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DYNAMICS OF SECRETORY
MEMBRANE TRAFFICKING,
SORTING, AND COMPARTMENTALIZATION
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Jennifer Lippincott-Schwartz, PhD, Head, Section on Organelle Biology George
Patterson, PhD, Staff Scientist Nihal
Altan-Bonnet, PhD, Postdoctoral Fellow Peter
Kim, PhD, Postdoctoral Fellow Wei
Liu, PhD, Postdoctoral Fellow Holger
Lorenz, PhD, Postdoctoral Fellow Manos
Mavrakis, PhD, Postdoctoral Fellow Erik Snapp, PhD, Postdoctoral Fellow Rachid
Sougrat, PhD, Postdoctoral Fellow Eileen
Whiteman, PhD, Postdoctoral Fellow Dale
Hailey, Student Manoj Raje, PhD, Guest Researcher |
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We investigate the global principles underlying
secretory membrane trafficking, sorting, and compartmentalization within
eukaryotic cells. We use live-cell imaging of green fluorescent protein (GFP)
fusion proteins in combination with photobleaching and photoactivation
techniques to investigate the subcellular localization, mobility, transport
routes, and binding interactions of a variety of proteins with important
roles in the organization and regulation of membrane traffic and
compartmentalization. Our section uses quantitative measurements of these
protein characteristics in kinetic modeling and simulation experiments to
test mechanistic hypotheses related to protein and organelle dynamics. The
topics currently under study include growth and maintenance of endoplasmic
reticulum and Golgi morphology in mammalian cells and developing Drosophila embryos; the mechanism(s)
of secretory protein transport into and out of the Golgi apparatus; membrane
binding/dissociation kinetics of trafficking machinery and its regulation; the
generation and maintenance of cell polarity; and organelle breakdown and
reassembly during mitosis. We have also recently developed a photoactivatable
GFP, whose mechanism of photoactivation is currently under investigation. Development
of green fluorescent protein technology Patterson, Snapp, Warda Over the past year, we have continued our
efforts to optimize numerous live-cell imaging approaches, including
fluorescence recovery after photobleaching (FRAP), fluorescence loss in
photobleaching (FLIP), and photoactivation, for analyzing the dynamics of
fluorescently labeled proteins expressed in living cells. In particular, we
have focused on the further characterization of photoactivatable GFP
(PA-GFP), which is a variant of wild-type GFP that exhibits an optical
enhancement of nearly two orders of magnitude after photoactivation. In
addition to its use to highlight specific protein or cell populations, PA-GFP
can be employed to measure protein turnover rates and is useful in FRAP and
FLIP experiments after a protein population has been photoactivated. We are
currently analyzing the mechanism of PA-GFP photoactivation, particularly the
identification of the amino acid residues in GFP responsible for
photoconversion. We are also investigating whether other photoactivatable
fluorescent proteins with spectral properties different from those of PA-GFP
can be developed to allow dual-color imaging of photoactivated proteins. Lippincott-Schwartz J, Altan-Bonnet N,
Patterson GH. Photobleaching and photoactivation: following protein dynamics
in living cells. Nat Rev Mol Cell Biol 2003;Suppl:S7-S14. Lippincott-Schwartz J, Patterson G.
Development and use of fluorescent protein markers in living cells. Science 2003;300:87-91. Lippincott-Schwartz J, Snapp E. Imaging of
membrane systems and membrane traffic in living cells. In: Spector D, ed. Live Cell Imaging: A Laboratory Manual. Woodbury: Patterson G, Lippincott-Schwartz J. Selective
photo-labeling of proteins using photoactivatable green fluorescent protein. Methods 2003;32:445-450. Snapp E, Altan-Bonnet N, Lippincott-Schwartz
J. Measuring protein mobility by photobleaching GFP-chimeras in living cells.
In: Bonfacino J, Dasso M, Harford J, Lippincott-Schwartz J, Yamada K, eds. Current Protocols in Cell Biology. Ward T, Lippincott-Schwartz J. The uses of GFP
in mammalian cells. In: Chalfie M, Kain S, eds. Green Fluorescent Protein: Properties, Applications and Protocols.
Lipid
raft dynamics and cell polarity Kenworthy,b Polishchukc;
in collaboration with Arias Lipid rafts are membrane microdomains enriched
with cholesterol and glycosphingolipids that serve as platforms for protein
segregation and signaling. Despite extensive research on rafts, it remains
unclear whether raft domains are mobile structures, if protein associations
with rafts are stable or transient, or how perturbations of raft structure
affect the dynamics of individual proteins. To address these questions, we have
used FRAP to test whether raft association affects a protein’s ability to
diffuse large distances laterally across the cell surface. We systematically
measured the diffusion coefficients (D)
of several types of raft and nonraft proteins under steady-state conditions
and in response to raft perturbations. We found that, under normal
conditions, raft proteins diffused freely over large distances (more than 4
micrometers) and exhibited Ds that
varied 10-fold, indicating that raft proteins do not undergo long-range
diffusion as part of discrete, stable raft domains. Perturbations reported to
affect lipid rafts in model membranes (including cholesterol depletion,
decreased temperature, and cholesterol loading) had similar effects on the
diffusional mobility of raft and nonraft proteins, indicating that raft
association is not the dominant factor in determining protein mobility at the
cell surface and ruling out several models for raft dynamics, including
stable immobile rafts and stable mobile rafts. Assuming that raft domains
exist, our data suggest that raft proteins must rapidly partition into and
out of them. It is thought that lipid rafts are important
for sorting of glycosylphosphatidylinositol (GPI)-anchored proteins to the
apical plasma membrane of polarized cells, with GPI-anchored proteins
segregating into raft-enriched carriers in the trans-Golgi network (TGN) and then trafficking directly to the apical
plasma membrane. This view relies on the localization of cholesterol-enriched
lipid rafts at both the TGN and apical plasma membrane and on the affinity of
GPI-anchored proteins for rafts. To test this model in living cells, we
studied the pathway for apical delivery of GPI-anchored proteins tagged with
GFP in polarized MDCK cells. We employed confocal microscopy and treatment
with tannic acid, a cell impermeant fixative that inhibits plasma membrane
fusion within seconds and does not pass through tight junctions. We found
that the GPI-anchored proteins followed an indirect, transcytotic route
rather than trafficking directly from the TGN to the apical plasma membrane,
as previously thought. The proteins first exited the TGN in membrane-bound
carriers that also contained basolateral cargo, although the two cargos were
laterally segregated. The carriers were then targeted to and fused with a
zone of lateral plasma membrane adjacent to tight junctions, a zone that is
known to contain the exocyst. Thereafter, the GPI-anchored proteins, but not
basolateral cargo, were rapidly internalized, together with endocytic tracer,
into clathrin-free transport intermediates that trans-cytosed to the apical
plasma membrane. The data revealed that apical sorting of GPI-anchored
proteins occurs at the plasma membrane rather than at the TGN and involves
apically directed trans-cytotic carriers derived from basolateral membranes. In addition to characterizing the polarized
trafficking of GPI-anchored proteins, we have used confocal imaging
techniques to study the trafficking itineraries of other apical and
basolateral cargo proteins in MDCK cells. We found that basolateral cargo
proteins such as VSVG traffic directly from the TGN to tight junctions, where
the exocyst is localized, and fuse with plasma membrane at this site before
diffusing laterally across the basolateral membrane. Apical cargo proteins
such as the neurotrophin receptor p75, which do not partition into rafts,
follow a direct pathway to apical plasma membranes and are never observed on
basolateral membranes. In work performed in collaboration with Irwin Arias’s
group, we also examined the trafficking of the bile salt export pump (ABCB11)
in polarized hepatocytes. As observed for the neurotrophin receptor, ABCB11
moved from the TGN to apical membranes (i.e., the bile canaliculus) in
carriers that never fused with basolateral membranes. Furthermore, the ABCB11
proteins cycled constitutively between the canalicular membrane and
rab11-positive endosomes, potentially serving as a mechanism for regulating
the expression levels of these proteins on canicular membranes. Kenworthy AK, Nichols BJ, Remmert CL, Hendrix
GM, Kumar M, Zimmerberg J, Lippincott-Schwartz J. Dynamics of lipid rafts at
the cell surface. J Cell Biol
2004;165:735-746. Polishchuk R, Di Pentima A,
Lippincott-Schwartz J. Delivery of raft-associated, GPI-anchored proteins to
the apical surface of polarized MDCK cells by a transcytotic pathway. Nat Cell Biol 2004;6:297-307. Wakabayashi Y, Lippincott-Schwartz J, Arias
IM. Intracellular trafficking of bile salt export pump (ABCB11) in polarized
hepatic cells: constitutive cycling between the canalicular membrane and
rab11-positive endosomes. Mol Biol Cell
2004;15:3485-3496. Dynamics
and differentiation of the endoplasmic reticulum Snapp, Frescas,d Lorentz,
Raje, Mavrakis, Hailey, Whiteman, Kim; in collaboration with Brandizzi, De Lotto, Hegde, Lilly There is a long tradition of research into the
molecular mechanisms underlying the architecture and dynamics of
membrane-bound organelles and the roles of motor proteins, coat proteins, and
matrix proteins. Our live-cell imaging studies with GFP-tagged endoplasmic
reticulum (ER) markers have revealed an unexpected new mechanism for the
regulation of organelle architecture involving weak homotypic interactions
between cytoplasmic domains of membrane proteins on apposing membranes. By
attaching a protein capable of low-affinity, head-to-tail dimerization (i.e.,
GFP) to the cytoplasmic domain of different resident ER membrane proteins and
expressing the construct at high levels in living cells, we observed the
formation of regular arrays of stacked ER membranes (including karmellae,
whorls, and crystalloid ER). Given that no such reorganization of ER was
observed with the use of a nondimerizing GFP variant, the data suggested that
weak homotypic interactions between cytoplasmic domains of membrane proteins
were sufficient to convert tubular elements of the ER into stacked cisternae.
These findings raise the possibility that weak transient interactions between
proteins on apposing membranes provide a general mechanism for the formation
of stacked organelle structures (e.g., the Golgi apparatus) within cells. The Drosophila
embryo and ovary provide two unique model systems for studying changes in the
organization and dynamics of the ER during development. To follow ER dynamics
in the syncytial blastoderm, a multinucleated single-cell embryo, we
expressed the GFP-tagged ER marker lysosome-KDEL in living Drosophila embryos and performed
time-lapse confocal microscopy. We found that cortical ER exists as a single
interconnected membrane system when nuclei are still localized in the embryo
interior. How this organization of ER changes upon nuclei arrival at the
embryo cortex and whether it is dependent on microtubules or actin are
questions currently under investigation. The answers should help clarify how
secretory membranes are equivalently partitioned among nuclei before
enclosure of each nucleus by plasma membrane at cellularization. To study ER
organization in the Drosophila ovary,
we visualized GFP-tagged reporters in ovarian cysts in collaboration with
Mary Lilly. We found that the ER included fusomal membranes (which associate
with cytoskeletal proteins and branch along spindle equators to physically
connect all cells within a cyst). We also found that ER proteins are capable
of diffusing freely throughout these membranes, indicating that the ER exists
as a single membrane system shared by all cystocytes in dividing ovarian
cysts. Many newly synthesized proteins that are misfolded
or unassembled in the ER undergo retrograde translocation into the cytoplasm,
where cytosolic proteases degrade them. To investigate this degradative
pathway, we tagged photoactivatable GFP to ER-associated degradation (ERAD)
substrates to visualize their fate in
vivo. The photoactivatable GFP is ideal for such analysis in that only
GFP molecules that have been photoactivated are visible within cells,
allowing protein turnover to be measured in the absence of new protein synthesis.
Using the PA-GFP–tagged ERAD substrates, we are studying the role of
ubiquitin ligases and other regulatory molecules involved in ERAD. We are
also studying the fate of ERAD substrates when proteasome activity is
inhibited. daSilva LLP, Snapp E, Denecke J,
Lippincott-Schwartz J, Hawes C, Brandizzi F. ER exit sites and Golgi bodies
in plant cells form mobile, secretory units. Plant Cell 2004;16:1753-1771. Snapp EL, Hegde RS, Francolini M, Lombardo F,
Colombo S, Pedranzzini E, Borgese N, Lippincott-Schwartz J. Formation of stacked
ER cisternae by low affinity protein interactions. J Cell Biol 2003;163:257-269. Snapp E, Iida T, Frescas D,
Lippincott-Schwartz J, Lilly M. The fusome mediates intercellular ER
connectivity in Drosophila ovarian
cysts. Mol Biol Cell
2004;15:4512-4521. Snapp E, Reinhart G, Bogert B,
Lippincott-Schwartz J, Hegde R. The organization of engaged and quiescent
translocons in the endoplasmic reticulum of mammalian cells. J Cell Biol 2004;164:997-1007. Molecular
basis for Golgi biogenesis and function Altan-Bonnet, Liu, Patterson, Sougrat,
Mavrakis; in collaboration with Hirschberg, Jackson, Phair The Golgi apparatus serves many essential
functions for cell growth and homeostasis, including protein sorting,
processing, and transport within the secretory pathway. In addition, the
Golgi acts as a membrane scaffold onto which diverse signaling, sorting, and
cytoskeleton proteins adhere. To understand how newly synthesized proteins
traffic through the Golgi apparatus, we have developed a protocol involving the
selective highlighting of GFP-tagged cargo proteins in the Golgi and the
monitoring of the distribution and export kinetics of these cargo molecules
as they pass through the Golgi apparatus. Using this approach combined with
conventional biochemical and ultrastructural methods, we are seeking to
obtain new information regarding the mechanism of transport of proteins
through the Golgi apparatus. We aim to determine whether the numerous
cisternae comprising the Golgi act as cargo carriers in a process of cisternal
maturation or whether the cisternae are stable elements with cargo
transferred between them by small vesicles or direct connections. Cytosolic coat proteins that bind reversibly
to membranes carry out a central role in membrane trafficking by concentrating
macromolecules into specialized membrane patches that deform into coated buds
to produce coated carriers. The coatomer (COPI)-type coat helps mediate
protein sorting and transport within the Golgi apparatus. Binding of COPI to
membranes is regulated by the small GTPase, Arf1, which in its GTP-bound
state is active and assembles coats and in its GDP-bound state is inactive
and disassembles coats. We are using confocal time-lapse imaging, FRAP, and
FLIP to investigate the in vivo
dynamics of GFP-tagged versions of COPI, Arf1, ARFGAP1, and Arf1GEF. We are
interested in learning whether membrane binding and release of COPI initiates
and stabilizes cargo sorting into membrane domains and determining the nature
of the functional interaction of ARFGAP1 (which controls how rapidly Arf1 is
released from membranes) with Arf1 and COPI on Golgi membranes. We also are
using the kinetic analysis tools of computational cell biology to test
different models of cargo transport mediated by COPI/Arf1 machinery. One model
incorporates reversible partitioning of cargo into membrane domains through
repeated cycling of Arf1 and COPI onto Golgi membranes to retrieve retrograde
cargo and its associated lipids, thus promoting the partitioning of
anterograde cargo into sphingolipid-rich domains. In mitosis, successful cell division depends
on the coordination of chromosome, cytoskeleton, and organelle dynamics. We
are currently investigating how such coordination occurs and have discovered
a major role of Arf1. Our data suggest that Arf1 helps orchestrate mitotic
Golgi breakdown, chromosome segregation, and cytokinesis. We found that,
early in mitosis, Arf1 becomes inactive and dissociates from Golgi membranes,
after which numerous Arf1-dependent peripheral Golgi proteins disperse,
including cullins, myosin IIa, tankyrase, spectrin, ankyrin, and Sak1
polo-like kinase, which each could have roles in coordinating mitotic
processes. If Arf1 is kept in an active state by treatment with the small
molecule H89 or expression of its GTP-locked form, intact Golgi membranes
with bound peripheral proteins persist throughout mitosis. Such cells enter
mitosis but exhibit gross defects in chromosome segregation and cytokinesis.
The findings suggest that mitotic Golgi disassembly is dependent on Arf1
inactivation and is used by the cell to disperse numerous peripheral Golgi
proteins for coordinating the behavior of Golgi membranes, chromosomes, and
cytoskeleton during mitosis. We propose that Arf1 serves as a cell cycle
regulator to coordinate Golgi dynamics with other cellular functions. Altan-Bonnet N, Phair RD, Polishchuk RS,
Weigart R, Lippincott-Schwartz J. Role of Arf1 in mitotic Golgi disassembly,
chromosome segregation and cytokinesis. Proc
Natl Acad Sci USA 2003;100:13314-13319. Altan-Bonnet N, Sougrat R, Lippincott-Schwartz
J. Molecular basis for Golgi maintenance and biogenesis. Curr Opin Cell Biol 2004;16:364-372. Bonifacino J, Lippincott-Schwartz J. Coat
proteins: shaping membrane transport.
Nat Rev Mol Cell Biol 2003;4:1-7. Lippincott-Schwartz J, Liu W. Membrane
trafficking: coat control by curvature. Nature
2003;426:507-508. aTheresa
Ward, PhD, former Visiting Fellow bAnne
Kenworthy, PhD, former Visiting Fellow cRoman Polishchuk, PhD, former Visiting Fellow dDave
Frescas, former Student COLLABORATORS Irwin Arias, MD, Cell Biology and Metabolism Branch, NICHD,
Federica Brandizzi, PhD, Robert De Lotto, PhD, Koret Hirschberg, PhD, Catherine Jackson, PhD, Cell Biology and Metabolism Branch, NICHD,
Mary Lilly, PhD, Cell Biology and Metabolism Branch, NICHD,
Robert Phair, PhD, BioInformatics, For further information, contact jlippin@helix.nih.gov |
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