bims-nucpor 2021-10-03 papers

Issue of 2021–10–03
five papers selected by
Sara Mingu, Johannes Gutenberg University

J Vis Exp. 2021 Sep 10.

Nuclei Isolation and Super-Resolution Structured Illumination Microscopy for Examining Nucleoporin Alterations in Human Neurodegeneration.

Alyssa N Coyne, Jeffrey D Rothstein.

The nuclear pore complex (NPC) is a complex macromolecular structure comprised of multiple copies of ~30 different nucleoporin proteins (Nups). Collectively, these Nups function to regulate genome organization, gene expression, and nucleocytoplasmic transport (NCT). Recently, defects in NCT and alterations to specific Nups have been identified as early and prominent pathologies in multiple neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), Alzheimer's Disease (AD)/Frontotemporal Dementia (FTD), and Huntington's Disease (HD). Advances in both light and electron microscopy allow for a thorough examination of sub-cellular structures, including the NPC and its Nup constituents, with increased precision and resolution. Of the commonly used techniques, super-resolution structured illumination microscopy (SIM) affords the unparalleled opportunity to study the localization and expression of individual Nups using conventional antibody-based labeling strategies. Isolation of nuclei prior to SIM enables the visualization of individual Nup proteins within the NPC and nucleoplasm in fully and accurately reconstructed 3D space. This protocol describes a procedure for nuclei isolation and SIM to evaluate Nup expression and distribution in human iPSC-derived CNS cells and postmortem tissues.

DOI: https://doi.org/10.3791/62789

Cells. 2021 Sep 21. pii: 2493. [Epub ahead of print]10(9):

Formation of Non-Nucleoplasmic Proteasome Foci during the Late Stage of Hyperosmotic Stress.

Jeeyoung Lee, Ly Thi Huong Luu Le, Eunkyoung Kim, Min Jae Lee.

Cellular stress induces the formation of membraneless protein condensates in both the nucleus and cytoplasm. The nucleocytoplasmic transport of proteins mainly occurs through nuclear pore complexes (NPCs), whose efficiency is affected by various stress conditions. Here, we report that hyperosmotic stress compartmentalizes nuclear 26S proteasomes into dense nuclear foci, independent of signaling cascades. Most of the proteasome foci were detected between the condensed chromatin mass and inner nuclear membrane. The proteasome-positive puncta were not colocalized with other types of nuclear bodies and were reversibly dispersed when cells were returned to the isotonic medium. The structural integrity of 26S proteasomes in the nucleus was slightly affected under the hyperosmotic condition. We also found that these insulator-body-like proteasome foci were possibly formed through disrupted nucleus-to-cytosol transport, which was mediated by the sequestration of NPC components into osmostress-responding stress granules. These data suggest that phase separation in both the nucleus and cytosol may be a major cell survival mechanism during hyperosmotic stress conditions.

Keywords: hyperosmotic stress; insulator body; liquid droplet; nuclear foci; nuclear pore complex; nucleocytoplasmic transport; phase separation; proteasome; stress granule

DOI: https://doi.org/10.3390/cells10092493

Mol Biol Cell. 2021 Sep 29. mbcE20090568

Photobleaching step analysis for robust determination of protein complex stoichiometries.

Johan Hummert, Klaus Yserentant, Theresa Fink, Jonas Euchner, Yin Xin Ho, Stanimir Asenov Tashev, Dirk-Peter Herten.

The counting of discrete photobleaching steps in fluorescence microscopy is ideally suited to study protein complex stoichiometry in situ. The counting range of photobleaching step analysis has significantly improved with more sophisticated algorithms for step detection, albeit at an increasing computational cost and with the necessity for high data quality. Here, we address concerns regarding robustness, automation, and experimental validation, optimizing both data acquisition and analysis. To make full use of the potential of photobleaching step analysis, we evaluate various labelling strategies with respect to their molecular brightness, photostability, and photoblinking. The developed analysis algorithm focuses on automation and computational efficiency. Moreover, we validate the developed methods with experimental data acquired on DNA origami labeled with defined fluorophore numbers, demonstrating counting of up to 35 fluorophores. Finally, we show the power of the combination of optimized trace acquisition and automated data analysis by counting labeled nucleoporin 107 in nuclear pore complexes of intact U2OS cells. The successful in situ application promotes this framework as a new resource enabling cell biologists to robustly determine the stoichiometries of molecular assemblies at the single-molecule level in an automated fashion.

DOI: https://doi.org/10.1091/mbc.E20-09-0568

Int J Cancer. 2021 Sep 30.

Nuclear transport proteins are secreted by cancer cells and identified as potential novel cancer biomarkers.

Pauline J van der Watt, Michael O Okpara, Andrew Wishart, M Iqbal Parker, Nelson C Soares, Jonathan M Blackburn, Virna D Leaner.

Previous studies have identified increased expression of members of the nuclear transport protein family in cancer cells. Recently, certain nuclear transport proteins have been reported to be secreted by cells and found in the serum. The aims of this study were to investigate the levels of multiple nuclear transport proteins secreted from cancer cells, and to determine their potential as diagnostic markers for cervical and oesophageal cancer. Mass spectrometry identified ten nuclear transport proteins in the secretome and exosomes of cultured cancer cells, and Western blot analysis confirmed increased secreted levels in cancer cells compared to normal. To investigate their presence in patient serum, ELISA assays were performed and revealed significantly increased levels of KPNβ1, CRM1, CAS, IPO5 and TNPO1 in cervical and oesophageal cancer patient serum compared to non-cancer controls. Significantly elevated KPNα2 and RAN levels were also identified in oesophageal cancer serum samples. Logistics regression analyses revealed IPO5 and TNPO1 to be the best performing individual candidate biomarkers in discriminating between cancer cases and controls. The combination of KPNβ1, CRM1, KPNα2, CAS, RAN, IPO5 and TNPO1 as a panel of biomarkers had the highest diagnostic capacity with an area under the curve (AUC) of 0.944 and 0.963, for cervical cancer and oesophageal cancer, and sensitivity of 92.5 % at 86.8 % specificity and 95.3 % sensitivity at 87.5 % specificity, respectively. These results suggest that nuclear transport proteins have potential as diagnostic biomarkers for cervical and oesophageal cancers, with a combination of protein family members being the best predictor. This article is protected by copyright. All rights reserved.

Keywords: cervical cancer; exosomes; nuclear transport proteins; oesophageal cancer; secretome

DOI: https://doi.org/10.1002/ijc.33832

J Chem Phys. 2021 Sep 28. 155(12): 125101

Phase separation vs aggregation behavior for model disordered proteins.

Ushnish Rana, Clifford P Brangwynne, Athanassios Z Panagiotopoulos.

Liquid-liquid phase separation (LLPS) is widely utilized by the cell to organize and regulate various biochemical processes. Although the LLPS of proteins is known to occur in a sequence-dependent manner, it is unclear how sequence properties dictate the nature of the phase transition and thereby influence condensed phase morphology. In this work, we have utilized grand canonical Monte Carlo simulations for a simple coarse-grained model of disordered proteins to systematically investigate how sequence distribution, sticker fraction, and chain length impact the formation of finite-size aggregates, which can preempt macroscopic phase separation for some sequences. We demonstrate that a normalized sequence charge decoration (SCD) parameter establishes a "soft" predictive criterion for distinguishing when a model protein undergoes macroscopic phase separation vs finite aggregation. Additionally, we find that this order parameter is strongly correlated with the critical density for phase separation, highlighting an unambiguous connection between sequence distribution and condensed phase density. Results obtained from an analysis of the order parameter reveal that at sufficiently long chain lengths, the vast majority of sequences are likely to phase separate. Our results suggest that classical LLPS should be the primary phase transition for disordered proteins when short-ranged attractive interactions dominate and suggest a possible reason behind recent findings of widespread phase separation throughout living cells.

DOI: https://doi.org/10.1063/5.0060046