A biochemical and immunological comparison of nuclear and cytoplasmic pore complexes

Abstract

Pore complexes are not confined to the nuclear envelope but can also be found in the cytoplasm of numerous cell types in the form of annulate lamellae (AL). We have induced formation of AL by exposure of rat cells (line RV) to sublethal doses of the antimitotic drug vinblastine sulfate, and compared the distribution of several nuclear pore complex proteins (nucleoporins) in the nuclear envelope and AL by immunocytochemistry, cytochemical lectin binding studies and immunoblot analyses of nuclear and AL-enriched fractions. All the antibodies used yielded punctate nuclear surface staining in immunofluorescence microscopy which is characteristic for nuclear pore complex components. When we applied antibodies against the nucleoporin p62, AL were visualized as numerous cytoplasmic dot-like structures. Immunogold electron microscopy confirmed the correspondence of the cytoplasmic bodies with stacks of AL. Antibodies to constituents of the cytoplasmic (nup180) and nucleoplasmic (nup153) filaments extending from both sides of nuclear pore complexes also stained the AL, indicating that pore complexes are intrinsically asymmetric assemblies independent of their specific intracellular topology. By contrast, AL were negative with five different antibodies against the transmembrane nuclear pore glycoprotein gp210 and the lectin concanavalin A (ConA) known to bind to the oligosaccharide side chains of gp210. Similarly, there was no staining of the AL with antibodies to the other nuclear pore membrane protein so far known in higher eukaryotes, POM121. Immunoblot analyses confirmed the presence of p62, nup180 and nup153 in both the nuclear and AL fractions and the absence of gp210 and POM121 from AL. Our results do not support the generally held view that gp210 and POM121 function in anchoring the pore complex scaffold to the pore membrane. Rather, they point to a role for these proteins in transport processes through the nuclear pore complexes. Since AL are not involved in nucleocytoplasmic transport processes they may lack components of the transport machinery.

The Cytoskeleton and Its Roles in Self-Organization Phenomena: Insights from Xenopus Egg Extracts

“…Indeed, addition of nocodazole, colcemid, as well as AS-2 (inhibitor of kinesin-mediated microtubule-dependent transport) did not prevent the formation of the nuclear envelope, but nuclei formed under these conditions lacked nuclear pore complexes and were therefore unable to accumulate karyophilic proteins. It is interesting to note that the assembly of annulate lamellae, a cytoplasmic structure forming stacks of membranes containing pore complexes similar to those of nuclear envelope but different in protein composition (studied in more details in mammalian cells [106,107]), was not affected by these same perturbations [105]. In addition, by combining Xenopus egg extracts with microfluidic devices to control the space occupied by microtubules around nuclei, Hara and Merten showed that dynein-based accumulation of membranes regulates nuclear expansion [27].…”