1988 — 1989 |
Lippincott-Schwartz, Jennifer |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Protein Sorting to a Nonlysosomal, Proteolytic Pathway @ U.S. National Institutes of Health |
1 |
2007 — 2016 |
Lippincott-Schwartz, Jennifer |
Z01Activity Code Description: Undocumented code - click on the grant title for more information. ZIAActivity Code Description: Undocumented code - click on the grant title for more information. |
Development of Green Fluorescent Protein Technology @ Child Health and Human Development
Point-localization superresolution techniques such as photoactivated localization microscopy (PALM) enable the imaging of fluorescent protein chimeras to reveal the organization of genetically-expressed proteins on the nanoscale with a density of molecules high enough to provide structural context. In PALM, serial photoactivation and subsequent bleaching of numerous sparse subsets of photoactivated fluorescent protein molecules is performed. Individual molecules are then localized at near molecular resolution by determining their centers of fluorescent emission via a statistical fit of their point-spread-function. The aggregate position information from all subsets is then assembled into a super-resolution image, in which individual fluorescent molecules are isolated at high molecular densities (up to 10,000 molecules/micron squared). While PALM is a powerful approach for investigating protein organization, tools for quantitative, spatial analysis of PALM datasets are largely missing. We continued to use and develop a pair-correlation analysis method with PALM (PC-PALM) that enables complex patterns of protein organization across the plasma membrane to be analyzed. The approach uses an algorithm to distinguish a single protein with multiple appearances from clusters of proteins. This enables quantification of different parameters of spatial organization, including the presence of protein clusters, their size, density and abundance in the plasma membrane. Using this method, we demonstrated distinct nanoscale organization of plasma-membrane proteins with different membrane anchoring and lipid partitioning characteristics. The ability to unambiguously distinguish more than a few different labels in a single fluorescence image has been severely hampered by the excitation cross-talk and emission bleed-through of fluorophores with highly overlapping spectra. To overcome this problem, we developed a cell labeling, image acquisition and image analysis approach to study the spatial distribution of six different organelles within eukaryotic cells. Cells were transfected with six fluorescent fusion protein markers of organelles or labeled with compartment-specific fluorescent chemical dyes to highlight the following six subcellular compartments: peroxisomes, lysosomes, ER, mitochondria, Golgi and lipid droplets. Live-cell, time-lapse images were acquired, and then linear unmixing algorithms were applied to every pixel in the image deconvolving spectrally-overlapping fluorophores. We also developed a novel image analysis pipeline for identifying regions within our images where two or more organelles contacted each other. We are using this approach to understand the full systems-level spatial organization of eukaryotic organelles under different physiological conditions.
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0.912 |
2007 |
Lippincott-Schwartz, Jennifer |
Z01Activity Code Description: Undocumented code - click on the grant title for more information. |
Dynamics of Secretory Membrane Trafficking, Sorting and Compartmentalization @ Child Health and Human Development
We have used fluorescent protein technology to investigate the characteristics of endomembrane organization in eukaryotic cells, including polarized cell monolayers in tissue culture and in living embryos. Our research has focused on the subcellular localization, mobility, transport routes and binding interactions of proteins with regulatory roles in the organization of membrane traffic and compartmentalization. [unreadable] [unreadable] In one project we have studied the biogenesis and inheritance of the Golgi apparatus. Golgi inheritance during mammalian cell division is known to occur through the disassembly, partitioning, and reassembly of Golgi membranes but the mechanisms responsible for these processes are poorly understood. To address these mechanisms, we examined the identity and behavior of Golgi proteins within mitotic membranes using dynamic cell imaging of Golgi and ER markers, electron microscopy, ER fragmentation with ionomycin, and ER entrapment through misfolding. Two overall conclusions were drawn from the data. First, that mitotic Golgi haze, seen in metaphase, represents recycled Golgi proteins trapped in the ER, a consequence that is likely related to the mitosis-specific disassembly of ER exit sites and inactivation of Arf1. Second, that mitotic Golgi fragments, seen in prometaphase and telophase, are not isolated breakdown products of the Golgi; rather, they are structures undergoing continuous exchange of their components through the ER and dispersed ER exit sites. These conclusions suggest a model in which the Golgi is inherited through the ER in mitosis and that mitotic Golgi disassembly/reassembly involves the inhibition and subsequent reactivation of cellular activities that control recycling of Golgi components into and out of the ER. Evidence supporting the first of these conclusions- that Golgi haze in metaphase cells represents Golgi proteins within the ER, came from three lines of evidence: 1) mitotic haze can be resolved into ER by high-resolution confocal microscopy, 2) it redistributes with ER into fragments upon ionomycin treatment, and 3) it displays quality control features characteristic of ER such as misfolding and retention of proteins. Evidence that mitotic Golgi fragments observed in prophase and telophase represent ER-derived structures through which Golgi proteins cycle rapidly (i.e., from ER exit sites to a fragment and then back again into the ER) came from fluorescence double-labeling, immunoelectron microscopy, and photobleaching recovery experiments. Live cell imaging of a single cell co-expressing Sec13-YFP and GalT-CFP revealed that mitotic Golgi fragments grow out from ER export domains at the end of mitosis, remain near these sites for a short period, and then undergo clustering into a Golgi ribbon. Immunoperoxidase electron microscopy of cells in prometaphase and telophase further showed that mitotic Golgi fragments were clusters of tubules/vesicles localized adjacent to ER export sites, and in some cases, were in direct continuity with ER export domains. Finally, when a mitotic Golgi fragment was photobleached in cells expressing GalT-YFP, fragment fluorescence rapidly recovered most of its original intensity within 2 min, indicating Golgi proteins continuously move in and out of mitotic fragments while maintaining steady-state pools in these fragments.[unreadable] In a second project we used live cell imaging approaches to investigate the organization of the secretory pathway in the fly embryo. We specifically asked how this pathway and its organelles become equally apportioned among the thousands of syncytial nuclei in preparation for cellularization. We found that the endoplasmic reticulum and Golgi apparatus became segregated among nuclei only when these nuclei migrated to the periphery of the embryo at nuclear division cycle 10. The nuclear-associated units of ER and Golgi across the syncytial blastoderom produced secretory products that were delivered to the plasma membrane in a spatially restricted fashion across the embryo. This occurred in the absence of plasma membrane boundaries between nuclei and was dependent on centrosome-derived microtubules. The emergence of secretory membranes that compartmentalize around individual nuclei in the syncytial blastoderm is likely to ensure that secretory organelles are equivalently partitioned among nuclei at cellularization and could play an important role in the establishment of localized gene and protein expression patterns within the early embryo.
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0.912 |
2008 — 2018 |
Lippincott-Schwartz, Jennifer |
Z01Activity Code Description: Undocumented code - click on the grant title for more information. ZIAActivity Code Description: Undocumented code - click on the grant title for more information. |
Organization and Dynamics of Endomembrane Pathways and Organelles @ Child Health and Human Development
One major area of investigation of our lab was the actomyosin cytoskeleton. Cells use the actomyosin cytoskeleton to modulate their size and shape, to crawl through different substrates, to extend out from cell masses, and to adapt to different tissue-specific environments. These processes are critical for the morphogenetic pathways underlying tissue regeneration and remodeling, as well as tissue invasion in cancer, so understanding actomyosin organization and dynamics could have multi-fold applications. To investigate the actomyosin cytoskeleton's role, we employed quantitative imaging, modeling and biophysical approaches. This included live cell 3D structured illumination microscopy; force spectrum microscopy; and, lattice light sheet microscopy. These investigations led to significant new mechanistic insights into how the actin cytoskeleton controls cell-cell interactions. In collaborative work with other labs, we were also able to demonstrate a role for actin contractile forces in mediating diffusive-like, non-thermal motion in the cytoplasm that causes intracellular movement of small and large cell components, as well as the motion of primary cilia. Related to actomyosin's role at cell-cell contacts, we used cytotoxic T lymphocytes (CTLs) as a model system. CTLs kill target cells by secreting granules containing perforin and granzymes into the immunological synapse (the site of contact formed between the CTL and target cell). We used spinning disk confocal and lattice light sheet microscopy to obtain unprecedented spatial and temporal resolution of the actin cytoskeleton and its role in both facilitating and limiting CTL secretion. We saw dynamic lamellapodial projections and a rearward flow of actin in migrating CTLs as these cells engaged a target cell. The synapse then formed in two stages: concentration of T cell receptors (TCRs) in the PM through lateral translocation (1 min), followed by vesicular delivery of intracellular TCRs as the centrosome reached the synapse (6 min). Prior to synapse formation, a continuous actin meshwork underlies the entire plasma membrane; however, local clearing occurred as both the centrosome and granules docked. After several vesicles fused, the actin meshwork reappeared and secretion stopped. Actin clearance and reappearance correlated with the loss and gain of PI(4,5)P2 in the contact zone. We concluded that the CTL contact zone is like a radially symmetric leading edge, with the distal region of protrusive actin polymerization being analogous to the lamellipodium, and the more central region, enriched in integrins and myosin IIA, analogous to the lamellum. The spatial-temporal regulation of actin in the contact zone serves to coordinate TCR docking and the timing of granule secretion. We also examined the role of the actin cytoskeleton in regulating overall motion within the cytoplasm. We reasoned that ensemble forces from actomyosin activity could have a large effect on global motion within the cytoplasm, making these forces a critical readout of the dynamic state of the cell. To quantify these forces and test how they control the motion of cytoplasmic components, we collaborated with physicist Dr. David Weitz, who devised a new methodology called force-spectrum-microscopy (FSM) to quantify force fluctuations within the cytoplasm. The technique combines measurements of the random motion of probe particles with independent micromechanical measurements of the cytoplasm. Increased cytoplasmic force fluctuations substantially enhanced intracellular movement of small and large components, including organelles. Cytoplasmic force fluctuations varied between cell types and were three times larger in malignant cells than in normal cells. Separately, in close collaboration with Christoph Schmidt, we found that force generation by the actin cytoskeleton surrounding the basal body causes previously undocumented active primary cilia movements, which could be important for tuning and calibrating ciliary sensory functions. The results of these studies reveal that actomyosin dynamics are a critical readout of proper cell health that have major effects on diverse cellular functions. A second major theme in the lab was related to mitochondrial dynamics, including mitochondrial interactions with other organelles within the cell. How these interactions impact the physiology of cells is not clear. To study mammalian cell adaptation to nutrient starvation, we examined the interplay between mitochondria fusion dynamics, autophagy, fatty acid (FA) trafficking and LDs. Given that cells appear to adapt to nutrient starvation by shifting their metabolism from reliance on glucose metabolism to utilization of mitochondrial FA oxidation, we developed an assay to investigate how FAs become mobilized and delivered to mitochondria. Using a pulse-chase labeling method to visualize movement of FAs in live cells, we demonstrated that starved cells primarily use LDs as a conduit to supply mitochondria with FAs for β-oxidation. This occurred through lipase-mediated FA mobilization from mitochondria associated LDs, rather than autophagy (contrary to the pathway used by yeast cells). Autophagy contributed to the altered metabolic scheme by recovering lipids from degraded organelles, which could be used to refill LDs. Notably, mitochondrial tubulation was essential for distribution of FAs throughout the mitochondria network. Defects in mitochondrial fusion led to massive alterations in cellular FA routing. Not only did non-metabolized FAs get redirected to and stored in LDs, they were excessively expulsed from cells. Given that FAs are toxic at high levels and serve as signaling molecules at low levels, these results suggest defects in mitochondria dynamics and FA trafficking pathways may underlie the pathologies of many metabolic diseases such as diabetes and obesity. In a different mitochondrial-related project, we uncovered new machinery regulating mitochondrial fission. Seminal work from others showed that before mitochondrial division by Drp1, ER tubules encircle and constrict mitochondria. Constriction results from actin polymerization controlled by the ER-localized formin protein, INF2. How ER tubules recognize mitochondria and facilitate fission, however, is unclear. In investigating this question, we discovered a novel mitochondria-localized actin-nucleating protein, Spire1C, which interacts with INF2 on the ER. Cooperation between Spire1C and INF2 enhanced actin assembly selectively at ER/mitochondria intersections, facilitating mitochondrial constriction. We are proposing, therefore, that during mitochondrial division a Spire1C-INF2 interaction tethers the ER to mitochondria and mediates actin polymerization, resulting in mitochondrial constriction.
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0.912 |