Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories

Hess, Samuel T.; Gould, Travis J.; Gudheti, Manasa V.; Maas, Sarah A.; Mills, Kevin D.; Zimmerberg, Joshua

doi:10.1073/pnas.0708066104

Cited by 354 publications

(344 citation statements)

References 31 publications

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“…Arrays of more than three detectors may be used, covering a larger observation area, and axial (z) sensitivity could be achieved by a tetrahedral arrangement (30)(31)(32) or similar. Employing photoswitchable probe molecules within this method to control the density of fluorescing entities could enable the study of dynamics of individual molecules at comparatively high concentrations (29,(39)(40)(41). Effects of molecular orientation were not observed in this study.…”

Section: Discussion Conclusion and Outlookmentioning

confidence: 72%

Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids

Sahl

Leutenegger

Hilbert

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

175

218

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We describe an optical method capable of tracking a single fluorescent molecule with a flexible choice of high spatial accuracy (∼10-20 nm standard deviation or ∼20-40 nm full-width-at-halfmaximum) and temporal resolution (<1 ms). The fluorescence signal during individual passages of fluorescent molecules through a spot of excitation light allows the sequential localization and thus spatio-temporal tracking of the molecule if its fluorescence is collected on at least three separate point detectors arranged in close proximity. We show two-dimensional trajectories of individual, small organic dye labeled lipids diffusing in the plasma membrane of living cells and directly observe transient events of trapping on <20 nm spatial scales. The trapping is cholesterolassisted and much more pronounced for a sphingo-than for a phosphoglycero-lipid, with average trapping times of ∼15 ms and <4 ms, respectively. The results support previous STED nanoscopy measurements and suggest that, at least for nontreated cells, the transient interaction of a single lipid is confined to macromolecular dimensions. Our experimental approach demonstrates that fast molecular movements can be tracked with minimal invasion, which can reveal new important details of cellular nano-organization. M any open questions in biology can be tackled only if the dynamics of individual molecules can be observed noninvasively in vivo and at the appropriate spatial and temporal scale (1-5). Over the years, specific labeling of the cell's constituent parts with fluorescent markers has enabled deeper understanding in many areas of cell biology and allowed, for example, the spatiotemporal tracking of single particles (6, 7). However, to reach the desired spatial and temporal accuracy, single-particle tracking often applies bright but large and clumsy signal markers, which potentially influence the system under study. One notable example is the dynamics of proteins and lipids in cellular membranes and their organization into nanodomains, so-called "lipid rafts" (8-12). Since their proposal, the question of the existence and functional role of such cholesterol-mediated lipid assemblies has caused much controversy (13-16). Owing to the lack of suitable noninvasive techniques to detect these nanodomains in living cells, their spatial extent has been estimated at somewhere in the range of 5-200 nm (17), that is between molecular dimensions and the resolution limit of conventional fluorescence microscopy. Camera-based tracking of single lipids could provide more detailed insight (18)(19)(20). However, so far it either lacked temporal resolution or it made use of rather large gold beads as lipid labels, implying that the measurements may not give comprehensive answers to the open questions. Recently, stimulated-emission-depletion (STED) nanoscopy (21, 22), delivering subdiffraction resolution in live cells, provided direct evidence that certain lipids are transiently trapped in cholesterol-assisted molecular complexes (23, 24). In those experiments, which are evaluate...

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Section: Discussion Conclusion and Outlookmentioning

confidence: 72%

Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids

Sahl

Leutenegger

Hilbert

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

175

218

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“…Already, serial photoactivated localization of single PA-FP tagged molecules has been used to track the diffusion of up to ~100× more protein molecules in the membranes of living cells than is possible with conventional fluorescent probes 13,15 . In vitro experiments can also be contemplated at much higher temporal resolution than demonstrated here, as many such experiments involve much lower molecular densities.…”

Section: Discussionmentioning

confidence: 99%

Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics

et al. 2008

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We demonstrate live-cell super-resolution imaging using photoactivated localization microscopy (PALM). The use of photon-tolerant cell lines in combination with the high resolution and molecular sensitivity of PALM permitted us to investigate the nanoscale dynamics within individual adhesion complexes (ACs) in living cells under physiological conditions for as long as 25 min, with half of the time spent collecting the PALM images at spatial resolutions down to ~60 nm and frame rates as short as 25 s. We visualized the formation of ACs and measured the fractional gain and loss of individual paxillin molecules as each AC evolved. By allowing observation of a wide variety of nanoscale dynamics, live-cell PALM provides insights into molecular assembly during the initiation, maturation and dissolution of cellular processes.A key advantage of using genetically expressed fluorescent proteins is that they permit the non-perturbative optical imaging of dynamic processes in living cells 1 . Although these markers are targeted to specific proteins with molecular precision, most of this information is lost when conventional, diffraction-limited live-cell imaging methods 2 are used. In response, several super-resolution imaging modalities have been developed to image fluorescent proteins in fixed cells [3][4][5] . Extension of these methods to living cells might at last lead to nanoscale imaging under true physiological conditions. Furthermore, by capturing many super-resolution images from a single living cell at different time points, the means by which macromolecular assembly drives cellular processes might be observed directly, as opposed to the much more indirect method of inferring "the rules of the game" 6 from a series of static images of different cells, each fixed at a different time.Nevertheless, reaching the longstanding goal of super-resolution live-cell imaging is far from trivial: the Nyquist criterion specifies that the image sampling interval must be smaller Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions

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“…We propose that the palmitoylation of hemagglutinin (maximum of three palmitates per trimer) provides a saturated lipid anchor that may itself induce the collection of other saturated-chain lipids and cholesterol when hemagglutinin is concentrated in the plasma membrane before budding (the viral pre-envelope 16 ) for the purpose of increasing viral stability during airborne transmission. Recent super-resolution microscopy studies 32 make clear that hemagglutinin remains mobile within Figure 6 Effect of temperature on lipid mixing assay of influenza virus fusion with RBC membranes. Virus labeled with R18 at self-quenching concentration was mixed with RBC membranes and incubated for 5 min to equilibrate temperature.…”

Section: Discussionmentioning

confidence: 99%

Progressive ordering with decreasing temperature of the phospholipids of influenza virus

Polozov¹,

Bezrukov²,

Gawrisch³

et al. 2008

Nat Chem Biol

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Using linewidth and spinning sideband intensities of lipid hydrocarbon chain resonances in proton magic angle spinning NMR spectra, we detected the temperature-dependent phase state of naturally occurring lipids of intact influenza virus without exogenous probes. Increasingly, below 41 1C ordered and disordered lipid domains coexisted for the viral envelope and extracts thereof. At 22 1C much lipid was in a gel phase, the fraction of which reversibly increased with cholesterol depletion. Diffusion measurements and fluorescence microscopy independently confirmed the existence of gel-phase domains. Thus the existence of ordered regions of lipids in biological membranes is now demonstrated. Above the physiological temperatures of influenza infection, the physical properties of viral envelope lipids, regardless of protein content, were indistinguishable from those of the disordered fraction. Viral fusion appears to be uncorrelated to ordered lipid content. Lipid ordering may contribute to viral stability at lower temperatures, which has recently been found to be critical for airborne transmission.Membranes of most enveloped viruses form by budding out from the plasma membranes of their host cells a highly select subset of plasma membrane components. In general, the selected membrane proteins are coded by the viral genome, whereas lipids are recruited from host membranes; however, the lipid composition of the viral envelope differs from that of the host membrane 1,2 and from other budding viruses 3 . The envelope of influenza contains higher amounts of both cholesterol 1 and glycosphingolipids 4 -lipids known to partition into the liquid ordered (l o ) phase. The l o phase is characterized by extended hydrocarbon chains having a reduced gauche-trans isomerization compared with those of the liquid disordered (l d ) phase, but having a similar rotational and translational mobility 5 . Liquid ordered phases are thought to be at the core of lipid rafts 6 , which are defined as transient membrane microdomains that are enriched in sphingolipids and cholesterol (1) 7 -a hypothesis that has generated much debate 8,9 .The influenza virus has played a pivotal role in the development of the raft hypothesis, starting with early studies that inferred ordered domains using spin probes 10-12 and fluorescence 13,14 and that suggested that an ordered lipid domain is selected in toto as the envelope during budding from the plasma membrane 15 . These lipids are either selected at the time of budding or pre-selected as the 'pre-envelope' suggested by clusters of the viral envelope protein hemagglutinin seen in immunoelectron microscopy 16 .Although detection of virus-sized domains of lipids (B100-nm diameter) is below the limit of resolution of fluorescent microscopy, large micrometer-sized membrane domains are reliably detected [17][18][19] . Recently, proton magic angle spinning NMR ( 1 H MAS NMR) has been used to determine the phase diagram of the same lipid compositions used to study large membrane domains in lipid bilayers and othe...

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Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories

Cited by 354 publications

References 31 publications

Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids

Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids

Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics

Progressive ordering with decreasing temperature of the phospholipids of influenza virus

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