The focus of the research in our laboratory is the nuclear pore complex, the immense structure that mediates highly selective, bi-directional transport between the nucleus and cytoplasm.  Recent work from ourselves and many others indicates that the nuclear pore is integrated with virtually all functions of the nucleus, at all stages of the cell cycle.  We take multiple approaches to address the various roles of the nuclear pore.  1.  Using GFP-fusion proteins in living cells together with confocal microscopy and photobleaching, we demonstrated that the nuclear pore is an unexpectedly dynamic structure. Several of the component proteins or nucleoporins move rapidly in and out of the complex.  We have shown that this mobility requires ongoing transcription and we are currently studying how the dynamics of the pore are linked to RNA transcription and export.   2.  We use extracts from the eggs of Xenopus frogs that can form nuclei in vitro.  By removal or modification of nuclear pore components in these extracts, we can study how individual nucleoporins are involved in nuclear assembly or other cell cycle dependent nuclear processes. 3. Another aspect of the lab is the structural analysis of nuclear pore proteins.  In collaboration with Alec Hodel, we determined the first structure of a domain of a nuclear pore protein, Nup98, and we continue to extend this work to other domains of Nup98 as well as other nuclear pore proteins.  4.  Nup98, a nucleoporin we have studied extensively, is a partner in chromosomal translocations found in Acute Myelogenous Leukemia.  We are using cell biology, biochemistry and genomic approaches to understand how alteration of this nucleoporin leads to the development of leukemia.

A Day in a Lab's Life: Of Xenopus and DNA, Emory Report October 1999

Summary of Published Research Results
For more complete information pertaining to these images, please refer to the cited publications.

I. Introduction

The vertebrate nuclear pore complex (NPC) is a 120 MDa structure that mediates traffic between the nucleus and cytoplasm of an eukaryotic cell. Each pore is composed of approximately 30 nuclear pore proteins, or nucleoporins (abbreviated Nup), that assemble to form three primary structures: the cytoplasmic filaments, a central eight-fold symmetrical ring, and a nuclear basket.

Figure 1A, Powers and Dasso (2004) Nat Cell Bio 2: 82-86.

Nup98 is a member of a subset of nucleoporins that contain a non-tandem FG (phenylalanine-glycine) amino acid repeat. FG repeats can come in several variations; FG, FxFG, or GLFG. Nup98 is the only vertebrate nucleoporin with the extended GLFG (glycine-leucine-phenylalanine-glycine) form. The GLFG motif is repeated numerous times in the central domain of nup98 and is believed to facilitate the movement of nuclear signal receptor proteins between the nucleus and the cytoplasm. Nup98 also has an amino terminal binding domain for the mRNA export protein Gle2 and a carboxyl terminal domain that is critical for targeting Nup98 to the nuclear pore complex.

The primary domains of Nup98
Figure 4A, Griffis et al. (2002) MBOC 13: 1282-1297.

II. Nup98's localization

Expression of GFP-Nup98 in living cells produces a distinct rim stain at the nuclear envelope and novel round structures in the nucleoplasm. These intranuclear structures, called GLFG bodies because their localization requires the presence of the GLFG domain, do not co-localize with any previously known nuclear structures, such as Cajal bodies, PML bodies or splicing factor speckles.

GLFG bodies in HeLa cells do not co-localize with
coilin, PML bodies, or splicing factor speckles.
Figure 2, Griffis et al. (2002) MBOC 13: 1282-1297.

Nup98 has previously been localized to the nuclear face of the pore and is known to directly bind to Nup96. We found by immunofluorescence and immunoelectron microscopy that Nup98 also localizes to the cytoplasmic face of the pore.

Localization of Nup98 after permeabilization by digitonin or triton X-100.
Figure 1A, Griffis et al. (2003) MBOC 14: 600-610.

Binding assays using GST-Nup98 and in vitro translated HA-tagged Nup88 and Nup214 indicate that at the cytoplasmic side of the pore, Nup98 interacts with Nup88.

Figure 3B, Griffis et al. (2003) MBOC 14: 600-610.

The autocatalytic cleavage of Nup98's final 8 kDa sequence at the c-terminus is critical for the binding of Nup98 with Nups 88 and 96. When Nup98 is mutagenzied at amino acid 864 so that a serine is replaced by an alanine, normal cleavage is eliminated and the protein no longer targets to the nuclear rim nor associates with Nups 88 and 96.

Figure 5B, Griffis et al. (2003) MBOC 14: 600-610.

Figure 5A, Griffis et al. (2003) MBOC 14: 600-610.

 

The crystal structure of the c-terminal domain of Nup98 confirmed the mechanism of cleavage and revealed a novel protein fold. This fold is the docking site for Nup98's 8 kDa cleavage peptide as well as Nup96 (on the nuclear side of the pore) or Nup88 (on the cytoplasmic side of the pore). However, if the peptide tail remains associated with this c-terminal region, it probably blocks Nup96 and Nup88 from binding at the same time.

A 3-D depiction of the c-terminus of Nup98 and the associated 8 kDa cleaved peptide
(positive charges in blue, negative charges in red).

Figure 4A, Hodel et al. (2002) Mol Cell 10: 347-358.

 

The same view of the internal architecture of the c-terminus of Nup98.

modified version of Figure 4B, Hodel et al. (2002) Mol Cell 10:347-358.

 

III. Nup98's mobility

FRAP (fluorescence recovery after photobleaching) experiments reveal that nup98 is a mobile nucleoporin, capable of moving on and off the nuclear rim as well as in and out of the GLFG bodies.

Figure 3A, Griffis et al. (2002) MBOC 13: 1282-1297.

click here to see a FRAP movie of the nuclear GLFG bodies

click here to see a FRAP movie of the nuclear rim

FLIP (fluorescence loss in photobleaching) experiments reveal that nup98 can shuttle between the nucleus and the cytoplasm.

Figure 5A, Griffis et al. (2002) MBOC MBOC 13: 1282-1297.

click here to see a FLIP movie of the nucleoplasm

IV. Nup98 and transcription

Treatment of GFP-98 expressing cells with the transcription inhibitors actinomycin D and DRB prevents recover after photobleaching at the nuclear rim and in the GLFG bodies, indicating that nup98's mobility is linked to ongoing transcription by RNA polymerases I and II.

Cells treated with actinomycin D
Figure 7A, Griffis et al. (2002) MBOC MBOC 13: 1282-1297.

Cells treated with DRB
Figure 7B, Griffis et al. (2002) MBOC 13: 1282-1297.