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ABSTRACT: Over the last decades there has been an explosion of new methodologies to study protein complexes. However, most of the approaches currently used are based on in vitro assays (e.g. nuclear magnetic resonance, X-ray, electron microscopy, isothermal titration calorimetry etc). The accurate measurement of parameters that define protein complexes in a physiological context has been largely limited due to technical constrains. Here, we present PICT (Protein interactions from Imaging of Complexes after Translocation), a new method that provides a simple fluorescence microscopy readout for the study of protein complexes in living cells. We take advantage of the inducible dimerization of FK506-binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain to translocate protein assemblies to membrane associated anchoring platforms in yeast. In this assay, GFP-tagged prey proteins interacting with the FRB-tagged bait will co-translocate to the FKBP-tagged anchor sites upon addition of rapamycin. The interactions are thus encoded into localization changes and can be detected by fluorescence live-cell imaging under different physiological conditions or upon perturbations. PICT can be automated for high-throughput studies and can be used to quantify dissociation rates of protein complexes in vivo. In this work we have used PICT to analyze protein-protein interactions from three biological pathways in the yeast Saccharomyces cerevisiae: Mitogen-activated protein kinase cascade (Ste5-Ste11-Ste50), exocytosis (exocyst complex) and endocytosis (Ede1-Syp1).
PLoS ONE 01/2013; 8(5):e62195. · 4.09 Impact Factor
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ABSTRACT: Endocytosis, like many dynamic cellular processes, requires precise temporal and spatial orchestration of complex protein machinery to mediate membrane budding. To understand how this machinery works, we directly correlated fluorescence microscopy of key protein pairs with electron tomography. We systematically located 211 endocytic intermediates, assigned each to a specific time window in endocytosis, and reconstructed their ultrastructure in 3D. The resulting virtual ultrastructural movie defines the protein-mediated membrane shape changes during endocytosis in budding yeast. It reveals that clathrin is recruited to flat membranes and does not initiate curvature. Instead, membrane invagination begins upon actin network assembly followed by amphiphysin binding to parallel membrane segments, which promotes elongation of the invagination into a tubule. Scission occurs on average 9 s after initial bending when invaginations are ∼100 nm deep, releasing nonspherical vesicles with 6,400 nm2 mean surface area. Direct correlation of protein dynamics with ultrastructure provides a quantitative 4D resource.
Cell 08/2012; 150(3):508-20. · 32.40 Impact Factor
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ABSTRACT: The application of fluorescence and electron microscopy to the same specimen allows the study of dynamic and rare cellular events at ultrastructural detail. Here, we present a correlative microscopy approach, which combines high accuracy of correlation, high sensitivity for detecting faint fluorescent signals, as well as robustness and reproducibility to permit large dataset collections. We provide a step-by-step protocol that allows direct mapping of fluorescent protein signals into electron tomograms. A localization precision of <100 nm is achieved by using fluorescent fiducial markers which are visible both in fluorescence images and in electron tomograms. We explain the critical details of the procedure, give background information on the individual steps, present results from test experiments carried out during establishment of the method, as well as information about possible modifications to the protocol, such as its application to 2D electron micrographs. This simple, robust, and flexible method can be applied to a large variety of cellular systems, such as yeast cell pellets and mammalian cell monolayers, to answer a broad spectrum of structure-function related questions.
Methods in cell biology 01/2012; 111:235-57. · 2.05 Impact Factor
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ABSTRACT: Correlative electron and fluorescence microscopy has the potential to elucidate the ultrastructural details of dynamic and
rare cellular events, but has been limited by low precision and sensitivity. Here we present a method for direct mapping of
signals originating from ∼20 fluorescent protein molecules to 3D electron tomograms with a precision of less than 100 nm.
We demonstrate that this method can be used to identify individual HIV particles bound to mammalian cell surfaces. We also
apply the method to image microtubule end structures bound to mal3p in fission yeast, and demonstrate that growing microtubule
plus-ends are flared in vivo. We localize Rvs167 to endocytic sites in budding yeast, and show that scission takes place halfway
through a 10-s time period during which amphiphysins are bound to the vesicle neck. This new technique opens the door for
direct correlation of fluorescence and electron microscopy to visualize cellular processes at the ultrastructural scale.
The Journal of Cell Biology 01/2011; 192(1):111-119. · 10.26 Impact Factor
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ABSTRACT: Correlative electron and fluorescence microscopy has the potential to elucidate the ultrastructural details of dynamic and rare cellular events, but has been limited by low precision and sensitivity. Here we present a method for direct mapping of signals originating from ∼20 fluorescent protein molecules to 3D electron tomograms with a precision of less than 100 nm. We demonstrate that this method can be used to identify individual HIV particles bound to mammalian cell surfaces. We also apply the method to image microtubule end structures bound to mal3p in fission yeast, and demonstrate that growing microtubule plus-ends are flared in vivo. We localize Rvs167 to endocytic sites in budding yeast, and show that scission takes place halfway through a 10-s time period during which amphiphysins are bound to the vesicle neck. This new technique opens the door for direct correlation of fluorescence and electron microscopy to visualize cellular processes at the ultrastructural scale.
The Journal of Cell Biology 01/2011; 192(1):111-9. · 10.26 Impact Factor
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Oriol Gallego,
Matthew J Betts,
Jelena Gvozdenovic-Jeremic,
Kenji Maeda,
Christian Matetzki,
Carmen Aguilar-Gurrieri,
Pedro Beltran-Alvarez,
Stefan Bonn,
Carlos Fernández-Tornero,
Lars Juhl Jensen,
Michael Kuhn,
Jamie Trott,
Vladimir Rybin,
Christoph W Müller,
Peer Bork, Marko Kaksonen,
Robert B Russell,
Anne-Claude Gavin
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ABSTRACT: Protein-metabolite networks are central to biological systems, but are incompletely understood. Here, we report a screen to catalog protein-lipid interactions in yeast. We used arrays of 56 metabolites to measure lipid-binding fingerprints of 172 proteins, including 91 with predicted lipid-binding domains. We identified 530 protein-lipid associations, the majority of which are novel. To show the data set's biological value, we studied further several novel interactions with sphingolipids, a class of conserved bioactive lipids with an elusive mode of action. Integration of live-cell imaging suggests new cellular targets for these molecules, including several with pleckstrin homology (PH) domains. Validated interactions with Slm1, a regulator of actin polarization, show that PH domains can have unexpected lipid-binding specificities and can act as coincidence sensors for both phosphatidylinositol phosphates and phosphorylated sphingolipids.
Molecular Systems Biology 11/2010; 6:430. · 8.63 Impact Factor
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Marko Kaksonen
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ABSTRACT: The formation of clathrin-coated endocytic vesicles is driven by a complex and highly dynamic molecular machinery. In this issue, Idrissi et al. (Idrissi, F.-Z., H. Grötsch, I.M. Fernández-Golbano, C. Presciatto-Baschong, H. Riezman, and M.-I. Geli. 2008. J. Cell Biol. 180:1219-1232) reveal some of the secrets of this machinery by analyzing the localizations of nine endocytic proteins during vesicle budding in yeast using quantitative immunoelectron microscopy.
The Journal of Cell Biology 04/2008; 180(6):1059-60. · 10.26 Impact Factor
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ABSTRACT: The lipid phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P(2)) appears to play an important role in endocytosis. However, the timing of its formation and turnover, and its specific functions at different stages during endocytic internalization, have not been established. In this study, Sla2 ANTH-GFP and Sjl2-3GFP were expressed as functional fusion proteins at endogenous levels to quantitatively explore PtdIns(4,5)P(2) dynamics during endocytosis in yeast. Our results indicate that PtdIns(4,5)P(2) levels increase and decline in conjunction with coat and actin assembly and disassembly, respectively. Live-cell image analysis of endocytic protein dynamics in an sjl1Delta sjl2Delta mutant, which has elevated PtdIns(4,5)P(2) levels, revealed that the endocytic machinery is still able to assemble and disassemble dynamically, albeit nonproductively. The defects in the dynamic behavior of the various endocytic proteins in this double mutant suggest that PtdIns(4,5)P(2) turnover is required for multiple stages during endocytic vesicle formation. Furthermore, our results indicate that PtdIns(4,5)P(2) turnover may act in coordination with the Ark1/Prk1 protein kinases in stimulating disassembly of the endocytic machinery.
The Journal of Cell Biology 05/2007; 177(2):355-67. · 10.26 Impact Factor
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ABSTRACT: The inherent resolution limit of the light microscope has been a limiting factor in investigations of many fields of cell biology. A recent paper in Science by Betzig and coworkers describes a new method that can push the limit significantly lower.
Developmental Cell 11/2006; 11(4):438-9. · 14.03 Impact Factor
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ABSTRACT: Endocytosis in budding yeast is thought to occur in several phases. First, the membrane invaginates and then elongates into a tube. A vesicle forms at the end of the tube, eventually pinching off to form a "free" vesicle. Experiments show that actin polymerization is an active participant in the endocytic process, along with a number of membrane-associated proteins. Here we investigate the possible roles of these components in driving vesiculation by constructing a quantitative model of the process beginning at the stage where the membrane invagination has elongated into a tube encased in a sheath of membrane-associated protein. This protein sheath brings about the scission step where the vesicle separates from the tube. When the protein sheath is dynamin, it is commonly assumed that scission is brought about by the constriction of the sheath. Here, we show that an alternative scenario can work as well: The protein sheath acts as a "filter" to effect a phase separation of lipid species. The resulting line tension tends to minimize the interface between the tube region and the vesicle region. Interestingly, large vesicle size can further facilitate the reduction of the interfacial diameter down to a few nanometers, small enough so that thermal fluctuations can fuse the membrane and pinch off the vesicle. To deform the membrane into the tubular vesicle shape, the membrane elastic resistance forces must be balanced by some additional forces that we show can be generated by actin polymerization and/or myosin I. These active forces are shown to be important in successful scission processes as well.
Proceedings of the National Academy of Sciences 08/2006; 103(27):10277-82. · 9.68 Impact Factor
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ABSTRACT: Actin polymerization often occurs at the plasma membrane to drive the protrusion of lamellipodia and filopodia at the leading edge of migrating cells. A role for actin polymerization in another cellular process that involves the reshaping of the plasma membrane--namely endocytosis--has recently been established. Live-cell imaging studies are shedding light on the order and timing of the molecular events and mechanisms of actin function during endocytosis.
Nature Reviews Molecular Cell Biology 07/2006; 7(6):404-14. · 39.12 Impact Factor
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ABSTRACT: Much progress defining the order and timing of endocytic internalization events has come as a result of real-time, live-cell fluorescence microscopy. Although the availability of numerous endocytic mutants makes yeast an especially valuable organism for functional analysis of endocytic dynamics, a serious limitation has been the lack of a fluorescent cargo for receptor-mediated endocytosis. We have now synthesized biologically active fluorescent mating-pheromone derivatives and demonstrated that receptor-mediated endocytosis in budding yeast occurs via the clathrin- and actin-mediated endocytosis pathway. We found that endocytic proteins first assemble into patches on the plasma membrane, and then alpha-factor associates with the patches. Internalization occurs next, concomitant with actin assembly at patches. Additionally, endocytic vesicles move toward early endosomes on actin cables. Early endosomes also associate with actin cables, and they actively move toward endocytic sites to capture vesicles being released from the plasma membrane. Thus, endocytic vesicle formation and capture of the newly released vesicles by early endosomes occur in a highly concerted manner, mediated by the actin cytoskeleton.
Proceedings of the National Academy of Sciences 05/2006; 103(15):5793-8. · 9.68 Impact Factor
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ABSTRACT: Endocytosis depends on an extensive network of interacting proteins that execute a series of distinct subprocesses. Previously, we used live-cell imaging of six budding-yeast proteins to define a pathway for association of receptors, adaptors, and actin during endocytic internalization. Here, we analyzed the effects of 61 deletion mutants on the dynamics of this pathway, revealing functions for 15 proteins, and we analyzed the dynamics of 8 of these proteins. Our studies provide evidence for four protein modules that cooperate to drive coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin/actin-mediated endocytosis. We found that clathrin facilitates the initiation of endocytic-site assembly but is not needed for membrane invagination or vesicle formation. Finally, we present evidence that the actin-meshwork assembly that drives membrane invagination is nucleated proximally to the plasma membrane, opposite to the orientation observed for previously studied actin-assembly-driven motility processes.
Cell 11/2005; 123(2):305-20. · 32.40 Impact Factor
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ABSTRACT: A variety of studies have implicated the lipid PtdIns(4,5)P2 in endocytic internalization, but how this lipid mediates its effects is not known. The AP180 N-terminal homology (ANTH) domain is a PtdIns(4,5)P2-binding module found in several proteins that participate in receptor-mediated endocytosis. One such protein is yeast Sla2p, a highly conserved actin-binding protein essential for actin organization and endocytic internalization. To better understand how PtdIns(4,5)P2 binding regulates actin-dependent endocytosis, we investigated the functions of Sla2p's ANTH domain. A liposome-binding assay revealed that Sla2p binds to PtdIns(4,5)P2 specifically through its ANTH domain and identified specific lysine residues required for this interaction. Mutants of Sla2p deficient in PtdIns(4,5)P2 binding showed significant defects in cell growth, actin organization, and endocytic internalization. These defects could be rescued by increasing PtdIns(4,5)P2 levels in vivo. Strikingly, mutant Sla2p defective in PtdIns(4,5)P2 binding localized with the endocytic machinery at the cell cortex, establishing that the ANTH-PtdIns(4,5)P2 interaction is not necessary for this association. In contrast, multicolor real-time fluorescence microscopy and particle-tracking analysis demonstrated that PtdIns(4,5)P2 binding is required during endocytic internalization. These results demonstrate that the interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 plays a key role in regulation of the dynamics of actin-dependent endocytic internalization.
Molecular Biology of the Cell 03/2005; 16(2):717-30. · 4.94 Impact Factor
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ABSTRACT: Until recently, the actin cytoskeleton and the endocytic machinery were thought to operate independently. However, the actin cytoskeleton is an integral part of the cell cortex and there is growing evidence in diverse eukaryotes that F-actin plays a direct role during endocytic internalization. Genetic studies in Saccharomyces cerevisiae have demonstrated that Arp2/3-mediated F-actin assembly is required specifically for the internalization step of endocytosis. Using real-time image analysis, we recently defined a pathway for receptor-mediated endocytosis in budding yeast. Many features of this pathway appear to be conserved widely, indicating that principles derived from our studies in yeast will be directly applicable in more complex eukaryotes. We are pursuing our yeast studies using a combined approach involving image analysis, functional genomics, proteomics and biochemistry. These ongoing studies are providing a broader and deeper understanding of the molecular events of endocytosis, of how forces for actin polymerization are harnessed, and of how steps in the pathway are regulated. Our studies in mammalian cells provide evidence that this pathway is conserved in more complex organisms for endocytic and Golgi trafficking events.
Novartis Foundation symposium 02/2005; 269:35-42; discussion 43-6, 223-30.
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ABSTRACT: Contributions of actin-related proteins (Arp) 2 and 3 nucleotide state to Arp2/3 complex function were tested using nucleotide-binding pocket (NBP) mutants in Saccharomyces cerevisiae. ATP binding by Arp2 and Arp3 was required for full Arp2/3 complex nucleation activity in vitro. Analysis of actin dynamics and endocytosis in mutants demonstrated that nucleotide-bound Arp3 is particularly important for Arp2/3 complex function in vivo. Severity of endocytic defects did not correlate with effects on in vitro nucleation activity, suggesting that a critical Arp2/3 complex function during endocytosis may be structural rather than catalytic. A separate class of Arp2 and Arp3 NBP mutants suppressed phenotypes of mutants defective for actin nucleation. An Arp2 suppressor mutant increased Arp2/3 nucleation activity. Electron microscopy of Arp2/3 complex containing this Arp2 suppressor identified a structural change that also occurs upon Arp2/3 activation by nucleation promoting factors. These data demonstrate the importance of Arp2 and Arp3 nucleotide binding for nucleating activity, and Arp3 nucleotide binding for maintenance of cortical actin cytoskeleton cytoarchitecture.
The Journal of Cell Biology 02/2005; 168(2):315-28. · 10.26 Impact Factor
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ABSTRACT: In budding yeast, many proteins involved in endocytic internalization, including adaptors and actin cytoskeletal proteins, are localized to cortical patches of differing protein composition. Using multicolor real-time fluorescence microscopy and particle tracking algorithms, we define an early endocytic pathway wherein an invariant sequence of changes in cortical patch protein composition correlates with changes in patch motility. Three Arp2/3 activators each showed a distinct behavior, suggesting distinct patch-related endocytic functions. Actin polymerization occurs late in the endocytic pathway and is required both for endocytic internalization and for patch disassembly. In cells lacking the highly conserved endocytic protein Sla2p, patch motility was arrested and actin comet tails associated with endocytic patch complexes. Fluorescence recovery after photobleaching of the actin comet tails revealed that endocytic complexes are nucleation sites for rapid actin polymerization. Attention is now focused on the mechanisms by which the order and timing of events in this endocytic pathway are achieved.
Cell 12/2003; 115(4):475-87. · 32.40 Impact Factor
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Mariko Sekiya-Kawasaki,
Aaron Chris Groen,
M Jamie T V Cope, Marko Kaksonen,
Hadiya A Watson,
Chao Zhang,
Kevan M Shokat,
Beverly Wendland,
Kent L McDonald,
J Michael McCaffery,
David G Drubin
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ABSTRACT: We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis. In vivo Prk1p inhibition blocked pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p, Sla2p, Pan1p, Sla1p, and Ent1p. Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly. Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps. Upon inhibitor washout, the actin clumps rapidly disassembled, and properly polarized actin patches reappeared. Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins. Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch-related process, and propose that Prk1p negatively regulates the actin assembly-stimulating activity of endocytic proteins.
The Journal of Cell Biology 10/2003; 162(5):765-72. · 10.26 Impact Factor
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Mariko Sekiya-Kawasaki,
Aaron Chris Groen,
M. Jamie T.V. Cope, Marko Kaksonen,
Hadiya A. Watson,
Chao Zhang,
Kevan M. Shokat,
Beverly Wendland,
Kent L. McDonald,
J. Michael McCaffery,
David G. Drubin
[show abstract]
[hide abstract]
ABSTRACT: We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian
GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis. In vivo Prk1p inhibition blocked
pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p,
Sla2p, Pan1p, Sla1p, and Ent1p. Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated
Arp2/3-stimulated actin assembly. Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane
marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps. Upon inhibitor washout, the actin clumps
rapidly disassembled, and properly polarized actin patches reappeared. Our results suggest that actin clumps result from blockage
at a normally transient step during which actin assembly is stimulated by endocytic proteins. Thus, we revealed tight phosphoregulation
of an intrinsically dynamic, actin patch–related process, and propose that Prk1p negatively regulates the actin assembly–stimulating
activity of endocytic proteins.
The Journal of Cell Biology 08/2003; 162(5):765-772. · 10.26 Impact Factor
