Nup133 belongs to the Y-complex, a key component of the nuclear pore complex 
(NPC) scaffold. Studies on a null mutation in mice previously revealed that 
Nup133 is essential for embryonic development but not for mouse embryonic stem 
cell (mESC) proliferation. Using single-pore detection and average 
NE-fluorescence intensity, we find that Nup133 is dispensable for interphase and 
postmitotic NPC scaffold assembly in pluripotent mESCs. However, loss of Nup133 
specifically perturbs the formation of the nuclear basket as manifested by the 
absence of Tpr in about half of the NPCs combined with altered dynamics of 
Nup153. We further demonstrate that its central domain mediates Nup133's role in 
assembling Tpr and Nup153 into a properly configured nuclear basket. Our 
findings thus revisit the role of the Y-complex in pore biogenesis and provide 
insights into the interplay between NPC scaffold architecture, nuclear basket 
assembly, and the generation of heterogeneity among NPCs.