Christian J. Malone Assistant Professor of Biochemistry and Molecular Biology 406 Althouse Laboratory, University Park, PA 16802 Phone: (814) 863 6668 Fax: (814) 863-7024 E-mail: cjm42@psu.edu B.A. in Biology , Albertson College of Idaho Ph.D. in Molecular, Cellular & Developmental Biology, University of Colorado, Boulder Postdoctoral, University of Wisconsin, Madison Malone Lab Web Site

Interaction of the Nucleus with the Cytoplasm

Nuclear interactions with the cytoplasm

A fundamental, yet unanswered question in cell biology concerns how the nucleus interacts structurally with other cellular components, and how this interaction is deployed during cell division and nuclear positioning. These interactions are important: mispositioned nuclei in muscle cells have been implicated in muscular dystrophy and nuclear migration is essential for fertilization and epithelial morphogenesis. In addition, our work has demonstrated that centrosomes and nuclei must be attached at the time of nuclear envelope breakdown for a functional mitotic spindle to form. Using genomic, traditional genetic and cell biological approaches, we are undertaking studies of centrosomal attachment, nuclear positioning, and nuclear migration. Using the model system C. elegans, we aim to understand the mechanism by which the nucleus can interact with cytoplasmic components during these biological processes and also determine how the proteins necessary for interaction are positioned on the outer membrane of the nuclear envelope.

Centrosomal attachment to the nucleus

An example of the type of interaction we study is the association between the centrosome and nucleus. These organelles are tightly attached during interphase in a variety of organisms and cell types, yet the significance and mechanism of this attachment have been poorly understood. To characterize the mechanism of centrosomal attachment, we identified and cloned the attachment gene zyg-12, which encodes multiple isoforms of Hook-like proteins. Hook family members in humans mediate the interaction of membrane-bound organelles such as the Golgi with the microtubule cytoskeleton. To characterize the role of ZYG-12 during centrosome attachment we determined that ZYG-12 isoforms localize to both the nuclear envelope and centrosomes if they include a transmembrane domain or just centrosomes if they do not. To investigate how isoforms of ZYG-12 localize to distinct cellular compartments, we identified additional proteins that are required for ZYG-12 localization. ZYG-12 localization to the centrosomes, but not the nuclear envelope, requires microtubules. Conversely, ZYG-12 localization to the nuclear envelope, but not the centrosome, requires SUN-1, a second protein required for centrosomal attachment, which was identified in a functional genomic screen.

To elucidate the role of ZYG-12 in centrosome attachment and identify proteins that function with ZYG-12, we performed two-hybrid and biochemical studies. ZYG-12 interacts with the DLI-1 light intermediate subunit of the microtubule dependent motor protein dynein in a two-hybrid screen. Additionally, ZYG-12 is required in vivo for dynein localization to the nuclear envelope but not to other structures. Also, ZYG-12 can bind to itself in vivo. Based on these data, we have proposed a two-step model of centrosome attachment to the nucleus. Initially, dynein, which localizes to the nuclear envelope via its interaction with ZYG-12, moves the centrosome towards the nucleus (Figure 1A). After proximity is established, ZYG-12 isoforms at both the nucleus and centrosome, acting independently of dynein, anchor the centrosome to the nucleus, perhaps through dimerization (Figure 1B). Having identified several proteins, including ZYG-12 and SUN-1 mentioned above, that are required for the interaction of the nucleus with the cytoplasm, we are focusing on testing mechanistic models of centrosomal attachment, nuclear migration and nuclear positioning.

Specifying the outer nuclear envelope

Our work has identified proteins that localize to the nuclear envelope, encode predicted transmembrane domains and have defined cytoplasmic functions. For example, ZYG-12 binds and localizes dynein and attaches the centrosome to the nucleus. These data suggest that the proteins reside in the outer nuclear envelope. This new class of proteins calls in to question the prevailing model that the outer nuclear envelope is indistinguishable from the ER. We propose, based on other studies of the inner nuclear envelope, that outer nuclear envelope proteins can be positioned by a diffusion retention mechanism. Given that ZYG-12 and other proteins depend on SUN proteins for their localization, we are investigating the role of SUNs in specifying the outer nuclear envelope.

Representative Publications:

• Malone CJ, Misner L, Le Bot N, Tsai MC, Campbell JM, Ahringer J, White JG. The C. elegans hook protein, ZYG-12, mediates the essential attachment between the centrosome and nucleus. Cell. 2003 Dec 26;115(7):825-36.
• Starr DA, Hermann GJ, Malone CJ, Fixsen W, Priess JR, Horvitz HR, Han M. unc-83 encodes a novel component of the nuclear envelope and is essential for proper nuclear migration. Development. 2001 Dec;128(24):5039-50.
• Strome S, Powers J, Dunn M, Reese K, Malone CJ, White J, Seydoux G, Saxton W. Spindle dynamics and the role of gamma-tubulin in early Caenorhabditis elegans embryos. Mol Biol Cell. 2001 Jun;12(6):1751-64.
• Malone CJ, Fixsen WD, Horvitz HR, Han M. UNC-84 localizes to the nuclear envelope and is required for nuclear migration and anchoring during C. elegans development. Development. 1999 Jun;126(14):3171-81.

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