Dynamics of Membrane Traficking, Sorting, and Compartmentalization Within Eukaryotic Cells

Jennifer Lippincott-Schwartz, PhD, Head, Section on Organelle Biology
George Patterson, PhD, Staff Scientist
Rachid Sougrat, PhD, Staff Scientist
Dylan Burnette, PhD, Postdoctoral Fellow
Jennifer Chua, PhD, Postdoctoral Fellow
Markus Elsner, PhD, Postdoctoral Fellow
Jennifer Gillette, PhD, Postdoctoral Fellow
Peter Kim, PhD, Postdoctoral Fellow
Suliana Manley, PhD, Postdoctoral Fellow
Manos Mavrakis, PhD, Postdoctoral Fellow
Kasturi Mitra, PhD, Postdoctoral Fellow
Carolyn Ott, PhD, Postdoctoral Fellow
Richa Rikhy, PhD, Postdoctoral Fellow
Prasanna Satpute, PhD, Postdoctoral Fellow
Prabuddha Sengupta, PhD, Postdoctoral Fellow
Christian Wunder, PhD, Postdoctoral Fellow
Dale Hailey, BA, Predoctoral Fellow

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 turnover of a variety of proteins with important roles in the organization and regulation of membrane trafficking and compartmentalization. To test mechanistic hypotheses about protein and organelle dynamics, we use quantitative measurements of these protein characteristics in kinetic modeling and simulation experiments. Among the topics under investigation are (1) membrane partitioning and its role in protein sorting and transport in the Golgi apparatus; (2) biogenesis and dynamics of unconventional organelles; (3) mitochondrial morphology and its regulation of cell cycle progression; and (4) cytoskeletal and endomembrane cross-talk in polarized epithelial cells, hematopoietic niche cells, and the developing Drosophila syncytial blastoderm embryo. We have also devoted considerable effort to developing new fluorescence microscopy for imaging fluorescently tagged proteins at near-molecular resolution.

Development of green fluorescent protein technology

Patterson, Manley, Gillette, Sougrat; in collaboration with Hess, Betzig

Super-resolution techniques such as photoactivated localization microscopy (PALM) permit the imaging of fluorescent protein chimeras, revealing the organization of genetically expressed proteins on the nanoscale with a density of molecules high enough to provide structural context. The PALM method involves serial photoactivation and subsequent bleaching of numerous sparse subsets of photoactivated fluorescent protein molecules. A statistical fit of the point-spread function of individual molecules’ centers of fluorescent emission then localizes the molecules at near-molecular resolution. The aggregate position information from all subsets is then assembled into a superresolution image that isolates individual fluorescent molecules at high molecular densities (up to 105 molecules/µm²). We have previously demonstrated PALM imaging of intracellular structures (including lysosome, Golgi apparatus, and mitochondria) in cryo-prepared thin sections as well as imaging of vinculin and actin in fixed cells with TIRF (total internal reflection fluorescence) excitation along with correlative PALM /transmission electron microscopy of a mitochondrial marker protein.

We helped develop a dual-label PALM assay system that uses two different photo-activatable molecules expressed within cells. In addition, using PALM, we have developed a system for singleparticle tracking in living cells, allowing protein diffusion and immobilization to be characterized at the single-molecule level. Called single-particle tracking PALM (sptPALM), the technique involves activating, localizing, and bleaching many subsets of photoactivated fluorescent protein chimeras in live cells. With sptPALM, we are able to image membrane proteins and obtain spatially resolved maps of single-molecule motions, providing several orders-of-magnitude more trajectories per cell than with traditional single-particle tracking. By probing distinct subsets of molecules, including Gag and vesicular stomatitis virus G, we demonstrated that sptPALM provides a powerful means for exploring the origin of spatial and temporal heterogeneities in membranes. Another fluorescent protein technique developed in our laboratory allows determination of a protein’s topology in living cells. Termed fluorescence protease protection (FPP), the assay provides a fluorescent readout in response to trypsin-induced destruction of GFP attached to a protein of interest before and after plasma membrane permeabilization. The FPP assay involves the attachment of a fluorescent protein to the N- or C-terminus of a protein of interest. Subsequently, cells expressing the fusion protein are exposed to trypsin either before or after plasma membrane permeabilization by digitonin. If the fluorescent protein moiety on the expressed protein faces the environment exposed to trypsin (that is, the cytoplasm), it loses its fluorescent signal. Conversely, if the fluorescent protein moiety on the expressed protein faces the environment protected from trypsin (that is, the lumen of a compartment), its fluorescence persists.

Given these outcomes and the fluorescent protein’s known engineered position within the protein, we are able to deduce the orientation of the protein across the lipid bilayer. We demonstrated the broad applicability of FPP by using it to define the topology of proteins localized to several organelles, including the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, peroxisomes, and autophagosomes.

Lorenz H, Hailey DW, Lippincott-Schwartz J. Addressing membrane protein topology using the fluorescence protease protection (FPP) assay. Methods Mol Biol 2008;440:227-233.
Manley S, Gillette JM, Patterson GH, Shroff H, Hess H, Betzig E, Lippincott-Schwartz J. High-density mapping of single molecule trajectories with photoactivated localization microscopy. Nat Methods 2008;5:155-157.
Patterson GH, Lippincott-Schwartz J. Fluorescent proteins for photoactivation experiments. Methods Cell Biol 2008;85:45-61.

Membrane partitioning and its role in protein sorting and transport within the Golgi apparatus

Patterson, Sougrat, Sengupta, Elsner; in collaboration with Phair, Hirschberg, Gerlich, Polishchuk

The Golgi apparatus processes and filters newly synthesized protein and lipid moving through the secretory pathway, but how it transfers secretory cargo through its 6 to 8 flattened cisternae has remained unclear. The most widely accepted model for intra-Golgi transport is cisternal progression. This classical model postulates that the stack of cisternae in the Golgi apparatus constitute a historical record of progression from entry at the cis face to exit at the trans face. Recently arrived cargo molecules are confined in the cis-most cisterna, undergo initial processing there, and await the arrival of enzymes delivered by retrograde vesicles from more distal cisternae for subsequent processing. Cargo molecules remain within a given cisterna as it passes, conveyor belt–like, through an average of seven locations within the Golgi stack on their way to the trans face; the molecules then exit from the Golgi through transport carriers. A key prediction of the model is that newly arrived cargo exhibits a discrete lag or transit time before export. To test such a prediction, we analyzed various classes of cargo molecules within the Golgi stack after they were fluorescently pulse-labeled and quantitatively visualized as they transited from the Golgi. Our results revealed an exponential loss of cargo from the entire Golgi rather than the linear pattern predicted by the classical model. Moreover, when transmembrane cargo entered the Golgi apparatus, it differentially partitioned between two membrane environments—processing domains enriched in Golgi enzymes and export domains capable of budding transport intermediates. Based on the results of our experiments, we constructed and tested a new model of intra-Golgi trafficking in which cargo molecules continuously partition between processing and export domains defined by different lipid compositions as they move up and down the Golgi stack. Cargo can be exported to the plasma membrane from within the export domain found within every cisterna. Simulation and experimental testing of this rapid partitioning model produced all the key characteristics of the Golgi apparatus, including polarized lipid and protein gradients, exponential cargo export kinetics, and cargo waves, thus representing a viable description of the mechanism of intra-Golgi transport.

Patterson G, Hirschberg K, Polishchuk R, Gerlich D, Phair RD, Lippincott-Schwartz J. Transport through the Golgi apparatus by rapid partitioning within a two-phase membrane system. Cell 2008;133:1055-1067.

Biogenesis and dynamics of unconventional organelles

Kim, Hailey, Ott

We explored the origin and dynamics of unconventional organelles, including autophagosomes, primary cilia, and peroxisomes. Autophagosomes form during autophagy, which is a highly conserved, bulk degradation pathway that is also involved in turnover of large aggregates and organelles within cells. In the initial step of autophagy, an isolation membrane forms in the cytoplasm through the activation of specific autophagy effectors. The membrane wraps around the protein aggregate or organelle to form a double membrane–bounded structure called the autophagosome. The autophagosome then targets to and fuses with the lysosome, where the sequestered materials are degraded by various hydrolytic enzymes and recycled as amino acids for macromolecule synthesis and energy production. While emerging results have revealed the importance of autophagy in various biological and pathological processes, such as cellular remodeling, tumorigenesis, and neurodegeneration, how this pathway operates is far from clear. We used various live cell–imaging and molecular-genetic approaches to investigate the membrane origin of autophagosomes and the signals that recruit substrates to this organelle. Our data revealed that the outer membrane of mitochondria serves as the membrane source during starvation-induced autophagy formation and maturation. Furthermore, ubiquitin modification acts as a targeting signal for delivery of small cytosolic proteins and larger organelles to autophagosomes.

The primary cilium is a chemosensory and mechanosensory organelle. The axoneme of a cilium is composed of nine doublets of microtubules that extend out from the triplet microtubules of the centrosome. Primary cilia have been described as antennae because they often project away from the cell surface and are able to receive signals (both chemical and mechanical) from the extracellular environment. Imaging this important organelle is essential to developing a better understanding of how it functions. To that end, we imaged live polarized epithelial cells and found that cilia of one cell can make direct contact (bridges) with cilia of adjacent or nearby cells. In imaging live cells expressing different cilia-localized fluorophores, we observed that the bridges are composed of cilia from individual cells; that is, they are not a continuation from one cell to the next. Cilia bridges can be stable over many hours. Trypsin and other protein-disrupting treatments did not disrupt cilia-cilia adhesion. Our findings suggest that cilia may do more than just passively sense the environment; they may be able to initiate and mediate cell-cell communication through direct contact. The peroxisome is involved in the oxidation of fatty acids, bile salts, and cholesterol and converts hydrogen peroxide to nontoxic forms. Using a photolabeling, pulse-chase strategy in living cells, we demonstrated that peroxisomal membranes originate from the ER. To investigate the mechanism for peroxisome turnover, we attached monoubiquitin to peroxisomal membrane components facing the cytosol and observed that the peroxisome containing these components was a substrate for autophagy; the peroxisome was engulfed by autophagosomal membranes and, upon autophagosome-lysosome fusion, was degraded. The process was dependent on the ubiquitin-binding protein p62. Our results suggest that peroxisomal turnover occurs by autophagy through a pathway involving ubiquitination of peroxisomal membranes and p62-mediated autophagosome targeting.

Mitochondrial morphology and its regulation of cell cycle progression

Mitra, Wunder, Sougrat

Mitochondria are important energy-producing organelles within cells. Their morphology, including fragmented elements and tubular networks, results from a balance between fission and fusion events. To determine whether changes occur in mitochondrial dynamics at different stages of the cell cycle, we carried out live-cell imaging experiments in cells stably expressing RFP (red fluorescent protein) targeted to the mitochondrial matrix. We found that mitochondria exhibit distinct morphological and physiological states at different stages of the cell cycle. In mitosis, mitochondria fragmented into hundreds of small units for partitioning into daughter cells at cytokinesis. Strikingly, at G1/S, mitochondria fused into a single huge, dynamic filamentous system, unlike at any other cell cycle stage. Photobleaching of an area across the filamentous system revealed that the mitochondrial matrix was continuous. In addition, the mitochondrial network was electrically coupled and had a higher membrane potential than mitochondria at all other stages of the cell cycle. When the filamentous network or its membrane potential was disrupted or its dynamics perturbed, cell cycle progression from G1 into S phase was arrested in a p53-dependent manner. Moreover, p21-overexpression, which induces a G1/S arrest, resulted in filamentous mitochondria with reduced matrix continuity and loss of electrical coupling. The data revealed that, during the cell cycle, mitochondrial dynamism and morphology undergo critical changes that are sensed by the cell at G1/S to control cell cycle progression.

Cytoskeletal and endomembrane cross-talk in polarized epithelial cells, hematopoietic niche cells, and the developing Drosophila syncytial blastoderm embryo

Chua, Gillette, Rikhy, Mavrakis

We studied cytoskeletal and endomembrane cross-talk between cells by using three cellular systems: polarized epithelial cells, hematopoietic niche cells, and developing Drosophila syncytial blastoderm embryos. In polarized MDC K-cell epithelial monolayers, we employed live-cell imaging approaches to probe dynamin’s role in integrating membranes and the cytoskeleton. Dynamin is a large GTPase, a regulatory mechanoenzyme that participates in nascent vesicle formation and actin cytoskeletal regulation. Expression of the nucleotide-free form of dynamin led to dramatic apical constriction of expressing cells in the epithelial monolayer. The phenotype was dependent on cortactin, actin, and myosin II but independent of conditions that affect Arp2/3 activity. General endocytic inhibitors and GTP -bound dynamin mutants did not cause apical constriction. Rather, the response was mediated by nucleotide-free/GDP -bound dynamin interacting with cortactin in an Erk kinase signaling pathway. Thus, nucleotide-free/GDP-bound dynamin modulates the actomyosin contractile system through cortactin and Erk kinase activation to induce apical constriction, a process that underlies epithelial sheet folding and invagination during development as well as the shaping of tubular organs such as kidney and lung.

Hematopoietic stem/progenitor cells (HSPC) reside in the bone marrow niche, where adhesive interactions with osteoblasts provide essential cues for the cells’ proliferation and survival. We used live-cell imaging approaches to characterize the site of contact between osteoblasts and hematopoietic progenitor cells (HPC) as well as events at the contact site that result in downstream signaling responses important for niche maintenance. HPCs made prolonged contact with the osteoblast surface via a specialized membrane domain. At the contact site, the osteoblast took up portions of the specialized domain and internalized them into long-lived, SARA (Smad anchor for receptor activation)-positive, signaling endosomes. This caused the osteoblasts to downregulate Smad signaling and to increase their production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HSPC homing to bone marrow. Targeted regulation of signaling and remodeling events within the osteoblastic niche microenvironment thus involves intercellular transfer—from HSPC to signaling endosomes within osteoblasts.

Patterning in the Drosophila embryo requires local activation and dynamics of proteins in the plasma membrane (PM). How these events are coordinated before cellularization—in the absence of PM barriers—remains unclear. We used in vivo fluorescence imaging to characterize the organization and diffusional properties of the PM in embryos expressing various PM proteins. Before cellularization, the PM was polarized into discrete domains with epithelial-like characteristics. One domain resided above individual nuclei and exhibited apical-like characteristics while the other domain was lateral to nuclei and contained markers associated with basolateral membranes and junctions. Pulse-chase photoconversion experiments showed that molecules can diffuse in each domain but do not exchange between PM regions above adjacent nuclei. Drug-induced F-actin depolymerization disrupted the localization of PM polarity markers and abolished the restricted diffusion pattern in the PM. Our findings suggest a new model of PM organization in the syncytial embryo, in which epithelial-like properties and an intact F-actin network compartmentalize the PM and shape morphogen gradients.

DeLotto R, Steward R, Lippincott-Schwartz J. Nucleocytoplasmic shuttling mediates the dynamic maintenance of nuclear Dorsal levels during Drosophila embryogenesis. Development 2007;134:4233-4241.
Mavrakis M, Rikhy R, Lilly M, Lippincott-Schwartz J. Fluorescence imaging techniques for studying Drosophila embryo development. Curr Protoc Cell Biol 2008;4:Unit 4.18.
Wakabayashi Y, Chua J, Larkin J, Lippincott-Schwartz J, Arias IM. Four-dimensional imaging of filter-grown polarized MDCK cells. J Histochem Cell Biol 2007;127:463-472.

Publications Related to Other Work

Below GA, Altan-Bonnet N, Lippincott-Schwartz J, Ehrenfeld E. Hijacking components of the cellular secretory pathway for replication of Poliovirus RNA. J Virology 2007;81:558-567.
Hanssen E, Sougrat R, Frankland S, Deed S, Klonis N, Lippincott-Schwartz J, Tilley L. Electron tomography of the Maurer’s cleft organelles of Plasmodium falciparum-infected erythrocytes reveals novel structural features. Mol Microbiol 2008;67:703-718.
Kanaani J, Patterson G, Lippincott-Schwartz J, Baekkeskov S. A palmitoylation cycle dynamically regulates partitioning of the GABA-synthesizing enzyme GAD65 between ER-Golgi and post-Golgi membranes. J Cell Sci 2008;121:437-449.
Li L, Hailey DW, Soetandyo N, Li W, Lippincott-Schwartz J, Shu HB, Ye Y. Localization of A20 to a lysosome-associated compartment and its role in NFkappa signaling. Biochim Biophys Acta 2008;1783:1140-1149.
Mavrakis M, Lippincott-Schwartz J, Stratakis CA, Bossis I. mTO R kinase and the regulatory subunit of protein kinase A (PRKAR1A) spatially and functionally interact during autophagosome maturation. Autophagy 2007;3:151-153.

Collaborators

Win Arias, MD, Cell Biology and Metabolism Program, NICHD, Bethesda, MD
Eric Betzig, PhD, Howard Hughes Medical Institute, Janelia Farm Campus, Ashburn, VA
Juan Bonifacino, PhD, Cell Biology and Metabolism Program, NICHD, Bethesda, MD
Michael Davidson, PhD, Florida State University, Tallahassee, FL
Robert DeLotto, PhD, Københavns Universiteit, Copenhagen, Denmark
Ellie Ehrenfeld, PhD, Laboratory of Infectious Diseases, NIAID, Bethesda, MD
Daniel Gerlich, PhD, Eidgenössische Technische Hochschule Zürich, Institute of Biochemistry, Zürich, Switzerland
Harald Hess, PhD, Howard Hughes Medical Institute, Janelia Farm Campus, Ashburn, VA
Robert Phair, PhD, BioInformatics, Rockville, MD
Roman Polishchuk, PhD, Consorzio “Mario Negri Sud” Santa Maria Imbaro, Chieti, Italy
Constantine Stratakis, MD, D(med)Sci, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD

For further information, contact lippincj@mail.nih.gov or visit http://lippincottschwartzlab.nichd.nih.gov