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

Jennifer Lippincott-Schwartz, PhD, Head, Section on Organelle Biology
Dylan Burnette, PhD, Postdoctoral Fellow
Jennifer Chua, PhD, Postdoctoral Fellow
Jennifer Gillette, PhD, Postdoctoral Fellow
Dale Hailey, BA, Predoctoral Fellow
Suliana Manley, PhD, Postdoctoral Fellow
Kasturi Mitra, PhD, Postdoctoral Fellow
Carolyn Ott, PhD, Postdoctoral Fellow
George Patterson, PhD, Staff Scientist
Angelika Rambold, PhD, Postdoctoral Fellow
Richa Rikhy, PhD, Postdoctoral Fellow
Prasanna Satpute, PhD, Postdoctoral Fellow
Prabuddha Sengupta, PhD, Postdoctoral Fellow
Rachid Sougrat, PhD, Staff Scientist
Tajana Talisman, Ph.D, Postdoctoral Fellow
Christian Wunder, PhD, Postdoctoral 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 techniques for imaging fluorescently tagged proteins at near-molecular resolution.

Development of green fluorescent protein technology

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 lysosomes, 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 recently helped develop a new photoactivatiable fluorescent protein called PA-mCherry that switches from dark to red upon UV illumination. Using PA-mCherry and PA-GFP, we demonstrated two-color photoactivation imaging. The two-color approach was also used for PALM imaging of two different proteins within cells. We also used PALM in a new technique for single particle tracking (sptPALM) in living cells. This allowed protein diffusion and immobilization to be characterized at the single-molecule level.

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

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. To gain insight into this question, we analyzed various classes of cargo molecules within the Golgi stack after they were fluorescently pulse-labeled and quantitatively visualized them 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 of cisternal maturation. 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, each defined by a distinct lipid composition, 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.

Biogenesis and dynamics of unconventional organelles

We explored the dynamics of several 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. While emerging results have revealed the importance of autophagy in various biological and pathological processes, 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 that receives signals (both chemical and mechanical) from the extracellular environment. We imaged live polarized epithelial cells and found that cilia of one cell can make direct contact (bridges), which can be stable over many hours, with cilia of adjacent or nearby cells. The bridges are composed of cilia from individual cells; that is, they are not a continuation from one cell to the next. Cilia can thus 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.

Mitochondrial morphology and its regulation of cell cycle progression

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 various 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. During mitosis, mitochondria fragment into hundreds of small units for partitioning into daughter cells at cytokinesis. Strikingly, at G1/S, mitochondria fuse into a single, huge, dynamic filamentous system, unlike at any other stage of the cell cycle. Photobleaching of an area across the filamentous system revealed that the mitochondrial matrix is continuous. In addition, the mitochondrial network is electrically coupled and has a higher membrane potential than mitochondria at any other stage 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

We studied cytoskeletal and endomembrane cross-talk between cells using three cellular systems: polarized epithelial cells, hematopoietic niche cells, and developing Drosophila syncytial blastoderm embryos. In polarized MDCK-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 mechano-enzyme that participates in nascent vesicle formation. 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. 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, in addition to its known roles in endocytosis, dynamin modulates the actomyosin contractile system.

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, causing the osteoblasts to downregulate Smad signaling and 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.

Additional Funding

IATAP (Intramural AIDS Targeted Antiviral Program)

Publications

Mitra K, Rassam B, Lin G, Lippincott-Schwartz J. A fused mitochondrial state with increased ATP production is linked to G1-S transition of the cell cycle. Proc Natl Acad Sci 2009 106:1190-1195.
Shtengel G, Galbraith JA, Galbraith CG, Lippincott-Schwartz J, Gillette JM, Manley S, Sougrat R, Waterman C, Kanchanawong A, Davidson MW, Fetter RD, Hess HF. Single photon fluorescence interferometry for 3D super-resolution microscopy. Proc Natl Acad Sci 2009 106:3125-3130.
Mavrakis M, Rikhy R, Lippincott-Schwartz J. Plasma membrane polarity and compartmentalization are established before cellularization in the fly embryo. Dev Cell 2009 16:93-104.
Gillette J, Lippincott-Schwartz J. Intercellular transfer to signalling endosomes for targeted regulation within the hemapoietic stem cell-marrow niche. Nat Cell Biol 2009 11:303-311.
Kim P, Hailey D, Mullen RT, Lippincott-Schwartz J. Ubiquitin-mediated targeting of cytosolic proteins and peroxisomes for degradation by autophagy. Proc Natl Acad Sci 2009 105:20567-20574.

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 on Developmental Endocrinology and Genetics, NICHD, Bethesda, MD

Contact

For more information, email lippincj@mail.nih.gov or visit lippincottschwartzlab.nichd.nih.gov.

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