The organization of the nuclear periphery is crucial for many nuclear functions. Nuclear lamins form dense network at the nuclear periphery and play a substantial role in chromatin organization, transcription regulation and in organization of nuclear pore complexes (NPCs). Here, we show that TPR, the protein located preferentially within the nuclear baskets of NPCs, associates with lamin B1. The depletion of TPR affects the organization of lamin B1 but not lamin A/C within the nuclear lamina as shown by stimulated emission depletion microscopy. Finally, reduction of TPR affects the distribution of NPCs within the nuclear envelope and the effect can be reversed by simultaneous knock-down of lamin A/C or the overexpression of lamin B1. Our work suggests a novel role for the TPR at the nuclear periphery: the TPR contributes to the organization of the nuclear lamina and in cooperation with lamins guards the interphase assembly of nuclear pore complexes.

• Keywords
• Image analysis, Lamina, Lamins, Nuclear pore complex, Nucleus, Super-resolution imaging, TPR, Translocated promoter region,
• MeSH
• HeLa Cells MeSH
• Nuclear Lamina metabolism   ultrastructure   MeSH
• Nuclear Envelope metabolism   ultrastructure   MeSH
• Nuclear Pore Complex Proteins antagonists & inhibitors   genetics   metabolism   MeSH
• Lamin Type A antagonists & inhibitors   genetics   metabolism   MeSH
• Lamin Type B genetics   metabolism   MeSH
• Humans MeSH
• RNA, Small Interfering genetics   metabolism   MeSH
• Molecular Imaging MeSH
• Proto-Oncogene Proteins antagonists & inhibitors   genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Signal Transduction MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Nuclear Pore Complex Proteins MeSH
• Lamin Type A MeSH
• Lamin Type B MeSH
• LMNA protein, human MeSH Browser
• RNA, Small Interfering MeSH
• Proto-Oncogene Proteins MeSH
• TPR protein, human MeSH Browser

BACKGROUND: In the present work, we provide an account of structured illumination microscopy (SIM) imaging of fixed and immunolabeled plant probes. We take advantage of SIM, to superresolve intracellular structures at a considerable z-range and circumvent its low temporal resolution capacity during the study of living samples. Further, we validate the protocol for the imaging of fixed transgenic material expressing fluorescent protein-based markers of different subcellular structures. RESULTS: Focus is given on 3D imaging of bulky subcellular structures, such as mitotic and cytokinetic microtubule arrays as well as on the performance of SIM using multichannel imaging and the quantitative correlations that can be deduced. As a proof of concept, we provide a superresolution output on the organization of cortical microtubules in wild-type and mutant Arabidopsis cells, including aberrant preprophase microtubule bands and phragmoplasts in a cytoskeletal mutant devoid of the p60 subunit of the microtubule severing protein KATANIN and refined details of cytoskeletal aberrations in the mitogen activated protein kinase (MAPK) mutant mpk4. We further demonstrate, in a qualitative and quantitative manner, colocalizations between MPK6 and unknown dually phosphorylated and activated MAPK species and we follow the localization of the microtubule associated protein 65-3 (MAP65-3) in telophase and cytokinetic microtubular arrays. CONCLUSIONS: 3D SIM is a powerful, versatile and adaptable microscopy method for elucidating spatial relationships between subcellular compartments. Improved methods of sample preparation aiming to the compensation of refractive index mismatches, allow the use of 3D SIM in the documentation of complex plant cell structures, such as microtubule arrays and the elucidation of their interactions with microtubule associated proteins.

• Keywords
• Immunofluorescence, Microtubule associated proteins, Microtubules, Structured illumination microscopy,
• Publication type
• Journal Article MeSH