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 V O LU M E T WO S E V E N T Y E I G H T

INTERNATIONAL REVIEW OF
CELL AND MOLECULAR
BIOLOGY
INTERNATIONAL REVIEW
OF CELL AND MOLECULAR
BIOLOGY
Series Editors

GEOFFREY H. BOURNE 1949–1988

JAMES F. DANIELLI 1949–1984
KWANG W. JEON 1967–
MARTIN FRIEDLANDER 1984–1992
JONATHAN JARVIK 1993–1995

Editorial Advisory Board

ISAIAH ARKIN KEITH LATHAM

PETER L. BEECH WALLACE F. MARSHALL
ROBERT A. BLOODGOOD BRUCE D. MCKEE
DEAN BOK MICHAEL MELKONIAN
KEITH BURRIDGE KEITH E. MOSTOV
HIROO FUKUDA ANDREAS OKSCHE
RAY H. GAVIN MANFRED SCHLIWA
MAY GRIFFITH TERUO SHIMMEN
WILLIAM R. JEFFERY ROBERT A. SMITH
 V O LU M E T WO S E V E N T Y E I G H T

INTERNATIONAL REVIEW OF
CELL AND MOLECULAR
BIOLOGY

EDITED BY

KWANG W. JEON
Department of Biochemistry
University of Tennessee
Knoxville, Tennessee

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ISBN: 978-0-12-374809-6

PRINTED AND BOUND IN USA

09 10 11 12 10 9 8 7 6 5 4 3 2 1
CONTENTS

Contributors ix

1. Macromolecular Trafficking and Immune Evasion

in African Trypanosomes 1
Mark C. Field, Jennifer H. Lumb, Vincent O. Adung’a,
Nicola G. Jones, and Markus Engstler
1. General Overview of the Trypanosome Life Cycle 3
2. Immune Evasion Mechanisms 5
3. Endocytic Pathways 16
4. Developmental Remodeling and Signaling 27
5. Sorting Signals 28
6. Secretory Protein Folding and Exocytosis 32
7. Golgi Apparatus; Functions and Replication 39
8. Ubiquitylation and Endocytosis of Trans-Membrane Domain Proteins 42
9. Evolution of the Trypanosome Endomembrane System 46
10. Conclusions and Future Perspectives 50
Acknowledgments 51
References 52

2. Biological and Biophysical Properties of Vascular

Connexin Channels 69
Scott Johnstone, Brant Isakson, and Darren Locke

1. Introduction 71
2. The Vasculature 72
3. Connexin Channels 75
4. Gap Junctional Communication Pathways in the Vasculature 82
5. Permeability of Vascular Connexin Channels 87
6. Involvement of Connexins in Vascular Diseases 101
7. Concluding Remarks 104
Acknowledgments 105
References 105

v
vi Contents

3. Genotype–Phenotype Mapping: Developmental Biology

Confronts the Toolkit Paradox 119
Joel Atallah and Ellen Larsen
1. Introduction 120
2. Understanding the Genotype–Phenotype Relationship 121
3. From Sequence to Form 125
4. Beyond Sequence 135
5. Case Study for Parsing Gene, Cell and Self-Organizing Roles
in Morphogenesis 138
6. Conclusion 142
Acknowledgments 142
References 142

4. Role of Spindle Asymmetry in Cellular Dynamics 149

Yves Barral and Dimitris Liakopoulos
1. Introduction 150
2. Spindle Asymmetry and Control of Cell Cycle 152
3. Spindle Asymmetry During Asymmetric Cell Divisions 174
4. Concluding Remarks 201
References 203

5. Cell Adhesion in Amphibian Gastrulation 215

Rudolf Winklbauer

1. Introduction 216
2. Characteristics of Amphibian Gastrulation 217
3. Gastrula Tissue as a Liquid: Cell Adhesion, Cell Sorting,
Boundary Formation, and Tissue Positioning 223
4. Collective Cell Migration, Cell Rearrangement,
and Intercellular Migration 249
5. Conclusion 260
Acknowledgments 261
References 261

6. Molecular and Cell Biology of Testicular Germ Cell Tumors 277

Paolo Chieffi, Renato Franco, and Giuseppe Portella

1. Introduction 278
2. Epidemiology and Risk Factors 279
3. Histopathology 280
4. Prognostic and Diagnostic Markers 294
Contents vii

5. Therapy 297
6. Conclusions and Perspectives 301
Acknowledgments 301
References 302

7. Polarity Proteins and Cell–Cell Interactions in the Testis 309

Elissa W.P. Wong and C. Yan Cheng
1. Introduction 310
2. Rho GTPases and Cell–Cell Interactions in the Testis: Cdc42 315
3. Polarity Proteins and Cell–Cell Interactions in the Testis 327
4. The Apical ES-BTB-Basement Membrane Functional Axis in the
Seminiferous Epithelium that Coordinates the Cellular Events
of Spermiation and BTB Restructuring During the Seminiferous
Epithelial Cycle of Spermatogenesis: The Role of Polarity Proteins
in Mediating the Apical ES-BTB-Basement Membrane Axis 335
5. Roles of Polarity Proteins in Coordinating the Opposing Effects
of Cytokines and Testosterone in Primary Preleptotene
Spermatocyte Transit at the BTB 339
6. Concluding Remarks and Future Perspectives 341
Acknowledgment 342
References 342

Index 355
CONTRIBUTORS

Vincent O. Adung’a
Department of Pathology, University of Cambridge, Cambridge CB2 1QT,
United Kingdom
Joel Atallah
Department of Cell and Systems Biology, University of Toronto, Toronto,
Ontario M5S 3G5, Canada
Yves Barral
Institute of Biochemistry, ETH Hönggerberg, HPM, CH-8093 Zurich,
Switzerland
C. Yan Cheng
Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for
Biomedical Research, Population Council, New York, New York 10065, USA
Paolo Chieffi
Dipartimento di Medicina Sperimentale, II Università di Napoli, 80138 Naples,
Italy
Markus Engstler
Lehrstuhl für Zell- und Entwicklungsbiologie, Biozentrum der Universitaet
Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany
Mark C. Field
Department of Pathology, University of Cambridge, Cambridge CB2 1QT,
United Kingdom
Renato Franco
Area Funzionale di Anatomia Patologica, Istituto Nazionale dei Tumori ‘‘Fondazione
G. Pascale,’’ 80131 Naples, Italy
Brant Isakson
Robert M. Berne Cardiovascular Research Center, University of Virginia School
of Medicine, Charlottesville, Virginia 29908, USA; and Department of Molecular
Physiology and Biological Physics, University of Virginia School of Medicine,
Charlottesville, Virginia 29908, USA
Scott Johnstone
Robert M. Berne Cardiovascular Research Center, University of Virginia School
of Medicine, Charlottesville, Virginia 29908, USA

ix
x Contributors

Nicola G. Jones
Lehrstuhl für Zell- und Entwicklungsbiologie, Biozentrum der Universitaet
Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany
Ellen Larsen
Department of Cell and Systems Biology, University of Toronto, Toronto,
Ontario M5S 3G5, Canada
Dimitris Liakopoulos
Biochemie Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg,
Germany
Darren Locke
Department of Pharmacology and Physiology, New Jersey Medical School,
University of Medicine and Dentistry of New Jersey, Newark, New Jersey
07103, USA
Jennifer H. Lumb
Department of Pathology, University of Cambridge, Cambridge CB2 1QT,
United Kingdom
Giuseppe Portella
Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli
‘‘Federico II,’’ 80131 Naples, Italy
Rudolf Winklbauer
Department of Cell and Systems Biology, University of Toronto, Toronto,
Ontario M5S 3G5, Canada
Elissa W.P. Wong
Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for
Biomedical Research, Population Council, New York, New York 10065, USA
 C H A P T E R O N E

Macromolecular Trafficking and

Immune Evasion in African
Trypanosomes
Mark C. Field,* Jennifer H. Lumb,* Vincent O. Adung’a,*
Nicola G. Jones,† and Markus Engstler†

Contents
1. General Overview of the Trypanosome Life Cycle 3
2. Immune Evasion Mechanisms 5
2.1. VSG and antigenic variation 5
2.2. Membrane dynamics and antibody clearance 7
2.3. Cellular immune responses 11
2.4. Innate immunity 13
3. Endocytic Pathways 16
3.1. Clathrin-mediated endocytosis 16
3.2. Cargo adaptors and sorting 18
3.3. Early and recycling endocytic compartments 20
3.4. Multivesicular bodies and late endocytosis 21
3.5. Lysosome 22
3.6. Involvement of the cytoskeleton in endocytosis 24
3.7. Complexity of endsomal sorting 25
3.8. Surface receptors and endocytic pathways 25
4. Developmental Remodeling and Signaling 27
5. Sorting Signals 28
5.1. Targeting at the ER 28
5.2. Endocytic and lysosomal targeting signals 29
5.3. Endomembrane sorting based on posttranslational
modification 30
5.4. Sorting to the flagellum 30
5.5. Sorting to the glycosome 31

* Department of Pathology, University of Cambridge, Cambridge CB2 1QT, United Kingdom

{
Lehrstuhl für Zell- und Entwicklungsbiologie, Biozentrum der Universitaet Wuerzburg, Am Hubland,
97074 Wuerzburg, Germany

International Review of Cell and Molecular Biology, Volume 278 # 2009 Elsevier Inc.
ISSN 1937-6448, DOI: 10.1016/S1937-6448(09)78001-3 All rights reserved.

1
2 Mark C. Field et al.

6. Secretory Protein Folding and Exocytosis 32

6.1. Membrane protein biosynthesis 32
6.2. Polypeptide delivery 32
6.3. Polypeptide folding and maturation 35
6.4. ER exit to the Golgi complex or ER-associated degradation
(ERAD) 37
7. Golgi Apparatus; Functions and Replication 39
7.1. Golgi replication by binary fission 39
7.2. Role of the cytoskeleton in Golgi complex replication 40
7.3. Cell-cycle and life-cycle coordination of Golgi complex
replication and function 41
7.4. Retrograde transport 42
8. Ubiquitylation and Endocytosis of Trans-Membrane Domain
Proteins 42
8.1. The VSG sorting problem 42
8.2. Ubiquitylation and endocytosis 43
8.3. ISGs are ubiquitylated 44
8.4. Late endocytosis and the multivesicular body 45
9. Evolution of the Trypanosome Endomembrane System 46
9.1. Changing views of the eukaryotic tree of life 46
9.2. Comparative genomics 47
9.3. G-protein signaling complexity and evolution 48
9.4. Convergent evolution 49
9.5. Evolutionary exploitation of the flagellar pocket 49
10. Conclusions and Future Perspectives 50
Acknowledgments 51
References 52

Abstract
Intracellular trafficking is a major mechanism contributing to maintenance of
the surface composition in most eukaryotic cells. In the case of unicellular
eukaryotic pathogens, the surface also represents the host–parasite interface.
Therefore, the parasite surface is both a critical player in immune recognition,
from the host’s point of view, or in immune evasion, from the pathogen’s point.
The African trypanosomes are remarkable in dwelling throughout their period in
the mammalian host within the bloodstream and tissue spaces, and have
evolved several mechanisms that facilitate chronic infection. Here, we discuss
current understanding of intracellular trafficking pathways of trypanosomes,
and relate these processes to immune evasion strategies by the parasite and
avoidance of immune responses from the host.
Key Words: Antigenic variation, Immune evasion, Intracellular trafficking,
Protein sorting, Trypanosome. ß 2009 Elsevier Inc.
Macromolecular Trafficking in Trypanosomes 3

1. General Overview of the Trypanosome

Life Cycle
The lifestyle of Trypanosoma brucei, the African trypanosome, is ven-
turesome, and unlike the vast majority of endoparasites, has exploited
mechanisms allowing survival without sequestration within host cells.
While thriving in the body fluids of vertebrate hosts, the parasites are
continuously attacked by the immune system and also endure harsh physi-
comechanical conditions that prevail in the mammalian circulation.
The mammalian host is infected when a tsetse fly carrying trypanosomes
(Glossina spp.) takes a blood meal (Vickerman, 1985; Vickerman et al.,
1988) (Fig. 1.1). The insect’s bite inoculates the parasites into the host’s
subcutaneous tissue, where they rapidly proliferate. Metabolic products, or
other undefined factors, cause local inflammation of the skin at the bite site,
producing a so-called chancre, which is the first sign of infection. From
here, rapidly dividing cells enter the draining lymphatic system and are

Host Vector
Antigenic Immune
variation response
+ Stumpy
−

Proliferation Cell cycle Proliferation

arrest

+
Slender Density
sensing

Figure 1.1 Partial life cycle of trypanosomes, emphasizing the transition from the
mammalian host to the insect vector. Left box (host): Proliferative slender forms (gray)
survive within the host bloodstream, lymphatics, and issue spaces. These cells exhibit
antigenic variation as the primary route to immune evasion, facilitating chronic infec-
tion. Additionally, a density sensing mechanism, mediated by stumpy induction factor,
causes the slender forms to exit from the cell cycle, preventing overgrowth of the host
by excessively high parasite numbers. Cell cycle exit is accompanied by morphological
changes, resulting in the stumpy form, as well as additional underlying metabolic and
proteome changes, preadapting the parasite to life in the tsetse fly. These cells are
sensitive to immune killing and do not exhibit antigenic variation. Right box (vector):
On entry to the tsetse midgut, stumpy form parasites reenter the cell cycle and begin to
proliferate. A fuller version of the parasite life cycle is detailed in Vickerman (1969).
4 Mark C. Field et al.

flushed into the circulation. An early symptom of infection is enlarged

cervical lymph nodes of the neck, the Winterbottom’s sign, but both of
these symptoms fade rapidly and have limited diagnostic use (Ormerod,
1991).
The parasites are well prepared for life in the mammalian host. The
quiescent, nondividing metacyclic stage, which awaits transmission in the
insect salivary glands, expresses a special version of the most conspicuous
weapon that trypanosomes have evolved to defend against host attack:
A dense layer of a single protein species, the variant surface glycoprotein
(VSG), shields the entire cell surface (Pays, 2006). In the skin tissue,
following injection the metacyclic trypanosomes reenter the cell cycle and
proliferate as long slender trypomastigotes (Fenn and Matthews, 2007).
Energy metabolism in this form is simplified and relies completely on
nonoxidative consumption of glucose, which is constantly provided by
the host. In the bloodstream, the trypanosome population oscillates
between proliferative slender forms and cell cycle-arrested stumpy forms.
The parasites secrete an, as yet, undefined low-molecular-weight factor
which triggers differentiation to the stumpy stage in a cell-density-dependent
manner. As parasitemia rises, more stumpy induction factor (SIF) accumu-
lates and the cells become quiescent (Reuner et al., 1997). Thereby, popula-
tion density is limited, allowing the parasites to persist for significant periods
without killing the host. Thus, one could argue that the stumpy stage
acts altruistically in sacrificing itself for the sake of population survival. In
fact, stumpy forms survive only for a rather short period (a few days) and
appear to be efficiently eliminated by the humoral immune response. How-
ever, the stumpy stage is crucial for life-cycle progression as only they
can successfully establish an infection in the transmitting insect vector
(Fig. 1.1). Stumpy forms are preadapted to a life within the fly, including
increased mitochondrial activity, reorganization of the endomembrane
system, and hypersensitivity to environmental cues such as cold shock and
citrate/cis-aconitate (Engstler and Boshart, 2004).
When the fly ingests stumpy form trypanosomes, the parasites immedi-
ately encounter a loss of temperature homeostasis. This has been suggested
to cause upregulation of the major insect stage surface proteins, procyclins
(Engstler and Boshart, 2004). The expression of procyclins is accompanied
by rapid loss of the VSG coat. How exactly the VSG is lost is not fully
understood, but the participation of both a metalloprotease and phospholi-
pase appears likely (Gruszynski et al., 2003). The expression of distinct
procyclins during the process of adaptation to the insect environment has
also been described in a series of elegant experiments, however, the function
of the new cell-surface coat remains unclear (Acosta-Serrano et al., 2001).
In fact, procyclins appear to be dispensable both in vitro and their expression
is not essential for successful passage through the tsetse fly (Vassella et al.,
2009).
Macromolecular Trafficking in Trypanosomes 5

In the insect, trypanosome metabolism switches from glycolysis to

cytochrome-mediated oxidative respiration. After establishing a midgut
infection, trypanosomes migrate to the fly salivary glands, to complete the
life cycle. Little is known about this journey and how trypanosomes navi-
gate through the fly. Having reached the salivary glands, the epimastigote
parasites attach to the microvilli of epithelial cells (Urwyler et al., 2007).
This anchoring is mediated by the trypanosome flagellum through an, as
yet, unknown mechanism. The density of the actively dividing epimasti-
gotes within the salivary glands can become rather high. Finally, the
attached epimastigotes give rise to quiescent (i.e., nondividing) metacyclic
trypanosomes, which reacquire the VSG coat. This completes the trypano-
some life cycle.
Although this basic itinerary for T. brucei transmission through host and
vector has been known for decades, the signals and molecular responses
underlying the alterations between proliferative and quiescent stages remain
enigmatic. While considerable effort and progress in understanding the
slender-to-stumpy and stumpy-to-procyclic differentiation events has
been made recently, and a molecular marker, PAD1, has now been identi-
fied for the stumpy stages, very little is known about transition between fly
stages (Dean et al., 2009). This is in part due to the fact that fly resources are
limited and few laboratories are experienced in maintaining and manipulat-
ing tsetse. This extensive research effort should be devoted to understanding
the fly–parasite interaction and development stages within the tsetse fly is
underlined by exciting recent findings showing that meiosis and the
exchange of genetic material occurs only within the fly; hence the tsetse
fly is formally the definitive host.

2. Immune Evasion Mechanisms

2.1. VSG and antigenic variation
The ability to survive in the vasculature of the mammalian host, despite
constant exposure to a highly sophisticated immune response, was a major
challenge to understanding of trypanosome virulence mechanisms. It is now
30 years since seminal work identified the VSG surface coat as the basis for
antigenic variation (Cross, 1977).
A 15 nm thick layer of 5
106 identical VSG homodimers covers each
trypanosome cell. This amazingly high concentration of a single species of
plasma membrane protein is unprecedented, and represents  90% of cell-
surface protein. The spacing between individual VSG dimers is 3–5 nm,
which effectively shields most of the VSG epitopes from antibody recogni-
tion (Overath and Engstler, 2004). The classical view proposes that other
6 Mark C. Field et al.

surface proteins are buried within the VSG coat and that the plasma
membrane is virtually untouchable by the immune system. However, a
full description of the mammalian host immune response remains to be
achieved and it is possible that additional determinants are recognized.
Evolution may have shaped the molecular structure of VSG for maxi-
mum efficiency as an immunological shield. Two extended a-helices proj-
ect perpendicular to the cell surface and provide the molecule with an
extended conformation (Freymann et al., 1990). VSG proteins generally
comprise two domains, a larger externally disposed N-terminus of 350–400
residues and a smaller C-terminal domain 40–80 residues that is proximal to
the plasma membrane (Chattopadhyay et al., 2005). Once packed into the
surface coat, only a restricted number of amino acids are accessible to
external probes. The exposed amino acid positions are highly variable,
while more conserved sites, especially several cysteines required for disulfide
bond formation and hence secondary structure, are less accessible (Field and
Boothroyd, 1996). While the crystal structure of two N-terminal domains
was solved some 20 years ago, the structure of the relatively conserved C-
terminal was described only recently (Chattopadhyay et al., 2005). NMR
analyses revealed that C-terminal domains display related core structures
consisting of two a- or 310-helices and two antiparallel b-sheets. However,
the length of the secondary structure elements and the loops connecting
these elements vary between different C-terminal domains. Up to now no
complete VSG structure has been solved, and the structure of the connec-
tion between the two domains remains unknown. More importantly, we
still have little structural appreciation of the linkage between the VSG C-
terminal domain and the hallmark of VSGs, the glycosylphosphatidylinosi-
tol (GPI) anchor.
The GPI anchor is rapidly added posttranslationally to the C-terminus of
VSG and ultimately anchors the protein to the outer leaflet of the plasma
membrane (Martin and Smith, 2006). The lipid anchoring of VSG has many
important implications for the physicochemistry of the trypanosome cell
surface and VSG sorting. A great many GPI-anchored proteins (GPI-APs)
with various functions have been described in many organisms (Field and
Menon, 1993). However, compared to trans-membrane proteins, GPI-APs
are rare or of low abundance in most taxa. As the insect stage dominant
antigens, procyclins and BARP, are also GPI anchored, African trypano-
somes link all dominant surface proteins via a GPI anchor to the plasma
membrane independent of the life-cycle stage (Engstler et al., 2004;
Urwyler et al., 2007).
The unique structure of VSG and its ability to support an extreme form
of molecular crowding on the cell surface would not help the parasites to
survive in the mammalian bloodstream unless coupled to antigenic varia-
tion. In fact, the immune system would actually become hyperactivated by
an almost crystalline array of protein epitopes, accurately displayed on the
Macromolecular Trafficking in Trypanosomes 7

surface of the trypanosome cell, and VSG is known to be an effective

immunogen. During rising parasitemia the majority of trypanosomes
belong to a particular antigenic type, giving rise to a strong antibody
response, which kills the large majority of trypanosomes bearing that
VSG. These parasites are apparently cleared from the bloodstream. How-
ever, antigenic variation replaces the VSG on the cell surface of a small
proportion of trypanosomes with another, and immune selection deter-
mines if these parasites expressing a new VSG can survive. Laboratory strains
switch rarely, with a rate of 10 6–10 7 per cell generation. Recent work
suggests that the antigenic switching rate in natural isolates is much higher,
up to 10 2–10 3 (Lythgoe et al., 2007). A repertoire of hundreds of
different VSGs that can potentially be expressed on the cell surface indicates
that the host immune response always lags behind, allowing trypanosomes
to survive for prolonged periods.
VSGs are transcribed from one of 20 telomeric expression sites (ESs)
(Cross, 1996; McCulloch, 2004; Pays, 2006). The VSG gene is located at
the end of a long polycistronic transcriptional unit, and transcribed by RNA
polymerase I. This transcription unit contains more than 10 genes, which
are termed as expression site associated genes (ESAGs). The function of
many ESAGs is still unknown. The active ES is localized to an extranu-
cleolar structure that is known as the ES body (Navarro and Gull, 2001).
Besides the ES-linked copies, the majority of VSG genes locate as nontran-
scribed basic copies to either large arrays in central regions of megabase
chromosomes or as single copies on mini chromosomes. The basic copies
are abundantly flanked by repeats that facilitate homologous recombination
with the active ES.
Multiple mechanisms for mobilizing VSG genes have been described.
Activation of a silent ES displaying another VSG is known as an in situ
switch. This form of switching not only activates another VSG, but also a
new array of ESAGs. It has been postulated that activation of alternative
ESAGs could be involved in adaptation to various host environments
(Gerrits et al., 2002). Telomere exchange between ESs or gene conversions
that introduce all or part of a basic copy VSG into the active ES are also
known. The potential for creation of mosaic VSG sequences from multiple
basic copy ORFs extends the repertoire hugely, while gene conversion
most probably dominates in field infections, and provides access to this near
limitless repository.

2.2. Membrane dynamics and antibody clearance

The VSG coat is endocytosed with unprecedented speed and fidelity
(Engstler et al., 2004). An area equivalent to the entire plasma membrane
is internalized and recycled once every 10 min. Compared to membrane
recycling in other organisms this is amazingly fast kinetics, especially when
8 Mark C. Field et al.

taking into account that all membrane traffic is routed via the tiny flagellar
pocket, which only accounts for  2% of surface membrane (Field and
Carrington, 2009). In trypanosomes, internalization of plasma membrane
and embedded proteins is restricted to clathrin-mediated endocytosis
(CME). Comparatively large clathrin vesicles, termed CCV class I, abun-
dantly bud from the flagellar pocket. Every second six new CCVs are
rapidly transported by an actin-dependent mechanism to early endosomes,
which are located between 2 and 4 mm away from the pocket. About 60–70
CCV class I can be found in the posterior part of the cell. CCV I fuse to
early endosomes that are easily detectable in trypanosomes as mostly circular
cisternal structures (Grünfelder et al., 2002). In bloodstream forms this also
appears independent of dynamin, but in the procyclic it is possible that
dynamin is required (Morgan et al., 2004; Chanez et al., 2006).
The next step on the itinerary of endocytosed membrane is the recycling
endosome, which appear to be the main sorting station of GPI-APs.
However, unlike yeast or mammalian cells, trafficking through recycling
endosomes in T. brucei is not rate limiting. In fact, the delivery from early
endosomes follows biphasic kinetics, with one part of VSG or reporter
proteins being delivered very rapidly to the recycling endosome, while
the other half arrives significantly later. The reason for this is a detour of
part of the internalized membrane and embedded proteins to late endo-
somes. Interestingly, this material is not delivered to the lyosome but is
redirected to recycling endosomes, albeit with comparatively slow kinetics
(Engstler et al., 2004). In trypanosomes, recycling endosomes are most
prominent endomembrane structures. In this ‘‘recycling factory,’’ ligands
uncouple from their receptors and are sorted together with endocytosed
fluid-phase cargo into smaller clathrin-coated vesicles (CCV class II), which
abundantly bud from the rims of the cisternal recycling endosome
(Grünfelder et al., 2003). CCV class II have two destinations within the
cell: late endosomes and the lysosome. They are devoid of VSG, transferrin
receptor (TfR), and some trans-membrane proteins (e.g., invariant surface
glycoproteins (ISGs)). Thus, by a negative mechanism, namely withdrawal
of membrane, GPI-APs are passively (i.e., by default) concentrated
(Overath and Engstler, 2004). How GPI-proteins are excluded from entry
into budding vesicles remains to be elucidated. The subregion of the
recycling endosome that carries the concentrated VSG eventually gives
rise to small, disk or cup-shaped carriers that fuse with the flagellar pocket.
These exocytic carriers (EXCs) are profusely found within the posterior part
of the cell (Grünfelder et al., 2003).
Why have trypanosomes evolved such a sophisticated and highly active
plasma membrane recycling machinery? The selective pressure for uptake of
sufficient nutrients from host blood is one obvious answer. However, it has
been shown that the vast majority of fluid-phase cargo is actually not trans-
ported to the lysosome, but is excreted by the cells (Engstler et al., 2004).
Macromolecular Trafficking in Trypanosomes 9

More than 20 years ago, it was reported that antibodies bound to the
trypanosome cell surface are internalized and most probably routed to the
lysosome. These early studies suggested that host antibodies are cleared from
the cell surface of bloodstream trypanosomes within 15–30 min (Russo
et al., 1993). A more recent report has revealed that VSG-bound IgGs
(VSG-IgG) accumulate at the flagellar pocket region within 20–40 s.
VSGs and bound antibodies are rapidly internalized via CME (Allen et al.,
2003; Engstler et al., 2007). 3D-fluorescence microscopy and quantitative
colocalization analyses with organelle-specific marker proteins have con-
firmed that a significant amount of antibody is routed via late endosomes to
the lysosome, while VSG is recycled to the cell surface.
The internalization of antibody-bound VSG from the trypanosome cell
surface comprises three consecutive steps, each displaying distinct tempera-
ture sensitivities. Initially, VSG–IgG complexes accumulate at the posterior
pole of the cell. The kinetics of this process is comparatively independent of
temperature; even at 12  C rapid antibody accumulation is observed.
In contrast, the rate of entry of VSG–IgG complexes into the flagellar
pocket is significantly decelerated at 24  C, and at 12  C the process almost
halts. Once arrived in the flagellar pocket, VSG–IgG is internalized by bulk
membrane uptake. The endocytosis of IgG-bound VSG exhibits similar
temperature sensitivity as the entry into the flagellar pocket. Hence, the
three-step antibody clearance process involves posterior accumulation of
antibodies as immediate event and passage through the flagellar pocket as
rate-limiting step. Posterior accumulation of VSG–IgG does not result from
VSG shedding and is independent of endocytosis.
Downregulation by RNAi of clathrin heavy chain causes a block of all
endocytic traffic. Although in clathrin-depleted cells endocytosis is stalled,
VSG–IgG accumulates at the flagellar pocket in a similar manner as in
control cells. Posterior accumulation of VSG–IgG requires energy as the
glycolytic inhibitor 2-deoxyglucose decreases posterior accumulation of
VSG–IgG complexes. When cellular motility is stalled by ATP depletion,
no antibody accumulation is observed, but cells remained uniformly coated
with immunoglobulin, suggesting a correlation between cellular motility
and antibody clearance (Engstler et al., 2007).
Bloodstream forms of T. brucei swim with an average speed of 20 mm s 1.
A directional, spiral trajectory is mediated by a single flagellum, which
emerges from the flagellar pocket, attaches to the cell body, and extends
beyond the anterior pole of the cell (Fig. 1.2). FlaI is required for the
connection of the flagellum to the cell body, and downregulation of FlaI
results in detachment of the flagellum and loss of directional motility. FlaI-
depleted trypanosomes retain their VSG coat and VSG-bound antibodies
are internalized from the flagellar pocket with similar kinetics as in control
cells. Strikingly, obstruction of directional swimming coincides with a loss
of accumulation of antibody-bound VSG at the posterior cell surface.
10 Mark C. Field et al.

Antibody capping

0s Trypanosome
motility vector

5s Net media flow

10 s

30 s Hydrodynamic flow drags

antibody toward posterior
of cell for endocytosis
60 s

120 s

Figure 1.2 Immune evasion by antibody capping; a role for hydrodynamic flow. Left
column: Antibody recognizing the surface variant surface glycoprotein (VSG) is rapidly
capped toward the posterior of the cell. The high rate of endocytosis facilitates efficient
uptake of the antibody, which is ultimately degraded; the uptake process, even in the
presence of high antibody titers, can be completed within 2 min. Right column:
Trypanosomes continuously swim, and thereby generate directional flow fields on
their cell surface. These flow forces become more significant when the surface VSG
is recognized by immunoglobulins. Antibody–VSG complexes are pulled by hydrody-
namic forces toward the rear of the cell, where they are endocytosed. This implies that
purely physical forces can sort proteins in the plane of the plasma membrane. The
schematic shows antibody in green coating VSG in gray. Note that the mechanism for
capping, or delivery of antibody to the flagellar pocket, is likely distinct from that which
operates in metazoan cells.

Conversely, a marked reduction in antibody concentration is observed in the

flagellar pocket area, which is explained by continuing membrane recycling
in the absence of antibody accumulation. Clearly, there is direct involvement
of cell motility and endocytosis in antibody clearance (Engstler et al., 2007).
The size, but not the nature of the protein bound to VSG, is critical for
accelerated removal from the cell surface; for example, IgM is removed much
more rapidly than Fab fragments. Mammals respond to trypanosome infec-
tions with elevated levels of VSG-specific IgM rather than IgG. Both immu-
noglobulins are internalized in a concentration-dependent manner but the
overall kinetics of IgM-uptake is significantly faster. This observation is
difficult to explain on the basis of specific recognition of antibody-bound
VSGs by cytoplasmic adapter proteins as VSG is anchored via a GPI-anchor.
A provocative alternative possibility is that hydrodynamic flow acts
on swimming trypanosomes and specifically drags VSG–Ig complexes
toward the flagellar pocket (Fig. 1.2). The most direct evidence for
Macromolecular Trafficking in Trypanosomes 11

hydrodynamic drag arises from downregulation of the dynein arm interme-

diate chain DNAI1, which reverses the trypanosome swimming direction.
Hydrodynamic drag predicts that the immunoglobulins would be pushed
toward the other cell pole in the DNAI1-supressed cells, and is exactly what
is observed (Engstler et al., 2007).
However, the role of antibody clearance for parasite survival in vivo, that
is, in the natural host, remains to be elucidated. Antibody removal could be
crucial for parasite survival during early parasitemia, when antibody titers
are still low, after differentiation to the cell cycle-arrested stumpy stage,
which is critically required for the successful completion of the parasite life
cycle or for removal of immunoglobulins recognizing invariant epitopes.
All of these possibilities remain to be fully investigated.

2.3. Cellular immune responses

The trypanosome lifestyle is provocative in its complete exposure to the
mammalian host, yet, at the population level they prosper in the face of
massive immune attack. VSG is highly immunogenic and trypanosomes
produce it in huge amounts. The immune system, when confronted with a
wave of parasites, aims at rapidly clearing the trypanosomes, and paradoxi-
cally this mechanism is essential for chronic infection. Rapid growth of
trypanosomes can overwhelm the host within days, and one mechanism to
avoid this is the continuous secretion of SIF, triggering differentiation from
long slender to stumpy stage. The importance of SIF is underscored by the
observation that strains that have become unresponsive to SIF kill their host
within a few days. Further, generation of VSG-specific antibodies is crucial
to early host survival and B cell-deficient mice poorly control trypanosome
infections (Baral et al., 2006).
Antibody-opsonized trypanosomes will be phagocytosed and destroyed
by hepatic Kupffer cells. The paracrystalline structure of the VSG coat, with
homogeneously organized epitopes, may explain why the early immune
response is largely T cell independent (Mansfield, 1994). The VSG-specific
antibody response is crucially insufficient for efficient protection during
early phases of infection and IFN-g appears to be required. The majority
of IFN-g is produced after T cell stimulation through antigen presenting
cells (Magez et al., 2006). The absence of MHC-II diminishes IFN-g
production and parasite control. Also natural killer cells have been proposed
to be involved in the production of IFN-g (Mansfield and Paulnock, 2005).
In the beginning, it was believed that IFN-g would bind directly to
trypanosomes stimulating their proliferation. Now it appears clear that
in vivo IFN-g is responsible for a proinflammatory reaction, which attacks
the parasites (Namangala et al., 2001). Mice deficient of either IFN-g or
IFN-g receptor reveal high-level parasitemia and die earlier than control
animals. The IFN-g-mediated control involves classic macrophage
12 Mark C. Field et al.

activation. The effector cells produce a range of antitrypanosome com-

pounds, including tumor necrosis factor alpha (TNF-a) as well as reactive
oxygen species, which cause the so-called host oxidative burst. Reactive
nitrogen, NO, is also involved. Interestingly, the role of NO as inflamma-
tory effector appears to be distinct for infections with different trypanosome
species (Vincendeau et al., 1992). Toll-like receptors are thought to be
involved in the activation of macrophages by T cell-derived IFN-g and
endogenous TNF-a. The toll-like receptors represent a steadily growing
family of proteins that can recognize conserved pathogen signatures, such as
the bacterial lipopolysaccharide, nonmethylated DNA, or GPI structures
(Coller et al., 2003). During the cyclical destruction of large trypanosome
populations, massive amounts of these molecules are released into the blood-
stream. Consequently, parasite control involves toll-like receptors that
recognize the VSG-GPI and free nonmethylated CpG DNA sequences.
Highly inflammatory reactions govern the initial phase of infections with
African trypanosomes. The long-lasting type I immune response leads to
many of the pathological signs associated with sleeping sickness, such as
anemia, fever, splenomegaly, liver damage and neurological disorder and
cachexia, from which any infected human or animal will finally die. There-
fore, having survived the first wave of parasitemia, the infected host needs to
remodel its immunological landscape to enhance survival chances. Although
TNF-a knockout mice are not able to control parasitemia efficiently, they
reveal prolonged survival (Magez et al., 1999). This is also true for mice
treated with anti-IFN-g antibodies. The initial proinflammatory phase ends
by secretion of cytokines and generation of alternatively activated macro-
phages. Interleukin 10 inhibits classically activated macrophages. The alter-
natively activated macrophages display anti-inflammatory properties and
could dampen the pathological consequences of type I immune response.
In addition to the induction of various immune pathological damage in
different tissues, immunosuppression is yet another consequence of try-
panosome infection and T cell suppression is severe. Obviously, this
leads to frequent opportunistic superinfections, which additionally weaken
the host. Early on, immunosuppression appears to be mediated by NO,
prostaglandin E2, and TNF-a from classically activated macrophages
(Mansfield and Paulnock, 2005; Sternberg and Mabbott, 1996). At later
stages, the immunosuppression is mediated by alternatively activated
macrophages, but our knowledge about the mechanisms behind this process
is rudimentary.
African trypanosomes are capable of manipulating and controlling the
highly sophisticated immune system of their vertebrate host. Apparently,
this hijacking can occur at very different levels. While some trypanosome
species kill their host rather quickly, other infections can last for months or
even years. In any case, most hosts will not survive their encounter with
trypanosomes.
Macromolecular Trafficking in Trypanosomes 13

2.4. Innate immunity

Fascinating exceptions to the fatal outcome are humans and primates, which
are fully protected against most trypanosome species. Human innate immu-
nity against African trypanosomes has been known for more than a century.
However, only recent extensive work of several laboratories has finally shed
light on the molecular mechanisms behind the trypanosome resistance.
Normal human serum (NHS) in vitro is cytotoxic for T. brucei. Neither
immunoglobulins, the complement activation pathway nor the blood clot-
ting system are responsible. Furthermore, NHS is not activated by trypano-
some-derived factors. Many different substances and macromolecules have
been put forward as candidate trypanosome lytic factor (TLF), one of
which, high-density lipoprotein (HDL), was postulated as early as 1960 to
be involved in trypanosome killing. The first systematic study on the nature
of TLF, demonstrated the involvement of HDL and revealed that trypano-
some lysis is a two-step process (Rifkin, 1978). Initially trypanosomes are
motile and intact but start swelling. After about an hour, lysis commences.
The kinetics of trypanosome killing depends on human serum concentra-
tion and temperature; at 4  C the process is totally inhibited but is optimal at
 37  C (Rifkin, 1984). Although this key concept of trypanosome lysis
by TLF was formulated in the mid-1980s, it required 20 years to identify
TLF, to understand the molecular mechanism of lysis and to unravel the
secret of resistance to TLF by human pathogenic trypanosomes, namely
T. brucei rhodesiense.
Since it was known that TLF was an HDL component, the obvious
candidate for the toxic factor was the major constituent of HDL itself,
namely apoA-1 (Gillett and Owen, 1991; Hajduk et al., 1989). However,
biochemical characterization of HDL revealed that TLF is only a minor
subset of human HDL. Furthermore, transgenic mice expressing human
apoA-1 were not significantly protected against trypanosome infection
(Rifkin, 1991). Fractionation of HDL and reconstitution revealed that
more than one protein was required for the assembly of the lytic particle,
suggesting that distinct TLF components may act cooperatively (Hajduk
et al., 1989). A more detailed analysis of TLF proteins suggested
paraoxonase–arylesterase and haptoglobin-related protein (Hpr) as potential
toxins. Since paraoxonase–arylesterase is present in nonlytic human HDL, it
could be excluded. The haptoglobin-related protein, on the other hand,
was selectively enriched in TLF and antibodies directed against the protein
were shown to inhibit lysis in a dose-dependent manner (Smith and
Hajduk, 1995).
Hpr is restricted to primates (Maeda, 1985). Although the physiological
function is unknown, it is evolutionarily derived from haptoglobin, an
abundant acute-phase protein forming a high-affinity complex with free
hemoglobin. Haptoglobin is critically involved in clearance of hemoglobin
14 Mark C. Field et al.

from the circulation after intravascular hemal lysis. The haptoglobin–

hemoglobin complex is recognized by macrophage surface receptors and
then degraded. Hpr is a dimer, and in contrast to haptoglobin, is associated
with apoA-1. Interestingly, although it was generally accepted that TLF was
part of HDL, reports suggesting an additional trypanolytic activity appeared.
This second lytic factor had a higher molecular mass than TLF and was
termed TLF-2 (Tomlinson et al., 1997). It was reported to be a lipid-poor
complex containing mainly IgM together with apoA-1 and Hpr, while
TLF-1 contained essentially apoA-1 and Hpr. Haptoglobin is a potent
inhibitor of TLF-1, but does not interfere with TLF-2-meditated lysis.
Apparently, the specific lytic activity of TLF-1 was much higher than that
of TLF-2 (Raper et al., 1999). Confusingly, haptoglobin levels below
physiological concentrations were shown to be sufficient to completely
inhibit TLF-1. However, purified TLF-1 protected mice in a dose-depen-
dent manner against trypanosome infections, and it was generally accepted
that Hpr was the actual toxin. Further, phylogenetic analyses of the primate
lineage revealed a direct correlation between the presence of Hpr and
trypanolytic potential. While sera from several old world monkeys
contained trypanolytic activity, this was not the case for chimpanzee
serum. The Hpr gene of chimpanzees was thought to be a pseudogene,
but recent data suggest that the coding region is intact (Lugli et al., 2004).
Trypanosome lysis by TLF/TLF-2 was initially believed to be initiated
at the plasma membrane (Rifkin, 1984). However, a detailed cellular study
showed that TLF rather affects the endocytic pathway after receptor-
mediated endocytosis (Hager et al., 1994). There are about 350 high-affinity
TLF binding sites within the flagellar pocket, and electron microscopy
revealed that gold-labeled TLF is endocytosed and transported to the
lysosome, where it accumulates and causes disintegration of the organelle.
The release of lysosomal proteases into the cytoplasm would be lethal to
the parasite. It was also suggested that free radical generation through the
Fenton reaction at low pH could cause peroxidation of lysosomal mem-
brane lipids, contributing to membrane disruption (Bishop et al., 2001).
A further mechanism was proposed whereby Hpr, or part of it, disrupted
the membrane directly (Molina Portela et al., 2000). The generation of
transgenic mice expressing Hpr added yet another level of complexity to the
problem. Although Hpr was properly incorporated into HDL, those mice
were not protected against trypanosomes (Hatada et al., 2002).
Two T. brucei subspecies are resistant against TLF and are the causative
agents of human sleeping sickness. It was known that TLF-resistant strains
become susceptible when maintained for long periods in mice, and when
exposed to human serum, resistant parasites arise from sensitive populations
at low frequency. This apparent high-frequency reversal was difficult to
reconcile with classical genetic views of resistance. In fact, it appears that
antigenic variation and resistance to human serum are linked processes.
Macromolecular Trafficking in Trypanosomes 15

The underlying gene responsible for TLF resistance was identified and
termed serum resistance associated gene (SRA) (de Greef and Hamers,
1994). Curiously, the SRA sequence closely resembles VSG, although the
region encoding surface exposed loops was deleted from SRA. Thus, SRA is
likely a truncated VSG (Campillo and Carrington, 2003). However, unlike
VSG, SRA is not expressed on the cell surface, but appears to be restricted to
endosomes and lysosome (Vanhamme et al., 2003). The exact localization
within the endosomes and the rate of recycling remains to be elucidated.
Importantly, when SRA was expressed in human serum sensitive trypano-
somes, the parasites became human infective (Xong et al., 1998).
SRA is expressed from just one expression site as an ESAG. The exact
mechanism by which SRA confirms human serum resistance is still not
unambiguously clear. Mutation analyses suggest that an N-terminal helix
could be involved in neutralizing the TLF toxin (Vanhamme et al., 2003).
Through interaction with immobilized SRA a fraction from normal human
serum was isolated. Surprisingly, Hpr did not bind to SRA, but apoL-1
revealed specific and strong binding. ApoL-1 is associated with HDL and its
physiological function is still unclear. Recombinant apoL-1 revealed an
SRA-dependent trypanolytic potential. ApoL-1 is found in both TLF-1
and TLF-2, and consequently, this lipoprotein was suggested to be the only
trypanosome toxin in human serum (Shiflett et al., 2005). Like Hpr, apoL-1
can be found in many primates, but is absent from the chimpanzee genome
(Poelvoorde et al., 2004).
It remains an open question whether apoL-1 and Hpr have to act
synergistically for full protective immunity, but reconstitution experiments
suggest that both proteins contribute to human serum resistance. While
apoL-1 is necessary and sufficient for lysis, Hpr may mediate binding of TLF
to a cognate receptor at the trypanosome cell surface. This receptor has
recently been identified as a glycoprotein modified by poly-N-acetyllacto-
samine (pNAL) residues and located in the flagellar pocket (Vanhollebeke
et al., 2008). In mice, the receptor binds the haptoglobin–hemoglobin (Hp–
Hb) complex with high affinity and is responsible for the uptake of sufficient
heme for incorporation into trypanosome hemoproteins. T. brucei appar-
ently cannot discriminate between Hp–Hb and TLF1–Hpr–Hb complexes.
Consequently, in human blood Hb-charged TLF1 complexes are targeted
to the trypanosome cell. Thus, it appears that Hpr is required for high-
affinity TLF-receptor binding, while apoL-1 is the actual toxin.
The exact mode of apoL-1-mediated lysis is still a matter of debate.
However, it appears likely that it acts by generating pores in the lysosome
membrane (Perez-Morga et al., 2005; Vanhollebeke et al., 2007). ApoL-1
reveals surprising homology to the pore-forming domain of bacterial coli-
cins. In fact, it was shown that the N-terminal domain of apoL-1 can
generate ionic pores in vitro and in vivo. Furthermore, a pH-sensitive domain
is thought to be involved in membrane targeting. Thus, a possible scenario
16 Mark C. Field et al.

for trypanolysis by human serum can be summarized as follows. In the

flagellar pocket, TLF1–Hpr–Hb particles bind to the TLF-receptor via
Hpr. Passage of the internalized complex through the endocytic pathway
is accompanied by progressive acidification. This induces conformational
changes in the apoL-1 membrane-tethering domain, which could facilitate
sequestration of apoL-1 from the HDL carrier. Free apoL-1 inserts into the
membrane of the acidic lysosome. Within the membrane, the two central
hydrophobic helices of the apoL-1 pore-forming domain open a pore,
leading to depolarization of the lysosomal membrane. Influx of chloride
ions from the cytoplasm into the lysosome generates osmotic pressure that
triggers lysosome swelling. The depletion of cytoplasmic chloride results in
compensatory chloride uptake from the extracellular environment. Even-
tually, the developing intracellular pressure compromises the plasma mem-
brane and the trypanosome is killed. While there are certain aspects of the
model that still await experimental confirmation, it provides the so far most
complete explanation of trypanolysis in human serum.

3. Endocytic Pathways
The endocytic system is an important component of the intracellular
trafficking system that modulates the composition of the cell surface
through the sorting of internalized proteins, lipids, and glycoconjugates
into recycling or degradative pathways, and thus play a crucial role in
many biological functions, including maintenance of the cell surface,
immune modulation, signal transduction, and nutrition (Piper and
Katzmann, 2007). Study of the uptake of various ligands and surface pro-
teins, coupled with development of subcompartment-specific markers has
allowed considerable delineation of the major endocytic routes in T. brucei
and the organism probably has the best characterized endocytosis apparatus
of any protist. Light and electron microscopy have revealed the presence of
morphologically distinct populations of early and late endosomes, based on
the kinetics in which they are loaded with material and the specific sets of
markers they contain (Engstler et al., 2004; Grünfelder et al., 2002, 2003).
Further, the later endocytic compartments, the multivesicular body and
lysosome have also been defined, and in part functionally investigated
(Leung et al., 2008; Peck et al., 2008).

3.1. Clathrin-mediated endocytosis

All endocytosis in African trypanosomes is CME and occurs solely at the
flagella pocket (FP; Allen et al., 2003; Overath et al., 1997) (Fig. 1.3). CME
involves assembly of receptor-bound cargo and GPI-anchored membrane
Macromolecular Trafficking in Trypanosomes 17

Bulk plasma membrane

Trans-membrane domain protein (ISG)

TbTIP1 GPI-anchored protein (VSG)

TbRab11
TbRab5B Flagellar
?U pocket Clathrin coats U Ubiquitin
?
Early endosome Recycling endosome Kinetoplast EpsinR
TbCHC

TbRab5A

TbVps23/28 TbRab7
p67
? U U

Early endosome
U TbCHC
MVB/late
Sorting endosome endosome Lysosome

Figure 1.3 A model of sorting of GPI-anchored and trans-membrane domain proteins

in trypanosomes. Trans-membrane domain proteins such as ISGs (green) are present at
low density at the cell surface in comparison to the dominant GPI-anchored VSG (red).
There is presently no evidence that there is selective partitioning of GPI versus trans-
membrane anchored proteins at the cell surface. Endocytosis requires the function of
clathrin (purple) at the flagellar pocket, and for ISG65 and VSG, this serves to target the
molecules to the Rab5A-positive early endosome. VSG is segregated at the sorting
endosome, and is excluded from a clathrin-tagged membrane microdomains; it is
hypothesized that clathrin may actively sort trans-membrane domain proteins at this
location, via recognition of ubiquitylated cargo (pink lozenge); this may involve the
trypanosome epsin-related protein, which interacts with clathrin (cyan lozenge).
Recycled molecules are returned to the cell surface via a Rab11-dependent pathway
that also involves a coiled-coil Rab11-interacting protein that likely serves as a docking
site at the flagellar pocket. Trans-membrane domain cargo is delivered to the lysosome
via the multivesicular body, and degraded. This latter pathway depends on functioning
of the ESCRT complex, including TbVps23. The site(s) where ubiquitin is added are
unknown. Also the model assumes that all GPI-anchored proteins are recycled and all
trans-membrane domain proteins are directed to the lysosome—this is unlikely to be the
case, but data concerning trafficking of additional factors are not available at this time.
Finally, the function of the Rab5B endosome remains mysterious as besides the pres-
ence of lactosamine-repeat determinants, the identity of the molecules transported via
this route are unidentified.

components into clathrin-coated pits, which are formed at the site of

endocytosis by recruitment and polymerization of assembly units of the
cytoplasmic protein, clathrin, composed of three heavy chains (CHC), each
tightly associated with a single light chain (CLC; Kirchhausen and Harrison,
1981). This structural organization results in a basket-like polyhedral lattice
(Brodsky et al., 2001) that assists in deformation of the underlying mem-
brane (Conner and Schmid, 2003) into coated pits. Subsequently, these pits
invaginate and pinch off, forming class I CCVs (Engstler et al., 2004;
18 Mark C. Field et al.

Grünfelder et al., 2003; Overath and Engstler, 2004). Class I CCVs rapidly
shed their coat and the resulting vesicles dock and fuse with an early
endosomal compartment. Subsequent intracellular sorting of components
designated for degradation occurs via a negative sorting mechanism
(Overath and Engstler, 2004). Here, class II CCVs containing components
for lysosomal degradation bud from early and recycling endosomes while
VSGs are concentrated in Rab11-positive, flat, disc-like structures desig-
nated EXCs that fuse with the FP (Grünfelder et al., 2003).
Mammalian forms of African trypanosomes exhibit extremely rapid
endocytosis and recycling compared to the insect forms. This upregulation
of  10-fold (Natesan et al., 2007) is suspected to be involved in immune
evasion in bloodstream forms (Morgan et al., 2002). A similar variation is
exhibited in clathrin expression levels (Morgan et al., 2001; Natesan et al.,
2007), evidence for developmental regulation of endocytosis. Clathrin
ablation is lethal due to a direct arrest of vesicular traffic from the FP leading
to an enlarged FP or ‘‘BigEye’’ phenotype (Allen et al., 2003; Hung et al.,
2004). This underscores the unique role of clathrin and CME as the sole
endocytic mechanism in African trypanosomes.
CCV-formation machinery in T. brucei is unusual (Fig. 1.4). First, pit
formation does not involve a concentration step (Grünfelder et al., 2003), a
hallmark of CME in other eukaryotes. Second, since the T. brucei genome
does not code for the key adaptor protein, AP-2 (Morgan et al., 2002), the
mechanism of clathrin recruitment to the plasma membrane is unknown,
despite clear ultrastructural evidence of clathrin-coated pits and CCVs
(Engstler et al., 2004). AP-2 is a heterodimeric complex that binds cargo
receptors and recruits clathrin in addition to providing a platform for
assembly of accessory proteins that stabilize the activated receptor/AP-2/
clathrin coat interaction. Reasons of this loss or its replacement are
unknown but its loss may be due to the extremely high levels of cell-surface
VSG (Field et al., 2007b) precluding further concentration. Significantly,
the absence of AP-2 is common to all salivarian trypanosomes, that is, those
possessing a VSG-mediated antigenic variation mechanism.

3.2. Cargo adaptors and sorting

In the absence of an AP-2 complex, it is unclear how clathrin is specifically
targeted to the flagellar pocket membrane or how the system interacts with
cargo (Fig. 1.4). One candidate for the latter is the trypanosome epsinR.
Various epsin isoforms have been characterized (Chen et al., 1998;
Rosenthal et al., 1999; Spradling et al., 2001) and epsinR or epsin-related
proteins (Ford et al., 2002; Hirst et al., 2003), also called clathrin interacting
protein, Clint (Kalthoff et al., 2002) or enthroprotin (Wasiak et al., 2002)
are also implicated in transport processes. The epsin family shares a similar
domain organization and mainly interacts with membrane lipids, clathrin,
Macromolecular Trafficking in Trypanosomes 19

Metazoa and fungi

AP-3 AP-4 AP-1 g -synergin SCAMP

ESCRTs
GGAs
ACK1/2

p619
PI3KC2a Clathrin
Hsc70

Synapto- Amphi-
Hrs Ankryin HiP1 LDL-R b-arrestin AP180 Epsin Auxillin
janin physin

Spectrin Actin Endophilin

AP-2 Syndapin
Arp2/3
N-WASP
Synapto- Dynamin
Numb tagmin PIP2
Stonin
Eps15 Intersectin

Trypanosoma brucei

AP-3 AP-4 AP-1 SCAMP

ESCRTs

PI3KC2a Clathrin
Hsc70

Synapto-
Ankryin AP180 Epsin
janin

Actin

Arp2/3

PIP2

Adaptin Clathrin-binding Phosphoinositide Dynamin Epsin Actin

After Lafer (2002)

Figure 1.4 Presence and connectivity between gene products in trypanosomes and
metazoa. In metazoan cells clathrin mediates only one of several endocytosis routes,
while in trypanosomes all evidence indicates exclusive use of the clathrin pathway.
A clear extensive level of connectivity is evident between the polypeptides of the
clathrin endocytosis pathway in metazoa, and many of these factors also participate in
additional clathrin-independent processes. In trypanosomes, the majority of these
factors are absent from the genome, or experimental evidence also precludes a direct
role in clathrin-mediated endocytosis. Moreover, the majority of connections shown
are hypothetical and have not been experimentally determined. Red, clathrin; black,
ESCRTs; brown, adaptins; gray, direct clathrin-binding partners; green, actin; orange,
phosphoinositide phosphates; purple, others. The diagram is based on the data origi-
nally given by Lafer (2002).
20 Mark C. Field et al.

and recruits additional proteins. At the N-terminus is a phosphatidylinositol

(4,5)-bisphosphate (PtdInsP2) binding epsin N-terminal homology
(ENTH) domain of  150 amino acids (de Camilli et al., 2002). The
remaining largely unstructured region includes an ubiquitin-interacting
motif (UIM), clathrin-binding box, and AP/GGA-binding motifs in the
central region. The epsinR forms lack the UIM but retain most of the other
sequence features.
The T. brucei genome has one ENTH-family gene, TbEpsinR; the
sequence has conserved lipid-binding residues in the ENTH domain,
lacks NPF and UIM motifs and multiple DPW motifs are replaced by
DxF, and together with phylogenetic reconstruction, placing this clearly
as an epsinR subfamily member (Gabernet-Castello et al., 2008). Like other
endocytic factors, it is located between the kinetoplast and nucleus, and
ablation is lethal. RNAi knockdown indicates a clear role in endocytosis,
turnover of ISGs, and a conserved interaction with clathrin. However,
knockdown of TbEpsinR does not prevent clathrin recruitment to the
membrane or emergence of a BigEye, which suggests that while TbEpsinR
and clathrin clearly function together in endocytosis, it is probable that
other factors help load clathrin onto the membrane. Further, TbEpsinR is
not a player in the mechanism of endocytosis per se, and clearly implicated as
a cargo adaptor. A related ENTH/ANTH domain protein, TbAP180, has
so far not been studied, and there is essentially no data on roles or locations
of phosphoinositides in trypanosomes.

3.3. Early and recycling endocytic compartments

Endocytic compartments progressively mature into degradative of recycling
pathways with a concomitant change in the markers they possess. Much
work in higher eukaryotes has focused on using Rab GTPases of the Ras
superfamily as such markers due to their integral roles in the regulation of
vesicle transport, thereby allowing the spatial coordination of vesicle target-
ing, docking, and possibly budding (Zerial and McBride, 2001). A similar
approach has also been applied to trypanosomes.
While endoyctosis in trypanosomes has a single mode of entry for
material, that is, CME (Allen et al., 2003; Overath and Engstler, 2004),
up to 16 Rabs are present in the T. brucei genome, reflecting an inherent
level of sophistication within the trafficking system (Ackers et al., 2005).
Sorting of material most likely occurs within internal endocytic compart-
ments and not at the plasma membrane. This is supported by the presence of
distinct, punctuate, early endocytic structures differentiated by the presence
of two Rab5 homologues, TbRab5A and TbRab5B, and containing
distinct cargo molecules (Field et al., 1998; Pal et al., 2002a). Selectivity
is apparent at this initial stage of uptake as the Rab5A endosome contains
IgG, transferrin, VSG, and ISG65, a type I trans-membrane protein
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