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08/08/17 | Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes.
Ritter AT, Kapnick SM, Murugesan S, Schwartzberg PL, Griffiths GM, Lippincott-Schwartz J
Proceedings of the National Academy of Sciences of the United States of America. 2017 Aug 08;114(32):E6585-E6594. doi: 10.1073/pnas.1710751114

CD8(+) cytotoxic T lymphocytes (CTLs) eliminate virally infected cells through directed secretion of specialized lytic granules. Because a single CTL can kill multiple targets, degranulation must be tightly regulated. However, how CTLs regulate the termination of granule secretion remains unclear. Previous work demonstrated that centralized actin reduction at the immune synapse precedes degranulation. Using a combination of live confocal, total internal reflection fluorescence, and superresolution microscopy, we now show that, after granule fusion, actin recovers at the synapse and no further secretion is observed. Depolymerization of actin led to resumed granule secretion, suggesting that recovered actin acts as a barrier preventing sustained degranulation. Furthermore, RAB27a-deficient CTLs, which do not secrete cytotoxic granules, failed to recover actin at the synapse, suggesting that RAB27a-mediated granule secretion is required for actin recovery. Finally, we show that both actin clearance and recovery correlated with synaptic phosphatidylinositol 4,5-bisphosphate (PIP2) and that alterations in PIP2 at the immunological synapse regulate cortical actin in CTLs, providing a potential mechanism through which CTLs control cortical actin density. Our work provides insight into actin-related mechanisms regulating CTL secretion that may facilitate serial killing during immune responses.

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07/17/17 | A consensus view of ESCRT-mediated Human Immunodeficiency Virus Type 1 abscission.
Lippincott-Schwartz J, Freed EO, van Engelenburg SB
Annual Review of Virology. 2017 Jul 17:. doi: 10.1146/annurev-virology-101416-041840

The strong dependence of retroviruses, such as human immunodeficiency virus type 1 (HIV-1), on host cell factors is no more apparent than when the endosomal sorting complex required for transport (ESCRT) machinery is purposely disengaged. The resulting potent inhibition of retrovirus release underscores the importance of understanding fundamental structure-function relationships at the ESCRT-HIV-1 interface. Recent studies utilizing advanced imaging technologies have helped clarify these relationships, overcoming hurdles to provide a range of potential models for ESCRT-mediated virus abscission. Here, we discuss these models in the context of prior work detailing ESCRT machinery and the HIV-1 release process. To provide a template for further refinement, we propose a new working model for ESCRT-mediated HIV-1 release that reconciles disparate and seemingly conflicting studies. Expected final online publication date for the Annual Review of Virology Volume 4 is September 29, 2017. Please see for revised estimates.

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06/29/17 | Rational engineering of photoconvertible fluorescent proteins for dual-color fluorescence nanoscopy enabled by a triplet-state mechanism of primed conversion.
Mohr MAlexander, Kobitski AYu, Sabater LRullan, Nienhaus K, Obara CJohn, Lippincott-Schwartz J, Nienhaus GUlrich, Pantazis P
Angewandte Chemie (International ed. in English). 2017 Jun 29:. doi: 10.1002/anie.201706121

Green-to-red photoconvertible fluorescent proteins (pcFPs) are powerful tools for super-resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion not only by the traditional 400-nm illumination but also by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far-red/near-infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive. Here, we uncover the molecular mechanism of primed conversion by reporting the intermediate "primed" state to be a triplet dark state formed by intersystem crossing. We show that formation of this state can be influenced by the introduction of serine or threonine at sequence position 69 (Eos notation) and use this knowledge to create "pr"- (for primed convertible) variants of most known green-to-red pcFPs.

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05/24/17 | Applying systems-level spectral imaging and analysis to reveal the organelle interactome.
Valm AM, Cohen S, Legant WR, Melunis J, Hershberg U, Wait E, Cohen AR, Davidson MW, Betzig E, Lippincott-Schwartz J
Nature. 2017 May 24:. doi: 10.1038/nature22369

The organization of the eukaryotic cell into discrete membrane-bound organelles allows for the separation of incompatible biochemical processes, but the activities of these organelles must be coordinated. For example, lipid metabolism is distributed between the endoplasmic reticulum for lipid synthesis, lipid droplets for storage and transport, mitochondria and peroxisomes for β-oxidation, and lysosomes for lipid hydrolysis and recycling. It is increasingly recognized that organelle contacts have a vital role in diverse cellular functions. However, the spatial and temporal organization of organelles within the cell remains poorly characterized, as fluorescence imaging approaches are limited in the number of different labels that can be distinguished in a single image. Here we present a systems-level analysis of the organelle interactome using a multispectral image acquisition method that overcomes the challenge of spectral overlap in the fluorescent protein palette. We used confocal and lattice light sheet instrumentation and an imaging informatics pipeline of five steps to achieve mapping of organelle numbers, volumes, speeds, positions and dynamic inter-organelle contacts in live cells from a monkey fibroblast cell line. We describe the frequency and locality of two-, three-, four- and five-way interactions among six different membrane-bound organelles (endoplasmic reticulum, Golgi, lysosome, peroxisome, mitochondria and lipid droplet) and show how these relationships change over time. We demonstrate that each organelle has a characteristic distribution and dispersion pattern in three-dimensional space and that there is a reproducible pattern of contacts among the six organelles, that is affected by microtubule and cell nutrient status. These live-cell confocal and lattice light sheet spectral imaging approaches are applicable to any cell system expressing multiple fluorescent probes, whether in normal conditions or when cells are exposed to disturbances such as drugs, pathogens or stress. This methodology thus offers a powerful descriptive tool and can be used to develop hypotheses about cellular organization and dynamics.

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04/07/17 | Defects in ER-endosome contacts impact lysosome function in hereditary spastic paraplegia.
Allison R, Edgar JR, Pearson G, Rizo T, Newton T, Günther S, Berner F, Hague J, Connell JW, Winkler J, Lippincott-Schwartz J, Beetz C, Winner B, Reid E
The Journal of Cell Biology. 2017 Apr 07:. doi: 10.1083/jcb.201609033

Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. We found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER-endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell-derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in spastin, strumpellin, or REEP1 cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. Our results implicate failure of the ER-endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration.

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04/04/17 | Optical measurement of receptor tyrosine kinase oligomerization on live cells.
Chung I
Biochimica et Biophysica Acta (BBA) - Biomembranes. 2017 Apr 04;1859(9):1436-44. doi: 10.1016/j.bbamem.2017.03.026

Receptor tyrosine kinases (RTK) are important cell surface receptors that transduce extracellular signals across the plasma membrane. The traditional view of how these receptors function is that ligand binding to the extracellular domains acts as a master-switch that enables receptor monomers to dimerize and subsequently trans-phosphorylate each other on their intracellular domains. However, a growing body of evidence suggests that receptor oligomerization is not merely a consequence of ligand binding, but is instead part of a complex process responsible for regulation of receptor activation. Importantly, the oligomerization dynamics and subsequent activation of these receptors are affected by other cellular components, such as cytoskeletal machineries and cell membrane lipid characteristics. Thus receptor activation is not an isolated molecular event mediated by the ligand-receptor interaction, but instead involves orchestrated interactions between the receptors and other cellular components. Measuring receptor oligomerization dynamics on live cells can yield important insights into the characteristics of these interactions. Therefore, it is imperative to develop techniques that can probe receptor movements on the plasma membrane with optimal temporal and spatial resolutions. Various microscopic techniques have been used for this purpose. Optical techniques including single molecule tracking (SMT) and fluorescence correlation spectroscopy (FCS) measure receptor diffusion on live cells. Receptor-receptor interactions can also be assessed by detecting Förster resonance energy transfer (FRET) between fluorescently-labeled receptors situated in close proximity or by counting the number of receptors within a diffraction limited fluorescence spot (stepwise bleaching). This review will describe recent developments of optical techniques that have been used to study receptor oligomerization on living cells. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

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12/30/16 | Live cell single molecule-guided Bayesian localization super resolution microscopy.
Xu F, Zhang M, He W, Han R, Xue F, Liu Z, Zhang F, Lippincott-Schwartz J, Xu P
Cell Research. 2016 Dec 30:. doi: 10.1038/cr.2015.160
12/14/16 | The nanoscale spatial organization of B cell receptors on IgM- and IgG-expressing human B cells.
Lee J, Sengupta P, Brzostowski J, Lippincott-Schwartz J, Pierce SK
Molecular Biology of the Cell. 2016 Dec 14:. doi: 10.1091/mbc.E16-06-0452

B cell activation is initiated by the binding of antigen to the B cell receptor (BCR). Here we used dSTORM super resolution imaging to characterize the nanoscale spatial organization of IgM and IgG BCRs on the surfaces of resting and antigen-activated human peripheral blood B cells. We provide insights into both the fundamental process of antigen-driven BCR clustering as well as differences in the spatial organization of IgM and IgG BCRs that may contribute to the characteristic differences in the responses of naïve and memory B cells to antigen. We provide evidence that although both IgM and IgG BCRs reside in highly heterogeneous protein islands that vary in both size and number of BCR single molecule localizations, both resting and activated B cells intrinsically maintain a high frequency of single isolated BCR localizations, which likely represent BCR monomers. IgG BCRs are more clustered than IgM BCRs on resting cells and form larger protein islands following antigen activation. Small dense BCR clusters likely formed via protein-protein interactions are present on the surface of resting cells and antigen activation induces these to come together to form less dense, larger islands, a process likely governed, at least in part, by protein-lipid interactions.

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12/08/16 | Sonic Hedgehog Pathway activation increases mitochondrial abundance and activity in hippocampal neurons.
Yao PJ, Manor U, Petralia RS, Brose RD, T Y Wu R, Ott C, Wang YX, Charnoff A, Lippincott-Schwartz J, Mattson MP
Molecular Biology of the Cell. 2016 Dec 08:. doi: 10.1091/mbc.E16-07-0553

Mitochondria are essential organelles whose biogenesis, structure, and function are regulated by many signaling pathways. In this study we present evidence that, in hippocampal neurons, activation of the Sonic hedgehog (Shh) signaling pathway impacts multiple aspects of mitochondria. Mitochondrial mass was increased significantly in neurons treated with Shh. Using biochemical and fluorescence imaging analyses, we show that Shh signaling activity reduces mitochondrial fission and promotes mitochondrial elongation, at least in part, via suppression of the mitochondrial fission protein dynamin-like GTPase Drp1. Mitochondria from Shh-treated neurons were more electron-dense as revealed by electron microscopy, and had higher membrane potential and respiratory activity. We further show that Shh protects neurons against a variety of stresses, including the mitochondrial poison rotenone, amyloid β-peptide, hydrogen peroxide, and high levels of glutamate. Collectively, our data suggest a link between Shh pathway activity and the physiological properties of mitochondria in hippocampal neurons.

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10/28/16 | AMPK activation prevents and reverses drug-induced mitochondrial and hepatocyte injury by promoting mitochondrial fusion and function.
Kang SWoo Sophie, Haydar G, Taniane C, Farrell G, Arias IM, Lippincott-Schwartz J, Fu D
PLoS One. 2016 Oct 31;11(10):e0165638. doi: 10.1371/journal.pone.0165638

Mitochondrial damage is the major factor underlying drug-induced liver disease but whether conditions that thwart mitochondrial injury can prevent or reverse drug-induced liver damage is unclear. A key molecule regulating mitochondria quality control is AMP activated kinase (AMPK). When activated, AMPK causes mitochondria to elongate/fuse and proliferate, with mitochondria now producing more ATP and less reactive oxygen species. Autophagy is also triggered, a process capable of removing damaged/defective mitochondria. To explore whether AMPK activation could potentially prevent or reverse the effects of drug-induced mitochondrial and hepatocellular damage, we added an AMPK activator to collagen sandwich cultures of rat and human hepatocytes exposed to the hepatotoxic drugs, acetaminophen or diclofenac. In the absence of AMPK activation, the drugs caused hepatocytes to lose polarized morphology and have significantly decreased ATP levels and viability. At the subcellular level, mitochondria underwent fragmentation and had decreased membrane potential due to decreased expression of the mitochondrial fusion proteins Mfn1, 2 and/or Opa1. Adding AICAR, a specific AMPK activator, at the time of drug exposure prevented and reversed these effects. The mitochondria became highly fused and ATP production increased, and hepatocytes maintained polarized morphology. In exploring the mechanism responsible for this preventive and reversal effect, we found that AMPK activation prevented drug-mediated decreases in Mfn1, 2 and Opa1. AMPK activation also stimulated autophagy/mitophagy, most significantly in acetaminophen-treated cells. These results suggest that activation of AMPK prevents/reverses drug-induced mitochondrial and hepatocellular damage through regulation of mitochondrial fusion and autophagy, making it a potentially valuable approach for treatment of drug-induced liver injury.

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10/28/16 | Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER.
Nixon-Abell J, Obara CJ, Weigel AV, Li D, Legant WR, Xu CS, Pasolli A, Harvey K, Hess HF, Betzig E, Blackstone C, Lippincott-Schwartz J
Science (New York, N.Y.). 2016 Oct 28;354(6311):433-46. doi: 10.1126/science.aaf3928

The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle that plays crucial roles in numerous cellular functions. We used emerging superresolution imaging technologies to clarify the morphology and dynamics of the peripheral ER, which contacts and modulates most other intracellular organelles. Peripheral components of the ER have classically been described as comprising both tubules and flat sheets. We show that this system consists almost exclusively of tubules at varying densities, including structures that we term ER matrices. Conventional optical imaging technologies had led to misidentification of these structures as sheets because of the dense clustering of tubular junctions and a previously uncharacterized rapid form of ER motion. The existence of ER matrices explains previous confounding evidence that had indicated the occurrence of ER “sheet” proliferation after overexpression of tubular junction–forming proteins.

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08/01/16 | Midbody remnant licenses primary cilia formation in epithelial cells.
Ott CM
The Journal of Cell Biology. 2016 Aug 1;214(3):237-9. doi: 10.1083/jcb.201607046

Tethered midbody remnants dancing across apical microvilli, encountering the centrosome, and beckoning forth a cilium-who would have guessed this is how polarized epithelial cells coordinate the end of mitosis and the beginning of ciliogenesis? New evidence from Bernabé-Rubio et al. (2016. J. Cell Biol supports this emerging model.

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07/29/16 | Bright photoactivatable fluorophores for single-molecule imaging.
Lavis LD, Grimm JB, English BP, Choi H, Muthusamy AK, Mehl BP, Dong P, Brown TA, Lippincott-Schwartz J, Liu Z, Lionnet T
bioRxiv. 2016 Jul 29:066779. doi: 10.1101/066779

Small molecule fluorophores are important tools for advanced imaging experiments. The development of self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in live-cell microscopy. We recently described a general method for improving the brightness and photostability of small, cell-permeable fluorophores, resulting in the novel azetidine-containing "Janelia Fluor" (JF) dyes. Here, we refine and extend the utility of the JF dyes by synthesizing photoactivatable derivatives that are compatible with live cell labeling strategies. These compounds retain the superior brightness of the JF dyes once activated, but their facile photoactivation also enables improved single-particle tracking and localization microscopy experiments.

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02/06/16 | Dynamin regulates metaphase furrow formation and plasma membrane compartmentalization in the syncytial Drosophila embryo.
Rikhy R, Mavrakis M, Lippincott-Schwartz J
Biology open. 2015;4(3):301-11. doi: 10.1242/bio.20149936

The successive nuclear division cycles in the syncytial Drosophila embryo are accompanied by ingression and regression of plasma membrane furrows, which surround individual nuclei at the embryo periphery, playing a central role in embryo compartmentalization prior to cellularization. Here, we demonstrate that cell cycle changes in dynamin localization and activity at the plasma membrane (PM) regulate metaphase furrow formation and PM organization in the syncytial embryo. Dynamin was localized on short PM furrows during interphase, mediating endocytosis of PM components. Dynamin redistributed off ingressed PM furrows in metaphase, correlating with stabilized PM components and the associated actin regulatory machinery on long furrows. Acute inhibition of dynamin in the temperature sensitive shibire mutant embryo resulted in morphogenetic consequences in the syncytial division cycle. These included inhibition of metaphase furrow ingression, randomization of proteins normally polarized to intercap PM and disruption of the diffusion barrier separating PM domains above nuclei. Based on these findings, we propose that cell cycle changes in dynamin orchestrate recruitment of actin regulatory machinery for PM furrow dynamics during the early mitotic cycles in the Drosophila embryo.

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02/03/16 | Intracellular and extracellular forces drive primary cilia movement.
Battle C, Ott CM, Burnette DT, Lippincott-Schwartz J, Schmidt CF
Proceedings of the National Academy of Sciences of the United States of America. 2015 Feb 3;112(5):1410-5. doi: 10.1073/pnas.1421845112

Primary cilia are ubiquitous, microtubule-based organelles that play diverse roles in sensory transduction in many eukaryotic cells. They interrogate the cellular environment through chemosensing, osmosensing, and mechanosensing using receptors and ion channels in the ciliary membrane. Little is known about the mechanical and structural properties of the cilium and how these properties contribute to ciliary perception. We probed the mechanical responses of primary cilia from kidney epithelial cells [Madin-Darby canine kidney-II (MDCK-II)], which sense fluid flow in renal ducts. We found that, on manipulation with an optical trap, cilia deflect by bending along their length and pivoting around an effective hinge located below the basal body. The calculated bending rigidity indicates weak microtubule doublet coupling. Primary cilia of MDCK cells lack interdoublet dynein motors. Nevertheless, we found that the organelles display active motility. 3D tracking showed correlated fluctuations of the cilium and basal body. These angular movements seemed random but were dependent on ATP and cytoplasmic myosin-II in the cell cortex. We conclude that force generation by the actin cytoskeleton surrounding the basal body results in active ciliary movement. We speculate that actin-driven ciliary movement might tune and calibrate ciliary sensory functions.

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05/23/15 | Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics.
Rambold AS, Cohen S, Lippincott-Schwartz J
Developmental Cell. 2015 Mar 23;32(6):678-92. doi: 10.1016/j.devcel.2015.01.029

Fatty acids (FAs) provide cellular energy under starvation, yet how they mobilize and move into mitochondria in starved cells, driving oxidative respiration, is unclear. Here, we clarify this process by visualizing FA trafficking with a fluorescent FA probe. The labeled FA accumulated in lipid droplets (LDs) in well-fed cells but moved from LDs into mitochondria when cells were starved. Autophagy in starved cells replenished LDs with FAs, increasing LD number over time. Cytoplasmic lipases removed FAs from LDs, enabling their transfer into mitochondria. This required mitochondria to be highly fused and localized near LDs. When mitochondrial fusion was prevented in starved cells, FAs neither homogeneously distributed within mitochondria nor became efficiently metabolized. Instead, FAs reassociated with LDs and fluxed into neighboring cells. Thus, FAs engage in complex trafficking itineraries regulated by cytoplasmic lipases, autophagy, and mitochondrial fusion dynamics, ensuring maximum oxidative metabolism and avoidance of FA toxicity in starved cells.

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03/03/15 | Profile of Eric Betzig, Stefan Hell, and W. E. Moerner, 2014 Nobel Laureates in Chemistry.
Lippincott-Schwartz J
Proceedings of the National Academy of Sciences of the United States of America. 2015 Mar 3;112(9):2630-2. doi: 10.1073/pnas.1500784112
02/16/15 | Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation.
Lu Q, Insinna C, Ott C, Stauffer J, Pintado PA, Rahajeng J, Baxa U, Walia V, Cuenca A, Hwang YS, Daar IO, Lopes S, Lippincott-Schwartz J, Jackson PK, Caplan S, Westlake CJ
Nature Cell Biology. 2015 Feb 16;17(4):228-40. doi: 10.1038/ncb3109

Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11–​Rab8 cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins ​EHD1 and ​EHD3, in association with the Rab11–​Rab8 cascade, function in early ciliogenesis. ​EHD1 and ​EHD3 localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle formation from smaller distal appendage vesicles (DAVs). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and ​IFT20. ​SNAP29, a SNARE membrane fusion regulator and ​EHD1-binding protein, is also required for DAV-mediated ciliary vesicle assembly. Interestingly, only after ciliary vesicle assembly is ​Rab8 activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step, whereby ​EHD1 and ​EHD3 reorganize the M-centriole and associated DAVs before coordinated ciliary membrane and axoneme growth.

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03/27/15 | Selective visualization of GLUT4 storage vesicles and associated Rab proteins using IRAP-pHluorin.
Chen Y, Lippincott-Schwartz J
Methods in molecular biology (Clifton, N.J.). 2015;1298:173-9. doi: 10.1007/978-1-4939-2569-8_14

Fluorescence microscopy and fluorescent protein (FP)-tagged GLUT4 molecule have been great tools to characterize GLUT4 localization and dynamics inside the cell. However, it was difficult to distinguish GLUT4 storage vesicles (GSVs) from other intracellular compartments containing GLUT4 in live cells. Here, we describe the use of IRAP-pHluorin and total internal reflection fluorescence (TIRF) microscopy to selectively visualize GSVs and Rab proteins that associate with GSVs. This assay is also valuable to further defining GSV identity by unraveling other GSV-associated proteins.

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02/05/15 | Deacetylation of nuclear LC3 drives autophagy initiation under starvation.
Huang R, Xu Y, Wan W, Shou X, Qian J, You Z, Liu B, Chang C, Zhou T, Lippincott-Schwartz J, Liu W
Molecular cell. 2015 Feb 5;57(3):456-66. doi: 10.1016/j.molcel.2014.12.013

Shuttling of macromolecules between different cellular compartments helps regulate the timing and extent of different cellular activities. Here, we report that LC3, a key initiator of autophagy that cycles between the nucleus and cytoplasm, becomes selectively activated in the nucleus during starvation through deacetylation by the nuclear deacetylase Sirt1. Deacetylation of LC3 at K49 and K51 by Sirt1 allows LC3 to interact with the nuclear protein DOR and return to the cytoplasm with DOR, where it is able to bind Atg7 and other autophagy factors and undergo phosphatidylethanolamine conjugation to preautophagic membranes. The association of deacetylated LC3 with autophagic factors shifts LC3's distribution from the nucleus toward the cytoplasm. Thus, an acetylation-deacetylation cycle ensures that LC3 effectively redistributes in an activated form from nucleus to cytoplasm, where it plays a central role in autophagy to enable the cell to cope with the lack of external nutrients.

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12/19/14 | Cell Biology. Fixing problems with cell lines.
Lorsch JR, Collins FS, Lippincott-Schwartz J
Science (New York, N.Y.). 2014 Dec 19;346(6216):1452-3. doi: 10.1126/science.1259110
11/05/14 | Quantitative cell biology: transforming the conceptual, theoretical, instrumental, and methodological approaches to cell biology.
Lippincott-Schwartz J
Molecular biology of the cell. 2014 Nov 5;25(22):3437. doi: 10.1091/mbc.E14-08-1297
12/15/14 | Superresolution imaging reveals structural features of EB1 in microtubule plus-end tracking.
Xia P, Liu X, Wu B, Zhang S, Song X, Yao PY, Lippincott-Schwartz J, Yao X
Molecular biology of the cell. 2014 Dec 15;25(25):4166-73. doi: 10.1091/mbc.E14-06-1133

Visualization of specific molecules and their interactions in real time and space is essential to delineate how cellular dynamics and the signaling circuit are orchestrated. Spatial regulation of conformational dynamics and structural plasticity of protein interactions is required to rewire signaling circuitry in response to extracellular cues. We introduce a method for optically imaging intracellular protein interactions at nanometer spatial resolution in live cells, using photoactivatable complementary fluorescent (PACF) proteins. Subsets of complementary fluorescent protein molecules were activated, localized, and then bleached; this was followed by the assembly of superresolution images from aggregate position of sum interactive molecules. Using PACF, we obtained precise localization of dynamic microtubule plus-end hub protein EB1 dimers and their distinct distributions at the leading edges and in the cell bodies of migrating cells. We further delineated the structure-function relationship of EB1 by generating EB1-PACF dimers (EB1(wt):EB1(wt), EB1(wt):EB1(mt), and EB1(mt):EB1(mt)) and imaging their precise localizations in culture cells. Surprisingly, our analyses revealed critical role of a previously uncharacterized EB1 linker region in tracking microtubule plus ends in live cells. Thus PACF provides a unique approach to delineating spatial dynamics of homo- or heterodimerized proteins at the nanometer scale and establishes a platform to report the precise regulation of protein interactions in space and time in live cells.

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10/24/14 | Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.
Chen BC, Legant WR, Wang K, Shao L, Milkie DE, Davidson MW, Janetopoulos C, Wu XS, Hammer JA, Liu Z, English BP, Mimori-Kiyosue Y, Romero DP, Ritter AT, Lippincott-Schwartz J, Fritz-Laylin L, Mullins D, Mitchell DM, Bembenek JN, Reymann AC, Böhme R, Grill SW, Wang JT, Seydoux G, Tulu S, Kiehart DP, Betzig E
Science. 2014 Oct 24;346(6208):1257998. doi: 10.1126/science.1257998

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.

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11/05/14 | Flat clathrin lattices: stable features of the plasma membrane.
Grove J, Metcalf DJ, Knight AE, Wavre-Shapton ST, Sun T, Protonotarios ED, Griffin LD, Lippincott-Schwartz J, Marsh M
Molecular biology of the cell. 2014 Nov 5;25(22):3581-94. doi: 10.1091/mbc.E14-06-1154

Clathrin-mediated endocytosis (CME) is a fundamental property of eukaryotic cells. Classical CME proceeds via the formation of clathrin-coated pits (CCPs) at the plasma membrane, which invaginate to form clathrin-coated vesicles, a process that is well understood. However, clathrin also assembles into flat clathrin lattices (FCLs); these structures remain poorly described, and their contribution to cell biology is unclear. We used quantitative imaging to provide the first comprehensive description of FCLs and explore their influence on plasma membrane organization. Ultrastructural analysis by electron and superresolution microscopy revealed two discrete populations of clathrin structures. CCPs were typified by their sphericity, small size, and homogeneity. FCLs were planar, large, and heterogeneous and present on both the dorsal and ventral surfaces of cells. Live microscopy demonstrated that CCPs are short lived and culminate in a peak of dynamin recruitment, consistent with classical CME. In contrast, FCLs were long lived, with sustained association with dynamin. We investigated the biological relevance of FCLs using the chemokine receptor CCR5 as a model system. Agonist activation leads to sustained recruitment of CCR5 to FCLs. Quantitative molecular imaging indicated that FCLs partitioned receptors at the cell surface. Our observations suggest that FCLs provide stable platforms for the recruitment of endocytic cargo.

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08/14/14 | Probing the stochastic, motor-driven properties of the cytoplasm using force spectrum microscopy.
Guo M, Ehrlicher AJ, Jensen MH, Renz M, Moore JR, Goldman RD, Lippincott-Schwartz J, Mackintosh FC, Weitz DA
Cell. 2014 Aug 14;158(4):822-32. doi: 10.1016/j.cell.2014.06.051

Molecular motors in cells typically produce highly directed motion; however, the aggregate, incoherent effect of all active processes also creates randomly fluctuating forces, which drive diffusive-like, nonthermal motion. Here, we introduce force-spectrum-microscopy (FSM) to directly quantify random forces within the cytoplasm of cells and thereby probe stochastic motor activity. This technique combines measurements of the random motion of probe particles with independent micromechanical measurements of the cytoplasm to quantify the spectrum of force fluctuations. Using FSM, we show that force fluctuations substantially enhance intracellular movement of small and large components. The fluctuations are three times larger in malignant cells than in their benign counterparts. We further demonstrate that vimentin acts globally to anchor organelles against randomly fluctuating forces in the cytoplasm, with no effect on their magnitude. Thus, FSM has broad applications for understanding the cytoplasm and its intracellular processes in relation to cell physiology in healthy and diseased states.

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07/31/14 | ER stress-induced clearance of misfolded GPI-anchored proteins via the secretory pathway.
Satpute-Krishnan P, Ajinkya M, Bhat S, Itakura E, Hegde RS, Lippincott-Schwartz J
Cell. 2014 Jul 31;158(3):522-33. doi: 10.1016/j.cell.2014.06.026

Proteins destined for the cell surface are first assessed in the endoplasmic reticulum (ER) for proper folding before release into the secretory pathway. This ensures that defective proteins are normally prevented from entering the extracellular environment, where they could be disruptive. Here, we report that, when ER folding capacity is saturated during stress, misfolded glycosylphosphatidylinositol-anchored proteins dissociate from resident ER chaperones, engage export receptors, and quantitatively leave the ER via vesicular transport to the Golgi. Clearance from the ER commences within minutes of acute ER stress, before the transcriptional component of the unfolded protein response is activated. These aberrant proteins then access the cell surface transiently before destruction in lysosomes. Inhibiting this stress-induced pathway by depleting the ER-export receptors leads to aggregation of the ER-retained misfolded protein. Thus, this rapid response alleviates the elevated burden of misfolded proteins in the ER at the onset of ER stress, promoting protein homeostasis in the ER.

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07/01/14 | MicroRNA binding to the HIV-1 Gag protein inhibits Gag assembly and virus production.
Chen AK, Sengupta P, Waki K, Van Engelenburg SB, Ochiya T, Ablan SD, Freed EO, Lippincott-Schwartz J
Proceedings of the National Academy of Sciences of the United States of America. 2014 Jul 1;111(26):E2676-83. doi: 10.1073/pnas.1408037111

MicroRNAs (miRNAs) are small, 18-22 nt long, noncoding RNAs that act as potent negative gene regulators in a variety of physiological and pathological processes. To repress gene expression, miRNAs are packaged into RNA-induced silencing complexes (RISCs) that target mRNAs for degradation and/or translational repression in a sequence-specific manner. Recently, miRNAs have been shown to also interact with proteins outside RISCs, impacting cellular processes through mechanisms not involving gene silencing. Here, we define a previously unappreciated activity of miRNAs in inhibiting RNA-protein interactions that in the context of HIV-1 biology blocks HIV virus budding and reduces virus infectivity. This occurs by miRNA binding to the nucleocapsid domain of the Gag protein, the main structural component of HIV-1 virions. The resulting miRNA-Gag complexes interfere with viral-RNA-mediated Gag assembly and viral budding at the plasma membrane, with imperfectly assembled Gag complexes endocytosed and delivered to lysosomes. The blockade of virus production by miRNA is reversed by adding the miRNA's target mRNA and stimulated by depleting Argonaute-2, suggesting that when miRNAs are not mediating gene silencing, they can block HIV-1 production through disruption of Gag assembly on membranes. Overall, our findings have significant implications for understanding how cells modulate HIV-1 infection by miRNA expression and raise the possibility that miRNAs can function to disrupt RNA-mediated protein assembly processes in other cellular contexts.

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01/01/15 | Photocontrollable fluorescent proteins for superresolution imaging.
Shcherbakova DM, Sengupta P, Lippincott-Schwartz J, Verkhusha VV
Annual review of biophysics. 2014;43:303-29. doi: 10.1146/annurev-biophys-051013-022836

Superresolution fluorescence microscopy permits the study of biological processes at scales small enough to visualize fine subcellular structures that are unresolvable by traditional diffraction-limited light microscopy. Many superresolution techniques, including those applicable to live cell imaging, utilize genetically encoded photocontrollable fluorescent proteins. The fluorescence of these proteins can be controlled by light of specific wavelengths. In this review, we discuss the biochemical and photophysical properties of photocontrollable fluorescent proteins that are relevant to their use in superresolution microscopy. We then describe the recently developed photoactivatable, photoswitchable, and reversibly photoswitchable fluorescent proteins, and we detail their particular usefulness in single-molecule localization-based and nonlinear ensemble-based superresolution techniques. Finally, we discuss recent applications of photocontrollable proteins in superresolution imaging, as well as how these applications help to clarify properties of intracellular structures and processes that are relevant to cell and developmental biology, neuroscience, cancer biology and biomedicine.

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04/14/14 | A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells.
Burnette DT, Shao L, Ott C, Pasapera AM, Fischer RS, Baird MA, Der Loughian C, Delanoe-Ayari H, Paszek MJ, Davidson MW, Betzig E, Lippincott-Schwartz J
Journal of Cell Biology. 2014 Apr 14;205(1):83-96. doi: 10.1083/jcb.201311104

How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers' attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.

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03/18/14 | LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes.
Homolya L, Fu D, Sengupta P, Jarnik M, Gillet JP, Vitale-Cross L, Gutkind S, Lippincott-Schwartz J, Arias IM
PloS one. 2014;9(3):e91921. doi: 10.1371/journal.pone.0091921

Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation.

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01/16/14 | Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits.
Van Engelenburg SB, Shtengel G, Sengupta P, Waki K, Jarnik M, Ablan SD, Freed EO, Hess HF, Lippincott-Schwartz J
Science. 2014 Jan 16;343(6171):653-6. doi: 10.1126/science.1247786

The human immunodeficiency virus (HIV) hijacks the endosomal sorting complexes required for transport (ESCRT) to mediate virus release from infected cells. The nanoscale organization of ESCRT machinery necessary for mediating viral abscission is unclear. Here, we applied three-dimensional superresolution microscopy and correlative electron microscopy to delineate the organization of ESCRT components at HIV assembly sites. We observed ESCRT subunits localized within the head of budding virions and released particles, with head-localized levels of CHMP2A decreasing relative to Tsg101 and CHMP4B upon virus abscission. Thus, the driving force for HIV release may derive from initial scaffolding of ESCRT subunits within the viral bud interior followed by plasma membrane association and selective remodeling of ESCRT subunits.

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01/13/14 | Superresolution imaging of biological systems using photoactivated localization microscopy.
Sengupta P, Van Engelenburg SB, Lippincott-Schwartz J
Chemical reviews. 2014 Mar 26;114(6):3189-202. doi: 10.1021/cr400614m
09/24/13 | Superresolution imaging with standard fluorescent probes.
Millis BA, Burnette DT, Lippincott-Schwartz J, Kachar B
Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]. 2013;60:Unit 21.8.. doi: 10.1002/0471143030.cb2108s60

For more than 100 years, the ultimate resolution of a light microscope (∼ 200 nm) has been constrained by the fundamental physical phenomenon of diffraction, as described by Ernst Abbe in 1873. While this limitation is just as applicable to today's light microscopes, it is the combination of high-end optics, clever methods of sample illumination, and computational techniques that has enabled researchers to access information at an order of magnitude greater resolution than once thought possible. This combination, broadly termed superresolution microscopy, has been increasingly practical for many labs to implement from both a hardware and software standpoint, but, as with many cutting-edge techniques, it also comes with limitations. One of the current drawbacks to superresolution microscopy is the limited number of probes and conditions that have been suitable for imaging. Here, a technique termed bleaching/blinking-assisted localization microscopy (BaLM) makes use of the inherent blinking and bleaching properties of almost all fluorophores as a means to generate superresolution images.

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12/05/13 | Incisive imaging and computation for cellular mysteries: lessons from abscission.
Elia N, Ott C, Lippincott-Schwartz J
Cell. 2013 Dec 5;155(6):1220-31. doi: 10.1016/j.cell.2013.11.011

The final cleavage event that terminates cell division, abscission of the small, dense intercellular bridge, has been particularly challenging to resolve. Here, we describe imaging innovations that helped answer long-standing questions about the mechanism of abscission. We further explain how computational modeling of high-resolution data was employed to test hypotheses and generate additional insights. We present the model that emerges from application of these complimentary approaches. Similar experimental strategies will undoubtedly reveal exciting details about other underresolved cellular structures.

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11/08/14 | Photohighlighting approaches to access membrane dynamics of the Golgi apparatus.
Sengupta P, Lippincott-Schwartz J
Methods in cell biology. 2013;118:217-34. doi: 10.1016/B978-0-12-417164-0.00013-6

By providing quantitative, visual data of live cells, fluorescent protein-based microscopy techniques are furnishing novel insights into the complexities of membrane trafficking pathways and organelle dynamics. In this chapter, we describe experimental protocols employing fluorescent protein-based photohighlighting techniques to quantify protein movement into and out of the Golgi apparatus, an organelle that serves as the central sorting and processing station of the secretory pathway. The methods allow kinetic characteristics of Golgi-associated protein trafficking to be deciphered, which can help clarify how the Golgi maintains itself as a steady-state structure despite a continuous flux of secretory cargo passing into and out of this organelle. The guidelines presented in this chapter can also be applied to examine the dynamics of other intracellular organelle systems, elucidating mechanisms for how proteins are maintained in specific organelles and/or circulated to other destinations within the cell.

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12/09/13 | Increased mitochondrial fusion and autophagy help isolated hepatocytes repolarize in collagen sandwich cultures.
Fu D, Lippincott-Schwartz J, Arias IM
Autophagy. 2013 Dec;9(12):2154-5. doi: 10.4161/auto.26167

Freshly isolated, depolarized rat hepatocytes can repolarize into bile canalicular networks when plated in collagen sandwich cultures. We studied the events underlying this repolarization process, focusing on how hepatocytes restore ATP synthesis and resupply biosynthetic precursors after the stress of being isolated from liver. We found that soon after being plated in collagen sandwich cultures, hepatocytes converted their mitochondria into highly fused networks. This occurred through a combination of upregulation of mitochondrial fusion proteins and downregulation of a mitochondrial fission protein. Mitochondria also became more active for oxidative phosphorylation, leading to overall increased ATP levels within cells. We further observed that autophagy was upregulated in the repolarizing hepatocytes. Boosted autophagy levels likely served to recycle cellular precursors, supplying building blocks for repolarization. Repolarizing hepatocytes also extensively degraded lipid droplets, whose fatty acids provide precursors for ?-oxidation to fuel oxidative phosphorylation in mitochondria. Thus, through coordination of mitochondrial fusion, autophagy, and lipid droplet consumption, depolarized hepatocytes are able to boost ATP synthesis and biosynthetic precursors to efficiently repolarize in collagen sandwich cultures.

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10/29/13 | Fast structural responses of gap junction membrane domains to AB5 toxins.
Majoul IV, Gao L, Betzig E, Onichtchouk D, Butkevich E, Kozlov Y, Bukauskas F, Bennett MVL, Lippincott-Schwartz J, Duden R
Proceedings of the National Academy of Sciences of the United States of America. 2013 Oct 29;110(44):E4125-33. doi: 10.1073/pnas.1315850110

Gap junctions (GJs) represent connexin-rich membrane domains that connect interiors of adjoining cells in mammalian tissues. How fast GJs can respond to bacterial pathogens has not been known previously. Using Bessel beam plane illumination and confocal spinning disk microscopy, we found fast ( 500 ms) formation of connexin-depleted regions (CDRs) inside GJ plaques between cells exposed to AB5 toxins. CDR formation appears as a fast redistribution of connexin channels within GJ plaques with minor changes in outline or geometry. CDR formation does not depend on membrane trafficking or submembrane cytoskeleton and has no effect on GJ conductance. However, CDR responses depend on membrane lipids, can be modified by cholesterol-clustering agents and extracellular K(+) ion concentration, and influence cAMP signaling. The CDR response of GJ plaques to bacterial toxins is a phenomenon observed for all tested connexin isoforms. Through signaling, the CDR response may enable cells to sense exposure to AB5 toxins. CDR formation may reflect lipid-phase separation events in the biological membrane of the GJ plaque, leading to increased connexin packing and lipid reorganization. Our data demonstrate very fast dynamics (in the millisecond-to-second range) within GJ plaques, which previously were considered to be relatively stable, long-lived structures.

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09/12/13 | Insulin triggers surface-directed trafficking of sequestered GLUT4 storage vesicles marked by Rab10.
Chen Y, Lippincott-Schwartz J
Small GTPases. 2013 Jul-Sep;4(3):193-7. doi: 10.4161/sgtp.26471

Understanding how glucose transporter isoform 4 (GLUT4) redistributes to the plasma membrane during insulin stimulation is a major goal of glucose transporter research. GLUT4 molecules normally reside in numerous intracellular compartments, including specialized storage vesicles and early/recycling endosomes. It is unclear how these diverse compartments respond to insulin stimulation to deliver GLUT4 molecules to the plasma membrane. For example, do they fuse with each other first or remain as separate compartments with different trafficking characteristics? Our recent live cell imaging studies are helping to clarify these issues. Using Rab proteins as specific markers to distinguish between storage vesicles and endosomes containing GLUT4, we demonstrate that it is primarily internal GLUT4 storage vesicles (GSVs) marked by Rab10 that approach and fuse at the plasma membrane and GSVs don't interact with endosomes on their way to the plasma membrane. These new findings add strong support to the model that GSV release from intracellular retention plays a major role in supplying GLUT4 molecules onto the PM under insulin stimulation.

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05/01/13 | Rab10 delivers GLUT4 storage vesicles to the plasma membrane.
Chen Y, Lippincott-Schwartz J
Communicative & integrative biology. 2013 May 1;6(3):e23779. doi: 10.4161/cib.23779

The glucose transporter, GLUT4, redistributes to the plasma membrane (PM) upon insulin stimulation, but also recycles through endosomal compartments. Different Rab proteins control these transport itineraries of GLUT4. However, the specific roles played by different Rab proteins in GLUT4 trafficking has been difficult to assess, primarily due to the complexity of endomembrane organization and trafficking. To address this problem, we recently performed advanced live cell imaging using total internal reflection fluorescence (TIRF) microscopy, which images objects ~150 nm from the PM, directly visualizing GLUT4 trafficking in response to insulin stimulation. Using IRAP-pHluorin to selectively label GSVs undergoing PM fusion in response to insulin, we identified Rab10 as the only Rab protein that binds this compartment. Rab14 was found to label transferrin-positive, endosomal compartments containing GLUT4. These also could fuse with the PM in response to insulin, albeit more slowly. Several other Rab proteins, including Rab4A, 4B and 8A, were found to mediate GLUT4 intra-endosomal recycling, serving to internalize surface-bound GLUT4 into endosomal compartments for ultimate delivery to GSVs. Thus, multiple Rab proteins regulate the circulation of GLUT4 molecules within the endomembrane system, maintaining optimal insulin responsiveness within cells.

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04/30/13 | Coordinated elevation of mitochondrial oxidative phosphorylation and autophagy help drive hepatocyte polarization.
Fu D, Mitra K, Sengupta P, Jarnik M, Lippincott-Schwartz J, Arias IM
Proceedings of the National Academy of Sciences of the United States of America. 2013 Apr 30;110(18):7288-93. doi: 10.1073/pnas.1304285110

Cell polarization requires increased cellular energy and metabolic output, but how these energetic demands are met by polarizing cells is unclear. To address these issues, we investigated the roles of mitochondrial bioenergetics and autophagy during cell polarization of hepatocytes cultured in a collagen sandwich system. We found that as the hepatocytes begin to polarize, they use oxidative phosphorylation to raise their ATP levels, and this energy production is required for polarization. After the cells are polarized, the hepatocytes shift to become more dependent on glycolysis to produce ATP. Along with this central reliance on oxidative phosphorylation as the main source of ATP production in polarizing cultures, several other metabolic processes are reprogrammed during the time course of polarization. As the cells polarize, mitochondria elongate and mitochondrial membrane potential increases. In addition, lipid droplet abundance decreases over time. These findings suggest that polarizing cells are reliant on fatty acid oxidation, which is supported by pharmacologic inhibition of β-oxidation by etomoxir. Finally, autophagy is up-regulated during cell polarization, with inhibition of autophagy retarding cell polarization. Taken together, our results describe a metabolic shift involving a number of coordinated metabolic pathways that ultimately serve to increase energy production during cell polarization.

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03/26/13 | Nanotools for neuroscience and brain activity mapping.
Alivisatos P, Andrews AM, Boyden ES, Chun M, Church GM, Deisseroth K, Donoghue JP, Fraser SE, Lippincott-Schwartz J, Looger LL, Masmanidis S, McEuen PL, Nurmikko AV, Park H, Peterka DS, Reid C, Roukes ML, Scherer A, Schnitzer M, Sejnowski TJ, Shepard KL, Tsao D, Turrigiano G, Weiss PS, Xu C, Yuste R, Zhuang X
ACS Nano. 2013 Mar 26;7(3):1850-66. doi: 10.1021/nn4012847

Neuroscience is at a crossroads. Great effort is being invested into deciphering specific neural interactions and circuits. At the same time, there exist few general theories or principles that explain brain function. We attribute this disparity, in part, to limitations in current methodologies. Traditional neurophysiological approaches record the activities of one neuron or a few neurons at a time. Neurochemical approaches focus on single neurotransmitters. Yet, there is an increasing realization that neural circuits operate at emergent levels, where the interactions between hundreds or thousands of neurons, utilizing multiple chemical transmitters, generate functional states. Brains function at the nanoscale, so tools to study brains must ultimately operate at this scale, as well. Nanoscience and nanotechnology are poised to provide a rich toolkit of novel methods to explore brain function by enabling simultaneous measurement and manipulation of activity of thousands or even millions of neurons. We and others refer to this goal as the Brain Activity Mapping Project. In this Nano Focus, we discuss how recent developments in nanoscale analysis tools and in the design and synthesis of nanomaterials have generated optical, electrical, and chemical methods that can readily be adapted for use in neuroscience. These approaches represent exciting areas of technical development and research. Moreover, unique opportunities exist for nanoscientists, nanotechnologists, and other physical scientists and engineers to contribute to tackling the challenging problems involved in understanding the fundamentals of brain function.

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02/10/13 | Accelerating 3B single-molecule super-resolution microscopy with cloud computing.
Hu YS, Nan X, Sengupta P, Lippincott-Schwartz J, Cang H
Nature methods. 2013 Feb;10(2):96-7. doi: 10.1038/nmeth.2335
02/08/13 | Quantifying spatial organization in point-localization superresolution images using pair correlation analysis.
Sengupta P, Jovanovic-Talisman T, Lippincott-Schwartz J
Nature protocols. 2013 Feb;8(2):345-54. doi: 10.1038/nprot.2013.005

The distinctive distributions of proteins within subcellular compartments both at steady state and during signaling events have essential roles in cell function. Here we describe a method for delineating the complex arrangement of proteins within subcellular structures visualized using point-localization superresolution (PL-SR) imaging. The approach, called pair correlation photoactivated localization microscopy (PC-PALM), uses a pair-correlation algorithm to precisely identify single molecules in PL-SR imaging data sets, and it is used to decipher quantitative features of protein organization within subcellular compartments, including the existence of protein clusters and the size, density and number of proteins in these clusters. We provide a step-by-step protocol for PC-PALM, illustrating its analysis capability for four plasma membrane proteins tagged with photoactivatable GFP (PAGFP). The experimental steps for PC-PALM can be carried out in 3 d and the analysis can be done in ∼6-8 h. Researchers need to have substantial experience in single-molecule imaging and statistical analysis to conduct the experiments and carry out this analysis.

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02/15/13 | NBR1 acts as an autophagy receptor for peroxisomes.
Deosaran E, Larsen KB, Hua R, Sargent G, Wang Y, Kim S, Lamark T, Jauregui M, Law K, Lippincott-Schwartz J, Brech A, Johansen T, Kim PK
Journal of cell science. 2013 Feb 15;126(Pt 4):939-52. doi: 10.1242/jcs.114819

Selective macro-autophagy is an intracellular process by which large cytoplasmic materials are selectively sequestered and degraded in the lysosomes. Substrate selection is mediated by ubiquitylation and recruitment of ubiquitin-binding autophagic receptors such as p62, NBR1, NDP52 and Optineurin. Although it has been shown that these receptors act cooperatively to target some types of substrates to nascent autophagosomes, their precise roles are not well understood. We examined selective autophagic degradation of peroxisomes (pexophagy), and found that NBR1 is necessary and sufficient for pexophagy. Mutagenesis studies of NBR1 showed that the amphipathic α-helical J domain, the ubiquitin-associated (UBA) domain, the LC3-interacting region and the coiled-coil domain are necessary to mediate pexophagy. Strikingly, substrate selectivity is partly achieved by NBR1 itself by coincident binding of the J and UBA domains to peroxisomes. Although p62 is not required when NBR1 is in excess, its binding to NBR1 increases the efficiency of NBR1-mediated pexophagy. Together, these results suggest that NBR1 is the specific autophagy receptor for pexophagy.

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12/11/12 | Visualizing cell structure and function with point-localization superresolution imaging.
Sengupta P, van Engelenburg S, Lippincott-Schwartz J
Developmental cell. 2012 Dec 11;23(6):1092-102. doi: 10.1016/j.devcel.2012.09.022

Fundamental to the success of cell and developmental biology is the ability to tease apart molecular organization in cells and tissues by localizing specific proteins with respect to one another in a native cellular context. However, many key cellular structures (from mitochondrial cristae to nuclear pores) lie below the diffraction limit of visible light, precluding analysis of their organization by conventional approaches. Point-localization superresolution microscopy techniques, such as PALM and STORM, are poised to resolve, with unprecedented clarity, the organizational principles of macromolecular complexes within cells, thus leading to deeper insights into cellular function in both health and disease.

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12/01/12 | Visualization of live primary cilia dynamics using fluorescence microscopy.
Ott C, Lippincott-Schwartz J
Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]. 2012 Dec;Chapter 4:Unit 4.26. doi: 10.1002/0471143030.cb0426s57

Methods useful for exploring the formation and functions of primary cilia in living cells are described here. First, multiple protocols for visualizing solitary cilia that extend away from the cell body are described. Primary cilia collect, synthesize, and transmit information about the extracellular space into the cell body to promote critical cellular responses. Problems with cilia formation or function can lead to dramatic changes in cell physiology. These methods can be used to assess cilia formation and length, the location of the cilium relative to other cellular structures, and localization of specific proteins to the cilium. The subsequent protocols describe how to quantify movement of fluorescent molecules within the cilium using kymographs, photobleaching, and photoconversion. The microtubules that form the structural scaffold of the cilium are also critical avenues for kinesin and dynein-mediated movement of proteins within the cilium. Assessing intraflagellar dynamics can provide insight into mechanisms of ciliary-mediated signal perception and transmission.

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10/30/12 | Plasticity of the asialoglycoprotein receptor deciphered by ensemble FRET imaging and single-molecule counting PALM imaging.
Renz M, Daniels BR, Vámosi G, Arias IM, Lippincott-Schwartz J
Proceedings of the National Academy of Sciences of the United States of America. 2012 Oct 30;109(44):E2989-97. doi: 10.1073/pnas.1211753109

The stoichiometry and composition of membrane protein receptors are critical to their function. However, the inability to assess receptor subunit stoichiometry in situ has hampered efforts to relate receptor structures to functional states. Here, we address this problem for the asialoglycoprotein receptor using ensemble FRET imaging, analytical modeling, and single-molecule counting with photoactivated localization microscopy (PALM). We show that the two subunits of asialoglycoprotein receptor [rat hepatic lectin 1 (RHL1) and RHL2] can assemble into both homo- and hetero-oligomeric complexes, displaying three forms with distinct ligand specificities that coexist on the plasma membrane: higher-order homo-oligomers of RHL1, higher-order hetero-oligomers of RHL1 and RHL2 with two-to-one stoichiometry, and the homo-dimer RHL2 with little tendency to further homo-oligomerize. Levels of these complexes can be modulated in the plasma membrane by exogenous ligands. Thus, even a simple two-subunit receptor can exhibit remarkable plasticity in structure, and consequently function, underscoring the importance of deciphering oligomerization in single cells at the single-molecule level.

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08/20/12 | Rab10 and myosin-Va mediate insulin-stimulated GLUT4 storage vesicle translocation in adipocytes.
Chen Y, Wang Y, Zhang J, Deng Y, Jiang L, Song E, Wu XS, Hammer JA, Xu T, Lippincott-Schwartz J
The Journal of cell biology. 2012 Aug 20;198(4):545-60. doi: 10.1083/jcb.201111091

Rab proteins are important regulators of insulin-stimulated GLUT4 translocation to the plasma membrane (PM), but the precise steps in GLUT4 trafficking modulated by particular Rab proteins remain unclear. Here, we systematically investigate the involvement of Rab proteins in GLUT4 trafficking, focusing on Rab proteins directly mediating GLUT4 storage vesicle (GSV) delivery to the PM. Using dual-color total internal reflection fluorescence (TIRF) microscopy and an insulin-responsive aminopeptidase (IRAP)-pHluorin fusion assay, we demonstrated that Rab10 directly facilitated GSV translocation to and docking at the PM. Rab14 mediated GLUT4 delivery to the PM via endosomal compartments containing transferrin receptor (TfR), whereas Rab4A, Rab4B, and Rab8A recycled GLUT4 through the endosomal system. Myosin-Va associated with GSVs by interacting with Rab10, positioning peripherally recruited GSVs for ultimate fusion. Thus, multiple Rab proteins regulate the trafficking of GLUT4, with Rab10 coordinating with myosin-Va to mediate the final steps of insulin-stimulated GSV translocation to the PM.

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05/29/12 | Multiscale diffusion in the mitotic Drosophila melanogaster syncytial blastoderm.
Daniels BR, Rikhy R, Renz M, Dobrowsky TM, Lippincott-Schwartz J
Proceedings of the National Academy of Sciences of the United States of America. 2012 May 29;109(22):8588-93. doi: 10.1073/pnas.1204270109

Despite the fundamental importance of diffusion for embryonic morphogen gradient formation in the early Drosophila melanogaster embryo, there remains controversy regarding both the extent and the rate of diffusion of well-characterized morphogens. Furthermore, the recent observation of diffusional "compartmentalization" has suggested that diffusion may in fact be nonideal and mediated by an as-yet-unidentified mechanism. Here, we characterize the effects of the geometry of the early syncytial Drosophila embryo on the effective diffusivity of cytoplasmic proteins. Our results demonstrate that the presence of transient mitotic membrane furrows results in a multiscale diffusion effect that has a significant impact on effective diffusion rates across the embryo. Using a combination of live-cell experiments and computational modeling, we characterize these effects and relate effective bulk diffusion rates to instantaneous diffusion coefficients throughout the syncytial blastoderm nuclear cycle phase of the early embryo. This multiscale effect may be related to the effect of interphase nuclei on effective diffusion, and thus we propose that an as-yet-unidentified role of syncytial membrane furrows is to temporally regulate bulk embryonic diffusion rates to balance the multiscale effect of interphase nuclei, which ultimately stabilizes the shapes of various morphogen gradients.

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05/16/12 | Computational model of cytokinetic abscission driven by ESCRT-III polymerization and remodeling.
Elia N, Fabrikant G, Kozlov MM, Lippincott-Schwartz J
Biophysical journal. 2012 May 16;102(10):2309-20. doi: 10.1016/j.bpj.2012.04.007

The endosomal sorting complex required for transport (ESCRT)-III complex, capable of polymerization and remodeling, participates in abscission of the intercellular membrane bridge connecting two daughter cells at the end of cytokinesis. Here, we integrate quantitative imaging of ESCRT-III during cytokinetic abscission with biophysical properties of ESCRT-III complexes to formulate and test a computational model for ESCRT-mediated cytokinetic abscission. We propose that cytokinetic abscission is driven by an ESCRT-III fission complex, which arises from ESCRT-III polymerization at the edge of the cytokinetic midbody structure, located at the center of the intercellular bridge. Formation of the fission complex is completed by remodeling and breakage of the ESCRT-III polymer assisted by VPS4. Subsequent spontaneous constriction of the fission complex generates bending deformation of the intercellular bridge membrane. The related membrane elastic force propels the fission complex along the intercellular bridge away from the midbody until it reaches an equilibrium position, determining the scission site. Membrane attachment to the dome-like end-cap of the fission complex drives membrane fission, completing the abscission process. We substantiate the model by theoretical analysis of the membrane elastic energy and by experimental verification of the major model assumptions.

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05/14/12 | DRP1-dependent mitochondrial fission initiates follicle cell differentiation during Drosophila oogenesis.
Mitra K, Rikhy R, Lilly M, Lippincott-Schwartz J
The Journal of cell biology. 2012 May 14;197(4):487-97. doi: 10.1083/jcb.201110058

Exit from the cell cycle is essential for cells to initiate a terminal differentiation program during development, but what controls this transition is incompletely understood. In this paper, we demonstrate a regulatory link between mitochondrial fission activity and cell cycle exit in follicle cell layer development during Drosophila melanogaster oogenesis. Posterior-localized clonal cells in the follicle cell layer of developing ovarioles with down-regulated expression of the major mitochondrial fission protein DRP1 had mitochondrial elements extensively fused instead of being dispersed. These cells did not exit the cell cycle. Instead, they excessively proliferated, failed to activate Notch for differentiation, and exhibited downstream developmental defects. Reintroduction of mitochondrial fission activity or inhibition of the mitochondrial fusion protein Marf-1 in posterior-localized DRP1-null clones reversed the block in Notch-dependent differentiation. When DRP1-driven mitochondrial fission activity was unopposed by fusion activity in Marf-1-depleted clones, premature cell differentiation of follicle cells occurred in mitotic stages. Thus, DRP1-dependent mitochondrial fission activity is a novel regulator of the onset of follicle cell differentiation during Drosophila oogenesis.

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03/23/12 | Quantitative analysis of photoactivated localization microscopy (PALM) datasets using pair-correlation analysis.
Sengupta P, Lippincott-Schwartz J
BioEssays : news and reviews in molecular, cellular and developmental biology. 2012 May;34(5):396-405. doi: 10.1002/bies.201200022

Pointillistic based super-resolution techniques, such as photoactivated localization microscopy (PALM), involve multiple cycles of sequential activation, imaging, and precise localization of single fluorescent molecules. A super-resolution image, having nanoscopic structural information, is then constructed by compiling all the image sequences. Because the final image resolution is determined by the localization precision of detected single molecules and their density, accurate image reconstruction requires imaging of biological structures labeled with fluorescent molecules at high density. In such image datasets, stochastic variations in photon emission and intervening dark states lead to uncertainties in identification of single molecules. This, in turn, prevents the proper utilization of the wealth of information on molecular distribution and quantity. A recent strategy for overcoming this problem is pair-correlation analysis applied to PALM. Using rigorous statistical algorithms to estimate the number of detected proteins, this approach allows the spatial organization of molecules to be quantitatively described.

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02/23/12 | Bone marrow homing and engraftment of human hematopoietic stem and progenitor cells is mediated by a polarized membrane domain.
Larochelle A, Gillette JM, Desmond R, Ichwan B, Cantilena A, Cerf A, Barrett J, Wayne AS, Lippincott-Schwartz J, Dunbar CE
Blood. 2012 Feb 23;119(8):1848-55. doi: 10.1182/blood-2011-08-371583

Manipulation of hematopoietic stem/progenitor cells (HSPCs) ex vivo is of clinical importance for stem cell expansion and gene therapy applications. However, most cultured HSPCs are actively cycling, and show a homing and engraftment defect compared with the predominantly quiescent noncultured HSPCs. We previously showed that HSPCs make contact with osteoblasts in vitro via a polarized membrane domain enriched in adhesion molecules such as tetraspanins. Here we show that increased cell cycling during ex vivo culture of HSPCs resulted in disruption of this membrane domain, as evidenced by disruption of polarity of the tetraspanin CD82. Chemical disruption or antibody-mediated blocking of CD82 on noncultured HSPCs resulted in decreased stromal cell adhesion, homing, and engraftment in nonobese diabetic/severe combined immunodeficiency IL-2γ(null) (NSG) mice compared with HSPCs with an intact domain. Most leukemic blasts were actively cycling and correspondingly displayed a loss of domain polarity and decreased homing in NSG mice compared with normal HSPCs. We conclude that quiescent cells, unlike actively cycling cells, display a polarized membrane domain enriched in tetraspanins that mediates homing and engraftment, providing a mechanistic explanation for the homing/engraftment defect of cycling cells and a potential new therapeutic target to enhance engraftment.

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04/19/12 | Intracellular spatial localization regulated by the microtubule network.
Chen J, Lippincott-Schwartz J, Liu J
PloS one. 2012;7(4):e34919. doi: 10.1371/journal.pone.0034919

The commonly recognized mechanisms for spatial regulation inside the cell are membrane-bounded compartmentalization and biochemical association with subcellular organelles. We use computational modeling to investigate another spatial regulation mechanism mediated by the microtubule network in the cell. Our results demonstrate that the mitotic spindle can impose strong sequestration and concentration effects on molecules with binding affinity for microtubules, especially dynein-directed cargoes. The model can recapitulate the essence of three experimental observations on distinct microtubule network morphologies: the sequestration of germ plasm components by the mitotic spindles in the Drosophila syncytial embryo, the asymmetric cell division initiated by the time delay in centrosome maturation in the Drosophila neuroblast, and the diffusional block between neighboring energids in the Drosophila syncytial embryo. Our model thus suggests that the cell cycle-dependent changes in the microtubule network are critical for achieving different spatial regulation effects. The microtubule network provides a spatially extensive docking platform for molecules and gives rise to a "structured cytoplasm", in contrast to a free and fluid environment.

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11/01/11 | Cellular mechanism of bile acid-accelerated hepatocyte polarity.
Fu D, Lippincott-Schwartz J, Arias IM
Small GTPases. 2011 Nov 1;2(6):314-317. doi: 10.4161/sgtp.18087

We recently discovered that the major mammalian bile acid, taurocholate, accelerated polarity in primary rat hepatocytes. Taurocholate increased cellular cAMP and signals through an Epac-Rap1-MEK-LKB1-AMPK pathway for its polarity effect. This review discusses possible mechanisms for how taurocholate affects different cell polarity factors, particularly AMPK, and thereby regulates events that generate polarity. These include tight junction formation, apical trafficking, recycling endosome dynamics, and cytoskeleton rearrangement. We also discuss whether the effects of taurocholate are mediated by other LKB1 downstream kinases, such as Par1 and NUAK1.

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11/01/11 | Fuse or die: Shaping mitochondrial fate during starvation.
Rambold AS, Kostelecky B, Lippincott-Schwartz J
Communicative & integrative biology. 2011 Nov 1;4(6):752-4

Mitochondria continuously change their shape and thereby influence different cellular processes like cell death or development. Recently, we showed that during starvation mitochondria fuse into a highly connected network. The change in mitochondrial shape was dependent on inactivation of the fission protein Drp1, through targeting of two different phosphorylation sites. This rapid inhibition of mitochondrial fission led to unopposed fusion, protecting mitochondria from starvation-induced degradation and enabling the cell to survive nutrient scarce conditions.

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12/27/11 | Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules.
Burnette DT, Sengupta P, Dai Y, Lippincott-Schwartz J, Kachar B
Proceedings of the National Academy of Sciences of the United States of America. 2011 Dec 27;108(52):21081-6. doi: 10.1073/pnas.1117430109

Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.

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12/04/11 | Bayesian localization microscopy reveals nanoscale podosome dynamics.
Cox S, Rosten E, Monypenny J, Jovanovic-Talisman T, Burnette DT, Lippincott-Schwartz J, Jones GE, Heintzmann R
Nature methods. 2012 Feb;9(2):195-200. doi: 10.1038/nmeth.1812

We describe a localization microscopy analysis method that is able to extract results in live cells using standard fluorescent proteins and xenon arc lamp illumination. Our Bayesian analysis of the blinking and bleaching (3B analysis) method models the entire dataset simultaneously as being generated by a number of fluorophores that may or may not be emitting light at any given time. The resulting technique allows many overlapping fluorophores in each frame and unifies the analysis of the localization from blinking and bleaching events. By modeling the entire dataset, we were able to use each reappearance of a fluorophore to improve the localization accuracy. The high performance of this technique allowed us to reveal the nanoscale dynamics of podosome formation and dissociation throughout an entire cell with a resolution of 50 nm on a 4-s timescale.

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12/01/11 | Mechanisms of mitochondria and autophagy crosstalk.
Rambold AS, Lippincott-Schwartz J
Cell cycle (Georgetown, Tex.). 2011 Dec 1;10(23):4032-8. doi: 10.4161/cc.10.23.18384

Autophagy is a cellular survival pathway that recycles intracellular components to compensate for nutrient depletion and ensures the appropriate degradation of organelles. Mitochondrial number and health are regulated by mitophagy, a process by which excessive or damaged mitochondria are subjected to autophagic degradation. Autophagy is thus a key determinant for mitochondrial health and proper cell function. Mitophagic malfunction has been recently proposed to contribute to progressive neuronal loss in Parkinson's disease. In addition to autophagy's significance in mitochondrial integrity, several lines of evidence suggest that mitochondria can also substantially influence the autophagic process. The mitochondria's ability to influence and be influenced by autophagy places both elements (mitochondria and autophagy) in a unique position where defects in one or the other system could increase the risk to various metabolic and autophagic related diseases.

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11/04/11 | Functional nanoscale organization of signaling molecules downstream of the T cell antigen receptor.
Sherman E, Barr V, Manley S, Patterson G, Balagopalan L, Akpan I, Regan CK, Merrill RK, Sommers CL, Lippincott-Schwartz J, Samelson LE
Immunity. 2011 Nov 23;35(5):705-20. doi: 10.1016/j.immuni.2011.10.004

Receptor-regulated cellular signaling often is mediated by formation of transient, heterogeneous protein complexes of undefined structure. We used single and two-color photoactivated localization microscopy to study complexes downstream of the T cell antigen receptor (TCR) in single-molecule detail at the plasma membrane of intact T cells. The kinase ZAP-70 distributed completely with the TCRζ chain and both partially mixed with the adaptor LAT in activated cells, thus showing localized activation of LAT by TCR-coupled ZAP-70. In resting and activated cells, LAT primarily resided in nanoscale clusters as small as dimers whose formation depended on protein-protein and protein-lipid interactions. Surprisingly, the adaptor SLP-76 localized to the periphery of LAT clusters. This nanoscale structure depended on polymerized actin and its disruption affected TCR-dependent cell function. These results extend our understanding of the mechanism of T cell activation and the formation and organization of TCR-mediated signaling complexes, findings also relevant to other receptor systems.

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11/22/11 | An evolving paradigm for the secretory pathway?
Lippincott-Schwartz J
Molecular biology of the cell. 2011 Nov;22(21):3929-32. doi: 10.1091/mbc.E11-05-0452

The paradigm that the secretory pathway consists of a stable endoplasmic reticulum and Golgi apparatus, using discrete transport vesicles to exchange their contents, gained important support from groundbreaking biochemical and genetic studies during the 1980s. However, the subsequent development of new imaging technologies with green fluorescent protein introduced data on dynamic processes not fully accounted for by the paradigm. As a result, we may be seeing an example of how a paradigm is evolving to account for the results of new technologies and their new ways of describing cellular processes.

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12/07/11 | Together we are stronger: fusion protects mitochondria from autophagosomal degradation.
Rambold AS, Kostelecky B, Lippincott-Schwartz J
Autophagy. 2011 Dec;7(12):1568-9

Starvation induces a protective process of self-cannibalization called autophagy that is thought to mediate nonselective degradation of cytoplasmic material. We recently reported that mitochondria escape autophagosomal degradation through extensive fusion into mitochondrial networks upon certain starvation conditions. The extent of mitochondrial elongation is dependent on the type of nutrient deprivation, with amino acid depletion having a particularly strong effect. Downregulation of the mitochondrial fission protein Drp1 was determined to be important in bringing about starvation-induced mitochondrial fusion. The formation of mitochondrial networks during nutrient depletion selectively blocked their autophagic degradation, presumably allowing cells to sustain efficient ATP production and thereby survive starvation.

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10/14/11 | Cell biology. SevERing mitochondria.
Rambold AS, Lippincott-Schwartz J
Science (New York, N.Y.). 2011 Oct 14;334(6053):186-7. doi: 10.1126/science.1214059
09/18/11 | Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis.
Sengupta P, Jovanovic-Talisman T, Skoko D, Renz M, Veatch SL, Lippincott-Schwartz J
Nature methods. 2011 Nov;8(11):969-75. doi: 10.1038/nmeth.1704

Photoactivated localization microscopy (PALM) is a powerful approach for investigating protein organization, yet tools for quantitative, spatial analysis of PALM datasets are largely missing. Combining pair-correlation analysis with PALM (PC-PALM), we provide a method to analyze complex patterns of protein organization across the plasma membrane without determination of absolute protein numbers. 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 demonstrate distinct nanoscale organization of plasma-membrane proteins with different membrane anchoring and lipid partitioning characteristics in COS-7 cells, and show dramatic changes in glycosylphosphatidylinositol (GPI)-anchored protein arrangement under varying perturbations. PC-PALM is thus an effective tool with broad applicability for analysis of protein heterogeneity and function, adaptable to other single-molecule strategies.

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07/19/11 | Bridging structure and process in developmental biology through new imaging technologies.
Lippincott-Schwartz J
Developmental cell. 2011 Jul 19;21(1):5-10. doi: 10.1016/j.devcel.2011.06.030

Many unexpected discoveries in developmental biology have depended on advancement of imaging technologies to visualize developmental processes as they unfold across multiple spatial and temporal scales. This essay surveys the recent advances in imaging, highlighting emerging capabilities with an eye toward those poised to have the greatest impact on developmental biology.

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07/21/11 | Tubular network formation protects mitochondria from autophagosomal degradation during nutrient starvation.
Rambold AS, Kostelecky B, Elia N, Lippincott-Schwartz J
Proceedings of the National Academy of Sciences of the United States of America. 2011 Jun 21;108(25):10190-5. doi: 10.1073/pnas.1107402108

Mitochondria are highly dynamic organelles that mediate essential cell functions such as apoptosis and cell-cycle control in addition to their role as efficient ATP generators. Mitochondrial morphology changes are tightly regulated, and their shape can shift between small, fragmented units and larger networks of elongated mitochondria. We demonstrate that mitochondrial elements become significantly elongated and interconnected shortly after nutrient depletion. This mitochondrial morphological shift depends on the type of starvation, with an additive effect observed when multiple nutrients are depleted simultaneously. We further show that starvation-induced mitochondrial elongation is mediated by down-regulation of dynamin-related protein 1 (Drp1) through modulation of two Drp1 phosphorylation sites, leading to unopposed mitochondrial fusion. Finally, we establish that mitochondrial tubulation upon nutrient deprivation protects mitochondria from autophagosomal degradation, which could permit mitochondria to maximize energy production and supply autophagosomal membranes during starvation.

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01/01/11 | Emerging in vivo analyses of cell function using fluorescence imaging (*).
Lippincott-Schwartz J
Annual review of biochemistry. 2011;80:327-32. doi: 10.1146/annurev-biochem-121010-125553

Understanding how cells of all types sense external and internal signals and how these signals are processed to yield particular responses is a major goal of biology. Genetically encoded fluorescent proteins (FPs) and fluorescent sensors are playing an important role in achieving this comprehensive knowledge base of cell function. Providing high sensitivity and immense versatility while being minimally perturbing to a biological specimen, the probes can be used in different microscopy techniques to visualize cellular processes on many spatial scales. Three review articles in this volume discuss recent advances in probe design and applications. These developments help expand the range of biochemical processes in living systems suitable for study. They provide researchers with exciting new tools to explore how cellular processes are organized and their activity regulated in vivo.

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04/13/11 | A role for actin arcs in the leading-edge advance of migrating cells.
Burnette DT, Manley S, Sengupta P, Sougrat R, Davidson MW, Kachar B, Lippincott-Schwartz J
Nature cell biology. 2011 Apr;13(4):371-81. doi: 10.1038/ncb2205

Epithelial cell migration requires coordination of two actin modules at the leading edge: one in the lamellipodium and one in the lamella. How the two modules connect mechanistically to regulate directed edge motion is not understood. Using live-cell imaging and photoactivation approaches, we demonstrate that the actin network of the lamellipodium evolves spatio-temporally into the lamella. This occurs during the retraction phase of edge motion, when myosin II redistributes to the lamellipodial actin and condenses it into an actin arc parallel to the edge. The new actin arc moves rearward, slowing down at focal adhesions in the lamella. We propose that net edge extension occurs by nascent focal adhesions advancing the site at which new actin arcs slow down and form the base of the next protrusion event. The actin arc thereby serves as a structural element underlying the temporal and spatial connection between the lamellipodium and the lamella during directed cell motion.

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03/22/11 | Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission.
Elia N, Sougrat R, Spurlin TA, Hurley JH, Lippincott-Schwartz J
Proceedings of the National Academy of Sciences of the United States of America. 2011 Mar 22;108(12):4846-51. doi: 10.1073/pnas.1102714108

The final stage of cytokinesis is abscission, the cutting of the narrow membrane bridge connecting two daughter cells. The endosomal sorting complex required for transport (ESCRT) machinery is required for cytokinesis, and ESCRT-III has membrane scission activity in vitro, but the role of ESCRTs in abscission has been undefined. Here, we use structured illumination microscopy and time-lapse imaging to dissect the behavior of ESCRTs during abscission. Our data reveal that the ESCRT-I subunit tumor-susceptibility gene 101 (TSG101) and the ESCRT-III subunit charged multivesicular body protein 4b (CHMP4B) are sequentially recruited to the center of the intercellular bridge, forming a series of cortical rings. Late in cytokinesis, however, CHMP4B is acutely recruited to the narrow constriction site where abscission occurs. The ESCRT disassembly factor vacuolar protein sorting 4 (VPS4) follows CHMP4B to this site, and cell separation occurs immediately. That arrival of ESCRT-III and VPS4 correlates both spatially and temporally with the abscission event suggests a direct role for these proteins in cytokinetic membrane abscission.

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02/15/11 | Principles and current strategies for targeting autophagy for cancer treatment.
Amaravadi RK, Lippincott-Schwartz J, Yin XM, Weiss WA, Takebe N, Timmer W, DiPaola RS, Lotze MT, White E
Clinical cancer research : an official journal of the American Association for Cancer Research. 2011 Feb 15;17(4):654-66. doi: 10.1158/1078-0432.CCR-10-2634

Autophagy is an evolutionarily conserved, intracellular self-defense mechanism in which organelles and proteins are sequestered into autophagic vesicles that are subsequently degraded through fusion with lysosomes. Cells, thereby, prevent the toxic accumulation of damaged or unnecessary components, but also recycle these components to sustain metabolic homoeostasis. Heightened autophagy is a mechanism of resistance for cancer cells faced with metabolic and therapeutic stress, revealing opportunities for exploitation as a therapeutic target in cancer. We summarize recent developments in the field of autophagy and cancer and build upon the results presented at the Cancer Therapy Evaluation Program (CTEP) Early Drug Development meeting in March 2010. Herein, we describe our current understanding of the core components of the autophagy machinery and the functional relevance of autophagy within the tumor microenvironment, and we outline how this knowledge has informed preclinical investigations combining the autophagy inhibitor hydroxychloroquine (HCQ) with chemotherapy, targeted therapy, and immunotherapy. Finally, we describe ongoing clinical trials involving HCQ as a first generation autophagy inhibitor, as well as strategies for the development of novel, more potent, and specific inhibitors of autophagy.

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01/25/11 | Bile acid stimulates hepatocyte polarization through a cAMP-Epac-MEK-LKB1-AMPK pathway.
Fu D, Wakabayashi Y, Lippincott-Schwartz J, Arias IM
Proceedings of the National Academy of Sciences of the United States of America. 2011 Jan 25;108(4):1403-8. doi: 10.1073/pnas.1018376108

This study describes a unique function of taurocholate in bile canalicular formation involving signaling through a cAMP-Epac-MEK-Rap1-LKB1-AMPK pathway. In rat hepatocyte sandwich cultures, polarization was manifested by sequential progression of bile canaliculi from small structures to a fully branched network. Taurocholate accelerated canalicular network formation and concomitantly increased cAMP, which were prevented by adenyl cyclase inhibitor. The cAMP-dependent PKA inhibitor did not prevent the taurocholate effect. In contrast, activation of Epac, another cAMP downstream kinase, accelerated canalicular network formation similar to the effect of taurocholate. Inhibition of Epac downstream targets, Rap1 and MEK, blocked the taurocholate effect. Taurocholate rapidly activated MEK, LKB1, and AMPK, which were prevented by inhibition of adenyl cyclase or MEK. Our previous study showed that activated-LKB1 and AMPK participate in canalicular network formation. Linkage between bile acid synthesis, hepatocyte polarization, and regulation of energy metabolism is likely important in normal hepatocyte development and disease.

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12/01/10 | Determining membrane protein topologies in single cells and high-throughput screening applications.
Wunder C, Lippincott-Schwartz J, Lorenz H
Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]. 2010 Dec;Chapter 5:Unit 5.7. doi: 10.1002/0471143030.cb0507s49

Correct localization and topology are crucial for a protein's cellular function. To determine topologies of membrane proteins, a new technique, called fluorescence protease protection (FPP) assay, has recently been established. The sole requirements for FPP are the expression of fluorescent-protein fusion proteins and the selective permeabilization of the plasma membrane, permitting a wide range of cell types and organelles to be investigated. Proteins topologies in organelles like endoplasmic reticulum, Golgi apparatus, mitochondria, peroxisomes, and autophagosomes have already been determined by FPP. Here, two different step-by-step protocols of the FPP assay are provided. First, we describe the FPP assay using fluorescence microscopy for single adherent cells, and second, we outline the FPP assay for high-throughput screening applications.

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11/01/10 | Imaging: visualizing the possibilities.
Lippincott-Schwartz J
Journal of cell science. 2010 Nov 1;123(Pt 21):3619-20. doi: 10.1242/jcs081539
10/01/10 | The long road: peering into live cells.
Lippincott-Schwartz J
Nature cell biology. 2010 Oct;12(10):918. doi: 10.1038/ncb1010-918
09/27/10 | Starved cells use mitochondria for autophagosome biogenesis.
Rambold AS, Lippincott-Schwartz J
Cell cycle (Georgetown, Tex.). 2010 Sep 15;9(18):3633-4. doi: 10.4161/cc.9.18.13170
10/01/10 | Regulation of bile canalicular network formation and maintenance by AMP-activated protein kinase and LKB1.
Fu D, Wakabayashi Y, Ido Y, Lippincott-Schwartz J, Arias IM
Journal of cell science. 2010 Oct 1;123(Pt 19):3294-302. doi: 10.1242/jcs.068098

AMP-activated protein kinase (AMPK), a cellular metabolic sensor, is essential in energy regulation and metabolism. Hepatocyte polarization during liver development and regeneration parallels increased metabolism. The current study investigates the effects of AMPK and its upstream activator LKB1 on polarity and bile canalicular network formation and maintenance in collagen sandwich cultures of rat hepatocytes. Immunostaining for the apical protein ABCB1 and the tight junction marker occludin demonstrated that canalicular network formation is sequential and is associated with activation of AMPK and LKB1. AMPK and LKB1 activators accelerated canalicular network formation. Inhibition of AMPK or LKB1 by dominant-negative AMPK or kinase-dead LKB1 constructs blocked canalicular network formation. AICAR and 2-deoxyglucose, which activate AMPK, circumvented the inhibitory effect of kinase-dead LKB1 on canalicular formation, indicating that AMPK directly affects canalicular network formation. After the canalicular network was formed, inhibition of AMPK and LKB1 by dominant-negative AMPK or kinase-dead LKB1 constructs resulted in loss of canalicular network, indicating that AMPK and LKB1 also participate in network maintenance. In addition, activation of AMPK and LKB1 prevented low-Ca(2+)-mediated disruption of the canalicular network and tight junctions. These studies reveal that AMPK and its upstream kinase, LKB1, regulate canalicular network formation and maintenance.

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01/01/10 | Single-particle tracking photoactivated localization microscopy for mapping single-molecule dynamics.
Manley S, Gillette JM, Lippincott-Schwartz J
Methods in enzymology. 2010;475:109-20. doi: 10.1016/S0076-6879(10)75005-9

Recent developments in single-molecule localization techniques using photoactivatable fluorescent proteins have allowed the probing of single-molecule motion in a living cell with high specificity, millisecond time resolution, and nanometer spatial resolution. Analyzing the dynamics of individual molecules at high densities in this manner promises to provide new insights into the mechanisms of many biological processes, including protein heterogeneity in the plasma membrane, the dynamics of cytoskeletal flow, and clustering of receptor complexes in response to signaling cues. Here we describe the method of single-molecule tracking photoactivated localization microscopy (sptPALM) and discuss how its use can contribute to a quantitative understanding of fundamental cellular processes.

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01/01/10 | Mitochondria supply membranes for autophagosome biogenesis during starvation.
Hailey DW, Rambold AS, Satpute-Krishnan P, Mitra K, Sougrat R, Kim PK, Lippincott-Schwartz J
Cell. 2010 May 14;141(4):656-67. doi: 10.1016/j.cell.2010.04.009

Starvation-induced autophagosomes engulf cytosol and/or organelles and deliver them to lysosomes for degradation, thereby resupplying depleted nutrients. Despite advances in understanding the molecular basis of this process, the membrane origin of autophagosomes remains unclear. Here, we demonstrate that, in starved cells, the outer membrane of mitochondria participates in autophagosome biogenesis. The early autophagosomal marker, Atg5, transiently localizes to punctae on mitochondria, followed by the late autophagosomal marker, LC3. The tail-anchor of an outer mitochondrial membrane protein also labels autophagosomes and is sufficient to deliver another outer mitochondrial membrane protein, Fis1, to autophagosomes. The fluorescent lipid NBD-PS (converted to NBD-phosphotidylethanolamine in mitochondria) transfers from mitochondria to autophagosomes. Photobleaching reveals membranes of mitochondria and autophagosomes are transiently shared. Disruption of mitochondria/ER connections by mitofusin2 depletion dramatically impairs starvation-induced autophagy. Mitochondria thus play a central role in starvation-induced autophagy, contributing membrane to autophagosomes.

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01/01/10 | Analysis of mitochondrial dynamics and functions using imaging approaches.
Mitra K, Lippincott-Schwartz J
Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]. 2010 Mar;Chapter 4:Unit 4.25.1-21. doi: 10.1002/0471143030.cb0425s46

Mitochondria are organelles that have been primarily known as the powerhouse of the cell. However, recent advances in the field have revealed that mitochondria are also involved in many other cellular activities like lipid modifications, redox balance, calcium balance, and even controlled cell death. These multifunctional organelles are motile and highly dynamic in shapes and forms; the dynamism is brought about by the mitochondria's ability to undergo fission and fusion with each other. Therefore, it is very important to be able to image mitochondrial shape changes to relate to the variety of cellular functions these organelles have to accomplish. The protocols described here will enable researchers to perform steady-state and time-lapse imaging of mitochondria in live cells by using confocal microscopy. High-resolution three-dimensional imaging of mitochondria will not only be helpful in understanding mitochondrial structure in detail but it also could be used to analyze their structural relationships with other organelles in the cell. FRAP (fluorescence recovery after photobleaching) studies can be performed to understand mitochondrial dynamics or dynamics of any mitochondrial molecule within the organelle. The microirradiation assay can be performed to study functional continuity between mitochondria. A protocol for measuring mitochondrial potential has also been included in this unit. In conclusion, the protocols described here will aid the understanding of mitochondrial structure-function relationship.

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01/01/10 | Superresolution imaging using single-molecule localization.
Patterson G, Davidson M, Manley S, Lippincott-Schwartz J
Annual review of physical chemistry. 2010;61:345-67. doi: 10.1146/annurev.physchem.012809.103444

Superresolution imaging is a rapidly emerging new field of microscopy that dramatically improves the spatial resolution of light microscopy by over an order of magnitude (approximately 10-20-nm resolution), allowing biological processes to be described at the molecular scale. Here, we discuss a form of superresolution microscopy based on the controlled activation and sampling of sparse subsets of photoconvertible fluorescent molecules. In this single-molecule-based imaging approach, a wide variety of probes have proved valuable, ranging from genetically encodable photoactivatable fluorescent proteins to photoswitchable cyanine dyes. These have been used in diverse applications of superresolution imaging: from three-dimensional, multicolor molecule localization to tracking of nanometric structures and molecules in living cells. Single-molecule-based superresolution imaging thus offers exciting possibilities for obtaining molecular-scale information on biological events occurring at variable timescales.

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01/01/10 | Lipids and cholesterol as regulators of traffic in the endomembrane system.
Lippincott-Schwartz J, Phair RD
Annual review of biophysics. 2010;39:559-78. doi: 10.1146/annurev.biophys.093008.131357

The endomembrane system of eukaryotic cells uses membrane-enclosed carriers to move diverse macromolecules among different membrane-bound compartments, a requirement for cells to secrete and take up molecules from their environment. Two recycling pathways-biosynthetic and endocytic, each with specific lipid components-make up this system, with the Golgi apparatus mediating transport between the two. Here, we integrate lipid-based mechanisms into the description of this system. A partitioning model of the Golgi apparatus is discussed as a working hypothesis to explain how membrane lipids and proteins that are segregated based on lateral lipid partitioning support the unique composition of the biosynthetic and endocytic recycling pathways in the face of constant trafficking of molecular constituents. We further discuss how computational modeling can allow for interpretation of experimental findings and provide mechanistic insight into these important cellular pathways.

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01/01/10 | Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.
Subach FV, Patterson GH, Renz M, Lippincott-Schwartz J, Verkhusha VV
Journal of the American Chemical Society. 2010 May 12;132(18):6481-91. doi: 10.1021/ja100906g

Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.

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01/01/10 | The open reading frame 3a protein of severe acute respiratory syndrome-associated coronavirus promotes membrane rearrangement and cell death.
Freundt EC, Yu L, Goldsmith CS, Welsh S, Cheng A, Yount B, Liu W, Frieman MB, Buchholz UJ, Screaton GR, Lippincott-Schwartz J, Zaki SR, Xu XN, Baric RS, Subbarao K, Lenardo MJ
Journal of virology. 2010 Jan;84(2):1097-109. doi: 10.1128/JVI.01662-09

The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.

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