Molecular mechanisms underlying nucleocytoplasmic shuttling of actinin-4.

In addition to its well-known role as a crosslinker of actin filaments at focal-adhesion sites, actinin-4 is known to be localized to the nucleus. In this study, we reveal the molecular mechanism underlying nuclear localization of actinin-4 and its novel interactions with transcriptional regulators. We found that actinin-4 is imported into the nucleus through the nuclear pore complex in an importin-independent manner and is exported by the chromosome region maintenance-1 (CRM1)-dependent pathway. Nuclear actinin-4 levels were significantly increased in the late G2 phase of the cell cycle and were decreased in the G1 phase, suggesting that active release from the actin cytoskeleton was responsible for increased nuclear actinin-4 in late G2. Nuclear actinin-4 was found to interact with the INO80 chromatin-remodeling complex. It also directs the expression of a subset of cell-cycle-related genes and interacts with the upstream-binding factor (UBF)-dependent rRNA transcriptional machinery in the M phase. These findings provide molecular mechanisms for both nucleocytoplasmic shuttling of proteins that do not contain a nuclear-localization signal and cell-cycle-dependent gene regulation that reflects morphological changes in the cytoskeleton

Nucleocytoplasmic Shuttling of Cytoskeletal Proteins: Molecular Mechanism and Biological Significance

Various nuclear functional complexes contain cytoskeletal proteins as regulatory subunits; for example, nuclear actin participates in transcriptional complexes, and actin-related proteins are integral to chromatin remodeling complexes. Nuclear complexes such as these are involved in both basal and adaptive nuclear functions. In addition to nuclear import via classical nuclear transport pathways or passive diffusion, some large cytoskeletal proteins spontaneously migrate into the nucleus in a karyopherin-independent manner. The balance of nucleocytoplasmic distribution of such proteins can be altered by several factors, such as import versus export, or capture and release by complexes. The resulting accumulation or depletion of the nuclear populations thereby enhances or attenuates their nuclear functions. We propose that such molecular dynamics constitute a form of cytoskeleton-modulated regulation of nuclear functions which is mediated by the translocation of cytoskeletal components in and out of the nucleus

Redox-Sensitive Cysteines Confer Proximal Control of the Molecular Crowding Barrier in the Nuclear Pore

酸化ストレスが細胞の核膜機能を変える機構を解明 --環境に応じて分子の「混み具合」が変わる仕組み--. 京都大学プレスリリース. 2020-12-16.The nuclear pore complex forms a highly crowded selective barrier with intrinsically disordered regions at the nuclear membrane to coordinate nucleocytoplasmic molecular communications. Although oxidative stress is known to alter the barrier function, the molecular mechanism underlying this adaptive control of the nuclear pore complex remains unknown. Here we uncover a systematic control of the crowding barrier within the nuclear pore in response to various redox environments. Direct measurements of the crowding states using a crowding-sensitive FRET (Förster resonance energy transfer) probe reveal specific roles of the nuclear pore subunits that adjust the degree of crowding in response to different redox conditions, by adaptively forming or disrupting redox-sensitive disulfide bonds. Relationships between crowding control and the barrier function of the nuclear pore are investigated by single-molecular fluorescence measurements of nuclear transport. Based on these findings, we propose a proximal control model of molecular crowding in vivo that is dynamically regulated at the molecular level

核内構造の動的制御 - 細胞内骨格タンパク質の同定と実験的検証

京都大学0048新制・課程博士博士(生命科学)甲第15545号生博第188号新制||生||25(附属図書館)28023京都大学大学院生命科学研究科統合生命科学専攻(主査)教授 竹安 邦夫, 教授 石川 冬木, 教授 上村 匡学位規則第4条第1項該当Ph.D. (Life Sciences)Kyoto UniversityDA

Structural Mechanism of Nuclear Transport Mediated by Importin β and Flexible Amphiphilic Proteins

タンパク質が核膜孔を通り抜ける際の構造変化を解明 -細胞核内部への遺伝子導入技術への応用に期待-. 京都大学プレスリリース. 2014-11-27.Karyopherin β family proteins mediate the nuclear/cytoplasmic transport of various proteins through the nuclear pore complex (NPC), although they are substantially larger than the size limit of the NPC. To elucidate the molecular mechanism underlying this paradoxical function, we focused on the unique structures called HEAT repeats, which consist of repetitive amphiphilic α helices. An in vitro transport assay and FRAP analyses demonstrated that not only karyopherin β family proteins but also other proteins with HEAT repeats could pass through the NPC by themselves, and serve as transport mediators for their binding partners. Biochemical and spectroscopic analyses and molecular dynamics simulations of purified HEAT-rich proteins revealed that they interact with hydrophobic groups, including phenyl and alkyl groups, and undergo reversible conformational changes in tertiary structures, but not in secondary structures. These results show that conformational changes in the flexible amphiphilic motifs play a critical role in translocation through the NPC

Dissecting in vivo steady-state dynamics of karyopherin-dependent nuclear transport

Karyopherin-dependent molecular transport through the nuclear pore complex is maintained by constant recycling pathways of karyopherins coupled with the Ran-dependent cargo catch-and-release mechanism. Although many studies have revealed the bidirectional dynamics of karyopherins, the entire kinetics of the steady-state dynamics of karyopherin and cargo is still not fully understood. In this study, we used fluorescence recovery after photobleaching and fluorescence loss in photobleaching on live cells to provide convincing in vivo proof that karyopherin-mediated nucleocytoplasmic transport of cargoes is bidirectional. Continuous photobleaching of the cytoplasm of live cells expressing NLS cargoes led to progressive decrease of nuclear fluorescence signals. In addition, experimentally obtained kinetic parameters of karyopherin complexes were used to establish a kinetic model to explain the entire cargo import and export transport cycles facilitated by importin β. The results strongly indicate that constant shuttling of karyopherins, either free or bound to cargo, ensures proper balancing of nucleocytoplasmic distribution of cargoes and establishes effective regulation of cargo dynamics by RanGTP

Antibody-based analysis reveals "filamentous vs. non-filamentous" and "cytoplasmic vs. nuclear" crosstalk of cytoskeletal proteins.

To uncover the molecular composition and dynamics of the functional scaffold for the nucleus, three fractions of biochemically-stable nuclear protein complexes were extracted and used as immunogens to produce a variety of monoclonal antibodies. Many helix-based cytoskeletal proteins were identified as antigens, suggesting their dynamic contribution to nuclear architecture and function. Interestingly, sets of antibodies distinguished distinct subcellular localization of a single isoform of certain cytoskeletal proteins; distinct molecular forms of keratin and actinin were found in the nucleus. Their nuclear shuttling properties were verified by the apparent nuclear accumulations under inhibition of CRM1-dependent nuclear export. Nuclear keratins do not take an obvious filamentous structure, as was revealed by non-filamentous cytoplasmic keratin-specific monoclonal antibody. These results suggest the distinct roles of the helix-based cytoskeletal proteins in the nucleus

Evolutionary dynamics of spliceosomal intron revealed by in silico analyses of the P-Type ATPase superfamily genes.

It has been long debated whether spliceosomal introns originated in the common ancestor of eukaryotes and prokaryotes. In this study, we tested the possibility that extant introns were inherited from the common ancestor of eukaryotes and prokaryotes using in silico simulation. We first identified 21 intron positions that are shared among different families of the P-Type ATPase superfamily, some of which are known to have diverged before the separation of prokaryotes and eukaryotes. Theoretical estimates of the expected number of intron positions shared by different genes suggest that the introns at those 21 positions were inserted independently. There seems to be no intron that arose from before the diversification of the P-Type ATPase superfamily. Namely, the present introns were inserted after the separation of eukaryotes and prokaryotes

Prolines in the α-helix confer the structural flexibility and functional integrity of importin-β

The karyopherin family of nuclear transport receptors is composed of a long array of amphiphilic α-helices and undergoes flexible conformational changes to pass through the hydrophobic crowding barrier of the nuclear pore. Here, we focused on the characteristic enrichment of prolines in the middle of the outer α-helices of importin-β. When these prolines were substituted with alanine, nuclear transport activity was reduced drastically in vivo and in vitro, and caused a severe defect in mitotic progression. These mutations did not alter the overall folding of the helical repeat or affect its interaction with cargo or the regulatory factor Ran. However, in vitro and in silico analyses revealed that the mutant lost structural flexibility and could not undergo rapid conformational changes when transferring from a hydrophilic to hydrophobic environment or vice versa. These findings reveal the essential roles of prolines in ensuring the structural flexibility and functional integrity of karyopherins

In vivo dynamics of the cortical actin network revealed by fast-scanning atomic force microscopy

Together with lamellipodia and stress fibers, a dynamic network of actin filaments in the cell cortex plays a major role in the maintenance of cell morphology and motility. In contrast to lamellipodia, which have been well studied in various motile cells, the dynamics of actin filaments in the cell cortex have not yet been clarified due to a lack of proper imaging techniques. Here, we utilized high-speed atomic force microscopy for live-cell imaging and analyzed cortical actin dynamics in living cells. We successfully measured the polymerization rate and the frequency of filament synthesis in living COS-7 cells, and examined the associated effects of various inhibitors and actin-binding proteins. Actin filaments are synthesized beneath the plasma membrane and eventually descend into the cytoplasm. The inhibitors, cytochalasin B inhibited the polymerization, while jasplakinolide, inhibited the turnover of actin filaments as well as descension of the newly synthesized filaments, suggesting that actin polymerization near the membrane drives turnover of the cortical actin meshwork. We also determined how actin turnover is maintained and regulated by the free G-actin pool and G-actin binding proteins such as profilin and thymosin β4, and found that only a small amount of free G-actin was present in the cortex. Finally, we analyzed several different cell types, and found that the mesh size and the orientation of actin filaments were highly divergent, indicating the involvement of various actin-binding proteins in the maintenance and regulation of cortical actin architecture in each cell type