The Nuclear Envelope: Structure and Function
The hallmark feature of eukaryotic cells is their nucleus, which contains the genetic material. In addition to the chromosomes, the nucleus also contains numerous protein complexes that control gene expression, DNA replication and repair, chromosome segregation and many nuclear processes are essential for genomic integrity and cell proliferation. The core of my lab's interests lies in elucidating the structure and geography of the nucleus, especially in terms of the nuclear envelope (NE).
The nuclear membrane is a double lipid bilayer. The outer nuclear membrane (ONM) is contiguous with the endoplasmic reticulum (ER) and shares a number of integral membrane components. In contrast, the inner nuclear membrane (INM) contains a distinct set of proteins and lipids, which is different from either the ONM or the ER. Proteomic analysis of the INM indicates that it is composed of at least 80 distinct proteins, most of which are uncharacterized. Mutants in genes encoding INM proteins underlie a broad spectrum of human diseases ranging from tissue specific diseases of muscle, bone and fat cells to multi-system diseases such as the premature aging syndrome progeria and cancer. However, which nuclear processes are altered and why different cell types are affected differently is not understood. Elucidating the mechanism by which INM proteins are localized and determining how they function in the three-dimensional organization of the nucleus will advance our understanding of human health and development.
Research in my laboratory uses a combination of genetic, cell biological and biochemical approaches in the budding yeast model system to study two basic questions related to NE structures and the genetic material contained within:
- How are microtubule organizing centers (MTOCs) associated with the NE and how do they function in mitotic spindle assembly and chromosome segregation?
- How do INM proteins function in chromosome organization within the eukaryotic nucleus?
Using spindle pole body assembly to understand NE remodeling
Figure 1. Yeast spindle pole body (SPB) assembly. The 18 SPB components are arranged into five sub-complexes: the y-tubulin complex that nucleates microtubules, the linker proteins that connect the y-tubulin complex to the cytoplasmic and nuclear face of the core SPB, the soluble core SPB/satellite components that form the foundation of the SPB and SPB precursor, the membrane anchors that tether the core SPB in the NE and the half-bridge components that are important for SPB assembly. Based on cytological analysis of SPB intermediates in wild-type and mutant yeast cells, SPB duplication can be divided into three steps: elongation of the half-bridge and formation of the satellite, which contains soluble precursors to the SPB; expansion of the satellite into a duplication plaque and fenestration of the NE; and insertion into the NE and assembly of nuclear components to create duplicated side-by-side SPBs.
The NE has evolved as a barrier to organize and protect the genome. Protein complexes associated with the NE play a critical role in the transport of macromolecules into and out of the nucleus, chromosome segregation and positioning, regulation of gene expression and ribosome assembly and nuclear structure and migration. One such complex is the spindle pole body (SPB) of Saccharomyces cerevisiae, which is the yeast MTOC and is the functional equivalent of the centrosome. Because the NE remains intact throughout the budding yeast cell cycle, the duplication of the SPB within an intact NE poses an issue in terms of both membrane insertion and mitotic spindle assembly. The solution employed by S. cerevisiae is to embed the SPB in the NE throughout the yeast lifecycle. Therefore, the SPB simultaneously nucleates nuclear microtubules involved in spindle assembly and cytoplasmic microtubules required to position the nucleus. Assembly of the SPB requires insertion of cytoplasmic precursors into the NE (Figure 1). How a roughly 0.5 gigadalton complex is inserted into the double lipid bilayer of the NE and how SPB duplication is restricted to once-and-only-once per cell cycle are key questions that my lab is interested in understanding at a molecular level. The SPB is an excellent model to study how the NE is remodeled to allow insertion of protein complexes because it occurs at a specific time (late G1) and place (at the distal tip of the half-bridge, approximately 100 nm from the pre-existing SPB) in the cell.
Analyzing the role of the NE in SPB assembly: two models.
As the sole site of microtubule nucleation in budding yeast, the SPB must be assembled and inserted into the NE once every cell cycle. Although the exact mechanism of SPB insertion is unknown, genetic analysis of SPB assembly has shown that the membrane anchors and SUN protein Mps3 play a role in this process. Therefore, we were surprised to find that the function of Mps3 in SPB assembly could be bypassed if specific subunits of the nuclear pore complex (NPC) were also deleted. It is difficult to envision how the SPB could duplicate in the absence of a structural protein such as Mps3, so is our goal to determine how SPBs are anchored in the NE and understand their relationship to NPCs by addressing the following:
- Do NE complexes such as the SPB and NPC share a common insertion factor? How are NE proteins distributed between the NPC and SPB?
- What is the composition and structure of the NE, and how does this change during SPB duplication or NPC insertion?
We are currently framing our approach to these questions in terms of the following two models: The first one focuses on a limiting protein that modulates the insertion of large protein complexes into the membrane; the second one focuses on changes in the lipid composition of the nuclear membrane to facilitate insertion. Either model could account for the Mps3-independent SPB duplication that we have observed. Our studies will elucidate the mechanism and control of SPB insertion. In addition, our work will lead to important discoveries regarding the physical structure of the NE, including lipid composition, INM-ONM spacing, NE integrity and the control of NE protein distribution in space and time.
Evolution of SPB components.
Our unexpected finding that Mps3 and other SPB components are non-essential in certain genetic backgrounds made us reconsider the overall function of SPB proteins and their role in duplication and microtubule nucleation. We wondered if the organelle might be more plastic than originally anticipated and if small changes in the primary sequence of cytoskeletal components could translate into large changes in the organization of the microtubule cytoskeleton, resulting in differences in morphology, nuclear movements, cell division and growth. To address this issue we turned to Ashyba gossypii, a multinucleated filamentous fungus that is closely related to the budding yeast S. cerevisiae. In collaboration with Dr. Peter Philippsen’s lab, we characterized the microtubule cytoskeleton of A. gossypii using both high-resolution real-time microscopy and electron microscopy. A. gossypii contains a single, nuclear-associated spindle pole body with an overall laminar structure similar to that observed in budding yeast, but it has distinct differences on the cytoplasmic side, which are important for cytoplasmic microtubule nucleation and nuclear movements. Mutation of A. gossypii SPB components did not mimic the analogous mutant in budding yeast most likely because nuclear migration into a bud once every cell cycle is not critical for cell viability. This suggests that the multinucleate growth mode of A. gossypii has resulted in different demands on the microtubule cytoskeleton that have driven evolution of SPB components and microtubule regulators to fit its unique life-style. Our findings are also consistent with the notion that the SPB may be able to duplicate by more than one mechanism; the requirement for certain SPB proteins might be highly dependent on proteins and other factors present in the nuclear membrane. Our ongoing studies of spindle structure and SPB duplication in Ashbya will allow us to test these ideas.
INM composition and function
The three-dimensional organization of chromosomes within the nucleus of eukaryotes is non-random and changes in chromosome position can have dramatic effects on gene expression by the establishment and maintenance of heritable transcriptionally repressive or active subdomains. The relationship between nuclear positioning and the epigenetic control of gene expression has been extensively characterized in S. cerevisiae at the telomeres, where their localization at the nuclear periphery is associated with transcriptional repression. Two partially redundant pathways lead to telomere tethering: the yKu70/yKu80 pathway and the silent-information regulator (Sir) pathway. We showed that Mps3 plays a role in telomere tethering and transcriptional regulation during S phase in budding yeast. At the time of our original study, Mps3 was the first INM protein known to be involved in telomere tethering. Following our demonstration that Mps3 functions as a tether for the Sir4 pathway, other labs provided evidence that Mps3 is important for telomere length maintenance and tethering via yKu70/80 binding to Est1/2. In collaboration with Dr. Craig Peterson’s lab, we showed that Mps3 is important for tethering certainly types of non-repairable or slowly repairable DNA breaks at the nuclear periphery to prevent spurious recombination from occurring.
What is the role of the NE in chromosome organization?
Despite evidence from virtually every eukaryote that chromosome domains associate with membrane-associated proteins and recent identification of a number of widely conserved INM proteins that play a role in chromosome organization, key questions remain. Do nuclear membrane proteins directly or indirectly interact with chromosomes? Is the mechanism of chromosome interaction identical at all heterochromatic regions (telomeres, centromeres and rDNA)? How are chromosome domains organized? Do different INM proteins form distinct chromosome domains or do they cooperate or coordinate chromosome organization? Does silencing precede tethering or vice versa? To resolve these questions we need to establish a system in which both silencing and chromosome position can be simultaneously monitored and manipulated. Our approach is a combination of live cell imaging and yeast genetics.
How do proteins localize to the INM?
Localization of proteins to the INM is critical for chromosome position, gene expression, recombination and chromosome segregation. The mechanism(s) by which proteins are localized to the INM is poorly understood in comparison to our knowledge of protein targeting to other subcellular compartments. Three general models of transport have been proposed: the first involves diffusion through the NPC and tethering by lamins or chromatin, the second involves an INM sorting motif and the third involves a classical nuclear localization sequence. Examination of the import of SUN proteins in yeast, worms and tissue culture cells has failed to identify a consensus pathway by which these proteins are targeted to the INM. This could be due to different requirements for nuclear transport in each system because of variations in nuclear structure and development, differences in the sequence of SUN proteins, or even differences in assays used for INM localization in each study. Given the importance of INM localization for genome integrity, it is our goal to systematically study INM localization in the budding yeast system. Our studies are aimed at addressing two primary questions:
- What is the role of the histone variant H2A.Z in localizing proteins to the INM?
- What are the protein components of the INM and how do they localize there?
As our inventory of INM proteins expands, we anticipate that our studies will expand to include topics such as nuclear shape, spacing between INM and ONM, distribution of NPCs and membrane synthesis. By understanding these processes at a basic molecular level, we will better understand how INM protein dysfunction leads to human disease.