Abstract
Summary
V
Chapter?Summary
Many membrane proteins are, after endocytic uptake, efficiently recycled back to the plasma
membrane. The aim of the studies presented in this thesis was to determine pathways and
molecular mechanisms that are involved in recycling.
Plasma membrane-derived clathrin-coated vesicles fuse, after uncoating, with sorting endo-somes.
In this organelle recycling proteins are sorted from lysosomally
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directed proteins.
Recycling is thought to occur either directly from sorting endosomes to the plasma membrane
or via a second compartment that is formed in the perinuclear area and is composed of tubular
recycling endosomes. The nature of the putative recycling vesicles that derive from these two
endosome populations was unknown, but it has been suggested that endocytosed membrane
constituents recycle by default. However, in a previous study we characterized a novel class of
clathrin coated buds on recycling endosomes (Stoorvogel et al., 1996. J. Cell Biol. 132, 21-33), and
proposed that clathrin-coated vesicles might be involved in the recycling pathway from sorting
endosomes.
In chapter 2 we now show that these endosome associated clathrin-coated buds contain
dynamin-2, a GTPase that already had an established function in the fission of clathrin-coated
vesicles at the plasma membrane. To study recycling processes, we monitored the trafficking of
the transferrin receptor (TfR), a prototype recycling protein, in cells which overexpress a tem-perature-
sensitive dynamin-1 mutant (dyn ts ). At the non-permissive temperature, ~30% of
endocytosed transferrin (Tf) was retained in recycling endosomes by dyn ts cells. At these same
conditions dynamin-labeled clathrin-coated buds accumulated on tubular TfR-containing endo-somes.
In addition, recycling endosomes formed a more elaborate tubular network, suggesting
that fission of TfR containing clathrin-coated vesicles from tubular endosomes requires func-tional
dynamin. In contrast, exit from sorting endosomes was normal in these cells. From these
results, it was concluded that TfR recycling from recycling endosomes to the plasma membrane
is mediated by clathrin-coated vesicles and requires a functional dynamin, while exit from sort-ing
endosomes is a dynamin-independent process.
In chapter 3 we made use of the reversible phosphatidyl inositide (PI) 3-kinase inhibitor
LY294002 to study the requirements for PI 3-kinase in TfR recycling. LY294002 did not inter-fere
with the entry of TfR into sorting endosomes, nor with transport to, or release from recy-cling
endosomes. However, egress of endocytosed Tf from sorting endosomes was significant-ly
delayed. LY294002 and dyn ts had synergistic effects on Tf recycling kinetics, indicating that
98?they interfered with two distinct recycling pathways which can partly compensate for each oth-ers
loss of function. The inhibitory effect of LY294002 on Tf recycling was reversed upon
removal of the drug, indicating that its inhibitory effect was not due to delayed transport from
sorting endosomes to recycling endosomes. In addition, the ultrastructure of recycling endo-somes
was unaffected by this drug, as was the assembly of clathrin/dynamin coated buds on this
compartment. In contrast, the diameter of sorting endosomes was significantly increased, indi-cating
accumulation of membrane at this compartment. Together, these data indicate that endo-99
Summary
SE
RE
plasma membrane
1 2
3
4
dynamin
PI 3-kinase
Ca 2+ Ca 2+
Ca 2+ ?
Figure 1. Model for TfR recycling
(1) TfR is endocytosed via clathrin-coated pits at the plasma membranes. 100-150 nm clathrin-coated vesicles
pinch off from the plasma membrane and, after being uncoated, fuse with sorting endosomes (SE). This fusion
may be Ca 2+ dependent. (2) TfR can recycle from sorting endosomes to the plasma membrane directly in a PI
3-kinase dependent manner. This transport is probably mediated by transport vesicles. Fusion of sorting endo-some-
derived vesicles with the plasma membrane requires an influx of extracellular Ca 2+ . (3) Alternatively, TfR
can be transported from sorting endosomes to recycling endosomes (RE). It is still unclear if this transport step
is vesicle mediated, or whether tubular extensions detach from sorting endosomes and migrate to the perinuclear
area where they can form the recycling compartment. (4) Transport from recycling endosomes to the plasma
membrane is mediated by clathrin-coated vesicles that pinch off from recycling endosomes in a dynamin-dependent
manner. The fusion of these vesicles with the plasma membrane is also dependent on an influx of
extracellular Ca 2+ .?cytosed TfR recycles to the plasma membrane directly from sorting endosomes in a PI 3-kinase
dependent manner and via recycling endosomes from where clathrin/dynamin coated vesicles
are involved in further transport to the plasma membrane.
In chapter 4 we show that Ca 2+ plays an important role in membrane fusion events along the
endocytic pathway of the TfR. The membrane permeable Ca 2+ chelator BAPTA/AM interfered
with the function of sorting endosomes, most likely with the fusion of plasma membrane
derived endocytic vesicles with this compartment. Recycling of endocytosed Tf was severely
affected, while there was an accumulation of 100-150 nm vesicles, with the morphological char-acteristics
of plasma membrane derived vesicles. These vesicles were primarily found in aggre-gates
or in association with sorting endosomes. Ca 2+ channel antagonists also severely inhibited
Tf recycling, both from sorting endosomes and from recycling endosomes. Instead, we
observed an accumulation of recycling vesicles underneath the plasma membrane, suggesting
that fusion of transport vesicles with the plasmamembrane was affected. Taken together, these
data suggest that local release of endosomal Ca 2+ may be required for the homotypic and het-erotypic
fusion of endocytic vesicles and sorting endosomes, while an influx of extracellular
Ca 2+ may be required for fusion of recycling vesicles with the plasma membrane.
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Chapter 5
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