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Foundational Publications

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Foundational Publications
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Foundational Publications
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GFP technological innovations

Our lab has helped develop several new GFP-based imaging technologies. We introduced confocal FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching) to highlight selective populations of fluorescent proteins in a sample. This enabled us to measure for the first time intracellular protein diffusion, protein binding/dissociation rates and transport kinetics in single cells. We also created a photoactivatable form of GFP (PA-GFP) that is invisible until activated by UV light when it becomes bright fluorescent green. This approach opened up an entire new field of research by making it possible to label specific protein populations and thereby measure protein turnover rates and determine the temporal expression pattern/behavior of proteins in living cells or organisms. Together with Dr. E. Betzig, we further introduced the super-resolution imaging technique of photoactivated localization microscopy (PALM), which uses serial photoactivation and subsequent bleaching of numerous sparse subsets of photactivated fluorescent protein molecules to determine the centers of fluorescent emission of individual molecules at mean separations of a few nanometers, overcoming the diffraction barrier in fluorescence microscopy. This technique enabled imaging of genetically-expressed fluorescent proteins on the nanoscale with a density of molecules high enough to provide structural context. By combining PALM with single particle tracking, we tracked and provided spatially-resolved maps of movement of thousands of single molecules in live cells. In further studies, we used PALM to analyze the cluster patterns of different proteins on the plasma membrane to clarify the structural organization of viral particles and to determine the stoichiometry of receptors having multiple subunits.

Cole, N.B., Smith, C., Sciaky, N., Terasaki, M., Edidin, M. and Lippincott-Schwartz, J. (1996) Diffusional mobility of Golgi proteins in membranes of living cells. Science 273: 797-801.

Nehls, S., Snapp, E., Cole, N., Zaal, K., Kenworthy, A., Roberts, T., Ellenberg, J., Presley, J., Siggia, E., and Lippincott-Schwartz, J. (2000) Dynamics and retention of misfolded proteins in native ER membrane. Nature Cell Biology 2: 288-295.

Patterson, G. and J. Lippincott-Schwartz (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297: 1873-1877.

Kenworthy, A.K., Nichols, B.J., Remmert, C.L., Hendrix, G.M., Kumar, M., Zimmerberg, J. and J. Lippincott-Schwartz (2004) Dynamics of lipid rafts at the cell surface. J. Cell Biol. 165: 735-746.

Betzig, E., Patterson, G.H., Sougrat, R., Lindwasser, O.W., Olenych, S., Bonifacino, J.S., Davidson, M.W., Lippincott-Schwartz, J., and H. F. Hess (2006) Imaging intracellular fluorescent proteins at near-molecular resolution. Science 313: 1642-1645.

Manley, S., Gillette, J.M., Patterson, G.H., Shroff, H., Hess, H., Betzig, E., and J. Lippincott-Schwartz (2008) High-density mapping of single molecule trajectories with photoactivated localization microscopy. Nature Methods 5:155-157.

Sengupta, P., Jovanovic-Talisman, T., Skoko, D., Renz, M., Veatch, S., and J. Lippincott-Schwartz (2011) Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis. Nat. Methods 8:969-975.

Renz, M., Daniels, B., Vamosi, G., Arias, I.M. and J. Lippincott-Schwartz (2012) Plasticity of the Asialoglycoprotein receptor deciphered by ensemble FRET and single molecule counting PALM. Proc Natl Acad Sci USA Plus, 109:2989-2997.

Daniels, B.R., Rikhy, R., Renz, M., Dobrowsky, T., and J. Lippincott-Schwartz (2012) Multiscale diffusion in the mitotic Drosophila melanogastor syncytial blastoderm. Proc. Natl. Acad. USA.109:8588-8593.

Membrane trafficking

Using the above and other imaging approaches, we obtained new findings important for shaping core ideas related to membrane trafficking. We provided the first temporal description of the origin, pathway and fate of secretory transport carriers. By imaging the carriers as they ferried cargo between secretory compartments, we demonstrated they are large pleiomorphic structures (~1.3 µm2), which sort cargo and require microtubules to track through the cytosol. By correlating the fluorescent signal from GFP to an actual number of molecules being imaged, we quantified for the first time: the number of cargo molecules carried by an individual carrier; the flux of cargo out of the ER, Golgi apparatus and plasma membrane over time; and, the residence time of cargo in different compartments. We demonstrated retrograde trafficking from Golgi to ER. We also used photobleaching and kinetic modeling approaches to dissect the membrane binding/release kinetics of different coat protein components responsible for cargo sorting into carriers (including COPI, Arf1, ArfGAP1, GBF1 and Sec13), demonstrating that they all undergo fast cytosol/membrane exchange irrespective of vesicle budding. 

Presley, J.F., Cole, N.B., Schroer, Hirschberg, Zaal, K.J.M., and Lippincott-Schwartz, J. (1997) ER to Golgi transport visualized in living cells. Nature 389: 81-85.

Sciaky, N., Presley, J., Smith, C., Zaal, K.J.M., Cole, N., Moreira, J.E., Terasaki, M., Siggia, E. and Lippincott-Schwartz, J. (1997) Golgi tubule traffic and the effects of brefeldin A visualized in living cells. J. Cell Biol. 139: 1137-1156

Cole, N.B., Ellenberg, J., Song, J., DiEuliis, and Lippincott-Schwartz, J. (1998) Retrograde transport of Golgi localized proteins to the ER. J. Cell Biol. 140: 1-15.

Hirschberg, K., Miller, C.M, Presley, J.F., Ellenberg, J., Zaal, K., Cole, N.B., Siggia, E., Phair, and Lippincott-Schwartz (1998) Kinetic and morphological analysis of secretory protein traffic in living cells. J. Cell Biol. 143: 1485-1503

Nichols, B.J., Kenworthy, A.K., Roberts, T.H., Hirschberg, K., Lodge, R., Phair, R.D., and Lippincott-Schwartz, J. (2001) Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J. Cell Biol. 153: 529-541

Presley, J.P., Ward, T, Miller, C., Siggia E, Phair, R.D., and J. Lippincott-Schwartz (2002) Dissection of COPI and Arf1 dynamics in vivo and role in Golgi membrane transport. Nature 417: 187-193. 

Polishchuk, R., Di Pentima, A. and J. Lippincott-Schwartz (2004) Delivery of raft-associated, GPI-anchored proteins to the apical surface of polarized MDCK cells by a transcytotic pathway. Nature Cell Biology 6: 297-307.

Liu, W., Moriyama, K., Phair, R., Duden, R., and J. Lippincott-Schwartz (2005) In vivo dynamics of ARFGAP1 and its functional interaction with Arf1 and coatomer on Golgi membranes. J. Cell Biol. 168: 1053-1063.

Sengupta P, Satpute-Krishnan P, Seo AY, Burnette DT, Patterson GH and Lippincott-Schwartz J (2015) ER trapping reveals Golgi enzymes continually revisit the ER through a recycling pathway that controls Golgi organization. Proc Natl Acad Sci USA 112:E6752-61 PMID:26598700.

Maintenance and biogenesis of organelles

We used live cell imaging approaches to study the maintenance, biogenesis and transport functions of the Golgi apparatus, ER, nuclear envelope, mitochondria, and peroxisomes. The results revealed new aspects of the dynamics of these organelles.

Ellenberg, J., Siggia, E.D., Moreira, J.E., Smith, C.L., Presley, J.F., Worman, H. J., and J. Lippincott-Schwartz (1997) Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis. J. Cell Biol. 138: 1193-1206. 

Nehls, S., Snapp, E., Cole, N., Zaal, K., Kenworthy, A., Roberts, T., Ellenberg, J., Presley, J., Siggia, E., and Lippincott-Schwartz, J. (2000) Dynamics and retention of misfolded proteins in native ER membrane. Nature Cell Biology 2: 288-295.

Zaal, K., Smith, C.L., Polishchuk, R.S., Altan, N., Cole, N., Ellenberg, J., Hirschberg, K., Presley, J., Roberts, T., Siggia, E., Phair, R., and Lippincott-Schwartz, J. (1999) Golgi membranes are absorbed into and re-emerge from the ER during mitosis. Cell 99: 589-601.

Ward, T., Polishchuk, R., Hirschberg, K., Barr, F., and J. Lippincott-Schwartz (2001) Maintenance of Golgi structure and function depends on the integrity of ER export. J. Cell Biol. 155: 557-570

Snapp, E.L., Hegde, R.S., Francolini, M., Lombardo, F., Colombo, S., Pedranzzini, E., Borgese, N. and J. Lippincott-Schwartz (2003) Formation of stacked ER cisternae by low affinity protein interactionsJ. Cell Biol. 163: 257-69.

Altan-Bonnet, N., Phair, R.D., Polishchuk, R.S., Weigart, R., and J. Lippincott-Schwartz (2003) Role of Arf1 in mitotic Golgi disassembly, chromosome segregation and cytokinesis. Proc. Natl Acad. Sci. 100: 13314-13319.

Altan-Bonnet, N., Sougrat, R., Snapp, E.L., Ward, T. and J. Lippincott-Schwartz (2006) Golgi inheritance in mammalian cells is mediated through ER export activities. Mol. Biol. Cell. 17: 990-1005.

Kim, PK, Mullen, RT, Schumann U, and J. Lippincott-Schwartz (2006) The origin and maintenance of mammalian peroxisomes involves a de novo PEX16-dependent pathway from the ERJ. Cell Biol. 173: 521-532

Organelle dynamics during disease and development: roles of autophagy, mitochondria and lipid droplets 

Kim, P., Hailey, D., Mullen, RT, and J. Lippincott-Schwartz (2009) Ubiquitin-mediated targeting of cytosolic proteins and peroxisomes for degradation by autophagy. Proc. Nat. Acad. Sci. 105, 20567-20574.

Rambold, A.S., Kostelecky, B., Elia, N., and J. Lippincott Schwartz (2011) Tubular network formation protects mitochondria from autophagosomal degradation during nutrient starvation. Proc. Natl. Acad. Sci. U.S.A 108:10190-10195.

Mitra, K., Rikhy, R., Lilly, M. and J. Lippincott-Schwartz (2012) DRP1-dependent mitochondrial fission initiates follicle cell differentiation during during Drosophila oogenesis. J. Cell Biol. 197:487-497.

Mavrakis, M., Rikhy, R., and J. Lippincott-Schwartz (2009) Plasma membrane polarity and compartmentalization are established before cellularization in the fly embryo. Developmental Cell, 16, 93-104.

Gillette, J. and J. Lippincott-Schwartz (2009) Intercellular transfer to signalling endosomes for targeted regulation within the hemapoietic stem cell-marrow niche Nature Cell Biology, 11, 303-311.

Mitra, K., Rassam, B., Lin, G., and J. Lippincott-Schwartz (2009) A fused mitochondrial state with increased ATP production is linked to G1-S transition of the cell cycle. Proc. Natl Acad. Sci. 106: 1190-1195.

Hailey, D. W., Peter, K., Mitra, K., Sougrat, R., and J. Lippincott-Schwartz (2010) Mitochondria supply membranes during the biogenesis of autophagosomes. Cell 141, 656-667.

Burnette, D., Manley, S., Sengupta, P., Sougrat, R., Davidson, M., Kachar, B., and J. Lippincott-Schwartz (2011) A role for actin arcs in the leading edge advance of migrating cells. Nat. Cell Biol., 13: 371-381

Elia, N., Sougrat, R., Spurlin, T.A., Hurley, J.H., and J. Lippincott-Schwartz (2011) Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission. Proc. Natl Acad. Sci. 108:4846-4851.