Investigating molecular crowding within nuclear pores using polarization-PALM.
Investigating molecular crowding within nuclear pores using polarization-PALM.
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DOI：
10.7554/elife.28716
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发表时间：
2017-09-26
期刊：
影响因子：
7.7
通讯作者：
Musser SM
中科院分区：
文献类型：
--
作者：
Fu G;Tu LC;Zilman A;Musser SM
The key component of the nuclear pore complex (NPC) controlling permeability, selectivity, and the speed of nucleocytoplasmic transport is an assembly of natively unfolded polypeptides, which contain phenylalanine-glycine (FG) binding sites for nuclear transport receptors. The architecture and dynamics of the FG-network have been refractory to characterization due to the paucity of experimental methods able to probe the mobility and density of the FG-polypeptides and embedded macromolecules within intact NPCs. Combining fluorescence polarization, super-resolution microscopy, and mathematical analyses, we examined the rotational mobility of fluorescent probes at various locations within the FG-network under different conditions. We demonstrate that polarization PALM (p-PALM) provides a rich source of information about low rotational mobilities that are inaccessible with bulk fluorescence anisotropy approaches, and anticipate that p-PALM is well-suited to explore numerous crowded cellular environments. In total, our findings indicate that the NPC’s internal organization consists of multiple dynamic environments with different local properties. Most of the genetic material inside an animal cell is enclosed within a compartment called the nucleus. This compartment is separated from the rest of the cell by the nuclear envelope, a double-membrane structure containing thousands of pores that selectively allow certain molecules (collectively referred to as cargo) to enter and exit the nucleus. The movement of cargo through the pores is controlled by large groups of proteins called nuclear pore complexes. The pore is at the center of the complex and is filled by a selective barrier made of an extensive network of flexible proteins known as the FG-network. Other proteins known as nuclear transport receptors bind to the proteins in the FG-network and carry cargos through the barrier. The properties of the nuclear pore barrier and how it rapidly selects the right cargos have been difficult to study, in part, because the barrier network is constantly changing and is crowded with hundreds of transport receptors. New techniques are needed to investigate such highly crowded environments inside cells. Now, Fu et al. use a technique called polarization photoactivated localization microscopy (p-PALM) to explore the molecular crowding within the nuclear pore barrier in human cells. This technique measures the freedom with which a single molecule embedded in the network can rotate, providing information about the local environment. In a crowded environment, it is harder for the probe molecule to rotate as it is more likely to bump into other molecules. Fu et al. found that there are different levels of crowding within the barrier. This is consistent with previous ideas of how the pore barrier could work, which propose that the nuclear transport receptors are less tightly packed in the center of the FG-network. This enables transport receptor and cargo complexes to move more rapidly through the center of the pore. The molecular crowding in the barrier of nuclear pores parallels that observed in other cellular compartments that also rely on assemblies of proteins with flexible structures. Thus, future work using p-PALM is expected to reveal more details about the biophysical properties of nuclear pores as well as those of other structures inside cells.
