Nucleo-cytoplasmic transport provides a key aspect of cellular regulation during both normal development and disease/cancer progression, and has been thought to take place exclusively through Nuclear Pore Complexes (NPCs). Many large Ribonucleoprotein (megaRNP) particles are larger than the NPC diameter and were proposed to undergo unfolding and/or remodeling to fit through the pore. However, ground-breaking work from the Budnik lab has shown that some larger RNPs, including those assembled for major developmental signaling pathways, exit the nucleus through an exciting new alternate nuclear export mechanism involving vesicle-mediated nucleo-cytoplasmic transport or Nuclear Envelope (NE-) budding. 

Nuclear Envelope Buds. (A-A”) Confocal micrographs of Drosophila salivary gland nucleus showing co-localization (arrows) of dFz2C protein (red) with lamin buds (green). Scale bar: 5 mm. (B-C) High magnification views of NE-buds outlined by lamin (green) and showing dFz2c puncta (red; B) or uniform Pavarotti (Pav; red; C) distributions within the NE-buds. Scale bar: 0.5 mm. n=nucleus, c=cytoplasm.

In this conserved pathway, large macromolecular complexes are enveloped by the inner nuclear membrane (INM) to form a 200-500nm NE bud, transverse the perinuclear space, and then exit through the outer nuclear membrane (ONM) to release their contents into the cytoplasm. The Budnik lab has shown that a C-terminal fragment (dFz2C) of the Drosophila Wingless receptor dFz2, enters the nucleus, localizes in large RNPs (megaRNPs) that form foci/buds along with Lamins at the nuclear periphery.

Proposed cellular events comprising the endogenous NE-budding pathway.

[1] RNP assembly and/or transport. The primary cargos of NE buds are thought to be functionally related and/or co-regulated sets of RNAs and proteins that are assembled together into single large particles (megaRNPs) for nuclear export and delivery to specific subcellular locations.

[2] Modification of the nuclear lamina. NE-budding has been proposed to occur at sites along the INM where the nuclear lamina is modified by atypical Protein Kinase C (aPKC) phosphorylation.

[3] NE-bud formation. Physical NE-bud formation begins with the INM and underlying lamina bending prior to megaRNP entry.

[4] MegaRNP encapsulation. The megaRNP enters the nascent NE bud.

[5] NE-bud scission.  After the envelopment of the megaRNP/other cargos by the IMN/lamina, scission or the pinching off of the nascent bud into the perinuclear space, occurs.

[6a, 7a] NE-bud fusion and megaRNP release. The INM surrounded vesicle is proposed to fuse with the ONM. This fusion releases the cargoes into the cytoplasm followed by remodeling of the ONM. Nothing is currently known about the regulation and/or machinery of this step.

[6b, 7b] Alternatively, megaRNPs may bud through the ONM such that they are surrounded by a bi-layer membrane [6b]; and then released as a vesicle into the cytoplasm [7b].

Cellular functions requiring NE-budding. NE budding plays an essential role in neuro-muscular junction integrity affecting synapse development and mitochondrial integrity leading to premature aging phenotypes, and has been implicated in the removal of obsolete macromolecular complexes/large protein aggregates from the nucleus. NE-budding also shares many features with nuclear egress mechanisms used by herpesviruses, wide-spread pathogens that cause or contribute to human diseases, and that are particularly problematic for immune-compromised individuals.

Physical machinery for NE budding. For NE budding to occur, there must be physical machineries to form the NE-bud, to get it through the ONM so that it can release its contents into the cytoplasm, and to remodel the ONM to restore its normal lipid/protein composition and organization. We have found that the Wiskott-Aldrich protein WASH, its four subunit regulatory complex (SHRC), capping protein, and Arp2/3 are required for the physical aspects of NE-bud formation. Using mass spec of nuclear WASH-containing complexes and proximity proteomics with a NE-budding cargo, we subsequently showed that the centralspindlin proteins Pavarotti (Pav) and Tumbleweed (Tum) are also involved in the physical/structural aspects of NE budding.

We are currently using a combination of cell biological, genetic, developmental, imaging, molecular, biochemical, optogenetic, and proteomic approaches to define the molecular machineries and mechanisms underlying the physical aspects of NE-budding (i.e. membrane bending, bud detachment, vesicle-membrane fusion, repair/remodeling of membrane ruptures)

Verboon JM, Nakamura M, Davidson KA, Decker JR, Nandakumar V and Parkhurst SM (2020). Wash and the WASH Regulatory Complex function in Nuclear Envelope budding. J. Cell Sci. 133(13): jcs243576. https://doi.org/10.1242/jcs.243576 [preprint posted on BioRxiv].

Davidson KA*, Nakamura M*, Verboon JM and Parkhurst SM (2023). The Centralspindlin proteins Pavarotti and Tumbleweed function in Nuclear Envelope budding. J. Cell Biol., 222(8): e202211074.  https://rupress.org/jcb/article/222/8/e202211074/214102/Centralspindlin-proteins-Pavarotti-and-Tumbleweed 

Sule K*, Nakamura M* and Parkhurst SM. (2023). (invited review). Nuclear Envelope Budding: Getting large macromolecular complexes out of the nucleus. BioEssays 46(2): e2300182.  https://onlinelibrary.wiley.com/doi/10.1002/bies.202300182