(Wash)ing the nuclear envelope

From the Parkhurst lab, Basic Sciences Division.

“Man, like other organisms, is so perfectly coordinated that he may easily forget, whether awake or asleep, that he is a colony of cells in action, and that it is the cells which achieve, through him, what he has the illusion of accomplishing himself. It is the cells which create and maintain in us, during the span of our lives, our will to live and survive, to search and experiment, and to struggle.”

Albert Claude – Nobel Lecture, 1974

Since the early days of optical microscopy, the nuclear envelope stood out as the defining feature of the nuclear boundary. Nonetheless, it wasn’t until recently that we came to appreciate the diverse roles of the nuclear envelope in the organization and structure of the nucleus, and how like its plasma membrane counterpart, it is the site for signal sensing, molecule trafficking, and inter-/intra-nuclear attachments. The Parkhurst lab (Basic Sciences Division) is interested in the molecular mechanisms of cellular and nuclear architecture that allows the movement of components, complexes, and machineries within and outside the nucleus. The lab uses multidisciplinary approaches to study these dynamic structural elements in various processes including wound repair and nuclear architecture/organization and using Drosophila as a model organism.

Organization of the cytoskeleton is essential for maintenance of cell shape and wound repair, among other processes. Key regulators of the cytoskeleton include Wiskott-Aldrich syndrome family (WAS) proteins, which regulate actin polymerization and are often deregulated in disease/cancer. In a previous study, they established a role for the Drosophila WAS protein Washout (Wash) in global nuclear organization. It was rather unexpected to find that Wash, an actin related protein, localizes to the nucleus. Jeffrey Verboon, a research associate in the Parkhurst lab who led the study explained that “discovering a role for Wash in this process was actually completely fortuitous. We noticed that Drosophila salivary gland nuclei had nuclear envelope buds, and that these buds were completely gone when wash was knocked-down or removed.”

Following up on their previous observations, Verboon and colleagues identified a new role for Wash in the nuclear envelope budding pathway. They summarized their findings in a recent BioRxiv preprint. The nuclear envelope budding pathway is an alternative pathway for nuclear egress, particularly for large complexes that would otherwise need to unfold to fit through the canonical Nuclear Pore Complexes. While factors have been identified both as cargos or actively involved in this recently described phenomenon, the underlying mechanism remained unknown. “Nuclear budding, while relatively new in terms of the number of publications, was actually first identified over 10 years ago. In the intervening time, most of the field’s progress has been focused on appreciating nuclear budding being important to many biological processes and developmental events. However, the underlying mechanism of how it physically is able to occur has been largely unstudied,” said Verboon.

To address this question, Verboon et al. used fluorescence and electron microscopy, biochemical and cell biological assays, and genetic perturbations in the Drosophila model. They identified Wash, its regulatory complex, and actin related proteins (Arp)2/3 as drivers of nuclear envelope budding. For the Parkhurst lab, these findings were important as Verboon explains: “we demonstrate that physical mechanics of this process are likely driven through an actin-based mechanism that is dependent on the branched actin nucleation factors Arp2/3, and Wash”.

Nuclear envelope buds at high magnification. Immunostaining for Lamin in green and dFz2C in red.
Nuclear envelope buds at high magnification. Immunostaining for Lamin in green and dFz2C in red. dFz2C is the C-terminal fragment of the fly Wingless receptor protein which is implicated in NE-budding . From publication

One of the most striking observations of the study is the uncanny similarity between this mechanism of nuclear budding and the nuclear egress mechanism used by herpesviruses. It is not uncommon for viruses to co-opt pre-existing host pathways for their livelihoods. As such, the very mechanisms that allows nuclei to transfer macromolecular complex beyond the capacity of the nuclear pores, may have afforded the herpesvirus nucleocapsids an evolutionary advantage over their hosts. Future research at the confluence of virology and cell biology will shed the light on the diverse roles of nuclear budding in both normal and disease biology.

The Parkhurst lab already has an idea on how to move forward. While their findings provide a new perspective for the mechanism of nuclear envelop budding, they are eager to understand more about the process. “We are going about this in a few ways,” said Verboon. “First, we are using emerging fluorescent nuclear actin and nuclear envelope budding markers combined with live imaging to understand this process in real-time. Second, we have developed complementary proteomics and high throughput screening assays to continue to identify new proteins/genes involved in this process. Stay tuned!” he added.

Verboon J, Nakamura M, Decker J, Davidson K, Nandakumar V, Parkhurst S. 2019. Wash and the WASH Regulatory Complex function in Nuclear Envelope budding. BioRxiv.

UW/Fred Hutch Cancer Consortium member Susan Parkhurst contributed to this work.

This study was made possible by funding from the National Institutes of Health and pilot project funds from the Cancer Center Support Grant.