Casting a wider net: Differentiating between inner nuclear envelope and outer nuclear envelope transmembrane proteins
Genre
ReviewJournal
Date
2019-11-01Author
Tingey, MMudumbi, KC
Schirmer, EC
Yang, W
Permanent link to this record
http://hdl.handle.net/20.500.12613/4300Metadata
Show full item recordDOI
10.3390/ijms20215248Abstract
© 2019 by the authors. Licensee MDPI, Basel, Switzerland. The nuclear envelope (NE) surrounds the nucleus with a double membrane in eukaryotic cells. The double membranes are embedded with proteins that are synthesized on the endoplasmic reticulum and often destined specifically for either the outer nuclear membrane (ONM) or the inner nuclear membrane (INM). These nuclear envelope transmembrane proteins (NETs) play important roles in cellular function and participate in transcription, epigenetics, splicing, DNA replication, genome architecture, nuclear structure, nuclear stability, nuclear organization, and nuclear positioning. These vital functions are dependent upon both the correct localization and relative concentrations of NETs on the appropriate membrane of the NE. It is, therefore, important to understand the distribution and abundance of NETs on the NE. This review will evaluate the current tools and methodologies available to address this important topic.Citation to related work
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Quantifying Nucleoporin Stoichiometry Inside Single Nuclear Pore Complexes In vivo© 2015, Nature Publishing Group. All rights reserved. The nuclear pore complex (NPC) is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring-scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. NPCs are composed of ∼30 different nucleoporins (Nups), averaged at 8, 16 or 32 copies per NPC. This estimate has not been confirmed for individual NPCs in living cells due to the inherent difficulty of counting proteins inside single supramolecular complexes. Here we used single-molecule SPEED microscopy to directly count the copy-number of twenty-four different Nups within individual NPCs of live yeast, and found agreement as well as significant deviation from previous estimates. As expected, we counted 8 copies of four peripheral Nups and 16 copies of fourteen scaffold Nups. Unexpectedly, we counted a maximum of 16 copies of Nsp1 and Nic96, rather than 32 as previously estimated; and found only 10-15 copies of six other Nups, rather than 8 or 16 copies as expected. This in situ molecular-counting technology can test structure-function models of NPCs and other supramolecular structures in cells.
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Nucleoplasmic signals promote directed transmembrane protein import simultaneously via multiple channels of nuclear pores© 2020, The Author(s). Roughly 10% of eukaryotic transmembrane proteins are found on the nuclear membrane, yet how such proteins target and translocate to the nucleus remains in dispute. Most models propose transport through the nuclear pore complexes, but a central outstanding question is whether transit occurs through their central or peripheral channels. Using live-cell high-speed super-resolution single-molecule microscopy we could distinguish protein translocation through the central and peripheral channels, finding that most inner nuclear membrane proteins use only the peripheral channels, but some apparently extend intrinsically disordered domains containing nuclear localization signals into the central channel for directed nuclear transport. These nucleoplasmic signals are critical for central channel transport as their mutation blocks use of the central channels; however, the mutated proteins can still complete their translocation using only the peripheral channels, albeit at a reduced rate. Such proteins can still translocate using only the peripheral channels when central channel is blocked, but blocking the peripheral channels blocks translocation through both channels. This suggests that peripheral channel transport is the default mechanism that was adapted in evolution to include aspects of receptor-mediated central channel transport for directed trafficking of certain membrane proteins.
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HIGH-SPEED SINGLE-MOLECULE STUDIES OF THE STRUCTURE AND FUNCTION OF NUCLEAR PORE COMPLEXThe nuclear pore complex (NPC) is a proteinaceous gateway embedded in the nuclear envelope (NE) that regulates nucleocytoplasmic transport of molecules in eukaryotes. The NPC is formed by hundreds of proteins that are classified into approximately thirty different types of proteins called nucleoporin (Nup), each presents in multiples of eight copies. These nucleoporins are divided into two categories: the scaffold Nups forming the main structure of the NPC and the phenylalanine-glycine (FG) Nups that contain multiple repeats of intrinsically disordered and hydrophobic FG domains. These FG-Nups constitute the selective permeability barrier in the central channel of the NPC, which mediates the nuclear import of proteins into the nucleus, and the nuclear export of mRNA and pre-ribosomal subunits out of the nucleus. However, the precise copies of these Nups and their specific roles in the nucleocytoplasmic transport mechanism remain largely unknown. Moreover, the dysfunctional nuclear transport and the mutations of Nups have been closely associated with numerous human diseases, such as cancer, tumor and liver cirrhosis. We have developed and employed live-cell high-speed single-molecule microscopy to elucidate these critical questions remained in the nuclear transport and provide the fundamental knowledge for developing therapies. In this dissertation, I will present my major findings for the following three research projects: 1) determine the dynamic components of FG-Nups in native NPCs; 2) track the nucleocytoplasmic transport of transcription factor Smad proteins under ligand-activated conditions; and 3) elucidate the relationship between the nuclear export of mRNA and the presence and absence of specific Nups in live cells.Determination of the dynamic components for FG-Nups in native NPCs. Scaffold Nups have been intensively studied with electron microscopy to reveal their spatial positions and architecture in the past decades. However, the spatial organization of FG-Nups remains obscure due to the challenge of probing these disordered and dynamic polypeptides in live NPCs. By employing high-speed single-molecule microscopy and a live cell HaloTag labeling technique, I have mapped the spatial distribution for all eleven known mammalian FG-Nups within individual NPCs. Results show that all FG-Nups within NPCs are distinct in conformations and organized to form a ~300nm long hourglass shaped toroidal channel through the nuclear envelope. Exceptionally, the two remaining Nups (Nup98 and hCG1) almost extend through the entire NPC and largely overlap with all other FG-Nups in their spatial distributions. These results provide a complete map of FG-Nup organization within the NPC and also offer structural and functional insights into nucleocytoplasmic transport models. Tracking of the nucleocytoplasmic transport of Smad proteins under ligand-activated conditions. The inducement of transforming growth factor β1 (TGF-β1) was reported to cause the nuclear accumulation of Smad2/Smad4 heterocomplexes. However, the relationship between nuclear accumulation and the nucleocytoplasmic transport kinetics of Smad proteins in the presence of TGF-β1 remains obscure. By combining a high-speed single-molecule tracking microscopy technique (FRET), I tracked the entire TGF-β1-induced process of Smad2/Smad4 heterocomplex formation, as well as their transport through nuclear pore complex in live cells. The FRET results have revealed that in TGF-β1-treated cells, Smad2/Smad4 heterocomplexes formed in the cytoplasm, imported through the nuclear pore complexes as entireties, and finally dissociated in the nucleus. Moreover, it was found that basal-state Smad2 or Smad4 cannot accumulate in the nucleus without the presence of TGF-β1, mainly because both of them have an approximately twofold higher nuclear export efficiency compared to their nuclear import. Remarkably and reversely, heterocomplexes of Smad2/Smad4 induced by TGF-β1 can rapidly concentrate in the nucleus because of their almost fourfold higher nuclear import rate in comparison with their nuclear export rate. Thus, these single-molecule tracking data elucidate the basic molecular mechanism to understand nuclear transport and accumulation of Smad protein. Elucidation of the relationship between the nuclear export of mRNA and the presence and absence of specific Nups in live cells. In addition to explore the dynamic organization of NPC, in vivo characterization of the exact copy number and the specific function of each nucleoporin in the nuclear pore complex (NPC) remains desirable and challenging. Using live-cell high-speed super-resolution single-molecule microscopy, we first quantify the native copies of nuclear basket FG-Nups (Nup153, Nup50 and Tpr). Second, with same imaging technique and the auxin-inducible degradation strategies, I track the nuclear export of mRNA through native NPCs in absence of these FG-Nups. I found that these FG-Nups proteins possess the stoichiometric ratio of 1:1:1 and play distinct roles in the nuclear export of mRNAs in live cells. Tpr’s absence in the NPC dominantly reduces nuclear mRNA’s probability of entering the NPC for export. Complete depletion of Nup153 causes mRNA’s successful nuclear export efficiency dropped approximately four folds. Remarkably, the relationship between mRNA’s successful export efficiency and the copy number of Nup153 is not linear but instead follows a sigmoid function, in which mRNA can gain its maximum successful export efficiency as Nup153 increased from zero to around half of their full copies in the NPC. Lastly, the absence of Tpr or Nup153 also alters mRNA’s export routes through the NPC, but the removal of only Nup50 has almost no impact upon mRNA export route and kinetics.
