosomal system has equally evolved . The technique that we now know as ` classical ' EM , i.e. thin sections of plastic-embedded samples of chemically fixed cells , allowed our first acquaintance with the fascinating complexity of the cell 's inner world . Immunocytochemistry added another dimension by allowing the localization of enzymatic activities to specific compartments -LRB- as illustrated for acid phosphatase -RRB- . The next step was the development of immunocytochemical procedures , which greatly increased the repertoire of proteins that can be visualized in the cell . One of the first published examples of immuno-EM also marked a major leap in our understanding of the endo-lysosomal system ; i.e. the identification of the early endosome as sorting device . In 1983 , a novel immuno-EM technique was introduced by Hans Geuze and Jan Slot , in which differentially sized gold particles were used to simultaneously localize two distinct proteins . By applying this technique to visualize a ligand-asialoglycoprotein -LRB- ASGP -RRB- , which is endocytosed by liver cells for transport to lysosomes-as well as its receptor -LRB- ASGPR , which recycles after endocytosis -RRB- , they for the first time visualized a protein-sorting event . The ASGPR was found in tubular membranes that lacked ASGP and were connected to vacuoles that were enriched for ASGP but depleted of ASGPR -LRB- Geuze et al. 1983 -RRB- -LRB- Fig. 6 , left panel -RRB- . The ASGPR-containing tubules were implicated in the recycling of the ASGPR to the plasma membrane , and the ASGP-containing vacuoles were defined as ` precursor compartments en route to the lysosome ' . The entire compartment was called ` CURL ' : ` compartment of uncoupling receptor and ligand ' . Fig. 6From CURL to tubular sorting endosome . Left panel reproduction of the first electron micrograph using double-immunogold labeling , showing the uncoupling of a ligand -LRB- asialoglycoprotein -LRB- ASGP -RRB- , indicated by 5 nm gold particles -RRB- from its receptor -LRB- indicated by 15 nm gold particles -RRB- in early endosomes , at that time called CURL -LRB- compartment for uncoupling receptor and ligand -RRB- . The ASGP receptor is found in tubules implicated in its recycling , whereas ASGP en route to lysosomes remains in the vacuole . Originally published by Geuze et al. -LRB- 1983 -RRB- . Right panel , reproduction from Peden et al. -LRB- 2004 -RRB- , showing by triple labeling that the adaptor protein AP-3 -LRB- 15 nm gold particles -RRB- , the lysosomal membrane protein LAMP-1 -LRB- 10 nm gold particles -RRB- and the recycling ASGP receptor -LRB- 5 nm gold particles -RRB- co-localize in the tubules emanating from endosomal vacuoles . This work gave rise to the concept of the tubular sorting endosome Over the years , it became clear that CURL was involved in the sorting of multiple types of proteins and the modern structural equivalent of CURL is the early endosome . Indeed , the early endosome is the major cellular site , where cargo destined for recycling is sorted from cargo en route to late endosomes and lysosomes . To indicate this , early endosomes are often subdivided into the globular ` vacuolar or sorting ' endosome -LRB- Fig. 3b -RRB- , whereas the associated tubules were indicated as ` recycling tubules ' or ` recycling endosomes ' . However , recent studies provided accumulating evidence that early endosome-associated tubules are more than just transport intermediates for recycling cargo ; they are also actively involved in sorting -LRB- Fig. 3b -RRB- . With the increasing number of antibodies against distinct types of cargoes and by optimizing immuno-EM techniques -LRB- Slot and Geuze 2007 -RRB- , it became possible to localize less abundant cargoes . This revealed that at steady state conditions minor amounts of LAMP-1 and LAMP-2 are found in the early endosome-associated tubules -LRB- Fig. 6 , right panel -RRB- . The importance of this localization became apparent from studies on the pigmentation-bleeding disorder Hermansky-Pudlak-syndrome-2 -LRB- HPS-2 -RRB- . Cells from HPS-2 patients lack a functional AP-3 adaptor complex and show an increased transport of lysosomal membrane proteins over the plasma membrane -LRB- dell ' Angelica et al. 1999 -RRB- . A combination of biochemical and immuno-EM approaches revealed that AP-3 mediates the exit of lysosomal membrane proteins from the early endosome-associated tubules , most likely for transport to late endosomes or lysosomes -LRB- dell ' Angelica et al. 1998 ; Peden et al. 2004 -RRB- -LRB- Fig. 3b -RRB- . Impairment of AP-3 function results in an increased cycling of lysosomal membrane proteins between early endosomes and plasma membrane -LRB- Peden et al. 2004 -RRB- , which explains the increased levels of lysosomal membrane proteins at the plasma membrane of HPS-2 cells . Importantly , early endosomes can be reached by lysosomal membrane proteins via the plasma membrane , but possibly also via a direct pathway from the TGN . This may explain why in the literature AP-3 has been implicated in the direct as well as indirect pathway to lysosomes -LRB- Ihrke et al. 2004 ; Rous et al. 2002 -RRB- . In melanocytes , AP-3 also mediates a pathway from early endosomes to melanosomes , which are lysosome-related organelles -LRB- Theos et al. 2005 -RRB- . Importantly , by multiple double - and triple immunogold labellings , it was shown that a particular early endosome-associated tubule could contain the recycling proteins Tf and ASGPR , the CD and CI-MPR , and LAMP-1 and LAMP-2 . In addition , one single tubule could display multiple budding profiles that were positive for either the adaptor complex AP-1 or AP-3 -LRB- Peden et al. 2004 -RRB- . AP-1 on endosomal tubules has been implicated in the retrograde transport of the CD-MPR and of the Shiga toxin B subunit to the TGN -LRB- Meyer et al. 2000 -RRB- , as well as recycling of internalized Tf -LRB- Mallard et al. 1998 ; van Dam and Stoorvogel 2002 -RRB- . The presence of distinct types of recycling proteins and the association of multiple adaptor complexes indicated that the early endosome-associated tubules mediate sorting of cargo proteins to distinct destinations in the cell , i.e. plasma membrane , TGN and late endosomes or lysosomes . Therefore , these AP-3 positive compartments were introduced as tubular sorting endosomes -LRB- Figs. 3b and 6 , right panel -RRB- -LRB- Peden et al. 2004 -RRB- . A recent review of Bonifacino and Rojas extends this concept -LRB- indicated here as ` tubular endosomal network ' -RRB- , by ascribing all protein machinery currently known to be involved in endosome-to-TGN transport to distinct exits of the tubular sorting endosome -LRB- Bonifacino and Rojas 2006 -RRB- . Together , the available data launch the concept of a two-step rocket . In the first step , at the vacuolar domain of early endosomes , proteins destined for late endosomes-lysosomes are sorted from recycling proteins that accumulate in the attached tubules . Then , in the second step , taking place in the tubular sorting endosome , recycling proteins are sorted out to distinct destinations in the cell . Within this model , the recycling endosomes are one type of transport intermediate that evolve from the tubular sorting endosomes , mediating the recycling of cargo to the plasma membrane -LRB- Fig. 3b -RRB- . Multiple sorting events at the early endosomal limiting membrane Despite the relatively simple and attractive concept of the two-step rocket , sorting at the early endosomes displays another level of complexity ; recycling carriers not only evolve from tubular sorting endosomes , but also from endosomal vacuoles . A recent study showed the formation of so-called endosome-to-TGN carriers -LRB- ETCs -RRB- from the limiting membrane of early endosomal vacuoles -LRB- Fig. 3b -RRB- -LRB- Mari et al. 2007 -RRB- . These ETCs are so far specifically involved in the endosome-to-TGN recycling of lysosomal protein receptors , i.e. the CD - and CI-MPRs and sortilin . Importantly , ETCs constitute an entirely different population of recycling carriers than those emanating from the tubular sorting endosome . With the exception of the MPRs , ETCs are devoid of any of the cargo or machinery proteins found in the tubular sorting endosome . The associated machinery proteins of ETCs are SNX1 , SNX2 -LRB- Carlton et al. 2004 , 2005 ; Mari et al. 2007 -RRB- and hVps26p -LRB- Arighi et al. 2004 ; Mari , personal communication -RRB- . ETCs also differ from tubular sorting endosomes by morphology . By 3D-tomographic analyses , ETCs were identified as non-branched , short tubules and vesicles , not organized in a reticulum . Apart from segregating proteins into tubular sorting endosomes and ETCs , the limiting membrane of early endosomal vacuoles is involved in a third protein sorting event ; i.e. segregation of proteins into a characteristic bilayered coated area that is involved in protein sorting into ILVs -LRB- Fig. 3b , see also below -RRB- -LRB- Bonifacino and Rojas 2006 ; Peden et al. 2004 ; Raiborg et al. 2002 ; Raposo et al. 2001 ; Sachse et al. 2002b -RRB- . Thus , proteins present in early endosomal vacuoles may be sorted to at least three types of specialized sorting domains : tubular sorting endosomes , ETC or the bilayered coat . The formation of two types of exits from the non-coated regions of the early endosomal vacuoles predicts the existence of complementary molecular machineries that specifically recruit cargo to either of these recycling compartments -LRB- i.e. ETC or tubular sorting endosome -RRB- . Future investigations are needed to further unveil the components of these machineries . Sorting to late endosomes and lysosomes The default pathway for molecules entering early endosomes is recycling back to the plasma membrane , a pathway that starts with the segregation of cargo into the tubular extensions of the early endosomes -LRB- Draye et al. 1988 ; Yamashiro et al. 1984 -RRB- . Endocytosed cargo from the plasma membrane that is not destined for recycling requires active sorting to late endosomes/lysosomes , a process mostly mediated by inclusion into ILVs -LRB- Gruenberg and Stenmark 2004 ; Katzmann et al. 2002 ; Sachse et al. 2002b -RRB- . Protein sorting into ILVs is critical for growth factors and their receptors , such as growth hormone -LRB- GH -RRB- -LRB- Sachse et al. 2002b -RRB- and epidermal growth factor -LRB- EGF -RRB- -LRB- Raiborg et al. 2003 ; Urbe et al. 2003 -RRB- . Sorting into ILVs segregates these ligand -- receptor complexes from the cytoplasm , thereby terminating signaling . ILV sorting requires ubiquitination and involves among others the cytoplasmic proteins Hrs and STAM -LRB- Bache et al. 2003b ; Raiborg et al. 2001 , 2002 ; Urbe et al. 2003 -RRB- . By immuno-EM , it was shown that at the limiting membrane of the early endosomal vacuole the GHR and EGFR are concentrated in flat , bilayered coated areas with a fuzzy outer layer of clathrin and a dense inner layer of unknown protein composition . These bilayered coated domains were enriched in Hrs -LRB- Sachse et al. 2002b -RRB- . These findings led to the proposition of the ` sorting-by-retention ' model for endosomal sorting -LRB- Sachse et al. 2002b -RRB- . This model describes that receptor -- ligand complexes prior to their incorporation into ILVs are retained and concentrated in the bilayered coated areas of endosomal vacuoles , whereas recycling proteins pass through the vacuolar-limiting membrane and follow the bulk flow route towards recycling endosomes . A recent study showed that the recruitment of clathrin is responsible for concentrating Hrs in these endosomal microdomains , reinforcing an active role for clathrin in protein retention -LRB- Raiborg et al. 2006 -RRB- . Downstream of Hrs , the targeting of ubiquitinated receptors into ILVs requires the ` endosomal sorting complexes required for transport ' -LRB- ESCRT -RRB- -1 , -2 and -3 in concert with Alix/AIP -4 and the AAA-ATPase -LRB- hVps4 -RRB- . hVps4 is required for disassembly and recycling of ESCRT complexes -LRB- Babst 2005 ; Bache et al. 2003a ; Katzmann et al. 2003 -RRB- and interestingly , also for the disassembly of the bilayered coat -LRB- Sachse et al. 2004 -RRB- . Impairment of hVps4p function gives rise to an elongated coat and decreased formation of ILVs , indicating that coat disassembly is required for the inward budding process to proceed -LRB- Sachse et al. 2004 -RRB- . Indeed , 3D-electron tomography of early endosomes showed that inward budding of ILVs is mostly seen at the edges of the coats -LRB- Murk et al. 2003 -RRB- . Notably , the sorting-by-retention model proposes a novel role for clathrin in addition to its role in the formation of transport vesicles , i.e. defining endosomal subdomains involved in protein retention . Future developments With the increasing number of molecular markers of endosomes and lysosomes , there is a growing awareness that distinct populations of endosomes and perhaps even lysosomes , with different functions , can coexist within one cell -LRB- Bökel et al. 2006 ; Lakadamyali et al. 2006 ; Möbius et al. 2003 ; White et al. 2006 -RRB- . For example , ILVs enriched in the lipid lysobisphosphatidic acid -LRB- LBPA -RRB- are found in distinct MVBs from those enriched in cholesterol -LRB- Möbius et al. 2003 -RRB- . Another example is illustrated in Fig. 7 showing three seemingly identical late endosomes , of which only two are reached by endocytosed BSA-gold after 3 h of uptake . One explanation is that cargo drives the formation of specific endocytic compartments . For example , addition of EGF to cells results in the specific increase of the type of MVBs that do not contain LBPA -LRB- White et al. 2006 -RRB- . Formation of cargo-dependent carriers can start at the plasma membrane . The recent identification of clathrin-independent endocytosis , as well as the discovery of cargo specific adaptors that lead to clathrin-coated pit formation-either in conjunction with or independent of AP-2-illustrates that alternative endocytic pathways can arise from the plasma membrane . If and how these pathways converge at the endosomal level is presently unclear -LRB- Benmerah and Lamaze 2007 -RRB- . EM is an indispensable tool to search for cargo-specific endosomal compartments , since by fluorescence microscopy only the labelled compartment is visible and a nearby compartment negative for the cargo under study will remain unseen . Fig. 7Electron micrograph illustrating the heterogeneity of late endosomes -LRB- LE -RRB- . HepG2 cells were allowed to internalize bovine serum albumin -LRB- BSA -RRB- conjugated to 5 nm gold particles -LRB- abundant labeling -RRB- for 3 h and processed for immuno-EM of cathepsin D -LRB- 10 nm gold particles , scarce labeling -RRB- . Three seemingly identical late endosomes containing multiple ILVs and partially degraded material are shown , two of which are heavily loaded with BSA-gold , whereas the upper one is devoid of the endocytic tracer . G Golgi stacks ; Bar 200 nm Another point of interest is that not all cargo might be recruited to ILVs by the same mechanism . Both in yeast -LRB- Bilodeau et al. 2002 ; Epple et al. 2003 -RRB- and mammalian cells -LRB- Hislop et al. 2004 -RRB- , examples of cargoes are described that do not require direct ubiquitination . In case of the melanosomal protein Pmel17 , which is also targeted to ILVs independently of ubiquitin and Hrs , two N-terminal lumenal subdomains seem necessary and sufficient for efficient transport -LRB- Theos et al. 2006 -RRB- . Possibly , these non-ubiquitinated cargoes associate with others for their association with the ESCRT machinery , but perhaps they use an alternative mechanism . Interesting in this respect is that LBPA can induce ILV formation in liposomes , in the absence of protein , suggesting that lipid-driven and ESCRT-dependent pathways may coexist -LRB- Matsuo et al. 2004 -RRB- . Also the lysosomal targeting of LAMP-1 and LAMP-2 remains an interesting open question , since these proteins are predominantly located on the limiting membranes of late endosomes and lysosomes , and incorporation into ILVs seems therefore not involved . By a more detailed characterization of the distinct endosomal maturation stages -LRB- Mari et al. 2007 -RRB- , it will be feasible to obtain a more accurate definition of the stage in which a specific cargo enters or exits the endo-lysosomal system . One of the questions that is currently addressed in our lab is whether lysosomal membrane proteins can travel directly to late endosomes or lysosomes , thereby bypassing the need of ESCRT sorting machinery . Finally , with the development of live cell imaging methods and correlative live cell imaging EM , a unique tool is provided to integrate dynamic parameters with ultrastructural protein localization studies , which opens a whole new field of analysis .

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

electron microscopy
endosomes
imaging
lysosomal membrane proteins
lysosomes
protein sorting

mannose 6-phosphate receptor
trans-golgi network