Picture the scene. You’re driving along the I-5 and a large convoy with flashing yellow lights and a big banner labeled ‘Oversized-Load’ trundles along past you at a slow and steady pace onwards to their specific destination. This transport of large goods is similar to how large macromolecules are trafficked out of the cell nucleus via an export pathway termed nuclear envelope budding. This process “allows for large nuclear macromolecules such as megaRNPs [ribonucleoproteins] or protein aggregates to be exported out of the nucleus by bypassing nuclear pores that limit the size of nuclear cargo exported,” reported Kerri Davidson, a former Research Technician in the Parkhurst Lab in Fred Hutch’s Basic Sciences Division and current MD/PhD student at Yale School of Medicine. Emerging research describes how nuclear envelope budding plays a critical role in the regulation of developmental processes such as maintenance of mitochondrial integrity, and “dysregulation of this transport is involved in various pathological conditions including cancer, viral infection, and neurodegenerative diseases. Despite the importance of nuclear envelope budding for aging and viral infection [in particular], only a few molecules required for this process have been identified so far,” highlighted Dr. Mitsutoshi Nakamura, a current post-doctoral fellow in the Parkhurst Lab.
Together with colleagues and under the mentorship of Dr. Susan Parkhurst, Davidson and Dr. Nakamura, sought to build on previous research from their group that discovered Wash (Wiskott-Aldrich Syndrome family protein) as a key structural component in nuclear envelope budding in Drosophila. They undertook a mass spectrometry screen to identify proteins that interact with Wash and that may aid in nuclear envelope budding. Their screen revealed Pavorotti (Pav), a nuclear protein that plays a role in regulating the formation of centralspindlin complexes, as a top hit. The researchers then hypothesized that Pav may similarly play important roles in nuclear envelope budding with Wash. In their study, recently published in Journal of Cell Biology, Davidson summarizes that they “not only identify new players involved in the formation of cargo within nuclear enveloped buds, but also uncover potential mechanical forces, such as the role of nuclear actin bundling in the scaffolding of nuclear envelope buds.”
While Pav was a top hit from their mass spectrometry screen and an additional proteomics screen, Tumbleweed (Tum), another nuclear protein involved in centralspindlin complex regulation, was not. Undeterred, the authors stained the Drosophila larval salivary glands for both Tum and Pav and discovered enrichment of Pav and Tum in the nuclear envelope buds. They then performed knockdown experiments of pav and tum which resulted in loss of nuclear envelope budding, further cementing the role of Pav and Tum. Next, they assessed the interaction between Pav, Tum and Wash, noting that Wash forms two distinct nuclear complexes with these proteins – one Wash-Pav-Tum complex, and an additional complex formed by binding between Tum and Wash’s regulatory complex SHRC (Wash-SHRC-Tum). By utilizing transgenic models with the Wash-Pav-Tum complex depleted, the authors were able to observe decreased mitochondrial activity in the indirect flight muscle of Drosophila, suggesting a critical role of the Wash-Pav-Tum complex in nuclear envelope budding and mitochondrial function. Expanding on these findings, Davidson said, “when Wash cannot bind to Pavarotti/Tumbleweed [Pav/Tum] it can no longer bundle actin or form nuclear envelope buds. Additionally, we observed Pavarotti and Tumbleweed often localized to the interior region of nuclear buds - we found Pavarotti’s actin bundling is required for nuclear envelope budding and believe the Wash-Pav-Tum complex likely plays a role in the organization of cargo within nuclear envelope buds. Surprisingly, we also identified Tumbleweed as working independently of Pavarotti in a Wash complex we had previously shown to be required for the physical formation of NE buds – the Wash and WaSH Regulatory Complex, SHRC.” Dr. Nakamura added that “actin nucleation by the Wash-Tum-SHRC complex generates the force to bend the inner nuclear envelope to initiate nuclear envelope budding. Then, actin bundling by the Wash-Tum-Pav complex organizes RNA/protein complexes within the buds.”
“Our results suggest that two different Wash complexes with Pav and/or Tum have sequential functions during nuclear envelope budding,” continued Dr. Nakamura. “How are these two Wash complexes organized spatially and temporally? To address this question, our next step is to find other members of Wash complexes and to develop an inducible live imaging approach, which will allow us to investigate the process of nuclear envelope budding from the beginning to the end.” According to Davidson, this approach of “live imaging coupled with fluorescently tagged budding markers such as Wash, Pavarotti and Tumbleweed as well as nuclear actin and chemical treatment of nuclei with actin polymerization inhibitors” will enable the authors “to see how the players we have identified work together and independently during the budding process.”
Looking forward towards the big picture significance of their findings from this study, Davidson expressed how she is “really excited about our identification of Pavarotti and Tumbleweed in this new role in nuclear envelope budding, because the two proteins have previously been implicated in cell proliferation, migration, cancer cell progression and processes analogous to the budding of retroviral material across plasma membranes suggesting further understanding of their role in budding may have larger implications in developmental biology and virology.”
This research was supported by funding from the National Institutes of Health, the Mark Groudine Chair for Outstanding Achievements in Science and Service, a Koss family donation to the Fred Hutch Basic Sciences Division, and a National Cancer Institute Cancer Center Support Grant.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Susan M. Parkhurst contributed to this work.
Davidson KA, Nakamura M, Verboon JM, Parkhurst SM. 2023. Centralspindlin proteins Pavarotti and Tumbleweed along with WASH regulate nuclear envelope budding. J Cell Biol. 222(8):e202211074.