Biomedical subjects

Siegfried M Musser

Publications and source records attributed to Siegfried M Musser.

5 recordsLinked to original sources

PubMed2006

Single giant vesicle rupture events reveal multiple mechanisms of glass-supported bilayer formation.

The formation of supported lipid bilayers (SLBs) on glass from giant unilamellar vesicles (GUVs) was studied using fluorescence microscopy. We show that GUV rupture occurs by at least four mechanisms, including 1), spontaneous rupture of isolated GUVs yielding almost heart-shaped bilayer patches (asymmetric rupture); 2), spontaneous rupture of isolated GUVs yielding circular bilayer patches (symmetric rupture); 3), induced rupture of an incoming vesicle when it contacts a planar bilayer edge; and 4), induced rupture of an adsorbed GUV when a nearby GUV spontaneously ruptures. In pathway 1, the dominant rupture pathway for isolated GUVs, GUVs deformed upon adsorption to the glass surface, and planar bilayer patch formation was initiated by rupture pore formation near the rim of the glass-bilayer interface. Expanding rupture pores led to planar bilayer formation in approximately 10-20 ms. Rupture probability per unit time depended on the average intrinsic curvature of the component lipids. The membrane leaflet adsorbed to the glass surface in planar bilayer patches originated from the outer leaflet of GUVs. Pathway 2 was rarely observed. We surmise that SLB formation is predominantly initiated by pathway 1 rupture events, and that rupture events occurring by pathways 3 and 4 dominate during later stages of SLB formation.

Computer Simulation↗

PubMed2006

Nuclear import time and transport efficiency depend on importin beta concentration.

Although many components and reaction steps necessary for bidirectional transport across the nuclear envelope (NE) have been characterized, the mechanism and control of cargo migration through nuclear pore complexes (NPCs) remain poorly understood. Single-molecule fluorescence microscopy was used to track the movement of cargos before, during, and after their interactions with NPCs. At low importin beta concentrations, about half of the signal-dependent cargos that interacted with an NPC were translocated across the NE, indicating a nuclear import efficiency of approximately 50%. At high importin beta concentrations, the import efficiency increased to approximately 80% and the transit speed increased approximately sevenfold. The transit speed and import efficiency of a signal-independent cargo was also increased by high importin beta concentrations. These results demonstrate that maximum nucleocytoplasmic transport velocities can be modulated by at least approximately 10-fold by the importin beta concentration and therefore suggest a potential mechanism for regulating the speed of cargo traffic across the NE.

Active Transport, Cell Nucleus↗

PubMed2006

Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy.

The utility of single molecule fluorescence (SMF) for understanding biological reactions has been amply demonstrated by a diverse series of studies over the last decade. In large part, the molecules of interest have been limited to those within a small focal volume or near a surface to achieve the high sensitivity required for detecting the inherently weak signals arising from individual molecules. Consequently, the investigation of molecular behavior with high time and spatial resolution deep within cells using SMF has remained challenging. Recently, we demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single cargo level. We describe here the methodological approach that yields 2 ms and approximately 15 nm resolution for a stationary particle. The spatial resolution for a mobile particle is inherently worse, and depends on how fast the particle is moving. The signal-to-noise ratio is sufficiently high to directly measure the time a single cargo molecule spends interacting with the nuclear pore complex. Particle tracking analysis revealed that cargo molecules randomly diffuse within the nuclear pore complex, exiting as a result of a single rate-limiting step. We expect that narrow-field epifluorescence microscopy will be useful for elucidating other binding and trafficking events within cells.

Active Transport, Cell Nucleus↗

PubMed2005

Effect of average phospholipid curvature on supported bilayer formation on glass by vesicle fusion.

The adsorption of large unilamellar vesicles composed of various combinations of phosphatidylcholine, phosphatidylethanolamine (PE), monomethyl PE, and dimethyl PE (PE-Me2) onto a glass surface was studied using fluorescence microscopy. The average lipid geometry within the vesicles, described mathematically by the average intrinsic curvature, C(0,ave), was methodically altered by changing the lipid ratios to determine the effect of intrinsic curvature on the ability of vesicles to rupture and form a supported lipid bilayer. We show that the ability of vesicles to create fluid planar bilayers is dependent on C(0,ave) and independent of the identity of the component lipids. When the C(0,ave) was approximately -0.1 nm(-1), the vesicles readily formed supported lipid bilayers with almost full mobility. In contrast, when the C(0,ave) ranged from approximately -0.2 to approximately -0.3 nm(-1), the adsorbed vesicles remained intact upon the surface. The results indicate that the average shape of lipid molecules within a vesicle (C(0,ave)) is essential for determining kinetically viable reactions that are responsible for global geometric changes.

Absorption↗

PubMed2004

Imaging of single-molecule translocation through nuclear pore complexes.

Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoprotein complexes across the double-membrane nuclear envelope. In vitro studies with purified transport cofactors have revealed a general scheme of cofactor-dependent transport energetically driven by the G protein Ran. However, the size and complexity of NPCs have made it difficult to clearly define the loci and kinetics of the cofactor-NPC interactions required for transport. We now report the use of single-molecule fluorescence microscopy to directly monitor a model protein substrate undergoing transport through NPCs in permeabilized cells. This substrate, NLS-2xGFP, interacts with NPCs for an average of 10 +/- 1 ms during transport. However, because the maximum nuclear accumulation rate of NLS-2xGFP was measured to be at least approximately 10(3) molecules per NPC per s, NPCs must be capable of transporting at least approximately 10 substrate molecules simultaneously. Molecular tracking reveals that substrate molecules spend most of their transit time randomly moving in the central pore of the NPC and that the rate-limiting step is escape from the central pore.

Antigens, Viral, Tumor↗