Dynamic properties of endosomal membranes: implications for endocytic sorting and viral infection

Thesis

Reference

Dynamic properties of endosomal membranes: implications for endocytic sorting and viral infection

LUYET, Pierre-Philippe

Abstract

Like other enveloped viruses, vesicular stomatitis virus infects cells through endosomes.

There, the viral envelope undergoes fusion with endosomal membranes, thereby releasing the nucleocapsid into the cytoplasm and allowing infection to proceed. Here, we report that the viral envelope fuses preferentially with the membrane of vesicles present within multivesicular endosomes. Then, these intra-endosomal vesicles (containing nucleocapsids) are transported to late endosomes, where back-fusion with the endosome limiting membrane delivers the nucleocapsid into the cytoplasm. Presumably, export of cargo proteins from within endosomes also occurs "via" back-fusion with the limiting membrane, so that they become available for subsequent transport to their final destination. We further find out that this back-fusion process is altered by cholesterol accumulation and is regulated by the ESCRT component Tsg101, the endosomal lipid lysobisphosphatidic acid under the control of Ali/Vps31p and by phosphatidylinositol-3-phosphate "via" SNX16.

LUYET, Pierre-Philippe. Dynamic properties of endosomal membranes: implications for endocytic sorting and viral infection. Thèse de doctorat : Univ. Genève, 2008, no. Sc. 3942

DOI : 10.13097/archive-ouverte/unige:101907 URN : urn:nbn:ch:unige-1019079

Available at:

http://archive-ouverte.unige.ch/unige:101907

Disclaimer: layout of this document may differ from the published version.

UNIVERSITE DE GENEVE Section de chimie et biochimie Departement de biochimie

FACULTE DES SCIENCES

Professeur Jean Gruenberg

Dynamic properties of endosomal membranes:

Implications for endocytic sorting and viral infection

THESE

presentee a la Faculte des sciences de l'Universite de Geneve pour obtenir le grade de Docteur es sciences, mention biologie

par

,,•,1·

Pierre-Phm.ppe LUYET de

Saviese (VS)

These N^° 3942

Geneve

Atelier de Reproduction de la Section de Physique 2008

UNIVERSITE DE GENEVE Section de chimie et biochimie Departement de biochimie

FACULTE DES SCIENCES

Professeur Jean Gruenberg

Dynamic properties of endosomal membranes:

Implications for endocytic sorting and viral infection

THESE

presentee a la Faculte des sciences de l'Universite de Geneve pour obtenir le grade de Docteur es sciences, mention biologie

par

Pierre-Philippe LUYET de

Saviese (VS)

These N^° 3942

Geneve

Atelier de Reproduction de la Section de Physique 2008

intitulee:

UNIVERSITE DE GENEVE

FACULTE DES SCIENCES

Doctorat es sciences mention biologie

These de Monsieur Pierre-Philippe LUYET

" Dynamic Properties of Endosomal Membranes: Implications for Endocytic Sorting and Viral Infection "

La Faculte des sciences, sur le preavis de Messieurs J. GRUENBERG, professeur

_"l

ordinaire et directeur de these (Departement de biochimie), D. PICARD, professeur ordinaire et co-directeur de these (Departement de biologie cellulaire), et H. STENMARK, professeur {The Norwegian Radium Hospital - Department of Biochemistry - Oslo, Norway), autorise 1'1mpression de la presente these, sans exprimer d'opinion sur les propositions qui y sont enoncees.

Geneve, le 29 janvier 2008

These - 3942 -

N.B. - La these doit porter la declaration precedente et remplir les conditions enumerees dons les "Informations relatives aux theses de doctorat

a

l'Universite de Geneve".

a a

REMERCIEMENTS

Tout au long de mon travail de these, j'ai pu compter sur l'appui de nombreuses personnes. Certaines m'ont apporte une aide scientifique, d'autre un soutien un peu moins technique, mais tout aussi important et apprecie. C'est en partie grace

a

^elles

que j'ai pu ecrire ces pages.

J'aimerais tout d'abord remercier toutes les personnes avec qui j'ai travaille dans le laboratoire du Professeur Jean Gruenberg durant ces demieres annees:

Jean, merci de m'avoir fait confiance pour mener ce projet de recherche. J'ai tout particulierement apprecie la liberte que tu nous laisses dans nos projets, ton enthousiasme envers la science (et surtout pour nos "mauvais" resultats), tes connaissances et ta creativite.

Isabelle, qui m'a initie aux joies de VSV. Bien que les debuts de notre collaboration aient ete un peu mouvementes, finalement, je trouve que l'on s'en est tres bien sorti.

Je pensc avoir beaucoup appris

a

tes cotes, tant au niveau scientifique que personnel.

Nathalie et Anne qui m'ont encadre

a

mes debuts dans le laboratoire.

Michy et Julien, mes deux "senior" PhDs. Les doc-meetings resteront a Jamais graves dans ma memoire, au meme titre que tes premiers encouragements Julien.

Micky, ce fut un plaisir de discuter ski et sujets "suisses" avec toi, au milieu de tous ces frarn;:ais.

Ben, Christin et Eleonora, les trois "junior" PhDs. Bonne chance et courage

a

^vous

trois pour la suite de votre these. Eh oui, il y a une fin

a

ce long tunnel.

Ele, have fun with VSV and RT-PCR! Ben, I promise you that I will try to stop drinking "Heineken", although ... Christin, as you are the only swiss left in the lab, it is now of your responsibility to explain them what is a cervelas, "le Jeune federal", ... .

Marie-Helene, Marie-Claire, Brigitte et Stefania, un grand grand merci pour votre disponibilite et votre aide qui ont grandement facilite mon travail.

Merci aussi aux membranes de la famille des Post-Docs: Vero, Karine, Zeina, Cameron, Etienne, Thomas et Fabrizio.

Je vous ai finalement rejoint dans ce monde merveilleux dans lequel les experiences fonctionnent du premier coup, ou les idees ne cessent de jaillir, ou la bibliographie apparait comme par miracle dans son cerveau et surtout, un monde sans doc­

meetings ! Je vous souhaite

a

tous, bonne chance et courage pour la suite de votre carriere scientifique.

Un merci tout particulier

a

Cameron, Etienne et Thomas qui ont relu et corrige mon manuscrit de these.

Je tiens aussi

a

remercier tous les membres du departement de Biochimie, de la plate­

forme de Bio-imagerie de Science II et des equipes du Professeur Gisou Van der Goot

a

Lausanne et du Professeur Rob Parton

a

Brisbane pour l' aide apportee.

M�rci aux Professeurs Harald Stenmark et Didier Picard d'avoir accepte de juger ce travail de these.

Pour finir, je voudrais dire un enonne merci

a

ma famille,

a

^{Anouk et}

a

mes amis pour leur soutien tout au long de mes etudes.

Merci

a

vous tous ...

Pierre

TABLE of CONTENTS

Chapter 1 - Summaries of the Thesis

⁴

1.1 English summary of the thesis 4

1.2 Resume de la these en frarn;ais 5

1.2.1 Introduction 5

1.2.2 Resultats 6

1.2.3 Conclusion 7

Chapter 2 - Membrane compartments in eukaryotic cells

⁸

2.1 The biosynthetic-secretory pathway 9

2.2 The endocytic pathway 10

2.2.1 Phagocytosis 11

2.2.2 Pinocytosis 12

2.2.2.1 Macropinocytosis 12

2.2.2.2 Clathrin-mediated endocytosis 13

2.2.2.2.1 Clathrin-Coated Vesicles assembly 14

Clathrin 14

Adaptor and accessory proteins 16

Adaptor Protein-2 16

Ubiquitin-intemalization motif 18 2.2.2.2.2 Clathrin-Coated Vesicles scission 20

Dynamin 20

2.2.2.2.3 Clathrin-Coated Vesicles uncoating 21 2.2.2.2.4 Actin network and clathrin-mediated endocytosis 21

2.2.2.3 Caveolin-mediated endocytosis 22

2.2.2.4 Clathrin- and caveolin- independent endocytic pathway 24

2.2.3 Endosomal compartments 25

2.2.3.1 Early endosomes 25

2.2.3.2 Multivesicular endosomes 28

2.2.3.2.1 Multi-Vesicular-Bodies 29

2.2.3.2.2 Late endosomes 30

Chapter 3 - Growth-Factor receptor downregulation

3.1 Internalization of the EGF receptor 3 .2 EGF receptor on endosomes

3.3 Ubiquitin-dependent endosomal-lysosomal sorting motifs 3.4 Ubiquitin-independent endosomal-lysosomal sorting motifs

Chapter 4 - Endosomal Membrane Dynamics

4.1 ESCRTs in endosome biogenesis

4.2 Recycling from endosomal lumenal membranes

4.2.1 LBP A and Alix in late endosome membrane dynamics 4.2.2 Fission and fusion within late endosomes

4.2.3 Relationships between LBP A, Alix and ESCR Ts 4.3 Endosome homeostasis

Chapter 5 - Enveloped virus entry into cells by endocytosis

5.1 Virus binding to the host cell surface 5.2 Virus and the endocytic pathway

5.2.1 Virus and the ubiquitin-ESCRT machinery

5.2.1.1 Ubiquitin-ESCRT machinery required for viral budding 5.2.1.2 Ubiquitin-ESCRT machinery in viral traffic

5.2.2 Viral fusion at low pH

5.2.2.1 Class I viral fusion proteins 5.2.2.2 Class II viral fusion proteins 5.2.2.3 VSV-G fusion protein 5.2.3 Virus and multivesicular endosomes 5.3 Virus as a tool to study cellular physiology

Chapter 6 - Results

6.1 Endosome-to-cytosol transport of viral nucleocapsids.

32 34 35 36 40

41 42 44 47 48 48 50

51 52 53 54 55 56 57 58 58 59 60 62

64 64 Isabelle Le Blanc, Pierre-Philippe Luyet, Veronique Pons, Charles Ferguson, Neil Emans, Anne Petiot, Nathalie Mayran, Nicolas Demaurex, Julien Faure, Remy Sadoul, Robert G. Parton, Jean Gruenberg.

Nat Cell Biol.; 7(7):653-64. (2005).

6.2 The ESCRT-1 subunit TsglOl controls endosome-to-cytosol release of

viral RNA. 82

Pierre-Philippe Luyet, Thomas Falguieres, Veronique Pons, Asit K.

Pattnaik and Jean Gruenberg.

6.3 Hrs and SNX3 functions in sorting and membrane invagination within

multivesicular bodies. 117

Veronique Pons, Pierre-Philippe Luyet, Laurence Abrami, F. Gisou van der Goot, Robert G. Parton and Jean Gruenberg.

6.4 Late endosomal cholesterol accumulation leads to impaired intra-

endosomal trafficking. 154

Komla Soho, Isabelle Le Blanc, Pierre-Philippe Luyet, Marc Fivaz, Charles Ferguson, Robert G. Parton, Jean Gruenberg and F. Gisou van der Goot.

PloS ONE; 2(9):e85 l. (2007).

Chapter 7 - Summary and brief discussions

166

Chapter 8 - Bibliography

171

CHAPTER 1 - Summaries of the Thesis

1.1 English summary of the thesis

Like other enveloped viruses, vesicular stomatitis virus (VSV) infects cells through the endocytic pathway. VSV infection requires transport beyond early endosomes, hence to multivesicular endosomes (MVBs ), and then the transcriptionally active nucleocapsid is delivered to the cytoplasm after the low pH-mediated fusion of the viral envelope with endosomal membranes. We observed that VSV envelope fusion and nucleocapsid release into the cytosol occurs sequentially at two successive steps of the endocytic pathway - and not concomitantly as it was thought. Our results indicate that the VSV envelope undergoes fusion with the membrane of intra­

endosomal vesicles contained within MVBs, thereby releasing the nucleocapsid into the lumen of these vesicles. Then, the capsid uses these intra-endosomal vesicles as a Trojan horse to reach late endosomes, from where the nucleocapsid is delivered to the cytoplasm. This latest step occurs via the back-fusion of the internal vesicles with the endosome limiting membrane. Presumably, proteins and lipids, which are transported from within late endosomes to other cellular destinations, also use this intra­

endosomal trafficking pathway.

We further find that this back-fusion process is altered by cholesterol accumulation and is regulated by the endosomal lipid lysobisphosphatidic acid under the control of AlixNps3 lp and by phosphatidylinositol-3-phosphate via SNX16. Moreover, TsglOl, a subunit of the ESCRT-I complex is required for VSV infection and back­

fusion, certainly regulating in conjunction with its partner Alix, the fusion of lumenal vesicles with the limiting membrane.

Altogether, our studies have lead to the characterization of VSV traffic along the endocytic pathway and to increased understanding of internal vesicles dynamics in the multivesicular late endosomes required for VSV infection and the retrieval of proteins and lipids from within the organelles.

1.2 Resume de la these en francais

Modifie de Luyet and Gruenberg, 2005

1.2.1 Introduction

L' endocytose est une v01e de trafic qui implique un reseau complexe de compartiments membranaires. Elle est necessaire a !'internalization d'elements nutritifs, a la regulation de l' expression en surface des recepteurs membranaires actives et au maintien de l' equilibre de la membrane plasmique. Les molecules intemalisees a partir de la membrane plasmique arrivent tout d' abord dans des compartiments formes de tubules et de corps vesiculaires au pH legerement acide (pH 6,2), les endosomes precoces. De la, elles sont soit recyclees vers la membrane plasmique via les endosomes de recyclage, soit dirigees vers les endosomes tardifs (pH 5,5) et les lysosomes pour y etre degradees. Le trafic entre endosomes precoces et tardifs est assure par des intermediaires de transport appeles vesicules endosomiques de transport ou endosomal carrier vesicles/multivesicular bodies (ECV/MVB). Les organelles de la voie de degradation, ECV /MVB et endosomes tardifs, comportent, contrairement aux autres organites de l' appareil vacuolaire, de nombreuses vesicules intemes. Ces demieres se forment au niveau des endosomes precoces ou les molecules destinees a etre degradees y sont intemalisees.

Depuis quelques annees, de plus en plus d'exemples montrent que, dans les cellules de mammifere, les molecules incorporees dans les membranes intemes ne sont pas forcement toutes degradees. C'est le cas des recepteurs du mannose- 6-phosphate qui transitent entre le reseau trans-golgien et les endosomes tardifs, ou encore des tetraspanines qui s'accumulent dans ces vesicules intemes. Un autre cas bien connu est celui des molecules du complexe majeur d'histocompatibilite de classe II. Ces proteines sont stockees dans les membranes intemes des endosomes tardifs des cellules dendritiques immatures. Une fois les cellules activees, les molecules sont transportees jusqu'a la membrane plasmique via la formation de tubules res�ltant probablement de la fusion entre membranes intemes et limitantes des endosomes.

Ainsi, les cellules de mammiferes semblent avoir developpe un systeme de tri et de recyclage tres efficace au niveau des endosomes tardifs.

Dans ce travail de these, nous nous sommes interesses aux mouvements membranaires intra-endosomaux impliques dans ces processus, qui bien qu'essentiel sont encore mal compris.

1.2.2 Resultats

Nous avons montre dans une premiere etude qu'un virus, le virus de la stomatite vesiculaire ou Vesicular Stomatitis Virus (VSV), a pris avantage des vesicules intemes de l'endosome pour infecter les cellules efficacement. VSV est un virus a enveloppe qui entre dans les cellules via la voie de l'endocytose. Une fois internalise, l' enveloppe du virus fusionne de fac;on dependante du pH avec la membrane des endosomes.

A

la suite de la fusion, le VSV libere sa nucleocapside (ARN et nucleoproteines) dans le cytosol de la cellule ou l'ARN est replique. Nous avons montre que, contrairement ace que l'on aurait pu penser, la fusion du virus avec les membranes endosomiques et la liberation de la nucleocapside dans le cytoplasme se deroulent en deux temps, a deux etapes differentes de l' endocytose. En effet, la fusion virale se produit deja dans les ECV/MVB tandis que la nucleocapside n'est liberee qu'au niveau des endosomes tardifs. Pour le virus, la fusion entre membranes intemes et limitantes lui permet d'eviter d'etre degrade dans les lysosomes. Ainsi, !'infection virale, comme le tri et le recyclage des proteines a partir des vesicules intemes des endosomes, est dependante de la dynamique membranaire intra-endosomale.

A l' aide d 'un essai in vitro reconstituant la sortie d' ARN viral a partir d' endosomes tardifs enrichis en nucleocapsides, nous avons pu ensuite dissequer la dynamique membranaire des endosomes tardifs. Deux lipides semblent etre impliques dans ces processus : l'acide lyso-biphosphatidique (LBPA) et le phosphatidylinositol-3- phosphate (Ptdlns(3)P). Le LBP A est un lipide peu degradable detecte dans les membranes intemes des endosomes tardifs et qui influence leur dynamique. De plus, ce lipide a la caracteristique de pouvoir deformer des membranes in vitro. Nous avons aussi pu montrer que la proteine Alix, un eff ecteur du LBP A, ainsi que l 'un de ses partenaires, la proteine Tsg 101, regulent la liberation d 'ARN viral. Le Ptdlns(3 )P est, lui, surtout present dans les endosomes precoces ou il a ete decrit comme etant necessaire a la formation des ECV et au tri des molecules dans les membranes intemes. L'ajout du domaine FYVE ou de proteines SNX-16, tous deux se liant au

Ptdlns(3 )P, diminuent la liberation d' ARN viral, impliquant le Ptdlns(3 )P dans la dynamique membranaire endosomique.

1.2.3 Conclusion

Nous sommes encore loin de comprendre toutes les subtilites des mecanismes de tri et de recyclage au niveau des endosomes tardifs. Neanmoins, dans ce travail, par la caracterisation du trafic du virus VSV le long de la voie de degradation, nous avons pu identifier plusieurs acteurs impliques dans la dynamique intra-endosomale : les phospholipides LBPA et Ptdlns(3)P qui jouent un role majeur via Alix, TsglOl et SNX-16 ..

CHAPTER 2 - Membrane compartments in eukaryotic cells

Eukaryotic cells are highly organized structures composed of many different membrane compartments. The subcellular organization of the cytoplasm enables cells to carry out at the same time and in a more efficient way many incompatible chemical reactions, such as proteolysis processing and proteins neosynthesis.

Subcellular organelles are distinguished from each other by their structure, biochemical composition and functions. A lipid membrane working as a barrier assures the homeostasis of the compartments. Indeed, this lipid barrier is impermeable to most hydrophilic molecules and so, can maintain a gradient between the organelle's content and that of the cytosol. Hence, the lumen of intracellular membrane-enclosed compartments is topologically similar and equivalent to the extracellular space.

Biological membranes are composed of two thin asymmetrical layers of lipids and proteins that are held together mainly by non-covalent interactions. The protein-lipid composition of the membrane differs from one organelle to another and further contributes to the identity and function of the compartment.

Both transmembrane and soluble lumenal cargo proteins as well as lipids traffic from compartment to compartment via small vesicular carriers forming the basis of several intracellular pathways. The coordinated exchange between the distinct intracellular organelles is highly regulated and selective in order to maintain the specific composition and function of each organelle. The efficiency of this vesicular transport critically depends on organelles being correctly recognized.

In mammalian cells, two mam intracellular pathways can be highlighted: the biosynthetic-secretory and the endocytic pathways, both of which consist of intracellular networks of membrane-enclosed compartments that are directly linked to the plasma membrane that forms the cell barrier (Fig. 1 ).

The ability of the cell membrane to regulate molecular traffic in and out of the cell is essential for the maintanance of cellular homeostasis. In addition, as it is position at the interface with the extracellular environment where most of the information are received and sent out, the plasma membrane is connected to many signalling cascades controlling fundamental cellular processes.

The biosynthetic-secretory and endocytic pathways continuously interact with the cell surface and hence, control the abundance and/or activity of the majority of proteins and lipids at the cell surface.

biosynthetic-secretory pathway

I /

---....::

...,. __

(modified from Gruenberg and Stenrnark, 2004)

Figure 1: The biosynthetic-secretory and endocytic pathways

endocytic pathway

The biosynthetic-secretory and endocytic pathways are two complex networks of organelles that intersect with each other. They regulate cell membrane homeostasis and functions.

2.1 The biosynthetic-secretory pathway

The biosynthetic-secretory pathway transports newly synthesized proteins and lipids from the endoplasmic reticulum (ER) via the Golgi apparatus to the cell surface. From there, they can be either secreted into the extracellular space, become resident components of the plasma membrane or further traffic back into the cell en route to intracellular organelles.

While cytosolic proteins are synthezied via polyribosomes primarily in the cytosol, membrane and secreted proteins are translated directly into the ER. Correctly folded proteins are transported from the ER to the Golgi stacks, where they can be derivatized by addition/modification of sugar moities. The cargo eventually reaches the Trans-Golgi network (TGN), which acts as a cargo sorting and packing station.

Indeed, from there, molecules are segregated into different vesicle-carriers that will

conduct cargo components towards plasma membrane or endosomes (Bard and Malhotra, 2006).

It should be noted that while constitutive secretion is common to all eukaryotic cells, regulated exocytosis has been only described in specialized cells that release neurotransmitters, hormones or digestive enzymes in response to extracellular stimuli.

2.2 The endocytic pathway

Endocytosis is the basic cellular process by which eukaryotic cells internalize the proteins and lipids resident in the plasma membrane, as well as exogenous extracellular components: soluble factors and ligands that bind to the cell surface. It is an essential pathway, which controls fundamental cellular processes such as the turnover of membrane components, cellular signalling, uptake of nutrients, maintenance of cell polarity and antigen presentation (Conner and Schmid, 2003). In addition, many pathogenic agents take advantage of these pathways to reach their target intracellular destinations (Gruenberg and van der Goot, 2006).

Internalization of proteins and lipids into cell starts with the invagination of the cell membrane followed by the release into the cytoplasm of nascent vesicles that contain molecules from both the extracellular space and the plasma membrane. Then, these vesicles reach endosomal compartments where their components are sorted and addressed to different targets.

Mammalian cells have developed several mechanisms to internalize the broad range of molecules that enter into the cell (Conner and Schmid, 2003). Endocytosis can be divided in phagocytosis or pinocytosis events classified by the size of the internalization vesicles, nature of cargo and mode of vesicle formation (Fig. 2).

Plnocy1Dsla

Clalhnr>­

madhrted endocytooia

(•120nm)

(from Conner and Schmid, 2003)

Figure 2: Multiple portals of entry into mammalian cells

Clalhnr>- ­ caveoi.lndapendent

'"1doaytoee (-110 nm)

Internalized molecules enter into the cells via several distinct routes that differ by the mechanism of vesicle formation and the nature and size of endocytosed cargo.

2.2.1 Phagocytosis

In mammals, phagocytosis is the process of the engulfment of solid large particles such as pathogens or apoptotic cells (Fig. 2). This process is an active and highly regulated mechanism.

At the cell surface, large particles are recognized by specialized cellular receptors, which in tum initiate the massive cytoskeletal and membane rearrangements that mediate macromolecules uptake. The particle is enclosed by active membrane protrusion processes forming lamellipodia that zipper up around the "prey" by repeated receptor-ligand interactions. These major membrane deformations involve dramatic and acute reorganization of the cellular actin cytoskeleton mediated by the Rho (Ras homology)-family GTPases signalling cascades and are associated with delivery of endomembranes to the site of particle engagement (Bajno et al., 2000;

Hall and Nobes, 2000). Eventually, internalized particles are completely surrounded by membrane and are trapped into a sealed intracelluar compartment called the phagosome.

During their trip into the cell, the phagosome undergoes a maturation processe by interacting with different intracellular organelles and finally, fusing with lysosomes forming a hybrid compartment called phagolysosomes. From there, the ingested particles are degraded via the highly acidic pH of the compartment, active NADPH oxidase complexes and lysosomal enzymes.

Phagocytosis in mammals occurs sporadically in certain specialized cells, such as macrophages, monocytes and neutrophils, which play key roles in the immune response and inflammation events by engulfing and digesting infectious agents and senescent cells. In addition, phagocytosis is also implicated in embryonic development and tissue remodeling for clearance of apoptotic cells (Aderem and Underhill, 1999).

2.2.2 Pinocytosis

Pinocytosis is common to all cells and corresponds to uptake of fluid and solute cargo via small vesicles ( < 0.2µm diameter). This process can be further divided in four different pathways in regards to the mechanism by which vesicles are formed and the characteristics of internalized components. To date, four different types of pits and vesicles have been described, corresponding to: 1) macropinocytosis, 2) clathrin­

mediated endocytosis, 3) caveolin-mediated endocytosis and 4) clathrin and caveolin­

independent endocytosis (Fig. 2).

2.2.2.1 Macropinocvtosis

Macropinocytosis refers to the bulk uptake of fluid and solid cargo into large endocytic vesicles called macropinosomes (Fig. 2). As in phagocytosis, vesicle formation requires rapid and extensive actin remodeling. Indeed, macropinocytosis starts by formation at the cell surface of actin-driven membrane ruffles mediated by activation of the Rho-family GTPases cascade in response to signalling events such as growth factor stimulation (Dharmawardhane et al., 2000). The membrane protrusions do not "zipper up" along internalized particles via receptor-ligand interactions as in phagocytosis, but simply collapse and fuse with the cell membrane resulting in macropinosomes. Then, macropinosomes either are trafficked to and fuse with lysosomes or directly regurgitate their content into the extracellular medium.

Formation of these goblet-shaped membrane invaginations allows the cell to internalized large amounts of extracellular fluid and plasma membrane. It has been implicated in nutrients uptake, downregulation of activated signalling-receptors, cell migration and in presentation of antigens in dendritic cells (Dharmawardhane et al., 2000; Mellman and Steinman, 2001; Ridley, 2001).

2.2.2.2 Clathrin-mediated endocytosis

In higher eukaryotes, clathrin-mediated endocytosis is the major pathway of receptor­

mediated endocytosis from plasma membrane and extracellular space (Fig. 2). By controlling endocytosis of transporters, pumps and si_gnalling receptors present at the cell surface, the clathrin route regulates cellular homeostasis and intracellular si_gnalling-transduction events. It is also involved in other cellular functions such as nutrient uptake, antigen presentation and the recycling of synaptic vesicles after neurotransmission (De Camilli and Takei, 1996; Hirst and Robinson, 1998; Takei and Haucke, 2001). Moreover, this route is an important entry door for many pathogens such as the Influenza virus, Semliki forest virus (SFV) and Vesicular Stomatitis Virus (VSV) (Marsh and Helenius, 2006; Veiga and Cossart, 2006).

In the classical model, clathrin-mediated endocytosis is initiated by the sorting and concentration of transmembrane proteins into clathrin assembly zones on the cell membrane, and the recruitment of accessory proteins and clathrin-coat molecules to the cytoplasmic leaflet of the plasma membrane (Fig. 3). This triggers formation of an invaginated coated bud called clathrin-coated pit (CCP). Then, additional propagation of the clathrin-coat further bends the membrane. Once deeply invaginated and narrowed at the neck, the CCP eventually pinches off into the cytoplasm generating a nascent clathrin-coated vesicle (CCV), which transports the concentrated receptor­

ligand complexes into the cytoplasm. (Fig. 3). The clathrin-coat of the vesicle is then rapidly disassembled and recycled, while the uncoated vesicle is delivered to endosomes (Fig. 3) (Royle, 2006).

It is noteworthy that clathrin-coated vesicles also can bud from the TGN and traffic cargo molecules to the plasma membrane or endosomes (Bard and Malhotra, 2006;

Kirchhausen, 2000a).

/ Recycling End090mt -- Ly,=al

Degradation

(from Prof. Volker Haucke webpage)

Figure 3: Clatbrin-coated vesicle formation and disassembly

At the cell surface, cargo molecules are gathered into a CCP, around which an open polyhedral clathrin lattice assembles and that begins to invaginate inward cytoplasm. Once deeply invaginated and narrowed at the neck, the CCP detaches from the plasma membrane thus generating a CCV. Then, the clathrin coat is removed and the vesicle reaches its acceptor membranes.

2.2.2.2. 1 Clathrin-Coated Vesicles assembly

Formation of CCVs is a highly complex process that requires spatial and temporal coordination of many proteins and lipids. The main players are the lipid phosphatidylinositol( 4,5)-bisphosphate (Ptdlns( 4,5)P2), the coat molecule clathrin, the multiple adaptor/accessory proteins and the GTPase protein dynamin.

Clathrin

Clathrin is the major protein component in the coat of CCPs and CCV s. The molecule is composed of three heavy (192 kDa) and three light chains (25-29 kDa) that take together the shape of a triskelion, a figure with three bent legs (Fig. 4a). This clathrin assembly unit has the ability to spontaneously polymerize into spherical lattice structure made of hexagons and pentagons (Fig. 4b-c) (Edeling et al., 2006b).

a b

(from Puertollano, 2004; Heuser et al., 1989 and Sachse et al., 2002)

Figure 4: Clathrin structure and organization

a) Schematic representation of a clathrin triskelion with the different domains.

b) Reconstitution of a clathrin-coated cage.

c) Scanning electron microscope (SEM) picture of clathrin lattices on the cytoplasmic face of the plasma membrane. Bar 100nm.

d) Bilayered coat on early endosome (EE) labeled for clathrin light chain (10-nm gold). Bar 200nm.

On CCPs, the clathrin-coat provides a scaffold around the pits that is necessary for receptor sorting, membrane deformation and vesicle budding (Fig. 4c) (Kirchhausen, 2000b). However, to date it is still unclear if the clathrin-coat contributes to the mechanical force necessary to deform membrane or works as a support to maintain membrane curvature (Maldonado-Baez and Wendland, 2006).

It is noteworthy that clathrin also can assemble in flat lattices on endosomes (Fig. 4d) (Raiborg et al., 2002; Raiborg et al., 200 1 ; Sachse et al., 2002).

Adaptor and accessory proteins

Assembly of CCV s requires cytosolic proteins referred to as adaptor or accessory proteins including molecules such as Adaptor Protein-2 (AP-2), 13-arrestin, Epsin, Amphiphysin, clathrin assembly lymphoid myeloid leukemia (CALM) and AP180.

Recruitment of these protein complexes to the cell surface is regulated in a spatial and temporal fashion by their interaction with transmembrane cargo and the lipid Ptdlns(4,5)P2 which is enriched on the plasma membrane (Di Paolo and De Camilli, 2006). On CCV s, associated proteins form an inner shell that is surrounding by clathrin-coat and function as adaptors proteins, regulatory proteins and mechanicaVassembly proteins (Fig. 5) (Lafer, 2002; Perrais and Merrifield, 2005;

Traub, 2005).

Adaptor Protein-2

Clathrin molecules show no affinity for biological membranes and thus are engaged at cell surface via their interaction with molecules such as the classical adaptor protein AP (Adaptor Proteins) (Collins et al., 2002; Edeling et al., 2006a). The family of the heterotetrameric AP includes four members (AP l -4) that all share a common structure, composed of two large subunits (y/j31, a./j32, o/j33 and E:/j34), a medium subunit (µ1-4) and a small chain (crl -4). Despite their similar organization, they show different subcellular localizations: APl ,3,4 have been associated with the TGN and endosomes, while AP-2 functions at the plasma membrane (Robinson and Bonifacino, 2001).

AP-2 is recruited to the cell surface via its affinity for Ptdins(4,5)P2 and recognition of transmembrane cargo proteins (Fig. 5) (Owen et al., 2004). Indeed, the cytosolic tail of cargo proteins contains different internalization motifs that trigger their concentration for clathrin-mediated uptake. Two major sorting motifs are known to interact with AP-2: the acidic di-leucine and tyrosine-base (Tyr-X-X-� where � corresponds to a bulky hydrophobic amino acid) motifs (Bonifacino and Traub, 2003;

Janvier et al., 2003; Mishra et al., 2005; Nesterov et al., 1999). In addition, AP-2 recruits several proteins important for clathrin-dependent endocytosis such as Epsin and AP180 (Marsh and McMahon, 1999; Traub, 2003).

Plasma membrane

Cargo proteins

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Figure 5: Organization of AP-2, clathrin, cargo and Ptdlns(4,5)P2 in CCV

AP-2 binds to and connects cargo contents, the lipid Ptdlns(4,5)P2 and the clathrin scaffold. It hence, has a pivotal position in CCV assembly and function.

The importance of AP-2 in clathrin-mediated endocytosis is highlighted by the lethality of mice embryos after disruption of genes encoding AP-2 subunits (Mitsunari et al., 2005). Moreover, although sile�cing of the AP-2 gene by RNA interference decreases the number of CCV s at the cell surface, only internalization of a subset of clathrin-dependent cargo is disrupted, suggesting existence of other adaptator proteins (Hinrichsen et al., 2003; Huang et al., 2004; Motley et al., 2003). A group of

"alternate adaptors" (alternate to AP-2) are the monomeric clathrin-associated sorting proteins (CLASP) which include proteins such as 13-arrestin (Traub, 2005). By binding to other endocytic-sorting motifs, these proteins increase the diversity of molecules internalized by this pathway.

At the cell surface, 13-arrestin detects serine phosphorylation on seven­

transmembrane-helix G-protein-coupled receptors and mediates their uptake into CCVs (Braun et al., 2003; Lefkowitz and Shenoy, 2005). Hence, this adaptor protein is a key component in regulation of G-protein-coupled receptors signalling (van Koppen and Jakobs, 2004). Upon association with activated receptors, 13-arrestin binds to clathrin, AP-2 and Ptdlns(4,5)P₂ (Gaidarov et al., 1999a; Moore et al., 2007;

Traub, 2005).

Ubiguitin-intemalization motif

Another endocytosis signal is the post-translational addition of ubiquitin moieties (Bonifacino and Traub, 2003; Traub and Lukacs, 2007). Ubiquitin is a highly conserved peptide of 7 6 amino acids that can be reversibly conjugated to lysine residues via a cascade of enzymatic reactions (Fig. 6) (Staub and Rotin, 2006). Mono­

and poly-ubiquitination have been shown to regulate many cellular processes including proteasomal proteolytic degradation, protein targeting and viral budding (Hicke, 2001; Mukhopadhyay and Riezman, 2007).

COOH ..,_ ,

DUbs Ubp Uch

·,.Ub/

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_Ub

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Ligases E3

Figure 6: Transfer and removal of ubiquitin peptides to substrate

Ubiquitin (ub) peptide binds to the El ubiquitin-activating enzyme via a thioester bound and in an A TP-dependent fashion. Then, the ubiquitin moiety is further transferred to an E2 ubiquitin­

conjugated enzyme otherwise known as a ubiquitin-carrier enzyme. Finally, the ubiquitin-E2 complex acts in concert with the E3 ubiquitin-protein ligase. The E3 enzyme carries out the important tasks of substrate specificity and transfer of ubiquitin onto lysine side chains (K) of the substrate. Sometimes, adaptor proteins (Ap) recruit E3 enzymes on the substrate. There are two groups ofE3 enzymes: RING finger E3 or Hect E3.

Ubiquitin is removed from substrate by deubiquitinating enzyme (DUBs).

Ubiquitin signal mediates uptake of plasma membrane signalling molecules, transporters, ion channels and immune molecules that are internalized in a clathrin­

dependent as well as clathrin-independent manner (Chen and De Camilli, 2005;

Sigismund et al., 2005; Traub and Lukacs, 2007).

Interestingly, in dendritic cells ubiquitination events regulate surface expression of Major Histocompatibility Complex Class II (MHCII) molecules. In immature dendritic cells, the complexes are transiently ubiquitinated mediating their uptake and their accumulation in late endosomes-lysosomes (Kleijmeer et al., 2001; Shin et al., 2006; van Niel et al., 2006). Upon dendritic cell maturation, ubiquitination of the complexes is prevented, resulting in antigenic peptide presentation at the cell surface (Shin et al., 2006; van Niel et al., 2006). Ubiquitin-dependent internalization and degradation of the Epidermal Growth Factor signalling receptor (EGF receptor) will be discussed in more details later.

Epsin 1/2 and epsl5/eps l5R (epidermal growth factor receptor substrate) contain an Ubiquitin-Interacting-Motif (UIM) and so, are putative adaptors that mediate internalization of ubiquitinated proteins (Traub and Lukacs, 2007). These proteins share an epsin N-terminal homology (ENTH) domain that interacts with Ptdlns(4,5)P2, and other clathrin and clathrin adaptor protein-binding motifs (Owen et al., 2004). Interestingly, Epsin interaction with clathrin molecules appears to negatively regulate its association with ubiquitinated-cargo (Chen and De Camilli, 2005).

Epsin and its isoforms have been proposed to drive membrane curvature by insertion of an amphipathic alpha-helix of the ENTH domain into the cytosolic face of the plasma membrane (Ford et al., 2002). Insertion of the helix generates an imbalance between the two membrane leaflets and results in membrane curvature. Hence, while the ENTH domain induces membrane deformation, other parts of the protein recruit essential components for clathrin coat assembly, thus stabilizing the membrane deformation (Horvath et al., 2007; McMahon and Gallop, 2005).

2.2.2.2.2 Clathrin-Coated Vesicles scission

Dynamin

The GTPase dynamin is an essential element of the budding of CCPs into the cytoplasm as well as mediating other vesicle fission events of different intracellular organelles and in other internalization routes.

Dynamin is directly recruited onto membranes via its pleckstrin homology (PH) domain that binds to Ptdlns( 4,5)P2 (Zheng et al., 1996). Lipid binding appears to stimulate the GTPase activity of the protein and vesicle formation process (Jost et al., 1998; Kinuta et al., 2002). Dynamin molecules can self-assemble and form rings and spirals around the neck of deeply invaginated CCPs (Takei et al., 1995). Moreover, in vitro experiments have shown that dynamin has the ability to deform membrane into narrow tubules and to fragment liposomes (Roux et al., 2006; Sweitzer and Hinshaw, 1998; Takei et al., 1998). However, the exact mechanism by which fission occurs is still not understood. It is currently thought that after dynamin polymerisation around the CCP neck and upon GTP hydrolysis, dynamin molecules either constrict the CCP neck (Chen et al., 2004) and/or work as a molecular spring that propels the CCP towards the cytosol (Stowell et al., 1999), in either case resulting in vesicle fission.

Dynamin GTPase activity and its capacity to deform liposomes appear to be regulated by amphiphysin, a binding partner of dynamin (Takei et al., 1999; Yoshida et al., 2004). This protein contains a BAR (BIN-Amphiphysin-Rvs) domain, which mediates dimerization and binding to acidic phospholipids. BAR domains have a banana-like structure in which the concave surface can interact with curved membranes and hence, has been proposed to work as membrane curvature-sensor or to stabilize membrane curvature by recruitment of scaffold proteins (Itoh and De Camilli, 2006;

Peter et al., 2004). The amphiphysin BAR domain works in combination with an N­

terminal amphipathic helix (N-BAR domain) (Farsad et al., 2003). The N-BAR domain can deform membrane via the insertion of an amphipathic helix resulting in membrane curvature stabilized by the BAR domain (McMahon and Gallop, 2005).

Amphiphysin certainly serves in generation of membrane curvature in concert with dynamin. The process occurs after the assembly of the clathrin coat but before the vesicle budding into the cytoplasm (McMahon and Gallop, 2005).

2.2.2.2.3 Clathrin-Coated Vesicles uncoating

Once released from the plasma membrane, coated vesicles must disassemble their clathrin lattice and assembly particles coats before fusing with early endosomes.

Uncoating is mediated by the cytosolic chaperon protein, heat-shock cognate 70 (Hsc70) and its co-factor proteins, the neuronally expressed auxilin or its ubiquitously expressed homolog, cyclin G-associated kinase (GAK or auxilin-2) (Eisenberg and Greene, 2007). These co-factors bind to Ptdlns, dynamin, AP-2, clathrin and hence, recruit the chaperon Hsc70 on membranes. Then, Hsc70 promotes the irreversible release of clathrin in an ATP-dependent fashion (Jiang et al., 2005). Interestingly, the protein remains transiently attached to dissociated clathrin to avoid inappropriate polymerization in the cytosol and to prime clathrin recycling to forming CCPs (Jiang et al., 2000).

The Ptdlns( 4,5)P2 phosphatase synaptojanin also appears to act in clathrin uncoating process (Cremona et al., 1999). Hydrolysis of Ptdlns(4,5)P₂ to Ptdins(4)P by synaptojanin is proposed to increase the affinity of the various co-factors for CCVs, resulting in a burst of Auxilin and GAK on the CCV s after dynamin recruitment thereby preventing an early disassembly of the clathrin coat (Lee et al., 2006; Massol et al., 2006).

2.2.2.2.4 Actin network and clathrin-mediated endocytosis

Several actin-regulatory proteins, including cortactin and profiling, have been implicated in clathrin-mediated endocytosis (Engqvist-Goldstein and Drubin, 2003;

Merrifield, 2004; Qualmann et al., 2000; Zhu et al., 2007). In mammalian cells, actin seems to facilitate endocytosis more than being an essential element. It provides a structural and mechanical support. Indeed, disruption of the actin cytoskeleton by drug treatment in mammalian cells shows partial or no effect on the rate of endocytosis although this is dependent on the cell types and growth conditions being studied (Fujimoto et al., 2000). In contrast, perturbation of the actin network in yeast by genetic or chemical means inhibits endocytosis completely (Engqvist-Goldstein and Drubin, 2003; Wendland et al., 1998).

To date, the actin cytoskeleton has been linked to different steps of CCV formation including lateral movement of coated patches at the cell surface, fission of vesicles

and vesicles movement into the cytoplasm (Engqvist-Goldstein and Drubin, 2003;

Gaidarov et al., 1999b; Qualmann et al., 2000; Y arar et al., 2005).

2.2.2.3 Caveolin-mediated endocytosis

Caveolae-mediated endocytosis is characterized by the formation of flask-shaped invaginations with a diameter of 60-80 nm called the caveolae pit (Fig. 2). This pathway mediates entry of lipid rafts components including cholesterol, glycosylphosphatidylinositol (GPI)-anchored proteins, signalling proteins and pathogens such as Simian virus-40 (SV 40) or cholera toxin (Parton and Richards, 2003; Pelkmans and Helenius, 2002; Shin et al., 2000). Moreover, in endothelial cells caveolae have been implicated, in addition to their function in endocytosis, in the transcytosis of macromolecules necessary to maintain tissue homeostasis (McIntosh et al., 2002; Schnitzer et al., 1994). In these cells caveolae can constitute up to 10-20%

of the cell surface.

The caveolae pits are characterized by high concentrations of gangliosides, sphingolipids and cholesterol linking this pathway to lipid rafts (Simons and Ikonen, 1997). Lipid rafts correspond to small, low-density specialized microdomains of membranes that are enriched in cholesterol and sphingolipids. Due to their lipid composition and organization, these domains show a highly specialized profile of proteins content compared to the rest of the cell membrane. GPI-anchored proteins and dually acylated proteins are usual components of the lipid rafts. Interestingly, depletion of the cell surface cholesterol causes caveolae to flatten (Rothberg et al., 1990).

Caveolae formation is driven by the oligomerization of the caveolin proteins, raft­

resident molecules that bind to cholesterol (Fig. 7) (Drab et al., 2001; Fra et al., 1995;

Galbiati et al., 2001; Murata et al., 1995). Three different caveolin proteins are described in mammalian cells: Caveolin-1, 2 and 3. Caveolin-1, 2 are mainly co­

expressed in non-muscle cells, while caveolin-3 is present in skeletal and cardiac muscles (Tang et al., 1996; Way and Parton, 1995). These molecules have a central intra-membrane domain that is inserted as a hairpin loop only in the inner leaflet of

the plasma membrane (Fig. 7). Hence, the protein interacts with the membrane in a stable and constitutive manner in opposite to the clathrin-coat molecules that are transiently recruited (Pelkrnans et al., 2004).

Caveolin-1 molecules self-associate in filamentous structures that are proposed to form a coat around the caveolae and to define their size and shape (Fernandez et al., 2002). It has been also suggested that by stabilizing the plasma membrane association of caveolae, caveolins can negatively regulate caveolae-mediated endocytosis (Le et al., 2002).

Figure 7: Caveolae pit and caveolin

Caveolae pits are driven by oligomerization of the raft­

resident protein caveolin. Caveolin is inserted into membrane with a hairpin intra-membrane domain and with its N- and C­

terminal domains exposed in the cytoplasm. Caveolin-1 is palmitoylated on its C-terminal domain. However, this post­

traductionnel modification does not seem to be required for localisation of the protein on caveolae pits (Dietzen et al., 1995).

(from Parton and Simons, 2007)

At the cell surface, caveolae structures show a relatively low motility (Thomsen et al., 2002). However, caveolae-mediated endocytosis appears to be regulated by cargo molecules (Conner and Schmid, 2003). Indeed, various agents such as SV40 virus, specific lipids and phosphatase inhibitors enhance the internalization rate via tyrosine­

phosphorylation of caveolae constituents (Kirkham et al., 2005; Minshall et al., 2000;

Parton et al., 1 994; Pelkrnans and Helenius, 2002; Tagawa et al., 2005).

Caveolae budding into the cytoplasm involves dynamin, protein kinase C, Src kinase and actin recruitment (Henley et al., 1 998; Oh et al., 1998; Parton and Simons, 2007).

Then, caveolar vesicles either recycle back to the cell surface to be reused or fuse with caveosomes or early endosomes. By contrast to clathrin, caveolin proteins are constitutively on membranes and are not removed from the vesicle.

The caveosome is an intracellular organelle positive for caveolin and, in contrast to endosomal compartments, with a neutral pH (Pelkmans et al., 2001). Their membranes are typically enriched in cholesterol and glycosphingolipids (Pelkmans and Helenius, 2002). To date, except for SV40 and cholera toxin, neither endosomal markers, components internalized via clathrin endocytosis, or fluid-phase marker such as dextran or horseradish peroxidase have been detected in these organelles. From caveosomes, caveolar units are recycled back to the cell surface while SV 40 and cholera toxin are further transported via carrier vesicles towards the endoplasmic reticulum and the Golgi complex respectively (Nichols, 2002; Pelkmans et al., 2001).

2.2.2.4 C/athrin- and caveolin- independent endocvtic pathway

The existence of pathways that do not depend on caveolae or clathrin have also been highlighted by studying the uptake of various molecules into cells expressing dominant-negative mutants, depleted for specific proteins or in cell lines devoid of caveolin such as lymphocytes (Fig. 2) (Damke et al., 1 995; Guha et al., 2003;

Kirkham and Parton, 2005; Lamaze et al., 2001; Mayor and Pagano, 2007;

Sabharanjak et al., 2002; Sauvonnet et al., 2005). However, to date these different internalization processes are still poorly understood.

Altogether, mammalian cells have developed several routes to internalize extracellular and cell surface components. The diversity of pathways reflects the broad range of endocytosed components and the necessity to tightly control their entry into cells in regards to their nature and function. Hence, molecular sorting of internalized material already begins at the plasma membrane.

Moreover, some molecules depending on their fate and function are differentially internalized (Chen and De Camilli, 2005; Di Guglielmo et al., 2003; Sigismund et al., 2005). For instance, the transforming growth factor � receptor is internalized via both clathrin- and caveolin- dependent routes. However, its signal is only transduced by receptors trafficked to clathrin-coated pits (Di Guglielmo et al., 2003). In addition, the EGF receptor, depending on its stimulation, enters into the cells via the clathrin- or caveolae- mediated endocytosis (Sigismund et al., 2005).

2.2.3 Endosomal compartments

Even if they use different internalized routes, most of the cargos converge at the early endosomes. The endocytic pathway is organized as a network of distinct compartments including early endosomes, recycling endosomes, multivesicular bodies (MVBs ), late endosomes and lysosomes (Fig. 8-9). Endosomal organelles differ in their lipid and protein composition, morphology, function and distribution in the cell (Gruenberg and Stenmark, 2004).

2.2.3.1 Early endosomes

Cargos form the cell surface and the biosynthetic pathway intersect at early endosomes. Early endosomes are an important sorting step in the endocytic pathway.

From there molecules are sorted to different destinations: housekeeping receptors are uncoupled from their ligand at the mildly acidic lumenal pH of early endosome (pH 6.2) (Fig. 8a) and then, recycled back to the cell surface for reutilization, while other cargos are directed towards the TGN and the biosynthetic pathway along the retrograde transport route. By contrast, downregulated signalling receptors as well as other endocytosed proteins and lipids are efficiently sorted away from recycling molecules within early endosomes, and are then routed into the MVB for further transport towards late endosomes-lysosomes for degradation (Gruenberg, 2001;

Johannes and Lamaze, 2002; Katzmann et al., 2002; Maxfield and McGraw, 2004).

However, cargo sorting might also occur even earlier in the pathway. Pre-early endosomal sorting events have been proposed to already begin during formation of CCVs (Lakadamyali et al., 2006). Indeed, CCVs themselves may not all be equal and contain different cargos destined for types of early endosomes. This study strongly argues for the existence of distinct populations of early endosomes showing different dynamic properties (Lakadamyali et al., 2006).

Extracellular

medium Plasma membrane

Cytosol

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Recycling pathway

Degradation pathway

Anti-CD63 Abs

Endocytosis

Early endosome

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Figure 8: Organization and electron-microscopy pictures of endosomal organelles

a) Endosomal compartments, including early endosomes, recycling endosomes, multivesicular bodies (MVBs), late endosomes and lysosomes, are organized as a complex network of membranes.

b) Early endosomes consist of cistemal regions from which large vesicles and thin tubules, in which recycled molecules such as transferrin receptors are segregated, emanate (SEM picture from J. Heuser, Washington University, USA ).

c) ECV/MVBs contain a multitude ofintralumenal vesicles and mediate transport between early and late endosomes in a microtubule dependent manner (Gruenberg et al., 1989).

VSV-G: Vesicular Stomatitis Virus-Glycoprotein

d) Late endosomes show a morphological heterogeneity: they can have a multivesicular and/or multilamellar structure (Kobayashi et al., 2002). Bar, 250 nm. CD63: Cluster of differentiation 63

e) Lysosomes, where degradation occurs, are electron-dense organelles (Dorothy Bainton et al., 1981, Picture from Daniel S. Friend)

Note that the size-ratio between the different organelles is not maintained.

Early endosomes are distributed mainly at the cell periphery and consist of cistemal regions from which thin tubules (60 run diameter) and large vesicles (300-400 run diameter) emanate (Fig. 8b) (Gruenberg, 2001). Internalized molecules that need to be recycled back to the cell surface, e.g. the transferrin receptor, are collected into the thin tubular domains that eventually detach and form, or mediate transport towards, recycling endosomes (Maxfield and McGraw, 2004). In contrast, proteins that must be transported towards late endosomes are targeted to the vesicular regions of early endosomes.

Molecules that regulate function and structure of the organelle are not randomly distributed on endosome, but form a mosaic of biochemically and functionally well­

defined domains (Gruenberg, 2001).

Proteins of the Rab (Ras-related small GTP binding protein) GTPase family and certain lipids such as phosphoinositides are key regulators of organellar identity and membrane organization (Gruenberg, 2001; Pfeffer, 200 1 ; Zerial and McBride, 2001).

They are selectively distributed on different organelles and their activation-states and metabolisms are tightly regulated (Fig. 9). Hence, these two components control in a spatio-temporal fashion the recruitment and functions of a wide variety of proteins including molecular motors and tethering factors.

Rab5 and Rab4 are found on early endosomes and delineate distinct dynamic membrane territories on endosomes (Fig. 9) (Sonnichsen et al., 2000). Rab4 also partitions with Rab 11 on recycling endosomes (Fig. 9) (Sonnichsen et al., 2000).

Rab5 effectors are selectively recruited to well-defined protein platforms that are involved in transport, membrane fusion, membrane budding and interaction with cytoskeleton (Zerial and McBride, 2001). Along the endocytic pathway, Rab5 is then replaced by Rab7, which is mainly enriched on late endosomes similar to Rab9 (Fig.

9) (Rink et al., 2005; Vonderheit and Helenius, 2005; Zerial and McBride, 2001). On late endosomal membranes, Rab7 and Rab9 occupy distinct domains (Barbero et al., 2002).

Ptdlns(3)P 1s constitutively synthesized on early endosome via the phosphatidylinositol 3-kinase (PI3K), a Rab5 effector which facilitates formation of Rab5-Ptdins(3)P enriched microdomains (Fig. 9) (Christoforidis et al., 1999;

Gruenberg, 2001; Zerial and McBride, 2001 ). Proteins containing lipid-binding domains such as FYVE (Fabl p, OTB, Vac l p and EEAl )- or PX (Phox homology)­

domains selectively bind to Ptdlns(3)P platforms (Simonsen et al., 2001). Some proteins in addition to their affinity for Ptdlns(3)P, are also Rab5 effectors, further increasing their selective recruitment (Zerial and McBride, 2001 ).

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Figure 9: Distribution of lipids and Rab proteins on endosomal compartments

Ptdlns(4,5)P₂ is predominantly found at the plasma membrane (PM) and mediates formation of CCPs. Ptdlns(3)P, generated by the Pl3K, is abundant on early endosomes (EE) and accumulates on the intralumenal vesicles of the multivesicular bodies (ECV/MVB). Ptdlns(3)P is then presumably degraded or transformed into Ptdlns(3,5)P₂• However, the exact distribution and function of Ptdlns(3,5)P₂ is unclear in mammalian cells. Lysobisphosphatidic acid (LBPA) is enriched in late endosomes (LE). Late endosomes contain different types of intralurnenal vesicles, positive for Ptdlns(3)P or LBPA. In addition, cholesterol-enriched membranes and rafts might also be present in late endosomes.

Each Rab protein is associated with a distinct subpopulation of endosomes. These proteins delineate specific territories on endosomal membranes.

RE: recycling endosome; Lys: lysosome

2.2.3.2 Multivesicular endosomes

Endosomal membranes can be deformed and vesiculate in two different directions, either towards the cytoplasm, generating carrier vesicles such as MVBs or inward from the cytoplasm releasing vesicles into the lumen of endosomal organelles (Fig.

10). In accordance with their different topologies, these two processes seem to require different molecular machineries.

Through membrane budding into the lumen of endosomes, the compartments acquire a characteristic multivesicular appearance exemplified by MVBs and late endosomes (Fig. 8c-d).

2.2.3.2.1 Multi-Vesicular-Bodies

MVBs also known as endosomal carrier vesicles (ECV s ), conduct transport between early and late endosomes in a microtubule- and motor- dependent manner (Aniento et al., 1993; Gruenberg, 2001). These compartments are relatively large in comparison to other transport intermediates (300-500 nm of diameter) and enclose multiple intralumenal vesicles (IL V s) (Fig. 8c ). MVBs have a similar lumenal pH to late endosomes (pH 5.5) (Fig. 8a).

Detachment -or maturation- of MVBs from early endosomes is supported by the recruitment of annexinA2 to cholesterol platforms and has also been suggested to be dependent on both the endosomal coatomer protein COP-I complex and ARFl (ADP­

ribosylation factor-I) (Daro et al., 1997; Gu and Gruenberg, 1999; Gu and Gruenberg, 2000; Mayran et al., 2003). In addition, Rab7 is also probably involved in this process (Press et al., 1998; Rink et al., 2005; Vonderheit and Helenius, 2005).

Membrane invagination into the lumen of the organelle involves Ptdins(3)P and the endosomal sorting complex required for transport (ESCRT) machinery (Fig. 10). The ESCRT machinery and formation of lLVs will be discussed in more detail later.

Importantly, IL V biogenesis and MVB release into the cytoplasm are two distinct processes that can be uncoupled and might occur sequentially (Gruenberg and Stenmark, 2004; Petiot et al., 2003).

Once detached, MVBs traffic along microtubules towards late endosomes with which they eventually dock and fuse with (Aniento et al., 1993; Gu and Gruenberg, 1999).