Super-Resolution Imaging of Nuclear Pore Responses to Mechanical Stress and Energy Depletion

HIV-1 entry into host cells culminates in integration of the reverse transcribed double-stranded viral DNA into host genes. Several lines of evidence suggest that intact, or nearly intact, HIV-1 cores-large, ~60 nm-wide structures-pass through the nuclear pore complex (NPC), and that this passage is associated with pore remodeling. Cryo-electron tomography studies support the dynamic nature of NPCs and their regulation by cytoskeleton and ATP-dependent processes. To explore NPC remodeling, we used super-resolution Stochastic Optical Reconstruction Microscopy (STORM) of U2OS cells endogenously expressing nucleoporin 96 tagged with SNAP. Single-molecule localization imaging and computational averaging resolved 8-fold symmetric nuclear pores with an average radius of ~51 nm. Depletion of cellular ATP using sodium azide or antimycin A, previously reported to reduce the size of yeast NPCs, did not significantly alter the nuclear pore radius in U2OS cells. Similarly, stressing the nuclear envelope by hypotonic or hypertonic conditions failed to induce detectable expansion or contraction of NPCs. These results indicate that the NPCs in U2OS cells do not respond to ATP depletion nor mechanical stresses on changes in pore morphology that can be resolved by STORM. Since these cells are infectable by HIV-1, we surmise that direct multivalent interactions between HIV-1 capsid and phenylalanine-glycine nucleoporins lining the pore's interior drive the core penetration into the nucleus and the associated changes in the pore structure.