Journal:

Article Title: SIGNIFICANT PROPORTIONS OF NUCLEAR TRANSPORT PROTEINS WITH REDUCED INTRACELLULAR MOBILITIES RESOLVED BY FLUORESCENCE CORRELATION SPECTROSCOPY

doi: 10.1016/j.jmb.2006.09.08

Figure Lengend Snippet: Molecular interactions during nuclear transport are shown for NLS-containing cargo molecule (orange), importin α (light green), importin β (dark green), Ran (red), NTF2 (cyan), CAS (blue), immobile binging partners (cross hatched), microtubules (cylinders) and NPC (black). Freely diffusing molecules are shown in unshaded regions, molecules bound to medium, slow or immobile partners are shown in shaded regions in nucleus and cytoplasm.

Article Snippet: Unincorporated dye was removed by gel filtration in PBS using Micro Bio-Spin P6 (for NTF2) or P30 (for all other proteins) spin columns (Bio-Rad Laboratories, Hercules, CA).

Techniques:

Journal:

Article Title: SIGNIFICANT PROPORTIONS OF NUCLEAR TRANSPORT PROTEINS WITH REDUCED INTRACELLULAR MOBILITIES RESOLVED BY FLUORESCENCE CORRELATION SPECTROSCOPY

doi: 10.1016/j.jmb.2006.09.08

Figure Lengend Snippet: FCS analysis of nuclear transport proteins in vitro

Article Snippet: Unincorporated dye was removed by gel filtration in PBS using Micro Bio-Spin P6 (for NTF2) or P30 (for all other proteins) spin columns (Bio-Rad Laboratories, Hercules, CA).

Techniques:

Journal:

Article Title: SIGNIFICANT PROPORTIONS OF NUCLEAR TRANSPORT PROTEINS WITH REDUCED INTRACELLULAR MOBILITIES RESOLVED BY FLUORESCENCE CORRELATION SPECTROSCOPY

doi: 10.1016/j.jmb.2006.09.08

Figure Lengend Snippet: Intrinsically fluorescent APC and Alexa Fluor 647 labeled importin β, importin α, NTF2, Ran, RanE70A, RanT24N and RanQ69L were microinjected into the cytoplasm of B104 cells. After 30–60 minutes the injected cells were imaged by confocal microscopy. Scale bar indicates 10 μm.

Article Snippet: Unincorporated dye was removed by gel filtration in PBS using Micro Bio-Spin P6 (for NTF2) or P30 (for all other proteins) spin columns (Bio-Rad Laboratories, Hercules, CA).

Techniques: Labeling, Injection, Confocal Microscopy

Journal:

Article Title: SIGNIFICANT PROPORTIONS OF NUCLEAR TRANSPORT PROTEINS WITH REDUCED INTRACELLULAR MOBILITIES RESOLVED BY FLUORESCENCE CORRELATION SPECTROSCOPY

doi: 10.1016/j.jmb.2006.09.08

Figure Lengend Snippet: Fluorescently labeled importin β, importin α, NTF2, Ran, RanE70A, RanT24N and RanQ69L labeled with Alexa Fluor 647 were microinjected into cytoplasm of B104 cells. After injected protein achieved steady state nucleocytoplasmic distribution, FCS measurements were performed with confocal volume positioned inside nucleus. For each protein at least 30 cells were analyzed. Autocorrelation data from all cells were globally fitted to resolve protein populations with different mobilities. Immobile fraction for each protein was determined from photobleaching amplitudes. Concentrations for each population were calculated based on total concentration and nucleocytoplasmic distribution of endogenous protein. Concentrations of each component are shown on bar graphs and in the table. Fast, medium, slow and immobile populations are labeled with red, green, blue, and black colors respectively. For mutant Ran proteins, the fraction of each population is shown compared with wt Ran. The table shows autocorrelation decay times (τD), fractions, and concentrations for each population. Molecular species comprising each population and calculated diffusion coefficients or off rates are listed.

Article Snippet: Unincorporated dye was removed by gel filtration in PBS using Micro Bio-Spin P6 (for NTF2) or P30 (for all other proteins) spin columns (Bio-Rad Laboratories, Hercules, CA).

Techniques: Labeling, Injection, Concentration Assay, Mutagenesis, Diffusion-based Assay

Journal:

Article Title: SIGNIFICANT PROPORTIONS OF NUCLEAR TRANSPORT PROTEINS WITH REDUCED INTRACELLULAR MOBILITIES RESOLVED BY FLUORESCENCE CORRELATION SPECTROSCOPY

doi: 10.1016/j.jmb.2006.09.08

Figure Lengend Snippet: Importin β, importin α, NTF2, Ran wt, RanE70A, RanT24N and RanQ69L labeled with Alexa Fluor 647 were microinjected into the cytoplasm of B104 cells. Cells were incubated for 30–60 minutes after injection and analyzed by FCS with observation volume positioned over the nuclear envelope. For each protein at least 20 cells were analyzed. To resolve protein populations specific to NPC and nuclear envelope, contributions from adjacent nucleoplasm and cytoplasm were subtracted by a global fitting procedure. Number of molecules per NPC for each protein component was calculated based on number of pores encompassed by FCS observation volume. Distributions of each protein between populations with different mobilities are shown on bar graphs and in the table. Fast, medium, slow and immobile populations are labeled with red, green, blue, and black colors respectively. For mutant Ran proteins, the fraction of each component compared with wt Ran is shown. The table shows autocorrelation decay times (τD), concentrations, numbers of molecules per NPC, or fractions for each population. Molecular species comprising each population and calculated diffusion coefficients or off rates are listed.

Article Snippet: Unincorporated dye was removed by gel filtration in PBS using Micro Bio-Spin P6 (for NTF2) or P30 (for all other proteins) spin columns (Bio-Rad Laboratories, Hercules, CA).

Techniques: Labeling, Incubation, Injection, Mutagenesis, Diffusion-based Assay